CaMKII dB and dC have Opposing roles in regulating fate

钙调蛋白依赖性蛋白激酶II(CaMKII)在哺乳动物细胞中广泛表达，其中d亚型主要在心肌细胞中表达。我们最近的研究表明，b1-肾上腺素能受体刺激通过非PKA依赖的CaMKII信号诱导心肌细胞凋亡。然而，目前尚不清楚CaMKII的激活是否足以引发心肌细胞凋亡，如果是，潜在的机制是什么。为了特异地操纵CaMKII-DC的活性，我们使用腺病毒基因转移(m.o.I.)过表达了CaMKII-DC的一个结构性活性突变体(CA-CaMKII-DC)。在培养的成年大鼠心肌细胞中，CaMKII活性在感染24小时后增加2倍。利用Western blotting和共聚焦免疫荧光成像技术，我们观察到CaMKII-DC在线粒体和胞浆中高表达。在感染的心肌细胞中，细胞凋亡以时间依赖的方式发生。CA-CaMKII诱导的TUNEL阳性染色细胞增多，DNA梯状条带显示DNA片段化。此外，用KN-93(1 MM)或膜通透性多肽抑制剂AIP(5 MM)抑制CaMKII，或通过腺病毒基因转移显性负性CaMKII-DC(m.o.I.完全阻止CA-CaMKII-DC介导的细胞凋亡，表明增强的CaMKII活性足以触发心肌细胞的凋亡。更重要的是，在AdV-CA-CaMKII-DC感染的心肌细胞中，CaMKII-DC诱导的细胞凋亡伴随着线粒体细胞色素C释放的增加，提示CaMKII信号的增强导致了线粒体凋亡途径的激活。与之形成鲜明对比的是，CaMKII-DB通过增加Akt的激活，保护新生和成年啮齿动物心肌细胞免受缺氧和氧化应激诱导的心肌细胞凋亡。因此，我们得出结论，CaMKII-DC的持续激活足以通过线粒体凋亡途径触发心肌细胞凋亡，而CaMKII-dB的增强激活则具有心脏保护作用。

Ca2+-Camodulin dependent protein kinase II (CaMKII) is widely expressed in mammalian cells, with the d isoform predominantly expressed in cardiomyocytes. Our recent studies have shown that b1-adrenergic receptor stimulation induces cardiomyocyte apoptosis via PKA-independent CaMKII signaling. It is unknown, however, whether activation of CaMKII is sufficient to trigger cardiomyocyte apoptosis and if so, what is the underlying mechanism. To specifically manipulate CaMKII-dC activity, we overexpressed a constitutively active mutant of CaMKII-dC (CA-CaMKII-dC) using adenoviral gene transfer (m.o.i. 100) in cultured adult rat cardiomyocytes where CaMKII activity was increased by 2 fold after 24 h infection. Using Western blotting and confocal immunofluorescence imaging, we observed overexpressed CaMKII-dC in mitochondria as well as cytosol. In the infected cardiomyocytes, apoptosis occurred in a time-dependent manner. This was evidenced by CA-CaMKII induced increase in TUNEL positive staining cells and DNA fragmentation detected with DNA ladder. Furthermore, inhibition of CaMKII with KN-93 (1 mM) or a membrane permeable peptide inhibitor AIP (5 mM), or by adenoviral gene transfer of a dominant negative-CaMKII-dC (m.o.i. of 100) fully prevented CA-CaMKII-dC mediated apoptosis, indicating that enhanced CaMKII activation is sufficient to trigger cardiomyocyte apoptosis. More importantly, CaMKII-dC induced apoptosis was accompanied by increased cytochrosome C release from mitochondria in cardiomyocytes infected by Adv-CA-CaMKII-dC, suggesting that enhanced CaMKII signaling leads to the activation of mitochondrial apoptotic pathway. In sharp contrast, CaMKII-dB protects both neonatal and adult rodent cardiomyocytes against hypoxia- and oxdative stress-induced apoptosis via increasing Akt activation. Thus, we conclude that sustained activation of CaMKII-dC is sufficient to trigger cardiomyocyte apoptosis likely via a mitochondrial apoptotic pathway, whereas enhanced activation of CaMKII-dB is cardiac protective.