Study Of The Role And Mechanisms Of20-HETE On Ischemic Reperfusion Injury And Apoptosis In Rats Heart

20-hydroxyeicosatetraenoic acid （20-HETE） is a metabolite of arachidonic acidcatalyzed by the ω-hydroxylase enzymes of the cytochrome P-450（CYP）.20-HETE plays animportant role in the regulation of vascular tone in the brain, kidney, heart and splanchnicbeds by activation of L-type Ca²⁺channels and inhibition of Ca²⁺sensitive K+channels invascular smooth muscle cells.20-HETE is a strong vascular constrictor in these vascular beds;thus, plays an important role in not only the physiological regulation of blood supply to thoseorgans but also in the development of ischemic diseases.The researches on20-HETE’s effects on vascular smooth muscle have widely beenconducted. However, so far, very few research reports have been made on the role andmechanisms of20-HETE on hearts. This paper studies the effects of20-HETE on cardiacfunction and mechanism from the following perspectives:Part I:20-HETE increases NADPH oxidase-derived ROS production andstimulates the L-type Ca²⁺channel via a PKC-dependent mechanism in cardiomyocytesThe production of20-hydroxyeicosatetraenoic acid （20-HETE） is increased duringischemia-reperfusion, and inhibition of20-HETE production has been shown to reduce infarctsize caused by ischemia. This study was aimed to discover the molecular mechanismunderlying the action of20-HETE in cardiac myocytes. The effect of20-HETE on L-typeCa²⁺currents （ICa,L） was examined in rat isolated cardiomyocytes by patch-clamp recordingin the whole cell mode. Superfusion of cardiomyocytes with20-HETE （10–100nM） resultedin a concentration-dependent increase in ICa,L, and this action of20-HETE was attenuated bya specific NADPH oxidase inhibitor, gp91ds-tat （5M）, or a superoxide scavenger,polyethylene glycol-superoxide dismutase （25U/ml）, suggesting thatNADPH-oxidase-derived superoxide is involved in the stimulatory action of20-HETE onICa,L. Treatment of cardiomyocytes with20-HETE （100nM） increased both NADPH oxidaseactivity and superoxide production by approximately twofold. To study the molecularmechanism mediating the20-HETE-induced increase in NADPH oxidase activity, PKCactivity was measured in cardiomyocytes. Incubation of the cells with20-HETE （100nM）significantly increased PKC activity, and pretreatment of cardiomyocytes with a selectivePKC inhibitor, GF-109203（1M）, attenuated the20-HETE-induced increases in ICa,L and inNADPH oxidase activity. In summary,20-HETE stimulates NADPH oxidase-derivedsuperoxide production, which activates L-type-Ca²⁺channels via a PKC-dependentmechanism in cardiomyocytes.20-HETE and20-HETE-producing enzymes could be noveltargets for the treatment of cardiac ischemic diseases. Part II:20-hydroxyeicosatetraenoic acid mediates the isolated heartischemia-reperfusion injury via increasing NADPH oxidase-derived ROS productionIn the studies of part1, we find that20-HETE increases NADPH oxidase-derived ROSproduction and stimulates the L-type Ca²⁺channel via a PKC-dependent mechanism incardiomyocytes, which can be the important reason for20HETE to aggravate the myocardialischemia reperfusion injury. In this part, we discuss and testify the the aforementioned celllevel discovering mechanisms in isolated rat heart.Experiments were performed in isolated rat heart subjected to35min of ischemiafollowed by40min of reperfusion on Langendorff preparations. Perfusion with HET0016, aninhibitor of20-HETE production, significantly improved I/R-induced reduction in cardiaccontractility, myocardial infarction, and myocardial apoptosis. In contrast, administration of20-HETE aggravated I/R-induced myocardial injury and enhanced apoptosis. I/R significantlyincreased reactive oxygen species （ROS） production and oxidative stresses, which weresignificantly inhibited by HET0016and enhanced by20-HETE administration. Apocynin, aninhibitor of NADPH oxidase, blocked20-HETE-induced ROS production in the I/R hearts.20-HETE increased the expression of gp91phoxand p22phoxthe subunits of NADPH oxidase;and stimulated NADPH oxidase activity. In addition, GF-109203significantly attenuated the20-HETE-induced increases in the NADPH oxidase expression and activity. Finally, in theLangendorff I/R preparation, both apocynin and tempol, a ROS scavenger, significantlyblocked20-HETE-induced myocardial dysfunction.All of the results demonstrate that20-HETE stimulates NADPH oxidase-derivedsuperoxide production, which aggravates I/R-induced myocardial injury via a PKC-dependentmechanism in isolated rat hearts.Part III:20-Hydroxyeicosatetraenoic Acid is involved in Angiotensin II-mediatedapoptosis in rat cardiac myocytesThrough the studies of the previous two parts we have preliminarily discussed the roleand mechanisms of20-HETE on IR. During that process, another important factor isapoptosis. According to our previous studies,20-HETE is able to induces apoptosis throughmitochondrial-dependent pathways. More importantly, this study, in its second part,discovers that during the course of IR,20-HETE obviously induces apoptosis. Some studiesshow that AngII plays an important role in inducing apoptosis. However, the crosstalkbetween20-HETE and Ang II in cardiomyocyte apoptosis process is unclear.In the current study, we examined apoptosis using flow cytometry in primary culturedneonatal rat ventricular myocytes treated with control, Ang II （100nM）, Ang II plus HET0016（a20-HETE formation inhibitor,10μM）, HET0016, or20-HETE （10nM） alone. The resultsdemonstrated that treatment with Ang II or20-HETE significantly increased apoptosis andthat Ang II-induced apoptosis were markedly attenuated by HET0016. In addition, Ang II-induced increases of caspase-3activity were significantly attenuated by20.9±3.4%afterco-treatment with HET0016. Our results also demonstrated that HET0016significantlysuppressed Ang II-induced increases in superoxide production by27.5±2.3%and AngII-induced decreases in mitochondrial membrane potential by64.5±6.3%. Ang II-inducednuclei crenation, chromatin condensation and fractionation were attenuated by73.6±8.5%with HET0016treatment. Finally, treatment cardiomyocytes with Ang II significantlyincreased CYP4A1expression and20-HETE production, measured by Western blot, real-timeRT-PCR, and mass spectrometric analysis.All results suggest that20-HETE may play a key role in Ang II-induce apoptosis incardiomyocytes.20-HETE and20-HETE-producing enzymes could be novel targets for thetreatment of Ang II related cardiac diseases.Part IV: δPKC mediates20-HETE-induced enhancement of ICa,Landdescreasement of IKFrom the results of part1, we demonstrate that20-HETE can stimulate NADPHoxidase-derived superoxide production, which activate L-type Ca²⁺channels via aPKC-dependent mechanism in cardiomyocytes. PKC family includes12subtypes, amongwhich δPKC plays an important role in inducing the cell apoptosis and aggravating themyocardial ischemia reperfusion injuries, however, the specific factors that activate δPKCstill remain unknown. Therefore, the research of this part majorly, through applying theresearch methods of patch clamp techniques, discusses the effects of δPKC in the role of20-HETE on the L-type calcium channels and whole-cell potassium channels.The results show that δPKC inhibitor Rottlerin inhibited the role of20-HETE onenhancement of ICa,Lwhile descreasement of IK. It proves that δPKC mediates andparticipates in the role of20-HETE on the electric currents inside those ion channels. All sucheffects of20-HETE may result in increased calcium influx, lead to intracellular calciumoverload. Moreover,20-HETE led to the decline in cardiac mechanical index bydetermination of myocardial function in vitro, and administration of δPKC inhibitor Rottlerin,attenuated the myocardial dysfunction induced by20-HETE.In summary, it is the first time to elaborate the roles and mechanisms of20-HETE onmyocardial ischemia reperfusion injuries and the induced cell apoptosis, making in-depthanalysis of the physiological and pathological mechanisms of the myocardial cell injuriescaused by20-HETE, providing applicable theoretic foundations for searching the effectivepretreatment medicines as the target spots to reduce the myocardial cell injuries caused by20-HETE, which can be applied to the clinical treatments of ischemic cardiomyopathy.