Background and Purpose: Heart failure can reflect impaired contractile function at the myofilament level. In healthy hearts, myofilaments become more sensitive to Ca 2+ as cells are stretched. This represents a fundamental property of the myocardium that contributes to the Frank-Starling response, although the molecular mechanisms underlying the effect remain unclear. Mavacamten, which binds to myosin, is under investigation as a potential therapy for heart disease. We investigated how mavacamten affects the sarcomere-length dependence of Ca 2+-sensitive isometric contraction to determine how mavacamten might modulate the Frank-Starling mechanism. Experimental Approach: Multicellular preparations from the left ventricular-free wall of hearts from organ donors were chemically permeabilized and Ca 2+ activated in the presence or absence of 0.5-μM mavacamten at 1.9 or 2.3-μm sarcomere length (37 C). Isometric force and frequency-dependent viscoelastic myocardial stiffness measurements were made. Key Results: At both sarcomere lengths, mavacamten reduced maximal force and Ca 2+ sensitivity of contraction. In the presence and absence of mavacamten, Ca 2+ sensitivity of force increased as sarcomere length increased. This suggests that the length-dependent activation response was maintained in human myocardium, even though mavacamten reduced Ca 2+ sensitivity. There were subtle effects of mavacamten reducing force values under relaxed conditions (pCa 8.0), as well as slowing myosin cross-bridge recruitment and speeding cross-bridge detachment under maximally activated conditions (pCa 4.5). Conclusion and Implications: Mavacamten did not eliminate sarcomere lengthdependent increases in the Ca 2+ sensitivity of contraction in myocardial strips from organ donors at physiological temperature. Drugs that modulate myofilament function may be useful therapies for cardiomyopathies. K E Y W O R D S cardiac muscle mechanics, human myosin, mavacamten, sarcomere length Abbreviations: (Pi), inorganic phosphate; (pCa), −log 10 [Ca 2+ ]; (F pas), passive stress under relaxed conditions; (F act), maximal Ca 2+-activated stress; (pCa 50), free Ca 2+ concentration required to develop half the maximum Ca 2+-activated stress; (nH), Hill coefficient.

Morbidity and mortality associated with heart disease is a growing threat to the global population and novel therapies are needed. Mavacamten (formerly called MYK-461) is a small molecule that binds to cardiac myosin and inhibits myosin ATPase. Mavacamten is currently in clinical trials for the treatment of obstructive hypertrophic cardiomyopathy (HCM), and it may provide benefits for treating other forms of heart disease. We investigated the effect of mavacamten on cardiac muscle contraction in two transgenic mouse lines expressing the human isoform of cardiac myosin regulatory light chain (RLC) in their hearts. Control mice expressed wild-type RLC (WT-RLC), and HCM mice expressed the N47K RLC mutation. In the absence of mavacamten, skinned papillary muscle strips from WT-RLC mice produced greater isometric force than strips from N47K mice. Adding 0.3 µM mavacamten decreased maximal isometric force and reduced Ca2+-sensitivity of contraction for both genotypes, but this reduction in pCa50 was nearly twice as large for WT-RLC vs. N47K. We also used stochastic length-perturbation analysis to characterize cross-bridge kinetics. The cross-bridge detachment rate was measured as a function of [MgATP] to determine the effect of mavacamten on myosin nucleotide handling rates. Mavacamten increased the MgADP release and MgATP binding rates for both genotypes, thereby contributing to faster cross-bridge detachment, which could speed myocardial relaxation during diastole. Our data suggest that mavacamten reduces isometric tension and Ca2+-sensitivity of contraction via decreased strong cross-bridge binding. Mavacamten may become a useful therapy for patients with heart disease, including some forms of HCM.

Background and Purpose: Heart failure can reflect impaired contractile function at the myofilament level. In healthy hearts, myofilaments become more sensitive to Ca2+ as cells are stretched. This represents a fundamental property of myocardium that contributes to the Frank-Starling response, although the molecular mechanisms underlying the effect remain unclear. Mavacamten is a drug that binds to myosin, which is under investigation as a potential therapy for cardiovascular disease. We tested how mavacamten affects the sarcomere-length dependence of Ca2+-sensitive isometric contraction to determine how mavacamten might modulate the Frank-Starling mechanism. Experimental Approach: Multicellular preparations from the left ventricular free wall of hearts procured from organ donors were chemically permeabilized and Ca2+-activated in the presence or absence of 0.5 μM mavacamten at 1.9 or 2.3 μm sarcomere length (37°C). Isometric force and frequencydependent viscoelastic myocardial stiffness measurements were made. Key Results: At both sarcomere lengths, mavacamten reduced maximal force and Ca2+-sensitivity of contraction. In the presence and absence of mavacamten, Ca2+-sensitivity of force increased as sarcomere length increased. This suggests that the length-dependent activation response was maintained in human myocardium, even though mavacamten reduced Ca2+-sensitivity. There were subtle effects of mavacamten reducing force values under relaxed conditions (pCa 8.0), as well as slowing myosin cross-bridge recruitment and speeding cross-bridge detachment under maximally activated conditions (pCa 4.5). Conclusion and Implications: Mavacamten did not eliminate sarcomere lengthdependent increases in the Ca2+-sensitivity of contraction in myocardial strips from organ donors at physiological temperature. Pharmaceuticals that modulate myofilament function may be useful therapies for cardiovascular disease.

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We have used spectroscopic and functional assays to evaluate the effects of actin-binding compounds on striated muscle protein structure and function. Actin is present in every human cell, and its interaction with multiple myosin isoforms and multiple actin-binding proteins is essential for cellular viability. Our high-throughput time-resolved fluorescence resonance energy transfer (TR-FRET) assay previously detected several compounds that bind to actin and affected actomyosin structure and function (Guhathakurta, et al. 2018, J Biol Chem 293:12288). To determine the muscle specificity of these compounds, we tested their effects on intact skeletal and cardiac myofibrils, which represent a more physiologically relevant environment. We found that the concentration-dependent responses of several compounds were different for skeletal and cardiac myofibrils, suggesting that the mode of action is different for the two muscle types. These compounds also affected the transition of monomeric G-actin to filamentous F-actin of different actin isoforms (skeletal and cardiac) by different degrees; further confirming the specificity of these compounds for a specific muscle type. We conclude that these compounds differentially affect skeletal and cardiac muscles, and these results set the stage to screen large chemical libraries for discovery of novel actin-binding drugs with specific therapeutic potential for treating disorders of cardiac or skeletal muscle. This work was supported by NIH grants to DDT (R01AR032961, R37AG26160).