Degradation of cardiac myosin light chain kinase by matrix metalloproteinase-2 contributes to myocardial contractile dysfunction during ischemia/reperfusion
Degradation of cardiac myosin light chain kinase by matrix metalloproteinase-2 contributes to myocardial contractile dysfunction during ischemia/reperfusion
复制标题
基质金属蛋白酶-2 降解心肌肌球蛋白轻链激酶导致缺血/再灌注期间心肌收缩功能障碍
DOI：
10.1016/j.yjmcc.2014.10.004
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发表时间：
2014-12-01
影响因子：
5
通讯作者：
Yang, Huang-Tian
中科院分区：
文献类型：
--
作者：
Gao, Ling;Zheng, Yan-Jun;Yang, Huang-Tian
尽管缺血/再灌注(I/R)引起的心肌收缩功能障碍与肌丝对钙的敏感性显著降低有关，但其潜在的机制尚未完全阐明。心肌肌球蛋白轻链2(MLC-2v)的磷酸化促进了肌动蛋白-肌球蛋白的相互作用并增强了肌球蛋白的收缩能力，然而，其在I/R心脏中的水平及其对心肌MLC激酶(CMLCK)和CMLC磷酸酶(CMLCP)的调节仍存在争议。本研究测定了MLC-2v磷酸化、cMLCK、cMLCP和蛋白水解酶在I/R过程中的水平和/或作用。整体心肌I/R抑制大鼠离体心功能的同时伴随着MLC-2v磷酸化、肌原纤维钙离子刺激的ATPase活性和cMLCK含量的降低，但不包括cMLCP蛋白。在分离的心肌细胞中，模拟I/R一贯地抑制细胞缩短、钙瞬变、MLC-2v磷酸化和肌丝对钙的敏感性。CMLCK的过表达逆转了上述观察结果，而短发夹状RNA(ShRNA)特异性的cMLCK敲除则具有相反的作用。此外，抑制基质金属蛋白酶-2(一种锌依赖的内肽酶)可逆转IR引起的cMLCK、MLC-2v磷酸化、肌原纤维钙激活的ATPase活性、心肌收缩功能和肌丝对钙的敏感性的降低，而ML-9或特异性shRNA抑制或敲除cMLCK则取消了MMP2抑制引起的心肌保护作用。最后，观察了基质金属蛋白酶-2和cMLCK在心肌细胞中的共定位和体内相互作用。纯化的重组大鼠cMLCK在体外可被大鼠MMP2以浓度和时间依赖的方式降解，这种降解可被MMP2的抑制所阻止。这些结果表明，I/R激活的基质金属蛋白酶-2导致cMLCK降解，导致MLC-2v磷酸化降低，肌原纤维钙激活的ATPase活性降低，从而通过降低肌丝对钙的敏感性而抑制心肌收缩功能。(C)2014爱思唯尔有限公司。保留所有权利。
Although ischemia/reperfusion (I/R)-induced myocardial contractile dysfunction is associated with a prominent decrease in myofilament Ca2+ sensitivity, the underlying mechanisms have not yet been fully clarified. Phosphorylation of ventricular myosin light chain 2 (MLC-2v) facilitates actin-myosin interactions and enhances contractility, however, its level and regulation by cardiac MLC kinase (cMLCK) and cMLC phosphatase (cMLCP) in I/R hearts are debatable. In this study, the levels and/or effects of MLC-2v phosphorylation, cMLCK, cMLCP, and proteases during I/R were determined. Global myocardial I/R-suppressed cardiac performance in isolated rat hearts was concomitant with decreases of MLC-2v phosphorylation, myofibrillar Ca2+-stimulated ATPase activity, and cMLCK content, but not cMLCP proteins. Consistently, simulated I/R in isolated cardiomyocytes inhibited cell shortening, Ca2+ transients, MLC-2v phosphorylation, and myofilament sensitivity to Ca2+. These observations were reversed by cMLCK overexpression, while the specific cMLCK knockdown by short hairpin RNA (shRNA) had the opposite effect. Moreover, the inhibition of matrix metalloproteinase-2 (MMP-2, a zinc-dependent endopeptidase) reversed IR-decreased cMLCK, MLC-2v phosphorylation, myofibrillar Ca2+-stimulated ATPase activity, myocardial contractile function, and myofilament sensitivity to Ca2+, while the inhibition or knockdown of cMLCK by ML-9 or specific shRNA abolished MMP-2 inhibition-induced cardioprotection. Finally, the co-localization in cardiomyocytes and interaction in vivo of MMP-2 and cMLCK were observed. Purified recombinant rat cMLCK was concentration- and time-dependently degraded by rat MMP-2 in vitro, and this was prevented by the inhibition of MMP-2. These findings reveal that the I/R-activated MMP-2 leads to the degradation of cMLCK, resulting in a reduction of MLC-2v phosphorylation, and myofibrillar Ca2+-stimulated ATPase activity, which subsequently suppresses myocardial contractile function through a decrease of myofilament Ca2+ sensitivity. (C) 2014 Elsevier Ltd. All rights reserved.