Abstract 3846: Hierarchical and Critical Functions of Ser282 Phosphorylation in Cardiac Myosin Binding Protein-C Phosphorylation and Cardiac Function
Background: Cardiac myosin binding protein-C (cMyBP-C) phosphorylation at Ser273, Ser282 and Ser302 regulates myocardial function and can be cardioprotective during heart failure. PKA, PKC and CaMKII phosphorylate these sites in vivo. The objective of this study was to differentiate the roles of the three sites in cMyBP-C phosphorylation and its consequences.
Hypothesis: Ser282 in cMyBP-C regulates Ser273 and Ser302 phosphorylation and myocardial function during β-adrenergic stimulation.
Methods: C1C2 peptides of cMyBP-C in which the three phosphorylatable sites were individually mutated to Ala, as well as a wild-type control, were used in conjunction with site-specific phospho antibodies to determine the targeting of these sites by PKA and CaMKII. To demonstrate regulation of the sites during heart failure, a transverse aortic constriction (TAC) mouse model was used. To determine the sites’ functional roles in vivo, three transgenic lines, which expressed cMyBP-C containing Ser273-Ala282-Ser302 (SAS), Ala273-Asp282-Ala302 (ADA) or Asp273-Ala282-Asp302 (DAD), were generated.
Results: Using mutant C1C2 peptides, we determined that CaMKII targets Ser302 in vitro. Strikingly, Ser302 phosphorylation by CaMKII was abolished by Ser282Ala replacement, demonstrating the hierarchical function of Ser282 phosphorylation. During TAC-induced heart failure, Ser282 phosphorylation was significantly decreased after 24hrs; in contrast, Ser273 and Ser302 phosphorylation decreased only after 8wks, suggesting phosphorylation of Ser273 and Ser302 may be cardioprotective. Transgenic SAS, ADA and DAD mice showed no morbidity and mortality. In vivo hemodynamic studies revealed that SAS, ADA and DAD hearts showed no differences in contractility at baseline, compared to controls. Importantly, ADA and SAS hearts had significantly decreased contractility and Mg2+-ATPase activity during β-agonist infusion, confirming the role of Ser282 phosphorylation in mediating subsequent phosphorylation of Ser273 and Ser302, followed by accelerated myocardial function.
Conclusion: Ser282 phosphorylation is a critical determinant of cMyBP-C phosphorylation in vitro and in vivo and regulates the contractile response to β-adrenergic stimulation in vivo.
This research has received full or partial funding support from the American Heart Association, National Center.