Mechanical and signaling responses of unloaded rat soleus muscle to chronically elevated ß-myosin activity.

It has been reported that muscle functional unloading is accompanied by an increase in motoneuronal excitability despite the elimination of afferent input. Thus, we hypothesized that pharmacological potentiation of spontaneous contractile soleus muscle activity during hindlimb unloading could activate anabolic signaling pathways and prevent the loss of muscle mass and strength. To investigate these aspects and underlying molecular mechanisms, we used ß-myosin allosteric effector Omecamtiv Mekarbil (OM). We found that OM partially prevented the loss of isometric strength and intrinsic stiffness of the soleus muscle after two weeks of disuse. Notably, OM was able to attenuate the unloading-induced decrease in the rate of muscle protein synthesis (MPS). At the same time, the use of drug neither prevented the reduction in the markers of translational capacity (18S and 28S rRNA) nor activation of the ubiquitin-proteosomal system, which is evidenced by a decrease in the cross-sectional area of fast and slow muscle fibers. These results suggest that chemically-induced increase in low-intensity spontaneous contractions of the soleus muscle during functional unloading creates prerequisites for protein synthesis. At the same time, it should be assumed that the use of OM is advisable with pharmacological drugs that inhibit the expression of ubiquitin ligases.

N-Terminal Fragment of Cardiac Myosin Binding Protein C Modulates Cooperative Mechanisms of Thin Filament Activation in Atria and Ventricles.

Cardiac myosin binding protein C (cMyBP-C) is one of the essential control components of the myosin cross-bridge cycle. The C-terminal part of cMyBP-C is located on the surface of the thick filament, and its N-terminal part interacts with actin, myosin, and tropomyosin, affecting both kinetics of the ATP hydrolysis cycle and lifetime of the cross-bridge, as well as calcium regulation of the actin-myosin interaction, thereby modulating contractile function of myocardium. The role of cMyBP-C in atrial contraction has not been practically studied. We examined effect of the N-terminal C0-C1-m-C2 (C0-C2) fragment of cMyBP-C on actin-myosin interaction using ventricular and atrial myosin in an in vitro motility assay. The C0-C2 fragment of cMyBP-C significantly reduced the maximum sliding velocity of thin filaments on both myosin isoforms and increased the calcium sensitivity of the actin-myosin interaction. The C0-C2 fragment had different effects on the kinetics of ATP and ADP exchange, increasing the affinity of ventricular myosin for ADP and decreasing the affinity of atrial myosin. The effect of the C0-C2 fragment on the activation of the thin filament depended on the myosin isoforms. Atrial myosin activates the thin filament less than ventricular myosin, and the C0-C2 fragment makes these differences in the myosin isoforms more pronounced.

Exploring the Super-Relaxed State of Human Cardiac ß-Myosin by Molecular Dynamics Simulations.

Human ß-cardiac myosin plays a critical role in generating the mechanical forces necessary for cardiac muscle contraction. This process relies on a delicate dynamic equilibrium between the disordered relaxed state (DRX) and the super-relaxed state (SRX) of myosin. Disruptions in this equilibrium due to mutations can lead to heart diseases. However, the structural characteristics of SRX and the molecular mechanisms underlying pathogenic mutations have remained elusive. To bridge this gap, we conducted molecular dynamics simulations and free energy calculations to explore the conformational changes in myosin. Our findings indicate that the size of the phosphate-binding pocket can serve as a valuable metric for characterizing the transition from the DRX to SRX state. Importantly, we established a global dynamic coupling network within the myosin motor head at the residue level, elucidating how the pathogenic mutation E483K impacts the equilibrium between SRX and DRX through allosteric effects. Our work illuminates molecular details of SRX and offers valuable insights into disease treatment through the regulation of SRX.

Homologous mutations in human ß, embryonic, and perinatal muscle myosins have divergent effects on molecular power generation.

Mutations at a highly conserved homologous residue in three closely related muscle myosins cause three distinct diseases involving muscle defects: R671C in ß-cardiac myosin causes hypertrophic cardiomyopathy, R672C and R672H in embryonic skeletal myosin cause Freeman-Sheldon syndrome, and R674Q in perinatal skeletal myosin causes trismus-pseudocamptodactyly syndrome. It is not known whether their effects at the molecular level are similar to one another or correlate with disease phenotype and severity. To this end, we investigated the effects of the homologous mutations on key factors of molecular power production using recombinantly expressed human ß, embryonic, and perinatal myosin subfragment-1. We found large effects in the developmental myosins but minimal effects in ß myosin, and magnitude of changes correlated partially with clinical severity. The mutations in the developmental myosins dramatically decreased the step size and load-sensitive actin-detachment rate of single molecules measured by optical tweezers, in addition to decreasing overall enzymatic (ATPase) cycle rate. In contrast, the only measured effect of R671C in ß myosin was a larger step size. Our measurements of step size and bound times predicted velocities consistent with those measured in an in vitro motility assay. Finally, molecular dynamics simulations predicted that the arginine to cysteine mutation in embryonic, but not ß, myosin may reduce pre-powerstroke lever arm priming and ADP pocket opening, providing a possible structural mechanism consistent with the experimental observations. This paper presents direct comparisons of homologous mutations in several different myosin isoforms, whose divergent functional effects are a testament to myosin's highly allosteric nature.

Inducing positive inotropy in human iPSC-derived cardiac muscle by gene editing-based activation of the cardiac α-myosin heavy chain.

Human induced pluripotent stem cells and their differentiation into cardiac myocytes (hiPSC-CMs) provides a unique and valuable platform for studies of cardiac muscle structure-function. This includes studies centered on disease etiology, drug development, and for potential clinical applications in heart regeneration/repair. Ultimately, for these applications to achieve success, a thorough assessment and physiological advancement of the structure and function of hiPSC-CMs is required. HiPSC-CMs are well noted for their immature and sub-physiological cardiac muscle state, and this represents a major hurdle for the field. To address this roadblock, we have developed a hiPSC-CMs (ß-MHC dominant) experimental platform focused on directed physiological enhancement of the sarcomere, the functional unit of cardiac muscle. We focus here on the myosin heavy chain (MyHC) protein isoform profile, the molecular motor of the heart, which is essential to cardiac physiological performance. We hypothesized that inducing increased expression of α-MyHC in ß-MyHC dominant hiPSC-CMs would enhance contractile performance of hiPSC-CMs. To test this hypothesis, we used gene editing with an inducible α-MyHC expression cassette into isogeneic hiPSC-CMs, and separately by gene transfer, and then investigated the direct effects of increased α-MyHC expression on hiPSC-CMs contractility and relaxation function. Data show improved cardiac functional parameters in hiPSC-CMs induced with α-MyHC. Positive inotropy and relaxation was evident in comparison to ß-MyHC dominant isogenic controls both at baseline and during pacing induced stress. This approach should facilitate studies of hiPSC-CMs disease modeling and drug screening, as well as advancing fundamental aspects of cardiac function parameters for the optimization of future cardiac regeneration, repair and re-muscularization applications.

Cryo-EM structure of the folded-back state of human ß-cardiac myosin.

To save energy and precisely regulate cardiac contractility, cardiac muscle myosin heads are sequestered in an 'off' state that can be converted to an 'on' state when exertion is increased. The 'off' state is equated with a folded-back structure known as the interacting-heads motif (IHM), which is a regulatory feature of all class-2 muscle and non-muscle myosins. We report here the human ß-cardiac myosin IHM structure determined by cryo-electron microscopy to 3.6 Å resolution, providing details of all the interfaces stabilizing the 'off' state. The structure shows that these interfaces are hot spots of hypertrophic cardiomyopathy mutations that are thought to cause hypercontractility by destabilizing the 'off' state. Importantly, the cardiac and smooth muscle myosin IHM structures dramatically differ, providing structural evidence for the divergent physiological regulation of these muscle types. The cardiac IHM structure will facilitate development of clinically useful new molecules that modulate IHM stability.

Binding pocket dynamics along the recovery stroke of human ß-cardiac myosin.

The druggability of small-molecule binding sites can be significantly affected by protein motions and conformational changes. Ligand binding, protein dynamics and protein function have been shown to be closely interconnected in myosins. The breakthrough discovery of omecamtiv mecarbil (OM) has led to an increased interest in small molecules that can target myosin and modulate its function for therapeutic purposes (myosin modulators). In this work, we use a combination of computational methods, including steered molecular dynamics, umbrella sampling and binding pocket tracking tools, to follow the evolution of the OM binding site during the recovery stroke transition of human ß-cardiac myosin. We found that steering two internal coordinates of the motor domain can recapture the main features of the transition and in particular the rearrangements of the binding site, which shows significant changes in size, shape and composition. Possible intermediate conformations were also identified, in remarkable agreement with experimental findings. The differences in the binding site properties observed along the transition can be exploited for the future development of conformation-selective myosin modulators.

Dilated cardiomyopathy mutation E525K in human beta-cardiac myosin stabilizes the interacting-heads motif and super-relaxed state of myosin.

The auto-inhibited, super-relaxed (SRX) state of cardiac myosin is thought to be crucial for regulating contraction, relaxation, and energy conservation in the heart. We used single ATP turnover experiments to demonstrate that a dilated cardiomyopathy (DCM) mutation (E525K) in human beta-cardiac myosin increases the fraction of myosin heads in the SRX state (with slow ATP turnover), especially in physiological ionic strength conditions. We also utilized FRET between a C-terminal GFP tag on the myosin tail and Cy3ATP bound to the active site of the motor domain to estimate the fraction of heads in the closed, interacting-heads motif (IHM); we found a strong correlation between the IHM and SRX state. Negative stain electron microscopy and 2D class averaging of the construct demonstrated that the E525K mutation increased the fraction of molecules adopting the IHM. Overall, our results demonstrate that the E525K DCM mutation may reduce muscle force and power by stabilizing the auto-inhibited SRX state. Our studies also provide direct evidence for a correlation between the SRX biochemical state and the IHM structural state in cardiac muscle myosin. Furthermore, the E525 residue may be implicated in crucial electrostatic interactions that modulate this conserved, auto-inhibited conformation of myosin.

T cells specific for α-myosin drive immunotherapy-related myocarditis.

Immune-related adverse events, particularly severe toxicities such as myocarditis, are major challenges to the utility of immune checkpoint inhibitors (ICIs) in anticancer therapy1. The pathogenesis of ICI-associated myocarditis (ICI-MC) is poorly understood. Pdcd1-/-Ctla4+/- mice recapitulate clinicopathological features of ICI-MC, including myocardial T cell infiltration2. Here, using single-cell RNA and T cell receptor (TCR) sequencing of cardiac immune infiltrates from Pdcd1-/-Ctla4+/- mice, we identify clonal effector CD8+ T cells as the dominant cell population. Treatment with anti-CD8-depleting, but not anti-CD4-depleting, antibodies improved the survival of Pdcd1-/-Ctla4+/- mice. Adoptive transfer of immune cells from mice with myocarditis induced fatal myocarditis in recipients, which required CD8+ T cells. The cardiac-specific protein α-myosin, which is absent from the thymus3,4, was identified as the cognate antigen source for three major histocompatibility complex class I-restricted TCRs derived from mice with fulminant myocarditis. Peripheral blood T cells from three patients with ICI-MC were expanded by α-myosin peptides. Moreover, these α-myosin-expanded T cells shared TCR clonotypes with diseased heart and skeletal muscle, which indicates that α-myosin may be a clinically important autoantigen in ICI-MC. These studies underscore the crucial role for cytotoxic CD8+ T cells, identify a candidate autoantigen in ICI-MC and yield new insights into the pathogenesis of ICI toxicity.

Genetic Association of Beta-Myosin Heavy-Chain Gene (MYH7) with Cardiac Dysfunction.

Cardiac dysfunction accelerates the risk of heart failure, and its pathogenesis involves a complex interaction between genetic and environmental factors. Variations in myosin affect contractile abilities of cardiomyocytes and cause structural and functional abnormalities in myocardium. The study aims to find the association of MYH7 rs121913642 (c.1594 T>C) and rs121913645 (c.667G>A) variants with cardiac dysfunction in the Punjabi Pakistani population. Patients with heart failure (n = 232) and healthy controls (n = 205) were enrolled in this study. MYH7 variant genotyping was performed using tetra ARMS-PCR. MYH7 rs121913642 TC genotype was significantly more prevalent in the patient group (p < 0.001). However, MYH7 rs121913645 genotype frequencies were not significantly different between the patient and control groups (p < 0.666). Regression analysis also revealed that the rs121913642 C allele increases the risk of cardiac failure by ~2 [OR:1.98, CI: 1.31−2.98, p < 0.001] in comparison to the T allele. High levels of the cardiac enzymes cardiac troponin I (cTnI) and CK-MB were observed in patients. There was also an increase in total cholesterol, LDL cholesterol, and uric acid in patients compared to the healthy control group (p < 0.001). In conclusion, the MYH7 gene variant rs121913642 is genetically associated with cardiac dysfunction and involved in the pathogenesis of HF.

Myosin essential light chain 1sa decelerates actin and thin filament gliding on ß-myosin molecules.

The ß-myosin heavy chain expressed in ventricular myocardium and the myosin heavy chain (MyHC) in slow-twitch skeletal Musculus soleus (M. soleus) type-I fibers are both encoded by MYH7. Thus, these myosin molecules are deemed equivalent. However, some reports suggested variations in the light chain composition between M. soleus and ventricular myosin, which could influence functional parameters, such as maximum velocity of shortening. To test for functional differences of the actin gliding velocity on immobilized myosin molecules, we made use of in vitro motility assays. We found that ventricular myosin moved actin filaments with ∼0.9 µm/s significantly faster than M. soleus myosin (0.3 µm/s). Filaments prepared from isolated actin are not the native interaction partner of myosin and are believed to slow down movement. Yet, using native thin filaments purified from M. soleus or ventricular tissue, the gliding velocity of M. soleus and ventricular myosin remained significantly different. When comparing the light chain composition of ventricular and M. soleus ß-myosin, a difference became evident. M. soleus myosin contains not only the "ventricular" essential light chain (ELC) MLC1sb/v, but also an additional longer and more positively charged MLC1sa. Moreover, we revealed that on a single muscle fiber level, a higher relative content of MLC1sa was associated with significantly slower actin gliding. We conclude that the ELC MLC1sa decelerates gliding velocity presumably by a decreased dissociation rate from actin associated with a higher actin affinity compared to MLC1sb/v. Such ELC/actin interactions might also be relevant in vivo as differences between M. soleus and ventricular myosin persisted when native thin filaments were used.

Hypertrophic cardiomyopathy mutations in the pliant and light chain-binding regions of the lever arm of human ß-cardiac myosin have divergent effects on myosin function.

Mutations in the lever arm of ß-cardiac myosin are a frequent cause of hypertrophic cardiomyopathy, a disease characterized by hypercontractility and eventual hypertrophy of the left ventricle. Here, we studied five such mutations: three in the pliant region of the lever arm (D778V, L781P, and S782N) and two in the light chain-binding region (A797T and F834L). We investigated their effects on both motor function and myosin subfragment 2 (S2) tail-based autoinhibition. The pliant region mutations had varying effects on the motor function of a myosin construct lacking the S2 tail: overall, D778V increased power output, L781P reduced power output, and S782N had little effect on power output, while all three reduced the external force sensitivity of the actin detachment rate. With a myosin containing the motor domain and the proximal S2 tail, the pliant region mutations also attenuated autoinhibition in the presence of filamentous actin but had no impact in the absence of actin. By contrast, the light chain-binding region mutations had little effect on motor activity but produced marked reductions in autoinhibition in both the presence and absence of actin. Thus, mutations in the lever arm of ß-cardiac myosin have divergent allosteric effects on myosin function, depending on whether they are in the pliant or light chain-binding regions.

Cardiac ventricular myosin and slow skeletal myosin exhibit dissimilar chemomechanical properties despite bearing the same myosin heavy chain isoform.

The myosin II motors are ATP-powered force-generating machines driving cardiac and muscle contraction. Myosin II heavy chain isoform-beta (ß-MyHC) is primarily expressed in the ventricular myocardium and in slow-twitch muscle fibers, such as M. soleus. M. soleus-derived myosin II (SolM-II) is often used as an alternative to the ventricular ß-cardiac myosin (ßM-II); however, the direct assessment of biochemical and mechanical features of the native myosins is limited. By employing optical trapping, we examined the mechanochemical properties of native myosins isolated from the rabbit heart ventricle and soleus muscles at the single-molecule level. We found purified motors from the two tissue sources, despite expressing the same MyHC isoform, displayed distinct motile and ATPase kinetic properties. We demonstrate ßM-II was approximately threefold faster in the actin filament-gliding assay than SolM-II. The maximum actomyosin (AM) detachment rate derived in single-molecule assays was also approximately threefold higher in ßM-II, while the power stroke size and stiffness of the "AM rigor" crossbridge for both myosins were comparable. Our analysis revealed a higher AM detachment rate for ßM-II, corresponding to the enhanced ADP release rates from the crossbridge, likely responsible for the observed differences in the motility driven by these myosins. Finally, we observed a distinct myosin light chain 1 isoform (MLC1sa) that associates with SolM-II, which might contribute to the observed kinetics differences between ßM-II and SolM-II. These results have important implications for the choice of tissue sources and justify prerequisites for the correct myosin heavy and light chains to study cardiomyopathies.

Nanosurfer assay dissects ß-cardiac myosin and cardiac myosin-binding protein C interactions.

Cardiac myosin-binding protein C (cMyBP-C) modulates cardiac contractility through putative interactions with the myosin S2 tail and/or the thin filament. The relative contribution of these binding-partner interactions to cMyBP-C modulatory function remains unclear. Hence, we developed a "nanosurfer" assay as a model system to interrogate these cMyBP-C binding-partner interactions. Synthetic thick filaments were generated using recombinant human ß-cardiac myosin subfragments (HMM or S1) attached to DNA nanotubes, with 14- or 28-nm spacing, corresponding to the 14.3-nm myosin spacing in native thick filaments. The nanosurfer assay consists of DNA nanotubes added to the in vitro motility assay so that myosins on the motility surface effectively deliver thin filaments to the DNA nanotubes, enhancing thin filament gliding probability on the DNA nanotubes. Thin filament velocities on nanotubes with either 14- or 28-nm myosin spacing were no different. We then characterized the effects of cMyBP-C on thin filament motility by alternating HMM and cMyBP-C N-terminal fragments (C0-C2 or C1-C2) on nanotubes every 14 nm. Both C0-C2 and C1-C2 reduced thin filament velocity four- to sixfold relative to HMM alone. Similar inhibition occurred using the myosin S1 construct, which lacks the myosin S2 region proposed to interact with cMyBP-C, suggesting that the cMyBP-C N terminus must interact with other myosin head domains and/or actin to slow thin filament velocity. Thin filament velocity was unaffected by the C0-C1f fragment, which lacks the majority of the M-domain, supporting the importance of this domain for inhibitory interaction(s). A C0-C2 fragment with phospho-mimetic replacement in the M-domain showed markedly less inhibition of thin filament velocity compared with its phospho-null counterpart, highlighting the modulatory role of M-domain phosphorylation on cMyBP-C function. Therefore, the nanosurfer assay provides a platform to precisely manipulate spatially dependent cMyBP-C binding-partner interactions, shedding light on the molecular regulation of ß-cardiac myosin contractility.

Two Classes of Myosin Inhibitors, Para-nitroblebbistatin and Mavacamten, Stabilize ß-Cardiac Myosin in Different Structural and Functional States.

In addition to a conventional relaxed state, a fraction of myosins in the cardiac muscle exists in a low-energy consuming super-relaxed (SRX) state, which is kept as a reserve pool that may be engaged under sustained increased cardiac demand. The conventional relaxed and the super-relaxed states are widely assumed to correspond to a structure where myosin heads are in an open configuration, free to interact with actin, and a closed configuration, inhibiting binding to actin, respectively. Disruption of the myosin SRX population is an emerging model in different heart diseases, such as hypertrophic cardiomyopathy, which results in excessive muscle contraction, and stabilizing them using myosin inhibitors is budding as an attractive therapeutic strategy. Here we examined the structure-function relationships of two myosin ATPase inhibitors, mavacamten and para-nitroblebbistatin, and found that binding of mavacamten at a site different than para-nitroblebbistatin populates myosin into the SRX state. Para-nitroblebbistatin, binding to a distal pocket to the myosin lever arm near the nucleotide-binding site, does not affect the usual myosin SRX state but instead appears to render myosin into a new, perhaps much more inhibited, 'ultra-relaxed' state. X-ray scattering-based rigid body modeling shows that both mavacamten and para-nitroblebbistatin induce novel conformations in human ß-cardiac heavy meromyosin that diverge significantly from the hypothetical open and closed states, and furthermore, mavacamten treatment causes greater compaction than para-nitroblebbistatin. Taken together, we conclude that mavacamten and para-nitroblebbistatin stabilize myosin in different structural states, and such states may give rise to different functional energy-sparing states.

Alpha and beta myosin isoforms and human atrial and ventricular contraction.

Human atrial and ventricular contractions have distinct mechanical characteristics including speed of contraction, volume of blood delivered and the range of pressure generated. Notably, the ventricle expresses predominantly ß-cardiac myosin while the atrium expresses mostly the α-isoform. In recent years exploration of the properties of pure α- & ß-myosin isoforms have been possible in solution, in isolated myocytes and myofibrils. This allows us to consider the extent to which the atrial vs ventricular mechanical characteristics are defined by the myosin isoform expressed, and how the isoform properties are matched to their physiological roles. To do this we Outline the essential feature of atrial and ventricular contraction; Explore the molecular structural and functional characteristics of the two myosin isoforms; Describe the contractile behaviour of myocytes and myofibrils expressing a single myosin isoform; Finally we outline the outstanding problems in defining the differences between the atria and ventricles. This allowed us consider what features of contraction can and cannot be ascribed to the myosin isoforms present in the atria and ventricles.

Ginkgolide B Protects Cardiomyocytes from Angiotensin II-Induced Hypertrophy via Regulation of Autophagy through SIRT1-FoxO1.

Ginkgolide B (GB) is an active ingredient extracted from Ginkgo biloba leaves. However, the effects of GB on cardiac hypertrophy remain unclear. The study is aimed at determining whether GB could alleviate cardiac hypertrophy and exploring its underlying molecular mechanism. Rat cardiomyocyte cell line H9c2 cells were pretreated with GB and incubated with angiotensin II (Ang II) to simulate an in vitro cardiac hypertrophy model. Cell viability, cell size, hypertrophy markers, and autophagy were determined in H9c2 cells after Ang II treatment. Proteins involved in autophagy and the SIRT1 pathway were determined by western blot. Our data demonstrated that GB attenuated Ang II-induced cardiac hypertrophy and reduced the mRNA expressions of hypertrophy marker, atrial natriuretic peptide (ANP), and ß-myosin heavy chain (ß-MHC). GB further increased Ang II-induced autophagy in H9c2 cells and modulated expressions of autophagy-related proteins Beclin1 and P62. Modulation of autophagy using autophagy inhibitor 3-methyladenine (3-MA) could abrogate GB-downregulated transcription of NPPA. We then showed that GB attenuated Ang II-induced oxidative stress and reduction in SIRT1 and FoxO1 protein expression. Finally, the effect of GB on autophagy and cardiac hypertrophy could be reversed by SIRT1 inhibitor EX-527. GB inhibits Ang II-induced cardiac hypertrophy by enhancing autophagy via the SIRT1-FoxO1 signaling pathway and might be a potential agent in treating pathological cardiac hypertrophy.

Investigation of the Recovery Stroke and ATP Hydrolysis and Changes Caused Due to the Cardiomyopathic Point Mutations in Human Cardiac ß Myosin.

Human cardiac ß 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 Dictyostelium 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 HPO42- with the stabilization of the metaphosphate intermediate. We also observed clear differences between the Dictyostelium myosin II and human cardiac ß myosin for ATP hydrolysis as well as predict the effect of the mutation I467T on the chemical step.

Hypertrophic cardiomyopathy ß-cardiac myosin mutation (P710R) leads to hypercontractility by disrupting super relaxed state.

Hypertrophic cardiomyopathy (HCM) is the most common inherited form of heart disease, associated with over 1,000 mutations, many in ß-cardiac myosin (MYH7). Molecular studies of myosin with different HCM mutations have revealed a diversity of effects on ATPase and load-sensitive rate of detachment from actin. It has been difficult to predict how such diverse molecular effects combine to influence forces at the cellular level and further influence cellular phenotypes. This study focused on the P710R mutation that dramatically decreased in vitro motility velocity and actin-activated ATPase, in contrast to other MYH7 mutations. Optical trap measurements of single myosin molecules revealed that this mutation reduced the step size of the myosin motor and the load sensitivity of the actin detachment rate. Conversely, this mutation destabilized the super relaxed state in longer, two-headed myosin constructs, freeing more heads to generate force. Micropatterned human induced pluripotent derived stem cell (hiPSC)-cardiomyocytes CRISPR-edited with the P710R mutation produced significantly increased force (measured by traction force microscopy) compared with isogenic control cells. The P710R mutation also caused cardiomyocyte hypertrophy and cytoskeletal remodeling as measured by immunostaining and electron microscopy. Cellular hypertrophy was prevented in the P710R cells by inhibition of ERK or Akt. Finally, we used a computational model that integrated the measured molecular changes to predict the measured traction forces. These results confirm a key role for regulation of the super relaxed state in driving hypercontractility in HCM with the P710R mutation and demonstrate the value of a multiscale approach in revealing key mechanisms of disease.