Abstract 536: Absence of Protein Kinase A Phosphorylation of Cardiac Myosin Binding Protein Causes Myocardial Dysfunction and Hypertrophy
Cardiac myosin binding protein C (cMyBP-C) is a thick filament regulatory protein and has three known protein kinase A (PKA) sites. We hypothesized that PKA phosphorylation of cMyBP-C regulates myocardial mechanics and that loss of this regulation would cause dysfunction. We tested this idea by assessing the effects on contraction kinetics due to introduction of a non-phosphorylatable form of cMyBP-C. Mutant cMyBP-C (m/−) with Ser to Ala mutations to prevent PKA phosphorylation was expressed on the cMyBP-C null background of the cMyBP-C knock-out (KO) mouse. KO with wild type cMyBP-C add-back (+/−) served as a control. Western blots were used to identify lines with similar levels of expression of protein, i.e., (m/−) at 74% and (+/−) at 72% expression vs. wild type (WT). Fluorescent conjugated antibodies verified incorporation of cMyBP-C into the C zone of myocardial sarcomeres. (m/−) mice exhibited similar hypertrophic heart to body weight ratio as KO. ProQ diamond phosphoprotein staining showed PKA does not phosphorylate (m/−) cMyBP-C, but as expected PKA phosphorylates troponin I in these mice. (m/−) myocardium had similar Ca-dependence of force and PKA-induced shift of pCa50 as WT myocardium. After an immediate stiffness response and transient relaxation of force, skinned myocardium responds to stretch with a delayed development of force, i.e., stretch activation. The rates of relaxation, krel (1/sec), and delayed force development, kdf (1/sec), in the stretch activation response are indicators of crossbridge detachment and attachment kinetics, respectively. (m/−) myocardium had baseline krel and kdf similar to WT, but unlike WT, (m/−) baseline kinetics were not accelerated by PKA. Thus, PKA phosphorylation of cMyBP-C accelerated kinetics. As indicated by the hypertrophic phenotype in (m/−) mice, the loss of regulation of contraction kinetics mediated by cMyBP-C phosphorylation leads to significant physiologic dysfunction.