Mechanisms of thin filament regulation by cardiac myosin-binding protein c in health and disease

The National Heart, Lung, and Blood Institute awarded Old Dominion University $1,630,808 on July 1, 2026, under the Cardiovascular Diseases Research program (CFDA 93.837) to investigate mechanisms of thin filament regulation by cardiac myosin-binding protein C in health and disease.

The award funds research on how mutations in MYBPC3, the gene encoding cardiac myosin-binding protein C (cMyBP-C), contribute to familial hypertrophic cardiomyopathy in over 40 percent of patients. cMyBP-C regulates cardiac contractility through phosphorylation, and its interactions with cardiac thin filaments represent a potential therapeutic target for heart failure and hypertrophic cardiomyopathy drug development. The research employs cryo-electron microscopy to map cMyBP-C interactions with thin filaments at physiological calcium levels and in the presence and absence of myosin fragments, identifying key residues mediating these interactions through fluorescence lifetime-detected fluorescence resonance energy transfer. A second aim defines the mechanistic pathology of HCM-linked cMyBP-C variants that increase binding affinity to thin filaments, correlating structural and kinetic findings with muscle mechanics and whole-heart physiology in mice.

Old Dominion University Research Foundation serves as the fiscal and administrative agent. Performance occurs in Norfolk, Virginia, with the period of performance running from July 1, 2026, through March 31, 2028. The award is classified as a Project Grant, an assistance type that funds discrete, time-limited research projects.

Abstract

Mutations in MYBPC3, the gene encoding cardiac myosin binding protein-c (cmybp-c), have been linked to familial hypertrophic cardiomyopathy (HCM) in more than 40% of patients. Cmybp-c is a vital regulator of a healthy heart. IT is involved in promoting enhanced contractility in response to inotropic stimuli that phosphorylate cmybp-c. As phosphorylation is associated with cardiac protection, IT is often decreased in patients with heart failure (HF). Since cmybp-c phosphorylation presumably decreases its interactions with cardiac thin filaments (ctfs), cmybp- c is a potential target for HF and HCM drugs that modify its interactions with the CTF. However, despite its significance to cardiac function, there is still remarkably little known regarding how cmybp-c mediates its functional effects or how HCM-linked mutations alter cmybp-c interactions with the CTF. Work from our labs and others shows that cmybp-c binds to ctfs through multiple interactions of its n-terminal domains (ntds), which together can directly regulate CTF activation by shifting tropomyosin (TM) from its off to its on position, similar to CA2+ and myosin cross- bridges. In aim 1, we leverage our unique expertise in cryo-EM to reveal the details of cmybp-c interactions with the CTF at physiological CA2+ levels, both in the presence/absence of the myosin fragment, which is comprised of two heads and tail regions that harbor binding sites for ntds. We will identify key residues that mediate interactions with ctfs and myosin and test these model predictions using fluorescence lifetime-detected fluorescence resonance energy transfer (FLT- fret). In aim 2, ROR the first time, we will define a mechanistic model of cmybp-c variants pathogenic for HCM that increase ntds' affinity to CTF. Structural and kinetic studies will be correlated with muscle mechanics and whole heart physiology in mice. In aim 3, we will utilize cryo-EM and FLT-fret to delineate the mechanism(s) by which small-molecule modulators of cmybp-c (two drug cluster scaffolds with sulfur-containing motifs) affect ntds in vitro binding to ctfs, in situ mechanical cross-bridge function in demembranated myocardium, and in vivo cardiac function of HCM variants in mice. This work will provide mechanistic insight into cmybp- c-based therapeutics to treat HCM and HF diseases from the atomic to cellular to organ levels. Integrated structural, kinetic, and functional studies will define how small-molecule modulators of cmybp-c affect normal and variant NTD structure and dynamics to accelerate mechanistic-based cmybp-c therapies for cardiomyopathy and catalyze proof-of-principle for the development of chemical derivatives and design of precise structure-based screens.