Introduction
Cardiovascular disease remains a leading cause of death world-wide. Heart failure occurs when a patient’s heart loses the capacity to adequately pump blood around their body (Mozaffarian et al., 2016). Heart failure is a growing health problem affecting ~30 million people worldwide, with 50% of heart failure patients dying within five years of diagnosis (Ambrosy et al., 2014; Mozaffarian et al., 2016). The causes of heart-failure are multi-factorial, but dysregulated myofilament function within the sarcomere is a leading contributor. Recently, multiple pharmaceutical compounds have been developed to directly influence myofilament protein function as potential new therapies for cardiac disease (Cleland et al., 2011; Malik et al., 2011; Green et al., 2016; Teerlink et al., 2016; Kawas et al., 2017; Grillo et al., 2018; Heitner et al., 2019).
Cardiac muscle contraction is powered by cyclic interactions between myosin cross-bridges along thick-filaments and actin binding sites along thin-filaments (Huxley and Hanson, 1954; Lymn and Taylor, 1971). Contractility is modulated by several well-known mechanisms including: i) Ca2+-regulation via the thin filament proteins troponin and tropomyosin, which modulates the number of available actin binding on a thin-filament as intracellular [Ca2+] rises and falls throughout a heartbeat; and ii) thick-to-thin filament overlap, which determines how many cross-bridges are close enough to bind with Ca2+-activated actin sites. A mechanosensitive thick-filament regulatory mechanism has been discovered in the last few years as well, whereby myosin heads transition between OFF (also called the super-relaxed state, or interacting heads motif) and ON states (also called the disordered relaxed state) (Hooijman et al., 2011; Campbell, 2017). Heads in the OFF state are unable to bind actin (Fig. 1), while those in the ON state can form cross-bridges by attaching to actin (Spudich, 2015; Anderson et al., 2018; Liu et al., 2018). OFF-ON transitions can be very dynamic (Fusi et al., 2017; Reconditi et al., 2017; Piazzesi et al., 2018) and equilibrium kinetics are known to be regulated by i) biochemical and steric interactions with thick-filament regulatory proteins [regulatory light chain (Kampourakis et al., 2016; Zhang et al., 2017) and cardiac myosin binding protein-C (McNamara et al., 2015, 2017)], as well as ii) myocardial force levels (Linari et al., 2015; Ait-Mou et al., 2016; Fusi et al., 2016; Kampourakis et al., 2016; Campbell et al., 2018). These multiple regulation pathways combine to influence length-dependent activation of contraction, wherein the myofilaments become more sensitive to Ca2+ as muscle cells are stretched. Length-dependent activation is an important cellular-level mechanism that underpins the Frank-Starling mechanism and enables the heart to increase cardiac output in response to elevated filling pressures.
Mavacamten (formerly known as MYK-461; MyoKardia Inc.) is a pharmaceutical under investigation to treat cardiac hypercontractility, a phenotype commonly associated with a form heart disease called hypertrophic cardiomyopathy (HCM) (Green et al., 2016; Stern et al., 2016). HCM affects ~1 in 300 people, and typically causes thickening and stiffening of the ventricular wall, thereby impairing ventricular filling and reducing cardiac output (Klein et al., 1965; Brandt et al., 1967; Wilson et al., 1967; Stewart et al., 1968; Maron et al., 1995; Semsarian et al., 2015). Mavacamten binds to myosin, inhibits actin-myosin ATPase activity, and stabilizes the myosin OFF state (Green et al., 2016; Anderson et al., 2018; Rohde et al., 2018; Toepfer et al., 2019b). Solution biochemistry, in vitro motility, and single molecule assays have shown that mavacamten slows the rates of inorganic phosphate (Pi) and ADP release (Green et al., 2016; Kawas et al., 2017; Rohde et al., 2018). Mavacamten also slows the rate of cross-bridge recruitment in skinned rodent myocardium (Mamidi et al., 2018). Increases in mavacamten concentration reduced sarcomere shortening, while increasing the speed of relaxation in isolated electrically paced myocytes (Toepfer et al., 2019b, 2019a). Although there are mixed findings with respect to mavacamten either reducing or not affecting Ca2+-sensitivity of contraction in skinned myocardial strips (Green et al., 2016; Mamidi et al., 2018), studies have shown that mavacamten consistently reduces maximal force production (Green et al., 2016; Anderson et al., 2018; Mamidi et al., 2018).
These combined effects of mavacamten on myosin force production and ATPase activity are starting to provide evidence that mavacamten may reduce hypercontractility at the level of the myosin motor (Heitner et al., 2019; Tuohy et al., 2020). However, prior studies have not provided a consistent interpretation by which mavacamten affects myosin cross-bridge kinetics, nor how mavacamten might influence length-dependent myocardial function (i.e. the cellular basis of the Frank-Starling law). Therefore, we tested the effect of mavacamten on Ca2+-activated force production at 1.9 and 2.3 µm sarcomere length in permeabilized myocardial strips from organ donors.