A Critical Function for Ser-282 in Cardiac Myosin Binding Protein-C Phosphorylation and Cardiac Function

Abstract

Rationale: Cardiac myosin-binding protein-C (cMyBP-C) phosphorylation at Ser-273, Ser-282, and Ser-302 regulates myocardial contractility. In vitro and in vivo experiments suggest the nonequivalence of these sites and the potential importance of Ser-282 phosphorylation in modulating the protein's overall phosphorylation and myocardial function. Objective: To determine whether complete cMyBP-C phosphorylation is dependent on Ser-282 phosphorylation and to define its role in myocardial function. We hypothesized that Ser-282 regulates Ser-302 phosphorylation and cardiac function during β-adrenergic stimulation. Methods and Results: Using recombinant human C1-M-C2 peptides in vitro, we determined that protein kinase A can phosphorylate Ser-273, Ser-282, and Ser-302. Protein kinase C can also phosphorylate Ser-273 and Ser-302. In contrast, Ca 2+ -calmodulin-activated kinase II targets Ser-302 but can also target Ser-282 at nonphysiological calcium concentrations. Strikingly, Ser-302 phosphorylation by Ca 2+ -calmodulin-activated kinase II was abolished by ablating the ability of Ser-282 to be phosphorylated via alanine substitution. To determine the functional roles of the sites in vivo, three transgenic lines, which expressed cMyBP-C containing either Ser-273-Ala-282-Ser-302 (cMyBP-C SAS ), Ala-273-Asp-282-Ala-302 (cMyBP-C ADA ), or Asp-273-Ala-282-Asp-302 (cMyBP-C DAD ), were generated. Mutant protein was completely substituted for endogenous cMyBP-C by breeding each mouse line into a cMyBP-C null (t/t) background. Serine-to-alanine substitutions were used to ablate the abilities of the residues to be phosphorylated, whereas serine-to-aspartate substitutions were used to mimic the charged state conferred by phosphorylation. Compared to control nontransgenic mice, as well as transgenic mice expressing wild-type cMyBP-C, the transgenic cMyBP-C SAS(t/t) , cMyBP-C ADA(t/t) , and cMyBP-C DAD(t/t) mice showed no increases in morbidity and mortality and partially rescued the cMyBP-C (t/t) phenotype. The loss of cMyBP-C phosphorylation at Ser-282 led to an altered β-adrenergic response. In vivo hemodynamic studies revealed that contractility was unaffected but that cMyBP-C SAS(t/t) hearts showed decreased diastolic function at baseline. However, the normal increases in cardiac function (increased contractility/relaxation) as a result of infusion of β-agonist was significantly decreased in all of the mutants, suggesting that competency for phosphorylation at multiple sites in cMyBP-C is a prerequisite for normal β-adrenergic responsiveness. Conclusions: Ser-282 has a unique regulatory role in that its phosphorylation is critical for the subsequent phosphorylation of Ser-302. However, each residue plays a role in regulating the contractile response to β-agonist stimulation.