The Role And Mechanism Of TRPV4 Channel During Hypoxia/Reoxygenation Injury In Cardiomyocytes

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The role of TRPV4 channel during H/R in cardiomyocytesObjective:Recent studies identified that the TRPV4 channel is highly expressed in the cardiocerebral vascular system,and playes important roles both in mice cerebral ventricle and myocardial I/R injury,but the specific mechanism still unknown.So,the purpose of this study is to investigate the role of TRPV4 in the H/R-indued injury in cardiomyocytes.Methods:To mimic myocardial I/R injury,we established a H/R model in H9C2 cells and neonatal rat ventricle myocytes?NRVMs?in vitro.TRPV4 mRNA and protein expression was confirmed in the H9C2 and NRVM,whereas functional TRPV4 activity was assessed by Ca²⁺influx response to a TRPV4 agonist GSK1016790A.And TRPV4 agonist and antagonist were given at the set of reoxygenation.The cell damage was detected by CCK-8 and LDH kits,and apoptosis was detected by AV/PI double staining method.In addition,specific TRPV4-siRNA silenced TRPV4 genes were prepared.Results:TRPV4 mRNA and protein expression were detected,and GSK1016790A induced robust concentration-dependent?100,300 and 500nM?Ca²⁺influx in H9C2 and NRVMs,which were almost blocked by pretreatment with a selective TRPV4 antagonist,HC-067047?1?M?.Morever,H/R enhanced the functional expression of TRPV4,Besides,H/R induced cell injury as evidenced by the cell viability,the release of lactate dehydrogenase?LDH?and apoptosis,which were further aggravated by GSK1016790A,and obviously alleviated by HC-067047 or specific TRPV4-siRNA.Conclusions:TRPV4 channel are functionally expressed in cardiomyocytes and significant enhanced during H/R,and play a key role in H/R.Part ? The mechanisms of TRPV4 channel in H/R injury of cardiomyocytes: involved calcium overload,oxidative stress and mitochondrial damageObjective: our study confirms that TRPV4 channel play an important role in mice myocardial I/R in vivo and H/R injury in vitro,but its detailed mechanism is unknown.And TRPV4 highly permeates calcium ions after activation.Previous study have confirmed TRPV4 activation mediated the calcium influx induced cytoplasm calcium overload,stimulated the production of ROS,leading to mitochondrial dysfunction mediated cell damage in the bladder epithelial cells,endothelial cells,nerve cells and others under pathological process.However,there is no relevant research reports in cardiomyocytes.Therefore,this study is to explore whether the mechanisms of TRPV4 channel during H/R in cardiomyocytes involved calcium overload,oxidative stress and mitochondrial dama ge.Methods: The intracellular Ca2+ concentration?[Ca2+]i?was assessed by relative Ca2+ fluorescences intensity.ROS production in H9C2 was detected using the Reactive Oxygen Species Assay Kit.In addition,Changes of mitochondrial membrane potential???m?were measured by staining with JC-1 and m PTP opening was assayed by measuring calcein fluorescence quenched by cobalt chloride as previously reported.Above fluorescences were detected with a fluorescent microscope or an Enspire Multimode Plate Reader.Besides,Ca2+ free medium and ROS scavenger NAC were also used during reoxygenation.Results: H/R increased the intracellular Ca2+ concentration?[Ca2+]i?were reversed by HC-067047 and TRPV4-si RNA but was further aggravated by GSK1016790 A.Moreover,HC067047 treatment significantly alleviated the increase of reactive oxygen species?ROS? generation,the depolarization of mitochondrial membrane potential???m?and the opening of mitochondrial permeability transition pore?m PTP?during H/R.On the contrary,GSK1016790 A exacerbated those effects.Meanwhile,increase in [Ca2+]i and ROS induced by activation of TRPV4 was almost abolished when cells were cultured in Ca2+ free medium.In addition,ROS scavenger NAC obviously reversed activation of TRPV4 induced changes of ??m and m PTP opening.Conclusions: Activation of TRPV4 induces Ca2+ influx in cardiomyocytes,with subsequent ROS release,depolarizing of ??m,opening m PTP,inducing injury and TRPV4 plays key roles during I/R via these pathways.Part ? TRPV4 channel mediated myocardial oxidative stress injury and its specific mechanismObjective : Antioxidative stress provided the cardioprotective effects in myocardial ischemia/reperfusion?I/R?.Transient receptor potential vanilloid 4?TRPV4?is a Ca2+-permeable nonselective cation channel and widely expressed in the cardiovascular system.We previously demonstrated TRPV4 activation induces Ca2+ influx and increases reactive oxygen species?ROS?release,and finally induces injury during hypoxia/reoxygenation?H/R?in cardiomyocytes.The current study focused on whether blockade of TRPV4 reduced the injury via the antioxidative activity during H/R and explored the molecular mechanisms in cardiomyocytes.Methods: H9C2 cardiomyocytes were subjected to H/R.The cell damage was detected by CCK-8 and LDH kits,and apoptosis was detected by AV/PI double staining and Tunel methods.ROS was detected by DCFH-DA and MDA was quantifed with MDA kits.The activity of CAT,GSH-PX1 and SOD were measured by relative enzyme activity test kit,respectively.Western-Blots reflected the protein expression of AKT and Nrf2,and Real-Time PCR indicated the m RNA expression of related antioxidant response element?ARE?mainly including Nrf2,SOD2,GSH-PX,HO-1,NQO1,CAT,GST and GCSH.And the translocation of Nrf2 was checked by a fluorescent microscope.Isolated rat hearts were subjected to 10 min H2O2 perfusion followed by 60 min KH buffer reperfusion and treated with the HC-067047.Results: Our results reveal that H/R induced cell injury as evidenced by the cell viability,the release of lactate dehydrogenase?LDH?and apoptosis,which were obviously alleviated by a selective TRPV4 blocker HC-067047 or specific TRPV4-si RNA.Moreover,H/R also increased the contents of ROS and malondialdehyde?MDA?and decreased the activity of superoxide dismutase?SOD?and glutathione peroxidase?GSH-PX?,which were reversed by HC-067047 or specific TRPV4-si RNA.Furthermore,HC-067047 treatment increased the expression of P-AKT,and the translocation of nuclear factor E2-related factor 2?Nrf2?and related antioxidant response element?ARE?mainly including SOD,GSH-PX and GST after H/R.In addition,the AKT inhibitor,LY294002,absolutely hampered these cardioprotective effects of HC-067047.Finally,we confirmed the antioxidative stress roles of blockade of TRPV4 in myocardial I/R or application of exogenous H2O2.Conclusions: Inhibition of TRPV4 exerts protective effects against oxidative stress induced cardiomyocyte injury possibly related to the up-regulated AKT/Nrf2/ARE pathway.