The Mechanism Of Activation Of TRPV4 Channel Aggravating Myocardial Ischemia-Reperfusion Injury

Aims:Blocking transient receptor potential vanilloid receptor subtype 4（TRPV4）channels can alleviate myocardial ischemia-reperfusion injury（MIRI）.However,the underlying mechanisms of TRPV4 channel activation exacerbates MIRI remains uncertain.The aims of this study are:（1）to confirm GSK1016790A（GSK101）,a TPRV4 specific agonist,can aggravate MIRI;（2）to verify whether the activation of TRPV4 channel can worsen calcium homeostasis dysregulation during ischemia-reperfusion（I/R）;（3）to clarify the mechanisms of GSK101 aggravating MIRI.Methods:The langendorff technique was applied to establish a mouse I/R model by global ischemia for 30 minutes followed by reperfusion for 60 minutes.GSK101（20 n M）was used to activate the TRPV4 channel.The expression of TRPV4 protein in myocardial tissue and cardiomyocytes were measured.Cardiac function indexes,including recovery ratio（RR）,left ventricular systolic pressure（LVSP）,left ventricular developmental pressure（LVDP）,and left ventricular end diastolic pressure（LVEDP）were recorded.The size of myocardical infarction area was detected by TTC staining;the levels of apoptosis（Bax/Bcl-2,Cyt c）were detected by Western-blotting.Meanwhile,to investigate the effect of TRPV4activation on mitochondrial function,HL-1 cells were exposed to hypoxia/reoxygention（H/R,Hypoxia 2h,Reoxygention 2h）,and then treated with GSK101（300 n M）.The level of reactive oxygen species（ROS）and cell viability were measured by fluorescence,and the changes of ATP production were observed by chemiluminescence technique.In addition,the optical mapping technique was employed to record the Time to peak,CaD80,and CaT recovery rate in mouse I/R model（15 minutes of global ischemia followed by 10minutes of reperfusion）,and the calcium homeostasis-related proteins（NCX1,Ry R2,SERCA-2a,PLN）in mouse myocardial tissue were determined by Western-blotting.To further elucidate the molecular mechanism of GSK101 in the aggravation of MIRI,the expression of TRPV4,JNK and CaMKⅡ was confirmed by immunofluorescence staining.The CaMKⅡ inhibitors KN93 as well as KN92 and JNK inhibitor SP600125 were used in the MIRI model,subsequently the indexes of RR,LVSP,LVDP and LVEDP were recorded.The size of myocardical infarction area was detected by TTC staining.The levels of apoptosis（Bax/Bcl-2,Cyt c）were also detected by Western-blotting.To further confirm whether JNK and CaMKⅡ were involved in the GSK101-induced impairment of mitochondrial function or not,HL-1 cells were treated with GSK101（300 n M）after hypoxia for 2 h and reoxygenation for 2 h,some groups were pretreated with TRPV4 channel-specific antagonist GSK219,JNK-specific inhibitor SP600125 or CaMKⅡ-specific inhibitor KN93,and inactive control KN92,30 min before GSK101 treatment,respectively.The level of ROS and cell viability were measured by fluorescence,and the changes of ATP production were observed by chemiluminescence technique.Furthermore,the effect of TRPV4 activation on intracellular calcium concentration([Ca²⁺]_i)in HL-1 cells was investigated by fluo-4,AM.The HL-1 cells were incubated with Fluo-4and AM for 30 min,then washed with PBS,after equilibration for 2 min,the HL-1 cells were pretreated with GSK101（10,100,300,900 n M）,and some of them were pretreated with GSK219（300 n M）30 min before GSK101 stimulation.In the Ca²⁺-free group,the medium was replaced with Ca²⁺-free medium.Then the intracellular Ca²⁺was detected and A23187 was added as a positive control.Finally,to confirm whether TRPV4 activation could induce JNK and CaMKⅡ activation in an extracellular Ca²⁺-dependent way.HL-1 cells were incubated with GSK101 for 5,10,20and 30 min,while the medium was replaced by Ca²⁺-free medium in the Ca²⁺-free group The cellular proteins were collected and the activation of JNK and CaMKⅡ was detected by Western-blotting.Results:The immunofluorescence results showed that the TRPV4 protein was expressed in cardiomyocytes（Troponin+）,cardiac vascular endothelial cells（CD31+）,and cardiac fibroblasts（Vimentin+）.The Western blot results also showed that the TRPV4 protein was being expressed in both HL-1 cells and cardiac tissue.TRPV4 agonist GSK101（20 n M）enhanced MIRI,as demonstrated by the further decrease RR,LVSP and LVDP and increase LVEDP,infarct size,Bax/Bcl-2,and Cyt c release.Furthermore,HL-1 cells that were pretreated with GSK101 and then exposed to HR showed a marked increase in mitochondrial dysfunction（higher levels of ROS,lower cell viability,and reduced ATP content）.Interestingly,the effects of GSK101 mentioned above were significantly reversed by blockade of TRPV4 or TRPV4 gene deletion.In addition,treatment with GSK 101 significantly prolonged the time to peak and CaD80and reduced Ry R2 refractory in isolated hearts following IR.Meanwhile,the phosphorylation levels of PLN-Thr17 and Ry R2-Ser2814 were further increased.All these changes were reversed by blockade of TRPV4.Moreover,GSK101 treatment increased the activation of JNK and CaMKⅡ,but had no effect on ERK or P38 in isolated hearts following I/R.More importantly,JNK inhibition（with SP600125）or CaMKⅡ inhibition（with KN93 or in transgenic AC3-I mice）prevented GSK101-induced myocardial injury during IR,as shown by improved cardiac function and decreased infarct size.Additionally,there was less apoptosis and mitochondrial dysfunction.Last,GSK101 increased[Ca²⁺]_i in a concentration-dependent manner and directly activated JNK and CaMKⅡ in HL-1 cells;however,this activity was inhihited by the removal extracelluar Ca²⁺or pretreatment with TRPV4 antogonists.JNK inhibition significantly inhibited the phosphorylation of CaMKⅡ induced by GSK101 but CaMKⅡ inhibition had no effect on JNK activation induced by GSK101.Conclusion:（1）TRPV4 activation can aggravate MIRI;（2）TRPV4 activation can promote calcium homeostasis dysregulation;（3）TRPV4 activation promotes Ca²⁺influx and subsequently activated JNK-CaMKⅡ signaling pathway,which resulted in detrimental myocardial effects.