title: Degradation of cardiac myosin light chain kinase by matrix metalloproteinase-2 contributes to myocardial contractile dysfunction during ischemia/reperfusion.
abstract: 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.
authors.affiliation: 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.
authors.forename: Ling
authors.initials: L
authors.lastname: Gao
authors.affiliation: 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.
authors.forename: Yan-Jun
authors.initials: YJ
authors.lastname: Zheng
authors.affiliation: 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.
authors.forename: Shan-Shan
authors.initials: SS
authors.lastname: Gu
authors.affiliation: 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.
authors.forename: Ji-Liang
authors.initials: JL
authors.lastname: Tan
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Cincinnati Medical Center, Cincinnati, OH, USA.
authors.forename: Christian
authors.initials: C
authors.lastname: Paul
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Cincinnati Medical Center, Cincinnati, OH, USA.
authors.forename: Yi-Gang
authors.initials: YG
authors.lastname: Wang
authors.affiliation: 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. Electronic address: htyang@sibs.ac.cn.
authors.forename: Huang-Tian
authors.initials: HT
authors.lastname: Yang
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D008297:Male; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism*; D009200:Myocardial Contraction; D017202:Myocardial Ischemia / Q000201:enzymology; D015428:Myocardial Reperfusion Injury / Q000201:enzymology* / Q000503:physiopathology; D009206:Myocardium / Q000201:enzymology / Q000473:pathology; D032383:Myocytes, Cardiac / Q000201:enzymology; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D059748:Proteolysis; D017207:Rats, Sprague-Dawley
chemical_list: D009219:Myosin-Light-Chain Kinase; D020778:Matrix Metalloproteinase 2; C522363:Mmp2 protein, rat
keywords: Cardiac myosin light chain kinase; Contraction; Ischemia/reperfusion injury; Matrix metalloproteinase-2; Ventricular myosin regulatory light chain