Methajit Methawasin

Interests

Research

My research seeks to understand pathophysiology of heart failure with preserved ejection fraction (HFpEF) and identify its possible therapeutic targets by using an integrative approach. I am experienced in cardiomyocyte mechanics as well as in-vivo cardiac functional techniques.My current work focuses on multiple diastolic contributors: titin, microtubular networks, and residual crossbridges, as an integrated system, and to study the progression of diastolic dysfunction in the aged heart at the single cell level.

Courses

Scholarly Contributions

Journals/Publications

• 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
• 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.
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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.
• 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.
• 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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  Aims: 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.Methods and results: Skeletal muscle tissue was obtained from HFpEF and control patients and from three different animal models including the respective controls-ZSF1 rat, Dahl salt-sensitive rat, and transverse aortic constriction surgery/deoxycorticosterone mouse. The development of HFpEF was verified by echocardiography. Protein expression and enzyme activity of selected markers were assessed in SM tissue homogenates. Protein expression between SM tissue obtained from HFpEF patients and the ZSF1 rats revealed similarities for protein markers involved in muscle atrophy (MuRF1 expression, protein ubiquitinylation, and LC3) and mitochondrial metabolism (succinate dehydrogenase and malate dehydrogenase activity, porin expression). The other two animal models exhibited far less similarities to the human samples.Conclusions: None of the three tested animal models mimics the condition in HFpEF patients completely, but among the animal models tested, the ZSF1 rat (ZSF1-lean vs. ZSF1-obese) shows the highest overlap to the human condition. Therefore, when studying therapeutic interventions to treat HFpEF and especially alterations in the SM, we suggest that the ZSF1 rat is a suitable model.Keywords: Animal models; Heart failure with preserved ejection fraction; Skeletal muscle.
• 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.
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  We thank Dr Mehmood for raising interesting points regarding the inhibition of phosphodiesterase in cardiac disease. Recently published studies have demonstrated upregulation of various phosphodiesterase isoforms in the failing hearts of animal models and patients (phosphodiesterases 1C, 2A, 5A, 9A1, and 10A) and revealed beneficial effects of selectively inhibiting phosphodiesterase activities on both cardiac remodeling and function. These studies show that inhibition of one specific phosphodiesterase at a time is sufficient to raise the targeted cyclic nucleotides and alter their corresponding biological activities. The effects of nonselective phosphodiesterase inhibitors or a combination of different phosphodiesterase inhibitors have not been evaluated in preclinical research. cGMP-stimulated PKG (protein kinase G) is known for various cardioprotective effects, whereas cAMP-dependent PKA (protein kinase A) augments inotropy in response to sympathetic stimulation and is known to improve contractility and affect titin’s stiffness.2 However, a sustained increase in cAMP is associated with harmful effects, for example, increased energy consumption, prohypertrophic, and arrhythmogenic effects,3 whereas reducing cAMP by moderate enhancing of phosphodiesterase 4B activity is cardioprotective.4 Thus, broad-spectrum phosphodiesterase inhibition could lead to a combination of positive and negative consequences. The more critical concern about the efficiency of the phosphodiesterase inhibitors is to verify the level of the phosphodiesterase activity since phosphodiesterase upregulation is likely to vary among individuals.1,5 To achieve the beneficial effect of phosphodiesterase inhibition, the synthesis of cyclic nucleotides has to be adequate, or a combined treatment of phosphodiesterase inhibitor with adenylate or guanylate cyclase activator should be considered.During the past decades, phosphodiesterses 5A and 9A have gained significant interest as potential targets for heart failure with preserved ejection fraction (HFpEF). However, the majority of the preclinical studies related to phosphodiesterses 5A and 9A focused on pathological remodeling and systolic dysfunction rather than diastolic dysfunction. The limitation of the HFpEF therapeutic studies is, in part, attributed to an incomplete understanding in its pathophysiology and lacking the in vivo and in vitro models that truly recapitulate human HFpEF phenotypes. Creating in vitro HFpEF models in cardiomyocyte-fibroblast culture systems through the administration of inflammatory cytokines may be worth considering. However, the most crucial point for both in vivo and in vitro models is to verify that the models indeed develop characteristic HFpEF features, which include delayed cardiomyocyte relaxation and increased cellular and extracellular matrix stiffness. Measurement of the mechanical properties of the myocardium and the cardiomyocyte is essential in HFpEF studies.Last but not least, in addition to targeting the phosphodiesterases and phosphorylation status, additional research should explore other types of post-translational modifications, particularly the oxidative post-translational modifications in response to systemic inflammation, which could be the upstream signaling that dysregulates cyclic nucleotides and phosphodiesterase activities.
• 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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  Background: 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.Methods: To examine the effect of chronic PDE9a inhibition, 2 diastolic dysfunction mouse models were studied: (1) TAC-deoxycorticosterone acetate and (2) Leprdb/db. PDE9a inhibitor (5 and 8 mg/kg per day) was administered to the mice via subcutaneously implanted osmotic minipumps for 28 days. The effect of acute PDE9a inhibition was investigated in intact cardiomyocytes isolated from TAC-deoxycorticosterone acetate mice. Atrial natriuretic peptide together with PDE9a inhibitor were administered to the isolated intact cardiomyocytes through the cell perfusate.Results: For acute inhibition, no cellular stiffness reduction was found, whereas chronic PDE9a inhibition resulted in reduced left ventricular chamber stiffness in TAC-deoxycorticosterone acetate, but not in Leprdb/db mice. Passive cardiomyocyte stiffness was reduced by chronic PDE9a inhibition, with no differences in myocardial fibrosis or cardiac morphometry. PDE9a inhibition increased the ventricular-arterial coupling ratio, reflecting impaired systolic function.Conclusions: Chronic PDE9a inhibition lowers left ventricular chamber stiffness in TAC-deoxycorticosterone acetate mice. However, the usefulness of PDE9a inhibition to treat high-diastolic stiffness may be limited as the required PDE9a inhibitor dose also impairs systolic function, observed as a decline in ventricular-arterial coordination, in this model.Keywords: constriction; diastole; guanosine monophosphate; heart failure; prevalence.
• 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. doi:10.1085/jgp.201812218
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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.
• 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. doi:10.1161/CIRCULATIONAHA.118.037588
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  BACKGROUND: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.METHODS:To study titin deficiency versus dysfunction, the authors generated and compared striated muscle specific knockouts (KOs) with progressive postnatal loss of the complete titin protein by removing exon 2 (E2-KO) or an M-band truncation that eliminates proper sarcomeric integration, but retains all other functional domains (M-band exon 1/2 [M1/2]-KO). The authors evaluated cardiac function, cardiomyocyte mechanics, and the molecular basis of the phenotype.RESULTS:Skeletal muscle atrophy with reduced strength, severe sarcomere disassembly, and lethality from 2 weeks of age were shared between the models. Cardiac phenotypes differed considerably: loss of titin leads to dilated cardiomyopathy with combined systolic and diastolic dysfunction-the absence of M-band titin to cardiac atrophy and preserved function. The elastic properties of M1/2-KO cardiomyocytes are maintained, while passive stiffness is reduced in the E2-KO. In both KOs, we find an increased stress response and increased expression of proteins linked to titin-based mechanotransduction (CryAB, ANKRD1, muscle LIM protein, FHLs, p42, Camk2d, p62, and Nbr1). Among them, FHL2 and the M-band signaling proteins p62 and Nbr1 are exclusively upregulated in the E2-KO, suggesting a role in the differential pathology of titin truncation versus deficiency of the full-length protein. The differential stress response is consistent with truncated titin contributing to the mechanical properties in M1/2-KOs, while low titin levels in E2-KOs lead to reduced titin-based stiffness and increased strain on the remaining titin molecules.CONCLUSIONS:Progressive depletion of titin leads to sarcomere disassembly and atrophy in striated muscle. In the complete knockout, remaining titin molecules experience increased strain, resulting in mechanically induced trophic signaling and eventually dilated cardiomyopathy. The truncated titin in M1/2-KO helps maintain the passive properties and thus reduces mechanically induced signaling. Together, these findings contribute to the molecular understanding of why titin mutations differentially affect cardiac growth and have implications for genotype-phenotype relations that support a personalized medicine approach to the diverse titinopathies.
• 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. doi:10.1085/jgp.201812259
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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.
• Yousefi, K., Irion, C. I., Takeuchi, L. M., Ding, W., Lambert, G., Eisenberg, T., Sukkar, S., Granzier, H. L., Methawasin, M., Lee, D. I., Hahn, V. S., Kass, D. A., Hatzistergos, K. E., Hare, J. M., Webster, K. A., & Shehadeh, L. A. (2019). Osteopontin Promotes Left Ventricular Diastolic Dysfunction Through a Mitochondrial Pathway. Journal of the American College of Cardiology, 73(21), 2705-2718. doi:10.1016/j.jacc.2019.02.074
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  Patients with chronic kidney disease (CKD) and coincident heart failure with preserved ejection fraction (HFpEF) may constitute a distinct HFpEF phenotype. Osteopontin (OPN) is a biomarker of HFpEF and predictive of disease outcome. We recently reported that OPN blockade reversed hypertension, mitochondrial dysfunction, and kidney failure in Col4a3 mice, a model of human Alport syndrome.
• Methawasin, M., & Granzier, H. (2018). Softening the Stressed Giant Titin in Diabetes Mellitus. Circulation research, 123(3), 315-317. doi:10.1161/CIRCRESAHA.118.313396
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  Comment onDiabetes-Induced Cardiomyocyte Passive Stiffening Is Caused by Impaired Insulin-Dependent Titin Modification and Can Be Modulated by Neuregulin-1. [Circ Res. 2018]
• 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. doi:10.1161/CIRCULATIONAHA.117.026955
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  Response to Lewis and Miller 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. 2017]
• 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. doi:10.1038/s41467-017-01144-9
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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.
• 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. doi:10.1161/CIRCRESAHA.116.308904
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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.
• 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. doi:10.1016/j.yjmcc.2016.04.013
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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.
• 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. doi:10.1161/CIRCULATIONAHA.116.023003
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  BACKGROUND: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.METHODS:Mice in which the RNA recognition motif (RRM) of one of the RNA binding motif-20 alleles was floxed and that expressed the MerCreMer transgene under control of the αMHC promoter (referred to as cRbm20ΔRRM mice) were used. Mice underwent transverse aortic constriction (TAC) surgery and deoxycorticosterone acetate (DOCA) pellet implantation. RRM deletion in adult mice was triggered by injecting raloxifene (cRbm20ΔRRM-raloxifene), with dimethyl sulfoxide (DMSO)-injected mice (cRbm20ΔRRM-DMSO) as the control. Diastolic function was investigated with echocardiography and pressure-volume analysis; passive stiffness was studied in LV muscle strips and isolated cardiac myocytes before and after elimination of titin-based stiffness. Treadmill exercise performance was also studied. Titin isoform expression was evaluated with agarose gels.RESULTS:cRbm20ΔRRM-raloxifene mice expressed large titins in the hearts, called supercompliant titin (N2BAsc), which, within 3 weeks after raloxifene injection, made up ≈45% of total titin. TAC/DOCA cRbm20ΔRRM-DMSO mice developed LV hypertrophy and a marked increase in LV chamber stiffness as shown by both pressure-volume analysis and echocardiography. LV chamber stiffness was normalized in TAC/DOCA cRbm20ΔRRM-raloxifene mice that expressed N2BAsc. Passive stiffness measurements on muscle strips isolated from the LV free wall revealed that extracellular matrix stiffness was equally increased in both groups of TAC/DOCA mice (cRbm20ΔRRM-DMSO and cRbm20ΔRRM-raloxifene). However, titin-based muscle stiffness was reduced in the mice that expressed N2BAsc (TAC/DOCAcRbm20ΔRRM-raloxifene). Exercise testing demonstrated significant improvement in exercise tolerance in TAC/DOCA mice that expressed N2BAsc.CONCLUSIONS:Inhibition of the RNA binding motif-20-based titin splicing system upregulates compliant titins, which improves diastolic function and exercise tolerance in the TAC/DOCA model. Titin holds promise as a therapeutic target for heart failure with preserved ejection fraction.
• 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. doi:10.3389/fphys.2016.00322
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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.
• 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. doi:10.1073/pnas.1411493111
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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.
• Methawasin, M., Hutchinson, K. R., Lee, E. J., 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. doi:10.1161/CIRCULATIONAHA.113.005610
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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.
• 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), 19-28. doi:10.1161/CIRCULATIONAHA.112.001268
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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.
• 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, 90-7. doi:10.1016/j.yjmcc.2012.11.012
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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.
• 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), 428-34. doi:10.1016/j.yjmcc.2011.06.006
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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.
• King, N. M., Methawasin, M., Nedrud, J., Harrell, N., Chung, C. S., Helmes, M., & Granzier, H. (2011). Mouse intact cardiac myocyte mechanics: cross-bridge and titin-based stress in unactivated cells. The Journal of general physiology, 137(1), 81-91. doi:10.1085/jgp.201010499
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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.
• Charoenphandhu, N., Teerapornpuntakit, J., Methawasin, M., Wongdee, K., Thongchote, K., & Krishnamra, N. (2008). Prolactin decreases expression of Runx2, osteoprotegerin, and RANKL in primary osteoblasts derived from tibiae of adult female rats. Canadian journal of physiology and pharmacology, 86(5), 240-8. doi:10.1139/y08-037
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  Hyperprolactinemia caused by physiological or pathological conditions, such as those occurring during lactation and prolactinoma, respectively, results in progressive osteopenia. The underlying mechanisms, however, are controversial. Prolactin (PRL) may directly attenuate the functions of osteoblasts, since these bone cells express PRL receptors. The present study therefore aimed to investigate the effects of PRL on the expression of genes related to the osteoblast functions by using quantitative real-time PCR technique. Herein, we used primary osteoblasts that were derived from the tibiae of adult rats and displayed characteristics of differentiated osteoblasts, including in vitro mineralization. Osteoblasts exposed for 48 h to 1000 ng/mL PRL, but not to 10 or 100 ng/mL PRL, showed decreases in the mRNA expression of Runx2, osteoprotegerin (OPG), and receptor activator of nuclear factor kappaBeta ligand (RANKL) by 60.49%, 72.74%, and 87.51%, respectively. Nevertheless, PRL did not change the RANKL/OPG ratio, since expression of OPG and RANKL were proportionally decreased. These concentrations of PRL had no effect on the mRNA expression of osteocalcin and osteopontin, nor on mineralization. High pathologic concentrations of PRL (1000 ng/mL) may downregulate expression of genes that are essential for osteoblast differentiation and functions. The present results explained the clinical findings of hyperprolactinemia-induced bone loss.

Presentations

• Methawasin, M. (2019, February). Increasing titin compliance as a therapeutic manipulation for Heart Failure with preserved Ejection Fraction (HFpEF).. CMM seminar. University of Arizona.
• Methawasin, M. (2019, May). A therapeutic approach for Heart Failure with preserved Ejection Fraction (HFpEF) through modification of titin’s phosphorylation: study a therapeutic effect of Phosphodiesterase 9a inhibitor.. Leducq investigators meeting. Cedar Key, Florida.
• Methawasin, M. (2019, September). Increasing titin compliance through inhibition of titin splicing factor RBM20 improves diastolic function in HFpEF-like condition. Arizona Joint Biology Retreat 2019. Biosphere, University of Arizona.
• Methawasin, M. (2018, June). A therapeutic approach for Heart Failure with preserved Ejection Fraction (HFpEF) through modification of titin’s phosphorylation: study a therapeutic effect of Phosphodiesterase 9a inhibitor.. Leducq investigators meeting. Boston, Massachusette.
• Methawasin, M. (2018, March). A therapeutic approach for Heart Failure with preserved Ejection Fraction (HFpEF) through modification of titin’s phosphorylation: study a therapeutic effect of Phosphodiesterase 9a inhibitor. Heart Development Conference. University of Arizona.
• Methawasin, M. (2017, February). A therapeutic approach for Heart Failure with preserved Ejection Fraction (HFpEF) through modification of titin’s phosphorylation: study a therapeutic effect of Phosphodiesterase 9a inhibitor. Heart Development Conference. University of Arizona.
• Methawasin, M. (2017, March). The role of PDE9A inhibitor in reducing diastolic dysfunction in HFpEF-like animal models.. Leducq investigators meeting. Augustine, Florida.
• Methawasin, M. (2017, September). A therapeutic approach for Heart Failure with preserved Ejection Fraction (HFpEF) through modification of titin’s phosphorylation: study a therapeutic effect of Phosphodiesterase 9a inhibitor.. Leducq investigators meeting. Berlin, Germany.
• Methawasin, M. (2016, March). Experimentally increasing titin’s compliance in a mouse model of HFpEF ameliorates diastolic dysfunction. Heart Development Conference. University of Arizona.
• Methawasin, M. (2016, October). The role of PDE9A inhibitor in reducing diastolic dysfunction in HFpEF-like animal models. Heart Development Conference. University of Arizona.
• Methawasin, M. (2016, October). Upregulating compliant titin in the heart attenuates left ventricular stiffness in a HFpEF-liked mouse model. Arizona Physiological Society Meeting. University of Arizona.
• Methawasin, M. (2015, March). Upregulating compliant titin in the heart as a potential therapeutic strategy for HFpEF: studies in the mouse model MCM;cRbm20ΔRRM. Heart Development Conference. University of Arizona.

Poster Presentations

• Methawasin, M., Farman, G. P., Granzier-Nakajima, S., Strom, J. G., Kiss, B., Smith, J. E., & Granzier, H. L. (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.
• Methawasin, M., Strom, J., Hourani, Z., Smith, J., & Granzier, H. (2019, July). The Effect Of Phosphodiesterase 9a Inhibition In 2 Mouse Models Of Diastolic Dysfunction. Basic Cardiovascular Sciences (BCVS) conference. Boston, Massachusette.
• Methawasin, M., Helmes, M., Strom, J., Kiss, B., & Granzier, H. (2018, May). A Cellular Work Loop Study of Single Intact Cardiomyocytes of Mouse Models with Disparate Titin’s Size and A Surgical Mouse Model of Diastolic Dysfunction.. Myofilament meeting. Madison, Wisconsin.
• Methawasin, M., Strom, J., Helmes, M., & Granzier, H. (2018, July). The Cardiomyocyte Diastolic Stiffness and Contractility are Inversely Related to the Size of Titin: A Cellular Work Loop Study of Single Intact Cardiomyocytes. Basic Cardiovascular Sciences (BCVS) meeting. San Antonio, Texas.
• Methawasin, M., Strom, J., Slater, R., & Granzier, H. (2016, July). Experimentally Increasing Titin’s Compliance in a Mouse Model of Heart Failure with Preserved Ejection Fraction Ameliorates Diastolic Dysfunction.. Basic Cardiovascular Sciences (BCVS) meeting. Phoenix Arizona.
• Methawasin, M., Strom, J., Smith, J., & Granzier, H. (2016, March). Upregulating compliant titin in the heart attenuates left ventricular stiffness in a mouse model with diastolic dysfunction. Biophysical Society 60th Annual Meeting. Los Angeles California.
• Methawasin, M., Smith, J., Tonino, P., & Granzier, H. (2015, July). The role of titin in diastolic function: studies in two genetically engineered mouse models with disparate titin size.. Basic Cardiovascular Sciences (BCVS) conference. New Orleans, Louisiana.