Effects of hypertrophic and dilated cardiomyopathy mutations on power output by human β-cardiac myosin

Abstract

Hypertrophic cardiomyopathy is the most frequently occurring inherited cardiovascular disease, with a prevalence of more than one in 500 individuals worldwide. Genetically acquired dilated cardiomyopathy is a related disease that is less prevalent. Both are caused by mutations in the genes encoding the fundamental forcegenerating protein machinery of the cardiac muscle sarcomere, including human β-cardiac myosin, the motor protein that powers ventricular contraction. Despite numerous studies, most performed with non-human or non-cardiac myosin, there is no clear consensus about the mechanism of action of these mutations on the function of human β-cardiac myosin. We are using a recombinantly expressed human β-cardiac myosin motor domain along with conventional and new methodologies to characterize the forces and velocities of the mutant myosins compared with wild type. Our studies are extending beyond myosin interactions with pure actin filaments to include the interaction of myosin with regulated actin filaments containing tropomyosin and troponin, the roles of regulatory light chain phosphorylation on the functions of the system, and the possible roles of myosin binding protein-C and titin, important regulatory components of both cardiac and skeletal muscles.

The myosin mesa and the basis of hypercontractility caused by hypertrophic cardiomyopathy mutations

“…This parameter may be key in fine-tuned regulation of the heart 30–33 , and, as previously hypothesized 25,28,29,34–37 , may be pivotal in determining the hypercontractility known to arise from HCM mutations in human β-cardiac myosin.…”

“…Our mesa hypothesis 36,37 states that interactions between S1 and proximal S2 and/or MyBP-C would sequester heads from being able to interact with actin. To set up a functional assay, we took advantage of observations that phosphorylation of Ser15 on the myosin regulatory light chain (RLC) has considerable effects on cardiac muscle force production, shortening velocity and peak power output, and is altered during cardiac disease states 46–48 .…”

“…We have proposed that the cluster of arginine residues on the mesa that are mutated in HCM are likely to act as a binding domain for another protein, thereby holding the myosin heads in an inactive form 36 . We noted two candidate binding partners near S1 in the sarcomere 36,37 : (i) the proximal 126 residues of the coiled-coil subfragment 2 (S2) tail region of myosin (proximal S2) and (ii) MyBP-C, which is in myosin thick filaments in a ratio of one MyBP-C to approximately six myosin molecules 38 . In comparison with cardiac myosin, most residues on the mesa are conserved in fast skeletal muscle, which has MyBP-C, whereas many mesa residues have been changed in smooth muscle myosin and nonmuscle myosin II, which lack MyBP-C 36 .…”

A Mixed-Kinetic Model Describes Unloaded Velocities of Smooth, Skeletal, and Cardiac Muscle Myosin Filaments in Vitro

“…For example, myosin subfragments such as S1 and heavy meromyosin (HMM), which lack all or a portion of the tail domain, can be studied using the A/M m assay. These subfragments are soluble and easier to express than full-length myosin and thus allow the study of the effects of mutations (7)(8)(9)(10)(11). In contrast, the M f /A assay requires myosin with a full-length tail domain, the C-terminal portion of which contains the structural requirements for self-assembly into filaments.…”

How to Measure Load-Dependent Kinetics of Individual Motor Molecules Without a Force-Clamp

“…The exponential change of rate provides a clue to the molecular basis of the force–velocity curve and the Fenn effect of contracting muscle. In a translational point-of-view, the method can be applied to human β-cardiac S1 bearing hypertrophic cardiomyopathy (HCM)-causing single-point mutations to understand the devastating heart disease at the single-molecule level (Spudich, 2014; Spudich et al, 2016). This method is not limited to our myosin study but can be further extended into other systems, with some modification and optimization.…”