Abstract 1441: Cardiac Myosin Binding Protein-C Phosphorylation Modulates Cardiac Contractile Function
Background: Cardiac myosin binding protein-C (cMyBP-C) has 3 phosphorylatable serines (Ser-273, -282 and -302) at its N-terminus and the residues’ phosphorylation states may alter thick filament structure and function. Recently, we showed that cMyBP-C phosphorylation was significantly decreased during the development of heart failure or pathologic hypertrophy. The goal of this study is to examine the effect(s) of total chronic phosphorylation of cMyBP-C at these three sites, as they represent convergent targets for adrenergic signaling in the heart.
Hypothesis: cMyBP-C phosphorylation modulates cardiac contractile function.
Methods: A cDNA was made, in which the three phosphorylation sites of mouse cMyBP-C were mutated to aspartic acid, mimicking constitutive phosphorylation (cMyBP-CAllP+). This cDNA was linked to the cardiomyocyte-specific α-myosin heavy chain promoter and used to generate transgenic mice with cardiac-specific expression. These mice were then bred with cMyBP-C nulls (cMyBP-C(t/t)) to ensure the absence of endogenous dephosphorylated cMyBP-C. An animal that expressed the normal cardiac isoform, cMyBP-CWT, was also bred to the nulls to serve as a control.
Results: The cMyBP-CAllP+ replacement in the cMyBP-C null background was complete and rescued the null phenotype. The protein was incorporated normally into the cardiac sarcomere and constitutive expression of the phosphorylation mimetic, cMyBP-CAllP+, was benign. Electron microscopy showed regular A- and I-bands and M-lines in the cMyBP-CAllP+/(t/t) sarcomeres. However, the thick filaments exhibited a relatively loose structure compared to controls. Furthermore, unlike the cMyBP-CWT/(t/t) and non-transgenic hearts, the cMyBP-CAllP+/(t/t) hearts were largely resistant to ischemia/reperfusion injury. Yeast-two-hybrid data and pull-down assays showed that phosphorylation of the residues effectively prevented interaction with the myosin heavy chain. In conclusion, these data together with our previous finding, demonstrate that cMyBP-C phosphorylation is essential in modulating cardiac contractility, functions in maintaining thick filament spacing and structure, and protects the myocardium from ischemic injury.