Key Laboratory of Stem Cell Biology and Laboratory of Molecular Cardiology, Institute of Health Sciences, Institutes for Biological Sciences (SIBS), Chinese Academy of Sciences (CAS) and Shanghai Jiao Tong University School of Medicine (SJTUSM), Shanghai, China.
Department of Pathology and Laboratory Medicine, University of Cincinnati Medical Center, Cincinnati, OH, USA.
J Mol Cell Cardiol. 2014 Dec;77:102-12. doi: 10.1016/j.yjmcc.2014.10.004. Epub 2014 Oct 18.
Although ischemia/reperfusion (I/R)-induced myocardial contractile dysfunction is associated with a prominent decrease in myofilament Ca(2+) 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 Ca(2+)-stimulated ATPase activity, and cMLCK content, but not cMLCP proteins. Consistently, simulated I/R in isolated cardiomyocytes inhibited cell shortening, Ca(2+) transients, MLC-2v phosphorylation, and myofilament sensitivity to Ca(2+). 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 Ca(2+)-stimulated ATPase activity, myocardial contractile function, and myofilament sensitivity to Ca(2+), 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 Ca(2+)-stimulated ATPase activity, which subsequently suppresses myocardial contractile function through a decrease of myofilament Ca(2+) sensitivity.
虽然缺血/再灌注（I/R）引起的心肌收缩功能障碍与肌球蛋白轻链 2（MLC-2v）的磷酸化水平降低有关。但是，其水平以及心脏肌球蛋白轻链激酶（cMLCK）和肌球蛋白轻链磷酸酶（cMLCP）在 I/R 心脏中的调节机制仍未完全阐明。在这项研究中，确定了 I/R 过程中 MLC-2v 磷酸化、cMLCK、cMLCP 和蛋白酶的水平和/或作用。整体心肌 I/R 抑制了离体大鼠心脏的心脏功能，同时降低了 MLC-2v 磷酸化、肌球蛋白轻链磷酸酶和肌球蛋白轻链对 Ca2+的刺激 ATP 酶活性以及 cMLCP 蛋白的水平。一致地，在分离的心肌细胞中模拟 I/R 抑制了细胞缩短、Ca2+瞬变、MLC-2v 磷酸化和肌球蛋白丝对 Ca2+的敏感性。这些观察结果通过 cMLCK 过表达而逆转，而短发夹 RNA（shRNA）的特异性 cMLCK 敲低则产生相反的效果。此外，基质金属蛋白酶-2（MMP-2，一种锌依赖性内肽酶）的抑制逆转了 I/R 降低的 cMLCK、MLC-2v 磷酸化、肌球蛋白轻链对 Ca2+的刺激 ATP 酶活性、心肌收缩功能和肌球蛋白丝对 Ca2+的敏感性，而 ML-9 或特异性 shRNA 抑制 cMLCK 则消除了 MMP-2 抑制诱导的心脏保护作用。最后，观察到 MMP-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 在体外被大鼠 MMP-2 浓度和时间依赖性降解，而 MMP-2 的抑制则阻止了这一降解。这些发现表明，I/R 激活的 MMP-2 导致 cMLCK 的降解，从而导致 MLC-2v 磷酸化和肌球蛋白轻链对 Ca2+的刺激 ATP 酶活性降低，进而通过降低肌球蛋白丝对 Ca2+的敏感性来抑制心肌收缩功能。
