Phosphorylation of cardiac myosin binding protein C (cMyBP-C) accelerates myocardial contraction, but neither the molecular or structural mechanism nor the in vivo significance of these effects is known. Our working model is that phosphorylation regulates twitch kinetics by regulating cross-bridge recruitment to the thin filament. In this dual-PI proposal we will use our complementary expertise in cardiac muscle physiology and myofilament structure to generate an integrated view of cMyBP-C function. This dual approach includes strict cross-checks of data over a range of spatial organization, from isolated filaments to working hearts in vivo. We will test the hypothesis that the mechanism by which phosphorylation of cMyBP-C?s N-terminus enhances contraction involves disruption of its binding to myosin and increased binding to actin, thereby increasing the rate of cross-bridge binding to the thin filament.
Aim 1 will test the idea that phosphorylation speeds the cooperative recruitment of cross-bridges in skinned myocardium by measuring rates of force development (kADP) following photolysis of caged ADP, which activates the cooperative recruitment process. N-terminal fragments will be used to determine the roles of charged residues within cMyBP-C?s M-domain in the regulation of force and rate of contraction. Studies will be extended to transgenic mice with the same residues mutated to disrupt cMyBP-C binding to either myosin or actin to determine the effects of these mutations on twitch characteristics in vivo; the possibility that phosphorylation of cTnI also contributes to adrenergic inotropy will be investigated using phosphomimetic cTnI mice. The alternative idea, that phosphorylation accelerates contraction by increasing the rates of cross-bridge transitions, will be investigated by characterizing the steps in the cross-bridge cycle corresponding to force development (Pi release) and relaxation (ADP release).
Aim 2 will combine cryo-electron microscopy and 3D reconstruction of thick filaments with X-ray diffraction of myocardium, to test our model structurally. We will determine whether cMyBP-C stabilizes the super-relaxed state of myosin heads on the thick filament, whether cMyBP-C phosphorylation disrupts this, and the role of M-domain charged residues in these effects. We will also test whether phosphorylation releases the cMyBP-C N-terminus from the thick filament backbone, facilitating its binding to actin. Since hypertrophic cardiomyopathies (HCM) due to mutations in cMyBP-C are typically associated with enhanced contraction, Aim 3 explores whether the hypercontractility involves altered interactions of the M- domain, such that binding to myosin is reduced and/or binding to actin is increased, as we propose with phosphorylation. These studies will test the idea that HCM mutations weaken cMyBP-C?s stabilization of myosin heads, accelerating their recruitment to the thin filament. This collaborative project takes advantage of our complementary expertise in myocardial function (Moss) and structure (Craig) and will lead to an in-depth, integrated understanding of cMyBP-C function/dysfunction which would not be possible by either lab alone.
Cardiac myosin-binding protein C (cMyBP-C) is a protein in heart muscle that plays a critical role in regulating the heart?s pumping action, and defects in cMyBP-C are among the major causes of inherited heart disease. Despite its importance, relatively little is known about how cMyBP-C functions. To address this deficiency we will determine specific mechanisms by which cMyBP-C regulates the strength and speed of contraction and how defects in cMyBP-C affect this process. The insights gained should be invaluable for understanding cMyBP-C function, and help guide the search for therapeutic agents to treat diseases such as heart failure.
| Moss, Richard L (2018) Novel Regulatory Elements within Myofilaments of Vertebrate Striated Muscles-Who Knew. Biophys J 115:1401-1402 |
