Discussion
This study contributes new biophysical observations about the effects of mavacamten on human cardiac muscle function at physiological temperature. With recent therapeutic treatments for heart failure showing promise at the sarcomere level (Teerlink et al., 2016; Heitner et al., 2019), there is a need to better define and identify molecular mechanisms of contraction that may ultimately be targeted to benefit patient care. Sarcomere length-dependent increases in Ca2+-sensitivity of contraction represent a key physiological relationship at the cellular and tissue level that underlies the Frank-Starling response in healthy hearts. Pharmaceuticals that compromised length-dependent activation may ultimately prove to be less effective therapies for heart disease (Gollapudi et al., 2017; Henze et al., 2019). Herein we show that mavacamten preserves sarcomere length-dependent increases in the Ca2+-sensitivity of force, while reducing maximum levels of force production.
These data have implications for mavacamten affecting systolic and diastolic aspects of cardiac muscle function. On the systolic side, HCM has typically been viewed as a hypercontractile phenotype due to a ‘gain of function’ mutation (Moore et al., 2012; Green et al., 2016). Thus, reducing the Ca2+-sensitivity of the myocardium with mavacamten treatment could reduce the hypercontractile state during systole, normalizing function and potentially leading to beneficial remodeling of the heart. Administering mavacamten (early in life) to transgenic mice carrying the myosin R403Q mutation, which typically displays a potent HCM phenotype, helped reduce ventricular hypertrophy, cardiomyocyte disarray, and myocardial fibrosis (Green et al., 2016). On the other side of a heartbeat, diastolic dysfunction leads to impaired relaxation and ventricular filling, which underlies roughly 50% of heart failure diagnoses (Aljaroudi et al., 2012; Lekavich et al., 2015; Mozaffarian et al., 2016). The findings that mavacamten reduced passive tension at both sarcomere lengths (Fig. 3B) and increased cross-bridge detachment rates under maximally activated conditions (Fig. 6B) suggests that mavacamten may improve relaxation.
Prior biophysical assays have shown that mavacamten slows cross-bridge recruitment, Pi release, and actin-associated ADP release, all of which underlie strong cross-bridge binding and force-generation (Green et al., 2016; Kawas et al., 2017; Rohde et al., 2018). In skinned myocardial mechanics and intact myocyte assays mavacamten reduced contractility in a dose-dependent manner, slowing the rate of force development (Mamidi et al., 2018) and increasing the relaxation rate (Toepfer et al., 2019b, 2019a). Our frequency-dependent viscoelastic stiffness data support these findings in human myocardium at physiological temperature (Fig. 5). Mavacamten shifted the negative viscous moduli-frequency relationships towards lower frequencies and reduced the amount of work generated by the myocardiaum (Fig. 5C-D), consistent with impaired force generation (Fig. 2) and slowed cross-bridge recruitment (Fig 6A). The frequency range where negative viscous moduli occur reflects the physiological range of heart rates within a species, and the diminished range for mavacamten-treated strips could indicate that mavacamten plays a limiting role at the upper end of this range. Our findings that mavacamten speeds cross-bridge detachment (Fig. 6B) agrees with data showing that mavacamten increases relaxation rates in human myofibrils (Scellini et al., 2020). Similar measurements by Scellini et al. show that mavacamten had no effect on relaxation rate in rabbit skeletal myofibrils, which suggests that the effects of mavacamten may vary with species and muscle type (Scellini et al., 2020). Additional studies will be required to more fully define the potential impacts of mavacamten on different myosin isoforms.
Previously, we used mathematical models to test how mechanosensitive contributions of the myosin OFF-ON equilibrium influence length-dependent activation of contraction (Campbell et al. 2018). These data showed that elevated force levels, from passive and/or active mechanisms, amplify cross-bridge recruitment and force production, more fully illustrating the fascinating (yet complicated) impacts of dynamic filament regulation (Fig. 1) that underly force production and relaxation throughout a heartbeat. Knowing that mavacamten stabilizes the OFF state (Anderson et al., 2018; Rohde et al., 2018), we wondered if sarcomere length-dependent increases in Ca2+-sensitivity would be amplified in mavacamten treated strips. Our data do not show this response in a statistically significant manner (Fig. 4A), although the pCa50 change between 1.9 µm and 2.3 µm was 3-fold greater for mavacamten treated strips (on average ΔpCa50=0.03 for control vs. 0.09 for mavacamten-treated strips). These findings differ from a preliminary report using permeabilized myocardial strips from pigs, where mavacamten suppressed length-dependent increases from OFF-to-ON and abolished length-dependent increases in Ca2+-sensitivity of contraction (Henze et al., 2019). The functional effects of the OFF-ON equilibrium are only starting to come to light for the muscle physiology research field, yet this equilibrium represents a critical mechanism underlying regulatory coupling between the thick- and thin-filaments and the Frank-Starling law of the heart. One interesting aspect of this OFF-ON relationship is the force-dependent recruitment of myosin from the OFF-to-ON state, which encompasses a force-feedback effect to amplify contractility as force develops during isometric contractions and when muscles are stretched (Ait-Mou et al., 2016; Campbell, 2017; Fusi et al., 2017; Reconditi et al., 2017; Zhang et al., 2017; Piazzesi et al., 2018; Brunello et al., 2020). Thus, any means of modulating length-dependent changes Ca2+-sensitivity leads to significant functional effects, because myofilaments are highly cooperative and subtle shifts in pCa50 can produce large change in force-production as muscle length changes. Prior to the discovery of force-dependent OFF-ON transitions, length-dependent changes in Ca2+-sensitivity were typically thought to reflect changes in thin filament function and/or the binding kinetics of individual myosin heads (Gordon et al., 2000). Therefore, ongoing and future research testing the effects of mavacamten to preserve or even amplify length-dependent activation in transgenic models of heart failure and human tissue samples from heart failure patients will be helpful for informing the potential scope of mavacamten as a therapeutic treatment for cardiac dysfunction. More generally, these findings also imply that the OFF-ON equilibrium of myosin may be a particularly important therapeutic target for modulating myocardial function.