Hendrikus L Granzier

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• Granzier, H. L., & Labeit, S. (2025). Discovery of Titin and Its Role in Heart Function and Disease. Circulation research, 136(1), 135-157.
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  This review examines the giant elastic protein titin and its critical roles in heart function, both in health and disease, as discovered since its identification nearly 50 years ago. Encoded by the TTN (titin gene), titin has emerged as a major disease locus for cardiac disorders. Functionally, titin acts as a third myofilament type, connecting sarcomeric Z-disks and M-bands, and regulating myocardial passive stiffness and stretch sensing. Its I-band segment, which includes the N2B element and the PEVK (proline, glutamate, valine, and lysine-rich regions), serves as a viscoelastic spring, adjusting sarcomere length and force in response to cardiac stretch. The review details how alternative splicing of titin pre-mRNA produces different isoforms that greatly impact passive tension and cardiac function, under physiological and pathological conditions. Key posttranslational modifications, especially phosphorylation, play crucial roles in adjusting titin's stiffness, allowing for rapid adaptation to changing hemodynamic demands. Abnormal titin modifications and dysregulation of isoforms are linked to cardiac diseases such as heart failure with preserved ejection fraction, where increased stiffness impairs diastolic function. In addition, the review discusses the importance of the A-band region of titin in setting thick filament length and enhancing Ca² sensitivity, contributing to the Frank-Starling Mechanism of the heart. TTN truncating variants are frequently associated with dilated cardiomyopathy, and the review outlines potential disease mechanisms, including haploinsufficiency, sarcomere disarray, and altered thick filament regulation. Variants in TTN have also been linked to conditions such as peripartum cardiomyopathy and chemotherapy-induced cardiomyopathy. Therapeutic avenues are explored, including targeting splicing factors such as RBM20 (RNA binding motif protein 20) to adjust isoform ratios or using engineered heart tissues to study disease mechanisms. Advances in genetic engineering, including CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats), offer promise for modifying TTN to treat titin-related cardiomyopathies. This comprehensive review highlights titin's structural, mechanical, and signaling roles in heart function and the impact of TTN mutations on cardiac diseases.
• Coulson, Z., Kolb, J., Sabha, N., Karimi, E., Hourani, Z., Ottenheijm, C., Granzier, H., & Dowling, J. J. (2024). Generation of a novel mouse model of nemaline myopathy due to recurrent exon 55 deletion. Research square.
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  Biallelic pathogenic variants in the nebulin () gene lead to the congenital muscle disease nemaline myopathy. In-frame deletion of exon 55 (ΔExon55) is the most common disease-causing variant in . Previously, a mouse model of was developed; however, it presented an uncharacteristically severe phenotype with a near complete reduction in transcript expression that is not observed in exon 55 patients. We identified by RNA sequencing that the cause of this unexpectedly severe presentation in mice is the generation of a pseudoexon containing two premature termination codons (and promoting nonsense mediated decay) at the exon 55 deletion site. To prove that this is the cause of the loss of transcript, and to generate a more faithful model of the human disease, we used CRISPR gene editing to remove the pseudoexon sequence and replace it with human intron 54 sequence containing a validated cas9 gRNA protospacer. The resulting "hmz" mice have a significant reduction in pseudoexon formation (93.6% reduction), and a re-introduction of stable transcript expression. This new model has the characteristic features of nemaline myopathy at the physiological, histological, and molecular levels. Importantly, unlike the existing exon 55 deletion mice (which die by age 7 days), it survives beyond the first months and exhibits obvious signs of neuromuscular dysfunction. It thus provides a new, robust model for studying pathomechanisms and developing therapies for related nemaline myopathy.
• Dominic, K. L., Schmidt, A. V., Granzier, H., Campbell, K. S., & Stelzer, J. E. (2024). Mechanism-based myofilament manipulation to treat diastolic dysfunction in HFpEF. Frontiers in physiology, 15, 1512550.
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  Heart failure with preserved ejection fraction (HFpEF) is a major public health challenge, affecting millions worldwide and placing a significant burden on healthcare systems due to high hospitalization rates and limited treatment options. HFpEF is characterized by impaired cardiac relaxation, or diastolic dysfunction. However, there are no therapies that directly treat the primary feature of the disease. This is due in part to the complexity of normal diastolic function, and the challenge of isolating the mechanisms responsible for dysfunction in HFpEF. Without a clear understanding of the mechanisms driving diastolic dysfunction, progress in treatment development has been slow. In this review, we highlight three key areas of molecular dysregulation directly underlying impaired cardiac relaxation in HFpEF: altered calcium sensitivity in the troponin complex, impaired phosphorylation of myosin-binding protein C (cMyBP-C), and reduced titin compliance. We explore how targeting these pathways can restore normal relaxation, improve diastolic function, and potentially provide new therapeutic strategies for HFpEF treatment. Developing effective HFpEF therapies requires precision targeting to balance systolic and diastolic function, avoiding both upstream non-specificity and downstream rigidity. This review highlights three rational molecular targets with a strong mechanistic basis and potential for therapeutic success.
• Fabian, L., Karimi, E., Farman, G. P., Gohlke, J., Ottenheijm, C. A., Granzier, H. L., & Dowling, J. J. (2024). Comprehensive phenotypic characterization of an allelic series of zebrafish models of NEB-related nemaline myopathy. Human molecular genetics, 33(12), 1036-1054.
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  Nemaline myopathy (NM) is a rare congenital neuromuscular disorder characterized by muscle weakness and hypotonia, slow gross motor development, and decreased respiratory function. Mutations in at least twelve genes, all of each encode proteins that are either components of the muscle thin filament or regulate its length and stability, have been associated with NM. Mutations in Nebulin (NEB), a giant filamentous protein localized in the sarcomere, account for more than 50% of NM cases. At present, there remains a lack of understanding of whether NEB genotype influences nebulin function and NM-patient phenotypes. In addition, there is a lack of therapeutically tractable models that can enable drug discovery and address the current unmet treatment needs of patients. To begin to address these gaps, here we have characterized five new zebrafish models of NEB-related NM. These mutants recapitulate most aspects of NEB-based NM, showing drastically reduced survival, defective muscle structure, reduced contraction force, shorter thin filaments, presence of electron-dense structures in myofibers, and thickening of the Z-disks. This study represents the first extensive investigation of an allelic series of nebulin mutants, and thus provides an initial examination in pre-clinical models of potential genotype-phenotype correlations in human NEB patients. It also represents the first utilization of a set of comprehensive outcome measures in zebrafish, including correlation between molecular analyses, structural and biophysical investigations, and phenotypic outcomes. Therefore, it provides a rich source of data for future studies exploring the NM pathomechanisms, and an ideal springboard for therapy identification and development for NEB-related NM.
• Flanigan, E. G., Farman, G. P., Dennis, M. R., Wollman, L., Van Den Berg, M., Granzier, H., Banek, C. T., & Fregosi, R. F. (2024). Developmental nicotine exposure alters cardiovascular structure and function in neonatal and juvenile rats. American journal of physiology. Heart and circulatory physiology, 327(6), H1442-H1454.
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  Here we test the hypothesis that continuous nicotine exposure throughout pre- and postnatal development (developmental nicotine exposure, DNE) alters the cardiovascular structure and function in neonatal and juvenile rats. Echocardiography showed that DNE reduced left ventricular mass, left ventricular outflow tract (LVOT) diameter, and posterior wall thickness, but only in females. Both male and female DNE rats had a lower end-systolic volume, higher ejection fraction, and increased fractional shortening, with unchanged stroke volume and cardiac output. Left ventricular single cardiac myocytes from male and female DNE animals exhibited increased calcium-evoked maximal tension with no effect on EC. Tail-cuff plethysmography in awake rats showed that DNE males had lower systolic blood pressure and higher heart rate than control males. No significant changes in preload, afterload, or the in vitro renal artery response to vasodilators were observed. The results suggest that DNE enhances myocyte tension-generating capacity, possibly compensating for an unknown developmental insult, which may differ in males and females. Although this adaptation maintains normal resting cardiac function, it may lead to reduced cardiac reserve, increased energy demand, and elevated oxidative stress, potentially compromising both short- and long-term cardiovascular health in developing neonates. Developmental nicotine exposure (DNE) induced cardiovascular changes in neonatal/juvenile rats. Relative to controls, females had reduced left ventricular mass and dimensions, while both sexes had increased ejection fraction and fractional shortening. DNE increased calcium-evoked tension in cardiac myocytes, suggesting an adaptive mechanism as resting cardiac output was preserved. Despite normal resting function, these changes may reduce cardiac reserve, potentially compromising long-term cardiovascular health. These novel findings highlight how DNE disrupts cardiovascular development and function.
• Gohlke, J., Lindqvist, J., Hourani, Z., Heintzman, S., Tonino, P., Elsheikh, B., Morales, A., Vatta, M., Burghes, A., Granzier, H., & Roggenbuck, J. (2024). Pathomechanisms of Monoallelic variants in TTN causing skeletal muscle disease. Human molecular genetics, 33(23), 2003-2023.
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  Pathogenic variants in the titin gene (TTN) are known to cause a wide range of cardiac and musculoskeletal disorders, with skeletal myopathy mostly attributed to biallelic variants. We identified monoallelic truncating variants (TTNtv), splice site or internal deletions in TTN in probands with mild, progressive axial and proximal weakness, with dilated cardiomyopathy frequently developing with age. These variants segregated in an autosomal dominant pattern in 7 out of 8 studied families. We investigated the impact of these variants on mRNA, protein levels, and skeletal muscle structure and function. Results reveal that nonsense-mediated decay likely prevents accumulation of harmful truncated protein in skeletal muscle in patients with TTNtvs. Splice variants and an out-of-frame deletion induce aberrant exon skipping, while an in-frame deletion produces shortened titin with intact N- and C-termini, resulting in disrupted sarcomeric structure. All variant types were associated with genome-wide changes in splicing patterns, which represent a hallmark of disease progression. Lastly, RNA-seq studies revealed that GDF11, a member of the TGF-β superfamily, is upregulated in diseased tissue, indicating that it might be a useful therapeutic target in skeletal muscle titinopathies.
• Gregorich, Z. R., Zhang, Y., Kamp, T. J., Granzier, H., & Guo, W. (2024). Mechanisms of RBM20 Cardiomyopathy: Insights From Model Systems. Circulation. Genomic and precision medicine, e004355.
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  RBM20 (RNA-binding motif protein 20) is a vertebrate- and muscle-specific RNA-binding protein that belongs to the serine-arginine-rich family of splicing factors. The gene was first identified as a dilated cardiomyopathy-linked gene over a decade ago. Early studies in knockout rodents implicated disrupted splicing of RBM20 target genes as a causative mechanism. Clinical studies show that pathogenic variants in are linked to aggressive dilated cardiomyopathy with early onset heart failure and high mortality. Subsequent studies employing pathogenic variant knock-in animal models revealed that variants in a specific portion of the arginine-serine-rich domain in RBM20 not only disrupt splicing but also hinder nucleocytoplasmic transport and lead to the formation of RBM20 biomolecular condensates in the sarcoplasm. Conversely, mice harboring a disease-associated variant in the RRM (RNA recognition motif) do not show evidence of adverse remodeling or exhibit sudden death despite disrupted splicing of RBM20 target genes. Thus, whether disrupted splicing, biomolecular condensates, or both contribute to dilated cardiomyopathy is under debate. Beyond this, additional questions remain, such as whether there is sexual dimorphism in the presentation of RBM20 cardiomyopathy. What are the clinical features of RBM20 cardiomyopathy and why do some individuals develop more severe disease than others? In this review, we summarize the reported observations and discuss potential mechanisms of RBM20 cardiomyopathy derived from studies employing in vivo animal models and in vitro human-induced pluripotent stem cell-derived cardiomyocytes. Potential therapeutic strategies to treat RBM20 cardiomyopathy are also discussed.
• Karimi, E., Gohlke, J., van der Borgh, M., Lindqvist, J., Hourani, Z., Kolb, J., Cossette, S., Lawlor, M. W., Ottenheijm, C., & Granzier, H. (2024). Characterization of NEB pathogenic variants in patients reveals novel nemaline myopathy disease mechanisms and omecamtiv mecarbil force effects. Acta neuropathologica, 147(1), 72.
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  Nebulin, a critical protein of the skeletal muscle thin filament, plays important roles in physiological processes such as regulating thin filament length (TFL), cross-bridge cycling, and myofibril alignment. Pathogenic variants in the nebulin gene (NEB) cause NEB-based nemaline myopathy (NEM2), a genetically heterogeneous disorder characterized by hypotonia and muscle weakness, currently lacking curative therapies. In this study, we examined a cohort of ten NEM2 patients, each with unique pathogenic variants, aiming to understand their impact on mRNA, protein, and functional levels. Results show that pathogenic truncation variants affect NEB mRNA stability and lead to nonsense-mediated decay of the mutated transcript. Moreover, a high incidence of cryptic splice site activation was found in patients with pathogenic splicing variants that are expected to disrupt the actin-binding sites of nebulin. Determination of protein levels revealed patients with either relatively normal or markedly reduced nebulin. We observed a positive relation between the reduction in nebulin and a reduction in TFL, or reduction in tension (both maximal and submaximal tension). Interestingly, our study revealed a pathogenic duplication variant in nebulin that resulted in a four-copy gain in the triplicate region of NEB and a much larger nebulin protein and longer TFL. Additionally, we investigated the effect of Omecamtiv mecarbil (OM), a small-molecule activator of cardiac myosin, on force production of type 1 muscle fibers of NEM2 patients. OM treatment substantially increased submaximal tension across all NEM2 patients ranging from 87 to 318%, with the largest effects in patients with the lowest level of nebulin. In summary, this study indicates that post-transcriptional or post-translational mechanisms regulate nebulin expression. Moreover, we propose that the pathomechanism of NEM2 involves not only shortened but also elongated thin filaments, along with the disruption of actin-binding sites resulting from pathogenic splicing variants. Significantly, our findings highlight the potential of OM treatment to improve skeletal muscle function in NEM2 patients, especially those with large reductions in nebulin levels.
• Laitila, J., Seaborne, R. A., Ranu, N., Kolb, J. S., Wallgren-Pettersson, C., Witting, N., Vissing, J., Vilchez, J. J., Zanoteli, E., Palmio, J., Huovinen, S., Granzier, H., & Ochala, J. (2024). Myosin ATPase inhibition fails to rescue the metabolically dysregulated proteome of nebulin-deficient muscle. The Journal of physiology, 602(20), 5229-5245.
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  Nemaline myopathy (NM) is a genetic muscle disease, primarily caused by mutations in the NEB gene (NEB-NM) and with muscle myosin dysfunction as a major molecular pathogenic mechanism. Recently, we have observed that the myosin biochemical super-relaxed state was significantly impaired in NEB-NM, inducing an aberrant increase in ATP consumption and remodelling of the energy proteome in diseased muscle fibres. Because the small-molecule Mavacamten is known to promote the myosin super-relaxed state and reduce the ATP demand, we tested its potency in the context of NEB-NM. We first conducted in vitro experiments in isolated single myofibres from patients and found that Mavacamten successfully reversed the myosin ATP overconsumption. Following this, we assessed its short-term in vivo effects using the conditional nebulin knockout (cNeb KO) mouse model and subsequently performing global proteomics profiling in dissected soleus myofibres. After a 4 week treatment period, we observed a remodelling of a large number of proteins in both cNeb KO mice and their wild-type siblings. Nevertheless, these changes were not related to the energy proteome, indicating that short-term Mavacamten treatment is not sufficient to properly counterbalance the metabolically dysregulated proteome of cNeb KO mice. Taken together, our findings emphasize Mavacamten potency in vitro but challenge its short-term efficacy in vivo. KEY POINTS: No cure exists for nemaline myopathy, a type of genetic skeletal muscle disease mainly derived from mutations in genes encoding myofilament proteins. Applying Mavacamten, a small molecule directly targeting the myofilaments, to isolated membrane-permeabilized muscle fibres from human patients restored myosin energetic disturbances. Treating a mouse model of nemaline myopathy in vivo with Mavacamten for 4 weeks, remodelled the skeletal muscle fibre proteome without any noticeable effects on energetic proteins. Short-term Mavacamten treatment may not be sufficient to reverse the muscle phenotype in nemaline myopathy.
• Mira Hernandez, J., Shen, E. Y., Ko, C. Y., Hourani, Z., Spencer, E. R., Smoliarchuk, D., Bossuyt, J., Granzier, H., Bers, D. M., & Hegyi, B. (2024). Differential sex-dependent susceptibility to diastolic dysfunction and arrhythmia in cardiomyocytes from obese diabetic HFpEF model. Cardiovascular research.
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  Sex-differences in heart failure with preserved ejection fraction (HFpEF) are important, but key mechanisms involved are incompletely understood. While animal models can inform about sex-dependent cellular and molecular changes, many previous preclinical HFpEF models have failed to recapitulate sex-dependent characteristics of human HFpEF. We tested for sex-differences in HFpEF using a two-hit mouse model (leptin receptor-deficient db/db mice plus aldosterone infusion for 4 weeks; db/db+Aldo).
• Smith, J. E., & Granzier, H. (2024). In vivo gene editing of CAMKIID: out with the bad and in with the good. The Journal of clinical investigation, 134(1).
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  The ability to change an organism's DNA through gene editing is of great importance for the prevention and treatment of genetic and acquired diseases. Rapid progress has been made during the last decade due to the discovery and refinement of CRISPR/Cas9 as an accurate, fast, and reliable genome editing technique. In this issue of the JCI, Lebek et al. present the culmination from a line of work in the Olson laboratory focused on in vivo gene editing of CAMK2D. The paper presents a combined state-of-the-art gene therapy approach that demonstrates how gene therapy can yield cardioprotection in a mouse model and takes notable steps toward potential applicability in patients.
• Strom, J., Bull, M., Gohlke, J., Saripalli, C., Methawasin, M., Gotthardt, M., & Granzier, H. (2024). Titin's cardiac-specific N2B element is critical to mechanotransduction during volume overload of the heart. Journal of molecular and cellular cardiology, 191, 40-49.
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  The heart has the ability to detect and respond to changes in mechanical load through a process called mechanotransduction. In this study, we focused on investigating the role of the cardiac-specific N2B element within the spring region of titin, which has been proposed to function as a mechanosensor. To assess its significance, we conducted experiments using N2B knockout (KO) mice and wildtype (WT) mice, subjecting them to three different conditions: 1) cardiac pressure overload induced by transverse aortic constriction (TAC), 2) volume overload caused by aortocaval fistula (ACF), and 3) exercise-induced hypertrophy through swimming. Under conditions of pressure overload (TAC), both genotypes exhibited similar hypertrophic responses. In contrast, WT mice displayed robust left ventricular hypertrophy after one week of volume overload (ACF), while the KO mice failed to undergo hypertrophy and experienced a high mortality rate. Similarly, swim exercise-induced hypertrophy was significantly reduced in the KO mice. RNA-Seq analysis revealed an abnormal β-adrenergic response to volume overload in the KO mice, as well as a diminished response to isoproterenol-induced hypertrophy. Because it is known that the N2B element interacts with the four-and-a-half LIM domains 1 and 2 (FHL1 and FHL2) proteins, both of which have been associated with mechanotransduction, we evaluated these proteins. Interestingly, while volume-overload resulted in FHL1 protein expression levels that were comparable between KO and WT mice, FHL2 protein levels were reduced by over 90% in the KO mice compared to WT. This suggests that in response to volume overload, FHL2 might act as a signaling mediator between the N2B element and downstream signaling pathways. Overall, our study highlights the importance of the N2B element in mechanosensing during volume overload, both in physiological and pathological settings.
• Van der Pijl, R. J., Ma, W., Lewis, C. T., Haar, L., Buhl, A., Farman, G. P., Rhodehamel, M., Jani, V. P., Nelson, O. L., Zhang, C., Granzier, H., & Ochala, J. (2024). Increased cardiac myosin super-relaxation as an energy saving mechanism in hibernating grizzly bears. Molecular metabolism, 92, 102084.
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  The aim of the present study was to define whether cardiac myosin contributes to energy conservation in the heart of hibernating mammals.
• de Winter, J. M., Molenaar, J. P., Yuen, M., van der Pijl, R., Shen, S., Conijn, S., van de Locht, M., Willigenburg, M., Bogaards, S. J., van Kleef, E. S., Lassche, S., Persson, M., Rassier, D. E., Sztal, T. E., Ruparelia, A. A., Oorschot, V., Ramm, G., Hall, T. E., Xiong, Z., , Johnson, C. N., et al. (2024). KBTBD13 is an actin-binding protein that modulates muscle kinetics. The Journal of clinical investigation, 134(3).
• van den Berg, M., Shi, Z., Claassen, W. J., Hooijman, P., Lewis, C. T., Andersen, J. L., van der Pijl, R. J., Bogaards, S. J., Conijn, S., Peters, E. L., Begthel, L. P., Uijterwijk, B., Lindqvist, J., Langlais, P. R., Girbes, A. R., Stapel, S., Granzier, H., Campbell, K. S., Ma, W., , Irving, T., et al. (2024). Super-relaxed myosins contribute to respiratory muscle hibernation in mechanically ventilated patients. Science translational medicine, 16(758), eadg3894.
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  Patients receiving mechanical ventilation in the intensive care unit (ICU) frequently develop contractile weakness of the diaphragm. Consequently, they may experience difficulty weaning from mechanical ventilation, which increases mortality and poses a high economic burden. Because of a lack of knowledge regarding the molecular changes in the diaphragm, no treatment is currently available to improve diaphragm contractility. We compared diaphragm biopsies from ventilated ICU patients ( = 54) to those of non-ICU patients undergoing thoracic surgery ( = 27). By integrating data from myofiber force measurements, x-ray diffraction experiments, and biochemical assays with clinical data, we found that in myofibers isolated from the diaphragm of ventilated ICU patients, myosin is trapped in an energy-sparing, super-relaxed state, which impairs the binding of myosin to actin during diaphragm contraction. Studies on quadriceps biopsies of ICU patients and on the diaphragm of previously healthy mechanically ventilated rats suggested that the super-relaxed myosins are specific to the diaphragm and not a result of critical illness. Exposing slow- and fast-twitch myofibers isolated from the diaphragm biopsies to small-molecule compounds activating troponin restored contractile force in vitro. These findings support the continued development of drugs that target sarcomere proteins to increase the calcium sensitivity of myofibers for the treatment of ICU-acquired diaphragm weakness.
• Barefield, D. Y., Tonino, P., Woulfe, K. C., Rahmanseresht, S., O'Leary, T. S., Burnham, H. V., Wasserstrom, J. A., Kirk, J. A., Previs, M. J., Granzier, H. L., & McNally, E. M. (2023). Myosin-binding protein H-like regulates myosin-binding protein distribution and function in atrial cardiomyocytes. Proceedings of the National Academy of Sciences of the United States of America, 120(51), e2314920120.
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  Mutations in atrial-enriched genes can cause a primary atrial myopathy that can contribute to overall cardiovascular dysfunction. encodes myosin-binding protein H-like (MyBP-HL), an atrial sarcomere protein that shares domain homology with the carboxy-terminus of cardiac myosin-binding protein-C (cMyBP-C). The function of MyBP-HL and the relationship between MyBP-HL and cMyBP-C is unknown. To decipher the roles of MyBP-HL, we used structured illumination microscopy, immuno-electron microscopy, and mass spectrometry to establish the localization and stoichiometry of MyBP-HL. We found levels of cMyBP-C, a major regulator of myosin function, were half as abundant compared to levels in the ventricle. In genetic mouse models, loss of MyBP-HL doubled cMyBP-C abundance in the atria, and loss of cMyBP-C doubled MyBP-HL abundance in the atria. Structured illumination microscopy showed that both proteins colocalize in the C-zone of the A-band, with MyBP-HL enriched closer to the M-line. Immuno-electron microscopy of mouse atria showed MyBP-HL strongly localized 161 nm from the M-line, consistent with localization to the third 43 nm repeat of myosin heads. Both cMyBP-C and MyBP-HL had less-defined sarcomere localization in the atria compared to ventricle, yet areas with the expected 43 nm repeat distance were observed for both proteins. Isometric force measurements taken from control and null single atrial myofibrils revealed that loss of accelerated the linear phase of relaxation. These findings support a mechanism where MyBP-HL regulates cMyBP-C abundance to alter the kinetics of sarcomere relaxation in atrial sarcomeres.
• Casey, J. G., Kim, E. S., Joseph, R., Li, F., Granzier, H., & Gupta, V. A. (2023). NRAP reduction rescues sarcomere defects in nebulin-related nemaline myopathy. Human molecular genetics, 32(10), 1711-1721.
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  Nemaline myopathy (NM) is a rare neuromuscular disorder associated with congenital or childhood-onset of skeletal muscle weakness and hypotonia, which results in limited motor function. NM is a genetic disorder and mutations in 12 genes are known to contribute to autosomal dominant or recessive forms of the disease. Recessive mutations in nebulin (NEB) are the most common cause of NM affecting about 50% of patients. Because of the large size of the NEB gene and lack of mutational hot spots, developing therapies that can benefit a wide group of patients is challenging. Although there are several promising therapies under investigation, there is no cure for NM. Therefore, targeting disease modifiers that can stabilize or improve skeletal muscle function may represent alternative therapeutic strategies. Our studies have identified Nrap upregulation in nebulin deficiency that contributes to structural and functional deficits in NM. We show that genetic ablation of nrap in nebulin deficiency restored sarcomeric disorganization, reduced protein aggregates and improved skeletal muscle function in zebrafish. Our findings suggest that Nrap is a disease modifier that affects skeletal muscle structure and function in NM; thus, therapeutic targeting of Nrap in nebulin-related NM and related diseases may be beneficial for patients.
• Fukuda, N., Granzier, H., Ishiwata, S., & Morimoto, S. (2023). Editorial: Recent Advances on Myocardium Physiology, Volume II. Frontiers in physiology, 14, 1170396.
• Karimi, E., van der Borgh, M., Lindqvist, J., Gohlke, J., Hourani, Z., Kolb, J., Cossette, S., Lawlor, M. W., Ottenheijm, C., & Granzier, H. (2023). Characterization of mutations in patients reveals novel nemaline myopathy disease mechanisms and omecamtiv mecarbil force effects. bioRxiv : the preprint server for biology.
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  Nebulin, a critical protein of the skeletal muscle thin filament, plays important roles in physiological processes such as regulating thin filament length (TFL), cross-bridge cycling, and myofibril alignment. Mutations in the nebulin gene ( ) cause NEB-based nemaline myopathy (NEM2), a genetically heterogeneous disorder characterized by hypotonia and muscle weakness, currently lacking therapies targeting the underlying pathological mechanisms. In this study, we examined a cohort of ten NEM2 patients, each with unique mutations, aiming to understand their impact on mRNA, protein, and functional levels. Results show that truncation mutations affect mRNA stability and lead to nonsense-mediated decay of the mutated transcript. Moreover, a high incidence of cryptic splice site activation was found in patients with splicing mutations which is expected to disrupt the actin-binding sites of nebulin. Determination of protein levels revealed patients with relatively normal nebulin levels and others with markedly reduced nebulin. We observed a positive relation between the reduction in nebulin and a reduction in TFL, and a positive relation between the reduction in nebulin level and the reduction in tension (both maximal and submaximal tension). Interestingly, our study revealed a duplication mutation in nebulin that resulted in a larger nebulin protein and longer TFL. Additionally, we investigated the effect of Omecamtiv mecarbil (OM), a small-molecule activator of cardiac myosin, on force production of type I muscle fibers of NEM2 patients. OM treatment substantially increased submaximal tension across all NEM2 patients ranging from 87-318%, with the largest effects in patients with the lowest level of nebulin. In summary, this study indicates that post-transcriptional or post-translational mechanisms regulate nebulin expression. Moreover, we propose that the pathomechanism of NEM2 involves not only shortened but also elongated thin filaments, along with the disruption of actin-binding sites resulting from splicing mutations. Significantly, our findings highlight the potential of OM treatment to improve skeletal muscle function in NEM2 patients, especially those with large reductions in nebulin levels.
• Lindqvist, J., & Granzier, H. (2023). Pharmacological Inhibition of Myostatin in a Mouse Model of Typical Nemaline Myopathy Increases Muscle Size and Force. International journal of molecular sciences, 24(20).
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  Nemaline myopathy is one of the most common non-dystrophic congenital myopathies. Individuals affected by this condition experience muscle weakness and muscle smallness, often requiring supportive measures like wheelchairs or respiratory support. A significant proportion of patients, approximately one-third, exhibit compound heterozygous nebulin mutations, which usually give rise to the typical form of the disease. Currently, there are no approved treatments available for nemaline myopathy. Our research explored the modulation of myostatin, a negative regulator of muscle mass, in combating the muscle smallness associated with the disease. To investigate the effect of myostatin inhibition, we employed a mouse model with compound heterozygous nebulin mutations that mimic the typical form of the disease. The mice were treated with mRK35, a myostatin antibody, through weekly intraperitoneal injections of 10 mg/kg mRK35, commencing at two weeks of age and continuing until the mice reached four months of age. The treatment resulted in an increase in body weight and an approximate 20% muscle weight gain across most skeletal muscles, without affecting the heart. The minimum Feret diameter of type IIA and IIB fibers exhibited an increase in compound heterozygous mice, while only type IIB fibers demonstrated an increase in wild-type mice. In vitro mechanical experiments conducted on intact extensor digitorum longus muscle revealed that mRK35 augmented the physiological cross-sectional area of muscle fibers and enhanced absolute tetanic force in both wild-type and compound heterozygous mice. Furthermore, mRK35 administration improved grip strength in treated mice. Collectively, these findings indicate that inhibiting myostatin can mitigate the muscle deficits in nebulin-based typical nemaline myopathy, potentially serving as a much-needed therapeutic option.
• Slick, R. A., Tinklenberg, J. A., Sutton, J., Zhang, L., Meng, H., Beatka, M. J., Vanden Avond, M., Prom, M. J., Ott, E., Montanaro, F., Heisner, J., Toro, R., Granzier, H., Geurts, A. M., Stowe, D. F., Hill, R. B., & Lawlor, M. W. (2023). Aberrations in Energetic Metabolism and Stress-Related Pathways Contribute to Pathophysiology in the Neb Conditional Knockout Mouse Model of Nemaline Myopathy. The American journal of pathology, 193(10), 1528-1547.
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  Nemaline myopathy (NM) is a genetically and clinically heterogeneous disease that is diagnosed on the basis of the presence of nemaline rods on skeletal muscle biopsy. Although NM has typically been classified by causative genes, disease severity or prognosis cannot be predicted. The common pathologic end point of nemaline rods (despite diverse genetic causes) and an unexplained range of muscle weakness suggest that shared secondary processes contribute to the pathogenesis of NM. We speculated that these processes could be identified through a proteome-wide interrogation using a mouse model of severe NM in combination with pathway validation and structural/functional analyses. A proteomic analysis was performed using skeletal muscle tissue from the Neb conditional knockout mouse model compared with its wild-type counterpart to identify pathophysiologically relevant biological processes that might impact disease severity or provide new treatment targets. A differential expression analysis and Ingenuity Pathway Core Analysis predicted perturbations in several cellular processes, including mitochondrial dysfunction and changes in energetic metabolism and stress-related pathways. Subsequent structural and functional studies demonstrated abnormal mitochondrial distribution, decreased mitochondrial respiratory function, an increase in mitochondrial transmembrane potential, and extremely low ATP content in Neb conditional knockout muscles relative to wild type. Overall, the findings of these studies support a role for severe mitochondrial dysfunction as a novel contributor to muscle weakness in NM.
• Zhang, Y., Gregorich, Z. R., Wang, Y., Braz, C. U., Zhang, J., Liu, Y., Liu, P., Shen, J., Aori, N., Hacker, T. A., Granzier, H., & Guo, W. (2023). Disruption of the nuclear localization signal in RBM20 is causative in dilated cardiomyopathy. JCI insight, 8(13).
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  Human patients carrying genetic mutations in RNA binding motif 20 (RBM20) develop a clinically aggressive dilated cardiomyopathy (DCM). Genetic mutation knockin (KI) animal models imply that altered function of the arginine-serine-rich (RS) domain is crucial for severe DCM. To test this hypothesis, we generated an RS domain deletion mouse model (Rbm20ΔRS). We showed that Rbm20ΔRS mice manifested DCM with mis-splicing of RBM20 target transcripts. We found that RBM20 was mis-localized to the sarcoplasm in Rbm20ΔRS mouse hearts and formed RBM20 granules similar to those detected in mutation KI animals. In contrast, mice lacking the RNA recognition motif showed similar mis-splicing of major RBM20 target genes but did not develop DCM or exhibit RBM20 granule formation. Using in vitro studies with immunocytochemical staining, we demonstrated that only DCM-associated mutations in the RS domain facilitated RBM20 nucleocytoplasmic transport and promoted granule assembly. Further, we defined the core nuclear localization signal (NLS) within the RS domain of RBM20. Mutation analysis of phosphorylation sites in the RS domain suggested that this modification may be dispensable for RBM20 nucleocytoplasmic transport. Collectively, our findings revealed that disruption of RS domain-mediated nuclear localization is crucial for severe DCM caused by NLS mutations.
• Hettige, P., Mishra, D., Granzier, H., Nishikawa, K., & Gage, M. J. (2022). Contributions of Titin and Collagen to Passive Stress in Muscles from Mice with a Small Deletion in Titin's Molecular Spring. International journal of molecular sciences, 23(16).
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  Muscular dystrophy with myositis () is a naturally occurring mutation in the mouse gene that results in higher passive stress in muscle fibers and intact muscles compared to wild-type (WT). The goal of this study was to test whether alternative splicing of titin exons occurs in muscles, which contain a small deletion in the N2A-PEVK regions of titin, and to test whether splicing changes are associated with an increase in titin-based passive tension. Although higher levels of collagen have been reported previously in muscles, here we demonstrate alternative splicing of titin in skeletal muscle fibers. We identified Z-band, PEVK, and C-terminus Mex5 exons as splicing hotspots in titin using RNA sequencing data and further reported upregulation in ECM-associated genes. We also treated skinned soleus fiber bundles with trypsin, trypsin + KCl, and trypsin + KCL + KI to degrade titin. The results showed that passive stress dropped significantly more after trypsin treatment in fibers (11 ± 1.6 mN/mm) than in WT fibers (4.8 ± 1 mN/mm; = 0.0004). The finding that treatment with trypsin reduces titin-based passive tension more in than in WT fibers supports the hypothesis that exon splicing leads to the expression of a stiffer and shorter titin isoform in fibers. After titin extraction by trypsin + KCl + KI, fibers (6.7 ± 1.27 mN/mm) had significantly higher collagen-based passive stress remaining than WT fibers (2.6 ± 1.3 mN/mm; = 0.0014). We conclude that both titin and collagen contribute to higher passive tension of muscles.
• Lin, Y. H., Major, J. L., Liebner, T., Hourani, Z., Travers, J. G., Wennersten, S. A., Haefner, K. R., Cavasin, M. A., Wilson, C. E., Jeong, M. Y., Han, Y., Gotthardt, M., Ferguson, S. K., Ambardekar, A. V., Lam, M. P., Choudhary, C., Granzier, H. L., Woulfe, K. C., & McKinsey, T. A. (2022). HDAC6 modulates myofibril stiffness and diastolic function of the heart. The Journal of clinical investigation, 132(10).
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  Passive stiffness of the heart is determined largely by extracellular matrix and titin, which functions as a molecular spring within sarcomeres. Titin stiffening is associated with the development of diastolic dysfunction (DD), while augmented titin compliance appears to impair systolic performance in dilated cardiomyopathy. We found that myofibril stiffness was elevated in mice lacking histone deacetylase 6 (HDAC6). Cultured adult murine ventricular myocytes treated with a selective HDAC6 inhibitor also exhibited increased myofibril stiffness. Conversely, HDAC6 overexpression in cardiomyocytes led to decreased myofibril stiffness, as did ex vivo treatment of mouse, rat, and human myofibrils with recombinant HDAC6. Modulation of myofibril stiffness by HDAC6 was dependent on 282 amino acids encompassing a portion of the PEVK element of titin. HDAC6 colocalized with Z-disks, and proteomics analysis suggested that HDAC6 functions as a sarcomeric protein deacetylase. Finally, increased myofibril stiffness in HDAC6-deficient mice was associated with exacerbated DD in response to hypertension or aging. These findings define a role for a deacetylase in the control of myofibril function and myocardial passive stiffness, suggest that reversible acetylation alters titin compliance, and reveal the potential of targeting HDAC6 to manipulate the elastic properties of the heart to treat cardiac diseases.
• Lindqvist, J., Kolb, J., de Winter, J., Tonino, P., Hourani, Z., Labeit, S., Ottenheijm, C., & Granzier, H. (2022). Removal of MuRF1 Increases Muscle Mass in Nemaline Myopathy Models, but Does Not Provide Functional Benefits. International journal of molecular sciences, 23(15).
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  Nemaline myopathy (NM) is characterized by skeletal muscle weakness and atrophy. No curative treatments exist for this debilitating disease. NM is caused by mutations in proteins involved in thin-filament function, turnover, and maintenance. Mutations in nebulin, encoded by NEB, are the most common cause. Skeletal muscle atrophy is tightly linked to upregulation of MuRF1, an E3 ligase, that targets proteins for proteasome degradation. Here, we report a large increase in MuRF1 protein levels in both patients with nebulin-based NM, also named NEM2, and in mouse models of the disease. We hypothesized that knocking out MuRF1 in animal models of NM with muscle atrophy would ameliorate the muscle deficits. To test this, we crossed MuRF1 KO mice with two NEM2 mouse models, one with the typical form and the other with the severe form. The crosses were viable, and muscles were studied in mice at 3 months of life. Ultrastructural examination of gastrocnemius muscle lacking MuRF1 and with severe NM revealed a small increase in vacuoles, but no significant change in the myofibrillar fractional area. MuRF1 deficiency led to increased weights of various muscle types in the NM models. However, this increase in muscle size was not associated with increased in vivo or in vitro force production. We conclude that knocking out MuRF1 in NEM2 mice increases muscle size, but does not improve muscle function.
• Methawasin, M., Farman, G. P., Granzier-Nakajima, S., Strom, J., Kiss, B., Smith, J. E., & Granzier, H. (2022). Shortening the thick filament by partial deletion of titin's C-zone alters cardiac function by reducing the operating sarcomere length range. Journal of molecular and cellular cardiology, 165, 103-114.
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  Titin's C-zone is an inextensible segment in titin, comprised of 11 super-repeats and located in the cMyBP-C-containing region of the thick filament. Previously we showed that deletion of titin's super-repeats C1 and C2 (Ttn model) results in shorter thick filaments and contractile dysfunction of the left ventricular (LV) chamber but that unexpectedly LV diastolic stiffness is normal. Here we studied the contraction-relaxation kinetics from the time-varying elastance of the LV and intact cardiomyocyte, cellular work loops of intact cardiomyocytes, Ca transients, cross-bridge kinetics, and myofilament Ca sensitivity. Intact cardiomyocytes of Ttn mice exhibit systolic dysfunction and impaired relaxation. The time-varying elastance at both LV and single-cell levels showed that activation kinetics are normal in Ttn mice, but that relaxation is slower. The slowed relaxation is, in part, attributable to an increased myofilament Ca sensitivity and slower early Ca reuptake. Cross-bridge dynamics showed that cross-bridge kinetics are normal but that the number of force-generating cross-bridges is reduced. In vivo sarcomere length (SL) measurements revealed that in Ttn mice the operating SL range of the LV is shifted towards shorter lengths. This normalizes the apparent cell and LV diastolic stiffness but further reduces systolic force as systole occurs further down on the ascending limb of the force-SL relation. We propose that the reduced working SLs reflect titin's role in regulating diastolic stiffness by altering the number of sarcomeres in series. Overall, our study reveals that thick filament length regulation by titin's C-zone is critical for normal cardiac function.
• Ranu, N., Laitila, J., Dugdale, H. F., Mariano, J., Kolb, J. S., Wallgren-Pettersson, C., Witting, N., Vissing, J., Vilchez, J. J., Fiorillo, C., Zanoteli, E., Auranen, M., Jokela, M., Tasca, G., Claeys, K. G., Voermans, N. C., Palmio, J., Huovinen, S., Moggio, M., , Beck, T. N., et al. (2022). NEB mutations disrupt the super-relaxed state of myosin and remodel the muscle metabolic proteome in nemaline myopathy. Acta neuropathologica communications, 10(1), 185.
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  Nemaline myopathy (NM) is one of the most common non-dystrophic genetic muscle disorders. NM is often associated with mutations in the NEB gene. Even though the exact NEB-NM pathophysiological mechanisms remain unclear, histological analyses of patients' muscle biopsies often reveal unexplained accumulation of glycogen and abnormally shaped mitochondria. Hence, the aim of the present study was to define the exact molecular and cellular cascade of events that would lead to potential changes in muscle energetics in NEB-NM. For that, we applied a wide range of biophysical and cell biology assays on skeletal muscle fibres from NM patients as well as untargeted proteomics analyses on isolated myofibres from a muscle-specific nebulin-deficient mouse model. Unexpectedly, we found that the myosin stabilizing conformational state, known as super-relaxed state, was significantly impaired, inducing an increase in the energy (ATP) consumption of resting muscle fibres from NEB-NM patients when compared with controls or with other forms of genetic/rare, acquired NM. This destabilization of the myosin super-relaxed state had dynamic consequences as we observed a remodeling of the metabolic proteome in muscle fibres from nebulin-deficient mice. Altogether, our findings explain some of the hitherto obscure hallmarks of NM, including the appearance of abnormal energy proteins and suggest potential beneficial effects of drugs targeting myosin activity/conformations for NEB-NM.
• Valero-Muñoz, M., Saw, E. L., Hekman, R. M., Blum, B. C., Hourani, Z., Granzier, H., Emili, A., & Sam, F. (2022). Proteomic and phosphoproteomic profiling in heart failure with preserved ejection fraction (HFpEF). Frontiers in cardiovascular medicine, 9, 966968.
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  Although the prevalence of heart failure with preserved ejection fraction (HFpEF) is increasing, evidence-based therapies for HFpEF remain limited, likely due to an incomplete understanding of this disease. This study sought to identify the cardiac-specific features of protein and phosphoprotein changes in a murine model of HFpEF using mass spectrometry. HFpEF mice demonstrated moderate hypertension, left ventricle (LV) hypertrophy, lung congestion and diastolic dysfunction. Proteomics analysis of the LV tissue showed that 897 proteins were differentially expressed between HFpEF and Sham mice. We observed abundant changes in sarcomeric proteins, mitochondrial-related proteins, and NAD-dependent protein deacetylase sirtuin-3 (SIRT3). Upregulated pathways by GSEA analysis were related to immune modulation and muscle contraction, while downregulated pathways were predominantly related to mitochondrial metabolism. Western blot analysis validated SIRT3 downregulated cardiac expression in HFpEF vs. Sham (0.8 ± 0.0 vs. 1.0 ± 0.0; < 0.001). Phosphoproteomics analysis showed that 72 phosphosites were differentially regulated between HFpEF and Sham LV. Aberrant phosphorylation patterns mostly occurred in sarcomere proteins and nuclear-localized proteins associated with contractile dysfunction and cardiac hypertrophy. Seven aberrant phosphosites were observed at the z-disk binding region of titin. Additional agarose gel analysis showed that while total titin cardiac expression remained unaltered, its stiffer N2B isoform was significantly increased in HFpEF vs. Sham (0.144 ± 0.01 vs. 0.127 ± 0.01; < 0.05). In summary, this study demonstrates marked changes in proteins related to mitochondrial metabolism and the cardiac contractile apparatus in HFpEF. We propose that SIRT3 may play a role in perpetuating these changes and may be a target for drug development in HFpEF.
• Wang, C., Zhang, Y., Methawasin, M., Braz, C. U., Gao-Hu, J., Yang, B., Strom, J., Gohlke, J., Hacker, T., Khatib, H., Granzier, H., & Guo, W. (2022). RBM20 mutation is a high genetic risk factor for premature death through RNA-protein condensates. Journal of molecular and cellular cardiology, 165, 115-129.
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  Dilated cardiomyopathy (DCM) is a heritable and genetically heterogenous disease often idiopathic and a leading cause of heart failure with high morbidity and mortality. DCM caused by RNA binding motif protein 20 (RBM20) mutations is diverse and needs a more complete mechanistic understanding. RBM20 mutation S637G (S639G in mice) is linked to severe DCM and early death in human patients. In this study, we generated a RBM20 S639G mutation knock-in (KI) mouse model to validate the function of S639G mutation and examine the underlying mechanisms. KI mice exhibited severe DCM and premature death with a ~ 50% mortality in two months old homozygous (HM) mice. KI mice had enlarged atria and increased ANP and BNP biomarkers. The S639G mutation promoted RBM20 trafficking and ribonucleoprotein (RNP) granules in the sarcoplasm. RNA Seq data revealed differentially expressed and spliced genes were associated with arrhythmia, cardiomyopathy, and sudden death. KI mice also showed a reduction of diastolic stiffness and impaired contractility at both the left ventricular (LV) chamber and cardiomyocyte levels. Our results indicate that the RBM20 S639G mutation leads to RNP granules causing severe heart failure and early death and this finding strengthens the novel concept that RBM20 cardiomyopathy is a RNP granule disease.
• Zhang, Y., Wang, C., Sun, M., Jin, Y., Braz, C. U., Khatib, H., Hacker, T. A., Liss, M., Gotthardt, M., Granzier, H., Ge, Y., & Guo, W. (2022). RBM20 phosphorylation and its role in nucleocytoplasmic transport and cardiac pathogenesis. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 36(5), e22302.
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  Arginine-serine (RS) domain(s) in splicing factors are critical for protein-protein interaction in pre-mRNA splicing. Phosphorylation of RS domain is important for splicing control and nucleocytoplasmic transport in the cell. RNA-binding motif 20 (RBM20) is a splicing factor primarily expressed in the heart. A previous study using phospho-antibody against RS domain showed that RS domain can be phosphorylated. However, its actual phosphorylation sites and function have not been characterized. Using middle-down mass spectrometry, we identified 16 phosphorylation sites, two of which (S638 and S640 in rats, or S637 and S639 in mice) were located in the RSRSP stretch in the RS domain. Mutations on S638 and S640 regulated splicing, promoted nucleocytoplasmic transport and protein-RNA condensates. Phosphomimetic mutations on S638 and S640 indicated that phosphorylation was not the major cause for RBM20 nucleocytoplasmic transport and condensation in vitro. We generated a S637A knock-in (KI) mouse model (Rbm20 ) and observed the reduced RBM20 phosphorylation. The KI mice exhibited aberrant gene splicing, protein condensates, and a dilated cardiomyopathy (DCM)-like phenotype. Transcriptomic profiling demonstrated that KI mice had altered expression and splicing of genes involving cardiac dysfunction, protein localization, and condensation. Our in vitro data showed that phosphorylation was not a direct cause for nucleocytoplasmic transport and protein condensation. Subsequently, the in vivo results reveal that RBM20 mutations led to cardiac pathogenesis. However, the role of phosphorylation in vivo needs further investigation.
• de Winter, J. M., Bouman, K., Strom, J., Methawasin, M., Jongbloed, J. D., van der Roest, W., Wijngaarden, J. V., Timmermans, J., Nijveldt, R., van den Heuvel, F., Kamsteeg, E. J., van Engelen, B. G., Galli, R., Bogaards, S. J., Boon, R. A., van der Pijl, R. J., Granzier, H., Koeleman, B., Amin, A. S., , van der Velden, J., et al. (2022). KBTBD13 is a novel cardiomyopathy gene. Human mutation, 43(12), 1860-1865.
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  KBTBD13 variants cause nemaline myopathy type 6 (NEM6). The majority of NEM6 patients harbors the Dutch founder variant, c.1222C>T, p.Arg408Cys (KBTBD13 p.R408C). Although KBTBD13 is expressed in cardiac muscle, cardiac involvement in NEM6 is unknown. Here, we constructed pedigrees of three families with the KBTBD13 p.R408C variant. In 65 evaluated patients, 12% presented with left ventricle dilatation, 29% with left ventricular ejection fraction
• van den Berg, M., Peters, E. L., van der Pijl, R. J., Shen, S., Heunks, L. M., Granzier, H. L., & Ottenheijm, C. A. (2022). Rbm20 Mice, Expressing a Titin Isoform with Lower Stiffness, Are Protected from Mechanical Ventilation-Induced Diaphragm Weakness. International journal of molecular sciences, 23(24).
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  Diaphragm weakness frequently develops in mechanically ventilated critically ill patients and is associated with increased morbidity, including ventilator weaning failure, mortality, and health care costs. The mechanisms underlying diaphragm weakness are incompletely understood but may include the elastic properties of titin, a giant protein whose layout in the muscle's sarcomeres makes it an ideal candidate to sense ventilation-induced diaphragm unloading, resulting in downstream signaling through titin-binding proteins. In the current study, we investigated whether modulating titin stiffness affects the development of diaphragm weakness during mechanical ventilation. To this end, we ventilated genetically engineered mice with reduced titin stiffness (Rbm20), and robust (Ttn) or severely (Ttn) increased titin stiffness for 8 h, and assessed diaphragm contractility and protein expression of titin-binding proteins. Mechanical ventilation reduced the maximum active tension of the diaphragm in WT, Ttn and Ttn mice. However, in Rbm20 mice maximum active tension was preserved after ventilation. Analyses of titin binding proteins suggest that muscle ankyrin repeat proteins (MARPs) 1 and 2 may play a role in the adaptation of the diaphragm to mechanical ventilation, and the preservation of diaphragm contractility in Rbm20 mice. Thus, Rbm20 mice, expressing titin isoforms with lower stiffness, are protected from mechanical ventilation-induced diaphragm weakness, suggesting that titin elasticity may modulate the diaphragm's response to unloading during mechanical ventilation.
• Biquand, A., Spinozzi, S., Tonino, P., Cosette, J., Strom, J., Elbeck, Z., Knöll, R., Granzier, H., Lostal, W., & Richard, I. (2021). Titin M-line insertion sequence 7 is required for proper cardiac function in mice. Journal of cell science, 134(18).
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  Titin is a giant sarcomeric protein that is involved in a large number of functions, with a primary role in skeletal and cardiac sarcomere organization and stiffness. The titin gene (TTN) is subject to various alternative splicing events, but in the region that is present at the M-line, the only exon that can be spliced out is Mex5, which encodes for the insertion sequence 7 (is7). Interestingly, in the heart, the majority of titin isoforms are Mex5+, suggesting a cardiac role for is7. Here, we performed comprehensive functional, histological, transcriptomic, microscopic and molecular analyses of a mouse model lacking the Ttn Mex5 exon (ΔMex5), and revealed that the absence of the is7 is causative for dilated cardiomyopathy. ΔMex5 mice showed altered cardiac function accompanied by increased fibrosis and ultrastructural alterations. Abnormal expression of excitation-contraction coupling proteins was also observed. The results reported here confirm the importance of the C-terminal region of titin in cardiac function and are the first to suggest a possible relationship between the is7 and excitation-contraction coupling. Finally, these findings give important insights for the identification of new targets in the treatment of titinopathies.
• Chan, B. Y., Roczkowsky, A., Cho, W. J., Poirier, M., Sergi, C., Keschrumrus, V., Churko, J. M., Granzier, H., & Schulz, R. (2021). MMP inhibitors attenuate doxorubicin cardiotoxicity by preventing intracellular and extracellular matrix remodelling. Cardiovascular research, 117(1), 188-200.
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  Heart failure is a major complication in cancer treatment due to the cardiotoxic effects of anticancer drugs, especially from the anthracyclines such as doxorubicin (DXR). DXR enhances oxidative stress and stimulates matrix metalloproteinase-2 (MMP-2) in cardiomyocytes. We investigated whether MMP inhibitors protect against DXR cardiotoxicity given the role of MMP-2 in proteolyzing sarcomeric proteins in the heart and remodelling the extracellular matrix.
• Cremo, C., Moss, R. L., & Granzier, H. (2021). Further progress in understanding of myofibrillar function in health and disease. The Journal of general physiology, 153(7).
• Fukuda, N., Granzier, H., Ishiwata, S., & Morimoto, S. (2021). Editorial: Recent Advances on Myocardium Physiology. Frontiers in physiology, 12, 697852.
• Gohlke, J., Tonino, P., Lindqvist, J., Smith, J. E., & Granzier, H. (2021). The number of Z-repeats and super-repeats in nebulin greatly varies across vertebrates and scales with animal size. The Journal of general physiology, 153(3).
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  Nebulin is a skeletal muscle protein that associates with the sarcomeric thin filaments and has functions in regulating the length of the thin filament and the structure of the Z-disk. Here we investigated the nebulin gene in 53 species of birds, fish, amphibians, reptiles, and mammals. In all species, nebulin has a similar domain composition that mostly consists of ∼30-residue modules (or simple repeats), each containing an actin-binding site. All species have a large region where simple repeats are organized into seven-module super-repeats, each containing a tropomyosin binding site. The number of super-repeats shows high interspecies variation, ranging from 21 (zebrafish, hummingbird) to 31 (camel, chimpanzee), and, importantly, scales with body size. The higher number of super-repeats in large animals was shown to increase thin filament length, which is expected to increase the sarcomere length for optimal force production, increase the energy efficiency of isometric force production, and lower the shortening velocity of muscle. It has been known since the work of A.V. Hill in 1950 that as species increase in size, the shortening velocity of their muscle is reduced, and the present work shows that nebulin contributes to the mechanistic basis. Finally, we analyzed the differentially spliced simple repeats in nebulin's C terminus, whose inclusion correlates with the width of the Z-disk. The number of Z-repeats greatly varies (from 5 to 18) and correlates with the number of super-repeats. We propose that the resulting increase in the width of the Z-disk in large animals increases the number of contacts between nebulin and structural Z-disk proteins when the Z-disk is stressed for long durations.
• Kellermayer, D., Kiss, B., Tordai, H., Oláh, A., Granzier, H. L., Merkely, B., Kellermayer, M., & Radovits, T. (2021). Increased Expression of N2BA Titin Corresponds to More Compliant Myofibrils in Athlete's Heart. International journal of molecular sciences, 22(20).
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  Long-term exercise induces physiological cardiac adaptation, a condition referred to as athlete's heart. Exercise tolerance is known to be associated with decreased cardiac passive stiffness. Passive stiffness of the heart muscle is determined by the giant elastic protein titin. The adult cardiac muscle contains two titin isoforms: the more compliant N2BA and the stiffer N2B. Titin-based passive stiffness may be controlled by altering the expression of the different isoforms or via post-translational modifications such as phosphorylation. Currently, there is very limited knowledge about titin's role in cardiac adaptation during long-term exercise. Our aim was to determine the N2BA/N2B ratio and post-translational phosphorylation of titin in the left ventricle and to correlate the changes with the structure and transverse stiffness of cardiac sarcomeres in a rat model of an athlete's heart. The athlete's heart was induced by a 12-week-long swim-based training. In the exercised myocardium the N2BA/N2B ratio was significantly increased, Ser11878 of the PEVK domain was hypophosphorlyated, and the sarcomeric transverse elastic modulus was reduced. Thus, the reduced passive stiffness in the athlete's heart is likely caused by a shift towards the expression of the longer cardiac titin isoform and a phosphorylation-induced softening of the PEVK domain which is manifested in a mechanical rearrangement locally, within the cardiac sarcomere.
• Methawasin, M., & Granzier, H. (2021). Response by Methawasin and Granzier to Letter Regarding Article, "Phosphodiesterase 9a Inhibition in Mouse Models of Diastolic Dysfunction". Circulation. Heart failure, 14(1), e007755.
• Moss, R. L., Cremo, C., & Granzier, H. L. (2021). Toward an understanding of myofibrillar function in health and disease. The Journal of general physiology, 153(3).
• van de Locht, M., Donkervoort, S., de Winter, J. M., Conijn, S., Begthel, L., Kusters, B., Mohassel, P., Hu, Y., Medne, L., Quinn, C., Moore, S. A., Foley, A. R., Seo, G., Hwee, D. T., Malik, F. I., Irving, T., Ma, W., Granzier, H. L., Kamsteeg, E. J., , Immadisetty, K., et al. (2021). Pathogenic variants in TNNC2 cause congenital myopathy due to an impaired force response to calcium. The Journal of clinical investigation, 131(9).
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  Troponin C (TnC) is a critical regulator of skeletal muscle contraction; it binds Ca2+ to activate muscle contraction. Surprisingly, the gene encoding fast skeletal TnC (TNNC2) has not yet been implicated in muscle disease. Here, we report 2 families with pathogenic variants in TNNC2. Patients present with a distinct, dominantly inherited congenital muscle disease. Molecular dynamics simulations suggested that the pathomechanisms by which the variants cause muscle disease include disruption of the binding sites for Ca2+ and for troponin I. In line with these findings, physiological studies in myofibers isolated from patients' biopsies revealed a markedly reduced force response of the sarcomeres to [Ca2+]. This pathomechanism was further confirmed in experiments in which contractile dysfunction was evoked by replacing TnC in myofibers from healthy control subjects with recombinant, mutant TnC. Conversely, the contractile dysfunction of myofibers from patients was repaired by replacing endogenous, mutant TnC with recombinant, wild-type TnC. Finally, we tested the therapeutic potential of the fast skeletal muscle troponin activator tirasemtiv in patients' myofibers and showed that the contractile dysfunction was repaired. Thus, our data reveal that pathogenic variants in TNNC2 cause congenital muscle disease, and they provide therapeutic angles to repair muscle contractility.
• van der Pijl, R. J., van den Berg, M., van de Locht, M., Shen, S., Bogaards, S. J., Conijn, S., Langlais, P., Hooijman, P. E., Labeit, S., Heunks, L. M., Granzier, H., & Ottenheijm, C. A. (2021). Muscle ankyrin repeat protein 1 (MARP1) locks titin to the sarcomeric thin filament and is a passive force regulator. The Journal of general physiology, 153(7).
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  Muscle ankyrin repeat protein 1 (MARP1) is frequently up-regulated in stressed muscle, but its effect on skeletal muscle function is poorly understood. Here, we focused on its interaction with the titin-N2A element, found in titin's molecular spring region. We show that MARP1 binds to F-actin, and that this interaction is stronger when MARP1 forms a complex with titin-N2A. Mechanics and super-resolution microscopy revealed that MARP1 "locks" titin-N2A to the sarcomeric thin filament, causing increased extension of titin's elastic PEVK element and, importantly, increased passive force. In support of this mechanism, removal of thin filaments abolished the effect of MARP1 on passive force. The clinical relevance of this mechanism was established in diaphragm myofibers of mechanically ventilated rats and of critically ill patients. Thus, MARP1 regulates passive force by locking titin to the thin filament. We propose that in stressed muscle, this mechanism protects the sarcomere from mechanical damage.
• Chiao, Y. A., Zhang, H., Sweetwyne, M., Whitson, J., Ting, Y. S., Basisty, N., Pino, L. K., Quarles, E., Nguyen, N. H., Campbell, M. D., Zhang, T., Gaffrey, M. J., Merrihew, G., Wang, L., Yue, Y., Duan, D., Granzier, H. L., Szeto, H. H., Qian, W. J., , Marcinek, D., et al. (2020). Late-life restoration of mitochondrial function reverses cardiac dysfunction in old mice. eLife, 9.
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  Diastolic dysfunction is a prominent feature of cardiac aging in both mice and humans. We show here that 8-week treatment of old mice with the mitochondrial targeted peptide SS-31 (elamipretide) can substantially reverse this deficit. SS-31 normalized the increase in proton leak and reduced mitochondrial ROS in cardiomyocytes from old mice, accompanied by reduced protein oxidation and a shift towards a more reduced protein thiol redox state in old hearts. Improved diastolic function was concordant with increased phosphorylation of cMyBP-C Ser282 but was independent of titin isoform shift. Late-life viral expression of mitochondrial-targeted catalase (mCAT) produced similar functional benefits in old mice and SS-31 did not improve cardiac function of old mCAT mice, implicating normalizing mitochondrial oxidative stress as an overlapping mechanism. These results demonstrate that pre-existing cardiac aging phenotypes can be reversed by targeting mitochondrial dysfunction and implicate mitochondrial energetics and redox signaling as therapeutic targets for cardiac aging.
• Goto, K., Schauer, A., Augstein, A., Methawasin, M., Granzier, H., Halle, M., Craenenbroeck, E. M., Rolim, N., Gielen, S., Pieske, B., Winzer, E. B., Linke, A., & Adams, V. (2020). Muscular changes in animal models of heart failure with preserved ejection fraction: what comes closest to the patient?. ESC heart failure.
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  Heart failure with preserved ejection fraction (HFpEF) is associated with reduced exercise capacity elicited by skeletal muscle (SM) alterations. Up to now, no clear medical treatment advice for HFpEF is available. Identification of the ideal animal model mimicking the human condition is a critical step in developing and testing treatment strategies. Several HFpEF animals have been described, but the most suitable in terms of comparability with SM alterations in HFpEF patients is unclear. The aim of the present study was to investigate molecular changes in SM of three different animal models and to compare them with alterations of muscle biopsies obtained from human HFpEF patients.
• Granzier, H. L. (2020). Catch a Tiny Fish by the Tail. Biophysical journal, 119(4), 721-723.
• Grogan, A., Coleman, A., Joca, H., Granzier, H., Russel, M. W., Ward, C. W., & Kontrogianni-Konstantopoulos, A. (2020). Deletion of obscurin immunoglobulin domains Ig58/59 leads to age-dependent cardiac remodeling and arrhythmia. Basic research in cardiology, 115(6), 60.
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  Obscurin comprises a family of giant modular proteins that play key structural and regulatory roles in striated muscles. Immunoglobulin domains 58/59 (Ig58/59) of obscurin mediate binding to essential modulators of muscle structure and function, including canonical titin, a smaller splice variant of titin, termed novex-3, and phospholamban (PLN). Importantly, missense mutations localized within the obscurin-Ig58/59 region that affect binding to titins and/or PLN have been linked to the development of myopathy in humans. To elucidate the pathophysiological role of this region, we generated a constitutive deletion mouse model, Obscn-ΔIg58/59, that expresses obscurin lacking Ig58/59, and determined the consequences of this manipulation on cardiac morphology and function under conditions of acute stress and through the physiological process of aging. Our studies show that young Obscn-ΔIg58/59 mice are susceptible to acute β-adrenergic stress. Moreover, sedentary Obscn-ΔIg58/59 mice develop left ventricular hypertrophy that progresses to dilation, contractile impairment, atrial enlargement, and arrhythmia as a function of aging with males being more affected than females. Experiments in ventricular cardiomyocytes revealed altered Ca cycling associated with changes in the expression and/or phosphorylation levels of major Ca cycling proteins, including PLN, SERCA2, and RyR2. Taken together, our work demonstrates that obscurin-Ig58/59 is an essential regulatory module in the heart and its deletion leads to age- and sex-dependent cardiac remodeling, ventricular dilation, and arrhythmia due to deregulated Ca cycling.
• Kiss, B., Gohlke, J., Tonino, P., Hourani, Z., Kolb, J., Strom, J., Alekhina, O., Smith, J. E., Ottenheijm, C., Gregorio, C., & Granzier, H. (2020). Nebulin and Lmod2 are critical for specifying thin-filament length in skeletal muscle. Science advances, 6(46).
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  Regulating the thin-filament length in muscle is crucial for controlling the number of myosin motors that generate power. The giant protein nebulin forms a long slender filament that associates along the length of the thin filament in skeletal muscle with functions that remain largely obscure. Here nebulin's role in thin-filament length regulation was investigated by targeting entire super-repeats in the gene; nebulin was either shortened or lengthened by 115 nm. Its effect on thin-filament length was studied using high-resolution structural and functional techniques. Results revealed that thin-filament length is strictly regulated by the length of nebulin in fast muscles. Nebulin's control is less tight in slow muscle types where a distal nebulin-free thin-filament segment exists, the length of which was found to be regulated by leiomodin-2 (Lmod2). We propose that strict length control by nebulin promotes high-speed shortening and that dual-regulation by nebulin/Lmod2 enhances contraction efficiency.
• Lanzicher, T., Zhou, T., Saripalli, C., Keschrumrus, V., Smith Iii, J. E., Mayans, O., Sbaizero, O., & Granzier, H. (2020). Single-Molecule Force Spectroscopy on the N2A Element of Titin: Effects of Phosphorylation and CARP. Frontiers in physiology, 11, 173.
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  Titin is a large filamentous protein that forms a sarcomeric myofilament with a molecular spring region that develops force in stretched sarcomeres. The molecular spring has a complex make-up that includes the N2A element. This element largely consists of a 104-residue unique sequence (N2A-Us) flanked by immunoglobulin domains (I80 and I81). The N2A element is of interest because it assembles a signalosome with CARP (Cardiac Ankyrin Repeat Protein) as an important component; CARP both interacts with the N2A-Us and I81 and is highly upregulated in response to mechanical stress. The mechanical properties of the N2A element were studied using single-molecule force spectroscopy, including how these properties are affected by CARP and phosphorylation. Three protein constructs were made that consisted of 0, 1, or 2 N2A-Us elements with flanking I80 and I81 domains and with specific handles at their ends for study by atomic force microscopy (AFM). The N2A-Us behaved as an entropic spring with a persistence length (Lp) of ∼0.35 nm and contour length (Lc) of ∼39 nm. CARP increased the Lp of the N2A-Us and the unfolding force of the Ig domains; force clamp experiments showed that CARP reduced the Ig domain unfolding kinetics. These findings suggest that CARP might function as a molecular chaperone that protects I81 from unfolding when mechanical stress is high. The N2A-Us was found to be a PKA substrate, and phosphorylation was blocked by CARP. Mass spectrometry revealed a PKA phosphosite (Ser-9895 in NP_001254479.2) located at the border between the N2A-Us and I81. AFM studies showed that phosphorylation affected neither the Lp of the N2A-Us nor the Ig domain unfolding force (F). Simulating the force-sarcomere length relation of a single titin molecule containing all spring elements showed that the compliance of the N2A-Us only slightly reduces passive force (1.4%) with an additional small reduction by CARP (0.3%). Thus, it is improbable that the compliance of the N2A element has a mechanical function . Instead, it is likely that this compliance has local effects on binding of signaling molecules and that it contributes thereby to strain- and phosphorylation- dependent mechano-signaling.
• Li, F., Kolb, J., Crudele, J., Tonino, P., Hourani, Z., Smith, J. E., Chamberlain, J. S., & Granzier, H. (2020). Correction to: Expressing a Z-disk nebulin fragment innebulin-deficient mouse muscle: effects on muscle structure and function. Skeletal muscle, 10(1), 9.
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  Following the publication of this paper [1], it was brought to the authors' attention that one of the contributing authors was left off of the paper. The authors apologize for the unfortunate oversight. In this correction paper, they have included Dr. Paola Tonino in the author list section.
• Li, F., Kolb, J., Crudele, J., Tonino, P., Hourani, Z., Smith, J. E., Chamberlain, J. S., & Granzier, H. (2020). Expressing a Z-disk nebulin fragment in nebulin-deficient mouse muscle: effects on muscle structure and function. Skeletal muscle, 10(1), 2.
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  Nebulin is a critical thin filament-binding protein that spans from the Z-disk of the skeletal muscle sarcomere to near the pointed end of the thin filament. Its massive size and actin-binding property allows it to provide the thin filaments with structural and regulatory support. When this protein is lost, nemaline myopathy occurs. Nemaline myopathy causes severe muscle weakness as well as structural defects on a sarcomeric level. There is no known cure for this disease.
• Lindqvist, J., Ma, W., Li, F., Hernandez, Y., Kolb, J., Kiss, B., Tonino, P., van der Pijl, R., Karimi, E., Gong, H., Strom, J., Hourani, Z., Smith, J. E., Ottenheijm, C., Irving, T., & Granzier, H. (2020). Triggering typical nemaline myopathy with compound heterozygous nebulin mutations reveals myofilament structural changes as pathomechanism. Nature communications, 11(1), 2699.
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  Nebulin is a giant protein that winds around the actin filaments in the skeletal muscle sarcomere. Compound-heterozygous mutations in the nebulin gene (NEB) cause typical nemaline myopathy (NM), a muscle disorder characterized by muscle weakness with limited treatment options. We created a mouse model with a missense mutation p.Ser6366Ile and a deletion of NEB exon 55, the Compound-Het model that resembles typical NM. We show that Compound-Het mice are growth-retarded and have muscle weakness. Muscles have a reduced myofibrillar fractional-area and sarcomeres are disorganized, contain rod bodies, and have longer thin filaments. In contrast to nebulin-based severe NM where haplo-insufficiency is the disease driver, Compound-Het mice express normal amounts of nebulin. X-ray diffraction revealed that the actin filament is twisted with a larger radius, that tropomyosin and troponin behavior is altered, and that the myofilament spacing is increased. The unique disease mechanism of nebulin-based typical NM reveals novel therapeutic targets.
• Methawasin, M., Strom, J., Borkowski, T., Hourani, Z., Runyan, R., Smith, J. E., & Granzier, H. (2020). Phosphodiesterase 9a Inhibition in Mouse Models of Diastolic Dysfunction. Circulation. Heart failure, 13(5), e006609.
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  Low myocardial cGMP-PKG (cyclic guanosine monophosphate-protein kinase G) activity has been associated with increased cardiomyocyte diastolic stiffness in heart failure with preserved ejection fraction. Cyclic guanosine monophosphate is mainly hydrolyzed by PDE (phosphodiesterases) 5a and 9a. Importantly, PDE9a expression has been reported to be upregulated in human heart failure with preserved ejection fraction myocardium and chronic administration of a PDE9a inhibitor reverses preestablished cardiac hypertrophy and systolic dysfunction in mice subjected to transverse aortic constriction (TAC). We hypothesized that inhibiting PDE9a activity ameliorates diastolic dysfunction.
• Perrin, A., Metay, C., Villanova, M., Carlier, R. Y., Pegoraro, E., Juntas Morales, R., Stojkovic, T., Richard, I., Richard, P., Romero, N. B., Granzier, H., Koenig, M., Malfatti, E., & Cossée, M. (2020). A new congenital multicore titinopathy associated with fast myosin heavy chain deficiency. Annals of clinical and translational neurology, 7(5), 846-854.
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  Congenital titinopathies are myopathies with variable phenotypes and inheritance modes. Here, we fully characterized, using an integrated approach (deep phenotyping, muscle morphology, mRNA and protein evaluation in muscle biopsies), two siblings with congenital multicore myopathy harboring three TTN variants predicted to affect titin stability and titin-myosin interactions. Muscle biopsies showed multicores, type 1 fiber uniformity and sarcomeric structure disruption with some thick filament loss. Immunohistochemistry and Western blotting revealed a marked reduction of fast myosin heavy chain isoforms. This is the first observation of a titinopathy suggesting that titin defect leads to secondary loss of fast myosin heavy chain isoforms.
• Shah, S. J., Borlaug, B. A., Kitzman, D. W., McCulloch, A. D., Blaxall, B. C., Agarwal, R., Chirinos, J. A., Collins, S., Deo, R. C., Gladwin, M. T., Granzier, H., Hummel, S. L., Kass, D. A., Redfield, M. M., Sam, F., Wang, T. J., Desvigne-Nickens, P., & Adhikari, B. B. (2020). Research Priorities for Heart Failure With Preserved Ejection Fraction: National Heart, Lung, and Blood Institute Working Group Summary. Circulation, 141(12), 1001-1026.
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  Heart failure with preserved ejection fraction (HFpEF), a major public health problem that is rising in prevalence, is associated with high morbidity and mortality and is considered to be the greatest unmet need in cardiovascular medicine today because of a general lack of effective treatments. To address this challenging syndrome, the National Heart, Lung, and Blood Institute convened a working group made up of experts in HFpEF and novel research methodologies to discuss research gaps and to prioritize research directions over the next decade. Here, we summarize the discussion of the working group, followed by key recommendations for future research priorities. There was uniform recognition that HFpEF is a highly integrated, multiorgan, systemic disorder requiring a multipronged investigative approach in both humans and animal models to improve understanding of mechanisms and treatment of HFpEF. It was recognized that advances in the understanding of basic mechanisms and the roles of inflammation, macrovascular and microvascular dysfunction, fibrosis, and tissue remodeling are needed and ideally would be obtained from (1) improved animal models, including large animal models, which incorporate the effects of aging and associated comorbid conditions; (2) repositories of deeply phenotyped physiological data and human tissue, made accessible to researchers to enhance collaboration and research advances; and (3) novel research methods that take advantage of computational advances and multiscale modeling for the analysis of complex, high-density data across multiple domains. The working group emphasized the need for interactions among basic, translational, clinical, and epidemiological scientists and across organ systems and cell types, leveraging different areas or research focus, and between research centers. A network of collaborative centers to accelerate basic, translational, and clinical research of pathobiological mechanisms and treatment strategies in HFpEF was discussed as an example of a strategy to advance research progress. This resource would facilitate comprehensive, deep phenotyping of a multicenter HFpEF patient cohort with standardized protocols and a robust biorepository. The research priorities outlined in this document are meant to stimulate scientific advances in HFpEF by providing a road map for future collaborative investigations among a diverse group of scientists across multiple domains.
• van der Pijl, R. J., Hudson, B., Granzier-Nakajima, T., Li, F., Knottnerus, A. M., Smith, J., Chung, C. S., Gotthardt, M., Granzier, H. L., & Ottenheijm, C. A. (2020). Deleting Titin's C-Terminal PEVK Exons Increases Passive Stiffness, Alters Splicing, and Induces Cross-Sectional and Longitudinal Hypertrophy in Skeletal Muscle. Frontiers in physiology, 11, 494.
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  The Proline, Glutamate, Valine and Lysine-rich (PEVK) region of titin constitutes an entropic spring that provides passive tension to striated muscle. To study the functional and structural repercussions of a small reduction in the size of the PEVK region, we investigated skeletal muscles of a mouse with the constitutively expressed C-terminal PEVK exons 219-225 deleted, the Ttn model (MGI: Ttn ). Based on this deletion, passive tension in skeletal muscle was predicted to be increased by ∼17% (sarcomere length 3.0 μm). In contrast, measured passive tension (sarcomere length 3.0 μm) in both soleus and EDL muscles was increased 53 ± 11% and 62 ± 4%, respectively. This unexpected increase was due to changes in titin, not to alterations in the extracellular matrix, and is likely caused by co-expression of two titin isoforms in Ttn muscles: a larger isoform that represents the Ttn N2A titin and a smaller isoform, referred to as N2A2. N2A2 represents a splicing adaption with reduced expression of spring element exons, as determined by titin exon microarray analysis. Maximal tetanic tension was increased in Ttn soleus muscle (WT 240 ± 9; Ttn 276 ± 17 mN/mm), but was reduced in EDL muscle (WT 315 ± 9; Ttn 280 ± 14 mN/mm). The changes in active tension coincided with a switch toward slow fiber types and, unexpectedly, faster kinetics of tension generation and relaxation. Functional overload (FO; ablation) and hindlimb suspension (HS; unloading) experiments were also conducted. Ttn mice showed increases in both longitudinal hypertrophy (increased number of sarcomeres in series) and cross-sectional hypertrophy (increased number of sarcomeres in parallel) in response to FO and attenuated cross-sectional atrophy in response to HS. In summary, slow- and fast-twitch muscles in a mouse model devoid of titin's PEVK exons 219-225 have high passive tension, due in part to alterations elsewhere in splicing of titin's spring region, increased kinetics of tension generation and relaxation, and altered trophic responses to both functional overload and unloading. This implicates titin's C-terminal PEVK region in regulating passive and active muscle mechanics and muscle plasticity.
• Gineste, C., Ogier, A. C., Varlet, I., Hourani, Z., Bernard, M., Granzier, H., Bendahan, D., & Gondin, J. (2019). In vivo characterization of skeletal muscle function in nebulin-deficient mice. Muscle & nerve.
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  The conditional nebulin knockout mouse is a new model mimicking nemaline myopathy, a rare disease characterized by muscle weakness and rods within muscle fibers. We investigated the impact of nebulin (NEB) deficiency on muscle function in vivo.
• Granzier, H. L., & Moss, R. L. (2019). Progress on the regulation of myofibrillar function: Part 2. The Journal of general physiology, 151(5), 609.
• Kellermayer, D., Smith, J. E., & Granzier, H. (2019). Titin mutations and muscle disease. Pflugers Archiv : European journal of physiology, 471(5), 673-682.
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  The introduction of next-generation sequencing technology has revealed that mutations in the gene that encodes titin (TTN) are linked to multiple skeletal and cardiac myopathies. The most prominent of these myopathies is dilated cardiomyopathy (DCM). Over 60 genes are linked to the etiology of DCM, but by far, the leading cause of DCM is mutations in TTN with truncating variants in TTN (TTNtvs) associated with familial DCM in ∼ 20% of the cases. Titin is a large (3-4 MDa) and abundant protein that forms the third myofilament type of striated muscle where it spans half the sarcomere, from the Z-disk to the M-line. The underlying mechanisms by which titin mutations induce disease are poorly understood and targeted therapies are not available. Here, we review what is known about TTN mutations in muscle disease, with a major focus on DCM. We highlight that exon skipping might provide a possible therapeutic avenue to address diseases that arise from TTNtvs.
• Lee, E. J., Kolb, J., Hwee, D. T., Malik, F. I., & Granzier, H. L. (2019). Functional Characterization of the Intact Diaphragm in a Nebulin-Based Nemaline Myopathy (NM) Model-Effects of the Fast Skeletal Muscle Troponin Activator. International journal of molecular sciences, 20(20).
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  Respiratory failure due to diaphragm dysfunction is considered a main cause of death in nemaline myopathy (NM) and we studied both isometric force and isotonic shortening of diaphragm muscle in a mouse model of nebulin-based NM (Neb cKO). A large contractile deficit was found in nebulin-deficient intact muscle that is frequency dependent, with the largest deficits at low-intermediate stimulation frequencies (e.g., a deficit of 72% at a stimulation frequency of 20 Hz). The effect of the fast skeletal muscle troponin activator (FSTA) on force was examined. had a negligible effect at maximal stimulation frequencies, but greatly reduced the force deficit of the diaphragm at sub-maximal stimulation levels with an effect that was largest in Neb cKO diaphragm. As a result, the force deficit of Neb cKO diaphragm fell (from 72% to 29% at 20 Hz). Similar effects were found in in vivo experiments on the nerve-stimulated gastrocnemius muscle complex. Load-clamp experiments on diaphragm muscle showed that increased the shortening velocity, and reduced the deficit in mechanical power by 33%. Thus, significantly improves muscle function in a mouse model of nebulin-based nemaline myopathy.
• Li, F., Barton, E. R., & Granzier, H. (2019). Deleting nebulin's C-terminus reveals its importance to sarcomeric structure and function and is sufficient to invoke nemaline myopathy. Human molecular genetics.
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  Nebulin is a large skeletal muscle protein wound around the thin filaments, with its C-terminus embedded within the Z-disk and its N-terminus extending out towards the thin filament pointed end. While nebulin's C-terminus has been implicated in both sarcomeric structure and function as well as the development of nemaline myopathy, the contributions of this region remain largely unknown. Additionally, the C-terminus is reported to contribute to muscle hypertrophy via the IGF-1 growth pathway. To study the functions of nebulin's C-terminus, we generated a mouse model deleting the final two unique C-terminal domains, the serine-rich region (SRR) and the SH3 domain (NebΔ163-165). Homozygous NebΔ163-165 mice that survive past the neonatal stage exhibit a mild weight deficit. Characterization of these mice revealed that the truncation caused a moderate myopathy phenotype reminiscent of nemaline myopathy despite the majority of nebulin being localized properly in the thin filaments. This phenotype included muscle weight loss, changes in sarcomere structure, as well as a decrease in force production. GST pull-down experiments found novel binding partners with the SRR, several of which are associated with myopathies. While the C-terminus does not appear to be a limiting step in muscle growth, the IGF-1 growth pathway remained functional despite the deleted domains being proposed to be essential for IGF-1 mediated hypertrophy. The NebΔ163-165 mouse model emphasizes that nebulin's C-terminus is necessary for proper sarcomeric development and shows that its loss is sufficient to induce myopathy.
• Li, K. L., Methawasin, M., Tanner, B. C., Granzier, H. L., Solaro, R. J., & Dong, W. J. (2019). Sarcomere length-dependent effects on Ca-troponin regulation in myocardium expressing compliant titin. The Journal of general physiology, 151(1), 30-41.
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  Cardiac performance is tightly regulated at the cardiomyocyte level by sarcomere length, such that increases in sarcomere length lead to sharply enhanced force generation at the same Ca concentration. Length-dependent activation of myofilaments involves dynamic and complex interactions between a multitude of thick- and thin-filament components. Among these components, troponin, myosin, and the giant protein titin are likely to be key players, but the mechanism by which these proteins are functionally linked has been elusive. Here, we investigate this link in the mouse myocardium using in situ FRET techniques. Our objective was to monitor how length-dependent Ca-induced conformational changes in the N domain of cardiac troponin C (cTnC) are modulated by myosin-actin cross-bridge (XB) interactions and increased titin compliance. We reconstitute FRET donor- and acceptor-modified cTnC(13C/51C)AEDANS-DDPM into chemically skinned myocardial fibers from wild-type and RBM20-deletion mice. The Ca-induced conformational changes in cTnC are quantified and characterized using time-resolved FRET measurements as XB state and sarcomere length are varied. The RBM20-deficient mouse expresses a more compliant N2BA titin isoform, leading to reduced passive tension in the myocardium. This provides a molecular tool to investigate how altered titin-based passive tension affects Ca-troponin regulation in response to mechanical stretch. In wild-type myocardium, we observe a direct association of sarcomere length-dependent enhancement of troponin regulation with both Ca activation and strongly bound XB states. In comparison, measurements from titin RBM20-deficient animals show blunted sarcomere length-dependent effects. These results suggest that titin-based passive tension contributes to sarcomere length-dependent Ca-troponin regulation. We also conclude that strong XB binding plays an important role in linking the modulatory effect of titin compliance to Ca-troponin regulation of the myocardium.
• Lindqvist, J., Lee, E. J., Karimi, E., Kolb, J., & Granzier, H. (2019). Omecamtiv mecarbil lowers the contractile deficit in a mouse model of nebulin-based nemaline myopathy. PloS one, 14(11), e0224467.
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  Nemaline myopathy (NEM) is a congenital neuromuscular disorder primarily caused by nebulin gene (NEB) mutations. NEM is characterized by muscle weakness for which currently no treatments exist. In NEM patients a predominance of type I fibers has been found. Thus, therapeutic options targeting type I fibers could be highly beneficial for NEM patients. Because type I muscle fibers express the same myosin isoform as cardiac muscle (Myh7), the effect of omecamtiv mecarbil (OM), a small molecule activator of Myh7, was studied in a nebulin-based NEM mouse model (Neb cKO). Skinned single fibers were activated by exogenous calcium and force was measured at a wide range of calcium concentrations. Maximal specific force of type I fibers was much less in fibers from Neb cKO animals and calcium sensitivity of permeabilized single fibers was reduced (pCa50 6.12 ±0.08 (cKO) vs 6.36 ±0.08 (CON)). OM increased the calcium sensitivity of type I single muscle fibers. The greatest effect occurred in type I fibers from Neb cKO muscle where OM restored the calcium sensitivity to that of the control type I fibers. Forces at submaximal activation levels (pCa 6.0-6.5) were significantly increased in Neb cKO fibers (~50%) but remained below that of control fibers. OM also increased isometric force and power during isotonic shortening of intact whole soleus muscle of Neb cKO mice, with the largest effects at physiological stimulation frequencies. We conclude that OM has the potential to improve the quality of life of NEM patients by increasing the force of type I fibers at submaximal activation levels.
• Lostal, W., Roudaut, C., Faivre, M., Charton, K., Suel, L., Bourg, N., Best, H., Smith, J. E., Gohlke, J., Corre, G., Li, X., Elbeck, Z., Knöll, R., Deschamps, J. Y., Granzier, H., & Richard, I. (2019). Titin splicing regulates cardiotoxicity associated with calpain 3 gene therapy for limb-girdle muscular dystrophy type 2A. Science translational medicine, 11(520).
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  Limb-girdle muscular dystrophy type 2A (LGMD2A or LGMDR1) is a neuromuscular disorder caused by mutations in the calpain 3 gene (). Previous experiments using adeno-associated viral (AAV) vector-mediated calpain 3 gene transfer in mice indicated cardiac toxicity associated with the ectopic expression of the calpain 3 transgene. Here, we performed a preliminary dose study in a severe double-knockout mouse model deficient in calpain 3 and dysferlin. We evaluated safety and biodistribution of AAV9-desmin-hCAPN3 vector administration to nonhuman primates (NHPs) with a dose of 3 × 10 viral genomes/kg. Vector administration did not lead to observable adverse effects or to detectable toxicity in NHP. Of note, the transgene expression did not produce any abnormal changes in cardiac morphology or function of injected animals while reaching therapeutic expression in skeletal muscle. Additional investigation on the underlying causes of cardiac toxicity observed after gene transfer in mice and the role of titin in this phenomenon suggest species-specific titin splicing. Mice have a reduced capacity for buffering calpain 3 activity compared to NHPs and humans. Our studies highlight a complex interplay between calpain 3 and titin binding sites and demonstrate an effective and safe profile for systemic calpain 3 vector delivery in NHP, providing critical support for the clinical potential of calpain 3 gene therapy in humans.
• Ma, W., Gong, H., Kiss, B., Lee, E. J., Granzier, H., & Irving, T. (2019). Response to: Thick Filament Length Changes in Muscle Have Both Elastic and Structural Components. Biophysical journal, 116(6), 985-986.
• Meurs, K. M., Friedenberg, S. G., Kolb, J., Saripalli, C., Tonino, P., Woodruff, K., Olby, N. J., Keene, B. W., Adin, D. B., Yost, O. L., DeFrancesco, T. C., Lahmers, S., Tou, S., Shelton, G. D., & Granzier, H. (2019). A missense variant in the titin gene in Doberman pinscher dogs with familial dilated cardiomyopathy and sudden cardiac death. Human genetics, 138(5), 515-524.
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  The dog provides a large animal model of familial dilated cardiomyopathy for the study of important aspects of this common familial cardiovascular disease. We have previously demonstrated a form of canine dilated cardiomyopathy in the Doberman pinscher breed that is inherited as an autosomal dominant trait and is associated with a splice site variant in the pyruvate dehydrogenase kinase 4 (PDK4) gene, however, genetic heterogeneity exists in this species as well and not all affected dogs have the PDK4 variant. Whole genome sequencing of a family of Doberman pinchers with dilated cardiomyopathy and sudden cardiac death without the PDK4 variant was performed. A pathologic missense variant in the titin gene located in an immunoglobulin-like domain in the I-band spanning region of the molecule was identified and was highly associated with the disease (p 
• Mijailovich, S. M., Stojanovic, B., Nedic, D., Svicevic, M., Geeves, M. A., Irving, T. C., & Granzier, H. L. (2019). Nebulin and titin modulate cross-bridge cycling and length-dependent calcium sensitivity. The Journal of general physiology, 151(5), 680-704.
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  Various mutations in the structural proteins nebulin and titin that are present in human disease are known to affect the contractility of striated muscle. Loss of nebulin is associated with reduced actin filament length and impairment of myosin binding to actin, whereas titin is thought to regulate muscle passive elasticity and is likely involved in length-dependent activation. Here, we sought to assess the modulation of muscle function by these sarcomeric proteins by using the computational platform muscle simulation code (MUSICO) to quantitatively separate the effects of structural changes, kinetics of cross-bridge cycling, and calcium sensitivity of the thin filaments. The simulations show that variation in thin filament length cannot by itself account for experimental observations of the contractility in nebulin-deficient muscle, but instead must be accompanied by a decreased myosin binding rate. Additionally, to match the observed calcium sensitivity, the rate of TnI detachment from actin needed to be increased. Simulations for cardiac muscle provided quantitative estimates of the effects of different titin-based passive elasticities on muscle force and activation in response to changes in sarcomere length and interfilament lattice spacing. Predicted force-pCa relations showed a decrease in both active tension and sensitivity to calcium with a decrease in passive tension and sarcomere length. We conclude that this behavior is caused by partial redistribution of the muscle load between active muscle force and titin-dependent passive force, and also by redistribution of stretch along the thin filament, which together modulate the release of TnI from actin. These data help advance understanding of how nebulin and titin mutations affect muscle function.
• Qiu, B., Ruston, J., Granzier, H., Justice, M. J., & Dowling, J. J. (2019). Failure to identify modifiers of -related nemaline myopathy in two pre-clinical models of the disease. Biology open, 8(9).
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  Nemaline myopathy is a rare neuromuscular disorder that affects 1 in 50,000 live births, with prevalence as high as 1 in 20,000 in certain populations. 13 genes have been linked to nemaline myopathy (NM), all of which are associated with the thin filament of the muscle sarcomere. Of the 13 associated genes, mutations in () accounts for up to 50% of all cases. Currently, the disease is incompletely understood and there are no available therapeutics for patients. To address this urgent need for effective treatments for patients affected by NM, we conducted a large scale chemical screen in a zebrafish model of -related NM and an N-ethyl-N-nitrosourea (ENU)-based genetic screen in a mouse model of exon 55 deletion, the most common mutation in NM patients. Neither screen was able to identify a candidate for therapy development, highlighting the need to transition from conventional chemical therapeutics to gene-based therapies for the treatment of NM.
• Radke, M. H., Polack, C., Methawasin, M., Fink, C., Granzier, H. L., & Gotthardt, M. (2019). Deleting Full Length Titin Versus the Titin M-Band Region Leads to Differential Mechanosignaling and Cardiac Phenotypes. Circulation, 139(15), 1813-1827.
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  Titin is a giant elastic protein that spans the half-sarcomere from Z-disk to M-band. It acts as a molecular spring and mechanosensor and has been linked to striated muscle disease. The pathways that govern titin-dependent cardiac growth and contribute to disease are diverse and difficult to dissect.
• Ross, J. A., Levy, Y., Ripolone, M., Kolb, J. S., Turmaine, M., Holt, M., Lindqvist, J., Claeys, K. G., Weis, J., Monforte, M., Tasca, G., Moggio, M., Figeac, N., Zammit, P. S., Jungbluth, H., Fiorillo, C., Vissing, J., Witting, N., Granzier, H., , Zanoteli, E., et al. (2019). Impairments in contractility and cytoskeletal organisation cause nuclear defects in nemaline myopathy. Acta neuropathologica, 138(3), 477-495.
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  Nemaline myopathy (NM) is a skeletal muscle disorder caused by mutations in genes that are generally involved in muscle contraction, in particular those related to the structure and/or regulation of the thin filament. Many pathogenic aspects of this disease remain largely unclear. Here, we report novel pathological defects in skeletal muscle fibres of mouse models and patients with NM: irregular spacing and morphology of nuclei; disrupted nuclear envelope; altered chromatin arrangement; and disorganisation of the cortical cytoskeleton. Impairments in contractility are the primary cause of these nuclear defects. We also establish the role of microtubule organisation in determining nuclear morphology, a phenomenon which is likely to contribute to nuclear alterations in this disease. Our results overlap with findings in diseases caused directly by mutations in nuclear envelope or cytoskeletal proteins. Given the important role of nuclear shape and envelope in regulating gene expression, and the cytoskeleton in maintaining muscle fibre integrity, our findings are likely to explain some of the hallmarks of NM, including contractile filament disarray, altered mechanical properties and broad transcriptional alterations.
• Slater, R. E., Strom, J. G., Methawasin, M., Liss, M., Gotthardt, M., Sweitzer, N., & Granzier, H. L. (2019). Metformin improves diastolic function in an HFpEF-like mouse model by increasing titin compliance. The Journal of general physiology, 151(1), 42-52.
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  Heart failure with preserved ejection fraction (HFpEF) is a complex syndrome characterized by a preserved ejection fraction but increased diastolic stiffness and abnormalities of filling. Although the prevalence of HFpEF is high and continues to rise, no effective therapies exist; however, the diabetic drug metformin has been associated with improved diastolic function in diabetic patients. Here we determine the therapeutic potential of metformin for improving diastolic function in a mouse model with HFpEF-like symptoms. We combine transverse aortic constriction (TAC) surgery with deoxycorticosterone acetate (DOCA) supplementation to obtain a mouse model with increased diastolic stiffness and exercise intolerance. Echocardiography and pressure-volume analysis reveal that providing metformin to TAC/DOCA mice improves diastolic function in the left ventricular (LV) chamber. Muscle mechanics show that metformin lowers passive stiffness of the LV wall muscle. Concomitant with this improvement in diastolic function, metformin-treated TAC/DOCA mice also demonstrate preserved exercise capacity. No metformin effects are seen in sham operated mice. Extraction experiments on skinned ventricular muscle strips show that the metformin-induced reduction of passive stiffness in TAC/DOCA mice is due to an increase in titin compliance. Using phospho-site-specific antibodies, we assay the phosphorylation of titin's PEVK and N2B spring elements. Metformin-treated mice have unaltered PEVK phosphorylation but increased phosphorylation of PKA sites in the N2B element, a change which has previously been shown to lower titin's stiffness. Consistent with this result, experiments with a mouse model deficient in the N2B element reveal that the beneficial effect of metformin on LV chamber and muscle stiffness requires the presence of the N2B element. We conclude that metformin offers therapeutic benefit during HFpEF by lowering titin-based passive stiffness.
• Tinklenberg, J. A., Siebers, E. M., Beatka, M. J., Fickau, B. A., Ayres, S., Meng, H., Yang, L., Simpson, P., Granzier, H. L., & Lawlor, M. W. (2019). Myostatin Inhibition Using ActRIIB-mFc Does Not Produce Weight Gain or Strength in the Nebulin Conditional KO Mouse. Journal of neuropathology and experimental neurology, 78(2), 130-139.
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  Mutations in at least 12 genes are responsible for a group of congenital skeletal muscle diseases known as nemaline myopathies (NMs). NMs are associated with a range of clinical symptoms and pathological changes often including the presence of cytoplasmic rod-like structures (nemaline bodies) and myofiber hypotrophy. Our recent work has identified a variable degree of behavioral benefit when treating 2 NM mouse models due to mutations in Acta1 with myostatin inhibition. This study is focused on the effects of delivering ActRIIB-mFc (Acceleron; a myostatin inhibitor) to the nebulin conditional knockout KO (Neb cKO) mouse model of NM. Treatment of Neb cKO mice with ActRIIB-mFc did not produce increases in weight gain, strength, myofiber size, or hypertrophic pathway signaling. Overall, our studies demonstrate a lack of response in Neb cKO mice to myostatin inhibition, which differs from the response observed when treating other NM models.
• Tonino, P., Kiss, B., Gohlke, J., Smith, J. E., & Granzier, H. (2019). Fine mapping titin's C-zone: Matching cardiac myosin-binding protein C stripes with titin's super-repeats. Journal of molecular and cellular cardiology, 133, 47-56.
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  Titin is largely comprised of serially-linked immunoglobulin (Ig) and fibronectin type-III (Fn3) domains. Many of these domains are arranged in an 11 domain super-repeat pattern that is repeated 11 times, forming the so-named titin C-zone in the A-band region of the sarcomere. Each super-repeat is thought to provide binding sites for thick filament proteins, such as cMyBP-C (cardiac myosin-binding protein C). However, it remains to be established which of titin's 11 C-zone super-repeats anchor cMyBP-C as titin contains 11 super-repeats and cMyBP-C is found in 9 stripes only. To study the layout of titin's C-zone in relation to MyBP-C, immunolabeling studies were performed on mouse skinned myocardium with antibodies to titin and cMyBP-C, using both immuno-electron microscopy and super-resolution optical microscopy. Results indicate that cMyBP-C locates near the interface between titin's C-zone super-repeats. Studies on a mouse model in which two of titin's C-zone repeats have been genetically deleted support that the first Ig domain of a super-repeat is important for anchoring cMyBP-C but also Fn3 domains located at the end of the preceding repeat. Furthermore, not all super-repeat interfaces are equal as the interface between super-repeat 1 and 2 (close to titin's D-zone) does not contain cMyBP-C. Finally, titin's C-zone does not extend all the way to the bare zone but instead terminates at the level of the second myosin crown. This study enhances insights in the molecular layout of the C-zone of titin, its relation to cMyBP-C, and its possible roles in cardiomyopathies.
• van Bastelaar, J., Granzier, R., van Roozendaal, L. M., van Kuijk, S. M., Lerut, A. V., Beets, G., Hadfoune, M., Olde Damink, S., & Vissers, Y. L. (2019). Analysis of TNF-α and interleukin-6 in seroma of patients undergoing mastectomy with or without flap fixation: is there a predictive value for seroma formation and its sequelae?. Surgical oncology, 28, 36-41.
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  Seroma formation is a common complication after mastectomy. Flap fixation has the potential to prevent seroma formation, but identifying patients that are at risk of developing seroma, remains challenging. The aim of this study was to assess the association between pro-inflammatory cytokines in seroma fluid one day after surgery and seroma formation and it sequelae.
• van der Pijl, R. J., Granzier, H. L., & Ottenheijm, C. A. (2019). Diaphragm contractile weakness due to reduced mechanical loading: role of titin. American journal of physiology. Cell physiology, 317(2), C167-C176.
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  The diaphragm, the main muscle of inspiration, is constantly subjected to mechanical loading. Only during controlled mechanical ventilation, as occurs during thoracic surgery and in the intensive care unit, is mechanical loading of the diaphragm arrested. Animal studies indicate that the diaphragm is highly sensitive to unloading, causing rapid muscle fiber atrophy and contractile weakness; unloading-induced diaphragm atrophy and contractile weakness have been suggested to contribute to the difficulties in weaning patients from ventilator support. The molecular triggers that initiate the rapid unloading atrophy of the diaphragm are not well understood, although proteolytic pathways and oxidative signaling have been shown to be involved. Mechanical stress is known to play an important role in the maintenance of muscle mass. Within the muscle's sarcomere, titin is considered to play an important role in the stress-response machinery. Titin is a giant protein that acts as a mechanosensor regulating muscle protein expression in a sarcomere strain-dependent fashion. Thus titin is an attractive candidate for sensing the sudden mechanical arrest of the diaphragm when patients are mechanically ventilated, leading to changes in muscle protein expression. Here, we provide a novel perspective on how titin and its biomechanical sensing and signaling might be involved in the development of mechanical unloading-induced diaphragm weakness.
• Brynnel, A., Hernandez, Y., Kiss, B., Lindqvist, J., Adler, M., Kolb, J., van der Pijl, R., Gohlke, J., Strom, J., Smith, J., Ottenheijm, C., & Granzier, H. L. (2018). Downsizing the molecular spring of the giant protein titin reveals that skeletal muscle titin determines passive stiffness and drives longitudinal hypertrophy. eLife, 7.
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  Titin, the largest protein known, forms an elastic myofilament in the striated muscle sarcomere. To establish titin's contribution to skeletal muscle passive stiffness, relative to that of the extracellular matrix, a mouse model was created in which titin's molecular spring region was shortened by deleting 47 exons, the model. RNA sequencing and super-resolution microscopy predicts a much stiffer titin molecule. Mechanical studies with this novel mouse model support that titin is the main determinant of skeletal muscle passive stiffness. Unexpectedly, the in vivo sarcomere length working range was shifted to shorter lengths in mice, due to a ~ 30% increase in the number of sarcomeres in series (longitudinal hypertrophy). The expected effect of this shift on active force generation was minimized through a shortening of thin filaments that was discovered in mice. Thus, skeletal muscle titin is the dominant determinant of physiological passive stiffness and drives longitudinal hypertrophy.
• Dos Remedios, C. G., Lal, S. P., Li, A., McNamara, J., Keogh, A., Macdonald, P. S., Cooke, R., Ehler, E., Knöll, R., Marston, S. B., Stelzer, J., Granzier, H., Bezzina, C., van Dijk, S., De Man, F., Stienen, G. J., Odeberg, J., Pontén, F., Linke, W. A., & van der Velden, J. (2018). Correction to: The Sydney Heart Bank: improving translational research while eliminating or reducing the use of animal models of human heart disease. Biophysical reviews, 10(3), 941.
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  In the original version of this article, the name of one of the authors is not correct. The correct name should be W. A. Linke, which is shown correctly in the authorgroup section above.
• Jeong, M. Y., Lin, Y. H., Wennersten, S. A., Demos-Davies, K. M., Cavasin, M. A., Mahaffey, J. H., Monzani, V., Saripalli, C., Mascagni, P., Reece, T. B., Ambardekar, A. V., Granzier, H. L., Dinarello, C. A., & McKinsey, T. A. (2018). Histone deacetylase activity governs diastolic dysfunction through a nongenomic mechanism. Science translational medicine, 10(427).
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  There are no approved drugs for the treatment of heart failure with preserved ejection fraction (HFpEF), which is characterized by left ventricular (LV) diastolic dysfunction. We demonstrate that ITF2357 (givinostat), a clinical-stage inhibitor of histone deacetylase (HDAC) catalytic activity, is efficacious in two distinct murine models of diastolic dysfunction with preserved EF. ITF2357 blocked LV diastolic dysfunction due to hypertension in Dahl salt-sensitive (DSS) rats and suppressed aging-induced diastolic dysfunction in normotensive mice. HDAC inhibitor-mediated efficacy was not due to lowering blood pressure or inhibiting cellular and molecular events commonly associated with diastolic dysfunction, including cardiac fibrosis, cardiac hypertrophy, or changes in cardiac titin and myosin isoform expression. Instead, ex vivo studies revealed impairment of cardiac myofibril relaxation as a previously unrecognized, myocyte-autonomous mechanism for diastolic dysfunction, which can be ameliorated by HDAC inhibition. Translating these findings to humans, cardiac myofibrils from patients with diastolic dysfunction and preserved EF also exhibited compromised relaxation. These data suggest that agents such as HDAC inhibitors, which potentiate cardiac myofibril relaxation, hold promise for the treatment of HFpEF in humans.
• Kawai, M., Karam, T. S., Kolb, J., Wang, L., & Granzier, H. L. (2018). Nebulin increases thin filament stiffness and force per cross-bridge in slow-twitch soleus muscle fibers. The Journal of general physiology, 150(11), 1510-1522.
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  Nebulin (Neb) is associated with the thin filament in skeletal muscle cells, but its functions are not well understood. For this goal, we study skinned slow-twitch soleus muscle fibers from wild-type (Neb) and conditional Neb knockout (Neb) mice. We characterize cross-bridge (CB) kinetics and the elementary steps of the CB cycle by sinusoidal analysis during full Ca activation and observe that Neb increases active tension 1.9-fold, active stiffness 2.7-fold, and rigor stiffness 3.0-fold. The ratio of stiffness during activation and rigor states is 62% in Neb fibers and 68% in Neb fibers. These are approximately proportionate to the number of strongly attached CBs during activation. Because the thin filament length is 15% shorter in Neb fibers than in Neb fibers, the increase in force per CB in the presence of Neb is ∼1.5 fold. The equilibrium constant of the CB detachment step (), its rate (), and the rate of the reverse force generation step () are larger in Neb fibers than in Neb fibers. The rates of the force generation step () and the reversal detachment step () change in the opposite direction. These effects can be explained by Le Chatelier's principle: Increased CB strain promotes less force-generating state(s) and/or detached state(s). Further, when CB distributions among the six states are calculated, there is no significant difference in the number of strongly attached CBs between fibers with and without Neb. These results demonstrate that Neb increases force per CB. We also confirm that force is generated by isomerization of actomyosin (AM) from the AM.ADP.Pi state (ADP, adenosine diphophate; Pi, phosphate) to the AM*ADP.Pi state, where the same force is maintained after Pi release to result in the AM*ADP state. We propose that Neb changes the actin (and myosin) conformation for better ionic and hydrophobic/stereospecific AM interaction, and that the effect of Neb is similar to that of tropomyosin.
• Kiss, B., Lee, E. J., Ma, W., Li, F. W., Tonino, P., Mijailovich, S. M., Irving, T. C., & Granzier, H. L. (2018). Nebulin stiffens the thin filament and augments cross-bridge interaction in skeletal muscle. Proceedings of the National Academy of Sciences of the United States of America, 115(41), 10369-10374.
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  Nebulin is a giant sarcomeric protein that spans along the actin filament in skeletal muscle, from the Z-disk to near the thin filament pointed end. Mutations in nebulin cause muscle weakness in nemaline myopathy patients, suggesting that nebulin plays important roles in force generation, yet little is known about nebulin's influence on thin filament structure and function. Here, we used small-angle X-ray diffraction and compared intact muscle deficient in nebulin (using a conditional nebulin-knockout, Neb cKO) with control (Ctrl) muscle. When muscles were activated, the spacing of the actin subunit repeat (27 Å) increased in both genotypes; when converted to thin filament stiffness, the obtained value was 30 pN/nm in Ctrl muscle and 10 pN/nm in Neb cKO muscle; that is, the thin filament was approximately threefold stiffer when nebulin was present. In contrast, the thick filament stiffness was not different between the genotypes. A significantly shorter left-handed (59 Å) thin filament helical pitch was found in passive and contracting Neb cKO muscles, as well as impaired tropomyosin and troponin movement. Additionally, a reduced myosin mass transfer toward the thin filament in contracting Neb cKO muscle was found, suggesting reduced cross-bridge interaction. We conclude that nebulin is critically important for physiological force levels, as it greatly stiffens the skeletal muscle thin filament and contributes to thin filament activation and cross-bridge recruitment.
• Lindqvist, J., van den Berg, M., van der Pijl, R., Hooijman, P. E., Beishuizen, A., Elshof, J., de Waard, M., Girbes, A., Spoelstra-de Man, A., Shi, Z. H., van den Brom, C., Bogaards, S., Shen, S., Strom, J., Granzier, H., Kole, J., Musters, R. J., Paul, M. A., Heunks, L. M., & Ottenheijm, C. A. (2018). Positive End-Expiratory Pressure Ventilation Induces Longitudinal Atrophy in Diaphragm Fibers. American journal of respiratory and critical care medicine, 198(4), 472-485.
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  Diaphragm weakness in critically ill patients prolongs ventilator dependency and duration of hospital stay and increases mortality and healthcare costs. The mechanisms underlying diaphragm weakness include cross-sectional fiber atrophy and contractile protein dysfunction, but whether additional mechanisms are at play is unknown.
• Ma, W., Gong, H., Kiss, B., Lee, E. J., Granzier, H., & Irving, T. (2018). Thick-Filament Extensibility in Intact Skeletal Muscle. Biophysical journal, 115(8), 1580-1588.
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  Myofilament extensibility is a key structural parameter for interpreting myosin cross-bridge kinetics in striated muscle. Previous studies reported much higher thick-filament extensibility at low tension than the better-known and commonly used values at high tension, but in interpreting mechanical studies of muscle, a single value for thick-filament extensibility has usually been assumed. Here, we established the complete thick-filament force-extension curve from actively contracting, intact vertebrate skeletal muscle. To access a wide range of tetanic forces, the myosin inhibitor blebbistatin was used to induce low tetanic forces in addition to the higher tensions obtained from tetanic contractions of the untreated muscle. We show that the force/extensibility curve of the thick filament is nonlinear, so assuming a single value for thick-filament extensibility at all force levels is not justified. We also show that independent of whether tension is generated passively by sarcomere stretch or actively by cross-bridges, the thick-filament extensibility is nonlinear. Myosin head periodicity, however, only changes when active tension is generated under calcium-activated conditions. The nonlinear thick-filament force-extension curve in skeletal muscle, therefore, reflects a purely passive response to either titin-based force or actomyosin-based force, and it does not include a thick-filament activation mechanism. In contrast, the transition of myosin head periodicity to an active configuration appears to only occur in response to increased active force when calcium is present.
• Methawasin, M., & Granzier, H. (2018). Softening the Stressed Giant Titin in Diabetes Mellitus. Circulation research, 123(3), 315-317.
• Oates, E. C., Jones, K. J., Donkervoort, S., Charlton, A., Brammah, S., Smith, J. E., Ware, J. S., Yau, K. S., Swanson, L. C., Whiffin, N., Peduto, A. J., Bournazos, A., Waddell, L. B., Farrar, M. A., Sampaio, H. A., Teoh, H. L., Lamont, P. J., Mowat, D., Fitzsimons, R. B., , Corbett, A. J., et al. (2018). Congenital Titinopathy: Comprehensive characterization and pathogenic insights. Annals of neurology, 83(6), 1105-1124.
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  Comprehensive clinical characterization of congenital titinopathy to facilitate diagnosis and management of this important emerging disorder.
• van der Pijl, R., Strom, J., Conijn, S., Lindqvist, J., Labeit, S., Granzier, H., & Ottenheijm, C. (2018). Titin-based mechanosensing modulates muscle hypertrophy. Journal of cachexia, sarcopenia and muscle, 9(5), 947-961.
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  Titin is an elastic sarcomeric filament that has been proposed to play a key role in mechanosensing and trophicity of muscle. However, evidence for this proposal is scarce due to the lack of appropriate experimental models to directly test the role of titin in mechanosensing.
• Dos Remedios, C. G., Lal, S. P., Li, A., McNamara, J., Keogh, A., Macdonald, P. S., Cooke, R., Ehler, E., Knöll, R., Marston, S. B., Stelzer, J., Granzier, H., Bezzina, C., van Dijk, S., De Man, F., Stienen, G. J., Odeberg, J., Pontén, F., Linke, W., & van der Velden, J. (2017). The Sydney Heart Bank: improving translational research while eliminating or reducing the use of animal models of human heart disease. Biophysical reviews, 9(4), 431-441.
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  The Sydney Heart Bank (SHB) is one of the largest human heart tissue banks in existence. Its mission is to provide high-quality human heart tissue for research into the molecular basis of human heart failure by working collaboratively with experts in this field. We argue that, by comparing tissues from failing human hearts with age-matched non-failing healthy donor hearts, the results will be more relevant than research using animal models, particularly if their physiology is very different from humans. Tissue from heart surgery must generally be used soon after collection or it significantly deteriorates. Freezing is an option but it raises concerns that freezing causes substantial damage at the cellular and molecular level. The SHB contains failing samples from heart transplant patients and others who provided informed consent for the use of their tissue for research. All samples are cryopreserved in liquid nitrogen within 40 min of their removal from the patient, and in less than 5-10 min in the case of coronary arteries and left ventricle samples. To date, the SHB has collected tissue from about 450 failing hearts (>15,000 samples) from patients with a wide range of etiologies as well as increasing numbers of cardiomyectomy samples from patients with hypertrophic cardiomyopathy. The Bank also has hearts from over 120 healthy organ donors whose hearts, for a variety of reasons (mainly tissue-type incompatibility with waiting heart transplant recipients), could not be used for transplantation. Donor hearts were collected by the St Vincent's Hospital Heart and Lung transplantation team from local hospitals or within a 4-h jet flight from Sydney. They were flushed with chilled cardioplegic solution and transported to Sydney where they were quickly cryopreserved in small samples. Failing and/or donor samples have been used by more than 60 research teams around the world, and have resulted in more than 100 research papers. The tissues most commonly requested are from donor left ventricles, but right ventricles, atria, interventricular system, and coronary arteries vessels have also been reported. All tissues are stored for long-term use in liquid N or vapor (170-180 °C), and are shipped under nitrogen vapor to avoid degradation of sensitive molecules such as RNAs and giant proteins. We present evidence that the availability of these human heart samples has contributed to a reduction in the use of animal models of human heart failure.
• Joureau, B., de Winter, J. M., Stam, K., Granzier, H., & Ottenheijm, C. A. (2017). Muscle weakness in respiratory and peripheral skeletal muscles in a mouse model for nebulin-based nemaline myopathy. Neuromuscular disorders : NMD, 27(1), 83-89.
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  Nemaline myopathy is among the most common non-dystrophic congenital myopathies, and is characterized by the presence of nemaline rods in skeletal muscles fibers, general muscle weakness, and hypotonia. Although respiratory failure is the main cause of death in nemaline myopathy, only little is known regarding the contractile strength of the diaphragm, the main muscle of inspiration. To investigate diaphragm contractility, in the present study we took advantage of a mouse model for nebulin-based nemaline myopathy that we recently developed. In this mouse model, exon 55 of Neb is deleted (NebΔExon55), a mutation frequently found in patients. Diaphragm contractility was determined in permeabilized muscle fibers and was compared to the contractility of permeabilized fibers from three peripheral skeletal muscles: soleus, extensor digitorum longus, and gastrocnemius. The force generating capacity of diaphragm muscle fibers of NebΔExon55 mice was reduced to 25% of wildtype levels, indicating severe contractile weakness. The contractile weakness of diaphragm fibers was more pronounced than that observed in soleus muscle, but not more pronounced than that observed in extensor digitorum longus and gastrocnemius muscles. The reduced muscle contractility was at least partly caused by changes in cross-bridge cycling kinetics which reduced the number of bound cross-bridges. The severe diaphragm weakness likely contributes to the development of respiratory failure in NebΔExon55 mice and might explain their early, postnatal death.
• Kellermayer, D., Smith, J. E., & Granzier, H. (2017). Novex-3, the tiny titin of muscle. Biophysical reviews, 9(3), 201-206.
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  The giant multi-functional striated muscle protein titin is the third most abundant muscle protein after myosin and actin. Titin plays a pivotal role in myocardial passive stiffness, structural integrity and stress-initiated signaling pathways. The complete sequence of the human titin gene contains three isoform-specific mutually exclusive exons [termed novel exons (novex)] coding for the I-band sequence, named novex-1 (exon 45), novex-2 (exon 46) and novex-3 (exon 48). Transcripts containing either the novex-1 or novex-2 exons code for the novex-1 and novex-2 titin isoforms. The novex-3 transcript contains a stop codon and polyA tail signal, resulting in an unusually small (∼700 kDa) isoform, referred to as novex-3 titin. This 'tiny titin' isoform extends from the Z-disc (N-terminus) to novex-3 (C-terminus) and is expressed in all striated muscles. Biochemical analysis of novex-3 titin in cardiomyocytes shows that obscurin, a vertebrate muscle protein, binds to novex-3 titin. The novex-3/obscurin complex localizes to the Z-disc region and may regulate calcium, and SH3- and GTPase-associated myofibrillar signaling pathways. Therefore, novex-3 titin could be involved in stress-initiated sarcomeric restructuring.
• Methawasin, M., & Granzier, H. (2017). Response by Methawasin and Granzier to Letter Regarding Article, "Experimentally Increasing the Compliance of Titin Through RNA Binding Motif-20 (RBM20) Inhibition Improves Diastolic Function in a Mouse Model of Heart Failure With Preserved Ejection Fraction". Circulation, 135(11), e681-e682.
• Slater, R. E., Strom, J. G., & Granzier, H. (2017). Effect of exercise on passive myocardial stiffness in mice with diastolic dysfunction. Journal of molecular and cellular cardiology, 108, 24-33.
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  Heart failure with preserved ejection fraction (HFpEF) is a complex syndrome, characterized by increased diastolic stiffness and a preserved ejection fraction, with no effective treatment options. Here we studied the therapeutic potential of exercise for improving diastolic function in a mouse model with HFpEF-like symptoms, the TtnΔIAjxn mouse model. TtnΔIAjxn mice have increased diastolic stiffness and reduced exercise tolerance, mimicking aspects of HFpEF observed in patients. We investigated the effect of free-wheel running exercise on diastolic function. Mechanical studies on cardiac muscle strips from the LV free wall revealed that both TtnΔIAjxn and wildtype (WT) exercised mice had a reduction in passive stiffness, relative to sedentary controls. In both genotypes, this reduction is due to an increase in the compliance of titin whereas ECM-based stiffness was unaffected. Phosphorylation of titin's PEVK and N2B spring elements were assayed with phospho-site specific antibodies. Exercised mice had decreased PEVK phosphorylation and increased N2B phosphorylation both of which are predicted to contribute to the increased compliance of titin. Since exercise lowers the heart rate we examined whether reduction in heart rate per se can improve passive stiffness by administering the heart-rate-lowering drug ivabradine. Ivabradine lowered heart rate in our study but it did not affect passive tension, in neither WT nor TtnΔIAjxn mice. We conclude that exercise is beneficial for decreasing passive stiffness and that it involves beneficial alterations in titin phosphorylation.
• Tonino, P., Kiss, B., Strom, J., Methawasin, M., Smith, J. E., Kolb, J., Labeit, S., & Granzier, H. (2017). The giant protein titin regulates the length of the striated muscle thick filament. Nature communications, 8(1), 1041.
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  The contractile machinery of heart and skeletal muscles has as an essential component the thick filament, comprised of the molecular motor myosin. The thick filament is of a precisely controlled length, defining thereby the force level that muscles generate and how this force varies with muscle length. It has been speculated that the mechanism by which thick filament length is controlled involves the giant protein titin, but no conclusive support for this hypothesis exists. Here we show that in a mouse model in which we deleted two of titin's C-zone super-repeats, thick filament length is reduced in cardiac and skeletal muscles. In addition, functional studies reveal reduced force generation and a dilated cardiomyopathy (DCM) phenotype. Thus, regulation of thick filament length depends on titin and is critical for maintaining muscle health.
• Birch, C. L., Behunin, S. M., Lopez-Pier, M. A., Danilo, C., Lipovka, Y., Saripalli, C., Granzier, H., & Konhilas, J. P. (2016). Sex dimorphisms of crossbridge cycling kinetics in transgenic hypertrophic cardiomyopathy mice. American journal of physiology. Heart and circulatory physiology, 311(1), H125-36.
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  Familial hypertrophic cardiomyopathy (HCM) is a disease of the sarcomere and may lead to hypertrophic, dilated, restrictive, and/or arrhythmogenic cardiomyopathy, congestive heart failure, or sudden cardiac death. We hypothesized that hearts from transgenic HCM mice harboring a mutant myosin heavy chain increase the energetic cost of contraction in a sex-specific manner. To do this, we assessed Ca(2+) sensitivity of tension and crossbridge kinetics in demembranated cardiac trabeculas from male and female wild-type (WT) and HCM hearts at an early time point (2 mo of age). We found a significant effect of sex on Ca(2+) sensitivity such that male, but not female, HCM mice displayed a decrease in Ca(2+) sensitivity compared with WT counterparts. The HCM transgene and sex significantly impacted the rate of force redevelopment by a rapid release-restretch protocol and tension cost by the ATPase-tension relationship. In each of these measures, HCM male trabeculas displayed a gain-of-function when compared with WT counterparts. In addition, cardiac remodeling measured by echocardiography, histology, morphometry, and posttranslational modifications demonstrated sex- and HCM-specific effects. In conclusion, female and male HCM mice display sex dimorphic crossbridge kinetics accompanied by sex- and HCM-dependent cardiac remodeling at the morphometric, histological, and cellular level.
• Bogomolovas, J., Fleming, J. R., Anderson, B. R., Williams, R., Lange, S., Simon, B., Khan, M. M., Rudolf, R., Franke, B., Bullard, B., Rigden, D. J., Granzier, H., Labeit, S., & Mayans, O. (2016). Exploration of pathomechanisms triggered by a single-nucleotide polymorphism in titin's I-band: the cardiomyopathy-linked mutation T2580I. Open biology, 6(9).
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  Missense single-nucleotide polymorphisms (mSNPs) in titin are emerging as a main causative factor of heart failure. However, distinguishing between benign and disease-causing mSNPs is a substantial challenge. Here, we research the question of whether a single mSNP in a generic domain of titin can affect heart function as a whole and, if so, how. For this, we studied the mSNP T2850I, seemingly linked to arrhythmogenic right ventricular cardiomyopathy (ARVC). We used structural biology, computational simulations and transgenic muscle in vivo methods to track the effect of the mutation from the molecular to the organismal level. The data show that the T2850I exchange is compatible with the domain three-dimensional fold, but that it strongly destabilizes it. Further, it induces a change in the conformational dynamics of the titin chain that alters its reactivity, causing the formation of aberrant interactions in the sarcomere. Echocardiography of knock-in mice indicated a mild diastolic dysfunction arising from increased myocardial stiffness. In conclusion, our data provide evidence that single mSNPs in titin's I-band can alter overall muscle behaviour. Our suggested mechanisms of disease are the development of non-native sarcomeric interactions and titin instability leading to a reduced I-band compliance. However, understanding the T2850I-induced ARVC pathology mechanistically remains a complex problem and will require a deeper understanding of the sarcomeric context of the titin region affected.
• Bull, M., Methawasin, M., Strom, J., Nair, P., Hutchinson, K., & Granzier, H. (2016). Alternative Splicing of Titin Restores Diastolic Function in an HFpEF-Like Genetic Murine Model (TtnΔIAjxn). Circulation research, 119(6), 764-72.
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  Patients with heart failure with preserved ejection fraction (HFpEF) experience elevated filling pressures and reduced ventricular compliance. The splicing factor RNA-binding motif 20 (RBM20) regulates the contour length of titin's spring region and thereby determines the passive stiffness of cardiomyocytes. Inhibition of RBM20 leads to super compliant titin isoforms (N2BAsc) that reduce passive stiffness.
• Evans, J. M., Cox, M. L., Huska, J., Li, F., Gaitero, L., Guo, L. T., Casal, M. L., Granzier, H. L., Shelton, G. D., & Clark, L. A. (2016). Exome sequencing reveals a nebulin nonsense mutation in a dog model of nemaline myopathy. Mammalian genome : official journal of the International Mammalian Genome Society, 27(9-10), 495-502.
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  Nemaline myopathy (NM) is a congenital muscle disorder associated with muscle weakness, hypotonia, and rod bodies in the skeletal muscle fibers. Mutations in 10 genes have been implicated in human NM, but spontaneous cases in dogs have not been genetically characterized. We identified a novel recessive myopathy in a family of line-bred American bulldogs (ABDs); rod bodies in muscle biopsies established this as NM. Using SNP profiles from the nuclear family, we evaluated inheritance patterns at candidate loci and prioritized TNNT1 and NEB for further investigation. Whole exome sequencing of the dam, two affected littermates, and an unaffected littermate revealed a nonsense mutation in NEB (g.52734272 C>A, S8042X). Whole tissue gel electrophoresis and western blots confirmed a lack of full-length NEB in affected tissues, suggesting nonsense-mediated decay. The pathogenic variant was absent from 120 dogs of 24 other breeds and 100 unrelated ABDs, suggesting that it occurred recently and may be private to the family. This study presents the first molecularly characterized large animal model of NM, which could provide new opportunities for therapeutic approaches.
• Gigli, M., Begay, R. L., Morea, G., Graw, S. L., Sinagra, G., Taylor, M. R., Granzier, H., & Mestroni, L. (2016). A Review of the Giant Protein Titin in Clinical Molecular Diagnostics of Cardiomyopathies. Frontiers in cardiovascular medicine, 3, 21.
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  Titin (TTN) is known as the largest sarcomeric protein that resides within the heart muscle. Due to alternative splicing of TTN, the heart expresses two major isoforms (N2B and N2BA) that incorporate four distinct regions termed the Z-line, I-band, A-band, and M-line. Next-generation sequencing allows a large number of genes to be sequenced simultaneously and provides the opportunity to easily analyze giant genes such as TTN. Mutations in the TTN gene can cause cardiomyopathies, in particular dilated cardiomyopathy (DCM). DCM is the most common form of cardiomyopathy, and it is characterized by systolic dysfunction and dilation of the left ventricle. TTN truncating variants have been described as the most common cause of DCM, while the real impact of TTN missense variants in the pathogenesis of DCM is still unclear. In a recent population screening study, rare missense variants potentially pathogenic based on bioinformatic filtering represented only 12.6% of the several hundred rare TTN missense variants found, suggesting that missense variants are very common in TTN and are frequently benign. The aim of this review is to understand the clinical role of TTN mutations in DCM and in other cardiomyopathies. Whereas TTN truncations are common in DCM, there is evidence that TTN truncations are rare in the hypertrophic cardiomyopathy (HCM) phenotype. Furthermore, TTN mutations can also cause arrhythmogenic right ventricular cardiomyopathy (ARVC) with distinct clinical features and outcomes. Finally, the identification of a rare TTN missense variant cosegregating with the restrictive cardiomyopathy (RCM) phenotype suggests that TTN is a novel disease-causing gene in this disease. Clinical diagnostic testing is currently able to analyze over 100 cardiomyopathy genes, including TTN; however, the size and presence of extensive genetic variation in TTN presents clinical challenges in determining significant disease-causing mutations. This review discusses the current knowledge of TTN genetic variations in cardiomyopathies and the impact of the diagnosis of TTN pathogenic mutations in the clinical setting.
• Hinze, F., Dieterich, C., Radke, M. H., Granzier, H., & Gotthardt, M. (2016). Reducing RBM20 activity improves diastolic dysfunction and cardiac atrophy. Journal of molecular medicine (Berlin, Germany), 94(12), 1349-1358.
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  Impaired diastolic filling is a main contributor to heart failure with preserved ejection fraction (HFpEF), a syndrome with increasing prevalence and no treatment. Both collagen and the giant sarcomeric protein titin determine diastolic function. Since titin's elastic properties can be adjusted physiologically, we evaluated titin-based stiffness as a therapeutic target. We adjusted RBM20-dependent cardiac isoform expression in the titin N2B knockout mouse with increased ventricular stiffness. A ~50 % reduction of RBM20 activity does not only maintain cardiac filling in diastole but also ameliorates cardiac atrophy and thus improves cardiac function in the N2B-deficient heart. Reduced RBM20 activity partially normalized gene expression related to muscle development and fatty acid metabolism. The adaptation of cardiac growth was related to hypertrophy signaling via four-and-a-half lim-domain proteins (FHLs) that translate mechanical input into hypertrophy signals. We provide a novel link between cardiac isoform expression and trophic signaling via FHLs and suggest cardiac splicing as a therapeutic target in diastolic dysfunction.
• Kolb, J., Li, F., Methawasin, M., Adler, M., Escobar, Y. N., Nedrud, J., Pappas, C. T., Harris, S. P., & Granzier, H. (2016). Thin filament length in the cardiac sarcomere varies with sarcomere length but is independent of titin and nebulin. Journal of molecular and cellular cardiology, 97, 286-94.
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  Thin filament length (TFL) is an important determinant of the force-sarcomere length (SL) relation of cardiac muscle. However, the various mechanisms that control TFL are not well understood. Here we tested the previously proposed hypothesis that the actin-binding protein nebulin contributes to TFL regulation in the heart by using a cardiac-specific nebulin cKO mouse model (αMHC Cre Neb cKO). Atrial myocytes were studied because nebulin expression has been reported to be most prominent in this cell type. TFL was measured in right and left atrial myocytes using deconvolution optical microscopy and staining for filamentous actin with phalloidin and for the thin filament pointed-end with an antibody to the capping protein Tropomodulin-1 (Tmod1). Results showed that TFLs in Neb cKO and littermate control mice were not different. Thus, deletion of nebulin in the heart does not alter TFL. However, TFL was found to be ~0.05μm longer in the right than in the left atrium and Tmod1 expression was increased in the right atrium. We also tested the hypothesis that the length of titin's spring region is a factor controlling TFL by studying the Rbm20(ΔRRM) mouse which expresses titins that are ~500kDa (heterozygous mice) and ~1000kDa (homozygous mice) longer than in control mice. Results revealed that TFL was not different in Rbm20(ΔRRM) mice. An unexpected finding in all genotypes studied was that TFL increased as sarcomeres were stretched (~0.1μm per 0.35μm of SL increase). This apparent increase in TFL reached a maximum at a SL of ~3.0μm where TFL was ~1.05μm. The SL dependence of TFL was independent of chemical fixation or the presence of cardiac myosin-binding protein C (cMyBP-C). In summary, we found that in cardiac myocytes TFL varies with SL in a manner that is independent of the size of titin or the presence of nebulin.
• Kovács, ., Fülöp, G. Á., Kovács, A., Csípő, T., Bódi, B., Priksz, D., Juhász, B., Beke, L., Hendrik, Z., Méhes, G., Granzier, H. L., Édes, I., Fagyas, M., Papp, Z., Barta, J., & Tóth, A. (2016). Renin overexpression leads to increased titin-based stiffness contributing to diastolic dysfunction in hypertensive mRen2 rats. American journal of physiology. Heart and circulatory physiology, 310(11), H1671-82.
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  Hypertension (HTN) is a major risk factor for heart failure. We investigated the influence of HTN on cardiac contraction and relaxation in transgenic renin overexpressing rats (carrying mouse Ren-2 renin gene, mRen2, n = 6). Blood pressure (BP) was measured. Cardiac contractility was characterized by echocardiography, cellular force measurements, and biochemical assays were applied to reveal molecular mechanisms. Sprague-Dawley (SD) rats (n = 6) were used as controls. Transgenic rats had higher circulating renin activity and lower cardiac angiotensin-converting enzyme two levels. Systolic BP was elevated in mRen2 rats (235.11 ± 5.32 vs. 127.03 ± 7.56 mmHg in SD, P < 0.05), resulting in increased left ventricular (LV) weight/body weight ratio (4.05 ± 0.09 vs. 2.77 ± 0.08 mg/g in SD, P < 0.05). Transgenic renin expression had no effect on the systolic parameters, such as LV ejection fraction, cardiomyocyte Ca(2+)-activated force, and Ca(2+) sensitivity of force production. In contrast, diastolic dysfunction was observed in mRen2 compared with SD rats: early and late LV diastolic filling ratio (E/A) was lower (1.14 ± 0.04 vs. 1.87 ± 0.08, P < 0.05), LV isovolumetric relaxation time was longer (43.85 ± 0.89 vs. 28.55 ± 1.33 ms, P < 0.05), cardiomyocyte passive tension was higher (1.74 ± 0.06 vs. 1.28 ± 0.18 kN/m(2), P < 0.05), and lung weight/body weight ratio was increased (6.47 ± 0.24 vs. 5.78 ± 0.19 mg/g, P < 0.05), as was left atrial weight/body weight ratio (0.21 ± 0.03 vs. 0.14 ± 0.03 mg/g, P < 0.05). Hyperphosphorylation of titin at Ser-12742 within the PEVK domain and a twofold overexpression of protein kinase C-α in mRen2 rats were detected. Our data suggest a link between the activation of renin-angiotensin-aldosterone system and increased titin-based stiffness through phosphorylation of titin's PEVK element, contributing to diastolic dysfunction.
• Leite, F. S., Minozzo, F. C., Kalganov, A., Cornachione, A. S., Cheng, Y., Leu, N. A., Han, X., Saripalli, C., Yates, J. R., Granzier, H., Kashina, A. S., & Rassier, D. E. (2016). Reduced passive force in skeletal muscles lacking protein arginylation. American journal of physiology. Cell physiology, 310(2), C127-35.
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  Arginylation is a posttranslational modification that plays a global role in mammals. Mice lacking the enzyme arginyltransferase in skeletal muscles exhibit reduced contractile forces that have been linked to a reduction in myosin cross-bridge formation. The role of arginylation in passive skeletal myofibril forces has never been investigated. In this study, we used single sarcomere and myofibril measurements and observed that lack of arginylation leads to a pronounced reduction in passive forces in skeletal muscles. Mass spectrometry indicated that skeletal muscle titin, the protein primarily linked to passive force generation, is arginylated on five sites located within the A band, an important area for protein-protein interactions. We propose a mechanism for passive force regulation by arginylation through modulation of protein-protein binding between the titin molecule and the thick filament. Key points are as follows: 1) active and passive forces were decreased in myofibrils and single sarcomeres isolated from muscles lacking arginyl-tRNA-protein transferase (ATE1). 2) Mass spectrometry revealed five sites for arginylation within titin molecules. All sites are located within the A-band portion of titin, an important region for protein-protein interactions. 3) Our data suggest that arginylation of titin is required for proper passive force development in skeletal muscles.
• Methawasin, M., Strom, J. G., Slater, R. E., Fernandez, V., Saripalli, C., & Granzier, H. (2016). Experimentally Increasing the Compliance of Titin Through RNA Binding Motif-20 (RBM20) Inhibition Improves Diastolic Function In a Mouse Model of Heart Failure With Preserved Ejection Fraction. Circulation, 134(15), 1085-1099.
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  Left ventricular (LV) stiffening contributes to heart failure with preserved ejection fraction (HFpEF), a syndrome with no effective treatment options. Increasing the compliance of titin in the heart has become possible recently through inhibition of the splicing factor RNA binding motif-20. Here, we investigated the effects of increasing the compliance of titin in mice with diastolic dysfunction.
• Pulcastro, H. C., Awinda, P. O., Methawasin, M., Granzier, H., Dong, W., & Tanner, B. C. (2016). Increased Titin Compliance Reduced Length-Dependent Contraction and Slowed Cross-Bridge Kinetics in Skinned Myocardial Strips from Rbm (20ΔRRM) Mice. Frontiers in physiology, 7, 322.
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  Titin is a giant protein spanning from the Z-disk to the M-band of the cardiac sarcomere. In the I-band titin acts as a molecular spring, contributing to passive mechanical characteristics of the myocardium throughout a heartbeat. RNA Binding Motif Protein 20 (RBM20) is required for normal titin splicing, and its absence or altered function leads to greater expression of a very large, more compliant N2BA titin isoform in Rbm20 homozygous mice (Rbm20 (ΔRRM) ) compared to wild-type mice (WT) that almost exclusively express the stiffer N2B titin isoform. Prior studies using Rbm20 (ΔRRM) animals have shown that increased titin compliance compromises muscle ultrastructure and attenuates the Frank-Starling relationship. Although previous computational simulations of muscle contraction suggested that increasing compliance of the sarcomere slows the rate of tension development and prolongs cross-bridge attachment, none of the reported effects of Rbm20 (ΔRRM) on myocardial function have been attributed to changes in cross-bridge cycling kinetics. To test the relationship between increased sarcomere compliance and cross-bridge kinetics, we used stochastic length-perturbation analysis in Ca(2+)-activated, skinned papillary muscle strips from Rbm20 (ΔRRM) and WT mice. We found increasing titin compliance depressed maximal tension, decreased Ca(2+)-sensitivity of the tension-pCa relationship, and slowed myosin detachment rate in myocardium from Rbm20 (ΔRRM) vs. WT mice. As sarcomere length increased from 1.9 to 2.2 μm, length-dependent activation of contraction was eliminated in the Rbm20 (ΔRRM) myocardium, even though myosin MgADP release rate decreased ~20% to prolong strong cross-bridge binding at longer sarcomere length. These data suggest that increasing N2BA expression may alter cardiac performance in a length-dependent manner, showing greater deficits in tension production and slower cross-bridge kinetics at longer sarcomere length. This study also supports the idea that passive mechanical characteristics of the myocardium influence ensemble cross-bridge behavior and maintenance of tension generation throughout the sarcomere.
• Winter, J. M., Joureau, B., Lee, E. J., Kiss, B., Yuen, M., Gupta, V. A., Pappas, C. T., Gregorio, C. C., Stienen, G. J., Edvardson, S., Wallgren-Pettersson, C., Lehtokari, V. L., Pelin, K., Malfatti, E., Romero, N. B., Engelen, B. G., Voermans, N. C., Donkervoort, S., Bönnemann, C. G., , Clarke, N. F., et al. (2016). Mutation-specific effects on thin filament length in thin filament myopathy. Annals of neurology, 79(6), 959-69.
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  Thin filament myopathies are among the most common nondystrophic congenital muscular disorders, and are caused by mutations in genes encoding proteins that are associated with the skeletal muscle thin filament. Mechanisms underlying muscle weakness are poorly understood, but might involve the length of the thin filament, an important determinant of force generation.
• Begay, R. L., Graw, S., Sinagra, G., Merlo, M., Slavov, D., Gowan, K., Jones, K. L., Barbati, G., Spezzacatene, A., Brun, F., Di Lenarda, A., Smith, J. E., Granzier, H. L., Mestroni, L., Taylor, M., & , F. C. (2015). Role of Titin Missense Variants in Dilated Cardiomyopathy. Journal of the American Heart Association, 4(11).
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  The titin gene (TTN) encodes the largest human protein, which plays a central role in sarcomere organization and passive myocyte stiffness. TTN truncating mutations cause dilated cardiomyopathy (DCM); however, the role of TTN missense variants in DCM has been difficult to elucidate because of the presence of background TTN variation.
• Granzier, H. L. (2015). Reply to Tskhovrebova et al.: Titin's IA junction does not control thick filament length. Proceedings of the National Academy of Sciences of the United States of America, 112(11), E1173.
• Granzier, H. L., & de Tombe, P. P. (2015). Myosin light chain phosphorylation to the rescue. Proceedings of the National Academy of Sciences of the United States of America, 112(30), 9148-9.
• Hutchinson, K. R., Saripalli, C., Chung, C. S., & Granzier, H. (2015). Increased myocardial stiffness due to cardiac titin isoform switching in a mouse model of volume overload limits eccentric remodeling. Journal of molecular and cellular cardiology, 79, 104-14.
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  We investigated the cellular and molecular mechanisms of diastolic dysfunction in pure volume overload induced by aortocaval fistula (ACF) surgery in the mouse. Four weeks of volume overload resulted in significant biventricular hypertrophy; protein expression analysis in left ventricular (LV) tissue showed a marked decrease in titin's N2BA/N2B ratio with no change in phosphorylation of titin's spring region. Titin-based passive tensions were significantly increased; a result of the decreased N2BA/N2B ratio. Conscious echocardiography in ACF mice revealed eccentric remodeling and pressure volume analysis revealed systolic dysfunction: reductions in ejection fraction (EF), +dP/dt, and the slope of the end-systolic pressure volume relationships (ESPVR). ACF mice also had diastolic dysfunction: increased LV end-diastolic pressure and reduced relaxation rates. Additionally, a decrease in the slope of the end diastolic pressure volume relationship (EDPVR) was found. However, correcting for altered geometry of the LV normalized the change in EDPVR and revealed, in line with our skinned muscle data, increased myocardial stiffness in vivo. ACF mice also had increased expression of the signaling proteins FHL-1, FHL-2, and CARP that bind to titin's spring region suggesting that titin stiffening is important to the volume overload phenotype. To test this we investigated the effect of volume overload in the RBM20 heterozygous (HET) mouse model, which exhibits reduced titin stiffness. It was found that LV hypertrophy was attenuated and that LV eccentricity was exacerbated. We propose that pure volume overload induces an increase in titin stiffness that is beneficial and limits eccentric remodeling.
• Li, F., Buck, D., De Winter, J., Kolb, J., Meng, H., Birch, C., Slater, R., Escobar, Y. N., Smith, J. E., Yang, L., Konhilas, J., Lawlor, M. W., Ottenheijm, C., & Granzier, H. L. (2015). Nebulin deficiency in adult muscle causes sarcomere defects and muscle-type-dependent changes in trophicity: novel insights in nemaline myopathy. Human molecular genetics, 24(18), 5219-33.
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  Nebulin is a giant filamentous protein that is coextensive with the actin filaments of the skeletal muscle sarcomere. Nebulin mutations are the main cause of nemaline myopathy (NEM), with typical adult patients having low expression of nebulin, yet the roles of nebulin in adult muscle remain poorly understood. To establish nebulin's functional roles in adult muscle, we studied a novel conditional nebulin KO (Neb cKO) mouse model in which nebulin deletion was driven by the muscle creatine kinase (MCK) promotor. Neb cKO mice are born with high nebulin levels in their skeletal muscles, but within weeks after birth nebulin expression rapidly falls to barely detectable levels Surprisingly, a large fraction of the mice survive to adulthood with low nebulin levels (
• Pappas, C. T., Mayfield, R. M., Henderson, C., Jamilpour, N., Cover, C., Hernandez, Z., Hutchinson, K. R., Chu, M., Nam, K., Valdez, J. M., Wong, P. K., Granzier, H. L., & Gregorio, C. C. (2015). Knockout of Lmod2 results in shorter thin filaments followed by dilated cardiomyopathy and juvenile lethality. Proceedings of the National Academy of Sciences of the United States of America, 112(44), 13573-8.
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  Leiomodin 2 (Lmod2) is an actin-binding protein that has been implicated in the regulation of striated muscle thin filament assembly; its physiological function has yet to be studied. We found that knockout of Lmod2 in mice results in abnormally short thin filaments in the heart. We also discovered that Lmod2 functions to elongate thin filaments by promoting actin assembly and dynamics at thin filament pointed ends. Lmod2-KO mice die as juveniles with hearts displaying contractile dysfunction and ventricular chamber enlargement consistent with dilated cardiomyopathy. Lmod2-null cardiomyocytes produce less contractile force than wild type when plated on micropillar arrays. Introduction of GFP-Lmod2 via adeno-associated viral transduction elongates thin filaments and rescues structural and functional defects observed in Lmod2-KO mice, extending their lifespan to adulthood. Thus, to our knowledge, Lmod2 is the first identified mammalian protein that functions to elongate actin filaments in the heart; it is essential for cardiac thin filaments to reach a mature length and is required for efficient contractile force and proper heart function during development.
• Perkin, J., Slater, R., Del Favero, G., Lanzicher, T., Hidalgo, C., Anderson, B., Smith, J. E., Sbaizero, O., Labeit, S., & Granzier, H. (2015). Phosphorylating Titin's Cardiac N2B Element by ERK2 or CaMKIIδ Lowers the Single Molecule and Cardiac Muscle Force. Biophysical journal, 109(12), 2592-601.
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  Titin is a large filamentous protein that is responsible for the passive force of the cardiac sarcomere. Titin's force is generated by its I-band region, which includes the cardiac-specific N2B element. The N2B element consists of three immunoglobulin domains, two small unique sequence insertions, and a large 575-residue unique sequence, the N2B-Us. Posttranslational modifications of the N2B element are thought to regulate passive force, but the underlying mechanisms are unknown. Increased passive-force levels characterize diastolic stiffening in heart-failure patients, and it is critical to understand the underlying molecular mechanisms and identify therapeutic targets. Here, we used single-molecule force spectroscopy to study the mechanical effects of the kinases calcium/calmodulin-dependent protein kinase II delta (CaMKIIδ) and extracellular signal-regulated kinase 2 (ERK2) on the single-molecule mechanics of the N2B element. Both CaMKIIδ and ERK2 were found to phosphorylate the N2B element, and single-molecule force spectroscopy revealed an increase in the persistence length (Lp) of the molecule, indicating that the bending rigidity of the molecule was increased. Experiments performed under oxidizing conditions and with a recombinant N2B element that had a simplified domain composition provided evidence that the Lp increase requires the N2B-Us of the N2B element. Mechanical experiments were also performed on skinned myocardium before and after phosphorylation. The results revealed a large (∼30%) passive force reduction caused by CaMKIIδ and a much smaller (∼6%) reduction caused by ERK2. These findings support the notion that the important kinases ERK2 and CaMKIIδ can alter the passive force of myocytes in the heart (although CaMKIIδ appears to be more potent) during physiological and pathophysiological states.
• Zile, M. R., Baicu, C. F., Ikonomidis, J. S., Stroud, R. E., Nietert, P. J., Bradshaw, A. D., Slater, R., Palmer, B. M., Van Buren, P., Meyer, M., Redfield, M. M., Bull, D. A., Granzier, H. L., & LeWinter, M. M. (2015). Myocardial stiffness in patients with heart failure and a preserved ejection fraction: contributions of collagen and titin. Circulation, 131(14), 1247-59.
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  The purpose of this study was to determine whether patients with heart failure and a preserved ejection fraction (HFpEF) have an increase in passive myocardial stiffness and the extent to which discovered changes depend on changes in extracellular matrix fibrillar collagen and cardiomyocyte titin.
• de Winter, J. M., Joureau, B., Sequeira, V., Clarke, N. F., van der Velden, J., Stienen, G. J., Granzier, H., Beggs, A. H., & Ottenheijm, C. A. (2015). Effect of levosimendan on the contractility of muscle fibers from nemaline myopathy patients with mutations in the nebulin gene. Skeletal muscle, 5, 12.
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  Nemaline myopathy (NM), the most common non-dystrophic congenital myopathy, is characterized by generalized skeletal muscle weakness, often from birth. To date, no therapy exists that enhances the contractile strength of muscles of NM patients. Mutations in NEB, encoding the giant protein nebulin, are the most common cause of NM. The pathophysiology of muscle weakness in NM patients with NEB mutations (NEB-NM) includes a lower calcium-sensitivity of force generation. We propose that the lower calcium-sensitivity of force generation in NEB-NM offers a therapeutic target. Levosimendan is a calcium sensitizer that is approved for use in humans and has been developed to target cardiac muscle fibers. It exerts its effect through binding to slow skeletal/cardiac troponin C. As slow skeletal/cardiac troponin C is also the dominant troponin C isoform in slow-twitch skeletal muscle fibers, we hypothesized that levosimendan improves slow-twitch muscle fiber strength at submaximal levels of activation in patients with NEB-NM.
• de Winter, J. M., Molenaar, J. P., Yuen, M., van der Pijl, R., Shen, S., Conijn, S., van de Locht, M., Willigenburg, M., Bogaards, S. J., van Kleef, E. S., Lassche, S., Persson, M., Rassier, D. E., Sztal, T. E., Ruparelia, A. A., Oorschot, V., Ramm, G., Hall, T. E., Xiong, Z., , Johnson, C. N., et al. (2020). KBTBD13 is an actin-binding protein that modulates muscle kinetics. The Journal of clinical investigation.
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  The mechanisms that modulate the kinetics of muscle relaxation are critically important for muscle function. A prime example of the impact of impaired relaxation kinetics is nemaline myopathy caused by mutations in KBTBD13 (NEM6). In addition to weakness, NEM6 patients have slow muscle relaxation, compromising contractility and daily life activities. The role of KBTBD13 in muscle is unknown, and the pathomechanism underlying NEM6 is undetermined. A combination of transcranial magnetic stimulation-induced muscle relaxation, muscle fiber- and sarcomere-contractility assays, low-angle x-ray diffraction, and superresolution microscopy revealed that the impaired muscle-relaxation kinetics in NEM6 patients are caused by structural changes in the thin filament, a sarcomeric microstructure. Using homology modeling and binding and contractility assays with recombinant KBTBD13, Kbtbd13-knockout and Kbtbd13R408C-knockin mouse models, and a GFP-labeled Kbtbd13-transgenic zebrafish model, we discovered that KBTBD13 binds to actin - a major constituent of the thin filament - and that mutations in KBTBD13 cause structural changes impairing muscle-relaxation kinetics. We propose that this actin-based impaired relaxation is central to NEM6 pathology.
• Buck, D., Smith, J. E., Chung, C. S., Ono, Y., Sorimachi, H., Labeit, S., & Granzier, H. L. (2014). Removal of immunoglobulin-like domains from titin's spring segment alters titin splicing in mouse skeletal muscle and causes myopathy. The Journal of general physiology, 143(2), 215-30.
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  Titin is a molecular spring that determines the passive stiffness of muscle cells. Changes in titin's stiffness occur in various myopathies, but whether these are a cause or an effect of the disease is unknown. We studied a novel mouse model in which titin's stiffness was slightly increased by deleting nine immunoglobulin (Ig)-like domains from titin's constitutively expressed proximal tandem Ig segment (IG KO). KO mice displayed mild kyphosis, a phenotype commonly associated with skeletal muscle myopathy. Slow muscles were atrophic with alterations in myosin isoform expression; functional studies in soleus muscle revealed a reduced specific twitch force. Exon expression analysis showed that KO mice underwent additional changes in titin splicing to yield smaller than expected titin isoforms that were much stiffer than expected. Additionally, splicing occurred in the PEVK region of titin, a finding confirmed at the protein level. The titin-binding protein Ankrd1 was highly increased in the IG KO, but this did not play a role in generating small titin isoforms because titin expression was unaltered in IG KO mice crossed with Ankrd1-deficient mice. In contrast, the splicing factor RBM20 (RNA-binding motif 20) was also significantly increased in IG KO mice, and additional differential splicing was reversed in IG KO mice crossed with a mouse with reduced RBM20 activity. Thus, increasing titin's stiffness triggers pathological changes in skeletal muscle, with an important role played by RBM20.
• Elhamine, F., Radke, M. H., Pfitzer, G., Granzier, H., Gotthardt, M., & Stehle, R. (2014). Deletion of the titin N2B region accelerates myofibrillar force development but does not alter relaxation kinetics. Journal of cell science, 127(Pt 17), 3666-74.
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  Cardiac titin is the main determinant of sarcomere stiffness during diastolic relaxation. To explore whether titin stiffness affects the kinetics of cardiac myofibrillar contraction and relaxation, we used subcellular myofibrils from the left ventricles of homozygous and heterozygous N2B-knockout mice which express truncated cardiac titins lacking the unique elastic N2B region. Compared with myofibrils from wild-type mice, myofibrils from knockout and heterozygous mice exhibit increased passive myofibrillar stiffness. To determine the kinetics of Ca(2+)-induced force development (rate constant kACT), myofibrils from knockout, heterozygous and wild-type mice were stretched to the same sarcomere length (2.3 µm) and rapidly activated with Ca(2+). Additionally, mechanically induced force-redevelopment kinetics (rate constant kTR) were determined by slackening and re-stretching myofibrils during Ca(2+)-mediated activation. Myofibrils from knockout mice exhibited significantly higher kACT, kTR and maximum Ca(2+)-activated tension than myofibrils from wild-type mice. By contrast, the kinetic parameters of biphasic force relaxation induced by rapidly reducing [Ca(2+)] were not significantly different among the three genotypes. These results indicate that increased titin stiffness promotes myocardial contraction by accelerating the formation of force-generating cross-bridges without decelerating relaxation.
• Granzier, H. L., Hutchinson, K. R., Tonino, P., Methawasin, M., Li, F. W., Slater, R. E., Bull, M. M., Saripalli, C., Pappas, C. T., Gregorio, C. C., & Smith, J. E. (2014). Deleting titin's I-band/A-band junction reveals critical roles for titin in biomechanical sensing and cardiac function. Proceedings of the National Academy of Sciences of the United States of America, 111(40), 14589-94.
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  Titin, the largest protein known, forms a giant filament in muscle where it spans the half sarcomere from Z disk to M band. Here we genetically targeted a stretch of 14 immunoglobulin-like and fibronectin type 3 domains that comprises the I-band/A-band (IA) junction and obtained a viable mouse model. Super-resolution optical microscopy (structured illumination microscopy, SIM) and electron microscopy were used to study the thick filament length and titin's molecular elasticity. SIM showed that the IA junction functionally belongs to the relatively stiff A-band region of titin. The stiffness of A-band titin was found to be high, relative to that of I-band titin (∼ 40-fold higher) but low, relative to that of the myosin-based thick filament (∼ 70-fold lower). Sarcomere stretch therefore results in movement of A-band titin with respect to the thick filament backbone, and this might constitute a novel length-sensing mechanism. Findings disproved that titin at the IA junction is crucial for thick filament length control, settling a long-standing hypothesis. SIM also showed that deleting the IA junction moves the attachment point of titin's spring region away from the Z disk, increasing the strain on titin's molecular spring elements. Functional studies from the cellular to ex vivo and in vivo left ventricular chamber levels showed that this causes diastolic dysfunction and other symptoms of heart failure with preserved ejection fraction (HFpEF). Thus, our work supports titin's important roles in diastolic function and disease of the heart.
• Hidalgo, C., Saripalli, C., & Granzier, H. L. (2014). Effect of exercise training on post-translational and post-transcriptional regulation of titin stiffness in striated muscle of wild type and IG KO mice. Archives of biochemistry and biophysics, 552-553, 100-7.
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  Exercise has beneficial effects on diastolic dysfunction but the underlying mechanisms are not well understood. Here we studied the effects of exercise on the elastic protein titin, an important determinant of diastolic stiffness, in both the left ventricle and the diaphragm. We used wild type mice and genetically engineered mice with HFpEF symptoms (IG KO mice), including diastolic dysfunction. In the diaphragm muscle, exercise increased the expression level of titin (increased titin:MHC ratio) which is expected to increase titin-based stiffness. This effect was absent in the LV. We also studied the constitutively expressed titin residues S11878 and S12022 that are known targets of CaMKIIδ and PKCα with increased phosphorylation resulting in an increase in titin-based passive stiffness. The phosphorylation level of S11878 was unchanged whereas S12022 responded to exercise with a reduction in the phosphorylation level in the LV and, interestingly, an increase in the diaphragm. These changes are expected to lower titin's stiffness in the LV and increase stiffness in the diaphragm. We propose that these disparate effects reflect the unique physiological needs of the two tissue types and that both effects are beneficial.
• Le Sueur, H., Mollon, J. D., Granzier, J., & Jordan, G. (2014). Counterphase modulation photometry: comparison of two instruments. Journal of the Optical Society of America. A, Optics, image science, and vision, 31(4), A34-7.
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  The ratio of long-wavelength to medium-wavelength sensitive cones varies significantly among people. In order to investigate the possible effect of this variation in large numbers of participants, a quick and efficient method to estimate the ratio is required. The OSCAR test has been utilized previously for this purpose, but it is no longer available commercially. Having access to one of the few remaining OSCAR instruments, we compared the observers' mean settings to those obtained with the Medmont C100, a newer but apparently similar device. We also obtained Rayleigh matches for each participant. One hundred volunteers took part in the study. Settings on the OSCAR test were highly correlated with those on the Medmont C100. Both tests appeared to be influenced not only by L∶M cone ratios but also by the spectral positions of the cone photopigments, since anomaloscope midmatch points accounted for a significant proportion of the variance. We conclude that the Medmont C100 can be used as a suitable replacement for the OSCAR test and has a role in the rapid estimation of L∶M cone ratios.
• Meng, H., Janssen, P. M., Grange, R. W., Yang, L., Beggs, A. H., Swanson, L. C., Cossette, S. A., Frase, A., Childers, M. K., Granzier, H., Gussoni, E., & Lawlor, M. W. (2014). Tissue triage and freezing for models of skeletal muscle disease. Journal of visualized experiments : JoVE.
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  Skeletal muscle is a unique tissue because of its structure and function, which requires specific protocols for tissue collection to obtain optimal results from functional, cellular, molecular, and pathological evaluations. Due to the subtlety of some pathological abnormalities seen in congenital muscle disorders and the potential for fixation to interfere with the recognition of these features, pathological evaluation of frozen muscle is preferable to fixed muscle when evaluating skeletal muscle for congenital muscle disease. Additionally, the potential to produce severe freezing artifacts in muscle requires specific precautions when freezing skeletal muscle for histological examination that are not commonly used when freezing other tissues. This manuscript describes a protocol for rapid freezing of skeletal muscle using isopentane (2-methylbutane) cooled with liquid nitrogen to preserve optimal skeletal muscle morphology. This procedure is also effective for freezing tissue intended for genetic or protein expression studies. Furthermore, we have integrated our freezing protocol into a broader procedure that also describes preferred methods for the short term triage of tissue for (1) single fiber functional studies and (2) myoblast cell culture, with a focus on the minimum effort necessary to collect tissue and transport it to specialized research or reference labs to complete these studies. Overall, this manuscript provides an outline of how fresh tissue can be effectively distributed for a variety of phenotypic studies and thereby provides standard operating procedures (SOPs) for pathological studies related to congenital muscle disease.
• Methawasin, M., Hutchinson, K. R., Lee, E., Smith, J. E., Saripalli, C., Hidalgo, C. G., Ottenheijm, C. A., & Granzier, H. (2014). Experimentally increasing titin compliance in a novel mouse model attenuates the Frank-Starling mechanism but has a beneficial effect on diastole. Circulation, 129(19), 1924-36.
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  Experimentally upregulating compliant titins has been suggested as a therapeutic for lowering pathological diastolic stiffness levels. However, how increasing titin compliance impacts global cardiac function requires in-depth study. We investigate the effect of upregulating compliant titins in a novel mouse model with a genetically altered titin splicing factor; integrative approaches were used from intact cardiomyocyte mechanics to pressure-volume analysis and Doppler echocardiography.
• Rain, S., Bos, D. d., Handoko, D., Westerhof, D., Stienen, D., Ottenheijm, D., Goebel, D., Dorfmüller, D., Guignabert, D., Humbert, D., Bogaard, D., Remedios, D., Saripalli, D., Hidalgo, D., Granzier, D., Vonk-Noordegraaf, D., van der Velden, D., & de Man, D. (2014). Protein changes contributing to right ventricular cardiomyocyte diastolic dysfunction in pulmonary arterial hypertension. Journal of the American Heart Association, 3(3), e000716.
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  Right ventricular (RV) diastolic function is impaired in patients with pulmonary arterial hypertension (PAH). Our previous study showed that elevated cardiomyocyte stiffness and myofilament Ca(2+) sensitivity underlie diastolic dysfunction in PAH. This study investigates protein modifications contributing to cellular diastolic dysfunction in PAH.
• Granzier, H., Anderson, B. R., & Granzier, H. L. (0). Titin-based tension in the cardiac sarcomere: molecular origin and physiological adaptations. Progress in biophysics and molecular biology, 110(2-3).
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  The passive stiffness of cardiac muscle plays a critical role in ventricular filling during diastole and is determined by the extracellular matrix and the sarcomeric protein titin. Titin spans from the Z-disk to the M-band of the sarcomere and also contains a large extensible region that acts as a molecular spring and develops passive force during sarcomere stretch. This extensible segment is titin's I-band region, and its force-generating mechanical properties determine titin-based passive tension. The properties of titin's I-band region can be modulated by isoform splicing and post-translational modification and are intimately linked to diastolic function. This review discusses the physical origin of titin-based passive tension, the mechanisms that alter titin stiffness, and titin's role in stress-sensing signaling pathways.
• Granzier, H., Anderson, B. R., Bogomolovas, J., Labeit, S., & Granzier, H. L. (2013). Single molecule force spectroscopy on titin implicates immunoglobulin domain stability as a cardiac disease mechanism. The Journal of biological chemistry, 288(8).
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  Titin plays crucial roles in sarcomere organization and cardiac elasticity by acting as an intrasarcomeric molecular spring. A mutation in the tenth Ig-like domain of titin's spring region is associated with arrhythmogenic cardiomyopathy, a disease characterized by ventricular arrhythmias leading to cardiac arrest and sudden death. Titin is the first sarcomeric protein linked to arrhythmogenic cardiomyopathy. To characterize the disease mechanism, we have used atomic force microscopy to directly measure the effects that the disease-linked point mutation (T16I) has on the mechanical and kinetic stability of Ig10 at the single molecule level. The mutation decreases the force needed to unfold Ig10 and increases its rate of unfolding 4-fold. We also found that T16I Ig10 is more prone to degradation, presumably due to compromised local protein structure. Overall, the disease-linked mutation weakens the structural integrity of titin's Ig10 domain and suggests an Ig domain disease mechanism.
• Granzier, H., Chung, C. S., Hutchinson, K. R., Methawasin, M., Saripalli, C., Smith, J. E., Hidalgo, C. G., Luo, X., Labeit, S., Guo, C., & Granzier, H. L. (2013). Shortening of the elastic tandem immunoglobulin segment of titin leads to diastolic dysfunction. Circulation, 128(1).
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  Diastolic dysfunction is a poorly understood but clinically pervasive syndrome that is characterized by increased diastolic stiffness. Titin is the main determinant of cellular passive stiffness. However, the physiological role that the tandem immunoglobulin (Ig) segment of titin plays in stiffness generation and whether shortening this segment is sufficient to cause diastolic dysfunction need to be established.
• Granzier, H., Hidalgo, C. G., Chung, C. S., Saripalli, C., Methawasin, M., Hutchinson, K. R., Tsaprailis, G., Labeit, S., Mattiazzi, A., & Granzier, H. L. (2013). The multifunctional Ca(2+)/calmodulin-dependent protein kinase II delta (CaMKIIδ) phosphorylates cardiac titin's spring elements. Journal of molecular and cellular cardiology, 54.
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  Titin-based passive stiffness is post-translationally regulated by several kinases that phosphorylate specific spring elements located within titin's elastic I-band region. Whether titin is phosphorylated by calcium/calmodulin dependent protein kinase II (CaMKII), an important regulator of cardiac function and disease, has not been addressed. The aim of this work was to determine whether CaMKIIδ, the predominant CaMKII isoform in the heart, phosphorylates titin, and to use phosphorylation assays and mass spectrometry to study which of titin's spring elements might be targeted by CaMKIIδ. It was found that CaMKIIδ phosphorylates titin in mouse LV skinned fibers, that the CaMKIIδ sites can be dephosphorylated by protein phosphatase 1 (PP1), and that under baseline conditions, in both intact isolated hearts and skinned myocardium, about half of the CaMKIIδ sites are phosphorylated. Mass spectrometry revealed that both the N2B and PEVK segments are targeted by CaMKIIδ at several conserved serine residues. Whether phosphorylation of titin by CaMKIIδ occurs in vivo, was tested in several conditions using back phosphorylation assays and phospho-specific antibodies to CaMKIIδ sites. Reperfusion following global ischemia increased the phosphorylation level of CaMKIIδ sites on titin and this effect was abolished by the CaMKII inhibitor KN-93. No changes in the phosphorylation level of the PEVK element were found suggesting that the increased phosphorylation level of titin in IR (ischemia reperfusion) might be due to phosphorylation of the N2B element. The findings of these studies show for the first time that titin can be phosphoryalated by CaMKIIδ, both in vitro and in vivo, and that titin's molecular spring region that determines diastolic stiffness is a target of CaMKIIδ.
• Granzier, H., Hidalgo, C., & Granzier, H. L. (2013). Tuning the molecular giant titin through phosphorylation: role in health and disease. Trends in cardiovascular medicine, 23(5).
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  Titin is a giant multi-functional filament that spans half of the sarcomere. Titin's extensible I-band region functions as a molecular spring that provides passive stiffness to cardiac myocytes. Elevated diastolic stiffness is found in a large fraction of heart failure patients and thus understanding the normal mechanisms and pathophysiology of passive stiffness modulation is clinically important. Here we provide first a brief general background on titin including what is known about titin isoforms and then focus on recently discovered post-translational modifications of titin that alter passive stiffness. We discuss the various kinases that have been shown to phosphorylate titin and address the possible roles of titin phosphorylation in cardiac disease, including heart failure with preserved ejection fraction (HFpEF).
• Granzier, H., Lee, E., De Winter, J. M., Buck, D., Jasper, J. R., Malik, F. I., Labeit, S., Ottenheijm, C. A., & Granzier, H. L. (2013). Fast skeletal muscle troponin activation increases force of mouse fast skeletal muscle and ameliorates weakness due to nebulin-deficiency. PloS one, 8(2).
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  The effect of the fast skeletal muscle troponin activator, CK-2066260, on calcium-induced force development was studied in skinned fast skeletal muscle fibers from wildtype (WT) and nebulin deficient (NEB KO) mice. Nebulin is a sarcomeric protein that when absent (NEB KO mouse) or present at low levels (nemaline myopathy (NM) patients with NEB mutations) causes muscle weakness. We studied the effect of fast skeletal troponin activation on WT muscle and tested whether it might be a therapeutic mechanism to increase muscle strength in nebulin deficient muscle. We measured tension-pCa relations with and without added CK-2066260. Maximal active tension in NEB KO tibialis cranialis fibers in the absence of CK-2066260 was ∼60% less than in WT fibers, consistent with earlier work. CK-2066260 shifted the tension-calcium relationship leftwards, with the largest relative increase (up to 8-fold) at low to intermediate calcium levels. This was a general effect that was present in both WT and NEB KO fiber bundles. At pCa levels above ∼6.0 (i.e., calcium concentrations
• Granzier, H., Lee, E., Nedrud, J., Schemmel, P., Gotthardt, M., Irving, T. C., & Granzier, H. L. (2013). Calcium sensitivity and myofilament lattice structure in titin N2B KO mice. Archives of biochemistry and biophysics, 535(1).
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  The cellular basis of the Frank-Starling "Law of the Heart" is the length-dependence of activation, but the mechanisms by which the sarcomere detects length changes and converts this information to altered calcium sensitivity has remained elusive. Here the effect of titin-based passive tension on the length-dependence of activation (LDA) was studied by measuring the tension-pCa relation in skinned mouse LV muscle at two sarcomere lengths (SLs). N2B KO myocardium, where the N2B spring element in titin is deleted and passive tension is elevated, was compared to WT myocardium. Myofilament lattice structure was studied with low-angle X-ray diffraction; the myofilament lattice spacing (d1,0) was measured as well as the ratio of the intensities of the 1,1 and 1,0 diffraction peaks (I1,1/I1,0) as an estimate of the degree of association of myosin heads with the thin filaments. Experiments were carried out in skinned muscle in which the lattice spacing was reduced with Dextran-T500. Experiments with and without lattice compression were also carried out following PKA phosphorylation of the skinned muscle. Under all conditions that were tested, LDA was significantly larger in N2B KO myocardium compared to WT myocardium, with the largest differences following PKA phosphorylation. A positive correlation between passive tension and LDA was found that persisted when the myofilament lattice was compressed with Dextran and that was enhanced following PKA phosphorylation. Low-angle X-ray diffraction revealed a shift in mass from thin filaments to thick filaments as sarcomere length was increased. Furthermore, a positive correlation was obtained between myofilament lattice spacing and passive tension and the change in I1,1/I1,0 and passive tension and these provide possible explanations for how titin-based passive tension might regulate calcium sensitivity.
• Granzier, H., Lewinter, M. M., & Granzier, H. L. (2013). Cardiac Titin and Heart Disease. Journal of cardiovascular pharmacology.
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  The giant sarcomeric protein titin is a key determinant of myocardial passive stiffness and stress sensitive signaling. Titin stiffness is modulated by isoform variation, phosphorylation by protein kinases and possibly oxidative stress through disulfide bond formation. Titin has also emerged as an important human disease gene. Early studies in patients with dilated cardiomyopathy (DCM) revealed shifts toward more compliant isoforms, an adaptation that offsets increases in passive stiffness based in the extracellular matrix. Similar shifts are observed in heart failure with preserved ejection fraction (HFpEF). In contrast, hypophosphorylation of PKA/G sites contributes to a net increase in cardiomyocyte resting tension in HFpEF. More recently, titin mutations have been recognized as the most common etiology of inherited DCM. In addition, some DCM-causing mutations affect RBM20, a titin splice factor. Titin mutations are a rare cause of hypertrophic cardiomyopathy (HCM) and also underlie some cases of arrhythmogenic right ventricular dysplasia. Finally, mutations of genes encoding proteins that interact with and/or bind to titin are responsible for both DCM and HCM. Targeting titin as a therapeutic strategy is in its infancy, but could potentially involve manipulation of isoforms, post-translational modifications, and up-regulation of normal protein in patients with disease causing mutations.
• Granzier, H., Chung, C. S., & Granzier, H. L. (2011). Contribution of titin and extracellular matrix to passive pressure and measurement of sarcomere length in the mouse left ventricle. Journal of molecular and cellular cardiology, 50(4).
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  It remains to be established to what degree titin and the extracellular matrix (ECM) contribute to passive pressure in the left ventricle (LV). Thus, we aimed to elucidate the contribution of major molecular determinants of passive pressure in the normal mouse LV. Furthermore, we determined the working sarcomere length (SL) range of the LV to bridge our findings to earlier work in skinned muscle fibers. We utilized Frank-Starling type protocols to obtain diastolic pressure-volume relationships (PVR) in Langendorff perfused isolated LVs. To quantify the molecular contribution of titin and ECM, we innovated on methods of fiber mechanics to chemically permeabilize intact LVs and measure a fully passive PVR. To differentially dissect the contributions of the ECM and titin, we utilized myofilament extraction techniques in permeabilized LVs, measuring passive PVRs at each stage in the protocol. Myofilament extraction suggests that titin contributes ~80% of passive pressures in the heart. Langendorff perfusion was also used to chemically fix passive and BaCl(2) activated hearts at specific volumes to determine that the maximal working SL range of the midwall LV fibers is approximately 1.8-2.2 μm. A model of the passive SL-volume relationship was then used to estimate the pressure-SL relationships, indicating that the ECM contribution does not exceed titin's contribution until large volumes with SLs >~2.2 μm. In conclusion, within physiological volumes, titin is the dominant contributor to LV passive pressure, and ECM-based pressures dominate at larger volumes.
• Granzier, H., Chung, C. S., Bogomolovas, J., Gasch, A., Hidalgo, C. G., Labeit, S., & Granzier, H. L. (2011). Titin-actin interaction: PEVK-actin-based viscosity in a large animal. Journal of biomedicine & biotechnology, 2011.
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  Titin exhibits an interaction between its PEVK segment and the actin filament resulting in viscosity, a speed dependent resistive force, which significantly influences diastolic filling in mice. While diastolic disease is clinically pervasive, humans express a more compliant titin (N2BA:N2B ratio ~0.5-1.0) than mice (N2BA:N2B ratio ~0.2). To examine PEVK-actin based viscosity in compliant titin-tissues, we used pig cardiac tissue that expresses titin isoforms similar to that in humans. Stretch-hold experiments were performed at speeds from 0.1 to 10 lengths/s from slack sarcomere lengths (SL) to SL of 2.15 μm. Viscosity was calculated from the slope of stress-relaxation vs stretch speed. Recombinant PEVK was added to compete off native interactions and this found to reduce the slope by 35%, suggesting that PEVK-actin interactions are a strong contributor of viscosity. Frequency sweeps were performed at frequencies of 0.1-400 Hz and recombinant protein reduced viscous moduli by 40% at 2.15 μm and by 50% at 2.25 μm, suggesting a SL-dependent nature of viscosity that might prevent SL "overshoot" at long diastolic SLs. This study is the first to show that viscosity is present at physiologic speeds in the pig and supports the physiologic relevance of PEVK-actin interactions in humans in both health and disease.
• Granzier, H., Chung, C. S., Methawasin, M., Nelson, O. L., Radke, M. H., Hidalgo, C. G., Gotthardt, M., & Granzier, H. L. (2011). Titin based viscosity in ventricular physiology: an integrative investigation of PEVK-actin interactions. Journal of molecular and cellular cardiology, 51(3).
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  Viscosity is proposed to modulate diastolic function, but only limited understanding of the source(s) of viscosity exists. In vitro experiments have shown that the proline-glutamic acid-valine-lysine (PEVK) rich element of titin interacts with actin, causing a viscous force in the sarcomere. It is unknown whether this mechanism contributes to viscosity in vivo. We tested the hypothesis that PEVK-actin interaction causes cardiac viscosity and is important in vivo via an integrative physiological study on a unique PEVK knockout (KO) model. Both skinned cardiomyocytes and papillary muscle fibers were isolated from wildtype (WT) and PEVK KO mice and passive viscosity was examined using stretch-hold-release and sinusoidal analysis. Viscosity was reduced by ~60% in KO myocytes and ~50% in muscle fibers at room temperature. The PEVK-actin interaction was not modulated by temperature or diastolic calcium, but was increased by lattice compression. Stretch-hold and sinusoidal frequency protocols on intact isolated mouse hearts showed a smaller, 30-40% reduction in viscosity, possibly due to actomyosin interactions, and showed that microtubules did not contribute to viscosity. Transmitral Doppler echocardiography similarly revealed a 40% decrease in LV chamber viscosity in the PEVK KO in vivo. This integrative study is the first to quantify the influence of a specific molecular (PEVK-actin) viscosity in vivo and shows that PEVK-actin interactions are an important physiological source of viscosity.
• Granzier, H., Hudson, B., Hidalgo, C., Saripalli, C., & Granzier, H. L. (2011). Hyperphosphorylation of mouse cardiac titin contributes to transverse aortic constriction-induced diastolic dysfunction. Circulation research, 109(8).
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  Mechanisms underlying diastolic dysfunction need to be better understood.
• Granzier, H., King, N. M., Methawasin, M., Nedrud, J., Harrell, N., Chung, C. S., Helmes, M., & Granzier, H. L. (2011). Mouse intact cardiac myocyte mechanics: cross-bridge and titin-based stress in unactivated cells. The Journal of general physiology, 137(1).
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  A carbon fiber-based cell attachment and force measurement system was used to measure the diastolic stress-sarcomere length (SL) relation of mouse intact cardiomyocytes, before and after the addition of actomyosin inhibitors (2,3-butanedione monoxime [BDM] or blebbistatin). Stress was measured during the diastolic interval of twitching myocytes that were stretched at 100% base length/second. Diastolic stress increased close to linear from 0 at SL 1.85 µm to 4.2 mN/mm(2) at SL 2.1 µm. The actomyosin inhibitors BDM and blebbistatin significantly lowered diastolic stress by ∼1.5 mN/mm(2) (at SL 2.1 µm, ∼30% of total), suggesting that during diastole actomyosin interaction is not fully switched off. To test this further, calcium sensitivity of skinned myocytes was studied under conditions that simulate diastole: 37°C, presence of Dextran T500 to compress the myofilament lattice to the physiological level, and [Ca(2+)] from below to above 100 nM. Mean active stress was significantly increased at [Ca(2+)] > 55 nM (pCa 7.25) and was ∼0.7 mN/mm(2) at 100 nM [Ca(2+)] (pCa 7.0) and ∼1.3 mN/mm(2) at 175 nM Ca(2+) (pCa 6.75). Inhibiting active stress in intact cells attached to carbon fibers at their resting SL and stretching the cells while first measuring restoring stress (pushing outward) and then passive stress (pulling inward) made it possible to determine the passive cell's mechanical slack SL as ∼1.95 µm and the restoring stiffness and passive stiffness of the cells around the slack SL each as ∼17 mN/mm(2)/µm/SL. Comparison between the results of intact and skinned cells shows that titin is the main contributor to restoring stress and passive stress of intact cells, but that under physiological conditions, calcium sensitivity is sufficiently high for actomyosin interaction to contribute to diastolic stress. These findings are relevant for understanding diastolic function and for future studies of diastolic heart failure.
• Granzier, H., Nedrud, J., Labeit, S., Gotthardt, M., & Granzier, H. L. (2011). Mechanics on myocardium deficient in the N2B region of titin: the cardiac-unique spring element improves efficiency of the cardiac cycle. Biophysical journal, 101(6).
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  Titin (also known as connectin) is an intrasarcomeric muscle protein that functions as a molecular spring and generates passive tension upon muscle stretch. The N2B element is a cardiac-specific spring element within titin's extensible region. Our goal was to study the contribution of the N2B element to the mechanical properties of titin, particularly its hypothesized role in limiting energy loss during repeated stretch (diastole)-shortening (systole) cycles of the heart. We studied energy loss by measuring hysteresis from the area between the stretch and release passive force-sarcomere length curves and used both wild-type (WT) mice and N2B knockout (KO) mice in which the N2B element has been deleted. A range of protocols was used, including those that mimic physiological loading conditions. KO mice showed significant increases in hysteresis. Most prominently, in tissue that had been preconditioned with a physiological stretch-release protocol, hysteresis increased significantly from 320 ± 46 pJ/mm(2)/sarcomere in WT to 650 ± 94 pJ/mm(2)/sarcomere in N2B KO myocardium. These results are supported by experiments in which oxidative stress was used to mechanically inactivate portions of the N2B-Us of WT titin through cysteine cross-linking. Studies on muscle from which the thin filaments had been extracted (using the actin severing protein gelsolin) showed that the difference in hysteresis between WT and KO tissue cannot be explained by filament sliding-based viscosity. Instead the results suggest that hysteresis arises from within titin and most likely involves unfolding of immunoglobulin-like domains. These studies support that the mechanical function of the N2B element of titin includes reducing hysteresis and increasing the efficiency of the heart.
• Granzier, H., Anderson, B. R., Bogomolovas, J., Labeit, S., & Granzier, H. L. (2010). The effects of PKCalpha phosphorylation on the extensibility of titin's PEVK element. Journal of structural biology, 170(2).
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  Post-translational modifications, along with isoform splicing, of titin determine the passive tension development of stretched sarcomeres. It was recently shown that PKCalpha phosphorylates two highly-conserved residues (S26 and S170) of the PEVK region in cardiac titin, resulting in passive tension increase. To determine how each phosphorylated residue affects myocardial stiffness, we generated three recombinant mutant PEVK fragments (S26A, S170A and S170A/S26A), each flanked by Ig domains. Single-molecule force spectroscopy shows that PKCalpha decreases the PEVK persistence length (from 0.99 to 0.68 nm); the majority of this decrease is attributable to phosphorylation of S26. Before PKCalpha, all three mutant PEVK fragments showed at least 40% decrease in persistence length compared to wildtype. Furthermore, Ig domain unfolding force measurements indicate that PEVK's flanking Ig domains are relatively unstable compared to other titin Ig domains. We conclude that phosphorylation of S26 is the primary mechanism through which PKCalpha modulates cardiac stiffness.
• Granzier, H., Buck, D., Hudson, B. D., Ottenheijm, C. A., Labeit, S., & Granzier, H. L. (2010). Differential splicing of the large sarcomeric protein nebulin during skeletal muscle development. Journal of structural biology, 170(2).
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  We studied differential splicing of nebulin, a giant filamentous F-actin binding protein (M(r) approximately 700-800kDa) that is found in skeletal muscle. Nebulin spans the thin filament length, its C-terminus is anchored in the Z-disc, and its N-terminal region is located toward the thin filament pointed end. Various lines of evidence indicate that nebulin plays important roles in thin filament and Z-disc structure in skeletal muscle. In the present work we studied nebulin in a range of muscle types during postnatal development and performed transcript studies with a mouse nebulin exon microarray, developed by us, whose results were confirmed by RT-PCR. We also performed protein studies with high-resolution SDS-agarose gels and Western blots, and structural studies with electron microscopy. We found during postnatal development of the soleus muscle major changes in splicing in both the super-repeat region and the Z-disc region of nebulin; interestingly, these changes were absent in other muscle types. Three novel Z-disc exons, previously described in the mouse gene, were upregulated during postnatal development of soleus muscle and this was correlated with a significant increase in Z-disc width. These findings support the view that nebulin plays an important role in Z-disc width regulation. In summary, we discovered changes in both the super-repeat region and the Z-disc region of nebulin, that these changes are muscle-type specific, and that they correlate with differences in sarcomere structure.
• Granzier, H., Fukushima, H., Chung, C. S., & Granzier, H. L. (2010). Titin-isoform dependence of titin-actin interaction and its regulation by S100A1/Ca2+ in skinned myocardium. Journal of biomedicine & biotechnology, 2010.
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  Titin, also known as connectin, is a large filamentous protein that greatly contributes to passive myocardial stiffness. In vitro evidence suggests that one of titin's spring elements, the PEVK, interacts with actin and that this adds a viscous component to passive stiffness. Differential splicing of titin gives rise to the stiff N2B and more compliant N2BA isoforms. Here we studied the titin-isoform dependence of titin-actin interaction and studied the bovine left atrium (BLA) that expresses mainly N2BA titin, and the bovine left ventricle (BLV) that expresses a mixture of both N2B and N2BA isoforms. For comparison we also studied mouse left ventricular (MLV) myocardium which expresses predominately N2B titin. Using the actin-severing protein gelsolin, we obtained evidence that titin-actin interaction contributes significantly to passive myocardial stiffness in all tissue types, but most in MLV, least in BLA, and an intermediate level in BLV. We also studied whether titin-actin interaction is regulated by S100A1/calcium and found that calcium alone or S100A1 alone did not alter passive stiffness, but that combined they significantly lowered stiffness. We propose that titin-actin interaction is a "viscous break" that is on during diastole and off during systole.
• Granzier, H., Hudson, B. D., Hidalgo, C. G., Gotthardt, M., & Granzier, H. L. (2010). Excision of titin's cardiac PEVK spring element abolishes PKCalpha-induced increases in myocardial stiffness. Journal of molecular and cellular cardiology, 48(5).
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  Protein kinase C-alpha (PKCalpha) was recently reported to increase myocardial stiffness, an effect that was proposed to be due to phosphorylation of two highly conserved sites (S11878 and S12022) within the proline-glutamic acid-valine-lysine (PEVK) rich spring element of titin. To test this proposal we investigated the effect of PKCalpha on phosphorylation and passive stiffness in a mouse model lacking the titin exons that contain these two phosphorylation sites, the PEVK knockout (KO). We used skinned, gelsolin-extracted, left ventricular myocardium from wildtype and PEVK KO mice. Consistent with previous work we found that PKCalpha increased passive stiffness in the WT myocardium by 27+/-6%. Importantly, this effect was completely abolished in KO myocardium. In addition, increases in the elastic and viscous moduli at a wide range of frequencies (properties important in diastolic filling) following PKCalpha incubation (27+/-3% and 20+/-4%, respectively) were also ablated in the KO. Back phosphorylation assays showed that titin phosphorylation following incubation with PKCalpha was significantly reduced by 36+/-12% in skinned PEVK KO myocardial tissues. The remaining phosphorylation in the KO suggests that PKCalpha sites exist in the titin molecule outside the PEVK region; these sites are not involved in increasing passive stiffness. Our results firmly support that the PEVK region of cardiac titin is phosphorylated by PKCalpha and that this increases passive tension. Thus, the PEVK spring element is the critical site of PKCalpha's involvement in passive myocardial stiffness.
• Granzier, H., Lee, E., Peng, J., Radke, M., Gotthardt, M., & Granzier, H. L. (2010). Calcium sensitivity and the Frank-Starling mechanism of the heart are increased in titin N2B region-deficient mice. Journal of molecular and cellular cardiology, 49(3).
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  Previous work suggests that titin-based passive tension is a factor in the Frank-Starling mechanism of the heart, by increasing length-dependent activation (LDA) through an increase in calcium sensitivity at long sarcomere length. We tested this hypothesis in a mouse model (N2B KO model) in which titin-based passive tension is elevated as a result of the excision of the N2B element, one of cardiac titin's spring elements. LDA was assessed by measuring the active tension-pCa (-log[Ca(2+)]) relationship at sarcomere length (SLs) of 1.95, 2.10, and 2.30 microm in WT and N2B KO skinned myocardium. LDA was positively correlated with titin-based passive tension due to an increase in calcium sensitivity at the longer SLs in the KO. For example, at pCa 6.0, the KO:WT tension ratio was 1.28+/-0.07 and 1.42+/-0.04 at SLs of 2.1 and 2.3 microm, respectively. There was no difference in protein expression or total phosphorylation of sarcomeric proteins. We also measured the calcium sensitivity after PKA treating the skinned muscle and found that titin-based passive tension was also now correlated with LDA, with a slope that was significantly increased compared to no PKA treatment. Finally, we performed isolated heart experiments and measured the Frank-Starling relation (slope of developed wall stress-LV volume relation) as well as diastolic stiffness (slope of diastolic wall stress-volume relation). The FSM was more pronounced in the N2B KO hearts and the slope of the FSM correlated with diastolic stiffness. These findings support that titin-based passive tension triggers an increase in calcium sensitivity at long sarcomere length, thereby playing an important role in the Frank-Starling mechanism of the heart.
• Granzier, H., Ottenheijm, C. A., & Granzier, H. L. (2010). Lifting the nebula: novel insights into skeletal muscle contractility. Physiology (Bethesda, Md.), 25(5).
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  Nebulin is a giant protein and a constituent of the skeletal muscle sarcomere. The name of this protein refers to its unknown (i.e., nebulous) function. However, recent rapid advances reveal that nebulin plays important roles in the regulation of muscle contraction. When these functions of nebulin are compromised, muscle weakness ensues, as is the case in patients with nemaline myopathy.
• Granzier, H., Ottenheijm, C. A., & Granzier, H. L. (2010). Role of titin in skeletal muscle function and disease. Advances in experimental medicine and biology, 682.
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  This review covers recent developments in the titin field. Most recent reviews have discussed titin's role in cardiac function: here we will mainly focus on skeletal muscle, and discuss recent advances in the understanding of titin's role in skeletal muscle function and disease.
• Granzier, H., Tonino, P., Pappas, C. T., Hudson, B. D., Labeit, S., Gregorio, C. C., & Granzier, H. L. (2010). Reduced myofibrillar connectivity and increased Z-disk width in nebulin-deficient skeletal muscle. Journal of cell science, 123(Pt 3).
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  A prominent feature of striated muscle is the regular lateral alignment of adjacent sarcomeres. An important intermyofibrillar linking protein is the intermediate filament protein desmin, and based on biochemical and structural studies in primary cultures of myocytes it has been proposed that desmin interacts with the sarcomeric protein nebulin. Here we tested whether nebulin is part of a novel biomechanical linker complex, by using a recently developed nebulin knockout (KO) mouse model and measuring Z-disk displacement in adjacent myofibrils of both extensor digitorum longus (EDL) and soleus muscle. Z-disk displacement increased as sarcomere length (SL) was increased and the increase was significantly larger in KO fibers than in wild-type (WT) fibers; results in 3-day-old and 10-day-old mice were similar. Immunoelectron microscopy revealed reduced levels of desmin in intermyofibrillar spaces adjacent to Z-disks in KO fibers compared with WT fibers. We also performed siRNA knockdown of nebulin and expressed modules within the Z-disk portion of nebulin (M160-M170) in quail myotubes and found that this prevented the mature Z-disk localization of desmin filaments. Combined, these data suggest a model in which desmin attaches to the Z-disk through an interaction with nebulin. Finally, because nebulin has been proposed to play a role in specifying Z-disk width, we also measured Z-disk width in nebulin KO mice. Results show that most Z-disks of KO mice were modestly increased in width (approximately 80 nm in soleus and approximately 40 nm in EDL fibers) whereas a small subset had severely increased widths (up to approximately 1 microm) and resembled nemaline rod bodies. In summary, structural studies on a nebulin KO mouse show that in the absence of nebulin, Z-disks are significantly wider and that myofibrils are misaligned. Thus the functional roles of nebulin extend beyond thin filament length regulation and include roles in maintaining physiological Z-disk widths and myofibrillar connectivity.
• Granzier, H., Chandra, M., Mamidi, R., Ford, S., Hidalgo, C., Witt, C., Ottenheijm, C., Labeit, S., & Granzier, H. L. (2009). Nebulin alters cross-bridge cycling kinetics and increases thin filament activation: a novel mechanism for increasing tension and reducing tension cost. The Journal of biological chemistry, 284(45).
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  Nebulin is a giant filamentous F-actin-binding protein ( approximately 800 kDa) that binds along the thin filament of the skeletal muscle sarcomere. Nebulin is one of the least well understood major muscle proteins. Although nebulin is usually viewed as a structural protein, here we investigated whether nebulin plays a role in muscle contraction by using skinned muscle fiber bundles from a nebulin knock-out (NEB KO) mouse model. We measured force-pCa (-log[Ca(2+)]) and force-ATPase relations, as well as the rate of tension re-development (k(tr)) in tibialis cranialis muscle fibers. To rule out any alterations in troponin (Tn) isoform expression and/or status of Tn phosphorylation, we studied fiber bundles that had been reconstituted with bacterially expressed fast skeletal muscle recombinant Tn. We also performed a detailed analysis of myosin heavy chain, myosin light chain, and myosin light chain 2 phosphorylation, which showed no significant differences between wild type and NEB KO. Our mechanical studies revealed that NEB KO fibers had increased tension cost (5.9 versus 4.4 pmol millinewtons(-1) mm(-1) s(-1)) and reductions in k(tr) (4.7 versus 7.3 s(-1)), calcium sensitivity (pCa(50) 5.74 versus 5.90), and cooperativity of activation (n(H) 3.64 versus 4.38). Our findings indicate the following: 1) in skeletal muscle nebulin increases thin filament activation, and 2) through altering cross-bridge cycling kinetics, nebulin increases force and efficiency of contraction. These novel properties of nebulin add a new level of understanding of skeletal muscle function and provide a mechanism for the severe muscle weakness in patients with nebulin-based nemaline myopathy.
• Granzier, H., Hidalgo, C., Hudson, B., Bogomolovas, J., Zhu, Y., Anderson, B., Greaser, M., Labeit, S., & Granzier, H. L. (2009). PKC phosphorylation of titin's PEVK element: a novel and conserved pathway for modulating myocardial stiffness. Circulation research, 105(7).
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  Protein kinase C (PKC) regulates contractility of cardiac muscle cells by phosphorylating thin- and thick- filament-based proteins. Myocardial sarcomeres also contain a third myofilament, titin, and it is unknown whether titin can be phosphorylated by PKC and whether it affects passive tension.
• Granzier, H., Ottenheijm, C. A., Hidalgo, C., Rost, K., Gotthardt, M., & Granzier, H. L. (2009). Altered contractility of skeletal muscle in mice deficient in titin's M-band region. Journal of molecular biology, 393(1).
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  We investigated the contractile phenotype of skeletal muscle deficient in exons MEx1 and MEx2 (KO) of the titin M-band by using the cre-lox recombination system and a multidisciplinary physiological approach to study skeletal muscle contractile performance. At a maximal tetanic stimulation frequency, intact KO extensor digitorum longus muscle was able to produce wild-type levels of force. However, at submaximal stimulation frequency, force was reduced in KO mice, giving rise to a rightward shift of the force-frequency curve. This rightward shift of the force-frequency curve could not be explained by altered sarcoplasmic reticulum Ca(2+) handling, as indicated by analysis of Ca(2+) transients in intact myofibers and expression of Ca(2)(+)-handling proteins, but can be explained by the reduced myofilament Ca(2+) sensitivity of force generation that we found. Western blotting experiments suggested that the excision of titin exons MEx1 and MEx2 did not result in major changes in expression of titin M-band binding proteins or phosphorylation level of the thin-filament regulatory proteins, but rather in a shift toward expression of slow isoforms of the thick-filament-associated protein, myosin binding protein-C. Extraction of myosin binding protein-C from skinned muscle normalized myofilament Ca(2+) sensitivity of the KO extensor digitorum longus muscle. Thus, our data suggest that the M-band region of titin affects the expression of genes involved in the regulation of skeletal muscle contraction.
• Granzier, H., Ottenheijm, C. A., Knottnerus, A. M., Buck, D., Luo, X., Greer, K., Hoying, A., Labeit, S., & Granzier, H. L. (2009). Tuning passive mechanics through differential splicing of titin during skeletal muscle development. Biophysical journal, 97(8).
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  During postnatal development, major changes in mechanical properties of skeletal muscle occur. We investigated passive properties of skeletal muscle in mice and rabbits that varied in age from 1 day to approximately 1 year. Neonatal skeletal muscle expressed large titin isoforms directly after birth, followed by a gradual switch toward progressively smaller isoforms that required weeks-to-months to be completed. This suggests an extremely high plasticity of titin splicing during skeletal muscle development. Titin exon microarray analysis showed increased expression of a large group of exons in neonatal muscle, when compared to adult muscle transcripts, with the majority of upregulated exons coding for the elastic proline-glutamate-valine-lysine (PEVK) region of titin. Protein analysis supported expression of a significantly larger PEVK segment in neonatal muscle. In line with these findings, we found >50% lower titin-based passive stiffness of neonatal muscle when compared to adult muscle. Inhibiting 3,5,3'-tri-iodo-L-thyronine and 3,5,3',5'-tetra-iodo-L-thyronine secretion did not alter isoform switching, suggesting no major role for thyroid hormones in regulating differential titin splicing during postnatal development. In summary, our work shows that stiffening of skeletal muscle during postnatal development occurs through a decrease in titin isoform size, due mainly to a marked restructuring of the PEVK region of titin.
• Granzier, H., Ottenheijm, C. A., Witt, C. C., Stienen, G. J., Labeit, S., Beggs, A. H., & Granzier, H. L. (2009). Thin filament length dysregulation contributes to muscle weakness in nemaline myopathy patients with nebulin deficiency. Human molecular genetics, 18(13).
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  Nemaline myopathy (NM) is the most common non-dystrophic congenital myopathy. Clinically the most important feature of NM is muscle weakness; however, the mechanisms underlying this weakness are poorly understood. Here, we studied the muscular phenotype of NM patients with a well-defined nebulin mutation (NM-NEB), using a multidisciplinary approach to study thin filament length regulation and muscle contractile performance. SDS-PAGE and western blotting revealed greatly reduced nebulin levels in skeletal muscle of NM-NEB patients, with the most prominent reduction at nebulin's N-terminal end. Muscle mechanical studies indicated approximately 60% reduced force generating capacity of NM-NEB muscle and a leftward-shift of the force-sarcomere length relation in NM-NEB muscle fibers. This indicates that the mechanism for the force reduction is likely to include shorter and non-uniform thin filament lengths in NM-NEB muscle compared with control muscle. Immunofluorescence confocal microscopy and electron microscopy studies indicated that average thin filament length is reduced from approximately 1.3 microm in control muscle to approximately 0.75 microm in NM-NEB muscle. Thus, the present study is the first to show a distinct genotype-functional phenotype correlation in patients with NM due to a nebulin mutation, and provides evidence for the notion that dysregulated thin filament length contributes to muscle weakness in NM patients with nebulin mutations. Furthermore, a striking similarity between the contractile and structural phenotypes of nebulin-deficient mouse muscle and human NM-NEB muscle was observed, indicating that the nebulin knockout model is well suited for elucidating the functional basis of muscle weakness in NM and for the development of treatment strategies.
• Granzier, H., Zhu, Y., Bogomolovas, J., Labeit, S., & Granzier, H. L. (2009). Single molecule force spectroscopy of the cardiac titin N2B element: effects of the molecular chaperone alphaB-crystallin with disease-causing mutations. The Journal of biological chemistry, 284(20).
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  The small heat shock protein alphaB-crystallin interacts with N2B-Us, a large unique sequence found in the N2B element of cardiac titin. Using single molecule force spectroscopy, we studied the effect of alphaB-crystallin on the N2B-Us and its flanking Ig-like domains. Ig domains from the proximal tandem Ig segment of titin were also studied. The effect of wild type alphaB-crystallin on the single molecule force-extension curve was determined as well as that of mutant alphaB-crystallins harboring the dilated cardiomyopathy missense mutation, R157H, or the desmin-related myopathy mutation, R120G. Results revealed that wild type alphaB-crystallin decreased the persistence length of the N2B-Us (from approximately 0.7 to approximately 0.2 nm) but did not alter its contour length. alphaB-crystallin also increased the unfolding force of the Ig domains that flank the N2B-Us (by 51 +/- 3 piconewtons); the rate constant of unfolding at zero force was estimated to be approximately 17-fold lower in the presence of alphaB-crystallin (1.4 x 10(-4) s(-1) versus 2.4 x 10(-3) s(-1)). We also found that alphaB-crystallin increased the unfolding force of Ig domains from the proximal tandem Ig segment by 28 +/- 6 piconewtons. The effects of alphaB-crystallin were attenuated by the R157H mutation (but were still significant) and were absent when using the R120G mutant. We conclude that alphaB-crystallin protects titin from damage by lowering the persistence length of the N2B-Us and reducing the Ig domain unfolding probability. Our finding that this effect is either attenuated (R157H) or lost (R120G) in disease causing alphaB-crystallin mutations suggests that the interaction between alphaB-crystallin and titin is important for normal heart function.
• Granzier, H., Ottenheijm, C. A., Fong, C., Vangheluwe, P., Wuytack, F., Babu, G. J., Periasamy, M., Witt, C. C., Labeit, S., & Granzier, H. L. (2008). Sarcoplasmic reticulum calcium uptake and speed of relaxation are depressed in nebulin-free skeletal muscle. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 22(8).
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  Previous work suggested that altered Ca(2+) homeostasis might contribute to dysfunction of nebulin-free muscle, as gene expression analysis revealed that the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)-inhibitor sarcolipin (SLN) is up-regulated >70-fold in nebulin knockout mice, and here we tested this proposal. We investigated SLN protein expression in nebulin-free and wild-type skeletal muscle, as well as expression of other Ca(2+)-handling proteins. Ca(2+) uptake capacity was determined in isolated sarcoplasmic reticulum vesicles and in intact myofibers by measuring Ca(2+) transients. Muscle contractile performance was determined in skinned muscle activated with exogenous Ca(2+), as well as in electrically stimulated intact muscle. We found profound up-regulation of SLN protein in nebulin-free skeletal muscle, whereas expression of other Ca(2+)-handling proteins was not (calsequestrin and phospholamban) or was minimally (SERCA) affected. Speed of Ca(2+) uptake was >3-fold decreased in sarcoplasmic reticulum vesicles isolated from nebulin-free muscle as well as in nebulin-free intact myofibers. Ca(2+)-activated stress in skinned muscle and stress produced by intact nebulin-free muscle were reduced to a similar extent compared with wild type. Half-relaxation time was significantly longer in nebulin-free compared with wild-type muscle. Thus, the present study demonstrates for the first time that nebulin might also be involved in physiological Ca(2+) handling of the SR-myofibrillar system.

Presentations

• Desai, A., Sprissler, R. S., Sotak, S., Granzier, H. L., Tardiff, J. C., Khalpey, Z. I., Sweitzer, N. K., Gupta, A., Nai, V., & Whitaker, M. E. (2015, November). dult Onset Non-Ischemic Dilated Cardiomyopathy: A Novel Titin Mutation and a Case of Complex Inheritance. American College of Physicians (Arizona Chapter) Scientific Meeting. Phoenix, AZ: American College of Physicians (Arizona Chapter).
• Granzier, H. L. (2015, July). Role of titin in Heart Failure with Preserved Ejection fraction-Invited talk. BCVS-invited talk. New Orleans: AHA.
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  Invited talk
• Granzier, H. L. (2015, March 2015). Giant proteins in skeletal muscle. Bob Wade Memorial Lecture at the University of Maryland School of Medicine.. University of Maryland School of Medicine.: University of Maryland School of Medicine..
• Granzier, H. L. (2015, March). Giant proteins in skeletal muscle -invited talk. Bob Wade Memorial Lecture at the University of Maryland School of Medicine - invited talkUniversity of Maryland School of Medicine..
• Granzier, H. L. (2015, November). Role of nebulin in skeletal muscle and disease-invited talk. Invited Seminar at University of Iowa-invited. Iowa City: University of Iowa.
• Granzier, H. L. (2015, November). Titin in heart function and disease-invited talk. AHA-invited talk. Orlando, Florida: AHA.

Poster Presentations

• Granzier, H. L., Smith, J. E., Kiss, B., Strom, J. G., Granzier-Nakajima, S., Farman, G. P., & Methawasin, M. (2021, September). Shortening the thick filament by partial deletion of titin's C-zone alters cardiac function by reducing the operating sarcomere length range. International society of heart research. Denver Colorado: American Heart Association.
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  Titin's C-zone is an inextensible segment in titin, comprised of 11 super-repeats and located in the cMyBP-C-containing region of the thick filament. Previously we showed that deletion of titin's super-repeats C1 and C2 (TtnΔC1-2 model) results in shorter thick filaments and contractile dysfunction of the left ventricular (LV) chamber but that unexpectedly LV diastolic stiffness is normal. Here we studied the contraction-relaxation kinetics from the time-varying elastance of the LV and intact cardiomyocyte, cellular work loops of intact cardiomyocytes, Ca2+ transients, cross-bridge kinetics, and myofilament Ca2+ sensitivity. Intact cardiomyocytes of TtnΔC1-2 mice exhibit systolic dysfunction and impaired relaxation. The time-varying elastance at both LV and single-cell levels showed that activation kinetics are normal in TtnΔC1-2 mice, but that relaxation is slower. The slowed relaxation is, in part, attributable to an increased myofilament Ca2+ sensitivity and slower early Ca2+ reuptake. Cross-bridge dynamics showed that cross-bridge kinetics are normal but that the number of force-generating cross-bridges is reduced. In vivo sarcomere length (SL) measurements revealed that in TtnΔC1-2 mice the operating SL range of the LV is shifted towards shorter lengths. This normalizes the apparent cell and LV diastolic stiffness but further reduces systolic force as systole occurs further down on the ascending limb of the force-SL relation. We propose that the reduced working SLs reflect titin's role in regulating diastolic stiffness by altering the number of sarcomeres in series. Overall, our study reveals that thick filament length regulation by titin's C-zone is critical for normal cardiac function.