Modulating Beta-Cardiac Myosin Function at the Molecular and Tissue Levels

Abstract

Inherited cardiomyopathies are a common form of heart disease that are caused by mutations in sarcomeric proteins with beta cardiac myosin (MYH7) being one of the most frequently affected genes. Since the discovery of the first cardiomyopathy associated mutation in beta-cardiac myosin, a major goal has been to correlate the in vitro myosin motor properties with the contractile performance of cardiac muscle. There has been substantial progress in developing assays to measure the force and velocity properties of purified cardiac muscle myosin but it is still challenging to correlate results from molecular and tissue-level experiments. Mutations that cause hypertrophic cardiomyopathy are more common than mutations that lead to dilated cardiomyopathy and are also often associated with increased isometric force and hyper-contractility. Therefore, the development of drugs designed to decrease isometric force by reducing the duty ratio (the proportion of time myosin spends bound to actin during its ATPase cycle) has been proposed for the treatment of hypertrophic cardiomyopathy. Para-Nitroblebbistatin is a small molecule drug proposed to decrease the duty ratio of class II myosins. We examined the impact of this drug on human beta cardiac myosin using purified myosin motor assays and studies of permeabilized muscle fiber mechanics. We find that with purified human beta-cardiac myosin para-Nitroblebbistatin slows actin-activated ATPase and in vitro motility without altering the ADP release rate constant. In permeabilized human myocardium, para-Nitroblebbistatin reduces isometric force, power, and calcium sensitivity while not changing shortening velocity or the rate of force development (ktr). Therefore, designing a drug that reduces the myosin duty ratio by inhibiting strong attachment to actin while not changing detachment can cause a reduction in force without changing shortening velocity or relaxation.

Controlling load-dependent contractility of the heart at the single molecule level

“…Interestingly, we find that the duty ratios of the activators and HCM mutations in general lie above those of the inhibitor and DCM mutations. This result is consistent with physiological expectations that HCM mutations and activators cause myosin to have a higher duty ratio, while DCM mutations and inhibitors result in the opposite [28][29][30]34,43 . The inhibitor F3345 (4) is an outlier whose curve lies among those of the activators/HCM's.…”

Optimized single molecule fluorescence sheds light on elusive enzymatic mechanisms

“…Reduced velocity ( Fig. 2a) can be attributed to the effect of DMSO (2 %), used as a vehicle for COT and NBA and known as reversible inhibitor of actomyosin function (25)(26)(27)(28)(29). Importantly, however, the fraction of motile filaments (FMFs) was not affected (Fig.…”

Myocardial Contractility: Historical and Contemporary Considerations

“…Of particular relevance to the current discussion is the identification and investigation of genetic mutations and compounds that change the heart's inotropic state by altering sarcomeric protein cross-bridge (XB) kinetics (Spudich, 2011(Spudich, , 2014Ait Mou et al, 2015;Tardiff et al, 2015;Tang et al, 2017;Nanasi et al, 2018;Wang et al, 2018). The small molecule myosin regulator, omecamtiv mecarbil, for example, enhances sarcomeric force development by increasing the number and synchrony of strongly bound myosin crossbridges (XB's), thereby increasing sarcomeric force independent of changes in intracellular calcium concentration [Ca +2 ] i , calcium transients, shortening velocity or oxygen consumption (Malik et al, 2011;Tardiff et al, 2015;Utter et al, 2015;Hashem et al, 2017;Planelles-Herrero et al, 2017;Swenson et al, 2017;Kaplinsky and Mallarkey, 2018;Spudich, 2019;Kieu et al, 2019).…”

“…Alternatively, compounds that reduce sarcomeric force without changing shortening velocity or the rate of myocardial relaxation (ex. para-nitroblebbistatin; mavacapten) are being studied as treatment for hypertrophic cardiomyopathy (HCM) (Kawas et al, 2017;Tang et al, 2017;Kampourakis et al, 2018;Spudich, 2019).…”