Cardiac myosin-binding protein C (cMyBP-C) is a sarcomeric thick filament associated protein that is essential to normal cardiac structure and function. The importance of cMyBP-C is emphasized by mutations to cMyBP-C being a leading cause of hypertrophic cardiomyopathy. Despite being a key regulator of cardiac contractility, the molecular mechanism by which cMyBP-C modulates actomyosin force and motion generation is far from certain. Although cMyBP-C's N-terminal domains can bind to actin and the myosin head region, it is not known which of these binding partners is physiologically relevant and whether these binding partner interactions modulate cardiac contractility by directly affecting actomyosin power generation or indirectly by altering Ca2+- dependent thin filament activation. With phosphorylation of cMyBP-C's N terminus occurring in response to ?- adrenergic stimulation, phosphorylation may offer a measure of cMyBP-C functional tunability in order to enhance cardiac contractility. To address these questions, we propose two specific aims.
Aim 1 will test the hypothesis that phosphorylation modulates cMyBP-C's N-terminal domain structure to influence its binding partner interactions (i.e. thin filament and myosin head region). We will use a novel mass-spectrometry technique and atomic force microscopy to characterize the molecular mechanics of cMyBP-C's N terminus that has been structurally altered due to phosphorylation or mutagenesis. The functional impact of these structural perturbations will be characterized in the context of cardiac myofibrils and native thick filaments to determine if cMyBP-C operates only where it exist in the thick filament and whether it can sequester cardiac myosin into a reserve pool of super-relaxed myosin heads. Thus, we will measure the location and time course of fluorescent-ATP turnover in single cardiac myofibrils and the force generated by native thick filaments in the laser trap in preparations from transgenic mice expressing phosphorylation and binding partner ablated mutant cMyBP-C.
In Aim 2 we will create DNA-based ?designer? thick filament nanotubes to define how the spatial relationships that normally exist in the thick filament between cMyBP-C and its myosin and actin binding partners are critical determinants of cMyBP-C's modes of operation. These DNA-nanotubes will allow exquisite nanometer spatial positioning of expressed cMyBP-C and human ?-cardiac myosin on the nanotube surface relative to each other. By this novel approach we can assign cMyBP C's modulation of actomyosin motility to binding of the myosin head and/or thin filament, as assessed by both thin filament motility and force generation using the laser trap. With the knowledge and understanding of cMyBP-C function derived from these collective studies, targeted therapies directed at cMyBP-C binding partner interactions may be developed to help modulate and to improve cardiac performance in the failing heart.
Pumping of the heart is generated by interactions between the thin and thick contractile protein filaments that make up cardiac muscle. Cardiac myosin-binding protein-C (cMyBP-C) is a thick filament component that regulates cardiac contraction in response to stimulation, and genetic defects in cMyBP-C result in heart failure and sudden death. Despite its importance, significant gaps in our understanding of cMyBP-C functionality remain. This proposal will define the molecular structure, function, and regulation of cMyBP-C. These studies and derived knowledge are precursors to the development of targeted therapies in order to correct the functional defects associated with genetic mutations to cMyBP-C.
