Activation Of Thermo-TRPs Alleviates Vascular Injury By Attenuating Mitochondrial Dysfunction

Background and objectives: Hypertension and coronary atherosclerosis are the common diseases in clinic.Environmental factors play important roles in the pathogenesis of hypertension and coronary atherosclerosis.Epidemiological studies confirm that cold environmental temperature is one of the most important environmental factors which contribute to the pathogenesis of hypertension.Meanwhile,high-fat diet intake impairs endothelium and lead to the pathogenesis of atherosclerosis.Thus,investigating the mechanism in which harmful environmental factors contribute to the cardiovascular dysfunction is of great importance to prevent and treat cardiovascular diseases like hypertension and coronary atherosclerosis.Mitochondrial dysfunction-induced vascular oxidative stress contributes to the pathogenesis of hypertension and atherosclerosis.Angiotensin Ⅱ and oxidized low density lipoprotein(ox-LDL)treatment could damage the function of mitochondria and induce excessive ROS generation.Mitochondrial ROS further triggers the Ca~2+ influx through L-type Ca~2+ channels in vascular smooth muscle cells(VSMCs).In turn,the increased Ca~2+ influx and ROS production activates Rho A/Rho kinases pathway-mediated vasoconstriction.Excessive ROS production in endothelium reduces nitric oxide(NO)bioavailability and impairs endothelium-dependent vasodilatation.Transient receptor potential(TRP)channels are a family of non-selective cation channels.TRP channels are ubiquitous expressed in mammal tissues.Previous studies of our own laboratory and others found that the activation of Trpv1 or Trpm8 could prevent environmental factors like high-salt and high glucose-induced cardiovascular damages through attenuating mitochondrial dysfunction.Thus,we proposed that the activation of Trpm8 could antagonize cold-induced vascular dysfunction and hypertension;activation of Trpv1 could alleviate ox-LDL-induced mitochondrial dysfunction in endothelial cells.This study was divided into two parts.Part one: the role of activation of Trpm8 in regulating mitochondrial function and antagonizing cold-induced hypertension.Part two: the effect of Trpv1 activation on alleviating ox-LDL induced mitochondrial dysfunction in endothelial cells.The aim of this study was to investigate the role of TRP channels’ activation on environmental factor-induced cardiovascular dysfunction.Materials and methods:The whole study includes in vitro and in vivo experiments.In vitro models included primary cultured vascular smooth muscle cells from mice thoracic aorta,thoracic aorta and mesenteric arteries from mice and human umbilical vein endothelial cells.In vivo models included cold and angiotensin Ⅱ-induced hypertensive mice fed on normal diet with or without menthol.1.The location of Trpm8 in primary cultured primary cultured vascular smooth muscle cells from mice thoracic aorta was determined by RT-PCR,immunofluorescence and western blots.The expression of Trpv1 in human umbilical vein endothelial cells was also determined.The location of Trpm8 in sarcoplasmic reticulum was determined by western blots and Ca~2+ activity measurement.2.Cold and angiotensin Ⅱ-induced hypertensive mice were fed on normal diet with or without menthol.The tail-cuff blood pressure was routinely determined.At the end of intervention,24-hour ambulatory blood pressure was monitored by telemetry.3.Cardiac structure and function was measured by echocardiography.Exercise endurance test was also performed.Mice thoracic aorta was freshly isolated to measure ROS levels.The mesenteric artery was used for myograph test.4.Calcium fluorescent probes were used to determine the effect of menthol or angiotensin Ⅱ on cytosolic Ca~2+ influx and mitochondrial Ca~2+ upatke in VSMCs.5.ROS fluorescent probes were used to determine the effect of menthol or angiotensin Ⅱ on ROS production in VSMCs.The effect of capsaicin or ox-LDL on ROS production in ECs was also evaluated.6.The effect of menthol or angiotensin Ⅱ on mitochondrial respiratory function and ROS production in VSMCs was examined by high resolution respiratory.7.The effect of capsaicin or ox-LDL on mitochondrial respiratory function in ECs was examined by high resolution respiratory.JC-I assay was used for mitochondrial membrane potential measurement.8.The effect of menthol or angiotensin Ⅱ on the protein expression which was engage in Rho A/Rho kinases pathway was detected by western blots.9.The protein expressions of PKA,p-PKA,UCP2 and NDUFA9 in ECs treated by capsaicin or ox-LDL were determined by western blots.Results:1.Trpm8 was expressed in cell membrane and SR of VSMCs.The activation of Trpm8 coupled SR Ca~2+ release and mitochondrial Ca~2+ uptake.2.Angiotensin Ⅱ treatment resulted in VSMCs mitochondrial dysfunction and excessive ROS generation,which further triggered Ca~2+ influx through L-type Ca~2+ channels and promoted mitochondrial Ca~2+ uptake.3.Activation of Trpm8 by menthol attenuated angiotensin Ⅱ-induced mitochondrial dysfunction,and ROS generation,which further blunted Ca~2+ influx through L-type Ca~2+ channels and mitochondrial Ca~2+ uptake.4.Increased expression ratios of Rho A-GTP /RhoA,p-MYPT-1/ MYPT-1 and p-MLC /MLC induced by angiotensin Ⅱ treatment could be reversed by the activation of Trpm8 by menthol.5.Chronic dietary menthol improved cold or angiotensin Ⅱ-induced cardiovascular dysfunction and hypertension.It also blunted ROS generation and the expression ratios of RhoA-GTP /RhoA,p-MYPT-1/ MYPT-1 and p-MLC /MLC in aortic tissues.These beneficial effects of menthol were absent in Trpm8-/-mice.6.Capsaicin treatment attenuated ox-LDL induced endothelial mitochondrial dysfunction and ROS generation.This effect of capsaicin was blunted by the inhibition of Trpv1/PKA/UCP2 by the treatment of iRTX,KT5720 or genipin.7.Capsaicin treatment increased the expression of p-PKA,UCP2 and NDUFA9 in ECs,which could be inhibited by the treatment of iRTX,KT5720 or genipin.Conclusion:1.Trpm8 was functionally expressed in SR of VSMCs.Trpm8 activation coupled SR Ca~2+ release and mitochondrial Ca~2+ uptake,which further regulated the mitochondrial function of VSMCs.2.Chronic activation of Trpm8 attenuated cold or angiotensin Ⅱ-induced cardiovascular dysfunction and hypertension.This effect of menthol was associated with the improvement of mitochondria and decreased ROS production in VSMCs,which further blunted Ca~2+ influx through L-type Ca~2+ channels and mitochondrial Ca~2+ uptake,and prevented the enhanced activation of Rho A/Rho kinase pathway.3.Capsaicin antagonized ox-LDL induced endothelial mitochondrial dysfunction and excessive ROS production.The mechanism was related to the upregulation of Trpv1/ PKA/UCP2 pathway.