Phosphoproteomics study based on in vivo inhibition reveals sites of calmodulin-dependent protein kinase II regulation in the heart.

The multifunctional ca(2+)- and calmodulin-dependent protein kinase ii (camkii) is a crucial mediator of cardiac physiology and pathology. increased expression and activation of camkii has been linked to elevated risk for arrhythmic events and is a hallmark of human heart failure. a useful approach to determining camkii's role therein is large-scale analysis of phosphorylation events by mass spectrometry. however,current large-scale phosphoproteomics approaches have proved inadequate for high-fidelity identification of kinase-specific roles. the purpose of this study was to develop a phosphoproteomics approach to specifically identify camkii's downstream effects in cardiac tissue. 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 β-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. our data set included known camkii substrates,as well as several new candidate proteins involved in functions not previously implicated in camkii signaling.