Department of Physiology, University of Toronto, Toronto, ON, Canada M5G 1A8.
Proc Natl Acad Sci U S A. 2010 Oct 26;107(43):18481-6. doi: 10.1073/pnas.1013555107. Epub 2010 Oct 11.
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 α-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.
心肌特异性过表达钙调神经磷酸酶 A 的组成型激活形式（CNA）直接导致 CNA 小鼠模型中的心肌肥大。由于心肌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特征可能会确定新的、广泛适用的治疗靶点。通过对 CNA 小鼠及其 WT 同窝仔鼠的心脏分离样本进行凝胶自由液相色谱与串联质谱分析，进行蛋白质组学分析。我们鉴定了 1918 种高可信度的蛋白质，其中 290 种表达差异。对相同组织进行的微阵列分析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都表明内质网应激的上调，我们通过一系列免疫印迹和 RT-PCR 分析验证了其在成年 CNA 心脏中的发生。内质网应激通常会导致细胞凋亡增加，但 CNA 心脏中的凋亡很少，这表明激活的钙调神经磷酸酶可能会保护细胞免于凋亡。事实上，在血清饥饿（一种凋亡触发因素）后，来自 CNA 小鼠的培养新生鼠心肌细胞（NCM）的活力高于 WT。蛋白质组学数据确定α-晶体蛋白 B（Cryab）为这种保护作用的潜在介质，我们表明通过慢病毒介导的 shRNA 转导在 NCM 中沉默 Cryab 会导致 NCM 活力显著降低，并且对凋亡的保护作用丧失。鉴定 Cryab 为钙调神经磷酸酶诱导的抗凋亡保护作用的下游效应子，将使阐明其在心脏凋亡中的作用及其作为治疗靶点的潜力成为可能。
