Yue T L, Wang C, Romanic A M, Kikly K, Keller P, DeWolf W E, Hart T K, Thomas H C, Storer B, Gu J L, Wang X, Feuerstein G Z
Department of Cardiovascular Pharmacology, SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406-0939, USA.
J Mol Cell Cardiol. 1998 Mar;30(3):495-507. doi: 10.1006/jmcc.1997.0614.
Cardiomyocyte apoptosis has been demonstrated in animal models of cardiac injury as well as in patients with congestive heart failure or acute myocardial infarction. Therefore, apoptosis has been proposed as an important process in cardiac remodeling and progression of myocardial dysfunction. However, the mechanisms underlying cardiac apoptosis are poorly understood. The present study was designed to determine whether the family of caspase proteases and stress-activated protein kinase (SAPK/JNK) are involved in cardiac apoptosis. Cultured rat neonatal cardiac myocytes were treated with staurosporine to induce apoptosis as evidenced by the morphological (including ultrastructural) characteristics of cell shrinkage, cytoplasmic and nuclear condensation, and fragmentation. Nucleosomal DNA fragmentation in myocytes was further identified by agarose gel electrophoresis (DNA ladder) as well as in situ nick end-labeling (TUNEL). Staurosporine-induced apoptosis in myocytes was a time- and concentration-(0.25-1 micro M)-dependent process. Staurosporine-induced apoptosis in myocytes was reduced by a cell-permeable, irreversible tripeptide inhibitor of caspases, ZVAD-fmk, but not by the ICE-specific inhibitor, Ac-YVAD-CHO. At 10, 50 and 100 muM of ZVAD-fmk, staurosporine-induced myocyte apoptosis was reduced by 5.8, 39.1 (P<0.01) and 53.8% (P<0.01), respectively. Staurosporine, at 0.25-1 micro M, increased caspase activity in cardiomyocytes by five- to eight-fold, peaking at 4-8 h after stimulation. Based on substrate specificity analysis, the major component of caspases activated in myocytes was consistent with caspase-3 (CPP32). Moreover, the appearance of the 17-kD subunit of active caspase-3 in staurosporine-treated myocytes was demonstrated by immunocytochemical analysis. In contrast, staurosporine induced a rapid and transient inhibition of SAPK/JNK in myocytes. The SAPK activity in myocytes was reduced by 68.3 and 58.3% (P<0.01 v basal) at 10 and 30 min after treatment with 1 micro M of staurosporine, respectively. Our results suggest that staurosporine-induced cardiac myocyte apoptosis involves activation of caspases, mainly caspase-3, but not activation of the SAPK signaling pathway.
在心脏损伤的动物模型以及充血性心力衰竭或急性心肌梗死患者中，已证实存在心肌细胞凋亡。因此，细胞凋亡被认为是心脏重塑和心肌功能障碍进展中的一个重要过程。然而，心脏细胞凋亡的潜在机制仍知之甚少。本研究旨在确定半胱天冬酶蛋白酶家族和应激激活蛋白激酶（SAPK/JNK）是否参与心脏细胞凋亡。用星形孢菌素处理培养的大鼠新生心肌细胞以诱导凋亡，细胞收缩、细胞质和细胞核浓缩以及碎片化的形态学（包括超微结构）特征可证明这一点。通过琼脂糖凝胶电泳（DNA梯带）以及原位缺口末端标记（TUNEL）进一步鉴定心肌细胞中的核小体DNA片段化。星形孢菌素诱导的心肌细胞凋亡是一个时间和浓度（0.25 - 1微摩尔）依赖性过程。细胞可渗透的、不可逆的半胱天冬酶三肽抑制剂ZVAD - fmk可减少星形孢菌素诱导的心肌细胞凋亡，但ICE特异性抑制剂Ac - YVAD - CHO则不能。在10、50和100微摩尔的ZVAD - fmk作用下，星形孢菌素诱导的心肌细胞凋亡分别减少了5.8%、39.1%（P<0.01）和53.8%（P<0.01）。0.25 - 1微摩尔的星形孢菌素使心肌细胞中的半胱天冬酶活性增加了五到八倍，在刺激后4 - 8小时达到峰值。基于底物特异性分析，心肌细胞中激活的半胱天冬酶的主要成分与半胱天冬酶 - 3（CPP32）一致。此外，免疫细胞化学分析证明了在星形孢菌素处理的心肌细胞中活性半胱天冬酶 - 3的17-kD亚基的出现。相反，星形孢菌素在心肌细胞中诱导了SAPK/JNK的快速和短暂抑制。在用1微摩尔星形孢菌素处理后10和30分钟，心肌细胞中的SAPK活性分别降低了68.3%和58.3%（与基础值相比，P<0.01）。我们的结果表明，星形孢菌素诱导的心肌细胞凋亡涉及半胱天冬酶的激活，主要是半胱天冬酶 - 3，但不涉及SAPK信号通路的激活。
