A novel function of nuclear nonmuscle myosin regulatory light chain in promotion of xanthine oxidase transcription after myocardial ischemia/reperfusion

Nuclear myosin regulates gene transcription and this novel function might be modulated through phosphorylation of the myosin regulatory light chain (p-MLC₂₀). Nonmuscle MLC₂₀ (nmMLC₂₀) is also present in the nuclei of cardiomyocytes and a potential nmMLC₂₀ binding sequence has been identified in the promoter of the xanthine oxidase (XO) gene. Thus, we investigated its function in the regulation of XO transcription after myocardial ischemia/reperfusion (IR). In a rat model of myocardial IR and a cardiomyocyte model of hypoxia/reoxygenation (HR) injury, the cardiac or cell injury, myosin light chain kinase (MLCK) content, XO expression and activity, XO-derived products, and level of nuclear p-nmMLC₂₀ were detected. Coimmunoprecipitation (co-IP), chromatin immunoprecipitation, DNA pull-down, and luciferase reporter gene assays were used to decipher the molecular mechanisms through which nmMLC₂₀ promotes XO expression. IR or HR treatment dramatically elevated nuclear p-nmMLC₂₀ level, accompanied by increased XO expression, activity, and products (H₂O₂ and uric acid), as well as the IR or HR injury; these effects were ameliorated by inhibition of MLCK or knockdown of nmMLC₂₀. Our findings from these experiments demonstrated that nuclear p-nmMLC₂₀ binds to the consensus sequence GTCGCC in the XO gene promoter, interacts with RNA polymerase II and transcription factor IIB to form a transcription preinitiation complex, and hence activates XO gene transcription. These results suggest that nuclear p-nmMLC₂₀ plays an important role in IR/HR injury by transcriptionally upregulating XO gene expression to increase oxidative stress in myocardium. Our findings demonstrate nuclear nmMLC₂₀ as a potential new therapeutic target to combat cardiac IR injury.