Constitutively active calcineurin induces cardiac endoplasmic reticulum stress and protects against apoptosis that is mediated by α-crystallin-B
Constitutively active calcineurin induces cardiac endoplasmic reticulum stress and protects against apoptosis that is mediated by α-crystallin-B
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DOI：
10.1073/pnas.1013555107
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发表时间：
2010-10-26
影响因子：
11.1
通讯作者：
Gramolini, Anthony O.
中科院分区：
文献类型：
--
作者：
Bousette, Nicolas;Chugh, Shaan;Gramolini, Anthony O.
在CNA小鼠模型中，钙调磷酸酶A（CNA）的组成型活性形式的心脏特异性过表达直接导致心脏肥大。由于心脏肥大是许多心肌病的突出特征，我们推断，从该模型中描绘心室组织的蛋白质组学谱可能会识别新的、广泛适用的治疗靶点。通过将来自CNA小鼠及其WT同窝小鼠的分级心脏样品进行与鸟枪串联质谱法连接的无凝胶液相色谱来进行蛋白质组学分析。我们以高置信度鉴定了1，918种蛋白质，其中290种差异表达。对同一组织的微阵列分析为我们提供了心室转录组的改变。由于蛋白质组和转录组的生物信息学分析表明内质网应激的上调，我们通过一系列免疫印迹和RT-PCR分析验证了其在成人CNA心脏中的发生。内质网应激通常导致细胞凋亡增加，但在CNA心脏中细胞凋亡是最小的，这表明激活的钙调磷酸酶可能保护细胞凋亡。事实上，来自CNA小鼠的培养的新生小鼠心肌细胞（NCM）的活力在血清饥饿（凋亡触发剂）后高于WT。蛋白质组学数据确定α-晶状体蛋白B（Cryab）作为这种保护作用的潜在介质，并且我们表明，通过慢病毒载体介导的NCM中shRNA的转导沉默Cryab导致NCM活力显著降低并丧失对细胞凋亡的保护。鉴定Cryab作为钙调神经磷酸酶诱导的抗细胞凋亡保护的下游效应物将允许阐明其在心脏细胞凋亡中的作用及其作为治疗靶点的潜力。
Cardiac-specific overexpression of a constitutively active form of calcineurin A (CNA) leads directly to cardiac hypertrophy in the CNA mouse model. Because cardiac hypertrophy is a prominent characteristic of many cardiomyopathies, we deduced that delineating the proteomic profile of ventricular tissue from this model might identify novel, widely applicable therapeutic targets. Proteomic analysis was carried out by subjecting fractionated cardiac samples from CNA mice and their WT littermates to gel-free liquid chromatography linked to shotgun tandem mass spectrometry. We identified 1,918 proteins with high confidence, of which 290 were differentially expressed. Microarray analysis of the same tissue provided us with alterations in the ventricular transcriptome. Because bioinformatic analyses of both the proteome and transcriptome demonstrated the up-regulation of endoplasmic reticulum stress, we validated its occurrence in adult CNA hearts through a series of immunoblots and RT-PCR analyses. Endoplasmic reticulum stress often leads to increased apoptosis, but apoptosis was minimal in CNA hearts, suggesting that activated calcineurin might protect against apoptosis. Indeed, the viability of cultured neonatal mouse cardiomyocytes (NCMs) from CNA mice was higher than WT after serum starvation, an apoptotic trigger. Proteomic data identified alpha-crystallin B (Cryab) as a potential mediator of this protective effect and we showed that silencing of Cryab via lentivector-mediated transduction of shRNAs in NCMs led to a significant reduction in NCM viability and loss of protection against apoptosis. The identification of Cryab as a downstream effector of calcineurin-induced protection against apoptosis will permit elucidation of its role in cardiac apoptosis and its potential as a therapeutic target.