Calpain-1-sensitive myofibrillar proteins of the human myocardium
Calpain-1-sensitive myofibrillar proteins of the human myocardium
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DOI：
10.1007/s11010-005-1370-7
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发表时间：
2005-10-01
影响因子：
4.3
通讯作者：
Papp, Z
中科院分区：
文献类型：
--
作者：
Barta, J;Tóth, A;Papp, Z
Calpain-1 是一种普遍存在的细胞内 Ca2+ 激活蛋白酶，它与通过肌原纤维蛋白降解导致缺血和再灌注后可逆性心肌抑制（即心肌顿抑）的发病机制有关。然而，人类心肌中这种降解过程的靶蛋白尚未确定。为了比较一组人肌原纤维蛋白（肌联蛋白、α-胞质蛋白、结蛋白、肌钙蛋白 T (cTnT)、肌钙蛋白 I (cTnI) 和 α-辅肌动蛋白）内 Calpain-1 的敏感性水平，将粗左心室组织匀浆在 Calpain-1 存在的情况下孵育 0.5、15、30、60 或 120 分钟（1 U 或 5 U）。随后使用银染 SDS 聚丙烯酰胺凝胶和蛋白质免疫印迹分析来评估蛋白质降解动力学和程度的差异。这些测定显示肌原纤维蛋白具有高（肌联蛋白和α-胞质蛋白）、中度（结蛋白和cTnT）或低（cTnI 和α-肌动蛋白）相对Calpain-1 敏感性。 cTnI 的磷酸化水平并不能解释其相对较低的 Calpain-1 敏感性。此外，由 Ca2+ 依赖性自蛋白水解作用产生的截短的 α-fodrin、desmin 和 cTnI 片段的分子量分布与其 Calpain-1 剪辑产物的分子量分布表现出明显的相似性。这些体外结果揭示了收缩装置内的许多结构蛋白（肌联蛋白、α-胞质蛋白、结蛋白和α-肌动蛋白）和调节蛋白（cTnT 和 cTnI）是 Calpain-1 的潜在靶标。它们的降解可能有助于人类心肌缺血后击晕的发生。
Calpain-1 is a ubiquitous intracellular Ca2+-activated protease, which has been implicated in the pathogenesis of reversible myocardial depression (i.e. myocardial stunning) that follows ischemia and reperfusion via myofibrillar protein degradation. However, the target proteins of this degradative process in the human myocardium have not yet been identified. In order to compare the levels of Calpain-1 susceptibility within a set of human myofibrillar proteins (titin, alpha-fodrin, desmin, troponin T (cTnT), troponin I (cTnI) and alpha-actinin), crude left ventricular tissue homogenates were incubated for 0.5, 15, 30, 60 or 120 min in the presence of Calpain-1 (1 U or 5 U). Differences in the kinetics and extents of protein degradation were subsequently evaluated by using silver-stained SDS-polyacrylamide gels and Western immunoblot analyses. These assays revealed myofibrillar proteins with high (titin and alpha-fodrin), moderate (desmin and cTnT), or low (cTnI and alpha-actinin) relative Calpain-1 susceptibilities. The level of phosphorylation of cTnI did not explain its relatively low Calpain-1 susceptibility. Moreover, the molecular mass distributions of the truncated alpha-fodrin, desmin and cTnI fragments resulting from Ca2+-dependent autoproteolysis exhibited marked similarities with those of their Calpain-1-clipped products. These in vitro results shed light on a number of structural (titin, alpha-fodrin, desmin and alpha-actinin) and regulatory (cTnT and cTnI) proteins within the contractile apparatus as potential targets of Calpain-1. Their degradation may contribute to the development of postischemic stunning in the human myocardium.