The Role And Mechanism Of TRPV3 Channel In Regulating Hearing And Arterial Blood Pressure

The Transient Receptor Potential channel Vanilloid type 3(TRPV3)is a member of non-selective cation TRPV channel that participates in many physiological processes.TRPV3 is expressed in many tissues.TRPV3 plays an important role in maintaining skin barrier,mediating itch sensation,promoting wound healing and hair growth,etc.TRPV3 is also related to feeding,anxiety and other behaviors.Previous studies have shown that TRPV channels play an important role in the formation and maintenance of normal hearing.TRPV1-V4 were expressed in hair cells(HCs)of Corti organs.The upregulation of TRPV1 and/or TRPV2 or the downregulation of TRPV4 results in hearing impairment.TRPV3 is co-expressed with TRPV1 and TRPV4,but its role in auditory function is unclear.TRPV3 is also expressed in vascular tissues.In skin,activation of TRPV3 leads to vasodilation of skin microvessels.This process was induced by the production of NO from nitrite metabolism in NOS-independent manner.In cerebral arteries,activation of TRPV3 can induce vascular vasodilation through the Ca2+-induced activation of IKCa and/or SKCa,by which results in hyperpolarization in smooth muscle cells.In radial artery in rats,both the endothelium NO-GC-PKG pathway and Ca2+-activated IKCa channel are involved in TRPV3 activation-induced arterial relaxation.Up to date,the expression and distribution of TRPV3 in blood vessels as well as the role and underlying mechanism in regulation of blood pressure(BP)remain unclear.This project is to study the role and mechanism of TRPV3 in hearing and blood pressure regulation.Part one: The role of TRPV3 in hearingObjective: To investigate the expression of TRPV3 channel in the inner ear and its role in hearingMethods: 1.TRPV3 wild type(V3WT)mice and TRPV3 knockout(V3KO)mice were used in this study.2.Immunofluorescence(IF)experiment was performed.The hair cell bodies and their stereocilia were labeled with Myosin ⅦA and Phalloidin,respectively.The laser scanning confocal microscopy(LSCM)was used to investigate the expression of TRPV3 in the inner ear HCs.The IF intensity of TRPV1 and TRPV4,the members of the same subfamily were also detected in the inner ear hair cells in V3 WT and V3 KO mice,respectively.3.The hearing detection system was used to measure the auditory brainstem response(ABR)and the distortion product otoacoustic emission(DPOAE)in V3 WT and V3 KO mice.4.Mice were given kanamycin(KM,1000 mg/kg,two weeks,two times per day)through intraperitoneal injection to induce the animal model of hearing loss.The auditory function of V3 WT and V3 KO mice were measured after administration of KM,respectively.TRPV1 and TRPV4 were detected in HCs in V3 WT and V3 KO mice after the administration of KM,respectively..Results: 1.The immunostaining showed that TRPV3 was predominantly expressed in HCs of the organ of Corti in the apical,middle,and basal turns of the cochlea,whereas no expression was detected in the stria vascularis or spiral ganglion(SG)neurons.In hair cells,the fluorescence of TRPV3 was mainly in the cell body of hair cells,while TRPV3 had almost no fluorescence in the stereocilia.The IF intensity and the number of cells expressed with TRPV3 in the outer hair cells(OHCs)were significantly higher than that in the inner hair cells(IHCs).2.V3 WT and V3 KO mice was identified with DNA assay.The IF of TRPV3 in hair cells were hardly detected in V3 KO mice.These results indicated that the TRPV3 gene in V3 KO mice had been knocked out and could be used in the following experiments.3.The ABR and DPOAE thresholds of V3 WT and V3 KO mice were measured.The majority(72%)of V3 KO mice remained normal hearing,while about 28% of V3 KO mice had impaired hearing.The immunostaining results showed that the morphology and number of HCs’ bodies and stereocilia in the V3 KO mice with normal hearing were not changed,while they were severely damaged in the V3 KO mice with impaired hearing.At the same time,the expression of TRPV4 in HCs in V3 KO mice with normal hearing was significantly higher than that of V3 WT mice,while there was no significant difference or even decreased in those V3 KO mice with impaired hearing.There was no significant difference in TRPV1 expression among V3 KO mice with normal,V3 KO mice with impaired hearing and V3 WT mice.4.After administration of KM,the ABR threshold of V3 WT mice significantly increased,while the ABR threshold of V3 KO mice remained normal.IF staining assay showed that the expression of TRPV4 in hair cells of V3 KO mice was significantly higher than that of V3 WT.Discussion: 1.TRPV3 was mainly expressed in the cell bodies of HCs in the inner ear in mice,and the expression of TRPV3 in OHCs was significantly higher than that of IHCs.2.About 70% of V3 KO mice had normal hearing while about 30% of V3 KO mice had impaired hearing.Compared with normal hearing V3 WT mice,the expression of TRPV4 in HCs of Organ of Corti was significantly increased in normal hearing V3 KO mice.In the hearing impaired V3 KO mice,the expression of TRPV4 in HCs was unchanged or decreased.The expression of TRPV1 in HCs of Organ of Corti in normal hearing was unchanged comparing to the hearing impaired V3 KO mice.These results indicated that the upregulation of TRPV4 expression in HCs might compensate for the deficiency of TRPV3,by which mechanism to maintain normal hearing in childhood.3.After KM treatment,the hearing of V3 WT mice was severely impaired,while the hearing of V3 KO mice was maintained normally.Meanwhile,the expression of TRPV4 in the HCs in V3 KO was also significantly increased compared with that of V3 WT mice.It was concluded that the TRPV subfamily members in mouse cochlear HCs,especially TRPV4 and TRPV3,could protect mouse hearing.In addition,upregulation of TRPV4 can compensate for the deficiency of TRPV3 to maintain normal hearing and protect hearing from the ototoxicity induced by KM.Part 2: the role and mechanism of TRPV3 in BP regulationObjective: to investigate the role and underlying mechanism of TRPV3 in BP regulationMethods: 1.TRPV3 wild type(V3WT)mice and TRPV3 knockout(V3KO)mice were used in this study.2.The BP of V3 KO mice and V3 WT mice was measured with non-invasive tail-cuff method and carotid artery intubation method.When BP was measured with arterial intubation method,the effect of opener and inhibitor of TRPV3 on BP was observed through femoral vein administration simultaneously.The level of plasma norepinephrine(NE)in mice was measured by ELISA.2.Thoracic aorta,abdominal aorta and mesenteric artery of mice,represented aorta,median and arteriole,respectively,were used to perform IF staining to observe the expression and distribution of TRPV3.3.The tension of aortic in V3 WT and V3 KO mice were measured with a microvascular tensiometer,respectively.And the vasoconstriction and dilation of blood vessels were observed in the presence of Phenylephrine(Phe)and Acetylcholine(ACh).4.The fluorescence probe DAF-FM DA of NO was used to measure NO in V3 WT and V3 KO mice.N-Nitro-L-Arginine(L-NNA,700 mg/kg),the nitric oxide synthase(NOS)inhibitor,was used to establish a hypertension model.Western blot technology was used to explore the expression of TRPV3 in vascular tissues.Results: 1.Compared with V3 WT mice,the systolic blood pressure(SBP)and diastolic BP(DBP)of V3 KO mice were significantly increased.Carvacrol,a selective agonist of TRPV3,significantly decreased the BP of V3 WT mice but not V3 KO mice.Ruthenium red(RR),a non-selective inhibitor of TRPV,increased the BP of V3 WT mice but not V3 KO mice.The level of plasma NE was significantly higher in V3 KO mice than that of V3 WT mice.3.The immunostaining showed that TRPV3 was expressed in thoracic aorta,abdominal aorta and mesenteric artery.The IF of TRPV3 was detected in the smooth muscle cells(SMCs)and endothelial cells in these areas.3.There was no significant difference in Phe-induced vasoconstriction and ACh-induced vasodilatation between V3 WT and V3 KO mice.4.The IF intensity of NO in vascular endothelial cells showed no significant difference between V3 WT and V3 KO mice.The expression of TRPV3 in the blood vessels in L-NNA-induced hypertensive mice was significantly higher than that of control mice.Discussion: 1.The BP of V3 KO mice was significantly increased.The plasma NE was also increased in V3 KO mice.These results indicate that TRPV3 plays an important role in BP regulation.TRPV3 may protect mammals from hypertension under physiological conditions.2.TRPV3 was abundantly expressed in blood vessels,including major arteries,middle and small arteries.In addition,both smooth muscle cells and endothelial cells expressed TRPV3.3.There was no difference in the basic vascular tension and the level of endothelial NO between V3 WT and V3 KO mice.The effect of TRPV3 on regulation of BP may not invole with NOS-NO pathway.Conclusion: 1.TRPV3 expressed in HCs in Organ of Corti.TRPV3 protects hearing in mice.The upregulation of TRPV4 expression can compensate for the deficiency of TRPV3 to maintain normal hearing and protect hearing from the ototoxicity induced by KM.2.TRPV3 expressed in arterial SMCs and endothelial cells in mice.TRPV3 protects mice from hypertension in physiology.Loss of TRPV3 results in hypertension but not due to the deficiency of endothelial NO.