CaMKII-dB and CaMKII-dC Oppositely Regulate Cardiomyocyte viability

有，至少有两个剪接变体的CaMK Ⅱ-d，dB和dC，分别位于不透明和细胞质隔室。 我们目前的体内和体外研究提供了多条证据，证明CaMKII-dB和CaMKII-dC在调节心肌细胞活力方面表现出相反的功能作用，CaMKII-dB具有保护作用，CaMKII-dC具有凋亡作用。 （一） CaMKII激活是b1 AR诱导的心肌细胞凋亡和体内凋亡所必需的。 持续的b1 AR刺激以时间依赖性方式显着增加成年小鼠心肌细胞中的CaMKII活性;这种作用被特异性CaMKII抑制剂AIP或KN 93消除，但不被特异性PKA抑制剂Zhu等人，J. Clin. Invest. 111：617-625，（2003）。重要的是，抑制CaMKII完全保护心肌细胞免受b1 AR诱导的凋亡。在与Mark安德森的合作中，我们进一步证明了CaMK II对于体内对过量儿茶酚胺刺激的凋亡反应是必需的。Am. J.Physiol.291：H3065-H3075，（2006）。 （二） CaMKII-dC活性增加足以引起心肌细胞凋亡。 我们分别通过腺病毒基因转移组成型活性（CA-CaMKII-dC）或显性失活CaMKII-dC突变体（DN-CaMKIIdC）来特异性地增强或抑制CaMKII-dC活性。CA-CaMKII-dC单独增强表达引起心肌细胞凋亡增加。细胞凋亡的严重程度与CA-CaMKII-dC蛋白丰度和激酶活性密切相关，提示CaMKII-dC的激活与心肌细胞凋亡之间存在因果关系。 （三） 各种刺激激活内源性CaMK Ⅱ引起心肌细胞凋亡 多种细胞死亡诱导刺激，如细胞内Ca 2+浓度增加、酸中毒和氧化应激，使内源性CaMKII活性增加23倍。这些刺激以CaMKII抑制剂敏感的方式显著触发心肌细胞凋亡。 为了确定CaMKII-dC与CaMKII-dB对观察到的细胞死亡的相对贡献，我们使用DN-CaMKII-dC抑制CaMKII-dC活性，并发现DN-CaMKII-dC的表达抑制激酶活性和相关的细胞死亡。这些结果表明，CaMKII-dC的激活构成了汇聚心肌细胞中多种刺激诱发的凋亡信号的共同途径Zhu et al.，J.Biol.Chem.282：10833-10839，（2007）。 （四） CaMKII-dB保护心肌细胞免于凋亡。 在大鼠缺血/再灌注（I/R）和心肌梗死（MI）模型中以及在培养的心肌细胞中受到过氧化氢的氧化应激时，CaMKII-dB的表达在mRNA和蛋白质水平上均显著减弱。MI和H2 O2对CaMKII-dB的抑制作用被ROS清除剂完全阻止，表明ROS构成CaMKII-dB基因表达的负调节剂。同时，MI和H2 O2显着增加心肌细胞凋亡在体内和培养中，分别通过Hoechst或TUNEL染色和caspase激活测定。最重要的是，使用腺病毒基因转移过度表达CaMKII-dB保护心脏细胞免受氧化应激诱导的凋亡，通过DNA梯状和PI染色测定。CaMKII-dB的保护作用是由HSP 70介导的信号通路介导的。 基于这些心脏CaMKII同种型的相反作用，我们设想，作为治疗心力衰竭或心律失常的潜在疗法，CaMKII-dB的激活与CaMKII-dC的抑制的组合应上级CaMKII的同种型非选择性抑制。

There are, at least, two splicing variants of CaMKII-d, dB and dC, located in unclear and cytosol compartments, respectively. Our present in vivo and in vitro studies have provided multiple lines of evidence to demonstrate that CaMKII-dB and CaMKII-dC exhibit opposing functional roles in regulating cardiomyocyte viability with CaMKII-dB protective and CaMKII-dC apoptotic. (1) CaMKII Activation Is Required for b1AR-Induced Apoptosis in Cardiomyocytes and In Vivo. Sustained b1AR stimulation markedly increased CaMKII activity in a time-dependent manner in adult mouse cardiomyocytes; this effect was abolished by a specific CaMKII inhibitors AIP or KN93 but not by specific PKA inhibitors Zhu et al.,J. Clin. Invest. 111:617-625, (2003). Importantly, inhibition of CaMKII fully protected myocytes from b1AR-induced apoptosis. In collaboration with Mark Anderson, we further demonstrated that CaMKII was essential for in vivo apoptotic response to excessive catecholamine stimulation Yang et al., Am. J. Physiol. 291:H3065-H3075, (2006). (2) Increased CaMKII-dC Activity Is Sufficient to Cause Heart Muscle Cell Apoptosis. We specifically enhanced or inhibited CaMKII-dC activity by adenoviral gene transfer of a constitutively active (CA- CaMKII-dC) or a dominant negative CaMKII-dC mutant (DN-CaMKIIdC), respectively. Enforced expression of CA-CaMKII-dC alone caused increased cardiac myocyte apoptosis. The severity of cell apoptosis was closely correlated with CA-CaMKII-dC protein abundance and the kinase activity, suggesting there is a causal relation between activation of CaMKII-dC and cardiomyocyte apoptosis. (3) Activation of Endogenous CaMKII by Various Stimuli That Trigger Myocyte Apoptosis Multiple cell death-inducing stimuli, such as increased intracellular Ca2+ concentration, acidosis, and oxidative stress, increased endogenous CaMKII activity by 23-fold over baseline. These stimuli markedly triggered myocyte apoptosis in CaMKII inhibitors sensitive manner. To define the relative contributions of CaMKII-dC versus that of CaMKII-dB to the observed cell death, we suppressed CaMKII-dC activity using the DN-CaMKII-dC, and found that expression of DN-CaMKII-dC inhibited the kinase activity and the associated cell death. These results indicate that activation of CaMKII-dC constitutes a common pathway converging multiple stimuli evoked apoptotic signals in cardiomyocytes Zhu et al., J. Biol. Chem. 282:10833-10839, (2007). (4) CaMKII-dB Protects Cardiomyocytes Against Apoptosis. 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 cardiomyocytes subjected to oxidative stress with H2O2. The inhibitory effects of MI and H2O2 on CaMKII-dB are fully prevented by 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 Hoechst or TUNEL staining and caspase activation. Most importantly, overexpression of CaMKII-dB using adenoviral gene transfer protects heart cells against oxidative stress-induced apoptosis, assayed by DNA laddering and PI staining. The CaMKII-dB protective effect is mediated by an HSP70-mediated signaling pathway. Based on the opposing effects of these cardiac CaMKII isoforms, we envision that a combination of activation of CaMKII-dB with inhibition of CaMKII-dC should be superior to isoform-nonselective inhibition of CaMKII as a potential therapy for the treatment of heart failure or cardiac arrhythmia.