Anthracyclines induce calpain-dependent titin proteolysis and necrosis in cardiomyocytes
Anthracyclines induce calpain-dependent titin proteolysis and necrosis in cardiomyocytes
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DOI：
10.1074/jbc.m308033200
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发表时间：
2004-02-27
影响因子：
4.8
通讯作者：
Sawyer, DB
中科院分区：
文献类型：
--
作者：
Lim, CC;Zuppinger, C;Sawyer, DB
肌丝蛋白是最大的肌丝蛋白，是肌节组装的模板，也是促进舒张期功能的分子弹簧。已知肌动蛋白在体外对钙依赖的蛋白水解酶非常敏感。我们假设Titin的降解是阿霉素引起的心脏损伤的早期事件，并且Titin的降解是通过激活钙依赖的蛋白水解酶，即钙调蛋白来实现的。用1或3摩尔/升阿霉素处理培养的成年大鼠心肌细胞24小时后，心肌细胞裂解物中的肌动蛋白降解，肌节I带的肌动蛋白弹力型(PEVK)结构域抗体免疫染色减弱。Titin的弹性结构域似乎最容易被蛋白质降解，因为与Titin抗体在M线上的联合免疫染色被保留，这表明Titin的弹性结构域具有靶向性的蛋白质降解。阿霉素处理1小时后，钙蛋白酶活性增加了3倍，在48小时后仍保持较高水平。与钙蛋白酶抑制剂联合处理后，肌动蛋白得以保留，肌原纤维紊乱减少，心肌细胞坏死减少，但不能导致细胞凋亡。与caspase抑制剂共处理不能阻止titin的降解，这排除了caspase-3作为titin蛋白分解的早期机制。我们得出的结论是，在阿霉素治疗心肌细胞后，钙蛋白酶的激活是一个早期事件，并且似乎以肌动蛋白的降解为靶点。肌动蛋白弹簧结构域的蛋白分解可能导致心肌细胞发生舒张期功能障碍、肌丝不稳定和细胞坏死。
Titin, the largest myofilament protein, serves as a template for sarcomere assembly and acts as a molecular spring to contribute to diastolic function. Titin is known to be extremely susceptible to calcium-dependent protease degradation in vitro. We hypothesized that titin degradation is an early event in doxorubicin-induced cardiac injury and that titin degradation occurs by activation of the calcium-dependent proteases, the calpains. Treatment of cultured adult rat cardiomyocytes with 1 or 3 mumol/liter doxorubicin for 24 h resulted in degradation of titin in myocyte lysates, which was confirmed by a reduction in immunostaining of an antibody to the spring-like (PEVK) domain of titin at the I-band of the sarcomere. The elastic domain of titin appears to be most susceptible to proteolysis because co-immunostaining with an antibody to titin at the M-line was preserved, suggesting targeted proteolysis of the springlike domain of titin. Doxorubicin treatment for 1 h resulted in similar to3-fold increase in calpain activity, which remained elevated at 48 h. Co-treatment with calpain inhibitors resulted in preservation of titin, reduction in myofibrillar disarray, and attenuation of cardiomyocyte necrosis but not apoptosis. Co-treatment with a caspase inhibitor did not prevent the degradation of titin, which precludes caspase-3 as an early mechanism of titin proteolysis. We conclude that calpain activation is an early event after doxorubicin treatment in cardiomyocytes and appears to target the degradation of titin. Proteolysis of the spring-like domain of titin may predispose cardiomyocytes to diastolic dysfunction, myofilament instability, and cell death by necrosis.