The Role And Mechanisms Of TRPC3 Channel In Regulating Mitochondrial Functions In Vascular Smooth Muscle During Hypertension

Background and Aim:Hypertension is a kind of complicated disease with the effects of polygenic inheritance and multiple environmental factors.It is the main reason for the onset of stroke,coronary heart disease and heart-kidney failure.Currently,the hypertensive subjects in our country are mainly diagnosed with primary hypertension,which was defined as elevated blood pressure with the interaction of multiple environmental and genetic influences and without accurate etiology.The pathogenesis of hypertension contains neuroendocrine mechanisms,changes of renal functions,disturbance of vascular functions and abnormal ion transport in the plasma membrane,etc.The disturbance of ion homeostasis plays dominant roles in the pathogenesis of primary hypertension.Therefore,investigating the functional changes of ion channels is important for the prevention of hypertension.Calcium is one of the most important signaling molecules in the vascular smooth muscle cells(VSMCs),which plays important roles in hypertension by regulating vascular smooth muscle contraction and proliferations.Intracellular Ca2+ could function directly by inducing vascular constrictions and indirectly by inducing signal transductions as second messenger.Current research has concentrated on the role of a new type ion channel-the transient receptor potential channel(TRP)on cardiometabolic diseases.TRP channels are a group of non-selective cation channels that are permeable to Na+,Ca2+ and other kinds of cations.In mammals,TRP channels include several subtypes,namely TRPV,TRPC,TRPP,TRPM and TRPA.TRPC3 is one of the members of TRPCs subfamily,which attracts much attention in cardiovascular diseases.Studies have shown that TRPC3 is distributed in the excitatory and non-excitatory neurons,sensory neurons,myocardial tissue,lung tissue,airway smooth muscle,vascular smooth muscle,the endothelia and the pituitary gland.Currently,little is known about the role of TRPC3 in hypertension,while our previous research has demonstrated that TRPC3 could regulate vascular tones by influencing calcium homeostasis in the vasculatures.Previously,we were the first to report that increased TRPC3 expression levels in the vasculatures of spontaneously hypertensive rats(SHRs),which facilitates calcium entry and vaomotions in the vasculatures.Inhibition of TRPC3 could suppress the vasomotions in mesenteric arteries.Ca2+ is the main regulator for ROS generation,while Ca2+ has functional interplay with ROS.Mitochondrion is the first-recognized calcium store,with its outer membrane highly permeable to ions and its inner membrane highly selective to ions.Extremely low mitochondrial Ca2+ leads to decreased ATP production,while Ca2+ overload promotes ROS generation.Mitochondrial Ca2+ and mitochondrial ROS are closely correlated with the pathogenesis of hypertension,which exhibits impaired cellular energetics and increased ROS,which could directly regulate the opening of L-type calcium channel and store-operated calcium entry(SOCE)-related molecules by transcriptional and post-translational regulation,thus influencing vascular functions.Recent study also indicated that TRPC3 be localized in the inner membrane of mitochondria from MEFs,participating in the regulation of calcium homeostasis.However,little is known about the expression and function of mitochondrial TRPC3 in VSMCs.Therefore,it is urgent to further investigate whether the expression and functions of mitochondrial TRPC3 changes during hypertension and whether mitochondrial TRPC3 could affect vascular functions by acting on mitochondrial calcium uptake and mitochondrial functions.Angiotensin II plays important roles in the pathogenesis of hypertension and other cardiovascular diseases.Apart from directly acting on AT1 R,angiotensin II could regulate cellular functions by acting on other potential receptors.Studies have shown that a potential receptor of angiotensin II might exist in the mitochondria,thus regulating mitochondrial functions by direct interaction.We have previously demonstrated that TRPC3 participated in the pathogenesis of hypertension.Angiotensin II could regulate the expression levels of TRPC3 and promote the calcium influx in VSMCs,whereas telmisartan treatment could partially attenu ate the effects of angiotensin II.But,whether angiotensin II and telmisartan could influence mitochondrial TRPC3 and calcium uptake,thus acting on mitochondrial ROS production and vascular tones.This is the key issue that needs to be investigated in this study.This study includes three parts: 1.The expression and function of TRPC3 in the mitochondria from VSMCs.2.The effects of TRPC3 in regulating mitochondrial functions on ROS generation and vasoconstrictions in VSMCs.3.The role and mechanism of long-term telmisartan intervention on mitochondrial TRPC3,vascular functions and blood pressure.This part contains two sections: the first is the role of TRPC3 in angiotensin II-infused hypertension,the second is the effects and mechanisms of long-term telmisartan intervention on mitochondrial TRPC3 expression,vascular functions and blood pressure.In order to verify these hypothesis,SHRs and its normal control WKY rats with primarily cultured VSMCs,accompanied with TRPC3 gene knockout mice were used to demonstrate the role of TRPC3 on primary hypertension via affecting mitochondrial calcium uptake and ROS productions.Meanwhile,we used long-term intervention of telmisartan to verify the effects on mitochondrial TRPC3 expression,mitochondrial calcium homeostasis,vascular functions and blood pressure.Materials and Methods:The experiments include in vivo and in vitro tests.In the first and second part,primary cultured VSMCs from SHR and WKY rats were used as main objects to verify the expression and function of mitochondrial TRPC3 in regulating mitochondrial functions and vasoconstrictions in SHR.The third part was in vivo experiments,TRPC3-/-mice and their littermates control WT mice were infused with chronic angiotensin II to make the hypertensive model,in order to verify the role of TRPC3 in the regulation of mitochondrial functions and vascular tones in the vasculatures.Meanwhile,SHRs and WKY rats were intervened with long-term telmisartan to investigate the role of telmisartan on mitochondrial TRPC3 expression,vascular functions and blood pressure.The main experimental methods include:1.Non-invasive tail-cuff and invasive telemetry transducers were used to measure the tail blood pressure and 24 h ambulatory blood pressures of rats and mice;2.Vascular constrictions and dilatations were measured using wire myograph after treatment with above factors in different models.3.Cellular and mitochondrial calcium uptake were measured using Fluorescence Master System and Multiskan Spectrum treated with above factors.4.Realtime quantitative PCR,immunofluorescence and immunoblotting were used to detect the expression and distribution of TRPC3,pyruvate dehydrogenase and proteins in RhoA/Rho kinase pathways.5.High resolution respirometry and other parameters of redox were used to measure the mitochondrial respiratory functions,ATP and ROS generations.Results:1.TRPC3 was expressed in the mitochondria of VSMCs,while enhanced mitochondrial TRPC3 was observed in vasculatures from SHR,which promotes mitochondrial calcium uptake.Telmisartan treatment could inhibit mitochondrial TRPC3 expression and mitochondrial calcium uptake in SHR VSMCs.2.Elevated mitochondrial TRPC3 in SHR VSMCs promoted mitochondrial ROS production,impaired ATP synthesis and blunted mitochondrial respiratory functions,these abnormalities lead to enhanced vasoconstrictions in SHR.Administration of Pyr3 and telmisartan partially attenuated these changes by inhibiting TRPC3;3.TRPC3-/-mice and their littermate control WT mice were infused with angiotensin II for 14 days,it showed that TRPC3-/-could partially attenuated angiotensin II-induced enhanced vasoconstrictions and elevated blood pressure.4.TRPC3-/-could partially attenuated angiotensin II-induced mitochondrial calcium overload,PDH inhibition and mitochondrial impairment.TRPC3-/-could inhibit angiotensin II-induced Rho A/Rho kinase activation and ROS generation.5.Long-term telmisartan intervention attenuated hypertension,mitochondrial dysfunctions,ROS overproductions and enhanced vasoconstrictions in SHRs,partially via inhibition of mitochondrial TRPC3 expression.Conclusions:1.TRPC3 is expressed in the mitochondria of VSMCs.Inhibition of TRPC3 suppresses mitochondrial calcium uptake in VSMCs.2.Enhanced mitochondrial TRPC3 is observed in VSMCs from SHR,which promotes mitochondrial calcium uptake,ROS production and vasoconstrictions in the vasculatures.These results indicate that TRPC3 in VSMCs participates in the pathogenesis of hypertension.3.TRPC3-/-could partially ameliorate angiotensin II-induced hypertension and enhanced vasoconstrictions.4.Long-term telmisartan treatment attenuated enhanced vasoconstrictions and hypertension partially via inhibition of mitochondrial TRPC3 expression.Inhibition of mitochondrial TRPC3 is a new mechanism of telmisartan in improving vascular functions and preventing hypetension.