Eikemo Hilde, Moltzau Lise Román, Hussain Rizwan I, Nguyen Cam H T, Qvigstad Eirik, Levy Finn Olav, Skomedal Tor, Osnes Jan-Bjørn
Department of Pharmacology, Institute of Clinical Medicine, University of Oslo and Oslo University Hospital, Oslo, Norway; K.G. Jebsen Cardiac Research Centre and Center for Heart Failure Research, Faculty of Medicine, University of Oslo, Oslo, Norway.
Department of Pharmacology, Institute of Clinical Medicine, University of Oslo and Oslo University Hospital, Oslo, Norway; K.G. Jebsen Cardiac Research Centre and Center for Heart Failure Research, Faculty of Medicine, University of Oslo, Oslo, Norway.
Biochem Biophys Res Commun. 2016 Feb 26;471(1):219-25. doi: 10.1016/j.bbrc.2016.01.132. Epub 2016 Jan 22.
The aim was to identify kinase activities involved in the phosphorylation of regulatory light chain (RLC) in situ in cardiomyocytes. In electrically stimulated rat cardiomyocytes, phosphatase inhibition by calyculin A unmasked kinase activities evoking an increase of phosphorylated RLC (P-RLC) from about 16% to about 80% after 80 min. The phosphorylation rate in cardiomyocytes was reduced by about 40% by the myosin light chain kinase (MLCK) inhibitor, ML-7. In rat ventricular muscle strips, calyculin A induced a positive inotropic effect that correlated with P-RLC levels. The inotropic effect and P-RLC elevation were abolished by ML-7 treatment. The kinase activities phosphorylating RLC in cardiomyocytes were reduced by about 60% by the non-selective kinase inhibitor staurosporine and by about 50% by the calmodulin antagonist W7. W7 eliminated the inhibitory effect of ML-7, suggesting that the cardiac MLCK is Ca(2+)/calmodulin (CaM)-dependent. The CaM-dependent kinase II (CaMKII) inhibitor KN-93 attenuated the calyculin A-induced RLC phosphorylation by about 40%, indicating a contribution from CaMKII. The residual phosphorylation in the presence of W7 indicated that also CaM-independent kinase activities might contribute. RLC phosphorylation was insensitive to protein kinase C inhibition. In conclusion, in addition to MLCK, CaMKII phosphorylates RLC in cardiomyocytes. Involvement of other kinases cannot be excluded.
目的是鉴定心肌细胞中参与调节性轻链（RLC）原位磷酸化的激酶活性。在电刺激的大鼠心肌细胞中，加丽素A抑制磷酸酶可揭示激酶活性，80分钟后可使磷酸化RLC（P-RLC）从约16%增加至约80%。肌球蛋白轻链激酶（MLCK）抑制剂ML-7可使心肌细胞中的磷酸化速率降低约40%。在大鼠心室肌条中，加丽素A诱导正性肌力作用，且该作用与P-RLC水平相关。ML-7处理可消除正性肌力作用和P-RLC升高。非选择性激酶抑制剂星形孢菌素可使心肌细胞中磷酸化RLC的激酶活性降低约60%，钙调蛋白拮抗剂W7可使其降低约50%。W7消除了ML-7的抑制作用，表明心脏MLCK是Ca（2+）/钙调蛋白（CaM）依赖性的。钙调蛋白依赖性激酶II（CaMKII）抑制剂KN-93可使加丽素A诱导的RLC磷酸化减弱约40%，表明CaMKII有作用。在存在W7的情况下的残余磷酸化表明，不依赖CaM的激酶活性也可能有作用。RLC磷酸化对蛋白激酶C抑制不敏感。总之，除了MLCK外，CaMKII也可使心肌细胞中的RLC磷酸化。不能排除其他激酶的参与。
