Phosphoproteomics Study Based on In Vivo Inhibition Reveals Sites of Calmodulin-Dependent Protein Kinase II Regulation in the Heart

approach to specifically identify CaMKII’s downstream effects in cardiac tissue. Methods and Results-—To identify putative downstream CaMKII targets in cardiac tissue, animals with myocardial-delimited expression of the specific peptide inhibitor of CaMKII (AC3-I) or an inactive control (AC3-C) were compared using quantitative phosphoproteomics. The hearts were isolated after isoproterenol injection to induce CaMKII activation downstream of b-adrenergic receptor agonist stimulation. Enriched phosphopeptides from AC3-I and AC3-C mice were differentially quantified using stable isotope dimethyl labeling, strong cation exchange chromatography and high-resolution LC-MS/MS. Phosphorylation levels of several hundred sites could be profiled, including 39 phosphoproteins noticeably affected by AC3-I-mediated CaMKII inhibition. Conclusions-—Our data set included known CaMKII substrates, as well as several new candidate proteins involved in functions not previously implicated in CaMKII signaling. (J Am Heart Assoc. 2013;2:e000318 doi: 10.1161/JAHA.113.000318)