Functional Study Of Myh14 In Auditory System

Myh14 encodes a nonmuscle myosin heavy chain 14(NMHC ? C),a member of the myosin family,which involved in many cellular motility processes such as ion channel gating,organelle translocation,and cytoskeleton rearrangement.Myh14 mutations can lead to a DFNA4 type deafness.Studies have shown that Myh14 is a candidate noise induced hearing loss(NIHL)susceptible gene.However,the detailed roles of Myhl4 in auditory system and NIHL are still unclear.In our study,the purpose of this research is to elucidate the roles of Myhl4 in the auditory system and NIHL.Thus,we hope to charify its biological function and molecular mechanism of deafness caused by noise,and provide scientific basis for the pathogenesis of DNFA4 type deafness and for noise deafness prevention and treatment.In our study,a Myh14 gene knockout model was successful established in CBA/CaJ strain mice using the CRISPR/Cas9 technology.Through the phenotypic analysis and functional study of Myh14 gene knockout mice,we investigated the roles of Myh 14 in the auditory system.In this study,we detected the localization of MYH14 in the epithelial cells of the cochlea by basal membrane immunofluorescence staining.The results showed that MYH 14 was located at the apical junctional complex between hair cells and adjacent cells.The morphological changes of the cochlea in the 3-month-old mice were observed by H&E staining of the paraffin embedded sections.The results showed that the morphology of Myh 14 gene knockout mice was normal.ABR measurements were performed in Myh 14 gene knockout mice and wild type mice.We found that Myh 14 knockout mice exhibit hearing loss at five month old.Immunofluorescence results showed loss of OHC in the cochlea basal turn of 5-month-old mutants.According to the outer hair cell loss percentage statistics,it showed that cochlear outer hair cell loss was statistically significant.The expression of MYH9 and MYH 10 protein in the cochlea was detected by Western Blot analysis.The expression of MYH10 in the Myh14 knockout mice was significantly increased and the expression of MYH9 was not changed.Through establishment the temporary threshold shift mouse model,we used the Western Blot to check the expression of MYH 14 protein in cochlea after noise at different time,indicating the MYH 14 expression is noise exposure dependent.We next examined the ABR thresholds of Myh14 knockout mice and wild type mice,results showed that the hearing function of Myh14 knockout mice failed to recover after two weeks of noise exposure,suggesting that Myh 14 knockout mice are more susceptible to high intensity noise compared to wild type mice.Immunofluorescence staining was observed in 2 weeks after the noise stimulation,data showed that OHC loss was significantly increased in Myh14 gene knockout mice.The result that the percentage of outer hair cell loss showed loss of outer hair cells was statistically significant in Myh14 knockout mice.This study revealed that the Myhl4 gene knockout mice are a good model to study the function of Myhl4 in the auditory system.Our results indicated that Myh14 gene knockout mice have slight high frequency hearing loss begainning at five months of age;it is likely that the Myh10 gene may compensate for some of its function.The expression of MYH14 is noise exposure dependent.Myh 14 may have a protective role on the cochlea after acoustic stimulation in CBA/CaJ strain of mice.Altogether,these results showed some evidence that Myh14 might be a NIHL susceptible gene.