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.