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Nuclear myosin regulates gene transcription and this novel function might be modulated through phosphorylation of the myosin regulatory light chain (p-MLC 20 ). Nonmuscle MLC 20 (nmMLC 20 ) is also present in the nuclei of cardiomyocytes and a potential nmMLC 20 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-nmMLC 20 were detected. Coimmunoprecipitation (co-IP), chromatin immunoprecipitation, DNA pull-down, and luciferase reporter gene assays were used to decipher the molecular mechanisms through which nmMLC 20 promotes XO expression. IR or HR treatment dramatically elevated nuclear p-nmMLC 20 level, accompanied by increased XO expression, activity, and products (H 2 O 2 and uric acid), as well as the IR or HR injury; these effects were ameliorated by inhibition of MLCK or knockdown of nmMLC 20 . Our findings from these experiments demonstrated that nuclear p-nmMLC 20 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-nmMLC 20 plays an important role in IR/HR injury by transcriptionally upregulating XO gene expression to increase oxidative stress in myocardium. Our findings demonstrate nuclear nmMLC 20 as a potential new therapeutic target to combat cardiac IR injury. [ABSTRACT FROM AUTHOR]

Iron overload triggers the ferroptosis in the heart following ischemia/reperfusion (I/R) and transferrin receptor 1 (TfR1) charges the cellular iron uptake. Bioinformatics analysis shows that the three molecules of ubiquitin-specific protease 7 (USP7), p53 and TfR1 form a unique pathway of USP7/p53/TfR1. This study aims to explore whether USP7/p53/TfR1 pathway promotes ferroptosis in rat hearts suffered I/R and the underlying mechanisms. The SD rat hearts were subjected to 1 h-ischemia plus 3 h-reperfusion, showing myocardial injury (increase in creatine kinase release, infarct size, myocardial fiber loss and disarray) and up-regulation of USP7, p53 and TfR1 concomitant with an increase of ferroptosis (reflecting by accumulation of iron and lipid peroxidation while decrease of glutathione peroxidase activity). Inhibition of USP7 activated p53 via suppressing deubiquitination, which led to down-regulation of TfR1, accompanied by the decreased ferroptosis and myocardial I/R injury. Next, H9c2 cells underwent hypoxia/reoxygenation (H/R) in vitro to mimic the myocardial I/R model in vivo. Consistent with the results in vivo, inhibition or knockdown of USP7 reduced the H/R injury (decrease of LDH release and necrosis) and enhanced the ubiquitination of p53 along with the decreased levels of p53 and TfR1 as well as the attenuated ferroptosis (manifesting as the decreased iron content and lipid peroxidation while the increased GPX activity). Knockdown of TfR1 inhibited H/R-induced ferroptosis without p53 deubiquitination. Based on these observations, we conclude that a novel pathway of USP7/p53/TfR1 has been identified in the I/R-treated rat hearts, where up-regulation of USP7promotes ferrptosis via activation of the p53/TfR1 pathway. Image 1 • The protein levels of USP7, p53 and TfR1 are all elevated in the I/R-treated rat hearts. • Suppression of USP7 mitigate the myocardial I/R injury via reduction of ferroptosis. • USP7 promotes ferroptosis in the I/R-treated hearts via activation of p53/TfR1 pathway. [ABSTRACT FROM AUTHOR]