A critical function for Ser-282 in cardiac myosin binding protein-C phosphorylation and cardiac function

Sakthivel Sadayappan, James Gulick, Hanna Osinska, David Yeomans Barefield, Friederike Cuello, Metin Avkiran, Valerie M. Lasko, John N. Lorenz, Marjorie Maillet, Jody L. Martin, Joan Heller Brown, Donald M. Bers, Jeffery D. Molkentin, Jeanne James, Jeffrey Robbins*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

84 Scopus citations

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-calmodulin-activated kinase II targets Ser-302 but can also target Ser-282 at nonphysiological calcium concentrations. Strikingly, Ser-302 phosphorylation by Ca-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), Ala-273-Asp-282-Ala-302 (cMyBP-C), or Asp-273-Ala-282-Asp-302 (cMyBP-C), 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, cMyBP-C, and cMyBP-C mice showed no increases in morbidity and mortality and partially rescued the cMyBP-C 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 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.

Original languageEnglish (US)
Pages (from-to)141-150
Number of pages10
JournalCirculation research
Volume109
Issue number2
DOIs
StatePublished - Jul 8 2011

Keywords

  • contractile function
  • myofilament
  • myosin-binding protein-C
  • phosphorylation

ASJC Scopus subject areas

  • Physiology
  • Cardiology and Cardiovascular Medicine

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