The Phenotype And Mechanism Of Gjb2-mutation-related Hearing Loss

OBJECTIVE:1)To identify the detailed phenotype of Gjb2 gene p.V37I mutation knock-in mice.2)To reveal the pathological mechanism of Gjb2 gene p.V37I mutation induced hearing loss.3)To explore the susceptibility of the p.V37I knock-in mice to environmental insults.METHODS:1)ABRs were tested in wild-type and homozygous mice at different time points,and the Wave I was analyzed.Morphology of connexins,OHCs and IHC ribbon synapses was observed using immunofluorescent staining imaging.Stria vascularis(SV)and SGNs were evaluated by H&E staining imaging of paraffin sections of cochleae.EPs were measured in two genotypes at different ages.2)FMTCs were tested to analyze the cochlear amplification.Patch clamp recording was used to evaluate the function of Prestin and IHC synaptosis.3)Band-pass noise,middle ear injection of KCl and intraperitoneal injection of furosemide were used to stress both genotypes to observe the differences of reactions between two groups.RESULTS:1)Homozygotes exhibit late-onset progressive hearing loss,accompanied with prolonged ABR Wave I latencies.Mutant GJs express well on the cell membrane,but it is significantly shorter than wild-type GJs.EPs of homozygotes significantly reduce since 3 weeks old and remain stable up to 100 weeks.There is no significant change in OHC,IHC ribbon synapses SV and SGNs.2)FMTC shows that the cochlear amplification reduces in homozygotes,but NLC results show normal function of Prestin.Homozygotes have larger IHC calcium current but no change in Ca²⁺efficiency in triggering exocytosis.3)Homozygotes present permanent hearing loss after the noise and KCl exposure,with a frequency dependent wave I latency change.EP does not differ,and IHC synapses only reduce at the basal turn after noise exposure.Homozygotes are also more sensitive to furosemide,manifesting as greater threshold shifts and EP reductions.CONCLUSION:1)Gjb2 gene p.V37I mutation induces late-onset progressive hearing loss with prolonged latencies and reduced EPs;2)Reduced cochlear amplifier caused by lower EP and IHCs excitotoxicity associated with potassium accumulation around hair cells are the major pathogenetic mechanism underlying the Gjb2 gene p.V37I mutation-related hearing loss;3)Environmental insults such as noise and furosemide exacerbate the phenotypes of Gjb2 gene p.V37I mutation knock-in mice by stressing the potassium cycling pathway.