Study On The Role Of CaMKⅡ Mediated 20-HETE-induced Apoptosis And Hypertrophy In Neonatal Rat Cardiomyocytes

20-hydroxyeicosatetraenoic acid（20-HETE）is a metabolite of arachidonic acid catalyzed by theω–hydroxylase enzymes of cytochrome P-450（CYP）,which was found by Oliw and other scholars in recent years.Recently,20-HETE and CYP4A/4F are identified in hearts from rat and dog,and the production of 20-HETE is significantly enhanced during myocardial ischemia-reperfusion injury.Inhibiting20-HETE production produce a profound reduction in myocardial infarction size.Our previous study has confirmed that 20-HETE induces cardiomyocyte apoptosis and mediates angiotensin II（Ang II）–induced apoptosis via a mitochondrial superoxide-dependent pathway in cultured cardiomyocytes.Other research has shown that 20-HETE also involve in the non-adaptive myocardial hypertrophy induced by isoproterenol,benzopyrene and angiotensin II.Although accumulating evidence suggests that 20-HETE plays an important role in the cardiomyocyte apoptosis and hypertrophy,the exact molecular mechanisms underlying the myocardial injury induced by 20-HETE remains to be further elucidated.Cytosolic Ca²⁺overload is one of the hallmarks of apoptosis and hypertrophy in cardiomyocytes.Of note,our previous studies have shown that 20-HETE stimulates the L-type Ca²⁺channel via a PKC-dependent mechanism,thus elevating the concentration of Ca²⁺by calcium influx in cardiomyocytes.Calmodulin-dependent protein kinase II（CaMK II）,a multifunctional serine/threonine kinase,is an important regulator of cardiomyocytes Ca²⁺homeostasis,which has been recognized as an important factor in cardiomyocyte apoptosis and hypertrophy.Interestingly,several studies in vascular smooth muscle cells（VSMC）have shown that CaMK II participates in coupling G-protein receptors to release of AA,which can be subsequently metabolized by CYP4A/F enzymes to 20-HETE and suggested that activated CaMK II might be responsible for the enhanced 20-HETE dependent vascular reactivity.However,whether CaMK II is involved in the action of 20-HETE in cardiac myocytes has not been investigated previously.Therefore,the aims of present study were to explore the underling mechanisms of 20-HETE-induced cardiac apoptosis and hypertrophy by examining its effect on intracellular Ca²⁺released,and the role of CaMK II in these actions of 20-HETE in cultured neonatal rat cardiomyocytes.Objective:1.To observe the effects of 20-HETE on apoptosis and hypertrophy in cultured neonatal rat cardiomyocytes.2.To study the role of CaMK II on the intracellular Ca²⁺concentration,apoptosis and hypertrophy in cardiomyocytes induced by 20-HETE.Methods:1.Neonatal SD rat cardiomyocytes（<3 d）were cultured and purified by 5×10⁵/well cell density,which was inoculated in culture flask for 48 h.The cultured cells were randomly divided into 4 groups:the control group,culture medium was cultured for24 hours without any drug treatment.20-HETE group,20-HETE of 100 nmol/L was added to culture medium for 24 hours.20-HETE+KN-93 group,KN-93（3μmol/L）was added to the medium for 30 minutes,then 100 nmol/L 20-HETE was added for24 hours.KN-93 group,3μmol/L KN-93 was added to the medium and cultured for24 hours.2.After 12 hours of serum-free cultured,different concentrations of 20-HETE（1nmol/L,3 nmol/L,10 nmol/L,50 nmol/L,100 nmol/L）were added to culture medium for 24 hours,then CCK-8 assay was used to detect the activity of cardiomyocytes.3.The apoptotic rate of cardiomyocytes was detected by TUNEL.The surface area of cardiomyocytes was detected by HE staining.The total protein content of cardiomyocytes was detected by BCA and the ANP gene expression of hypertrophic marker was detected by RT-qPCR.4.Flou-3/AM labelled assay was applied to measure the concentration of intracellular calcium.5.Western blot was performed to measure the expressions of RyR2,SERCA2a,CaMK II and phospho-CaMK II.Result:1.20-HETE decreased the activity of cardiomyocytes by a concentration-and time-dependent manner.After treated with 100 nmol/L 20-HETE for 24 h,the activity of cardiomyocytes significantly decreased to 58.3±4.22%（P<0.05）;2.The results of TUNEL staining showed that the number of staining positive cells increased significantly from 2.0±1.05%in the control group to 49.8±2.3%（P<0.05）in the 20-HETE group,while KN-93,an inhibitor of CaMK II,blocked the effects induced by 20-HETE（23.1±4.12%,P<0.05）,and the number of staining positive cells were not affected by the treatment of KN-93（P>0.05）;3.The results of HE staining showed that the surface area of cardiomyocytes by treatment with 20-HETE increased significantly from（501.2±20.5）μm²to（956.7±85.5）μm²（P<0.05）,the total protein content increased from（1.61±0.03）mg/m L to（2.46±0.05）mg/mL（P<0.05）,the mRNA expression of hypertrophic biomarkers ANF significantly increased by 53.9%（P<0.05）,whereas co-treatment with KN-93（3μmol/L）significantly attenuated the effects of 20-HETE-induced hypertrophy.KN-93 treatment alone did not alter the surface area,total protein content or ANP expression compared with control groups（P>0.05）;4.Flou-3/AM fluorescent probe test showed that,compared with the control group,20-HETE could significantly increase the intracellular Ca²⁺concentration in cardiomyocytes by 75%（P<0.05）.KN-93 could significantly reduce the increasing of intracellular Ca²⁺concentration caused by 20-HETE（P<0.05）,and the intracellular Ca²⁺concentration were not affected by the treatment of KN-93（P>0.05）;5.Compared with control group,20-HETE can significantly up-regulated the expression of RyR2 by 3.8 times（P<0.05）,and down-regulated the expression of SERCA2a by 21%（P<0.05）,which could be blocked by KN-93.The intracellular Ca²⁺concentration were not affected by the treatment of KN-93（P>0.05）;6.Compared with control group,treatment with 20-HETE can significantly increase the protein expression of CaMK II and phospho-CaMK II in cardiomyocytesthe.Conclusion:1.20-HETE activates the CaMK II signaling pathway;2.20-HETE induced the increasing of intracellular Ca²⁺concentration via activating the CaMK II signaling pathway;3.CaMK II mediated the apoptosis and hypertrophy of cultured neonatal rat cardiomyocytes induced by 20-HETE.