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

BACKGROUND: 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. 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 ß-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.

Diabetes increases mortality after myocardial infarction by oxidizing CaMKII.

Diabetes increases oxidant stress and doubles the risk of dying after myocardial infarction, but the mechanisms underlying increased mortality are unknown. Mice with streptozotocin-induced diabetes developed profound heart rate slowing and doubled mortality compared with controls after myocardial infarction. Oxidized Ca(2+)/calmodulin-dependent protein kinase II (ox-CaMKII) was significantly increased in pacemaker tissues from diabetic patients compared with that in nondiabetic patients after myocardial infarction. Streptozotocin-treated mice had increased pacemaker cell ox-CaMKII and apoptosis, which were further enhanced by myocardial infarction. We developed a knockin mouse model of oxidation-resistant CaMKIIδ (MM-VV), the isoform associated with cardiovascular disease. Streptozotocin-treated MM-VV mice and WT mice infused with MitoTEMPO, a mitochondrial targeted antioxidant, expressed significantly less ox-CaMKII, exhibited increased pacemaker cell survival, maintained normal heart rates, and were resistant to diabetes-attributable mortality after myocardial infarction. Our findings suggest that activation of a mitochondrial/ox-CaMKII pathway contributes to increased sudden death in diabetic patients after myocardial infarction.

Genetic inhibition of Na+-Ca2+ exchanger current disables fight or flight sinoatrial node activity without affecting resting heart rate.

RATIONALE: The sodium-calcium exchanger 1 (NCX1) is predominantly expressed in the heart and is implicated in controlling automaticity in isolated sinoatrial node (SAN) pacemaker cells, but the potential role of NCX1 in determining heart rate in vivo is unknown. OBJECTIVE: To determine the role of Ncx1 in heart rate. METHODS AND RESULTS: We used global myocardial and SAN-targeted conditional Ncx1 knockout (Ncx1(-/-)) mice to measure the effect of the NCX current on pacemaking activity in vivo, ex vivo, and in isolated SAN cells. We induced conditional Ncx1(-/-) using a Cre/loxP system. Unexpectedly, in vivo and ex vivo hearts and isolated SAN cells showed that basal rates in Ncx1(-/-) (retaining ≈20% of control level NCX current) and control mice were similar, suggesting that physiological NCX1 expression is not required for determining resting heart rate. However, increases in heart rate and SAN cell automaticity in response to isoproterenol or the dihydropyridine Ca(2+) channel agonist BayK8644 were significantly blunted or eliminated in Ncx1(-/-) mice, indicating that NCX1 is important for fight or flight heart rate responses. In contrast, the pacemaker current and L-type Ca(2+) currents were equivalent in control and Ncx1(-/-) SAN cells under resting and isoproterenol-stimulated conditions. Ivabradine, a pacemaker current antagonist with clinical efficacy, reduced basal SAN cell automaticity similarly in control and Ncx1(-/-) mice. However, ivabradine decreased automaticity in SAN cells isolated from Ncx1(-/-) mice more effectively than in control SAN cells after isoproterenol, suggesting that the importance of NCX current in fight or flight rate increases is enhanced after pacemaker current inhibition. CONCLUSIONS: Physiological Ncx1 expression is required for increasing sinus rates in vivo, ex vivo, and in isolated SAN cells, but not for maintaining resting heart rate.

MyD88 mediated inflammatory signaling leads to CaMKII oxidation, cardiac hypertrophy and death after myocardial infarction.

The toll-like receptors (TLR) and myocardial infarction (MI) promote NF-κB-dependent inflammatory transcription and oxidative injury in myocardium. The multifunctional Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is activated by oxidation and contributes to NF-κB-dependent transcription, myocardial hypertrophy and post-MI death. The myeloid differentiation protein 88 (MyD88) is an adapter protein critical for many TLR functions, but downstream targets for TLR/MyD88 signaling in MI are not well understood. We asked if CaMKII and TLR/MyD88 pathways are interconnected and if TLR/MyD88 contributes to adverse outcomes after MI. Here we show that TLR-4 activation by lipopolysaccharide (LPS) induces CaMKII oxidation (ox-CaMKII) in cardiomyocytes. MI enhances ox-CaMKII in wild type (WT) hearts but not in MyD88(-/-) hearts that are defective in MyD88-dependent TLR signaling. In post-MI WT hearts expression of pro-inflammatory genes TNF-α (Tnfa), complement factor B (Cfb), myocyte death and fibrosis were significantly increased, but increases were significantly less in MyD88(-/-) hearts after MI. MyD88(-/-) cardiomyocytes were defective in NF-κB activation by LPS but not by the MyD88-independent TLR agonist poly(I:C). In contrast, TNF-α induced Cfb gene expression was not deficient in MyD88(-/-) cardiomyocytes. Several hypertrophy marker genes were upregulated in both WT and MyD88(-/-) hearts after MI, but Acta1 was significantly attenuated in MyD88(-/-) hearts, suggesting that MyD88 selectively affects expression of hypertrophic genes. Post-MI cardiac hypertrophy, inflammation, apoptosis, ox-CaMKII expression and mortality were significantly reduced in MyD88(-/-) compared to WT littermates. These data suggest that MyD88 contributes to CaMKII oxidation and is important for adverse hypertrophic and inflammatory responses to LPS and MI.