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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.

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 Ca2+ binding strength and the energy required to remove Ca2+ 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 Ca2+ 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 Ca2+ 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.

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.

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.

Heart failure is a systemic disease in which neuroendocrine activation, inflammation and metabolic changes can impair cardiac function. In addition, variants in genes encoding sarcomeric proteins can predispose individuals to develop heart failure. The 2022 Gordon Research Conference on Cardiac Regulatory Mechanisms aims to identify novel treatment targets by applying high-resolution approaches to complex cardiac disorders.

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.

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.

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.

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.

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.