Abstract 1579: Critical Role of the CaMKII-site (Ser282) in Cardiac Myosin Binding Protein-C Phosphorylation and Cardiac Function
Cardiac myosin binding protein-C (cMyBP-C) plays a major role as both a structural and regulatory protein in the heart. Three of the serines in cMyBP-C (Ser273, Ser282 and Ser302) are known to be substrates for PKA, PKC and CaMKII. In vitro studies showed that CaMKII first phosphorylates Ser282, followed by the other phosphorylation sites in a hierarchical fashion. However, the differential role(s) of the three sites in cardiac function in vivo remains unclear. The loss of cMyBP-C phosphorylation at Ser282 will alter cardiac function during β-adrenergic (β-AR) stimulation. To determine the phosphorylation pattern of the three sites during normal and failing conditions, phospho-specific antibodies were raised against Ser273, Ser282 and Ser302, affinity purified and tested for specificity. To demonstrate that phosphorylation of the CaMKII-site in cMyBP-C is required for the subsequent phosphorylation of Ser273 and Ser302 during β-AR stimulation, transgenic mice were generated in which the Ser282 of cMyBP-C was mutated to Ala, mimicking constitutive phosphoablation (cMyBP-C282A). Western blot analyses, using myofilament proteins from a transverse aortic constriction (TAC) induced pressure-overload model, showed that Ser273 and Ser302 phosphorylation was significantly increased after 5min and decreased after 4wks of TAC along with reduced fractional shortening and increased HW/BW ratio. In contrast, Ser282 phosphorylation was significantly increased after 5min of TAC, but decreased after 24hrs, suggesting that the CaMKII-site has a unique functional role as it is post-translationally modified. cMyBP-C282A transgenic mice showed no morbidity and mortality. In vivo hemodynamic studies revealed no differences in dP/dtmax and dP/dtmin at baseline in the cMyBP-C282A hearts, compared to controls. However, dP/dtmax and dP/dtmin were significantly decreased, 28% and 39% respectively, during β-agonist infusion resulting in reduced Ser273 and Ser302 phosphorylation in the cMyBP-C282A hearts. Phosphorylation of CaMKII-site in cMyBP-C is a necessary prerequisite for the subsequent phosphorylation of Ser273 and Ser302 to accelerate myocardial contractility during β-AR stimulation.