title: Nuclear cardiac myosin light chain 2 modulates NADPH oxidase 2 expression in myocardium: a novel function beyond muscle contraction.
abstract: Recent studies demonstrated that NADPH oxidase 2 (NOX2) expression in myocardium after ischemia-reperfusion (IR) is significantly upregulated. However, the underlying mechanisms remain unknown. This study aims to determine if nuclear cardiac myosin light chain 2 (MYL2), a well-known regulatory subunit of myosin, functions as a transcription factor to promote NOX2 expression following myocardial IR in a phosphorylation-dependent manner. We examined the phosphorylation status of nuclear MYL2 (p-MYL2) in a rat model of myocardial IR (left main coronary artery subjected to 1 h ligation and 3 h reperfusion) injury, which showed IR injury and upregulated NOX2 expression as expected, accompanied by elevated H₂O₂ and nuclear p-MYL2 levels; these effects were attenuated by inhibition of myosin light chain kinase (MLCK). Next, we explored the functional relationship of nuclear p-MYL2 with NOX2 expression in H9c2 cell model of hypoxia-reoxygenation (HR) injury. In agreement with our in vivo findings, HR treatment increased apoptosis, NOX2 expression, nuclear p-MYL2 and H₂O₂ levels, and the increases were ameliorated by inhibition of MLCK or knockdown of MYL2. Finally, molecular biology techniques including co-immunoprecipitation (Co-IP), chromatin immunoprecipitation (ChIP), DNA pull-down and luciferase reporter gene assay were utilized to decipher the molecular mechanisms. We found that nuclear p-MYL2 binds to the consensus sequence AGCTCC in NOX2 gene promoter, interacts with RNA polymerase II and transcription factor IIB to form a transcription preinitiation complex, and thus activates NOX2 gene transcription. Our results demonstrate that nuclear MYL2 plays an important role in IR injury by transcriptionally upregulating NOX2 expression to enhance oxidative stress in a phosphorylation-dependent manner.
authors.affiliation: Department of Pharmacology, School of Pharmaceutical Sciences, Central South University, Changsha, 410078, China.
authors.forename: Yi-Shuai
authors.initials: YS
authors.lastname: Zhang
authors.forename: Bin
authors.initials: B
authors.lastname: Liu
authors.forename: Xiu-Ju
authors.initials: XJ
authors.lastname: Luo
authors.forename: Ting-Bo
authors.initials: TB
authors.lastname: Li
authors.forename: Jie-Jie
authors.initials: JJ
authors.lastname: Zhang
authors.forename: Jing-Jie
authors.initials: JJ
authors.lastname: Peng
authors.forename: Xiao-Jie
authors.initials: XJ
authors.lastname: Zhang
authors.forename: Qi-Lin
authors.initials: QL
authors.lastname: Ma
authors.forename: Chang-Ping
authors.initials: CP
authors.lastname: Hu
authors.forename: Yuan-Jian
authors.initials: YJ
authors.lastname: Li
authors.forename: Jun
authors.initials: J
authors.lastname: Peng
authors.forename: Qingjie
authors.initials: Q
authors.lastname: Li
mesh_terms: D000818:Animals; D024722:Cardiac Myosins / Q000032:analysis / Q000502:physiology*; D002467:Cell Nucleus / Q000737:chemistry; D002478:Cells, Cultured; D008297:Male; D008562:Membrane Glycoproteins / Q000235:genetics*; D015428:Myocardial Reperfusion Injury / Q000517:prevention & control; D009206:Myocardium / Q000378:metabolism*; D018994:Myosin Light Chains / Q000032:analysis / Q000502:physiology*; D009219:Myosin-Light-Chain Kinase / Q000037:antagonists & inhibitors; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000235:genetics*; D018384:Oxidative Stress; D010766:Phosphorylation; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D008562:Membrane Glycoproteins; D018994:Myosin Light Chains; C107184:myosin light chain 2; C500593:Cybb protein, rat; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; D009219:Myosin-Light-Chain Kinase; D024722:Cardiac Myosins