Jil C Tardiff

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• Greenberg, M. J., & Tardiff, J. C. (2021). Complexity in genetic cardiomyopathies and new approaches for mechanism-based precision medicine. The Journal of general physiology, 153(3).
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  Genetic cardiomyopathies have been studied for decades, and it has become increasingly clear that these progressive diseases are more complex than originally thought. These complexities can be seen both in the molecular etiologies of these disorders and in the clinical phenotypes observed in patients. While these disorders can be caused by mutations in cardiac genes, including ones encoding sarcomeric proteins, the disease presentation varies depending on the patient mutation, where mutations even within the same gene can cause divergent phenotypes. Moreover, it is challenging to connect the mutation-induced molecular insult that drives the disease pathogenesis with the various compensatory and maladaptive pathways that are activated during the course of the subsequent progressive, pathogenic cardiac remodeling. These inherent complexities have frustrated our ability to understand and develop broadly effective treatments for these disorders. It has been proposed that it might be possible to improve patient outcomes by adopting a precision medicine approach. Here, we lay out a practical framework for such an approach, where patient subpopulations are binned based on common underlying biophysical mechanisms that drive the molecular disease pathogenesis, and we propose that this function-based approach will enable the development of targeted therapeutics that ameliorate these effects. We highlight several mutations to illustrate the need for mechanistic molecular experiments that span organizational and temporal scales, and we describe recent advances in the development of novel therapeutics based on functional targets. Finally, we describe many of the outstanding questions for the field and how fundamental mechanistic studies, informed by our more nuanced understanding of the clinical disorders, will play a central role in realizing the potential of precision medicine for genetic cardiomyopathies.
• Baldo, A. P., Tardiff, J. C., & Schwartz, S. D. (2020). Mechanochemical Function of Myosin II: Investigation into the Recovery Stroke and ATP Hydrolysis. The journal of physical chemistry. B, 124(45), 10014-10023.
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  Myosin regulates muscle function through a complex cycle of conformational rearrangements coupled with the hydrolysis of adenosine triphosphate (ATP). The recovery stroke reorganizes the myosin active site to hydrolyze ATP and cross bridge with the thin filament to produce muscle contraction. Engineered mutations K84M and R704E in myosin have been designed to specifically inhibit the recovery stroke and have been shown to indirectly affect the ATPase activity of myosin. We investigated these mutagenic perturbations to the recovery stroke and generated thermodynamically correct and unbiased trajectories for native ATP hydrolysis with computationally enhanced sampling methods. Our methodology was able to resolve experimentally observed changes to kinetic and equilibrium dynamics for the recovery stroke with the correct prediction in the severity of these changes. For ATP hydrolysis, the sequential nature along with the stabilization of a metaphosphate intermediate was observed in agreement with previous studies. However, we observed glutamate 459 being utilized as a proton abstractor to prime the attacking water instead of a lytic water, a phenomenon not well categorized in myosin but has in other ATPases. Both rare event methodologies can be extended to human myosin to investigate isoformic differences from and scan cardiomyopathic mutations to see differential perturbations to kinetics of other conformational changes in myosin such as the power stroke.
• Chowdhury, S. A., Warren, C. M., Simon, J. N., Ryba, D. M., Batra, A., Varga, P., Kranias, E. G., Tardiff, J. C., Solaro, R. J., & Wolska, B. M. (2020). Modifications of Sarcoplasmic Reticulum Function Prevent Progression of Sarcomere-Linked Hypertrophic Cardiomyopathy Despite a Persistent Increase in Myofilament Calcium Response. Frontiers in physiology, 11, 107.
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  Hypertrophic cardiomyopathy (HCM) is a genetic disorder caused by mutations in different genes mainly encoding myofilament proteins and therefore called a "disease of the sarcomere." Despite the discovery of sarcomere protein mutations linked to HCM almost 30 years ago, the cellular mechanisms responsible for the development of this disease are not completely understood and likely vary among different mutations. Moreover, despite many efforts to develop effective treatments for HCM, these have largely been unsuccessful, and more studies are needed to better understand the cellular mechanisms of the disease. In experiments reported here, we investigated a mouse model expressing the mutant cTnT-R92Q, which is linked to HCM and induces an increase in myofilament Ca sensitivity and diastolic dysfunction. We found that early correction of the diastolic dysfunction by phospholamban knockout (PLNKO) was able to prevent the development of the HCM phenotype in troponin T (TnT)-R92Q transgenic (TG) mice. Four groups of mice in FVB/N background were generated and used for the experiments: (1) non-transgenic (NTG)/PLN mice, which express wild-type TnT and normal level of PLN; (2) NTG/PLNKO mice, which express wild-type TnT and no PLN; (3) TG/PLN mice, which express TnT-R92Q and normal level of PLN; (4) TG/PLNKO mice, which express TnT-R92Q and no PLN. Cardiac function was determined using both standard echocardiographic parameters and speckle tracking strain measurements. We found that both atrial morphology and diastolic function were altered in TG/PLN mice but normal in TG/PLNKO mice. Histological analysis showed a disarray of myocytes and increased collagen deposition only in TG/PLN hearts. We also observed increased Ca/calmodulin-dependent protein kinase II (CaMKII) phosphorylation only in TG/PLN hearts but not in TG/PLNKO hearts. The rescue of the HCM phenotype was not associated with differences in myofilament Ca sensitivity between TG/PLN and TG/PLNKO mice. Moreover, compared to standard systolic echo parameters, such as ejection fraction (EF), speckle strain measurements provided a more sensitive approach to detect early systolic dysfunction in TG/PLN mice. In summary, our results indicate that targeting diastolic dysfunction through altering Ca fluxes with no change in myofilament response to Ca was able to prevent the development of the HCM phenotype and should be considered as a potential additional treatment for HCM patients.
• Powers, J. D., Kooiker, K. B., Mason, A. B., Teitgen, A. E., Flint, G. V., Tardiff, J. C., Schwartz, S. D., McCulloch, A. D., Regnier, M., Davis, J., & Moussavi-Harami, F. (2020). Modulating the tension-time integral of the cardiac twitch prevents dilated cardiomyopathy in murine hearts. JCI insight, 5(20).
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  Dilated cardiomyopathy (DCM) is often associated with sarcomere protein mutations that confer reduced myofilament tension-generating capacity. We demonstrated that cardiac twitch tension-time integrals can be targeted and tuned to prevent DCM remodeling in hearts with contractile dysfunction. We employed a transgenic murine model of DCM caused by the D230N-tropomyosin (Tm) mutation and designed a sarcomere-based intervention specifically targeting the twitch tension-time integral of D230N-Tm hearts using multiscale computational models of intramolecular and intermolecular interactions in the thin filament and cell-level contractile simulations. Our models predicted that increasing the calcium sensitivity of thin filament activation using the cardiac troponin C (cTnC) variant L48Q can sufficiently augment twitch tension-time integrals of D230N-Tm hearts. Indeed, cardiac muscle isolated from double-transgenic hearts expressing D230N-Tm and L48Q cTnC had increased calcium sensitivity of tension development and increased twitch tension-time integrals compared with preparations from hearts with D230N-Tm alone. Longitudinal echocardiographic measurements revealed that DTG hearts retained normal cardiac morphology and function, whereas D230N-Tm hearts developed progressive DCM. We present a computational and experimental framework for targeting molecular mechanisms governing the twitch tension of cardiomyopathic hearts to counteract putative mechanical drivers of adverse remodeling and open possibilities for tension-based treatments of genetic cardiomyopathies.
• Sadayappan, S., Tardiff, J. C., & Wold, L. E. (2020). Basic Cardiovascular Sciences Scientific Sessions 2020: Emerging Opportunities in Cardiovascular Diseases. Circulation research, 127(11), 1459-1467.
• Abdullah, S., Lynn, M. L., McConnell, M. T., Klass, M. M., Baldo, A. P., Schwartz, S. D., & Tardiff, J. C. (2019). FRET-based analysis of the cardiac troponin T linker region reveals the structural basis of the hypertrophic cardiomyopathy-causing Δ160E mutation. The Journal of biological chemistry, 294(40), 14634-14647.
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  Mutations in the cardiac thin filament (TF) have highly variable effects on the regulatory function of the cardiac sarcomere. Understanding the molecular-level dysfunction elicited by TF mutations is crucial to elucidate cardiac disease mechanisms. The hypertrophic cardiomyopathy-causing cardiac troponin T (cTnT) mutation Δ160Glu (Δ160E) is located in a putative "hinge" adjacent to an unstructured linker connecting domains TNT1 and TNT2. Currently, no high-resolution structure exists for this region, limiting significantly our ability to understand its role in myofilament activation and the molecular mechanism of mutation-induced dysfunction. Previous regulated motility data have indicated mutation-induced impairment of weak actomyosin interactions. We hypothesized that cTnT-Δ160E repositions the flexible linker, altering weak actomyosin electrostatic binding and acting as a biophysical trigger for impaired contractility and the observed remodeling. Using time-resolved FRET and an all-atom TF model, here we first defined the WT structure of the cTnT-linker region and then identified Δ160E mutation-induced positional changes. Our results suggest that the WT linker runs alongside the C terminus of tropomyosin. The Δ160E-induced structural changes moved the linker closer to the tropomyosin C terminus, an effect that was more pronounced in the presence of myosin subfragment (S1) heads, supporting previous findings. Our model fully supported this result, indicating a mutation-induced decrease in linker flexibility. Our findings provide a framework for understanding basic pathogenic mechanisms that drive severe clinical hypertrophic cardiomyopathy phenotypes and for identifying structural targets for intervention that can be tested and .
• Deranek, A. E., Klass, M. M., & Tardiff, J. C. (2019). Moving beyond simple answers to complex disorders in sarcomeric cardiomyopathies: the role of integrated systems. Pflugers Archiv : European journal of physiology, 471(5), 661-671.
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  The classic clinical definition of hypertrophic cardiomyopathy (HCM) as originally described by Teare is deceptively simple, "left ventricular hypertrophy in the absence of any identifiable cause." Longitudinal studies, however, including a seminal study performed by Frank and Braunwald in 1968, clearly described the disorder much as we know it today, a complex, progressive, and highly variable cardiomyopathy affecting ~ 1/500 individuals worldwide. Subsequent genetic linkage studies in the early 1990s identified mutations in virtually all of the protein components of the cardiac sarcomere as the primary molecular cause of HCM. In addition, a substantial proportion of inherited dilated cardiomyopathy (DCM) has also been linked to sarcomeric protein mutations. Despite our deep understanding of the overall function of the sarcomere as the primary driver of cardiac contractility, the ability to use genotype in patient management remains elusive. A persistent challenge in the field from both the biophysical and clinical standpoints is how to rigorously link high-resolution protein dynamics and mechanics to the long-term cardiovascular remodeling process that characterizes these complex disorders. In this review, we will explore the depth of the problem from both the standpoint of a multi-subunit, highly conserved and dynamic "machine" to the resultant clinical and structural human phenotype with an emphasis on new, integrative approaches that can be widely applied to identify both novel disease mechanisms and new therapeutic targets for these primary biophysical disorders of the cardiac sarcomere.
• Liu, Y., Afzal, J., Vakrou, S., Greenland, G. V., Talbot, C. C., Hebl, V. B., Guan, Y., Karmali, R., Tardiff, J. C., Leinwand, L. A., Olgin, J. E., Das, S., Fukunaga, R., & Abraham, M. R. (2019). Differences in microRNA-29 and Pro-fibrotic Gene Expression in Mouse and Human Hypertrophic Cardiomyopathy. Frontiers in cardiovascular medicine, 6, 170.
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  Hypertrophic cardiomyopathy (HCM) is characterized by myocyte hypertrophy and fibrosis. Studies in two mouse models (R92W-TnT/R403Q-MyHC) at early HCM stage revealed upregulation of endothelin (ET1) signaling in both mutants, but TGFβ signaling only in TnT mutants. Dysregulation of miR-29 expression has been implicated in cardiac fibrosis. But it is unknown whether expression of miR-29a/b/c and profibrotic genes is commonly regulated in mouse and human HCM. In order to understand mechanisms underlying fibrosis in HCM, and examine similarities/differences in expression of miR-29a/b/c and several profibrotic genes in mouse and human HCM, we performed parallel studies in rat cardiac myocyte/fibroblast cultures, examined gene expression in two mouse models of () HCM (R92W-TnT, R403Q-MyHC)/controls at early (5 weeks) and established (24 weeks) disease stage, and analyzed publicly available mRNA/miRNA expression data from -HCM patients undergoing septal myectomy/controls (unused donor hearts). Myocyte cultures: ET1 increased superoxide/HO, stimulated TGFβ expression/secretion, and suppressed miR-29a expression in myocytes. The effect of ET1 on miR-29 and TGFβ expression/secretion was antagonized by N-acetyl-cysteine, a reactive oxygen species scavenger. Fibroblast cultures: ET1 had no effect on pro-fibrotic gene expression in fibroblasts. TGFβ1/TGFβ2 suppressed miR-29a and increased collagen expression, which was abolished by miR-29a overexpression. Mouse and human HCM: Expression of miR-29a/b/c was lower, and /collagen gene expression was higher in TnT mutant-LV at 5 and 24 weeks; no difference was observed in expression of these genes in MyHC mutant-LV and in human myectomy tissue. expression was higher in LV of both mutant mice and human myectomy tissue. , a negative regulator of the renin-angiotensin-aldosterone system, was the most upregulated transcript in human myectomy tissue. Pathway analysis predicted upregulation of the anti-hypertrophic/anti-fibrotic liver X receptor/retinoid X receptor (LXR/RXR) pathway only in human myectomy tissue. Our studies suggest that activation of ET1 signaling in cardiac myocytes increases reactive oxygen species and stimulates TGFβ secretion, which downregulates miR-29a and increases collagen in fibroblasts, thus contributing to fibrosis. Our gene expression studies in mouse and human HCM reveal allele-specific differences in miR-29 family/profibrotic gene expression in mouse HCM, and activation of anti-hypertrophic/anti-fibrotic genes and pathways in human HCM.
• Szatkowski, L., Lynn, M. L., Holeman, T., Williams, M. R., Baldo, A. P., Tardiff, J. C., & Schwartz, S. D. (2019). Proof of Principle that Molecular Modeling Followed by a Biophysical Experiment Can Develop Small Molecules that Restore Function to the Cardiac Thin Filament in the Presence of Cardiomyopathic Mutations. ACS omega, 4(4), 6492-6501.
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  This article reports a coupled computational experimental approach to design small molecules aimed at targeting genetic cardiomyopathies. We begin with a fully atomistic model of the cardiac thin filament. To this we dock molecules using accepted computational drug binding methodologies. The candidates are screened for their ability to repair alterations in biophysical properties caused by mutation. Hypertrophic and dilated cardiomyopathies caused by mutation are initially biophysical in nature, and the approach we take is to correct the biophysical insult prior to irreversible cardiac damage. Candidate molecules are then tested experimentally for both binding and biophysical properties. This is a proof of concept study-eventually candidate molecules will be tested in transgenic animal models of genetic (sarcomeric) cardiomyopathies.
• Lehman, S. J., Tal-Grinspan, L., Lynn, M. L., Strom, J., Benitez, G. E., Anderson, M. E., & Tardiff, J. C. (2019). Chronic Calmodulin-Kinase II Activation Drives Disease Progression in Mutation-Specific Hypertrophic Cardiomyopathy. Circulation.
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  Although the genetic causes of Hypertrophic Cardiomyopathy (HCM) are widely recognized, considerable lag in the development of targeted therapeutics has limited interventions to symptom palliation. This is in part due to an incomplete understanding of how point mutations trigger pathogenic remodeling. As a further complication, similar mutations within sarcomeric genes can result in differential disease severity, highlighting the need to understand the mechanism of progression at the molecular level. One pathway commonly linked to HCM progression is calcium homeostasis dysregulation, though how specific mutations disrupt calcium homeostasis remains unclear.
• Lynn, M. L., Lehman, S. J., & Tardiff, J. C. (2018). Biophysical Derangements in Genetic Cardiomyopathies. Heart failure clinics, 14(2), 147-159.
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  This article focuses on three "bins" that comprise sets of biophysical derangements elicited by cardiomyopathy-associated mutations in the myofilament. Current therapies focus on symptom palliation and do not address the disease at its core. We and others have proposed that a more nuanced classification could lead to direct interventions based on early dysregulation changing the trajectory of disease progression in the preclinical cohort. Continued research is necessary to address the complexity of cardiomyopathic progression and develop efficacious therapeutics.
• Vakrou, S., Fukunaga, R., Foster, D. B., Sorensen, L., Liu, Y., Guan, Y., Woldemichael, K., Pineda-Reyes, R., Liu, T., Tardiff, J. C., Leinwand, L. A., Tocchetti, C. G., Abraham, T. P., O'Rourke, B., Aon, M. A., & Abraham, M. R. (2018). Allele-specific differences in transcriptome, miRNome, and mitochondrial function in two hypertrophic cardiomyopathy mouse models. JCI insight, 3(6).
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  Hypertrophic cardiomyopathy (HCM) stems from mutations in sarcomeric proteins that elicit distinct biophysical sequelae, which in turn may yield radically different intracellular signaling and molecular pathologic profiles. These signaling events remain largely unaddressed by clinical trials that have selected patients based on clinical HCM diagnosis, irrespective of genotype. In this study, we determined how two mouse models of HCM differ, with respect to cellular/mitochondrial function and molecular biosignatures, at an early stage of disease. We show that hearts from young R92W-TnT and R403Q-αMyHC mutation-bearing mice differ in their transcriptome, miRNome, intracellular redox environment, mitochondrial antioxidant defense mechanisms, and susceptibility to mitochondrial permeability transition pore opening. Pathway analysis of mRNA-sequencing data and microRNA profiles indicate that R92W-TnT mutants exhibit a biosignature consistent with activation of profibrotic TGF-β signaling. Our results suggest that the oxidative environment and mitochondrial impairment in young R92W-TnT mice promote activation of TGF-β signaling that foreshadows a pernicious phenotype in young individuals. Of the two mutations, R92W-TnT is more likely to benefit from anti-TGF-β signaling effects conferred by angiotensin receptor blockers and may be responsive to mitochondrial antioxidant strategies in the early stage of disease. Molecular and functional profiling may therefore serve as aids to guide precision therapy for HCM.
• Williams, M. R., Tardiff, J. C., & Schwartz, S. D. (2018). Mechanism of Cardiac Tropomyosin Transitions on Filamentous Actin As Revealed by All-Atom Steered Molecular Dynamics Simulations. The journal of physical chemistry letters, 9(12), 3301-3306.
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  The three-state model of tropomyosin (Tm) positioning along filamentous actin allows for Tm to act as a gate for myosin head binding with actin. The blocked state of Tm prevents myosin binding, while the open state allows for strong binding. Intermediate to this transition is the closed state. The details of the transition from the blocked to the closed state and then finally to the open state by Tm have not been fully accessible to experiment. Utilizing steered molecular dynamics, we investigate the work required to move the Tm strand through the extant set of proposed transitions. We find that an azimuthal motion around the actin filament by Tm is most probable in spite of increased initial energy barrier from the topographical landscape of actin.
• Coppini, R., Mazzoni, L., Ferrantini, C., Gentile, F., Pioner, J. M., Laurino, A., Santini, L., Bargelli, V., Rotellini, M., Bartolucci, G., Crocini, C., Sacconi, L., Tesi, C., Belardinelli, L., Tardiff, J., Mugelli, A., Olivotto, I., Cerbai, E., & Poggesi, C. (2017). Ranolazine Prevents Phenotype Development in a Mouse Model of Hypertrophic Cardiomyopathy. Circulation. Heart failure, 10(3).
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  Current therapies are ineffective in preventing the development of cardiac phenotype in young carriers of mutations associated with hypertrophic cardiomyopathy (HCM). Ranolazine, a late Na+ current blocker, reduced the electromechanical dysfunction of human HCM myocardium in vitro.
• Ferrantini, C., Coppini, R., Pioner, J. M., Gentile, F., Tosi, B., Mazzoni, L., Scellini, B., Piroddi, N., Laurino, A., Santini, L., Spinelli, V., Sacconi, L., De Tombe, P., Moore, R., Tardiff, J., Mugelli, A., Olivotto, I., Cerbai, E., Tesi, C., & Poggesi, C. (2017). Pathogenesis of Hypertrophic Cardiomyopathy is Mutation Rather Than Disease Specific: A Comparison of the Cardiac Troponin T E163R and R92Q Mouse Models. Journal of the American Heart Association, 6(7).
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  In cardiomyocytes from patients with hypertrophic cardiomyopathy, mechanical dysfunction and arrhythmogenicity are caused by mutation-driven changes in myofilament function combined with excitation-contraction (E-C) coupling abnormalities related to adverse remodeling. Whether myofilament or E-C coupling alterations are more relevant in disease development is unknown. Here, we aim to investigate whether the relative roles of myofilament dysfunction and E-C coupling remodeling in determining the hypertrophic cardiomyopathy phenotype are mutation specific.
• Lynn, M. L., Tal Grinspan, L., Holeman, T. A., Jimenez, J., Strom, J., & Tardiff, J. C. (2017). The structural basis of alpha-tropomyosin linked (Asp230Asn) familial dilated cardiomyopathy. Journal of molecular and cellular cardiology, 108, 127-137.
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  Recently, linkage analysis of two large unrelated multigenerational families identified a novel dilated cardiomyopathy (DCM)-linked mutation in the gene coding for alpha-tropomyosin (TPM1) resulting in the substitution of an aspartic acid for an asparagine (at residue 230). To determine how a single amino acid mutation in α-tropomyosin (Tm) can lead to a highly penetrant DCM we generated a novel transgenic mouse model carrying the D230N mutation. The resultant mouse model strongly phenocopied the early onset of cardiomyopathic remodeling observed in patients as significant systolic dysfunction was observed by 2months of age. To determine the precise cellular mechanism(s) leading to the observed cardiac pathology we examined the effect of the mutation on Ca2+ handling in isolated myocytes and myofilament activation in vitro. D230N-Tm filaments exhibited a reduced Ca2+ sensitivity of sliding velocity. This decrease in sensitivity was coupled to increase in the peak amplitude of Ca2+ transients. While significant, and consistent with other DCMs, these measurements are comprised of complex inputs and did not provide sufficient experimental resolution. We then assessed the primary structural effects of D230N-Tm. Measurements of the thermal unfolding of D230N-Tm vs WT-Tm revealed an increase in stability primarily affecting the C-terminus of the Tm coiled-coil. We conclude that the D230N-Tm mutation induces a decrease in flexibility of the C-terminus via propagation through the helical structure of the protein, thus decreasing the flexibility of the Tm overlap and impairing its ability to regulate contraction. Understanding this unique structural mechanism could provide novel targets for eventual therapeutic interventions in patients with Tm-linked cardiomyopathies.
• McConnell, M., Tal Grinspan, L., Williams, M. R., Lynn, M. L., Schwartz, B. A., Fass, O. Z., Schwartz, S. D., & Tardiff, J. C. (2017). Clinically Divergent Mutation Effects on the Structure and Function of the Human Cardiac Tropomyosin Overlap. Biochemistry, 56(26), 3403-3413.
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  The progression of genetically inherited cardiomyopathies from an altered protein structure to clinical presentation of disease is not well understood. One of the main roadblocks to mechanistic insight remains a lack of high-resolution structural information about multiprotein complexes within the cardiac sarcomere. One example is the tropomyosin (Tm) overlap region of the thin filament that is crucial for the function of the cardiac sarcomere. To address this central question, we devised coupled experimental and computational modalities to characterize the baseline function and structure of the Tm overlap, as well as the effects of mutations causing divergent patterns of ventricular remodeling on both structure and function. Because the Tm overlap contributes to the cooperativity of myofilament activation, we hypothesized that mutations that enhance the interactions between overlap proteins result in more cooperativity, and conversely, those that weaken interaction between these elements lower cooperativity. Our results suggest that the Tm overlap region is affected differentially by dilated cardiomyopathy-associated Tm D230N and hypertrophic cardiomyopathy-associated human cardiac troponin T (cTnT) R92L. The Tm D230N mutation compacts the Tm overlap region, increasing the cooperativity of the Tm filament, contributing to a dilated cardiomyopathy phenotype. The cTnT R92L mutation causes weakened interactions closer to the N-terminal end of the overlap, resulting in decreased cooperativity. These studies demonstrate that mutations with differential phenotypes exert opposite effects on the Tm-Tn overlap, and that these effects can be directly correlated to a molecular level understanding of the structure and dynamics of the component proteins.
• Tardiff, J. C. (2017). Assessing the Phenotypic Burden of Preclinical Sarcomeric Hypertrophic Cardiomyopathy-New Assessments to Guide Diagnosis and Management. JAMA cardiology, 2(4), 428-429.
• Behunin, S. M., Lopez-Pier, M. A., Mayfield, R. M., Danilo, C. A., Lipovka, Y., Birch, C., Lehman, S., Tardiff, J. C., Gregorio, C. C., & Konhilas, J. P. (2016). Liver Kinase B1 complex acts as a novel modifier of myofilament function and localizes to the Z-disk in cardiac myocytes. Archives of biochemistry and biophysics, 601, 32-41.
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  Contractile perturbations downstream of Ca(2+) binding to troponin C, the so-called sarcomere-controlled mechanisms, represent the earliest indicators of energy remodeling in the diseased heart [1]. Central to cellular energy "sensing" is the adenosine monophosphate-activated kinase (AMPK) pathway, which is known to directly target myofilament proteins and alter contractility [2-6]. We previously showed that the upstream AMPK kinase, LKB1/MO25/STRAD, impacts myofilament function independently of AMPK [5]. Therefore, we hypothesized that the LKB1 complex associated with myofilament proteins and that alterations in energy signaling modulated targeting or localization of the LKB1 complex to the myofilament. Using an integrated strategy of myofilament mechanics, immunoblot analysis, co-immunoprecipitation, mass spectroscopy, and immunofluorescence, we showed that 1) LKB1 and MO25 associated with myofibrillar proteins, 2) cellular energy stress re-distributed AMPK/LKB1 complex proteins within the sarcomere, and 3) the LKB1 complex localized to the Z-Disk and interacted with cytoskeletal and energy-regulating proteins, including vinculin and ATP Synthase (Complex V). These data represent a novel role for LKB1 complex proteins in myofilament function and myocellular "energy" sensing in the heart.
• Crocini, C., Ferrantini, C., Scardigli, M., Coppini, R., Mazzoni, L., Lazzeri, E., Pioner, J. M., Scellini, B., Guo, A., Song, L. S., Yan, P., Loew, L. M., Tardiff, J., Tesi, C., Vanzi, F., Cerbai, E., Pavone, F. S., Sacconi, L., & Poggesi, C. (2016). Novel insights on the relationship between T-tubular defects and contractile dysfunction in a mouse model of hypertrophic cardiomyopathy. Journal of molecular and cellular cardiology, 91, 42-51.
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  Abnormalities of cardiomyocyte Ca(2+) homeostasis and excitation-contraction (E-C) coupling are early events in the pathogenesis of hypertrophic cardiomyopathy (HCM) and concomitant determinants of the diastolic dysfunction and arrhythmias typical of the disease. T-tubule remodelling has been reported to occur in HCM but little is known about its role in the E-C coupling alterations of HCM. Here, the role of T-tubule remodelling in the electro-mechanical dysfunction associated to HCM is investigated in the Δ160E cTnT mouse model that expresses a clinically-relevant HCM mutation. Contractile function of intact ventricular trabeculae is assessed in Δ160E mice and wild-type siblings. As compared with wild-type, Δ160E trabeculae show prolonged kinetics of force development and relaxation, blunted force-frequency response with reduced active tension at high stimulation frequency, and increased occurrence of spontaneous contractions. Consistently, prolonged Ca(2+) transient in terms of rise and duration are also observed in Δ160E trabeculae and isolated cardiomyocytes. Confocal imaging in cells isolated from Δ160E mice reveals significant, though modest, remodelling of T-tubular architecture. A two-photon random access microscope is employed to dissect the spatio-temporal relationship between T-tubular electrical activity and local Ca(2+) release in isolated cardiomyocytes. In Δ160E cardiomyocytes, a significant number of T-tubules (>20%) fails to propagate action potentials, with consequent delay of local Ca(2+) release. At variance with wild-type, we also observe significantly increased variability of local Ca(2+) transient rise as well as higher Ca(2+)-spark frequency. Although T-tubule structural remodelling in Δ160E myocytes is modest, T-tubule functional defects determine non-homogeneous Ca(2+) release and delayed myofilament activation that significantly contribute to mechanical dysfunction.
• Jian, Z., Chen, Y. J., Shimkunas, R., Jian, Y., Jaradeh, M., Chavez, K., Chiamvimonvat, N., Tardiff, J. C., Izu, L. T., Ross, R. S., & Chen-Izu, Y. (2016). In Vivo Cannulation Methods for Cardiomyocytes Isolation from Heart Disease Models. PloS one, 11(8), e0160605.
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  Isolation of high quality cardiomyocytes is critically important for achieving successful experiments in many cellular and molecular cardiology studies. Methods for isolating cardiomyocytes from the murine heart generally are time-sensitive and experience-dependent, and often fail to produce high quality cells. Major technical difficulties can be related to the surgical procedures needed to explant the heart and to cannulate the vessel to mount onto the Langendorff system before in vitro reperfusion can begin. During this period, transient hypoxia and ischemia may damage the heart, resulting in low yield and poor quality of cells, especially for heart disease models that have fragile cells. We have developed novel in vivo cannulation methods to minimize hypoxia and ischemia, and fine-tuned the entire protocol to produce high quality ventricular myocytes. The high cell quality has been confirmed using important structural and functional criteria such as morphology, t-tubule structure, action potential morphology, Ca2+ signaling, responsiveness to beta-adrenergic agonist, and ability to have robust contraction under mechanically loaded condition. Together these assessments show the preservation of the cardiac excitation-contraction machinery in cells isolated using this technique. The in vivo cannulation method enables consistent isolation of high-quality cardiomyocytes, even from heart disease models that were notoriously difficult for cell isolation using traditional methods.
• Tardiff, J. C. (2016). The Role of Calcium/Calmodulin-Dependent Protein Kinase II Activation in Hypertrophic Cardiomyopathy. Circulation, 134(22), 1749-1751.
• Williams, M. R., Lehman, S. J., Tardiff, J. C., & Schwartz, S. D. (2016). Atomic resolution probe for allostery in the regulatory thin filament. Proceedings of the National Academy of Sciences of the United States of America, 113(12), 3257-62.
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  Calcium binding and dissociation within the cardiac thin filament (CTF) is a fundamental regulator of normal contraction and relaxation. Although the disruption of this complex, allosterically mediated process has long been implicated in human disease, the precise atomic-level mechanisms remain opaque, greatly hampering the development of novel targeted therapies. To address this question, we used a fully atomistic CTF model to test both Ca(2+) binding strength and the energy required to remove Ca(2+) from the N-lobe binding site in WT and mutant troponin complexes that have been linked to genetic cardiomyopathies. This computational approach is combined with measurements of in vitro Ca(2+) dissociation rates in fully reconstituted WT and cardiac troponin T R92L and R92W thin filaments. These human disease mutations represent known substitutions at the same residue, reside at a significant distance from the calcium binding site in cardiac troponin C, and do not affect either the binding pocket affinity or EF-hand structure of the binding domain. Both have been shown to have significantly different effects on cardiac function in vivo. We now show that these mutations independently alter the interaction between the Ca(2+) ion and cardiac troponin I subunit. This interaction is a previously unidentified mechanism, in which mutations in one protein of a complex indirectly affect a third via structural and dynamic changes in a second to yield a pathogenic change in thin filament function that results in mutation-specific disease states. We can now provide atom-level insight that is potentially highly actionable in drug design.
• Duncker, D. J., Bakkers, J., Brundel, B. J., Robbins, J., Tardiff, J. C., & Carrier, L. (2015). Animal and in silico models for the study of sarcomeric cardiomyopathies. Cardiovascular research, 105(4), 439-48.
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  Over the past decade, our understanding of cardiomyopathies has improved dramatically, due to improvements in screening and detection of gene defects in the human genome as well as a variety of novel animal models (mouse, zebrafish, and drosophila) and in silico computational models. These novel experimental tools have created a platform that is highly complementary to the naturally occurring cardiomyopathies in cats and dogs that had been available for some time. A fully integrative approach, which incorporates all these modalities, is likely required for significant steps forward in understanding the molecular underpinnings and pathogenesis of cardiomyopathies. Finally, novel technologies, including CRISPR/Cas9, which have already been proved to work in zebrafish, are currently being employed to engineer sarcomeric cardiomyopathy in larger animals, including pigs and non-human primates. In the mouse, the increased speed with which these techniques can be employed to engineer precise 'knock-in' models that previously took years to make via multiple rounds of homologous recombination-based gene targeting promises multiple and precise models of human cardiac disease for future study. Such novel genetically engineered animal models recapitulating human sarcomeric protein defects will help bridging the gap to translate therapeutic targets from small animal and in silico models to the human patient with sarcomeric cardiomyopathy.
• Gollapudi, S. K., Tardiff, J. C., & Chandra, M. (2015). The functional effect of dilated cardiomyopathy mutation (R144W) in mouse cardiac troponin T is differently affected by α- and β-myosin heavy chain isoforms. American journal of physiology. Heart and circulatory physiology, 308(8), H884-93.
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  Given the differential impact of α- and β-myosin heavy chain (MHC) isoforms on how troponin T (TnT) modulates contractile dynamics, we hypothesized that the effects of dilated cardiomyopathy (DCM) mutations in TnT would be altered differently by α- and β-MHC. We characterized dynamic contractile features of normal (α-MHC) and transgenic (β-MHC) mouse cardiac muscle fibers reconstituted with a mouse TnT analog (TnTR144W) of the human DCM R141W mutation. TnTR144W did not alter maximal tension but attenuated myofilament Ca(2+) sensitivity (pCa50) to a similar extent in α- and β-MHC fibers. TnTR144W attenuated the speed of cross-bridge (XB) distortion dynamics (c) by 24% and the speed of XB recruitment dynamics (b) by 17% in α-MHC fibers; however, both b and c remained unaltered in β-MHC fibers. Likewise, TnTR144W attenuated the rates of XB detachment (g) and tension redevelopment (ktr) only in α-MHC fibers. TnTR144W also decreased the impact of strained XBs on the recruitment of new XBs (γ) by 30% only in α-MHC fibers. Because c, b, g, ktr, and γ are strongly influenced by thin filament-based cooperative mechanisms, we conclude that the TnTR144W- and β-MHC-mediated changes in the thin filament interact to produce a less severe functional phenotype, compared with that brought about by TnTR144W and α-MHC. These observations provide a basis for lower mortality rates of humans (β-MHC) harboring the TnTR141W mutant compared with transgenic mouse studies. Our findings strongly suggest that some caution is necessary when extrapolating data from transgenic mouse studies to human hearts.
• Hill, M. G., Sekhon, M. K., Reed, K. L., Anderson, C. F., Borjon, N. D., Tardiff, J. C., & Barber, B. J. (2015). Intrauterine Treatment of a Fetus with Familial Hypertrophic Cardiomyopathy Secondary to MYH7 Mutation. Pediatric cardiology, 36(8), 1774-7.
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  There is no clear consensus on optimal management of fetuses affected by familial hypertrophic cardiomyopathy (HCM). Intrauterine treatment of the condition has not been attempted in any standardized fashion. We report the case of a fetus treated by maternal propranolol during the third trimester after septal hypertrophy and diastolic dysfunction was diagnosed on fetal echocardiogram. The pregnancy went successfully to term, and fetal septal hypertrophy was noted to improve prior to delivery.
• Sequeira, V., Najafi, A., McConnell, M., Fowler, E. D., Bollen, I. A., Wüst, R. C., dos Remedios, C., Helmes, M., White, E., Stienen, G. J., Tardiff, J., Kuster, D. W., & van der Velden, J. (2015). Synergistic role of ADP and Ca(2+) in diastolic myocardial stiffness. The Journal of physiology, 593(17), 3899-916.
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  Diastolic dysfunction in heart failure patients is evident from stiffening of the passive properties of the ventricular wall. Increased actomyosin interactions may significantly limit diastolic capacity, however, direct evidence is absent. From experiments at the cellular and whole organ level, in humans and rats, we show that actomyosin-related force development contributes significantly to high diastolic stiffness in environments where high ADP and increased diastolic [Ca(2+) ] are present, such as the failing myocardium. Our basal study provides a mechanical mechanism which may partly underlie diastolic dysfunction. Heart failure (HF) with diastolic dysfunction has been attributed to increased myocardial stiffness that limits proper filling of the ventricle. Altered cross-bridge interaction may significantly contribute to high diastolic stiffness, but this has not been shown thus far. Cross-bridge interactions are dependent on cytosolic [Ca(2+) ] and the regeneration of ATP from ADP. Depletion of myocardial energy reserve is a hallmark of HF leading to ADP accumulation and disturbed Ca(2+) handling. Here, we investigated if ADP elevation in concert with increased diastolic [Ca(2+) ] promotes diastolic cross-bridge formation and force generation and thereby increases diastolic stiffness. ADP dose-dependently increased force production in the absence of Ca(2+) in membrane-permeabilized cardiomyocytes from human hearts. Moreover, physiological levels of ADP increased actomyosin force generation in the presence of Ca(2+) both in human and rat membrane-permeabilized cardiomyocytes. Diastolic stress measured at physiological lattice spacing and 37°C in the presence of pathological levels of ADP and diastolic [Ca(2+) ] revealed a 76 ± 1% contribution of cross-bridge interaction to total diastolic stress in rat membrane-permeabilized cardiomyocytes. Inhibition of creatine kinase (CK), which increases cytosolic ADP, in enzyme-isolated intact rat cardiomyocytes impaired diastolic re-lengthening associated with diastolic Ca(2+) overload. In isolated Langendorff-perfused rat hearts, CK inhibition increased ventricular stiffness only in the presence of diastolic [Ca(2+) ]. We propose that elevations of intracellular ADP in specific types of cardiac disease, including those where myocardial energy reserve is limited, contribute to diastolic dysfunction by recruiting cross-bridges, even at low Ca(2+) , and thereby increase myocardial stiffness.
• Tardiff, J. C., Carrier, L., Bers, D. M., Poggesi, C., Ferrantini, C., Coppini, R., Maier, L. S., Ashrafian, H., Huke, S., & van der Velden, J. (2015). Targets for therapy in sarcomeric cardiomyopathies. Cardiovascular research, 105(4), 457-70.
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  To date, no compounds or interventions exist that treat or prevent sarcomeric cardiomyopathies. Established therapies currently improve the outcome, but novel therapies may be able to more fundamentally affect the disease process and course. Investigations of the pathomechanisms are generating molecular insights that can be useful for the design of novel specific drugs suitable for clinical use. As perturbations in the heart are stage-specific, proper timing of drug treatment is essential to prevent initiation and progression of cardiac disease in mutation carrier individuals. In this review, we emphasize potential novel therapies which may prevent, delay, or even reverse hypertrophic cardiomyopathy caused by sarcomeric gene mutations. These include corrections of genetic defects, altered sarcomere function, perturbations in intracellular ion homeostasis, and impaired myocardial energetics.
• van der Velden, J., Ho, C. Y., Tardiff, J. C., Olivotto, I., Knollmann, B. C., & Carrier, L. (2015). Research priorities in sarcomeric cardiomyopathies. Cardiovascular research, 105(4), 449-56.
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  The clinical variability in patients with sarcomeric cardiomyopathies is striking: a mutation causes cardiomyopathy in one individual, while the identical mutation is harmless in a family member. Moreover, the clinical phenotype varies ranging from asymmetric hypertrophy to severe dilatation of the heart. Identification of a single phenotype-associated disease mechanism would facilitate the design of targeted treatments for patient groups with different clinical phenotypes. However, evidence from both the clinic and basic knowledge of functional and structural properties of the sarcomere argues against a 'one size fits all' therapy for treatment of one clinical phenotype. Meticulous clinical and basic studies are needed to unravel the initial and progressive changes initiated by sarcomere mutations to better understand why mutations in the same gene can lead to such opposing phenotypes. Ultimately, we need to design an 'integrative physiology' approach to fully realize patient/gene-tailored therapy. Expertise within different research fields (cardiology, genetics, cellular biology, physiology, and pharmacology) must be joined to link longitudinal clinical studies with mechanistic insights obtained from molecular and functional studies in novel cardiac muscle systems. New animal models, which reflect both initial and more advanced stages of sarcomeric cardiomyopathy, will also aid in achieving these goals. Here, we discuss current priorities in clinical and preclinical investigation aimed at increasing our understanding of pathophysiological mechanisms leading from mutation to disease. Such information will provide the basis to improve risk stratification and to develop therapies to prevent/rescue cardiac dysfunction and remodelling caused by sarcomere mutations.
• Coppini, R., Ho, C. Y., Ashley, E., Day, S., Ferrantini, C., Girolami, F., Tomberli, B., Bardi, S., Torricelli, F., Cecchi, F., Mugelli, A., Poggesi, C., Tardiff, J., & Olivotto, I. (2014). Clinical phenotype and outcome of hypertrophic cardiomyopathy associated with thin-filament gene mutations. Journal of the American College of Cardiology, 64(24), 2589-2600.
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  Mild hypertrophy but increased arrhythmic risk characterizes the stereotypic phenotype proposed for hypertrophic cardiomyopathy (HCM) caused by thin-filament mutations. However, whether such clinical profile is different from more prevalent thick-filament-associated disease is unresolved.
• Jian, Z., Han, H., Zhang, T., Puglisi, J., Izu, L. T., Shaw, J. A., Onofiok, E., Erickson, J. R., Chen, Y. J., Horvath, B., Shimkunas, R., Xiao, W., Li, Y., Pan, T., Chan, J., Banyasz, T., Tardiff, J. C., Chiamvimonvat, N., Bers, D. M., , Lam, K. S., et al. (2014). Mechanochemotransduction during cardiomyocyte contraction is mediated by localized nitric oxide signaling. Science signaling, 7(317), ra27.
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  Cardiomyocytes contract against a mechanical load during each heartbeat, and excessive mechanical stress leads to heart diseases. Using a cell-in-gel system that imposes an afterload during cardiomyocyte contraction, we found that nitric oxide synthase (NOS) was involved in transducing mechanical load to alter Ca(2+) dynamics. In mouse ventricular myocytes, afterload increased the systolic Ca(2+) transient, which enhanced contractility to counter mechanical load but also caused spontaneous Ca(2+) sparks during diastole that could be arrhythmogenic. The increases in the Ca(2+) transient and sparks were attributable to increased ryanodine receptor (RyR) sensitivity because the amount of Ca2(+) in the sarcoplasmic reticulum load was unchanged. Either pharmacological inhibition or genetic deletion of nNOS (or NOS1), but not of eNOS (or NOS3), prevented afterload-induced Ca2(+) sparks. This differential effect may arise from localized NO signaling, arising from the proximity of nNOS to RyR, as determined by super-resolution imaging. Ca(2+)-calmodulin-dependent protein kinase II (CaMKII) and nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) also contributed to afterload-induced Ca(2+) sparks. Cardiomyocytes from a mouse model of familial hypertrophic cardiomyopathy exhibited enhanced mechanotransduction and frequent arrhythmogenic Ca(2+) sparks. Inhibiting nNOS and CaMKII, but not NOX2, in cardiomyocytes from this model eliminated the Ca2(+) sparks, suggesting mechanotransduction activated nNOS and CaMKII independently from NOX2. Thus, our data identify nNOS, CaMKII, and NOX2 as key mediators in mechanochemotransduction during cardiac contraction, which provides new therapeutic targets for treating mechanical stress-induced Ca(2+) dysregulation, arrhythmias, and cardiomyopathy.
• Moore, R. K., Abdullah, S., & Tardiff, J. C. (2014). Allosteric effects of cardiac troponin TNT1 mutations on actomyosin binding: a novel pathogenic mechanism for hypertrophic cardiomyopathy. Archives of biochemistry and biophysics, 552-553, 21-8.
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  The majority of hypertrophic cardiomyopathy mutations in (cTnT) occur within the alpha-helical tropomyosin binding TNT1 domain. A highly charged region at the C-terminal end of TNT1 unwinds to create a flexible "hinge". While this region has not been structurally resolved, it likely acts as an extended linker between the two cTnT functional domains. Mutations in this region cause phenotypically diverse and often severe forms of HCM. Mechanistic insight, however, has been limited by the lack of structural information. To overcome this limitation, we evaluated the effects of cTnT 160-163 mutations using regulated in vitro motility (R-IVM) assays and transgenic mouse models. R-IVM revealed that cTnT mutations Δ160E, E163R and E163K disrupted weak electrostatic actomyosin binding. Reducing the ionic strength or decreasing Brownian motion rescued function. This is the first observation of HCM-linked mutations in cTnT disrupting weak interactions between the thin filament and myosin. To evaluate the in vivo effects of altering weak actomyosin binding we generated transgenic mice expressing Δ160E and E163R mutant cTnT and observed severe cardiac remodeling and profound myofilament disarray. The functional changes observed in vitro may contribute to the structural impairment seen in vivo by destabilizing myofilament structure and acting as a constant pathophysiologic stress.
• Moore, R. K., Grinspan, L. T., Jimenez, J., Guinto, P. J., Ertz-Berger, B., & Tardiff, J. C. (2013). HCM-linked ∆160E cardiac troponin T mutation causes unique progressive structural and molecular ventricular remodeling in transgenic mice. Journal of molecular and cellular cardiology, 58, 188-98.
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  Hypertrophic cardiomyopathy (HCM) is a primary disease of the cardiac muscle, and one of the most common causes of sudden cardiac death (SCD) in young people. Many mutations in cardiac troponin T (cTnT) lead to a complex form of HCM with varying degrees of ventricular hypertrophy and ~65% of all cTnT mutations occur within or flanking the elongated N-terminal TNT1 domain. Biophysical studies have predicted that distal TNT1 mutations, including Δ160E, cause disease by a novel, yet unknown mechanism as compared to N-terminal mutations. To begin to address the specific effects of this commonly observed cTnT mutation we generated two independent transgenic mouse lines carrying variant doses of the mutant transgene. Hearts from the 30% and 70% cTnT Δ160E lines demonstrated a highly unique, dose-dependent disruption in cellular and sarcomeric architecture and a highly progressive pattern of ventricular remodeling. While adult ventricular myocytes isolated from Δ160E transgenic mice exhibited dosage-independent mechanical impairments, decreased sarcoplasmic reticulum calcium load and SERCA2a calcium uptake activity, the observed decreases in calcium transients were dosage-dependent. The latter findings were concordant with measures of calcium regulatory protein abundance and phosphorylation state. Finally, studies of whole heart physiology in the isovolumic mode demonstrated dose-dependent differences in the degree of cardiac dysfunction. We conclude that the observed clinical severity of the cTnT Δ160E mutation is caused by a combination of direct sarcomeric disruption coupled to a profound dysregulation of Ca(2+) homeostasis at the cellular level that results in a unique and highly progressive pattern of ventricular remodeling.
• Ford, S. J., Mamidi, R., Jimenez, J., Tardiff, J. C., & Chandra, M. (2012). Effects of R92 mutations in mouse cardiac troponin T are influenced by changes in myosin heavy chain isoform. Journal of molecular and cellular cardiology, 53(4), 542-51.
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  One limitation in understanding how different familial hypertrophic cardiomyopathy (FHC)-related mutations lead to divergent cardiac phenotypes is that such mutations are often studied in transgenic (TG) mouse hearts which contain a fast cycling myosin heavy chain isoform (α-MHC). However, the human heart contains a slow cycling MHC isoform (β-MHC). Given the physiological significance of MHC-troponin interplay effects on cardiac contractile function, we hypothesized that cardiac troponin T (cTnT) mutation-mediated effects on contractile function depend on the type of MHC isoform present in the sarcomere. We tested our hypothesis using two variants of cTnT containing mutations at FHC hotspot R92 (R92L or R92Q), expressed against either an α-MHC or β-MHC background in TG mouse hearts. One finding from our study was that R92L attenuated the length-dependent increase in tension and abolished the length-dependent increase in myofilament Ca(2+) sensitivity only when β-MHC was present. In addition, α- and β-MHC isoforms differentially affected how R92 mutations altered crossbridge (XB) recruitment dynamics. For example, the rate of XB recruitment was faster in R92L or R92Q fibers when β-MHC was present, but was unaffected when α-MHC was present. The R92Q mutation sped XB detachment in the presence of β-MHC, but not in the presence of α-MHC. R92Q affected the XB strain-dependent influence on XB recruitment dynamics, an effect not observed for R92L. Our findings have major implications for understanding not only the divergent effects of R92 mutations on cardiac phenotype, but also the distinct effects of MHC isoforms in determining the outcome of mutations in cTnT.
• He, H., Hoyer, K., Tao, H., Rice, R., Jimenez, J., Tardiff, J. C., & Ingwall, J. S. (2012). Myosin-driven rescue of contractile reserve and energetics in mouse hearts bearing familial hypertrophic cardiomyopathy-associated mutant troponin T is mutation-specific. The Journal of physiology, 590(21), 5371-88.
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  The thin filament protein troponin T (TnT) is a regulator of sarcomere function. Whole heart energetics and contractile reserve are compromised in transgenic mice bearing missense mutations at R92 within the tropomyosin-binding domain of cTnT, despite being distal to the ATP hydrolysis domain of myosin. These mutations are associated with familial hypertrophic cardiomyopathy (FHC). Here we test the hypothesis that genetically replacing murine αα-MyHC with murine ββ-MyHC in hearts bearing the R92Q cTnT mutation, a particularly lethal FHC-associated mutation, leads to sufficiently large perturbations in sarcomere function to rescue whole heart energetics and decrease the cost of contraction. By comparing R92Q cTnT and R92L cTnT mutant hearts, we also test whether any rescue is mutation-specific. We defined the energetic state of the isolated perfused heart using (31)P-NMR spectroscopy while simultaneously measuring contractile performance at four work states. We found that the cost of increasing contraction in intact mouse hearts with R92Q cTnT depends on the type of myosin present in the thick filament. We also found that the salutary effect of this manoeuvre is mutation-specific, demonstrating the major regulatory role of cTnT on sarcomere function at the whole heart level.
• Leinwand, L. A., Tardiff, J. C., & Gregorio, C. C. (2012). Mutations in the sensitive giant titin result in a broken heart. Circulation research, 111(2), 158-61.
• Manning, E. P., Guinto, P. J., & Tardiff, J. C. (2012). Correlation of molecular and functional effects of mutations in cardiac troponin T linked to familial hypertrophic cardiomyopathy: an integrative in silico/in vitro approach. The Journal of biological chemistry, 287(18), 14515-23.
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  Nearly 70% of all of the known cTnT mutations that cause familial hypertrophic cardiomyopathy fall within the TNT1 region that is critical to cTn-Tm binding. The high resolution structure of this domain has not been determined, and this lack of information has hindered structure-function analysis. In the current study, a coupled computational experimental approach was employed to correlate changes in cTnT dynamics to basic function using the regulated in vitro motility assay (R-IVM). An in silico approach to calculate forces in terms of a bending coordinate was used to precisely identify decreases in bending forces at residues 105 and 106 within the proposed cTnT "hinge" region. Significant functional changes were observed in multiple functional properties, including a decrease in the cooperativity of calcium activation, the calcium sensitivity of sliding speed, and maximum sliding speed. Correlation of the computational and experimental findings revealed an association between TNT1 flexibility and the cooperativity of thin filament calcium activation where an increase in flexibility led to a decrease in cooperativity. Further analysis of the primary sequence of the TNT1 region revealed a unique pattern of conserved charged TNT1 residues altered by the R92W and R92L mutations and may represent the underlying "structure" modulating this central functional domain. These data provide a framework for further integrated in silico/in vitro approaches that may be extended into a high-throughput predictive screen to overcome the current structural limitations in linking molecular phenotype to genotype in thin filament cardiomyopathies.
• Manning, E. P., Tardiff, J. C., & Schwartz, S. D. (2012). Molecular effects of familial hypertrophic cardiomyopathy-related mutations in the TNT1 domain of cTnT. Journal of molecular biology, 421(1), 54-66.
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  Familial hypertrophic cardiomyopathy (FHC) is one of the most common genetic causes of heart disease. Approximately 15% of FHC-related mutations are found in cTnT [cardiac troponin (cTn) T]. Most of the cTnT FHC-related mutations are in or flanking the N-tail TNT1 domain that directly interacts with overlapping tropomyosin (Tm). We investigate two sets of cTnT mutations at opposite ends of TNT1, mutations in residue 92 in the Tm-Tm overlap region of TNT1 and mutations in residues 160 and 163 in the C-terminal portion of TNT1 adjacent to the cTnT H1-H2 linker. Though all the mutations are located within TNT1, they have widely different phenotypes clinically and biophysically. Using a complete atomistic model of the cTn-Tm complex, we identify mechanisms by which the effects of TNT1 mutations propagate to the cTn core and site II of cTnC, where calcium binding and dissociation occurs. We find that mutations in TNT1 alter the flexibility of TNT1, which is inversely proportional to the cooperativity of calcium activation of the thin filament. Further, we identify a pathway of propagation of structural and dynamic changes from TNT1 to site II of cTnC, including TNT1, cTnT linker, I-T arm, regulatory domain of cTnI, the D-E linker of cTnC, and site II cTnC. Mutationally induced changes at site II of cTnC alter calcium coordination that corresponds to biophysical measurements of calcium sensitivity. Finally, we compare this pathway of mutational propagation with that of the calcium activation of the thin filament and find that they are identical but opposite in direction.
• Tardiff, J. C. (2012). It's never too early to look: subclinical disease in sarcomeric dilated cardiomyopathy. Circulation. Cardiovascular genetics, 5(5), 483-6.
• Jimenez, J., & Tardiff, J. C. (2011). Abnormal heart rate regulation in murine hearts with familial hypertrophic cardiomyopathy-related cardiac troponin T mutations. American journal of physiology. Heart and circulatory physiology, 300(2), H627-35.
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  Mutations in cardiac troponin T (cTnT), Δ160E and R92Q, have been linked to familial hypertrophic cardiomyopathy (FHC), and some studies have indicated that these mutations can lead to a high incidence of sudden cardiac death in the relative absence of significant ventricular hypertrophy. Alterations in autonomic function have been documented in patients with hypertrophic cardiomyopathy. We hypothesize that alterations in autonomic function may contribute to mutation-specific clinical phenotypes in cTnT-related FHC. Heart rate (HR) variability (HRV) has been used to assess autonomic function from an electrocardiograph. Nontransgenic, Δ160E, or R92Q mice were implanted with radiofrequency transmitters to obtain continuous electrocardiograph recordings during 24-h baseline and 30-min recordings after β-adrenergic receptor drug injections. Although Δ160E mice did not differ from nontransgenic mice for any 24-h HRV measurements, R92Q mice had impaired HR regulation, as measured by a decrease in the SD of the R-R interval, a decrease in the low frequency-to-high frequency ratio, a decrease in normalized low frequency, and an increase in normalized high frequency. β-Adrenergic receptor density measurements and HRV analysis after drug injections did not reveal any significant differences for Δ160E or R92Q mice versus nontransgenic mice. Arrhythmia analysis revealed both an increased incidence of heart block in R92Q mice at baseline and frequency of premature ventricular contractions after isoproterenol injections in Δ160E and R92Q mice. In addition, Δ160E and R92Q mice exhibited a prolonged P duration after drug injections. Therefore, between two independent and clinically severe cTnT mutations within the same functional domain, only R92Q mice exhibited altered autonomic function, whereas both mutations demonstrated abnormalities in conduction and ventricular ectopy.
• Manning, E. P., Tardiff, J. C., & Schwartz, S. D. (2011). A model of calcium activation of the cardiac thin filament. Biochemistry, 50(34), 7405-13.
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  The cardiac thin filament regulates actomyosin interactions through calcium-dependent alterations in the dynamics of cardiac troponin and tropomyosin. Over the past several decades, many details of the structure and function of the cardiac thin filament and its components have been elucidated. We propose a dynamic, complete model of the thin filament that encompasses known structures of cardiac troponin, tropomyosin, and actin and show that it is able to capture key experimental findings. By performing molecular dynamics simulations under two conditions, one with calcium bound and the other without calcium bound to site II of cardiac troponin C (cTnC), we found that subtle changes in structure and protein contacts within cardiac troponin resulted in sweeping changes throughout the complex that alter tropomyosin (Tm) dynamics and cardiac troponin--actin interactions. Significant calcium-dependent changes in dynamics occur throughout the cardiac troponin complex, resulting from the combination of the following: structural changes in the N-lobe of cTnC at and adjacent to sites I and II and the link between them; secondary structural changes of the cardiac troponin I (cTnI) switch peptide, of the mobile domain, and in the vicinity of residue 25 of the N-terminus; secondary structural changes in the cardiac troponin T (cTnT) linker and Tm-binding regions; and small changes in cTnC-cTnI and cTnT-Tm contacts. As a result of these changes, we observe large changes in the dynamics of the following regions: the N-lobe of cTnC, the mobile domain of cTnI, the I-T arm, the cTnT linker, and overlapping Tm. Our model demonstrates a comprehensive mechanism for calcium activation of the cardiac thin filament consistent with previous, independent experimental findings. This model provides a valuable tool for research into the normal physiology of cardiac myofilaments and a template for studying cardiac thin filament mutations that cause human cardiomyopathies.
• Tardiff, J. C. (2011). Thin filament mutations: developing an integrative approach to a complex disorder. Circulation research, 108(6), 765-82.
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  Sixteen years ago, mutations in cardiac troponin (Tn)T and α-tropomyosin were linked to familial hypertrophic cardiomyopathy, thus transforming the disorder from a disease of the β-myosin heavy chain to a disease of the cardiac sarcomere. From the outset, studies suggested that mutations in the regulatory thin filament caused a complex, heterogeneous pattern of ventricular remodeling with wide variations in clinical expression. To date, the clinical heterogeneity is well matched by an extensive array of nearly 100 independent mutations in all components of the cardiac thin filament. Significant advances in our understanding of the biophysics of myofilament activation, coupled to the emerging evidence that thin filament linked cardiomyopathies are progressive, suggests that a renewed focus on the most proximal events in both the molecular and clinical pathogenesis of the disease will be necessary to achieve the central goal of using genotype information to manage affected patients. In this review, we examine the existing biophysical and clinical evidence in support of a more proximal definition of thin filament cardiomyopathies. In addition, new high-resolution, integrated approaches are presented to help define the way forward as the field works toward developing a more robust link between genotype and phenotype in this complex disorder.
• Rice, R., Guinto, P., Dowell-Martino, C., He, H., Hoyer, K., Krenz, M., Robbins, J., Ingwall, J. S., & Tardiff, J. C. (2010). Cardiac myosin heavy chain isoform exchange alters the phenotype of cTnT-related cardiomyopathies in mouse hearts. Journal of molecular and cellular cardiology, 48(5), 979-88.
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  Familial hypertrophic cardiomyopathy, FHC, is a clinically heterogeneous, autosomal-dominant disease of the cardiac sarcomere leading to extensive remodeling at both the whole heart and molecular levels. The remodeling patterns are mutation-specific, a finding that extends to the level of single amino acid substitutions at the same peptide residue. Here we utilize two well-characterized transgenic FHC mouse models carrying independent amino acid substitutions in the TM-binding region of cardiac troponin T (cTnT) at residue 92. R92Q and R92L cTnT domains have mutation-specific average peptide conformation and dynamics sufficient to alter thin filament flexibility and cross-bridge formation and R92 mutant myocytes demonstrate mutation-specific temporal molecular remodeling of Ca(2+) kinetics and impaired cardiac contractility and relaxation. To determine if a greater economy of contraction at the crossbridge level would rescue the mechanical defects caused by the R92 cTnT mutations, we replaced the endogenous murine alpha-myosin heavy chain (MyHC) with the beta-MyHC isoform. While beta-MyHC replacement rescued the systolic dysfunction in R92Q mice, it failed to rescue the defects in diastolic function common to FHC-associated R92 mutations. Surprisingly, a significant component of the whole heart and molecular contractile improvement in the R92Q mice was due to improvements in Ca(2+) homeostasis including SR uptake, [Ca2+](i) amplitude and phospholamban phosphorylation. Our data demonstrate that while genetically altering the myosin composition of the heart bearing a thin filament FHC mutation is sufficient to improve contractility, diastolic performance is refractory despite improved Ca(2+) kinetics. These data reveal a previously unrecognized role for MyHC isoforms with respect to Ca(2+) homeostasis in the setting of cardiomyopathic remodeling and demonstrate the overall dominance of the thin filament mutation in determining the degree of diastolic impairment at the myofilament level.
• Tardiff, J. C. (2010). Tropomyosin and dilated cardiomyopathy: revenge of the actinomyosin "gatekeeper". Journal of the American College of Cardiology, 55(4), 330-2.
• Guinto, P. J., Haim, T. E., Dowell-Martino, C. C., Sibinga, N., & Tardiff, J. C. (2009). Temporal and mutation-specific alterations in Ca2+ homeostasis differentially determine the progression of cTnT-related cardiomyopathies in murine models. American journal of physiology. Heart and circulatory physiology, 297(2), H614-26.
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  Naturally occurring mutations in cardiac troponin T (cTnT) result in a clinical subset of familial hypertrophic cardiomyopathy. To determine the mechanistic links between thin-filament mutations and cardiovascular phenotypes, we have generated and characterized several transgenic mouse models carrying cTnT mutations. We address two central questions regarding the previously observed changes in myocellular mechanics and Ca(2+) homeostasis: 1) are they characteristic of all severe cTnT mutations, and 2) are they primary (early) or secondary (late) components of the myocellular response? Adult left ventricular myocytes were isolated from 2- and 6-mo-old transgenic mice carrying missense mutations at residue 92, flanking the TNT1 NH(2)-terminal tail domain. Results from R92L and R92W myocytes showed mutation-specific alterations in contraction and relaxation indexes at 2 mo with improvements by 6 mo. Alterations in Ca(2+) kinetics remained consistent with mechanical data in which R92L and R92W exhibited severe diastolic impairments at the early time point that improved with increasing age. A normal regulation of Ca(2+) kinetics in the context of an altered baseline cTnI phosphorylation suggested a pathogenic mechanism at the myofilament level taking precedence for R92L. The quantitation of Ca(2+)-handling proteins in R92W mice revealed a synergistic compensatory mechanism involving an increased Ser16 and Thr17 phosphorylation of phospholamban, contributing to the temporal onset of improved cellular mechanics and Ca(2+) homeostasis. Therefore, independent cTnT mutations in the TNT1 domain result in primary mutation-specific effects and a differential temporal onset of altered myocellular mechanics, Ca(2+) kinetics, and Ca(2+) homeostasis, complex mechanisms which may contribute to the clinical variability in cTnT-related familial hypertrophic cardiomyopathy mutations.
• Guinto, P. J., Manning, E. P., Schwartz, S. D., & Tardiff, J. C. (2007). Computational Characterization of Mutations in Cardiac Troponin T Known to Cause Familial Hypertrophic Cardiomyopathy. Journal of theoretical & computational chemistry, 6(3), 413.
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  Cardiac Troponin T (cTnT) is a central modulator of thin filament regulation of myofilament activation. The lack of structural data for the TNT1 tail domain, a proposed α-helical region, makes the functional implications of the FHC mutations difficult to determine. Studies have suggested that flexibility of TNT1 is important in normal protein-protein interactions within the thin filament. Our groups have previously shown through Molecular Dynamics (MD) simulations that some FHC mutations, Arg92Leu(R92L) and Arg92Trp(R92W), result in increased flexibility at a critical hinge region 12 residues distant from the mutation. To explain this distant effect and its implications for FHC mutations, we characterized the dynamics of wild type and mutational segments of cTnT using MD. Our data shows an opening of the helix between residues 105-110 in mutants. Consequently, the dihedral angles of these residues correspond to non-α-helical regions on Ramachandran plots. We hypothesize the removal of a charged residue decreases electrostatic repulsion between the point mutation and surrounding residues resulting in local helical compaction. Constrained ends of the helix and localized compaction results in expansion within the nearest non-polar helical turn from the mutation site, residues 105-109.
• Haim, T. E., Dowell, C., Diamanti, T., Scheuer, J., & Tardiff, J. C. (2007). Independent FHC-related cardiac troponin T mutations exhibit specific alterations in myocellular contractility and calcium kinetics. Journal of molecular and cellular cardiology, 42(6), 1098-110.
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  Mutations in cardiac troponin T (cTnT) are linked to a severe form of Familial Hypertrophic Cardiomyopathy. Patients carrying mutations flanking the tropomyosin-binding domain of cTnT (R92L and Delta160E) develop distinct clinical syndromes. In order to better understand the cellular pathophysiology underlying these clinically relevant differences, we studied isolated adult left ventricular myocytes from independent transgenic cTnT mouse lines carrying either a 35% (Delta160E) or 50% (R92L) replacement of the endogenous cTnT with the mutant forms. Measurement of baseline myocellular contraction revealed that the Delta160E cells had significant decreases in the peak rate of contraction and percent shortening as compared to either R92L or Non-TG myocytes. In addition, while both Delta160E and R92L myocytes demonstrated a decrease in the peak rate of relaxation as compared to Non-TG, the magnitude of the difference was significantly greater in Delta160E cells. Concurrent myocyte [Ca2+](i) transient measurements revealed that while the alterations in the peak rates and times of the rise and decline of the [Ca2+](i) transient were similar to the changes in the respective measures of sarcomeric mechanics, R92L cells also exhibited reduced rates of the rise and decline of the [Ca2+](i) transient but did not exhibit these reductions in terms of sarcomeric mechanics. Of note, only Delta160E, and not R92L myocytes, demonstrated significant reductions in SR Ca2+ load and uptake, corresponding to the impairments seen in the [Ca2+](i) and mechanical transients. Finally, Western analysis revealed a significant Delta160E-specific reduction in the SERCA2a/PLB ratio, which may well underlie the observed alterations in Ca2+ homeostasis. Therefore, independent cTnT mutations result in significant mutation-specific effects in Ca2+ handling that may, in part, contribute to the observed clinical variability in cTnT-related FHC.
• He, H., Javadpour, M. M., Latif, F., Tardiff, J. C., & Ingwall, J. S. (2007). R-92L and R-92W mutations in cardiac troponin T lead to distinct energetic phenotypes in intact mouse hearts. Biophysical journal, 93(5), 1834-44.
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  It is now known that the flexibility of the troponin T (TnT) tail determines thin filament conformation and hence cross-bridge cycling properties, expanding the classic structural role of TnT to a dynamic role regulating sarcomere function. Here, using transgenic mice bearing R-92W and R-92L missense mutations in cardiac TnT known to alter the flexibility of the TnT tropomyosin-binding domain, we found mutation-specific differences in the cost of contraction at the whole heart level. Compared to age- and gender-matched sibling hearts, mutant hearts demonstrate greater ATP utilization measured using (31)P NMR spectroscopy as decreases in [ATP] and [PCr] and |DeltaG(~ATP)| at all workloads and profound systolic and diastolic dysfunction at all energetic states. R-92W hearts showed more severe energetic abnormalities and greater contractile dysfunction than R-92L hearts. The cost of increasing contraction was abnormally high when [Ca(2+)] was used to increase work in mutant hearts but was normalized with supply of the beta-adrenergic agonist dobutamine. These results show that R-92L and R-92W mutations in the TM-binding domain of cardiac TnT alter thin filament structure and flexibility sufficiently to cause severe defects in both whole heart energetics and contractile performance, and that the magnitude of these changes is mutation specific.
• Tardiff, J. C. (2006). Cardiac hypertrophy: stressing out the heart. The Journal of clinical investigation, 116(6), 1467-70.
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  The question of what differentiates physiological from pathological cardiac hypertrophy remains one of the most clinically relevant questions in basic cardiovascular research. The answer(s) to this question will have far-ranging importance in the fight against hypertrophic heart disease and failure. In this issue of the JCI, Perrino et al. have used a unique model system to mimic the pathophysiologic effects of an intermittent pressure overload on the heart--in effect, to examine the basic issue of what determines an in vivo pathogenic stimulus (see the related article beginning on page 1547). Their findings clearly show that it is the nature of the inciting stimulus, as opposed to chronicity, that establishes the initial pathogenic response and that a distinct disruption of the beta-adrenergic system is centrally involved in the earliest alterations of myocellular physiology. These results suggest both a new paradigm for treatment options in hypertrophic cardiac disease and novel methodologies for further studies.
• Chandra, M., Tschirgi, M. L., & Tardiff, J. C. (2005). Increase in tension-dependent ATP consumption induced by cardiac troponin T mutation. American journal of physiology. Heart and circulatory physiology, 289(5), H2112-9.
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  How different mutations in cardiac troponin T (cTnT) lead to distinct secondary downstream cellular remodeling in familial hypertrophic cardiomyopathy (FHC) remains elusive. To explore the molecular basis for the distinct impact of different mutations in cTnT on cardiac myocytes, we studied mechanical activity of detergent-skinned muscle fiber bundles from different lines of transgenic (TG) mouse hearts that express wild-type cTnT (WTTG), R92W cTnT, R92L cTnT, and Delta-160 cTnT (deletion of amino acid 160). The amount of mutant cTnT is approximately 50% of the total myocellular cTnT in both R92W and R92L TG mouse hearts and approximately 35% in Delta-160 TG mouse hearts. Myofilament Ca2+ sensitivity was enhanced in all mutant cTnT TG cardiac muscle fibers. Compared with the WTTG fibers, Ca2+ sensitivity increased significantly at short sarcomere length (SL) of 1.9 microm (P < 0.001) in R92W TG fibers by 2.2-fold, in R92L by 2.0-fold, and in Delta-160 by 1.3-fold. At long SL of 2.3 microm, Ca2+ sensitivity increased significantly (P < 0.01) in a similar manner (R92W, 2.5-fold; R92L, 1.9-fold; Delta-160, 1.3-fold). Ca2+-activated maximal tension remained unaltered in all TG muscle fibers. However, tension-dependent ATP consumption increased significantly in Delta-160 TG muscle fibers at both short SL (23%, P < 0.005) and long SL (37%, P < 0.0001), suggesting a mutation-induced change in cross-bridge detachment rate constant. Chronic stresses on relative cellular ATP level in cardiac myocytes may cause a strain on energy-dependent Ca2+ homeostatic mechanisms. This may result in pathological remodeling that we observed in Delta-160 TG cardiac myocytes where the ratio of sarco(endo)plasmic reticulum Ca2+-ATPase 2/phospholamban decreased significantly. Our results suggest that different types of stresses imposed on cardiac myocytes would trigger distinct cellular signaling, which leads to remodeling that may be unique to some mutants.
• Ertz-Berger, B. R., He, H., Dowell, C., Factor, S. M., Haim, T. E., Nunez, S., Schwartz, S. D., Ingwall, J. S., & Tardiff, J. C. (2005). Changes in the chemical and dynamic properties of cardiac troponin T cause discrete cardiomyopathies in transgenic mice. Proceedings of the National Academy of Sciences of the United States of America, 102(50), 18219-24.
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  Cardiac troponin T (cTnT) is a central component of the regulatory thin filament. Mutations in cTnT have been linked to severe forms of familial hypertrophic cardiomyopathy. A mutational "hotspot" that leads to distinct clinical phenotypes has been identified at codon 92. Although the basic functional and structural roles of cTnT in modulating contractility are relatively well understood, the mechanisms that link point mutations in cTnT to the development of this complex cardiomyopathy are unknown. To address this question, we have taken a highly interdisciplinary approach by first determining the effects of the residue 92 mutations on the molecular flexibility and stability of cTnT by means of molecular dynamics simulations. To test whether the predicted alterations in thin filament structure could lead to distinct cardiomyopathies in vivo, we developed transgenic mouse models expressing either the Arg-92-Trp or Arg-92-Leu cTnT proteins in the heart. Characterization of these models at the cellular and whole-heart levels has revealed mutation-specific early alterations in transcriptional activation that result in distinct pathways of ventricular remodeling and contractile performance. Thus, our computational and experimental results show that changes in thin filament structure caused by single amino acid substitutions lead to differences in the biophysical properties of cTnT and alter disease pathogenesis.
• Tardiff, J. C. (2005). Sarcomeric proteins and familial hypertrophic cardiomyopathy: linking mutations in structural proteins to complex cardiovascular phenotypes. Heart failure reviews, 10(3), 237-48.
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  Hypertrophic Cardiomyopathy (HCM) is a relatively common primary cardiac disorder defined as the presence of a hypertrophied left ventricle in the absence of any other diagnosed etiology. HCM is the most common cause of sudden cardiac death in young people which often occurs without precedent symptoms. The overall clinical phenotype of patients with HCM is broad, ranging from a complete lack of cardiovascular symptoms to exertional dyspnea, chest pain, and sudden death, often due to arrhythmias. To date, 270 independent mutations in nine sarcomeric protein genes have been linked to Familial Hypertrophic Cardiomyopathy (FHC), thus the clinical variability is matched by significant genetic heterogeneity. While the final clinical phenotype in patients with FHC is a result of multiple factors including modifier genes, environmental influences and genotype, initial screening studies had suggested that individual gene mutations could be linked to specific prognoses. Given that the sarcomeric genes linked to FHC encode proteins with known functions, a vast array of biochemical, biophysical and physiologic experimental approaches have been applied to elucidate the molecular mechanisms that underlie the pathogenesis of this complex cardiovascular disorder. In this review, to illustrate the basic relationship between protein dysfunction and disease pathogenesis we focus on representative gene mutations from each of the major structural components of the cardiac sarcomere: the thick filament (beta MyHC), the thin filament (cTnT and Tm) and associated proteins (MyBP-C). The results of these studies will lead to a better understanding of FHC and eventually identify targets for therapeutic intervention.
• Tardiff, J. C. (2004). Myosin at the heart of the problem. The New England journal of medicine, 351(5), 424-6.
• Javadpour, M. M., Tardiff, J. C., Pinz, I., & Ingwall, J. S. (2003). Decreased energetics in murine hearts bearing the R92Q mutation in cardiac troponin T. The Journal of clinical investigation, 112(5), 768-75.
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  The thin filament protein cardiac troponin T (cTnT) is an important regulator of myofilament activation. Here we report a significant change in cardiac energetics in transgenic mice bearing the missense mutation R92Q within the tropomyosin-binding domain of cTnT, a mutation associated with a clinically severe form of familial hypertrophic cardiomyopathy. This functional domain of cTnT has recently been shown to be a crucial modulator of contractile function despite the fact that it does not directly interact with the ATP hydrolysis site in the myosin head. Simultaneous measurements of cardiac energetics using 31P NMR spectroscopy and contractile performance of the intact beating heart revealed both a decrease in the free energy of ATP hydrolysis available to support contractile work and a marked inability to increase contractile performance upon acute inotropic challenge in hearts from R92Q mice. These results show that alterations in thin filament protein structure and function can lead to significant defects in myocardial energetics and contractile reserve.
• Chandra, M., Rundell, V. L., Tardiff, J. C., Leinwand, L. A., De Tombe, P. P., & Solaro, R. J. (2001). Ca(2+) activation of myofilaments from transgenic mouse hearts expressing R92Q mutant cardiac troponin T. American journal of physiology. Heart and circulatory physiology, 280(2), H705-13.
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  The functional consequences of the R92Q mutation in cardiac troponin T (cTnT), linked to familial hypertrophic cardiomyopathy in humans, are not well understood. We have studied steady- and pre-steady-state mechanical activity of detergent-skinned fiber bundles from a transgenic (TG) mouse model in which 67% of the total cTnT in the heart was replaced by the R92Q mutant cTnT. TG fibers were more sensitive to Ca(2+) than nontransgenic (NTG) fibers [negative logarithm of half maximally activating molar Ca(2+) (pCa(50)) = 5.84 +/- 0.01 and 6.12 +/- 0.01 for NTG and TG fibers, respectively]. The shift in pCa(50) caused by increasing the sarcomere length from 1.9 to 2.3 microm was significantly higher for TG than for NTG fibers (DeltapCa(50) = 0.13 +/- 0.01 and 0.29 +/- 0.02 for NTG and TG fibers, respectively). The relationships between rate of ATP consumption and steady-state isometric tension were linear, and the slopes were the same in NTG and TG fibers. Rate of tension redevelopment was more sensitive to Ca(2+) in TG than in NTG fibers (pCa(50) = 5.71 +/- 0.02 and 6.07 +/- 0.02 for NTG and TG fibers, respectively). We concluded that overall cross-bridge cycling kinetics are not altered by the R92Q mutation but that altered troponin-tropomyosin interactions could be responsible for the increase in myofilament Ca(2+) sensitivity in TG myofilaments.
• Montgomery, D. E., Tardiff, J. C., & Chandra, M. (2001). Cardiac troponin T mutations: correlation between the type of mutation and the nature of myofilament dysfunction in transgenic mice. The Journal of physiology, 536(Pt 2), 583-92.
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  1. The heterogenic nature of familial hypertrophic cardiomyopathy (FHC) in humans suggests a link between the type of mutation and the nature of patho-physiological alterations in cardiac myocytes. Exactly how FHC-associated mutations in cardiac troponin T (cTnT) lead to impaired cardiac function is unclear. 2. We measured steady-state isometric force and ATPase activity in detergent-skinned cardiac fibre bundles from three transgenic (TG) mouse hearts in which 50, 92 and 6 % of the native cTnT was replaced by the wild type (WT) cTnT, R92Q mutant cTnT (R92Q) and the C-terminal deletion mutant of cTnT (cTnT(DEL)), respectively. 3. Normalized pCa-tension relationships of R92Q and cTnT(DEL) fibres demonstrated a significant increase in sensitivity to Ca2+ at short (2.0 microm) and long (2.3 microm) sarcomere lengths (SL). At short SL, the pCa50 values, representing the midpoint of the pCa-tension relationship, were 5.69 +/- 0.01, 5.96 +/- 0.01 and 5.81 +/- 0.01 for WT, R92Q and cTnT(DEL) fibres, respectively. At long SL, the pCa50 values were 5.81 +/- 0.01, 6.08 +/- 0.01 and 5.95 +/- 0.01 for WT, R92Q and cTnT(DEL) fibres, respectively. 4. The difference in pCa required for half-maximal activation (DeltapCa50) at short and long SL was 0.12 +/- 0.01 for the R92Q (92 %) TG fibres, which is significantly less than the previously reported DeltapCa50 value of 0.29 +/- 0.02 for R92Q (67 %) TG fibres. 5. At short SL, Ca2+-activated maximal tension in both R92Q and cTnT(DEL) fibres decreased significantly (24 and 21 %, respectively; P < 0.005), with no corresponding decrease in Ca2+-activated maximal ATPase activity. Therefore, at short SL, the tension cost in R92Q and cTnT(DEL) fibres increased by 35 and 29 %, respectively (P < 0.001). 6. The fibre bundles reconstituted with the recombinant mutant cTnT(DEL) protein developed only 37 % of the Ca2+-activated maximal force developed by recombinant WT cTnT reconstituted fibre bundles, with no apparent changes in Ca2+ sensitivity. 7. Our data indicate that an important mutation-linked effect on cardiac function is the result of an inefficient use of ATP at the myofilament level. Furthermore, the extent of the mutation-induced dysfunction depends not only on the nature of the mutation, but also on the concentration of the mutant protein in the sarcomere.
• Tardiff, J. C., Hewett, T. E., Factor, S. M., Vikstrom, K. L., Robbins, J., & Leinwand, L. A. (2000). Expression of the beta (slow)-isoform of MHC in the adult mouse heart causes dominant-negative functional effects. American journal of physiology. Heart and circulatory physiology, 278(2), H412-9.
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  Alpha- and beta-myosin heavy chain (MHC), the two MHC isoforms expressed in the mammalian heart, differ quantitatively in their enzymatic activities. The MHC composition of the heart can change dramatically in response to numerous stimuli, leading to the hypothesis that changes in cardiac function can be caused by myosin isoform shifts. However, this hypothesis has remained unproven because the stimuli used to generate these shifts are complex and accompanied by many additional physiological changes, including alterations in cardiac mass and geometry. Adult mouse ventricles normally express only alpha-MHC (the faster motor). To determine whether genetic alteration of the MHC isoform composition in the adult mouse heart would result in changes in cardiac chamber mass and contractility, we established transgenic mouse lines that express a Myc-tagged beta-MHC molecule (the slower motor) in adult ventricular tissue, one of which expresses 12% of its myosin as the transgene. There is no evidence of hypertrophy, induction of hypertrophic markers, and no histopathology. Myofibrillar Ca(2+)-activated ATPase activity is decreased by 23%, and Langendorff preparations demonstrate a significant 15% decrease in systolic function in transgenic hearts. These results suggest that even small shifts in the myosin isoform composition of the myocardium can result in physiologically significant changes in cardiac contractility and could be relevant to cardiovascular disease.
• Tardiff, J. C., Hewett, T. E., Palmer, B. M., Olsson, C., Factor, S. M., Moore, R. L., Robbins, J., & Leinwand, L. A. (1999). Cardiac troponin T mutations result in allele-specific phenotypes in a mouse model for hypertrophic cardiomyopathy. The Journal of clinical investigation, 104(4), 469-81.
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  Multiple mutations in cardiac troponin T (cTnT) can cause familial hypertrophic cardiomyopathy (FHC). Patients with cTnT mutations generally exhibit mild or no ventricular hypertrophy, yet demonstrate a high frequency of early sudden death. To understand the functional basis of these phenotypes, we created transgenic mouse lines expressing 30%, 67%, and 92% of their total cTnT as a missense (R92Q) allele analogous to one found in FHC. Similar to a mouse FHC model expressing a truncated cTnT protein, the left ventricles of all R92Q lines are smaller than those of wild-type. In striking contrast to truncation mice, however, the R92Q hearts demonstrate significant induction of atrial natriuretic factor and beta-myosin heavy chain transcripts, interstitial fibrosis, and mitochondrial pathology. Isolated cardiac myocytes from R92Q mice have increased basal sarcomeric activation, impaired relaxation, and shorter sarcomere lengths. Isolated working heart data are consistent, showing hypercontractility and diastolic dysfunction, both of which are common findings in patients with FHC. These mice represent the first disease model to exhibit hypercontractility, as well as a unique model system for exploring the cellular pathogenesis of FHC. The distinct phenotypes of mice with different TnT alleles suggest that the clinical heterogeneity of FHC is at least partially due to allele-specific mechanisms.
• Tardiff, J. C., Factor, S. M., Tompkins, B. D., Hewett, T. E., Palmer, B. M., Moore, R. L., Schwartz, S., Robbins, J., & Leinwand, L. A. (1998). A truncated cardiac troponin T molecule in transgenic mice suggests multiple cellular mechanisms for familial hypertrophic cardiomyopathy. The Journal of clinical investigation, 101(12), 2800-11.
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  Mutations in multiple cardiac sarcomeric proteins including myosin heavy chain (MyHC) and cardiac troponin T (cTnT) cause a dominant genetic heart disease, familial hypertrophic cardiomyopathy (FHC). Patients with mutations in these two genes have quite distinct clinical characteristics. Those with MyHC mutations demonstrate more significant and uniform cardiac hypertrophy and a variable frequency of sudden death. Patients with cTnT mutations generally exhibit mild or no hypertrophy, but a high frequency of sudden death at an early age. To understand the basis for these distinctions and to study the pathogenesis of the disease, we have created transgenic mice expressing a truncated mouse cTnT allele analogous to one found in FHC patients. Mice expressing truncated cTnT at low (< 5%) levels develop cardiomyopathy and their hearts are significantly smaller (18-27%) than wild type. These animals also exhibit significant diastolic dysfunction and milder systolic dysfunction. Animals that express higher levels of transgene protein die within 24 h of birth. Transgenic mouse hearts demonstrate myocellular disarray and have a reduced number of cardiac myocytes that are smaller in size. These studies suggest that multiple cellular mechanisms result in the human disease, which is generally characterized by mild hypertrophy, but, also, frequent sudden death.
• Tardiff, J., & Krauter, K. S. (1998). Divergent expression of alpha1-protease inhibitor genes in mouse and human. Nucleic acids research, 26(16), 3794-9.
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  The alpha1-protease inhibitor proteins of laboratory mice are homologous in sequence and function to human alpha1-antitrypsin and are encoded by a highly conserved multigene family comprised of five members. In humans, the inhibitor is expressed in liver and in macrophages and decreased expression or inhibitory activity is associated with a deficiency syndrome which can result in emphysema and liver disease in affected individuals. It has been proposed that macrophage expression may be an important component of the function of human alpha1-antitrypsin. Clearly, it is desirable to develop a mouse model of this deficiency syndrome, however, efforts to do this have been largely unsuccessful. In this paper, we report that aside from the issues of potentially redundant gene function, the mouse may not be a suitable animal for such studies, because there is no significant expression of murine alpha1-protease inhibitor in the macrophages of mice. This difference between the species appears to result from an absence of a functional macrophage-specific promoter in mice.
• Montgomery, K. T., Tardiff, J., Reid, L. M., & Krauter, K. S. (1990). Negative and positive cis-acting elements control the expression of murine alpha 1-protease inhibitor genes. Molecular and cellular biology, 10(6), 2625-37.
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  The alpha 1-protease inhibitor (alpha 1-PI) proteins of mice are encoded by a group of genes whose members are expressed coordinately in a liver-abundant pattern and are regulated primarily at the transcriptional level. To better understand the developmental and tissue-specific regulation of this gene family, one member that is analogous to the human alpha 1-antitrypsin gene was chosen for study. Deletional analysis of the upstream regulatory region of this gene was performed, spanning from -10 kilobases to -80 base pairs relative to the transcriptional start site. Two functional positive cis-acting elements within the 522 bases immediately upstream of the start site for transcription were shown to modulate the level of expression from this promoter when introduced into human or mouse hepatoma cells, and a third region acted as a negative regulatory element in that its deletion resulted in a two- to sixfold increase of expression of a transfected minigene construct. Sequence comparison between the regulatory domains of two mouse alpha 1-PI genes and the human alpha 1-antitrypsin gene showed that the mouse gene contains a novel positive cis-acting element which is absent in human gene and that a specific eight-base-pair difference between species results in a strong positive cis-acting element in the human gene acting as a negative element in the mouse gene. An enhancer located approximately 3,000 base pairs upstream of the major start site for transcription was also identified. This element is position and orientation independent. Several different DNA-protein binding assays were used to demonstrate that each DNA segment with functional significance in transfection assays interacts specifically with proteins found in adult mouse liver nuclei. The major positive-acting element appeared to be specifically recognized by nuclear proteins found only in tissues that express alpha 1-PI, while the negative element binding proteins were ubiquitous. Thus, the distal regulatory domain including bases -3500 to -133 of this murine alpha 1-PI gene family member is more complex than was previously demonstrated. It is composed of a set of at least three additional functional cis-acting regulatory elements besides those which have been mapped by others and has a far upstream enhancer.
• Kiss, I., Beaton, A. H., Tardiff, J., Fristrom, D., & Fristrom, J. W. (1988). Interactions and developmental effects of mutations in the Broad-Complex of Drosophila melanogaster. Genetics, 118(2), 247-59.
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  The 2B5 region on the X chromosome of Drosophila melanogaster forms an early ecdysone puff at the end of the third larval instar. The region contains a complex genetic locus, the Broad-Complex (BR-C) composed of four groups of fully complementing (br, rbp, l(1)2Bc, and l(1)2Bd) alleles, and classes of noncomplementing (npr 1) and partially noncomplementing l(1)2Bab alleles. BR-C mutants prevent metamorphosis, including the morphogenesis of imaginal discs. Results are presented that indicate that the BR-C contains two major functional domains. One, the br domain is primarily, if not exclusively, involved in the elongation and eversion of appendages by imaginal discs. The second, the l(1)2Bc domain, is primarily involved in the fusion of discs to form a continuous adult epidermis. Nonetheless, the two domains may encode products with related functions because in some situations mutants in both domains appear to affect similar developmental processes.
• Kraft, R., Tardiff, J., Krauter, K. S., & Leinwand, L. A. (1988). Using mini-prep plasmid DNA for sequencing double stranded templates with Sequenase. BioTechniques, 6(6), 544-6, 549.

Presentations

• Tardiff, J. C. (2020, October). Navigating the Transition: Mid Career Challenges and Strategies. International Society for Heart Research: North American Session Meeting. Virtual: International Society for Heart Research.
• Tardiff, J. C. (2019, April 25). Community Management of Hypertrophic Cardiomyopathy in 2019: Challenges and the Need for New Therapeutics. Novartis HCM Advisory Board Meeting. Boston, MA: Novartis.
• Tardiff, J. C. (2019, April 6). The Role of Allostery in the Pathogenesis of Hypertrophic Cardiomyopathy: New Mechanisms and Targets. UCLA Cardiovascular Theme: Distinguished Seminar Series. Los Angeles, CA: UCLA College of Medicine, Cardiology Theme.
• Tardiff, J. C. (2019, Jan 10). Hypertrophic Cardiomyopathy in 2019: Embrace the Complexity. Cedars-Sinai: Cardiology Grand Rounds. Cedars Sinai Medical Center, Los Angeles: Cedars Sinai Medical Center, Los Angeles.
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  Cardiovascular Ground Rounds (Clinical talk)
• Tardiff, J. C. (2019, Jan 10). The Role of Allostery in the Pathogenesis of Hypertrophic Cardiomyopathy: New Mechanisms and Targets. Smidt Heart Institute Research Seminar. Cedars Sinai Medical Center, Los Angeles: Smidt Heart Institute.
• Tardiff, J. C. (2019, June 15). Fetal cTnT Acts as an Intrinsic Modifier of Thin Filament Structure, Dynamics and Function in Health and Disease. International Society for Heart Research: World Congress, Beijing. Beijing, China: International Society for Heart Research.
• Tardiff, J. C. (2019, March 13). Hypertrophic Cardiomyopathy in 2019: Embrace the Complexity. Vanderbilt Department of Medicine: Cardiology Grand Rounds. Nashville TN: Vanderbiilt University College of Medicine.
• Tardiff, J. C. (2019, March 23). Hypertrophic Cardiomyopathy in 2019: Embrace the Complexity. Novartis Invited Research Seminar. Boston, MA: Novartis.
• Tardiff, J. C. (2019, March 30). Hypertropic Cardiomyopathy: Treatment in 2019. 2019 2019 Cardiology Symposium: Cardiology in the Desert. Phoenix, AZ: Banner University Medical Center Heart Institute.
• Tardiff, J. C. (2019, Nov 17). Myofilaments in Hypertrophic Cardiomyopathy. American Heart Association Scientific Sessions. Philadelphia, PA: American Heart Association.
• Tardiff, J. C. (2019, Nov 22). Intrinsic Modulators of Thin Filament Function: The Potential for Novel Targeted Agents. Cytokinetics Research Seminar. South San Francisco, CA: Cytokinetics.
• Tardiff, J. C. (2019, Sept 19). An Integrative Approach to Sarcomeric Cardiomyopathies. Gunnar Lecture: UIC CVRC Research Day. Chicago, IL: UIC Cardiovascular Research Center.
• Tardiff, J. C. (2019, Sept 8). Keynote: Walking a Thin Line: Intrinsic and Extrinsic Modulators of Cardiac Thin Filament Function in Health and Disease. European Muscle Conference Annual Meeting. Kent, Great Britain: European Muscle Society.
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  Meeting Keynote Presentation
• Tardiff, J. C. (2018, April). The Role of Allostery in the Pathogenesis of Hypertrophic Cardiomyopathy: Redefining our Approach to a Complex Disorder. • The Ohio State University School of Medicine, Invited T32 Lecturer.
• Tardiff, J. C. (2018, Fall). Developing a Fully Integrative Approach to Define the Molecular Pathogenesis of Hypertrophic Cardiomyopathy. Case Western Reserve University School of Medicine, Department of Physiology and Biophysics, Invited Speaker. Cleveland, OH: Case Western Reserve University School of Medicine.
• Tardiff, J. C. (2018, Fall). Linking Genotype to Phenotype in Sarcomeric Cardiomyopathies: Are we there yet?. Tufts Medical School Cardiovascular Research Institute Retreat, Distinguished Professor Presentation. Boston, MA: Tufts Medical School.
• Tardiff, J. C. (2018, Fall). The Next Frontier for HCM, Can we Prevent Disease?. American Heart Association Scientific Sessions, Session on State of the Art in Hypertrophic Cardiomyopathy. Chicago, IL: American Heart Association.
• Tardiff, J. C. (2018, Fall). The Role of Allostery and Dynamics in the Pathogenesis of Sarcomeric Cardiomyopathies: New Mechanisms and Targets. Washington University in St. Louis School of Medicine, Department of Biochemistry and Molecular Biophysics, Invited Speaker. St. Louis, MO: Washington University in St. Louis School of Medicine.
• Tardiff, J. C. (2018, June). Incorporating flexibility and dynamics as triggers for cardiomyopathic remodeling in hypertrophic cardiomyopathy. International Society for Heart Research - North American Conference. New London, Canada.
• Tardiff, J. C. (2018, March). “The Role of Allostery in the Pathogenesis of Hypertrophic Cardiomyopathy: Redefining the Approach to A Complex Disorder”. Texas A&M University, Institute of Biosciences & Technology Distinguished Lecture.
• Tardiff, J. C. (2018, May). Incorporating Flexibility and Dynamics as Triggers for Cardiomyopathic Remodeling in Hypertrophic Cardiomyopathy. Biannual Myofilament Meetings. Madison, WI.
• Tardiff, J. C. (2018, May). The Role of Allostery in Sarcomeric Cardiomyopathies. 1st Olympiad in Cardiovascular Medicine. Athens, Greece: University of Athens.
• Tardiff, J. C. (2018, SUmmer). The Role of Allostery in the Pathogenesis of Sarcomeric Cardiomyopathies: New Mechanisms and Targets. Annual AHA Basic Cardiovascular Sciences (BCVS) Meeting. San Antonio, TX: American Heart Association.
• Tardiff, J. C. (2017, December). An Integrative Approach to Hypertrophic Cardiomyopathy. Cardiology Grand Rounds. Seattle: University of Washington College of Medicine, Department of Medicine, Cardiology Division.
• Tardiff, J. C. (2017, December). An Integrative Approach to Sarcomeric Cardiomyopathies. Cardiology Grand Rounds, University of Illinois at Chicago. Chicago: University of Illinois at Chicago College of Medicine, Department of Medicine, Cardiology Division.
• Tardiff, J. C. (2017, December). The Role of Allostery in the Pathogenesis of Sarcomeric Cardiomyopathies: Redefining the Approach to a Complex Disorder. University of Washington Visiting Professorship - Cardiovascular T32. Seattle: University of Washington School of Medicine.
• Tardiff, J. C. (2016, April). From Computational Biophysics to Animal Models: Developing a New Paradigm and Eventual Targeted Treatments for Sarcomeric Cardiomyopathies. Invited Talk. Chicago, IL: Northwestern University Medical School.
• Tardiff, J. C. (2016, June). Integrative Approaches to Linking Genotype to Phenotype in Sarcomeric Cardiomyopathies: Are we there yet?. Biennial Myofilament Meeting - Plenary. Madison, Wisconsin: University of Wisconsin at Madison.
• Tardiff, J. C. (2016, June). Session on Myofilament Proteins in Disease. Cardiac Regulatory Mechanisms Gordon Research Conference. New London, NH: Gordon Research Conferences.
• Tardiff, J. C. (2016, March). From Computational Biophysics to Animal Models: Developing a New Paradigm and Eventual Targeted Treatments for Sarcomeric Cardiomyopathies. Invited talk at Novartis Institutes for BioMedical Research. Boston, MA: Novartis.
• Tardiff, J. C. (2016, Summer). Complex Genetic Cardiomyopathies: from Biophysical Triggers to LV Remodeling. Invited talk. Birmingham, AL: University of Alabama at Birmingham School of Medicine.
• Tardiff, J. C. (2015, Feb). An Integrative Approach to Thin Filament Cardiomyopathies: From Molecular and Computational Biophysics to Mice. Biophysical Society Meeting. Baltimore, MD.
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  Plenary Talk
• Tardiff, J. C. (2015, February). From Computational Biophysics to Animal Models: Forging an Integrative Approach to Sarcomeric Cardiomyopathies. Cardiology Ground Rounds. Johns Hopkins Medical School.
• Tardiff, J. C. (2015, June). Cardiac Thin Filament Models: High Resolution Tools for Studying Genetic Cardiomyopathies. International Society for Heart Research. Seattle, Washington: University of Washington, Seattle.
• Tardiff, J. C. (2015, June). Integrative Approaches to Sarcomeric Cardiomyopathies: From Computation to Animal Models. Minnesota Muscle Symposium. Minneapolis, Minnesota: University of Minnesota- Wellstone Muscular Dystrophy Center.
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  Invited Plenary Speaker
• Tardiff, J. C. (2015, March). Is Hypertrophic Cardiomyopathy Really a "Straightforward" Monogeneic Disorder?. Genetics Ground Rounds. U of A Tucson.
• Tardiff, J. C. (2015, November). From Computational Biophysics to Animal Models: An Integrative Approach to Sarcomeric Cardiomyopathies. Cardiology Research Grand Rounds. Boston, MA: Harvard Medical School, Beth-Israel Deaconess.
• Tardiff, J. C. (2015, October). Dissecting sarcomere function and dysfunction: Calcium Regulation in Hypertrophic Cardiomyopathy. III Florence International Symposium on Advances in Cardiomyopathies. Florence, Italy: University of Florence, European Society of Cardiology.
• Tardiff, J. C. (2015, Sept). Integrative Approaches to Genetic Cardiomyopathies: From Computation to Animal Models. Research Seminar - Department of Biomedical Engineering. New Haven, CT: Yale University.
• Whitaker, M. E., Nai, V., Gupta, A., Sweitzer, N. K., Khalpey, Z. I., Tardiff, J. C., Granzier, H. L., Sotak, S., Sprissler, R. S., & Desai, A. (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).