Role Of Nuclear Myosin In Nox2-mediated Cardiac Ischemia Injury And The Underlying Mechanisms

BackgroundCardiac ischemia/reperfusion (IR) injury is a phenomeon which occurs in ischemia area with more severe injury after reperfusion.Multiple mechanisms are reported to be involved myocardial IR injury. Oxidative stress is one of them. Oxidative stress injury is caused by the excessive production of reactive oxidative species (ROS). As a vital injury factor in oxidative stress, ROS are mainly derived from myocardial NADPH oxidase (Nox) in cardiac IR injury. In cardiomyocyte there are two subtypes of Nox, Nox2and Nox4, which produce ROS to induce cardiac IR injury. The immediate product of Nox is superoxide anion. However, the direct involvement of the superoxide anion itself in IR injury might not be significant, because superoxide anion is considered as a rather "sluggish" ROS, not reactive enough to be an important player on its own. Once generated, the superoxide anion dismutates into hydrogen peroxide (H2O2). H2O2is further catalyzed by peroxidases to form hypochlorous acid (HOCl), which provokes cell death and apoptosis. As a novel peroxidase, vascular peroxidase (VPO) can utilize H2O2generated from Nox to produce HOCl with stronger oxidative activity. VPO1is the major isoform of VPO expressed in heart. Therefore, we speculate that through the link of H2O2, Nox2and VPO1can form a novle pathway (Nox2/VPO1) to medicate oxidative injury in myocardium following IR. In the present study, by using a rat model of cardiac IR injury and cardiac cell model of hypoxia/reoxygenation (HR) injury, we explored the role of Nox/VPO1in cardiac oxidative injury following IR. This study will provide novel evidence for the theory of oxidative stress in mediation of IR injury and also provide novel ideas for development of new drugs in protection of heart against IR injury.MethodsFirst, we used a rat model of myocardial IR injury together with Nox inhibitor (apocynin or DPI) to evaluate the role of Nox/VPO1pathway in IR oxidative injury and the possible link between Nox2and VPO1. Then, we used cardiac cell model of HR injury together with drug interference and gene silence to further elucidate the mechanisms for Nox2/VPO1pathway-mediated oxidative injury following IR.Triphenyltetrazolium chloride (TTC) staining for infarct area; spectrophotometry for measurements of creatine kinase (CK), Nox, caspase-3, SOD activities and H2O2level, hematoxylin-eosin (HE) staining for morphological analysis; TUNEL assay and hochest staining for cellular apoptosis; immunohistochemistry and immunofluorescencefor measurements of caspase-3level and Nox2, VPO1expression; realtime PCR for Nox2, VPO1mRNA expression; Western blot for Nox2, VPO1,p-JNK, p-p38protein expression; fluorescence probe mark for HOCl level.ResultsIn animal experiments, IR treatment increased myocardial infarct size, CK release and myocardial apoptosis accompanied by the increased H2O2level, Nox activity and the upregulation of Nox2and VPO1expression. There was no significant change in Nox4mRNA expression following IR. DPI treatment significantly attenuated the effects of IR on myocardial infarction, H2O2production, Nox activity, Nox2and VPO1expression. Apocynin treatment obtained similar results to that of DPI treatment except that it did not affect Nox2expression. In cell experiments, HR treatment significantly increased cellular apoptosis, Nox activity and levels of H2O2and HOCl concomitantly with upregulation of Nox2and VPO1expression, which were significantly attenuated by Nox2gene silence. Apocynin treatment or VPOl gene silence obtained similar results to that of Nox2silence except that apocynin treatment did not affect Nox2expression while VPO1silence did not affect Nox2expression, Nox activity and H2O2production. Co-silence of Nox2and VPO1achieved similar results to that of Nox2or VPO1silence alone. SOD inhibitor did not display significant effect on H2O2production. H2O2treatment can induce VPO1expression in a concentration-dependent manner, which was attenuated in the presence of JNK or p38MAPK inhibitor.ConclusionDuring cardiac IR, Nox2and VPO1can form a novel pathway of Nox2/VPO1via the link of H2O2, which participates in the mediation of IR oxidative injury through cooperation between Nox2and VPO1. BackgroundIn our work before, we have demonstrated that Nox2and VPO1expression is significantly upregulated following cardiac ischemia/reperfusion (IR). Nox2and VPO1form a pathway of Nox2/VPO1via the link of H2O2to mediate the IR oxidative injury. Nox2-derived H2O2can induce VPO1expression via JNK/p38MAPK pathway, and then VPO1plays an important role in cardiac IR injury. However, as the upstream of VPO1effect the regulatory mechanisms underlying Nox2expression remain largely unknown.Myosin is a cytoskeleton and motor protein, which consists of heavy chains, essential light chains and regulatory light chains (MLC20).Except for participating in muscle contraction, myosin plays an important role in regulation of cellular migration, adhesion and vascular endothelial permeability. It has been found that MLC20exists in cellular nucleus. It can form the transcription preinitiation complex with RNA polymerase II (RNA pol) holoenzyme and transcription factor IIB (TF IIB) to participate in regulation of ICAM-1gene expression through a manner of phosphorylation/dephosphorylation in colonic circular smooth muscle cells. Whether myosin also exists in nucleus of cardiomyocytes? Except for mediation of myocardial contraction, if myosin has other functions in cardiomyocytes? Nox2plays an important role in myocardial IR injury, whether myosin takes a part in the regulation of Nox2expression? We have found that there exist binding site for MLC20in the upstream of Nox2promoter by bioinformatics analysis. Therefore, by using an in vivo rat cardiac IR and H9c2cell line hypoxia/reoxygenation (HR) models we will evaluate the role of nuclear myosin in cardiac IR injury and the underlying mechanism.MethodsFirst, by using a rat model of myocardial IR together with with MLCK inhibitor ML-7, we evaluated the role of MLC2o in IR injury and the correlation between MLC2o and Nox2expression. Then, by using a cardiac cell model of HR together with drug interference, gene silence, co-immunoprecipitation and chromatin immunoprecipitation, we further elucidated the mechanisms for MLC2o in regulation of Nox2gene expression.Triphenyltetrazolium chloride (TTC) staining for infarct area; spectrophotometry for measurements of CK, Nox, caspase-3, SOD activities and H2O2level, hematoxylin-eosin (HE) staining for morphological analysis; TUNEL assay and hochest staining for cellular apoptosis; immunohistochemistry for caspase-3level and cellular immunofluorescence for p-MLC20; realtime PCR for Nox2mRNA expression; Western blot for Nox2, nuclear or cytoplasmic p-MLC2o and MLC20protein expression; co-immunoprecipitation for examination of the interaction between MLC2o and RNA pol â…¡ or TF IIB; cHRomatin immunoprecipitation assay for confirmation of the binding between p-MLC20and the specific DNA sequences in the promoter area of target gene (Nox2).ResultsIn animal experiments, IR treatment increased myocardial infarct size, CK release and myocardial apoptosis accompanied by the increased H2O2level, Nox activity, the upregulated Nox2expression and phorsphorylation level of nuclear MLC20, which was impaired by MLCK inhibitor (ML-7). There was no significant change in level of cytoplasmic and nuclear MLC20among different groups. In cell experiments, HR treatment significantly increased cardiomyocyte apoptosis concomitantly with the increased H2O2and HOCl levels, nuclear MLC20phorsphorylation level and the upregulated Nox2expression, which was attenuated in the presence of ML-7or by MLC20gene silence. Compared with control or HR group MLC20gene silencing could downregulate the level of cytoplasmic and nuclear MLC20. The results of co-immunoprecipitation showed p-MLC20could interact with RNA pol â…¡ or TF IIB. Chromatin immunoprecipitation assay confirmed that HR could facilitate nuclear p-MLC20to bind to specific DNA sequence (AGCTCC) within the upstream areas of Nox2promoter, which was reversed by interference with MLCK inhibitor.ConclusionAs a nuclear transcription factor in heart nuclear myosin contributes to myocardial IR injury via upregulation of Nox2expression, which is related to phorsphorylation of MLC20.