Calnexin Silencing in Mouse Neonatal Cardiomyocytes Induces Ca2+ Cycling Defects, ER Stress, and Apoptosis
Calnexin Silencing in Mouse Neonatal Cardiomyocytes Induces Ca2+ Cycling Defects, ER Stress, and Apoptosis
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DOI：
10.1002/jcp.24459
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发表时间：
2014-03-01
影响因子：
5.6
通讯作者：
Gramolini, Anthony O.
中科院分区：
文献类型：
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作者：
Bousette, Nicolas;Abbasi, Cynthia;Gramolini, Anthony O.
Calnexin(CNX)是一种内质网(ER)质量控制伴侣蛋白，与内质网应激有关。内质网应激是包括心血管疾病在内的各种病理状态的显著病理特征。然而，CNX和内质网应激在心脏中的作用尚未被研究。在本研究中，我们旨在从内质网应激、细胞凋亡和心肌细胞钙循环等方面研究cnx在心肌细胞生理学中的作用。我们发现，通过Lenti载体介导的靶向shRNAs转导，CNX的mRNA和蛋白水平显著降低。CNX沉默的心肌细胞表现出内质网应激，表现为GRP78和ATF6蛋白水平增加，XBP1剪接mRNA、ASK-1、ERO1a和CHOP mRNA水平增加。CNX沉默还导致caspase-3和-9的显著激活。这种半胱氨酸天冬氨酸氨基转移酶的激活与细胞凋亡的标志性形态特征有关，包括肌节组织和核完整性的丧失。活体细胞的钙离子成像显示，CNX沉默导致钙瞬变的幅度明显增加，但频率和钙摄取速率在基础状态下降低。有趣的是，在对照组和CNX沉默的心肌细胞中，5 mM咖啡因刺激的钙瞬变相似。最后，我们证明了CNx沉默诱导了L型电压依赖性钙通道(CAV1.2)的表达，但降低了肌浆网ATPase(SERCA2a)的表达。综上所述，这是首次通过CNX对内质网应激、细胞凋亡和钙通道表达的影响，证明CNX在心肌细胞活力和钙循环中具有特定作用。J.细胞。物理。229：374-383,2014。(C)2013年威利期刊公司。
Calnexin (CNX) is an endoplasmic reticulum (ER) quality control chaperone that has been implicated in ER stress. ER stress is a prominent pathological feature of various pathologic conditions, including cardiovascular diseases. However, the role of CNX and ER stress has not been studied in the heart. In the present study, we aimed to characterize the role of CNX in cardiomyocyte physiology with respect to ER stress, apoptosis, and cardiomyocyte Ca2+ cycling. We demonstrated significantly decreased CNX mRNA and protein levels by LentiVector mediated transduction of targeting shRNAs. CNX silenced cardiomyocytes exhibited ER stress as evidenced by increased GRP78 and ATF6 protein levels, increased levels of spliced XBP1 mRNA, ASK-1, ERO1a, and CHOP mRNA levels. CNX silencing also led to significant activation of caspases-3 and -9. This activation of caspases was associated with hallmark morphological features of apoptosis including loss of sarcomeric organization and nuclear integrity. Ca2+ imaging in live cells showed that CNX silencing resulted in Ca2+ transients with significantly larger amplitudes but decreased frequency and Ca2+ uptake rates in the basal state. Interestingly, 5mM caffeine stimulated Ca2+ transients were similar between control and CNX silenced cardiomyocytes. Finally, we demonstrated that CNX silencing induced the expression of the L-type voltage dependent calcium channel (CAV1.2) but reduced the expression of the sarcoplasmic reticulum ATPase (SERCA2a). In conclusion, this is the first study to demonstrate CNX has a specific role in cardiomyocyte viability and Ca2+ cycling through its effects on ER stress, apoptosis and Ca2+ channel expression. J. Cell. Physiol. 229: 374-383, 2014. (c) 2013 Wiley Periodicals, Inc.