CaMKII-aB Protects the Heart Against Oxidative-Stress In

钙/钙调蛋白依赖的蛋白激酶II(CaMKII)是一种由钙和钙调蛋白的复合体激活的多功能蛋白激酶。CaMKII介导了一系列广泛的靶蛋白的磷酸化，这些蛋白参与了许多细胞过程，如钙离子处理、细胞生长和细胞死亡。CaMKII家族的D亚型主要在心肌细胞中表达。至少有两个CaMKII-d的剪接变异体，Db和Dc，分别位于不清晰和胞质间隔室。我们以前的研究表明，增强CaMKII-DC的激活对1AR诱导的心肌细胞凋亡既是必要的，也是充分的，此外，它还具有调节心肌钙调节蛋白的磷酸化从而调节心脏兴奋-收缩偶联的功能。然而，尽管CaMKII-dB与心肌肥厚有关，但其功能作用仍不明确。 本研究的目的是研究CaMKII-dB在心脏中的潜在生理和病理功能作用，并探讨其临床意义。我们已经证明，在大鼠缺血/再灌注(I/R)和心肌梗死(MI)模型以及培养的心肌细胞对过氧化氢氧化应激的反应中，CaMKII-dB在mRNA和蛋白水平的表达均显著减弱。MI和H_2O_2对CaMKII-dB的抑制作用可被ROS清除剂完全阻断，表明ROS是CaMKII-dB基因表达的负调控因子。同时，通过DNA梯带、Hoechst或TUNEL染色和caspase活性检测，MI和H_2O_2显著增加活体和培养心肌细胞的凋亡率。最重要的是，通过腺病毒基因转移过表达CaMKII-dB可以有效地保护心肌细胞免受缺血或氧化应激诱导的细胞凋亡。此外，一些证据表明，CaMKII-dB的心脏保护作用是由Akt依赖的途径介导的。首先，CaMKII-dB的过表达导致Akt磷酸化水平的强劲上升。其次，通过Akt介导的机制，CaMKII-Db的激活也显著增加了一种新的凋亡抑制因子ARC的表达，因为用LY 294002抑制PI3K/AKT信号通路可以显著阻断CaMKII-Db诱导的ARC的上调和保护作用。这些体内和体外数据表明，CaMKII-Db通过Akt和ARC依赖的途径在保护心脏免受缺血和氧化损伤中发挥关键作用，标志着CaMKII-Db成为治疗心肌缺血疾病的新靶点，预计到2020年，心肌缺血疾病将成为世界上人数最多的杀手。

Ca2+/calmodulin-dependent kinase II (CaMKII) is a multifunctional protein kinase activated by the complex of Ca2+ and calmodulin. CaMKII mediates phosphorylation of a wild range of target proteins involved in a multitude of cellular processes such as Ca2+ handling, cell growth, and cell death. The d isoform of CaMKII family is predominantly expressing in cardiac myocytes. There are, at least, two splicing variants of CaMKII-d, dB and dC, located in unclear and cytosol compartments, respectively. Our previous studies have shown that enhanced CaMKII-dC activation is both necessary and sufficient for a1AR-induced cardiomyocyte apoptosis, in addition to its well-established functions in regulating phosphorylation of cardiac Ca2+ handling proteins and thus modulating cardiac excitation-contraction coupling. However, the functional role of CaMKII-dB remains elusive, although it has been implicated in cardiac hypertrophy. The aim of the present study is to investigate the potential physiological and pathological functional roles of CaMKII-dB in the heart and explore its clinical implications. We have demonstrated that CaMKII-dB expression is remarkably attenuated at both mRNA and protein levels in rat ischemia/reperfusion (I/R) and myocardium infarction (MI) models and in cultured cardiac myocytes in response to oxidative stress with H2O2. The inhibitory effects of MI and H2O2 on CaMKII-dB are fully prevented by reactive oxygen species (ROS) scavengers, indicating ROS constitutes a negative regulator of CaMKII-dB gene expression. Concurrently, MI and H2O2 markedly increase myocyte apoptosis in vivo and in culture, respectively, assayed by DNA laddering, Hoechst or TUNEL staining, and caspase activation. Most importantly, overexpression of CaMKII-dB using adenoviral gene transfer substantially protects heart cells against ischemia- or oxidative stress-induced apoptosis. Moreover, several lines of evidence indicates that the CaMKII-dB cardiac protective effect is mediated by a Akt-dependent pathway. First, overexpression of CaMKII-dB leads to a robust elevation in Akt phosphorylation. Second, increased CaMKII-dB activation also profoundly augments the expression of a novel apoptosis repressor, ARC (apoptosis repressor with caspase recruitment domain), via a Akt-mediated mechanism, because inhibition of the PI3K/Akt signaling pathway with LY 294002 can substantially block CaMKII-dB induced upregulation of ARC and the protective effect. These in vivo and in vitro data indicate that CaMKII-dB plays a pivotal role in protecting the heart against ischemia and oxidative insults through an Akt and ARC-dependent pathway, marking CaMKII-dB as a promising novel therapeutic target for the treatment of myocardial ischemic disease, the predicted number killer in the world by 2020.