Cardiac Muscle: Functional Role of Myosin - Binding Protein C in Contraction

点击翻译按钮获取中文摘要

DESCRIPTION (provided by applicant): Mutations in the thick filament associated protein cMyBP-C are a major cause of familial hypertrophic cardiomyopathy. The pathological effects of mutations in cardiac myosin binding protein-C are consistent with increasing evidence that it plays both a structural and a regulatory role in myocardial contraction. Although the evidence suggesting a regulatory role for cMyBP-C in myocardial contraction appears compelling, the mechanism of action is far from clear. Most of the research on the functional role of cMyBPC and the effect of its phosphorylation has been done in rat or mouse myocardium which contains primarily the a-myosin heavy chain, despite evidence that thick filaments containing the (3-myosin heavy chain (such as in humans) may differ in the changes shown upon phosphorylation of cMyBP-C. This suggests the importance of investigating in more detail the effect of phosphorylation of cMyBP-C on the structure of the myocardial thick filament in species other than the rat; including species with the (3-myosin heavy chain such as the rabbit. In addition, increasing evidence suggests that N-terminus fragments of cMyBP-C may be able effect myocardial contractility independent of a tethering of the myosin S2 region to the backbone of the filament by cMyBP-C, and may also be able to directly bind actin. These studies emphasize the need to look at the effects of binding of these N-terminus fragments on the cross bridge arrangement and structure of the isolated cardiac thick filament; and further assess by electron microscopy the binding potential of these Nterminus fragments of cMyBP-C to actin. In addition in order to fully understand how cMyBP-C functions in the thick filament, it is vital to have higher resolution 30-reconstructions of the cardiac thick filaments than are currently available. The specfic aims of the research are to: 1) Determine the effect of phosphorylation of cMyBP-C on the crossbridge arrangement of isolated mouse, rat, and rabbit cardiac thick filaments; 2) Determine the effect of the C1-C2 and CO-C1 N-terminus fragments of cMyBP-C on the crossbridge arrangement of the isolated thick filaments from rabbit heart, and both wildtype and cMyBP-C"'" knockout mouse hearts; 3) Assess the of binding of C1-C2 and CO-C2 N-terminus fragments of cMyBP-C to actin; and 4) Compute higher resolution 3D-reconstructions of the cardiac thick filaments from both mouse and rabbit thick filaments by single particle analysis to precisely locate the myosin heads and cMyBP-C.