No abstract

The increased expression of hsp27 and alphaB-crystallin through an adenovirus vector system protects against ischemic injury in adult cardiomyocytes. Likewise, the overexpression of alphaB-crystallin protects against ischemic damage in neonatal cardiomyocytes. Decreasing the high levels of endogenous hsp25 present in neonatal cardiomyocytes renders them more susceptible to damage caused by simulated ischemia.

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 (β-AR) 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 Cε can also phosphorylate Ser-273 and Ser-302. In contrast, Ca2+-calmodulin-activated kinase II (CaMKII) targets Ser-302 but can also target Ser-282 at non-physiological calcium concentrations. Strikingly, Ser-302 phosphorylation by CaMKII was abolished by ablating Ser-282's ability to be phosphorylated via alanine substitution. To determine the sites’ functional roles in vivo, three transgenic lines, which expressed cMyBP-C containing either Ser-273-Ala-282-Ser-302 (cMyBP-CSAS), Ala-273-Asp-282-Ala-302 (cMyBP-CADA) or Asp-273-Ala-282-Asp-302 (cMyBP-CDAD), 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 residues’ abilities to be phosphorylated while serine to aspartate substitutions were used to mimic the charged state conferred by phosphorylation. Compared to control non-transgenic mice, as well as transgenic mice expressing wild-type cMyBP-C, the transgenic cMyBP-CSAS(t/t), cMyBP-CADA(t/t) and cMyBP-CDAD(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-CSAS(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.