Down-regulation Of Ca_V1.3 In Auditory Pathway Promotes Age-related Hearing Loss By Enhancing Calcium-mediated Oxidative Stress

Part One Age-related expression of Ca_V1.3 in auditory pathwayObjective:Exploring the age-related expression of Ca_V1.3 in the auditory pathway of C57BL/6J male mice,and to evaluate whether the expression of Ca_V1.3 in the auditory pathway is correlated with age.Methods:The age-related expression of Ca_V1.3 in cochlea and auditory pathway of C57BL/6J male mice was evaluated by immunofluorescence,PCR,western-blotting and flow cytometry.The expression of Ca_V1.3 in auditory cortex neurons was further confirmed by using flow cytometry to specifically label neurons as NeuN+.Results:Immunofluorescence results showed that the expression of Ca_V1.3 in cochlear hair cells and spiral ganglion neurons gradually decreased with age.PCR,western-blotting and flow cytometry results showed that the total expression in the auditory cortex was increased before 16 weeks and decreased after 16 weeks.After specific labeling of neurons by flow cytometry,Ca_V1.3 expression in neurons of auditory cortex was found to have no significant difference among different age groups.PCR results showed that the mRNA expression levels of Ca_V1.3 in the inferior colliculus and cochlear nucleus had no significant difference with age.Conclusion:The expression of Ca_V1.3 level in the cochlea gradually decreased with ageing,which indicates that the down-regulation of Ca_V1.3 may be related to age-related hearing loss.This provides a new research idea for the further study of age-related hearing loss and provides a potential target for the prevention of presbycusis.Part Two Effects of Ca_V1.3 down-regulation on hair cell senescenceObjective: To investigate underlying role played by Ca_V1.3 in the processes of cell senescence,a in vitro senescence model and Ca_V1.3 KO cell line is needed for the further study about function of Ca_V1.3 and underlying mechanism related to senescence.Using tissue culture of organ of Corti technology and AAV transfection technique in vivo experiments to verify the effects of Ca_V1.3 down-regulation on hair cells and age-related hearing loss.Methods: Crisper /Cas9 knockout technique was used to construct stable Ca_V1.3 knockout HEI-OC1 cell line,and the knockout effect was confirmed by flow cytometry.Patch clamp technique was used to detect the changes of MP and average NLCmax after Ca_V1.3 knockout.The senescence induction model of HEI-OC1 hair cell line was established by H₂O₂.Then the senescence induction effect was verified by P53 detection,C₁₂ FDG staining and β-galactosidase staining.After the treatment of H₂O₂ on wild-type HEI-OC1 and Ca_V1.3 KO type HEI-OC1 cell lines,C₁₂ FDG staining and β-galactosidase staining were used to detect the effect of cell senescence induction,CFSE staining and Red Dot staining were used to detect the cell proliferation,LDH and Caspase-3/7-AAD staining were used to detect the cell apoptosis and death.Transbullar injection with AAV was used to estabilish Ca_V1.3 knockdown mice.In this experiment,wild-type and Ca_V1.3-knock down C57BL/6J mice were induced with D-Galactose to build aging models.Auditory brainstem response（ABR）was evaluated,cochlea basilar membrane stretched preparation was used to observe the loss of hair cells.The organ of Corti（OC）of newborn mice（p3-p5）were isolated and cultured in vitro,after induction of Ca_V1.3 knocked down AAV,the organ were treated with H₂O₂ to induce aging,then loss of hair cells was observed by staining.Results: Flow cytometry showed that the expression of Ca_V1.3 in the Ca_V1.3 knockout HEI-OC1 cell line constructed by Crisper /Cas9 knockout technology was significantly lower than that in the wild-type HEI-OC1 cell line.Patch clamp results showed that the cell membrane potential decreased and the average NLCmax increased after Ca_V1.3 knockout.P53 protein level detecting,C₁₂ FDG staining and β-galactosidase staining confirmed the effect of cell senescence induction.Flow flow results showed that the expression of Ca_V1.3 increased in senescent cells.Compared with wild-type HEI-OC1,（1）C₁₂FDG staining and β-galactosidase staining showed that the number of senescence cells in Ca_V1.3 KO type HEI-OC1 cells was significantly increased after low-dose H₂O₂ induction.（2）CFSE staining of Ca_V1.3 KO-type HEI-OC1 cells showed enhanced cell proliferation arrest,and Red Dot staining showed significantly increased S-phase;（3）the released LDH was significantly increased in the supernatant of KO-type HEI-OC1 cells,which was further confirmed by Caspase-3/7-AAD staining.After the administration of transbullar injection with AAV,the immunofluorescence results showed that the fluorescence intensity of Ca_V1.3 in the knock down group was significantly decreased compared with the control group.The ABR results showed that the characteristic waveform of ABR were decreased after aging induction,and Ca_V1.3 knockdown aggravated this effect,while knockdown alone did not affect the ABR results.Cochlea basilar membrane stretched preparation results showed that slight hair cell losses could be observed after senescence induction,and the loss of hair cells increased after Ca_V1.3 knockdown.The tissue culture of the organ of Corti verified this effect.Conclusion: This study successfully constructed the Ca_V1.3-knockout HEI-OC1 cell lines,the mature H₂O₂-induced cell secenescence model was established.Related in vitro cell experiments found that,after apoptosis induced by oxidation of hydrogen,compared with wild-type HEI-OC1,Ca_V1.3 knock out aggravated senescence of hair cells accompanied by enhanced proliferation arrest,and increased S phase.This suggests that the down-regulation or knockout of Ca_V1.3 can increase the susceptibility of hair cells to ROS injury.The results of in vitro experiments further verified that Ca_V1.3 knockdown itself did not cause hair cell loss,and the down-regulation of Ca_V1.3 could aggravate hair cell loss after senescence induction,thus aggravating hearing loss.Part Three The underlying mechanism of Ca_V1.3 down-regulation in promoting hair cell senescenceObjective: Mitochondrial dysfunction and oxidative stress play a major role in aging.Age-related hearing loss can also be caused by reactive oxygen species and mitochondrial dysfunction.Oxidative stress,altered levels of antioxidant enzymes,and decreased activities of complex Ⅰ,Ⅱ,and Ⅳ can induce apoptosis.Based on this,this study further explored the specific mechanism of the down-regulation of Ca_V1.3 in promoting hair cell aging.Methods: After Ca_V1.3 knockout,we used DCFH-DA probe to detecte intracellular ROS changes in HEI-OC1 cells.After the administration of Ionmycin,3-AM probe and DCFH-DA probe was used to detecte intracellular calcium concentration and the changes of ROS,Caspase-3/7-AAD staining method was used to detect cell apoptosis,C₁₂ FDG staining test was used to evaluate cell senescence.Mito Tracker and mito SOX co-staining were performed to detecte the changes of mitochondria derived ROS after Ca_V1.3 knockout.After treatment with complex Ⅰ inhibitor Retenone and complex Ⅲ inhibitor antimycin A at different gradient concentrations,intracellular ROS levels were detected by DCFH-DA probe staining.Results: In this study,it was found that Ca2+ concentration in HEI-OC1 cells decreased after Ca_V1.3 knockout,and intracellular ROS was significantly up-regulated.However,Ionmycin,a calcium ion carrier,partially restored intracellular calcium ion levels,accompanied by a decrease in intracellular ROS.ROS-mediated apoptosis and senescence were also partially alleviated.Mitotracker and Mito Sox co-staining results showed that mitochondrial derived ROS increased after Ca_V1.3 knockout.Dose-dependent ROS reduction was observed in Ca_V1.3 knockout hair cells after treatment with the complex Ⅰ inhibitor Retenone,while antimycin A,a complex Ⅲ inhibitor,did not affect Ca_V1.3 knockout induced ROS upregulation.Conclusion: Ca_V1.3 knockout induced ROS upregulation partly due to the decrease of intracellular calcium ions.Ca_V1.3 knockout leads to a decrease in intracellular calcium ion concentration,which in turn leads to reduced inactivation of complex Ⅰ derived ROS in hair cells,and ultimately leads to an increase in intracellular ROS and promotes cell senescence.Ca_V1.3 knockdown aggravates the loss of hair cells after aging induction,thereby promoting age-related hearing loss,which may provide a potential target for the prevention of ARHL.