CaMKII-dB and CaMKII-dC Oppositely Regulate Cardiomyocyte viability

CaMKII-dB 和 CaMKII-dC 相反地调节心肌细胞活力

• 批准号: 7591974
• 负责人: Rui-Ping Xiao
• 金额: $ 65.47万
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7591974
• 关键词: Acceleration; Acidosis; Address; Adrenergic Receptor; Adult; Amino Acid Sequence; Apoptosis; Apoptotic; Arrhythmia; Artificial cardiac pacemaker; Attenuated; Biochemical Feedback Stimulation; Biological Assay; Biology; Brain; Cardiac; Cardiac Myocytes; Cardiomyopathies; Cardiovascular system; Caspase; Catecholamines; Cell Death; Cell physiology; Cells; Cessation of life; Chronic; Collaborations; Condition; Coupling; Cultured Cells; Cytosol; DNA; Dominant-Negative Mutation; Exhibits; Family; Frequencies; Functional disorder; Gene Expression; Gene Transfer; Genes; Goals; Heart; Heart Hypertrophy; Heart failure; Human; Hydrogen Peroxide; In Situ Nick-End Labeling; In Vitro; Infarction; Ischemia; Localized; Mediating; Medicine; Messenger RNA; Modeling; Molecular; Mus; Muscle Cells; Myocardial Infarction; Myocardium; Nature; Nuclear; Numbers; Oryctolagus cuniculus; Oxidative Stress; PKA inhibitor; Pathogenesis; Pathway interactions; Phosphorylation; Phosphotransferases; Physiological reperfusion; Play; Protein Isoforms; Protein Overexpression; Protein-Serine-Threonine Kinases; Proteins; RNA Splicing; Range; Rattus; Regulation; Relative (related person); Relaxation; Reperfusion Therapy; Research; Role; Severities; Signal Pathway; Signal Transduction; Staining method; Stains; Stimulus; Time; Variant; Yang; base; calmodulin-dependent protein kinase II; cell growth; clinically relevant; heart cell; in vivo; inhibitor/antagonist; member; mutant; prevent; programs; protective effect; response

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.

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。我们目前的体内和体外研究已经提供了多条证据表明，CaMKII-dB和CaMKII-DC在调节心肌细胞活力方面表现出相反的功能，而CaMKII-dB具有保护作用，CaMKII-DC具有凋亡作用。 (1)b1AR诱导的心肌细胞和活体细胞的凋亡需要CaMKII的激活。 持续的b1AR刺激以时间依赖的方式显著增加成年小鼠心肌细胞的CaMKII活性；这种作用可被特定的CaMKII抑制剂AIP或KN93取消，但不能被特定的PKA抑制剂朱等人，J.Clin。投资。111：617-625，(2003)。重要的是，抑制CaMKII完全保护心肌细胞免受b1AR诱导的细胞凋亡。在与Mark Anderson的合作中，我们进一步证明了CaMKII在过量儿茶酚胺刺激的体内细胞凋亡反应中是必不可少的J.Physiol.291：H3065-H3075，(2006)。 (2)CaMKII-DC活性升高足以引起心肌细胞凋亡。 我们通过腺病毒基因转移分别增强或抑制了CaMKII-DC的活性(CA-CaMKII-DC)和显性负性CaMKII-DC突变体(DN-CaMKIIdC)。单独表达CA-CaMKII-DC可导致心肌细胞凋亡增加。细胞凋亡的严重程度与CA-CaMKII-DC的蛋白丰度和激酶活性密切相关，提示CaMKII-DC的激活与心肌细胞的凋亡之间存在因果关系。 (3)内源性CaMKII被多种刺激激活，引发心肌细胞凋亡 多种细胞死亡诱导刺激，如细胞内钙离子浓度升高、酸中毒和氧化应激，使内源性CaMKII活性比基线增加23倍。这些刺激以CaMKII抑制剂敏感的方式显著启动心肌细胞的凋亡。 为了确定CaMKII-DC和CaMKII-dB在观察到的细胞死亡中的相对贡献，我们使用dN-CaMKII-DC抑制了CaMKII-DC的活性，发现dN-CaMKII-DC的表达抑制了激酶活性和相关的细胞死亡。这些结果表明，CaMKII-DC的激活构成了多种刺激引起的心肌细胞凋亡信号汇聚的共同途径。化学。10833-10839，(2007年)。 (4)CaMKII-DB对心肌细胞有保护作用。 在大鼠缺血/再灌注(I/R)、心肌梗死(MI)模型和过氧化氢氧化应激培养的心肌细胞中，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，Hoechst或TUNEL染色和半胱氨酸天冬氨酸氨基转移酶(Caspase)活性检测显示，MI和H_2O_2显著增加在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可以保护心肌细胞免受氧化应激诱导的细胞凋亡，DNA阶梯和PI染色检测到这一点。CaMKII-Db的保护作用是由HSP70介导的信号通路介导的。 基于这些心脏CaMKII亚型的相反作用，我们设想激活CaMKII-db和抑制CaMKII-DC作为一种潜在的治疗心力衰竭或心律失常的方法，应该优于非选择性抑制CaMKII。