Jil C Tardiff

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Books

• Tardiff, J. C., & Solaro, R. J. (2013). Biophysics of the failing heart. Springer New York. doi:10.1007/978-1-4614-7678-8
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  Biophysics of the failing heart : , Biophysics of the failing heart : , کتابخانه دیجیتال جندی شاپور اهواز

Chapters

• Simon, J. N., Tardiff, J. C., & Wolska, B. M. (2013). Sarcomeres and the Biophysics of Heart Failure. In Biophysics of the Failing Heart. Springer, New York, NY. doi:10.1007/978-1-4614-7678-8_11
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  Changes in the function of the sarcomere play a significant role in the development of cardiac dysfunction underlying heart failure. These changes in sarcomeric properties are the result of either alterations in isoform expression, post-translational modification of the sarcomeric proteins, or gene mutations linked to hypertrophic or dilated cardiomyopathy. These alterations act to modulate the contractile state of the heart via direct effects on the biophysical properties of the cardiac sarcomere. Coupling a deeper understanding of the primary biophysical causes of changes in contractile function to a more complete understanding of the resultant pathogenic ventricular remodeling that occurs over time will allow for both significant advances in disease management and new points of therapeutic intervention.
• Tardiff, J. C., & Solaro, R. J. (2013). Introduction to the Biophysics of the Failing Heart. In Biophysics of the Failing Heart. Springer, New York, NY. doi:10.1007/978-1-4614-7678-8_1
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  Heart failure is a primary, progressive disorder of cardiac muscle that leads to remodeling of the ventricles. While there has been much focus on the end-stage ventricular phenotype from the clinical standpoint, a better understanding of the molecular mechanisms provides significant insight regarding pathways involved in the preclinical states (the so-called Class I-II HF) where novel interventions may be targeted. Thus chapters in this book emphasize many of the biophysical components of the ventricle and working cardiac myocytes that contribute to molecular mechanisms determining its structure and function. While topics covered in each of the chapters may provide insights into the primary causes of ventricular remodeling, it is important to take into account that many of these processes (metabolic, cytoskeletal, sarcomere, and channel remodeling) are set in motion as a response to a primary stimulus and thus can be compensatory in the primary preclinical stages of disease. As illuminated in the book, it is common that the primary stimulus is biophysical in nature involving altered mechanics and stress–strain properties, for example. The transition from compensatory to pathogenic (or unregulated response) is likely to drive a significant component of the loss of function that defines the later stages of heart failure. Many of these components of cardiovascular structure and function work in concert. Yet in the early stages only subsets of the molecular framework are activated with end-stage disease representing the so-called common endpoint where all compensatory mechanisms have been exhausted. Understanding the relative role and contribution of each of these components/pathways (coupled to their specific pathogenic stimuli) in the early preclinical stages of ventricular remodeling provides both novel biomarkers and indicates new specific avenues of therapeutic intervention with a goal of altering the natural history of the disease process. As described in this book, many of the approaches and basic tools are in-hand to accomplish this understanding.

Journals/Publications

• Day, S. M., Tardiff, J. C., & Ostap, E. M. (2022). Myosin modulators: emerging approaches for the treatment of cardiomyopathies and heart failure. The Journal of clinical investigation, 132(5).
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  Myosin modulators are a novel class of pharmaceutical agents that are being developed to treat patients with a range of cardiomyopathies. The therapeutic goal of these drugs is to target cardiac myosins directly to modulate contractility and cardiac power output to alleviate symptoms that lead to heart failure and arrhythmias, without altering calcium signaling. In this Review, we discuss two classes of drugs that have been developed to either activate (omecamtiv mecarbil) or inhibit (mavacamten) cardiac contractility by binding to β-cardiac myosin (MYH7). We discuss progress in understanding the mechanisms by which the drugs alter myosin mechanochemistry, and we provide an appraisal of the results from clinical trials of these drugs, with consideration for the importance of disease heterogeneity and genetic etiology for predicting treatment benefit.
• Keyt, L. K., Duran, J. M., Bui, Q. M., Chen, C., Miyamoto, M. I., Silva Enciso, J., Tardiff, J. C., & Adler, E. D. (2022). Thin filament cardiomyopathies: A review of genetics, disease mechanisms, and emerging therapeutics. Frontiers in cardiovascular medicine, 9, 972301.
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  All muscle contraction occurs due to the cyclical interaction between sarcomeric thin and thick filament proteins within the myocyte. The thin filament consists of the proteins actin, tropomyosin, Troponin C, Troponin I, and Troponin T. Mutations in these proteins can result in various forms of cardiomyopathy, including hypertrophic, restrictive, and dilated phenotypes and account for as many as 30% of all cases of inherited cardiomyopathy. There is significant evidence that thin filament mutations contribute to dysregulation of Ca within the sarcomere and may have a distinct pathomechanism of disease from cardiomyopathy associated with thick filament mutations. A number of distinct clinical findings appear to be correlated with thin-filament mutations: greater degrees of restrictive cardiomyopathy and relatively less left ventricular (LV) hypertrophy and LV outflow tract obstruction than that seen with thick filament mutations, increased morbidity associated with heart failure, increased arrhythmia burden and potentially higher mortality. Most therapies that improve outcomes in heart failure blunt the neurohormonal pathways involved in cardiac remodeling, while most therapies for hypertrophic cardiomyopathy involve use of negative inotropes to reduce LV hypertrophy or septal reduction therapies to reduce LV outflow tract obstruction. None of these therapies directly address the underlying sarcomeric dysfunction associated with thin-filament mutations. With mounting evidence that thin filament cardiomyopathies occur through a distinct mechanism, there is need for therapies targeting the unique, underlying mechanisms tailored for each patient depending on a given mutation.
• Maack, C., & Tardiff, J. C. (2022). Targeted therapies for cardiac diseases. Nature reviews. Cardiology, 19(6), 343-344.
• Mason, A. B., Tardiff, J. C., & Schwartz, S. D. (2022). Free-Energy Surfaces of Two Cardiac Thin Filament Conformational Changes during Muscle Contraction. The journal of physical chemistry. B, 126(21), 3844-3851.
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  The troponin core is an important regulatory complex in cardiac sarcomeres. Contraction is initiated by a calcium ion binding to cardiac troponin C (cTnC), initiating a conformational shift within the protein, altering its interactions with cardiac troponin I (cTnI). The change in cTnC-cTnI interactions prompts the C-terminal domain of cTnI to dissociate from actin, allowing tropomyosin to reveal myosin-binding sites on actin. Each of the concerted movements in the cardiac thin filament (CTF) is crucial for allowing the contraction of cardiomyocytes, yet little is known about the free energy associated with each transition, which is vital for understanding contraction on a molecular level. Using metadynamics, we calculated the free-energy surface of two transitions in the CTF: cTnC opening in the presence and absence of Ca and cTnI dissociating from actin with both open and closed cTnC. These results not only provide the free-energy surface of the transitions but will also be shown to determine if the order of transitions in the contraction cycle is important. From our calculations, we found that the calcium ion helps stabilize the open conformation of cTnC and that the C-terminus of cTnI is stabilized by cTnC in the open conformation when dissociating from the actin surface.
• Pioner, J. M., Vitale, G., Gentile, F., Scellini, B., Piroddi, N., Cerbai, E., Olivotto, I., Tardiff, J., Coppini, R., Tesi, C., Poggesi, C., & Ferrantini, C. (2022). Genotype-Driven Pathogenesis of Atrial Fibrillation in Hypertrophic Cardiomyopathy: The Case of Different Mutations. Frontiers in physiology, 13, 864547.
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  Atrial dilation and atrial fibrillation (AF) are common in Hypertrophic CardioMyopathy (HCM) patients and associated with a worsening of prognosis. The pathogenesis of atrial myopathy in HCM remains poorly investigated and no specific association with genotype has been identified. By re-analysis of our cohort of thin-filament HCM patients (Coppini et al. 2014) AF was identified in 10% of patients with sporadic mutations in the cardiac Troponin T gene (), while AF occurrence was much higher (25-75%) in patients carrying specific "hot-spot" mutations. To determine the molecular basis of arrhythmia occurrence, two HCM mouse models expressing human variants (a "hot-spot" one, R92Q, and a "sporadic" one, E163R) were selected according to the different pathophysiological pathways previously demonstrated in ventricular tissue. Echocardiography studies showed a significant left atrial dilation in both models, but more pronounced in the R92Q. In E163R atrial trabeculae, in line with what previously observed in ventricular preparations, the energy cost of tension generation was markedly increased. However, no changes of twitch amplitude and kinetics were observed, and there was no atrial arrhythmic propensity. R92Q atrial trabeculae, instead, displayed normal ATP consumption but markedly increased myofilament calcium sensitivity, as previously observed in ventricular preparations. This was associated with reduced inotropic reserve and slower kinetics of twitch contractions and, importantly, with an increased occurrence of spontaneous beats and triggered contractions that represent an intrinsic arrhythmogenic mechanism promoting AF. The association of specific mutations with AF occurrence depends on the mutation-driven pathomechanism (i.e., increased atrial myofilament calcium sensitivity rather than increased myofilament tension cost) and may influence the individual response to treatment.
• Tardiff, J. C., Argirò, A., Ho, C., Day, S. M., van der Velden, J., Cerbai, E., Saberi, S., Lakdawala, N. K., & Olivotto, I. (2022). Sex‐Related Differences in Genetic Cardiomyopathies. Journal of the American Heart Association, 11(9). doi:10.1161/jaha.121.024947
• Tardiff, J. C., Chakraborti, A., & Schwartz, S. D. (2022). Insights into the Mechanism of the Cardiac Drug Omecamtiv Mecarbil─A Computational Study. The Journal of Physical Chemistry B, 126(48), 10069-10082. doi:10.1021/acs.jpcb.2c06679
• Tardiff, J. C., Schwartz, S. D., Baldo, A. P., Deranek, A. E., & Lynn, M. L. (2022). Structure and Dynamics of the Flexible Cardiac Troponin T Linker Domain in a Fully Reconstituted Thin Filament. Biochemistry, 61(13), 1229-1242. doi:10.1021/acs.biochem.2c00091
• Baldo, A. P., Tardiff, J. C., & Schwartz, S. D. (2021). A Proposed Mechanism for the Initial Myosin Binding Event on the Cardiac Thin Filament: A Metadynamics Study. The journal of physical chemistry letters, 12(14), 3509-3513.
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  The movement of tropomyosin over filamentous actin regulates the cross-bridge cycle of the thick with thin filament of cardiac muscle by blocking and revealing myosin binding sites. Tropomyosin exists in three, distinct equilibrium states with one state blocking myosin-actin interactions (blocked position) and the remaining two allowing for weak (closed position) and strong myosin binding (open position). However, experimental information illuminating how myosin binds to the thin filament and influences tropomyosin's transition across the actin surface is lacking. Using metadynamics, we mimic the effect of a single myosin head binding by determining the work required to pull small segments of tropomyosin toward the open position in several distinct regions of the thin filament. We find differences in required work due to the influence of cardiac troponin T lead to preferential binding sites and determine the mechanism of further myosin head recruitment.
• Baldo, A., Klass, M. M., Tardiff, J. C., Schwartz, S. D., Lynn, M. L., & Castillo, R. (2021). Calcium Dissociation of HCM Causative R21C Troponin I Mutation. Biophysical Journal, 120(3). doi:10.1016/j.bpj.2020.11.985
• Baldo, A., Tardiff, J. C., Schwartz, S. D., & Deranek, A. E. (2021). Mapping the Cardiac Troponin T Linker Region to Actin and Determining Pathogenic Point Mutational Effects. Biophysical Journal, 120(3). doi:10.1016/j.bpj.2020.11.1627
• Baldo, A., Tardiff, J. C., Schwartz, S. D., Deranek, A. E., & Baldo, A. (2021). Mapping the Cardiac Troponin T Linker Region to Actin and Determining Pathogenic Point Mutational Effects. Biophysical Journal, 120(3), 250a. doi:10.1016/j.bpj.2020.11.1627
• Chakraborti, A., Baldo, A. P., Tardiff, J. C., & Schwartz, S. D. (2021). Investigation of the Recovery Stroke and ATP Hydrolysis and Changes Caused Due to the Cardiomyopathic Point Mutations in Human Cardiac β Myosin. The journal of physical chemistry. B, 125(24), 6513-6521.
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  Human cardiac β myosin undergoes the cross-bridge cycle as part of the force-generating mechanism of cardiac muscle. The recovery stroke is considered one of the key steps of the kinetic cycle as it is the conformational rearrangement required to position the active site residues for hydrolysis of ATP and interaction with actin. We explored the free-energy surface of the transition and investigated the effect of the genetic cardiomyopathy causing mutations R453C, I457T, and I467T on this step using metadynamics. This work extends previous studies on myosin II with engineered mutations. Here, like previously, we generated an unbiased thermodynamic ensemble of reactive trajectories for the chemical step using transition path sampling. Our methodologies were able to predict the changes to the dynamics of the recovery stroke as well as predict the pathway of breakdown of ATP to ADP and HPO with the stabilization of the metaphosphate intermediate. We also observed clear differences between the myosin II and human cardiac β myosin for ATP hydrolysis as well as predict the effect of the mutation I467T on the chemical step.
• 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.
• Klass, M. M., Tardiff, J. C., Schwartz, S. D., Lynn, M. L., Klass, M. M., Castillo, R., & Baldo, A. (2021). Calcium Dissociation of HCM Causative R21C Troponin I Mutation. Biophysical Journal, 120(3), 128a-129a. doi:10.1016/j.bpj.2020.11.985
• Lehman, S. J., Tardiff, J. C., Klass, M. M., Kanassatega, R., Davis, J. P., & Colson, B. A. (2021). Exploring the Effects of Mutations and Thick Filament Proteins on Myofilament Calcium Kinetics Via Stopped-Flow. Biophysical Journal, 120(3), 54a-55a. doi:10.1016/j.bpj.2020.11.566
• Lehman, S. J., Tardiff, J. C., Klass, M. M., Kanassatega, R., Davis, J. P., & Colson, B. A. (2021). Exploring the Effects of Mutations and Thick Filament Proteins on Myofilament Calcium Kinetics Via Stopped-Flow. Biophysical Journal, 120(3). doi:10.1016/j.bpj.2020.11.566
• Mason, A. B., Lynn, M. L., Baldo, A. P., Deranek, A. E., Tardiff, J. C., & Schwartz, S. D. (2021). Computational and biophysical determination of pathogenicity of variants of unknown significance in cardiac thin filament. JCI insight, 6(23).
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  Point mutations within sarcomeric proteins have been associated with altered function and cardiomyopathy development. Difficulties remain, however, in establishing the pathogenic potential of individual mutations, often limiting the use of genotype in management of affected families. To directly address this challenge, we utilized our all-atom computational model of the human full cardiac thin filament (CTF) to predict how sequence substitutions in CTF proteins might affect structure and dynamics on an atomistic level. Utilizing molecular dynamics calculations, we simulated 21 well-defined genetic pathogenic cardiac troponin T and tropomyosin variants to establish a baseline of pathogenic changes induced in computational observables. Computational results were verified via differential scanning calorimetry on a subset of variants to develop an experimental correlation. Calculations were performed on 9 independent variants of unknown significance (VUS), and results were compared with pathogenic variants to identify high-resolution pathogenic signatures. Results for VUS were compared with the baseline set to determine induced structural and dynamic changes, and potential variant reclassifications were proposed. This unbiased, high-resolution computational methodology can provide unique structural and dynamic information that can be incorporated into existing analyses to facilitate classification both for de novo variants and those where established approaches have provided conflicting information.
• Mattiazzi, A., Tardiff, J. C., & Kranias, E. G. (2021). Stress Seats a New Guest at the Table of PLN/SERCA and Their Partners. Circulation research, 128(4), 471-473.
• Schwartz, S. D., Tardiff, J. C., & Baldo, A. P. (2021). A Proposed Mechanism for the Initial Myosin Binding Event on the Cardiac Thin Filament: A Metadynamics Study. The Journal of Physical Chemistry Letters, 12(14), 3509-3513. doi:10.1021/acs.jpclett.1c00223
• Schwartz, S. D., Tardiff, J. C., Baldo, A. P., & Chakraborti, A. (2021). Investigation of the Recovery Stroke and ATP Hydrolysis and Changes Caused Due to the Cardiomyopathic Point Mutations in Human Cardiac β Myosin. The Journal of Physical Chemistry B, 125(24), 6513-6521. doi:10.1021/acs.jpcb.1c03144
• Tardiff, J. C., & Greenberg, M. J. (2021). Complexity in genetic cardiomyopathies and new approaches for mechanism-based precision medicine. Journal of General Physiology, 153(3). doi:10.1085/jgp.202012662
• Tardiff, J. C., Mattiazzi, A., & Kranias, E. G. (2021). Stress Seats a New Guest at the Table of PLN/SERCA and Their Partners. Circulation Research, 128(4), 471-473. doi:10.1161/circresaha.121.318742
• Baldo, A. P., Tardiff, J. C., & Schwartz, S. D. (2020). Computational Evaluation of Point Mutation Perturbations to the Recovery Stroke of Dictyostelium Myosin II with Metadynamics. Biophysical Journal, 118(3), 436a. doi:10.1016/j.bpj.2019.11.2446
• 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.
• Baldo, A. P., Tardiff, J. C., Smith, A. B., & Schwartz, S. D. (2020). Classification of Genetic Cardiac Mutations Using Computational Chemistry. Biophysical Journal, 118(3), 426a-427a. doi:10.1016/j.bpj.2019.11.2398
• Baldo, A., Chakraborti, A., Tardiff, J. C., & Schwartz, S. D. (2020). Utilization of Transition Path Sampling to Perform Dynamically Unbiased Simulations of ATP Hydrolysis in Two Isoforms of Myosin II. Biophysical Journal, 118(3), 437a. doi:10.1016/j.bpj.2019.11.2450
• Baldo, A., Vasquez, C., Tardiff, J. C., Schwartz, S. D., & Deranek, A. E. (2020). Defining the Flexible Cardiac Troponin T Linker Region in Relationship to Actin and Determining Effects of Pathogenic Point Mutations. Biophysical Journal, 118(3), 423a-424a. doi:10.1016/j.bpj.2019.11.2385
• Chakraborti, A., Tardiff, J. C., & Schwartz, S. D. (2020). Computational Study of the Effect of Point Mutations Perturbing the Recovery Stroke of Human Cardiac Beta-Myosin using Metadynamics. Biophysical Journal, 118(3), 435a-436a. doi:10.1016/j.bpj.2019.11.2444
• 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.
• Klass, M. M., Lehman, S. J., Tardiff, J. C., Heffernon, G., Hauck, G. T., & Davis, J. P. (2020). Stopped-Flow Calcium Kinetics of Hypertrophic Cardiomyopathy-Associated Troponin T Mutations. Biophysical Journal, 118(3), 327a. doi:10.1016/j.bpj.2019.11.1834
• 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.
• Schwartz, S. D., Tardiff, J. C., & Baldo, A. P. (2020). Mechanochemical Function of Myosin II: Investigation into the Recovery Stroke and ATP Hydrolysis. The Journal of Physical Chemistry B, 124(45), 10014-10023. doi:10.1021/acs.jpcb.0c05762
• Tardiff, J. C., Sadayappan, S., & Wold, L. E. (2020). Basic Cardiovascular Sciences Scientific Sessions 2020: Emerging Opportunities in Cardiovascular Diseases. Circulation Research, 127(11), 1459-1467. doi:10.1161/circresaha.120.318257
• Tardiff, J. C., Schwartz, S. D., & Munusamy, E. (2020). A Molecular Dynamics Study of Small Molecules Bound to a Full Atomistic Model of Cardiac Thin Filament as a Method to Identify Possible Treatments for Genetic Cardiomyopathies. Biophysical Journal, 118(3), 326a. doi:10.1016/j.bpj.2019.11.1830
• Tudor, J., Tardiff, J. C., Regnier, M., Moussavi-harami, F., Kooiker, K. B., Freeman, J., & Branley, C. E. (2020). Two Myofilament-Based Approaches to Prevent Genetic Dilated Cardiomyopathy. Biophysical Journal, 118(3), 594a. doi:10.1016/j.bpj.2019.11.3216
• Vasquez, C., Tardiff, J. C., Lynn, M. L., Jin, J., Holeman, T. A., & Grinspan, L. T. (2020). Intrinsic Modifier Effect of cTnT Isoform Switching in Sarcomeric Cardiomyopathies. Biophysical Journal, 118(3), 427a. doi:10.1016/j.bpj.2019.11.2400
• Wolska, B. M., Warren, C. M., Tardiff, J. C., Solaro, R. J., Marszalek, R., Halas, M., Chowdhury, S. A., & Batra, A. (2020). CORONARY INSUFFICIENCY UNDERLIES FIBROTIC HCM PROGRESSION. Journal of the American College of Cardiology, 75(11), 1045. doi:10.1016/s0735-1097(20)31672-7
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  Despite the extensive clinical characterization of hypertrophic cardiomyopathy (HCM), the pathobiology remains unclear. Our efforts aim at deducing the relevant pathological processes using a transgenic mouse harboring a common human troponin T mutation (R92Q) in FVB/n background. We hypothesized
• Yob, J., Tardiff, J. C., Szczesniak, K., Szczesniak, D. L., Herrera, J. J., Goddard, R., Day, S. M., Yob, J., Tardiff, J. C., Szczesniak, K., Szczesniak, D. L., Herrera, J. J., Goddard, R., & Day, S. M. (2020). Cardiac Remodeling Following High Intensity Exercise Training In A Preclinical HCM Mouse Model: 597 Board #2 May 27 1:00 PM - 3:00 PM. Medicine and Science in Sports and Exercise, 52(7S), 151-151. doi:10.1249/01.mss.0000675108.57577.74
• 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 .
• Baldo, A., Tardiff, J. C., Schwartz, S. J., & Deranek, A. E. (2019). Computational and Experimental Investigation of Cardiac Troponin T R173Q, R173W and Δ160E Mutation Specific Correlates to Disease. Biophysical Journal, 116(3), 265a. doi:10.1016/j.bpj.2018.11.1438
• 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.
• Tardiff, J. C., Regnier, M., Powers, J. D., Moussavi-harami, F., Flint, G. V., & Davis, J. (2019). Predicting and Preventing Myocardial Remodeling in a Murine Model of Dilated Cardiomyopathy. Biophysical Journal, 116(3), 264a-265a. doi:10.1016/j.bpj.2018.11.1436
• Tardiff, J. C., Regnier, M., Powers, J. D., Moussavi-harami, F., Kooiker, K. B., & Davis, J. (2019). Engineered Thin Filament Mutation to Increase Calcium Sensitivity of Force in Tropomyosin Mutation of Dilated Cardiomyopathy. Biophysical Journal, 116(3), 115a-116a. doi:10.1016/j.bpj.2018.11.651
• Tardiff, J. C., Richards, A. M., Lynn, M. L., & Desai, S. H. (2019). Multi-Timepoint RNA-Sequencing Reveals Differential Gene Expression of Transgenic Mouse Models of Hypertrophic and Dilated Cardiomyopathies. Biophysical Journal, 116(3), 266a. doi:10.1016/j.bpj.2018.11.1441
• Tardiff, J. C., Tal-grinspan, L., Lynn, M. L., Holeman, T. A., & Deranek, A. E. (2019). The Mechanistic Role of Tropomyosin Overlap Dysregulation in Early Cardiomyopathic Disease Progression. Biophysical Journal, 116(3), 261a-262a. doi:10.1016/j.bpj.2018.11.1423
• Yob, J., Tardiff, J. C., Szczesniak, K., Szczesniak, D. L., Herrera, J. J., & Day, S. M. (2019). Translationally Designed HIIT Protocol Improves Peak VO2 In A Preclinical HCM Model Without Adverse Events: 2405 Board #69 May 31 9:30 AM - 11:00 AM. Medicine and Science in Sports and Exercise, 51(6S), 658-659. doi:10.1249/01.mss.0000562467.94535.ee
• Baldo, A., Williams, M. R., Tardiff, J. C., Schwartz, S. D., Mcconnell, M. T., Lynn, M. L., & Deranek, A. E. (2018). Computational and Experimental Investigation of Tropomyosin D230N and S215l Mutation Specific Correlates to Disease. Biophysical Journal, 114(3), 499a. doi:10.1016/j.bpj.2017.11.2731
• Baldo, A., Williams, M., Tardiff, J. C., Schwartz, S. D., Lynn, M. L., Deranek, A. E., & Abdullah, S. (2018). Computational and Experimental Studies of Divergent Clinical Effects in Proximate Thin Filament Mutations. Biophysical Journal, 114(3), 568a-569a. doi:10.1016/j.bpj.2017.11.3110
• Klass, M. M., Lehman, S. J., & Tardiff, J. C. (2018). Stopped-Flow Calcium Association Kinetics of Hypertrophic Cardiomyopathy Associated Troponin T Mutations. Biophysical Journal, 114(3), 501a-502a. doi:10.1016/j.bpj.2017.11.2742
• 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.
• Schwartz, S. D., Tardiff, J. C., & Williams, M. R. (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. doi:10.1021/acs.jpclett.8b00958
• Tardiff, J. C., Lynn, M. L., & Holeman, T. A. (2018). Unique Structural and Functional Effects of Alpha-Tropomyosin Mutations in HCM and DCM. Biophysical Journal, 114(3), 498a. doi:10.1016/j.bpj.2017.11.2725
• Tardiff, J. C., Regnier, M., Powers, J. D., Moussavi-harami, F., & Davis, J. (2018). Engineered Thin Filament Mutations to Study the Sarcomere Length Dependence of Cardiac Muscle Contractility. Biophysical Journal, 114(3), 313a-314a. doi:10.1016/j.bpj.2017.11.1769
• Tardiff, J. C., Regnier, M., Razumova, M. V., Moussavi-harami, F., Klaiman, J. M., & Cheng, Y. (2018). Abstract 439: Elevating 2-deoxy Adenosine Triphosphate Improves Contraction in a Mouse Model of Genetic Dilated Cardiomyopathy. Circulation Research, 123(Suppl_1). doi:10.1161/res.123.suppl_1.439
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  Several studies have identified reduced tension and Ca 2+ sensitivity as key regulators that precipitate dilated cardiomyopathy (DCM). We are developing a novel therapy to increase tension using adeno-associated viral (AAV) vectors that increase intracellular 2 deoxy-ATP (dATP) via overexpression of the enzyme ribonucleotide reductase (RNR). We have reported that dATP substitution for ATP increases the magnitude and rate of contraction and that RNR overexpression increases intracellular dATP and enhances cardiac function. Here we use a multiscale approach to test the effect of dATP in a genetic model of dilated cardiomyopathy (DCM) with a missense mutation (D230N) in alpha-tropomyosin (Tm). Using recombinant proteins in the in vitro motility assay, we found that filament velocity was lower with D230N compared to WT Tm (2.03±0.04 vs. 2.44±0.04 μm/s). This deficit was corrected when dATP was substituted for ATP (2.90±0.04 μm/s). Next, we used transgenic D230N mice that show progressive ventricular dilation and systolic dysfunction. Intact and demembranated trabeculae from these mice have decreased tension and calcium sensitivity of force, and loss of length-dependent activation (LDA) compared to WT mice. Substitution of dATP for ATP, increased Ca 2+ sensitivity in D230N Tm myocardium (pCa 50 =5.48±0.02 vs. 5.57±0.02) and restored LDA. Complete replacement of ATP with dATP resulted in an ~70% increase in force at submaximal [Ca 2+ ] and force at submaximal Ca 2+ was significantly increased by replacement of only 5% of the nucleotide pool with dATP. For isolated myofibrils from D230N Tm and WT mice, tension at submaximal Ca 2+ (pCa=5.6) was lower in D230N mice compared to WT mice (51±4 vs. 75±8 mN/mm 2 ) and this deficit was improved with dATP (68±5 mN/mm 2 ). Similarly, the rate of activation in D230N myofibrils was slower than in WT mice (2.1±0.2 vs.3.3±0.2 s -1 ) and partially corrected with dATP (2.7±0.2 s -1 ). These results demonstrate that enhanced cross bridge binding, via increasing dATP, can recover lost contractile function in a thin filament mutation model of DCM. In ongoing studies, we are testing the ability of elevated dATP, via AAV- RNR, to protect against reduced systolic function and progressive ventricular dilation in 2-3 week old D230N mice .
• Tardiff, J. C., Tal-grinspan, L., Mcconnell, M. T., Lynn, M. L., Holeman, T. A., & Benitez, G. E. (2018). The Role of cTnT Isoform Switching in Modulating Sarcomeric Cardiomyopathies. Biophysical Journal, 114(3), 497a. doi:10.1016/j.bpj.2017.11.2721
• 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.
• Wolska, B. M., Tardiff, J. C., Solaro, R. J., Simon, J. N., Ryba, D. M., Kranias, E. G., Chowdhury, S. A., & Begum, N. (2018). Long-term rescue of a familial hypertrophic cardiomyopathy caused by a mutation in troponin T, via reduced expression of phospholamban. Journal of Molecular and Cellular Cardiology, 124, 115-116. doi:10.1016/j.yjmcc.2018.07.097
• Yob, J., Tardiff, J. C., Szczesniak, K., Louzon, S., Herrera, J. J., & Day, S. M. (2018). Impaired Exercise Capacity In cTnT-delta160E Mice Validates Pre-clinical Model To Assess Exercise Interventions For HCM: 3402 Board # 271 June 2 9 30 AM - 11 00 AM. Medicine and Science in Sports and Exercise, 50(5S), 848-849. doi:10.1249/01.mss.0000538794.71399.c8
• 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.
• Lehman, S. J., Tardiff, J. C., Grinspan, L. T., & Anderson, M. E. (2017). Differential CaMK-II Activation in the Progression of HCM in cTnT Mutations. Biophysical Journal, 112(3), 2-6. doi:10.1016/j.bpj.2016.11.1396
• 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. doi:10.1001/jamacardio.2016.5677
• 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.
• Tardiff, J. C., Lehman, S., Grinspan, L., & Anderson, M. E. (2017). Abstract 383: Modulating Calcium Dysregulation in Troponin T Linked Hypertrophic Cardiomyopathy. Circulation Research, 121(suppl_1). doi:10.1161/res.121.suppl_1.383
• Tardiff, J. C., Lynn, M. L., Jin, J. P., Holeman, T. A., & Grinspan, L. T. (2017). cTnT isoform Switching in the Development of Early Childhood Tropomyosin-Linked DCM. Biophysical Journal, 112(3), 256a. doi:10.1016/j.bpj.2016.11.1397
• Tardiff, J. C., Regnier, M., Razumova, M. V., Powers, J. D., & Moussavi-harami, F. (2017). Abstract 226: Elucidating the Mechanism of Reduced Length-dependent Activation Due to a Dilated Cardiomyopathy-associated Mutation in Tropomyosin. Circulation Research, 121(suppl_1). doi:10.1161/res.121.suppl_1.226
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  At the subcellular level, the Frank-Starling law of the heart is described by an increase in calcium sensitivity and force with increased sarcomere length (SL). We examine how this relationship is ...
• Tardiff, J. C., Regnier, M., Razumova, M. V., Powers, J. D., & Moussavi-harami, F. (2017). The Frank-Starling Mechansim is Attenuated by a Dilated Cardiomyopathy-Associated Tropomyosin Mutation. Biophysical Journal, 112(3), 120a. doi:10.1016/j.bpj.2016.11.673
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  Sarcomere length-dependent activation (LDA) of force development in cardiac muscle is the cellular basis of the Frank-Starling mechanism, and is often blunted in heart failure. In patients with dilated cardiomyopathy (DCM), LDA may be affected differently depending on the sarcomeric protein mutation underlying the disorder, thus complicating strategies for therapeutic intervention. We investigated the effects of the DCM-associated tropomyosin mutation D230N (TpmD230N) on LDA by measuring force generation and twitch kinetics at short (∼2.0μm) and long (∼2.3μm) sarcomere lengths (SL) of intact and demembranated trabeculae/papillary muscles from hearts of a transgenic murine model (>6 months of age) containing the TpmD230N mutation. Both types of preparations were mounted between a force transducer and linear motor. Intact trabeculae were perfused with oxygenated physiological solution (30°C) and stimulated at 2Hz. Demembranated muscle preparations were bathed in physiological solutions (15°C) containing varying Ca2+ concentrations and allowed to generate steady-state force. As expected, for intact WT trabeculae, the twitch force (Tp) and maximum rate of force development increased and the time to peak tension decreased when muscles were stretched from short to long SL (p
• Tardiff, J. C., Schwartz, S. D., Schwartz, B. A., Williams, M. R., McConnell, M., Tal Grinspan, L., Lynn, M. L., & Fass, O. Z. (2017). Clinically Divergent Mutation Effects on the Structure and Function of the Human Cardiac Tropomyosin Overlap. Biochemistry, 56(26), 3403-3413. doi:10.1021/acs.biochem.7b00266
• Tardiff, J. C., Steczina, S., Regnier, M., Olafsson, S., Moussavi-harami, F., & Flint, G. V. (2017). Recovery of Calcium Activity and Contraction in Models of Dilated Cardiomyopathy. Biophysical Journal, 112(3), 165a. doi:10.1016/j.bpj.2016.11.910
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  Dilated Cardiomyopathy (DCM) is the most common form of systolic heart failure, causing progressive ventricular dilation and loss of systolic function. A transgenic mouse model of DCM, with a missense mutation in alpha-tropomyosin (D230N-Tm), is characteristic of the defects in contraction and calcium handling. Calcium transients and contraction are also depressed in cardiomyocytes from infarcted hearts. We have previously shown that dATP improves contraction of heart muscle from dogs with DCM. We have also shown that small increases in intracellular dATP, achieved by over-expressing ribonucleotide reductase (RNR) via an adeno-associated virus type 6 (AAV6-RNR), significantly improves cardiomyocyte contraction and left ventricular function of infarcted rodent and pig hearts. This study evaluated whether RNR over-expression can improve contraction and calcium handling in the D230N-Tm and infarct models of DCM. Four-month-old D230N-Tm mice were systemically injected with AAV6-RNR. Four months post-injection, cardiomyocytes were isolated to measure contraction, calcium handling, and dATP content using LC-MS. Western blots confirmed over-expression of RNR. We found a significant increase in sarcomere fractional shortening of cells isolated from injected mice compared to their transgenic, un-injected littermates (8.62±0.4 vs. 6.99±0.4 %, P
• Tardiff, J., 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., 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). doi:10.1161/jaha.116.005407
• Tesi, C., Tardiff, J. C., Scellini, B., Poggesi, C., Piroddi, N., Pioner, J. M., Olivotto, I., Morelli, C., Gentile, F., Ferrantini, C., Coppini, R., & Cerbai, E. (2017). Atrial Remodeling in Hypertrophic Cardiomyopathy. Biophysical Journal, 112(3), 556a. doi:10.1016/j.bpj.2016.11.3000
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  Changes in myofilament function related to HCM-associated mutations contribute to the diastolic dysfunction observed in the in vivo patient heart and in intact ventricular preparations from patient samples. HCM mutations that are ubiquitously expressed in the heart (e.g. cMyBP-C or cTnT) could also affect atrial function. Here we investigate whether HCM-associated atrial myopathy is a consequence of mutation-driven sarcomere dysfunction or results from atrial remodeling due to the diastolic dysfunction and increased LV filling pressures. In one HCM patient carrying the Lys814del cMyB-C mutation, changes in sarcomere function (increased myofilament Ca2+ sensitivity and increased cross bridges detachment rate under isometric conditions) were similar in atrial and ventricular myofibrils compared to donor preparations. However, isometric twitch mechanics and kinetics of intact trabeculae from the right atrium of 4 cMyB-C-mutant patients were unaffected as compared to trabeculae from non-HCM patients (N=8), or mutation negative HCM patients (N=3), or HCM patients carrying mutations in beta-myosin (N=2). We extended the study to HCM mouse models carrying mutations in cTnT. In the E163R mouse, atrial and ventricular sarcomere kinetics and energetics were similarly altered compared to WT mice. Isometric ATPase, both at rest and at maximal Ca2+-activation and the energy cost of tension generation were increased in both atrial and ventricular preparations of E163R vs WT. However, isometric twitch kinetics were prolonged in intact ventricular trabeculae of E163R mice vs WT while they were unaffected in atrial trabeculae. In the R92Q mouse model, that is associated with a much more severe degree of LV diastolic dysfunction and left atrial dilatation compared to the E163R, left atrial trabeculae showed prolonged twitch contractions, increased spontaneous activity and a number of E-C coupling alterations that resemble those observed in ventricular preparations. HCM-mutations in cMyBP-C and cTnT induce similar alterations in both atrial and ventricular sarcomeres. However, likely due to the different working conditions of the two chambers, sarcomere dysfunction can significantly alter the mechanics and kinetics of the intact myocardium only in the ventricles. Atrial muscle dysfunction in HCM is induced by remodeling processes that depend on the increased filling pressures.
• 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.
• Holeman, T. A., Tardiff, J. C., Tal-grinspan, L., Lynn, M. L., & Jin, J. P. (2016). Abstract 257: Determining the Potential Role of cTnT Isoform Switching in the Development of Early Childhood Tropomyosin-linked DCM. Circulation Research, 119(suppl_1). doi:10.1161/res.119.suppl_1.257
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  An oft noted component of sarcomeric DCM is the observation that patients within families carrying the same primary mutation exhibit significant phenotypic variability. This lack of a distinct link...
• 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.
• Lehman, S. J., Tardiff, J. C., Tal-grinspan, L., Lynn, M. L., & Anderson, M. E. (2016). Differential Effects of CaMKII Activity in HCM-Linked TNT Mutations. Biophysical Journal, 110(3), 3-6. doi:10.1016/j.bpj.2015.11.1974
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  Mutations in human cardiac troponin T (hcTnT) account for approximately 5-10% of all Hypertrophic Cardiomyopathy (HCM). Residue 92 (Arg) in cTnT is a mutational hotspot. Despite differing by only a single amino acid, Arg92Leu (R92L) and Arg92Trp (R92W) are associated with varying clinical presentations. In the course of studying the differential effects of the R92 mutations on Ca2+ homeostasis, an increase in phospholamban (PLB) phosphorylation at Thr17 was observed only in R92W mice, suggesting a potential mutation-specific role for CaMKII activation in cTnT-linked HCM. To directly test this hypothesis we crossed CaMKII peptide inhibitor mice (AC3I) to both R92W and R92L transgenic mice to evaluate whether a partial inhibition of CaMKII activity could specifically alter the natural history of HCM via modulation of SERCA2 activity. In the AC3IxR92W mice, a compensatory increase in PKA-mediated PLB-Ser16 phosphorylation led to an increase SERCA2 activity coupled with a decrease in atrial mass, indicating improved cardiac function. AC3IxR92L mice did not show any change in SERCA2 activity or morphology, suggesting minimal role of CaMKII in the progression of R92L-linked HCM. R92W has been shown to cause an increase in the Ca2+ dissociation rate from the thin filament, leading to an increase in resting Ca2+ concentration. This increase in Ca2+ initiates aberrant and likely pathophysiologic CaMKII activity, contributing to the progression of HCM in these patients. Thus, targeting the activity of CaMKII may be a potential drug therapy for patients expressing the R92W mutation. Ongoing work aims to elucidate the effects of conventional drug therapies on mice expressing these two mutations to differentiate treatment for patients.
• Schwartz, B. J., Tardiff, J. C., Schwartz, B. J., Mcconnell, M. T., Jayasundar, J. J., Grinspan, L. T., & Fass, O. Z. (2016). A FRET Investigation on the Effects of Tropomyosin D230N and Cardiac Troponin T R92L Mutants on the Tropomyosin Overlap Structure. Biophysical Journal, 110(3), 464a. doi:10.1016/j.bpj.2015.11.2484
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  Cardiac thin filament protein mutants cause changes in protein structure and dynamics. This results in the pathological tissue remodeling seen in patients with hypertrophic (HCM) and dilated (DCM) cardiomyopathies. We propose that two mutations, alpha-tropomyosin (Tm) D230N, which is known to cause DCM, and cardiac troponin T (cTnT) R92L, which causes HCM, differentially affect the structure of the Tm overlap. To investigate the effects of these mutations on this important domain, we used time resolved Forster Resonance Energy Transfer (FRET). Fully reconstituted thin filaments were labeled with probes measuring the distance between cTnT and Tm.Given that the baseline structure is still unknown in this region, the results inform the interactions of the five helices that contribute to the Tm overlap. The cTnT R92L and Tm D230N mutants result in opposite effects on measured distances at the overlap region when compared to wild-type. This occurred between residues 100 and 127 of cTnT and residues 271 and 279 of Tm. The effects are located at the C-terminus of Tm, near a conserved hydrophobic core and a cTnT binding region. These opposing effects on distances at the overlap reflect an altered primary interaction between cTnT and Tm. Ongoing experiments investigating the N-terminal portion of Tm will be informative about propagation of effects across the overlap domain.
• Schwartz, S. D., Tardiff, J. C., Lehman, S. J., & Williams, M. R. (2016). Atomic resolution probe for allostery in the regulatory thin filament. Proceedings of the National Academy of Sciences, 113(12), 3257-3262. doi:10.1073/pnas.1519541113
• Tardiff, J. C. (2016). The Role of Calcium/Calmodulin-Dependent Protein Kinase II Activation in Hypertrophic Cardiomyopathy. Circulation, 134(22), 1749-1751.
• Tardiff, J. C. (2016). The Role of Calcium/Calmodulin-Dependent Protein Kinase II Activation in Hypertrophic Cardiomyopathy. Circulation, 134(22), 1749-1751. doi:10.1161/circulationaha.116.025455
• Tardiff, J. C., Deranek, A., McConnell, M., Tal-Grinspan, L., Lynn, M., Schwartz, B., Fass, O., Farah, H., & Jayasundar, J. J. (2016). Abstract 58: Differential Ventricular Remodeling Induced by Thin Filament Mutational Effects on the Tropomyosin Overlap Structure. Circulation Research, 119(suppl_1). doi:10.1161/res.119.suppl_1.58
• Tesi, C., Tardiff, J. C., Scellini, B., Poggesi, C., Pioner, J. M., Gentile, F., Ferrantini, C., & Coppini, R. (2016). Mechanical Remodeling of Atrial Myocardium in HCM Mouse Models Carrying CTNT Mutations. Biophysical Journal, 110(3), 599a-600a. doi:10.1016/j.bpj.2015.11.3201
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  In hypertrophic cardiomyopathy (HCM) atrial dilatation (AD) and fibrillation (AF) are very common and associated with worse outcome. The cellular and molecular basis of atrial remodeling in HCM remain undefined. We previously characterized (Coppini et al. ABS Biophysical Journal 2015) the changes in sarcomere function and E-C coupling that occur in ventricular myocardium of two HCM mouse models carrying different mutations in cTnT (R92Q and E163R). Both models exhibited diastolic dysfunction that was, however, related to different mechanisms i.e. E-C coupling abnormalities in R92Q and sarcomere changes in E163R. Here we employ these mouse models to study whether atrial remodeling is a consequence of diastolic dysfunction or is also influenced by the specific underlying mutation.Echocardiographic measurements of left atrial (LA) dimensions showed that LA area was severely increased in R92Q hearts while it was only mildly increased in E163R (in mm2 : 6.73±0.5 in R92Q, 4.82±0.16 in E163R vs 3.97±0.26 in WT). Left atrial trabeculae were dissected and mounted isometrically to record twitch tension. We studied the steady-state force-frequency relationship and the response to positive inotropic stimuli such as Isoproterenol 10-7 mM (ISO) and 8 mM extracellular [Ca2+]. Compared to WT, R92Q atrial trabeculae showed: (i) slower kinetics of both force development and relaxation (e.g. at 1 Hz, 50% relaxation was prolonged by 35%), (ii) impaired twitch amplitude at high pacing rates (50% reduction), (iii) depressed rested-state contractions and (iv) blunted increase of twitch tension in ISO and high [Ca2+]. None of these changes were observed in intact E163R atrial trabeculae. These findings suggest that atrial remodeling in R92Q is more pronounced compared to E163R, and related to E-C coupling alterations. Supported by the Italian Ministry of Health (WFR GR-2011-02350583).
• 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.
• Jin, J. P., Tardiff, J. C., Tal-grinspan, L., Lynn, M. L., & Jin, J. P. (2015). Abstract 62: Cardiac Troponin T Isoform Switching in Early Childhood Tropomyosin-linked Dilated Cardiomyopathy. Circulation Research, 117(suppl_1). doi:10.1161/res.117.suppl_1.62
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  An oft-noted component of sarcomeric DCM is the observation that patients within families carrying the same primary mutation exhibit significant phenotypic variability. This lack of a distinct link between genotype and phenotype has complicated clinical management. In a recent study of two unrelated multigenerational families with the tropomyosin (Tm) mutation Asp230Asn (D230N), a striking “bimodal” distribution of severity was observed. In these families, many children (
• Lehman, S. J., Williams, M. R., Tardiff, J. C., & Schwartz, S. D. (2015). An Explicitly Solvated Full Atomistic Model of the Cardiac Thin Filament and Application on the Calcium Binding Affinity Effects from Familial Hypertrophic Cardiomyopathy Linked Mutations. Biophysical Journal, 108(2), 447a. doi:10.1016/j.bpj.2014.11.2441
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  The former version of our cardiac thin filament model consisted of the troponin complex (cTn), two coiled-coil dimers of tropomyosin (Tm), and twenty-nine actin subunits. We now present the newest revision of the model to include both solvation and ionization. The model was developed to continue our study of genetic mutations in the cardiac thin filament proteins which are linked to familial hypertrophic cardiomyopathies. Binding of calcium to the cardiac troponin C subunit (cTnC) causes subtle conformational changes to propagate through the cTnC to the inhibitor subunit (cTnI) which then detaches from actin. Conformational changes propagate through to the cTnT subunit, which allow for the movement of Tm into the open position along actin. Myosin heads can bind to the seven open binding sites on actin, which upon hydrolysis of ATP leads to muscle contraction. Calcium disassociation allows for the reverse to occur, which results in muscle relaxation. Alterations in the calcium binding affinity can disrupt the natural processes of the heart. The inclusion of explicit TIP3 water solvation and an ionic concentration of 0.15 mol/L allows for the model to mimic the true conditions that the cardiac thin filament would feel. The move from implicit to explicit solvation allows us to get better individual local solvent to protein interactions; which are important when observing the N-lobe calcium binding pocket of the cTnC. We are able to compare in silica and in vitro experimental results to better understand the physiological effects from mutants, such as the R92L/W and F110V/I of the cTnT, on the calcium binding affinity compared to the wild type.
• 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. (2015). An Integrative Approach to Thin Filament Cardiomyopathies: From Molecular and Computational Biophysics to Mice. Biophysical Journal, 108(2), 34a-35a. doi:10.1016/j.bpj.2014.11.216
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  In many ways, sarcomeric cardiomyopathies represent the most “biophysical” of disorders in that many of the known mutations, especially those linked to the components of the regulatory thin filament alter the biophysical properties of the sarcomere, leading to a complex and often severe human cardiomyopathy. Despite extensive research, developing robust mechanistic links between sarcomeric dysfunction caused by independent mutations and cardiac performance has proven surprisingly elusive. On the biophysical side of the question, newer approaches including computation and structural methodologies performed on fully reconstituted systems now provide more resolution into the inherently dynamic, allosteric effects of mutations on the sarcomeric machinery. On the clinical side, in recent years it has become clear that the lack of mechanistic insight is due, in part, to an oversimplification of the disease process as a static end state (e.g. “hypertrophy vs dilation”), where it is now clear that cardiac remodeling in sarcomeric cardiomyopathies is a complex, progressive process that includes an early “preclinical” stage prior to overt disease. It is this preclinical stage that defines or represents the biophysical basis of sarcomeric cardiomyopathies and where approaches to alter the natural history of this disorder are focusing. Developing these novel therapeutic approaches will require understanding of atom-level changes in sarcomeric dynamics coupled to precise in vitro methods to measure sarcomeric performance in biologically relevant complexes and, importantly, coherent animal models with which to study the progressive outcomes of molecular interventions at the whole organ level. In this talk the development and application of our integrative in silico-in vitro-in vivo approach to real-world clinical questions will be explored.
• 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.
• Tardiff, J. C., Gollapudi, S. K., & 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-H893. doi:10.1152/ajpheart.00528.2014
• Tardiff, J. C., Mcconnell, M. T., & Grinspan, L. T. (2015). Structural Effects of Cardiac Troponin T R92L and Tropomyosin D230N Mutants in the Cardiac Thin Filament. Biophysical Journal, 108(2), 295a. doi:10.1016/j.bpj.2014.11.1606
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  Introduction/Methods: Inherited mutations in cardiac thin filament proteins lead to changes in protein structure and dynamics. This results in the pathological tissue remodeling seen in patients with hypertrophic (HCM) and dilated (DCM) cardiomyopathies. Our group is interested in the alpha-Tm D230N mutation known to cause DCM and the cardiac troponin T (cTnT) mutation R92L which causes HCM. To investigate effects of these mutations on the tropomyosin (Tm) head-to-tail overlap we used Forster Resonance Energy Transfer (FRET). For both wild-type and mutants, Tm was labeled at site 271 with DDPM, cTnT was labeled with IAEDANS at site 100. Labeled cTnT was reconstituted into troponin complex (Tn) and combined with Tm along with actin to form the reconstituted thin filament. Time-resolved data was acquired and analyzed.Results/Conclusions: Both R92L and D230N mutants result in a change in distance between labeled sites of cTnT and Tm when compared to wild-type. Transfer efficiency decreased from 0.78 to 0.66 upon addition of the D230N mutation, representing an increased distance between the labeled sites. However, the transfer efficiency increased from 0.78 to 0.80 in the presence of the R92L mutation, supporting a decreased distance between the labeled sites. This differential modulation of the distance may reflect alterations in the interaction between the cTnT and Tm proteins due to these mutations. This supports the hypothesis that these mutations which cause different clinical phenotypes are having opposite effects in the region of interest. Ongoing experiments utilizing site 60 of cTnT, and sites 262 and 279 of Tm will provide additional information about the similar and differential effects of these mutants on the Tm overlap region.
• Tardiff, J., 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., Kuster, D. W., & van der Velden, J. (2015). Synergistic role of ADP and Ca²⁺in diastolic myocardial stiffness: Cross-bridging the gap between energetics and Ca²⁺. The Journal of Physiology, 593(17), 3899-3916. doi:10.1113/jp270354
• Teichman, S. L., Tardiff, J. C., Regnier, M., Razumova, M. V., Odom, G. L., Murry, C. M., Moussavi-harami, F., Hauschka, S. D., Cheng, Y., & Chamberlain, J. S. (2015). 11. Gene Therapy Mediated Increase in dATP Improves Cardiac Performance in Models of Systolic Heart Failure. Molecular Therapy, 23, S5. doi:10.1016/s1525-0016(16)33615-2
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  Heart Failure results in more deaths and hospitalizations than almost any other single cause. We are exploring gene therapy treatment strategies that increase 2 deoxy-adenosine triphosphate (dATP) levels via cardiac specific expression of ribonucleotide reductase (RNR) in heart muscle. dATP is produced by RNR. In vitro studies show it increases the magnitude & rate of contraction in rodent, pig & failing human heart muscle without altering calcium handling or slowing relaxation. RNR expression in heart muscle increases intracellular dATP, cardiomyocyte contraction & cardiac performance in rodents. To do this we ligated a cDNA encoding both human RNR subunits to miniaturized cardiac specific enhancer/promoter portions of human cardiac troponin T and packaged the therapeutic “BB-R12” gene in AAV6. In the current study expanded the types of heart failure treated by BB-R12 by testing the effect of dATP on contractile function in a transgenic mouse model of familial dilated cardiomyopathy (DCM). These mice carry and phenocopy a missense mutation (D230N) in alpha-tropomyosin (Tm) that was identified in two DCM family cohorts that exhibit progressive ventricular dilation and loss of systolic function. We measured the effect of dATP on the Ca2+ sensitivity of force in demembranated trabeculae from young adult D230N & WT mice (3 mice & 8-10 trabeculae per group). pCa50 was decreased (lower Ca2+ sensitivity) for D230N Tm (5.47±0.01) compared to WT (5.59±0 0.03) mice, and substituting dATP for ATP rescued this defect (5.55±0.03). dATP also increased isometric force ~20% at pCa=5.8, the approximate level of intracellular Ca2+ seen in a cardiomyocyte twitch. We also measured force and the kinetics of activation & relaxation of isolated myofibrils. Force at pCa=5.6 decreased from 85±14 mN/mm2 in WT myofibrils to 52±7 mN/mm2 (p
• Tosi, B., Tesi, C., Tardiff, J. C., Scellini, B., Poggesi, C., Piroddi, N., Pioner, M. J., Mazzoni, L., Gentile, F., Ferrantini, C., Coppini, R., & Cerbai, E. (2015). Myocardial Dysfunction in Hypertrophic Cardiomyopathy: Primary Effects of Sarcomeric Mutations Versus Secondary EC-Coupling Remodelling. Biophysical Journal, 108(2), 293a. doi:10.1016/j.bpj.2014.11.1596
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  In cardiac muscle from HCM patients primary changes in myofilament function, related to the presence of disease-causing mutations in sarcomeric proteins, are always associated with secondary abnormalities due to adverse remodeling of cardiomyocyte EC-coupling(Coppini et al,Circulation 2013). The latter are likely major contributors of the mechanical dysfunction and arrhythmogeneity of HCM human hearts. Here we characterize the changes in sarcomere function and EC-coupling that occur in two HCM mouse models carrying different mutations in cTnT (R92Q and E163R). Echocardiography showed LV hypertrophy, enhanced contractility, diastolic dysfunction and enlarged left atria in both HCM models; the phenotype was more pronounced in the R92Q mice. In E163R ventricular myofibrils, in spite of a significant increase in the rate of the initial isometric slow phase of relaxation, overall relaxation from maximal activation was impaired and prolonged vs WT and R92Q myofibrils that exhibited similar relaxation kinetics. Resting tension was higher in the E163Q compared to WT and R92Q myofibrils. Isometric ATPase both at rest and at maximal Ca2+-activation and the energy cost of tension generation were increased in E163R vs WT and R92Q skinned trabeculae. Myofilament Ca2+-sensitivity was increased in both mutant lines compared to WT; the change was larger in the R92Q preparations. R92Q intact cardiomyocytes and trabeculae compared to WT and E163R preparations showed blunted response to inotropic interventions, reduced amplitude and slower decay of Ca2+-transients with reduced SERCA function. Twitch kinetics were prolonged in both HCM mouse models, despite Ca2+-transient kinetics was faster and SERCA function unchanged in the E163R mice. Intact preparations of both HCM mouse models showed increased probability of arrhythmogenic behavior that increased in response to isoproterenol. The results suggest that similar HCM phenotypes can be generated through different pathogenic pathways. Grant Telethon-GGP13162.
• 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.
• Jin, J. P., Tardiff, J. C., Tal-grinspan, L., Lynn, M. L., & Jin, J. P. (2014). Abstract 197: Determining the Potential Role of cTnT Isoform Switching In the Development of Early Childhood Tropomyosin-Linked DCM. Circulation Research, 115(suppl_1). doi:10.1161/res.115.suppl_1.197
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  An oft-noted component of sarcomeric HCM and DCM is the observation that patients within families, carrying the same primary mutation, often exhibit significant phenotypic variability. This lack of a distinct link between genotype and phenotype has greatly complicated clinical management. In a recent study of two large unrelated multigenerational families carrying the tropomyosin (Tm) mutation Asp230Asn (D230N), a striking “bimodal” distribution of severity was observed. In these families, many children (
• Lehman, S. J., Tardiff, J. C., Schwartz, S. D., & Manning, E. P. (2014). Computational Prediction and Experimental Verification of Differential Calcium Affinity in Thin Filament Mutants Known to Cause Hypertrophic Cardiomyopathy. Biophysical Journal, 106(2), 349a. doi:10.1016/j.bpj.2013.11.1985
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  Alterations in the calcium affinity of cardiac troponin C (cTnC) with eventual effects on cardiac physiology have been known to result from thin filament mutations in cardiac Troponin T (cTnT) that cause hypertrophic cardiomyopathy (HCM). In this work we report on first principles computational predictions of calcium binding affinity as predicted by coordinating oxygen distance. These calculations are in both a phosphorylated and non-phosphorylated state at serines in positions 23 and 24 in the inhibitory protein of the troponin complex (cTnI,) and so represent the lowest level myofilament effects of adrenergic signaling. The predictions are made using an all atom molecular model of the troponin complex and tropomyosin developed in our two research groups. In order to test the validity of these predictions, IAANS measurements of calcium affinity in fully recombinant thin filaments were performed using a phosphomemetic cTnI (cTnI-DD). Preliminary steady-state results with the wild-type (phosphomimetic) cTnI replicated the predicted decrease in Ca2+ sensitivity. In the same fashion, atomistic calculations showed longer distances between the coordinating oxygens in the phosphorylated state as compared to the unphosphorylated state. These longer distances correspond to weaker Ca2+ binding and decreased sensitivity. The effects of substitutions in cTnT are in progress and will be presented along with the computational work. This work represents the beginning of ab initio prediction of disease effects at the molecular level via the use of validated computer simulation.
• Lehman, S. J., Williams, M. R., Tardiff, J. C., Jayasundar, J. J., & Brooks, K. S. (2014). The Cardiac Troponin T R92L Hcm Mutation Alters Cardiac Troponin I Dynamics and PKA Phosphorylation Potential. Biophysical Journal, 106(2), 3-8. doi:10.1016/j.bpj.2013.11.1983
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  Mutations in cardiac troponin T (cTnT) have been linked to hypertrophic cardiomyopathy (HCM). Previous experiments in our lab utilizing a novel cTnT R92L transgenic mouse model revealed an allosterically mediated decrease in the PKA phosphorylation potential of cardiac troponin I (cTnI). We hypothesized the cTnT R92L mutation causes a change in the dynamics of the N-terminal extension, hindering the availability of PKA substrates S23/S24 on cTnI. In the current study this hypothesis was tested both in silico and in vitro. First, molecular dynamics (MD) simulations using our all-atom thin filament model in the Ca2+ saturated state were performed. MD predicted an approximately 7A narrowing between the N-terminal extension of cTnI and cardiac troponin C (cTnC). To experimentally evaluate the in-silico predictions, Forster Resonance Energy Transfer (FRET) experiments were conducted on fully reconstituted thin filaments. The cTnI was labeled with the FRET donor AEDANS at three sites on the N-terminal extension of cTnI. The acceptor DDPM, was labeled on six sites on cTnC. Steady state and time resolved FRET experiments were performed in the presence and absence of the TnT R92L mutation. Both theoretical and experimental approaches revealed that in the presence of the cTnT R92L mutation the dynamics of cTnI was allosterically altered. The N-terminal extension of cardiac troponin I moved closer to the N-domain of cTnC. This anharmonic fluctuation likely hinders the availability of the PKA substrate sites and leads to the observed blunting of the beta-adrenergic response in animal models and human patients.
• 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.
• Schwartz, B. A., Tardiff, J. C., Mcconnell, M. T., Jayasundar, J. J., Grinspan, L. T., & Fass, O. Z. (2014). Dynamic Effects of Tropomyosin D230N Mutation and Fetal Troponin T on the Tropomyosin Overlap Region. Biophysical Journal, 106(2), 32a. doi:10.1016/j.bpj.2013.11.250
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  Introduction: Mutations in the cardiac thin filament cause changes in protein structure and dynamics. These alterations result in the complex tissue remodeling seen in patients with hypertrophic and dilated cardiomyopathies. Our group is investigating the effects of these mutations on the tropomyosin (Tm) head-to-tail overlap domain. In particular, we are investigating the alpha-Tm D230N mutation that causes a unique bimodal distribution of remodeling. We hypothesize that this is caused by the modulatory effects of the fetal form of cTnT (cTnT1) on overlap structure and function.Methods: Tm was modified to have one cysteine at residue 271 for both wildtype and D230N mutations, and was labeled with the FRET acceptor DDPM. cTnT1 or adult cardiac troponin T (cTnT3) were modified to have one cysteine at residue 100 and labeled with the FRET donor AEDANS. Labeled cTnT1 or cTnT3 were reconstituted into the troponin complex and combined with labeled Tm of either wildtype or D230N. Steady-state and lifetime data was collected.Results: The distance between the labeled sites in the wildtype complex increased with calcium activation. In contrast, the calcium activated state of the D230N complex resulted in a shorter measured distance. cTnT1 also resulted in a closer distance. cTnT1 in addition to D230N mutation resulted in a closer orientation compared D230N or cTnT1 alone. Differences in measured distances caused by the variants were more pronounced in the calcium activated state than in the low calcium state.Conclusion: The increased distance measured with wildtype complex supported the increased flexibility of Tm accompanying thin filament activation. The decreased distance measured at the Tm overlap supports the hypothesis that cTnT1 interacts with D230N in a manner that amplifies the effects of the mutation alone at the Tm overlap region.
• Tardiff, J. C., Jian, Z., Han, H., Zhang, T., Puglisi, J., Izu, L. T., Shaw, J. A., Onofiok, E., Erickson, J. R., Chen, Y., Horvath, B., Shimkunas, R., Xiao, W., Li, Y., Pan, T., Chan, J., Banyasz, T., 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). doi:10.1126/scisignal.2005046
• Tosi, B., Tesi, C., Tardiff, J. C., Poggesi, C., Mazzoni, L., Gentile, F., Ferrara, C., Ferrantini, C., Coppini, R., & Cerbai, E. (2014). 291Myocardial dysfunction in hypertrophic cardiomyopathy: primary effects of sarcomeric mutations versus secondary cardiomyocyte remodeling?. Cardiovascular Research, 103(suppl 1), S53.2-S53. doi:10.1093/cvr/cvu087.5
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  Introduction: In HCM human tissue, primary functional alterations at the level of the sarcomeres are associated with secondary changes of Ca2+ handling and membrane EP, leading to a pro-arrhythmogenic phenotype and alteration of myocardial relaxation (Circulation. 2013 Feb 5;127(5):575-84). The relative contribution of primary vs secondary alterations is still unknown. Methods: Here we aim to study these changes in intact trabeculae, single cardiomyocytes and skinned preparations from the ventricles of transgenic mouse models aged 6 months carrying HCM-related mutations of cTnT (R92Q, E163R). Results: Compared to WT, R92Q trabeculae showed: (i) preserved peak isometric twitch tension at low inotropic level with reduced contractile reserve; (ii) prolonged relaxation kinetics associated with reduced SERCA function; (iii) faster mechanical restitution, indicating shorter RyR2 refractoriness; (iv) Frequent after-contractions or spontaneous beats during pauses, which increased in response to isoproterenol. Compared to WT, R92Q cardiomyocytes showed: (i) prolonged action potentials due to ionic current remodeling; (ii) reduced amplitude and slower decay rate of Ca2+transients; (iii) elevated diastolic [Ca2+]I and (iv) spontaneous Ca2+ waves and Ca2+transients under isoproterenol. In E163R vs. WT trabeculae and cells, peak isometric tension and Ca2+ transient amplitude were preserved in all conditions. Interestingly, the kinetics of force development and relaxation was prolonged, despite Ca2+ transient kinetics was faster and SERCA function unchanged. E163R myocardium showed faster mechanical restitution and increased spontaneous activity. Further, E163R myofibrils showed a prolongation of the overall relaxation, with incomplete inactivation in the absence of Ca2+. Energy cost of contraction, measured with an enzymatic assay of sarcomeric ATPase activity, as well as myofilaments Ca2+ sensitivity, were increased in E163R vs. WT skinned trabeculae. Conclusions: Primary changes of myofilaments function (increased Ca2+ sensitivity), previously described in R92Q hearts, are associated with a large spectrum of EC-coupling and membrane EP changes, which appear to be a major contributor to the observed mechanical dysfunction and arrhythmogeneicity in this mouse line, resembling advanced human disease. In E163R instead, impairment of myofilament function appear to be the leading element determining mechanical abnormalities. In the absence of major EC-coupling changes, the increased arrhythmogeneicity in E163R myocardium may be a direct consequence of the increased myofilaments Ca2 sensitivity.
• Tosi, B., Tesi, C., Tardiff, J. C., Poggesi, C., Pioner, J. M., Mugelli, A., Moore, R. K., Mazzoni, L., Ferrantini, C., Coppini, R., & Cerbai, E. (2014). E-C Coupling Alterations and Spontaneous Activity in Mice Carrying Cardiac Troponin T Mutations. Biophysical Journal, 106(2), 644a. doi:10.1016/j.bpj.2013.11.3567
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  Ca2+ handling abnormalities are an early-onset pathogenic element in HCM. Here we characterize pro-arrhythmogenic changes in E-C coupling that occur in intact trabeculae and cardiomyocytes from cTnT mutant mouse models of HCM (R92Q, E163R and Δ160E) and test the effects of specific pharmacological interventions. Compared to WT, R92Q trabeculae ([Ca2+]o 2 mM, 30°C) showed (i) preserved peak isometric twitch tension and prolonged relaxation kinetics associated to decreased SERCA levels, (ii) faster mechanical restitution, further accelerated by isoproterenol (Iso) 100nM, (iii) decreased Ca2+-recirculation fraction markedly increased by Iso (iv) frequent after-contractions or regular spontaneous beats during stimulation pauses that increased in response to Iso. Compared to WT, R92Q cardiomyocytesshowed (i) prolonged action potentials associated with ionic current remodeling, (ii) slower rate of Ca2+transient decay, (iii) elevated diastolic [Ca2+]i, (iv) spontaneous Ca2+ waves during stimulation pauses. In R92Q preparations, the late-Na+ current blocker Ranolazine (Ran 10 μM) (i) reduced the rate of spontaneous beats and spontaneous Ca2+ waves, (ii) hastened Ca2+ transient kinetics and reduced diastolic Ca2+, (iii) reduced and reversed the acceleration of mechanical restitution and the increase in Ca2+ recirculation fraction induced by Iso.Compared to R92Q, occurrence of spontaneous contractions was similar in E163R but less pronounced in Δ160E. Iso and Ran showed similar effects in all three mouse models, in spite of some quantitative differences. The results are consistent with those recently reported in human HCM myocytes (Coppini et al, Circulation 2013) and suggest that remodeling and dysfunction of NCX and RyR2 contribute to the pro-arrhythmogenic E-C coupling abnormalities observed in HCM.
• Williams, M. R., Tardiff, J. C., & Schwartz, S. J. (2014). A Revised Atomistic Model of the Cardiac Thin Filament and Application to a Specific Disease Causing Mutation. Biophysical Journal, 106(2), 32a. doi:10.1016/j.bpj.2013.11.247
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  We have previously proposed an atomistic model of the thin filament which includes the troponin compex (cTn) and tropomyosin (Tm). We here discuss a newly revised model which includes twenty-nine actin subunits, four Tm chains, and the three cTn subunits. In addition, this model includes a corrected region of the Tm overlap based on more recent information. We develop this model to study genetic mutations in the proteins of the cardiac thin filament which can lead to familial hypertrophic cardiomyopathies. When calcium binds to the cardiac troponin C subunit (cTnC), subtle conformational changes propagate through the cTnC to the inhibitor subunit (cTnI), which detaches from actin. The detachment along with conformational propagation through to the cTnT subunit, moves Tm into the open position on actin. When Tm is in the open position, myosin binds to the seven open binding sites on actin, which upon hydrolysis of ATP eventually leads to muscle contraction. Molecular dynamics simulations of the full atomistic model reveal the conformational changes upon calcium activation of the cTnC. Comparison of the wildtype and the Tm D230N mutation sheds light the mutational effects in the Tm overlap region. It is critical to include actin in the model when studying this region. Simulations show mutation results in increased splaying of the Tm C-terminus end in the overlap region, in accord with recent experimental results from one of our labs.
• 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.
• Tardiff, J. C., Tal, L., Michael, J. J., & Chandra, M. (2013). Pseudophosphorylation of Cardiac Troponin I Residues 23/24 Decreases Myofilament Ca2+ Sensitivity in Transgenic Mice Containing D230N Mutation in α-Tropomyosin. Biophysical Journal, 104(2), 482a. doi:10.1016/j.bpj.2012.11.2663
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  The role of sarcomeric mutations in pathological cardiac-remodeling is not well understood. Furthermore, the constant modulation of myofilament contractility by post-translational modifications (PTM) of regulatory proteins, to adapt to a frequently changing circulatory demand, adds to the complexity. Therefore, we investigated how such a PTM in cardiac troponin I (cTnI) interacted with D230N (a dilated cardiomyopathy-related α-tropomyosin mutation), to alter the contractile properties of the myofilament. We reconstituted the pseudophosphorylated form of cTnI (cTnID23/D24) into detergent-skinned papillary muscle fibers from non-transgenic (NTG) and transgenic D230N mice, to mimic PKA-mediated phosphorylation in response to β-adrenergic stimulation. We carried out mechanical and dynamic studies on cTnID23/D24-D230N and control fibers at sarcomere length 2.3 μm. The pCa-tension relationship revealed that cTnID23/D24 decreased the pCa50 (-log10 [Ca2+]free required for half maximal activation) from 5.61±0.02 to 5.34±0.01 in NTG fibers and from 5.49±0.02 to 5.22±0.01 in D230N fibers. Thus, cTnID23/D24 uniformly decreased pCa50 by 0.27 pCa units in both NTG and D230N fibers. Interestingly, D230N mutation also uniformly decreased pCa50 by 0.12 pCa units, regardless of the phosphorylation status of cTnI. This indicates that cTnID23/D24 and D230N act independently to exert an additive effect of decreasing myofilament Ca2+ sensitivity. Therefore, under sub-maximal concentrations of Ca2+, the amount of force produced would be greatly impaired, resulting in systolic dysfunction.
• 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.
• Tardiff, J. C., Leinwand, L. A., & Gregorio, C. C. (2012). Mutations in the Sensitive Giant Titin Result in a Broken Heart. Circulation Research, 111(2), 158-161. doi:10.1161/res.0b013e3182635ca2
• Tardiff, J. C., Tal, L., & Jimenez, J. (2012). DCM-Linked D230N Tropomyosin Mutation Results in Early Dilatation and Systolic Dysfunction in Mice. Biophysical Journal, 102(3), 356a. doi:10.1016/j.bpj.2011.11.1945
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  Recently, a study in two large multi-generational families described a familial dilated cardiomyopathy (DCM) caused by a single amino acid substitution Asp230Asn (D230N) in tropomyosin. These families demonstrated a unique bimodal disease distribution in which infants presented with a severe form of DCM, while adults presented with a mild to moderate clinical phenotype. To determine the biophysical consequences of this mutation on tropomyosin and its effects on regulatory function in the sarcomere, we employed circular dichroism and the regulated in vitro motility assay. We found that while this mutation does not affect overall thermal stability of tropomyosin, it has a profound effect on regulatory function. As previously shown in solution, the presence of the D230N mutation decreases the maximal velocity of filament sliding and calcium sensitivity of thin filament activation compared to wild type filaments. Additionally, the D230N mutation increases the cooperativity of myofilament activation. In order to further explore our biophysical observations and the physiologic effects of the D230N mutation, we created a transgenic murine model. In mice carrying the D230N tropomyosin mutation we found evidence of early dilatation and systolic dysfunction by echocardiogram in the absence of histological changes such as fibrosis or inflammatory cell invasion. Ultrastructural analysis of transgenic left ventricular tissue demonstrated z-disk alterations. Finally, preliminary studies on isolated myocytes from transgenic mice loaded with fura-2AM demonstrate no discernible differences in calcium transients compared to non-transgenic siblings suggesting that functional impairments are not due to calcium handling defects. Collectively, these studies suggest that the D230N mutation in tropomyosin is responsible for alterations in structure and function of the thin filament that result in a primary dilatation of the cardiac left ventricle. This work is supported by funding from the Children's Cardiomyopathy Foundation.
• Tardiff, J. C., Tal, L., Michael, J. J., Mamidi, R., Gollapudi, S. K., & Chandra, M. (2012). D230N Mutation in Tropomyosin and R92L Mutation in Cardiac Troponin T have Strikingly Different Impact on Calcium-Regulated Activation of Cardiac Myofilaments. Biophysical Journal, 102(3), 358a. doi:10.1016/j.bpj.2011.11.1956
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  Dilated Cardiomyopathy (DCM) and Familial Hypertrophic Cardiomyopathy (FHC) are pathological heart conditions mainly associated with sarcomeric mutations that lead to contractile dysfunction. Despite the identification of several mutations associated with FHC and DCM, the role of these mutations in pathological cardiac-remodeling is still elusive. Therefore, we studied length-dependent contractile parameters of murine hearts expressing DCM-associated mutation (D230N in alpha-tropomyosin) and FHC-associated mutation (R92L in cTnT). Mechanical studies were carried out on detergent-skinned cardiac muscle fibers at sarcomere length (SL) 1.9 and 2.3 μm. Our preliminary results show that myofilament Ca2+ sensitivity and cooperativity are affected differently in both mutants, irrespective of SL. Ca2+ sensitivity was decreased in the D230N fibers, but increased in R92L fibers. Ca2+-activated maximal tension was unaltered in both examples. Cooperativity of myofilament activation was significantly decreased in R92L fibers and significantly increased in D230N fibers. Our results suggest that single amino acid substitution mutations in Tm (D230N) and TnT (R92L) cause diverse functional effects, which may correlate with varied pathological remodeling. Further mechano-dynamic studies are planned to determine if other aspects of myofilament activation may be involved in the evolution of complications associated with DCM and FHC.
• 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.
• Tardiff, J. C., & Jimenez, J. (2011). Abstract P072: Blunted β-Adrenergic Response in R92L Cardiac Troponin T Mutant Hearts Occurs via Decreased Accessibility to PKA-Mediated Phosphorylation Sites at Serine 22/23 Residues of Cardiac Troponin I. Circulation Research, 109.
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  While diastolic dysfunction due to impaired relaxation is a classic finding in patients with Familial Hypertrophic Cardiomyopathy (FHC), the primary cellular mechanisms remain undefined. We have previously demonstrated impaired relaxation in our transgenic mouse models of FHC carrying the cTnT R92L mutation. We hypothesize that the impaired relaxation in RL is a result of an allosterically mediated, diminished structural accessibility to the PKA phosphorylation sites on cTnI. Protein levels of phosphorylated TnI at baseline and following stimulation with isoproterenol were significantly lower for RL compared to non-transgenic (NT) mice while protein levels of phosphorylated phospholamban (PLB), TnI, and PLB were the same between both groups. These results indicate that PKA signaling is intact and uncoupling occurs at the myofilament level. Next, we crossed RL mice with phosphomimetic mice that have had their cTnI residues S 22 S 23 changed to D 22 D 23 to generate the double transgenic RL/DD. Preliminary isovolumic studies demonstrated improved contractility and relaxation for RL/DD compared to RL alone but did not reach levels observed for NT following graded doses of dobutamine (Figures A and B). In Ca 2+ transient measurements of isolated adult cardiac myocytes, DD cells demonstrated enhanced peak rates of Ca 2+ rise and fall as well as accelerated times to 50% and 90% Ca 2+ declines compared to NT and RL. However, RL/DD mice did not show improvement in any of these parameters. Collectively, these results indicate that the diastolic dysfunction observed in R92L is caused by direct impairment of the myofilament axis in the beta adrenergic signaling cascade.
• Tardiff, J. C., & Tal, L. (2011). Abstract P248: The DCM-Causing D230N Tropomyosin Mutation Produces an Age-Dependent Phenotype in Mice that Is Influenced by Transgene Dosage. Circulation Research, 109.
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  A recent study in two large multigenerational families demonstrated a novel mutation in tropomyosin (Tm), Asp230Asn (D230N) that caused DCM with a unique natural history resulting in a striking bimodal disease distribution. Infants and young children with the D230N Tm mutation presented with a severe, often fatal DCM while adults developed a mild to moderate clinical phenotype. Position 230 in tropomyosin is proximal to the C-terminal unwinding of tropomyosin that is essential for the Tm-Tm overlap and for the Tm-cardiac troponin T (cTnT) interaction. We hypothesized that the age-dependent remodeling is a result of temporal isoform switches in the closely linked Tm-binding partner cTnT. To directly address this hypothesis, we used the regulated in vitro motility assay to determine the biophysical effects of the D230N Tm mutation as well as the effects of cTnT isoform changes on the regulatory function of thin filaments. We have found that both wild type and D230N Tm filaments inhibit filament sliding at low calcium indicating that the D230N mutation does not completely disrupt regulation. However in the presence of high calcium the D230N Tm mutation significantly decreases the maximal velocity of filament sliding (3.790 ± 0.1333 microns/sec, n=98) as compared to the wild type filaments (4.900 ± 0.1044, microns/sec n=76). These findings support an altered Tm-cTnT interaction because at maximal calcium concentrations, the myofilament activation at the level of cardiac troponin is not properly transmitted to tropomyosin thus decreasing the activity of myosin-ATPase. In order to extend our biophysical observations and to provide translational insight, we generated a D230N Tm transgenic mouse model. In preliminary studies we demonstrate that at two months the D230N transgenic mice have evidence for early dilatation. In addition, the differences in ventricular remodeling were highly dependent on transgene dose. At six months, we observed dilatation of the ventricle and thinning of the walls compared to non-transgenic siblings. However, the differences were not as marked as those at the two-month time point. Collectively, these findings support an age-dependent DCM phenotype that is modulated by transgene dosage.
• Tardiff, J. C., Mamidi, R., Jimenez, J. J., Ford, S. J., & Chandra, M. (2011). Effects of R92 Mutations on Cardiac Contractile Function are Influenced by Changes in Myosin Heavy Chain Isoform. Biophysical Journal, 100(3), 127a. doi:10.1016/j.bpj.2010.12.898
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  Mutations of cardiac troponin T (cTnT), many of which are found at its N-terminus (cT1), are associated with various forms of human heart disease. Within cT1, an amino acid at residue 92 (Arginine, R92) is a hotspot for point mutations. Previous studies using transgenic mouse models have shown that disease-related mutations R92 to Leucine (R92L) or Glutamine (R92Q) each influence contractile behavior of cardiac myofilaments. Because such cTnT mutations are often accompanied by an increase β-myosin heavy chain (MHC) expression in the failing human heart, we sought to determine whether the R92 mutation effects on cardiac contractile function are further influenced by a shift in MHC isoform content. Detergent-skinned papillary muscle fiber bundles were harvested from transgenic mice expressing R92L or R92Q against native α-MHC (R92L/α-MHC or R92Q/α-MHC) and transgenic mice expressing R92L or R92Q against predominantly β-MHC (R92L/β-MHC or R92Q/β-MHC). Constantly-activated fiber bundles from R92 transgenic and nontransgenic controls (α-MHC and β-MHC) were used to study how differences in MHC influence the effects of R92 mutations on Ca2+- and length-dependent contractile activation. Our study shows that, concomitant with previous findings, R92 mutations against α-MHC result in a decrease in ATPase activity, increase in myofilament Ca2+-sensitivity, and a decrease in cooperativity of myofilament force production. Furthermore, our study suggest that mutant R92 effects cardiac contractile dynamics in such a way that R92Q/α-MHC slows rates of crossbridge recruitment and crossbridge detachment and blunts the nonlinear effect that crossbridge distortion has on crossbridge recruitment. Interestingly, many of these R92 mutant effects were significantly influenced by expression of β-MHC. Collectively, these novel findings indicate a strong interaction effect and suggest that MHC structure further influences how cTnT regulates functional and dynamical aspects of cardiac contractile activation.
• Tardiff, J. C., Payne, C. E., Khabbaz, S., Izu, L. T., Edelmann, S. E., Chen-izu, Y., & Banyasz, T. (2011). Increased Myofilament Ca2+ Sensitivity Decreases Sarcomere Length and Increases Spark-Spark Interactions. Biophysical Journal, 100(3), 560a. doi:10.1016/j.bpj.2010.12.3253
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  People with familial hypertrophic cardiomyopathy (FHC) harboring mutations of cardiac troponin T (cTnT) are often at a high risk of sudden cardiac death. Transgenic mice harboring some of these cTnT mutations show increased myofilament sensitivity to Ca2+ and also shortened diastolic sarcomere length (SL). Our computational studies predicted that decreasing the distances between Ca2+ release units (CRUs) of the sarcoplasmic reticulum (SR) by decreasing SL can destabilize the Ca2+ control system and increase the probability of spontaneous Ca2+ waves. Destabilization results from enhanced crosstalk between neighboring CRUs. In this study we mimic the greater myofilament Ca2+ sensitivity conferred by cTnT mutations using the myofilament Ca2+ sensitizer EMD 57033 (EMD). At concentrations up to 3 μM, EMD had no effect on either the peak Ca2+ transient or the diastolic Ca2+ levels and did not alter the SR Ca2+ load. To test the prediction that SL shortening increases the coupling between CRUs, we loaded myocytes with Di8-ANEPPS and Fluo-4 and simultaneously measured SL and Ca2+ sparks in 2 spatial dimensions using the Zeiss 5 Live high-speed 2-D scanning confocal microscope. EMD (1.5 μM) decreased SL significantly compared to the control cells in normal Tyrode (1.58 μm vs.1.69 μm, p
• Tardiff, J. C., Schwartz, S. D., & Manning, E. P. (2011). Computational Modeling of Cardiac Troponin Dynamics: Elucidating a Regulatory Mechanism for Calcium Activation of the Thin Filament. Biophysical Journal, 100(3), 225a. doi:10.1016/j.bpj.2010.12.1440
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  Calcium activation of the thin filament regulates contraction of skeletal and cardiac muscle via the protein complex troponin and its interaction with tropomyosin. The mechanism of this regulation remains mostly unknown. A complete dynamic, atomistic model of cardiac troponin is developed, constructed from various structures and models. The mechanism underlying cardiac troponin regulation of the thin filament is investigated using molecular dynamics simulations of the model in two states: calcium bound and not bound to site II of the N-lobe of cardiac troponin C. Significant changes in dynamics of various regions are observed throughout the cardiac troponin complex and in the overlapping tropomyosins. This leads us to believe that calcium-dependent alterations in dynamics propagate throughout the cardiac troponin complex and alter tropomyosin dynamics, resulting in calcium-dependent activation of the thin filament.
• Tardiff, J. C., Tal, L., Moore, R. K., & Dowell-martino, C. (2011). Pathophysiologic Changes Induced by Mutations in the TNT1 Domain of cTnT that Cause FHC. Biophysical Journal, 100(3), 452a. doi:10.1016/j.bpj.2010.12.2665
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  Familial Hypertrophic Cardiomyopathy (FHC) is a primary cardiac muscle disorder and one of the most common causes of sudden cardiac death in young people. A majority of Cardiac Troponin T (cTnT) mutations are located in the TNT1 domain and cluster at its N- and C-termini. We are investigating the cTnT deletion of glutamine 160 (delta-160E) that is known to be a severe mutation located in a predicted hinge region at the C-terminal end of TNT1. Previous in vitro motility studies in our laboratory showed that mutations in this region disrupt weak electrostatic interactions between the thin filament and myosin necessary for strong crossbridge formation. In the current study, we aim to examine the downstream pathophysioloic consequences of this mutation. Cardiac myocytes isolated from hearts of transgenic mice expressing delta-160E cTnT with 35% and 70% replacement and non-transgenic siblings were used to study mechanical function and calcium transients. Our study shows impairments in myocellular mechanics during contraction and relaxation and in the rise and decline of the calcium transient. Furthermore, the alterations in calcium kinetics were dose-dependent. These results support the progressive nature of delta-160E FHC suggested by electron micrographs that demonstrate ultrastructural sarcomeric disarray that increase with transgene expression. In addition, we determined downstream effects of the mutation on expression and function of calcium handling proteins in transgenic mouse hearts using functional assays and immunoblotting. We found that the delta-160E cTnT mutation causes secondary alterations in calcium handling, leading to decreased SR calcium uptake, increased NCX expression, and increased diastolic leak through RyR2. Collectively, these novel findings indicate a phenotype that is distinct from other cTnT mutations and support the need to establish genotype-phenotype links in order to better design molecular therapies to treat FHC.
• 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.
• Tardiff, J. C., Moore, R. K., Guinto, P. J., & Dowell-martino, C. (2010). Functional and Structural Changes Induced By cTNT-Related FHC Mutations in TNT1 Alter Actomyosin Binding Interactions. Biophysical Journal, 98(3), 6a. doi:10.1016/j.bpj.2009.12.038
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  Familial Hypertrophic Cardiomyopathy (FHC) is a primary disease of the cardiac sarcomere. Many FHC mutations in hcTnT are found within the TNT1 domain, with a mutational hotpot at residues 160-163. These residues fall within a highly charged region (158-RREEEENRR-166), which may create a flexible hinge necessary for function, the structure and function of which is affected by FHC mutations. We are investigating the effects of these hotspot mutations using in vitro motility (IVM) assays, SDSL-EPR, and transgenic mouse models. IVM data indicate that mutations Δ160E and E163R disrupt actin binding to heavy meromyosin under standard assay conditions. By reducing the ionic strength of the motility solutions, thin filament binding and sliding are restored suggesting that mutations in this region cause disease by disrupting the weak electrostatic interactions between the thin filament and myosin necessary for crossbridge formation. CW-EPR spectra show an increase in spin label isotropic rotational rate at hcTnT residue 153 (upstream of the putative hinge region) between Troponin ternary complexes containing Δ160E verses WT hcTnT, suggesting an increase in flexibility due to backbone changes induced by the mutation. We are expanding our SDSL-EPR experiments with additional cysteine substitutions superimposed onto 160-163 mutant proteins to provide further data regarding secondary structural changes imposed by these mutations. These results correspond with our Δ160E mouse model showing dose dependant myofilament disarray. Preliminary observations of an E163R model suggest that this mutant is less severely affected, tolerating a higher transgene dose. The structural and functional changes observed in vitro may contribute to the structural impairment seen in vivo. By correlating our IVM and SDSL-EPR findings with in vivo data generated from the Δ160E and E163R models, a mechanism of disease for these hotspot mutations can be determined.
• Wilson, J., Wilson, J. O., Tardiff, J. C., Moore, R. K., Guinto, P. J., & Gerfen, G. J. (2010). Structural and Functional Characterization of the TNT1 Domain of Cardiac Troponin T. Biophysical Journal, 98(3), 358a. doi:10.1016/j.bpj.2009.12.1934
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  Familial Hypertrophic Cardiomyopathy (FHC) is a primary cardiac muscle disorder and a common cause of sudden cardiac death among young people in the field. The majority of disease-causing mutations in the thin filament protein hcTnT are found within the TNT1 domain. This domain has not been crystallized and its structural details are poorly defined, limiting our ability to understand the mechanism of disease for these mutations. A highly charged region is found at the C-terminal end of TNT1 (158-RREEEENRR-166) in which this highly alpha helical domain may unwind to create a flexible hinge that is necessary for normal function. We aim to determine the structural details and function of this region using SDSL-EPR and regulated in vitro motility (R-IVM) assays. The purpose of our R-IVM experiments is two-fold: to functionally analyze our spin labeled proteins and to gain insight into the function of TNT1 in the presence of cysteine substitutions. R-IVM data shows a progressive increase in the severity of the functional effects of cysteine substitution and spin labeling across the putative hinge region, suggesting that this region is dynamically important and may be making critical interactions with other components of the sarcomere. CW-EPR spectra of spin labeled hcTnT in Troponin ternary complexes show an increase in spin label mobility from residue 153 to 157 and 177, consistent with a decrease in alpha helical character across the putative hinge region. Preliminary doubly labeled CW-EPR experiments show that interspin distance between hcTnT residues 157 and 177 exceed 25A. Interspin distance measurements using doubly labeled hcTnT will further elucidate the secondary and tertiary structure of this region. Additional spin label pairs are currently being investigated using both CW-EPR and DEER techniques to determine the structural details of this important region.
• 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.
• Tardiff, J. C., Schwartz, S. D., Moore, R. K., Manning, E. P., & Guinto, P. J. (2009). Structural and Functional Characterization of Cardiac Troponin T Mutations in the TNT1 Domain That Cause Familial Hypertrophic Cardiomyopathy. Biophysical Journal, 96(3), 4-9. doi:10.1016/j.bpj.2008.12.2575
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  FHC is a primary cardiac muscle disorder that is one of the most common causes of sudden death in young people. FHC “hotspot” mutations at residue 92 in cardiac troponin T (cTnT) flank the proposed α-helical TNT1 tail domain whose flexibility has been suggested to be important in normal protein-protein interactions within the thin filament. Through Molecular Dynamics (MD) simulations, we showed that FHC mutations Arg92Leu, Arg92Trp, and Arg92Gln cause local α-helical structural changes and increased flexibility at a critical hinge region 18 Angstroms distant from the mutation. We have extended this MD analysis via the use of a self-defined coordinate to measure localized bends in the helix and found that forces acting on this bending coordinate are lower in mutants than wildtype. This quantitatively suggests a less restrictive bending motion in mutants explaining the increased flexibility of the hinge region. To determine how primary biophysical changes induced by these mutations cause complex cardiomyopathies we hypothesize that flexibility alterations and changes in force within compaction-expansion regions in mutational segments lead to electrostatic perturbations, possibly interfering with cTnT-TM complex formation and thin filament function. In vitro motility assays with wildtype cTnT and hotspot FHC-cTnT mutants are in progress to directly correlate predicted alterations in electrostatic properties with resultant functional changes. Moreover, contractile and Ca2+ transient measurements on isolated myocytes address downstream myocellular responses to the mutation's primary perturbation on structure and function. Data showed normal percent shortening in Arg92Leu myocytes while Arg92Trp percent shortening was significantly impaired compared to Non-Tg (4.740 + 1.165 vs. 6.971 + 2.098, p
• Wilson, J., Wilson, J. O., Tardiff, J. C., Riegelhaupt, M., Moore, R. K., Guinto, P. J., & Gerfen, G. J. (2009). Structural and Functional Characterization of cTnT in Familial Hypertrophic Cardiomyopathy. Biophysical Journal, 96(3), 499a. doi:10.1016/j.bpj.2008.12.2576
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  Familial Hypertrophic Cardiomyopathy (FHC) is a primary disease of the cardiac sarcomere. Many disease-causing mutations in the thin filament protein cTnT are found within the TNT1 region. Residues 160-163 represent a mutational hotspot within a highly charged region (158-RREEEENRR-166). In this region, this highly alpha helical domain may unwind to create a flexible hinge that is necessary for function, the structure and dynamics of which may be affected by FHC mutations. We are investigating the structure and function of this region using in vitro motility (IVM) assays and SDSL-EPR. The purpose of our IVM experiments is two-fold: to functionally analyze our spin labeled proteins and to gain insight into the function of TNT1 in the presence of cysteine substitutions and FHC mutations. Preliminary IVM data shows a progressive increase in the severity of the functional effects of cysteine substitution and spin labeling across the putative hinge region (153
• 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. (2021, August 2021). A Physician Scientist's Journey: Linearity is Overrated. Keynote for Early Stage Investigator's Symposium at the AHA Basic Cardiovascular Sciences Meeting. Virtual: American Heart Association.
• Tardiff, J. C. (2021, August 2021). Allosteric Modulation of Myofilament Ca2+ Kinetics as a Potential Mechanism for Impaired Relaxation in Thin Filament Linked HCM. American Heart Association Basic Cardiovascular Sciences Meeting. Virtual: American Heart Association.
• Tardiff, J. C. (2021, Dec 2021). Modifying the Cardiac Thin Filament: New Approaches to Identifying Potential Targets. Invited Lecture for Bristol Myers Squibb. Virtual: Bristol Myers Squibb / MyoKardia - Connecticut.
• Tardiff, J. C. (2021, March 2021). Hypertrophic Cardiomyopathy in 2021: Linking Molecular Mechanisms to Patient Management, What a Long Strange Trip its Been. Northwestern Medical Center Cardiovascular Grand Rounds. Virtual: Northwestern Unversity Medical School.
• Tardiff, J. C. (2021, March 2021). Intrinsic and Extrinsic Modulators of Thin Filament Function: The Potential for Novel Therapeutic Targets in HCM. Invited Talk University of Minnesota Department of Integrative Biology and Physiology. Virtual: University of Minnesota.
• Tardiff, J. C. (2021, March 2021). Intrinsic and Extrinsic Modulators of Thin Filament Function: The Potential for Novel Therapeutic Targets in HCM. Northeast Ohio Medical University Invited Lecture. Virtual: Northeast Ohio Medical University.
• Tardiff, J. C. (2021, Sept 2021). Allosteric Regulation of Myofilament Ca2+ Kinetics in Thin Filament HCM. International Society for Heart Research, North American Section Meeting. Denver, CO: International Society for Heart Research.
• 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).