MYH1 deficiency disrupts outer hair cell electromotility, resulting in hearing loss

Abstract

Myh1 is a mouse deafness gene with an unknown function in the auditory system. Hearing loss in Myh1-knockout mice is characterized by an elevated threshold for the auditory brainstem response and the absence of a threshold for distortion product otoacoustic emission. Here, we investigated the role of MYH1 in outer hair cells (OHCs), crucial structures in the organ of Corti responsible for regulating cochlear amplification. Direct whole-cell voltage-clamp recordings of OHCs revealed that prestin activity was lower in Myh1-knockout mice than in wild-type mice, indicating abnormal OHC electromotility. We analyzed whole-exome sequencing data from 437 patients with hearing loss of unknown genetic causes and identified biallelic missense variants of MYH1 in five unrelated families. Hearing loss in individuals harboring biallelic MYH1 variants was non-progressive, with an onset ranging from congenital to childhood. Three of five individuals with MYH1 variants displayed osteopenia. Structural prediction by AlphaFold2 followed by molecular dynamic simulations revealed that the identified variants presented structural abnormalities compared with wild-type MYH1. In a heterogeneous overexpression system, MYH1 variants, particularly those in the head domain, abolished MYH1 functions, such as by increasing prestin activity and modulating the membrane traction force. Overall, our findings suggest an essential function of MYH1 in OHCs, as observed in Myh1-deficient mice, and provide genetic evidence linking biallelic MYH1 variants to autosomal recessive hearing loss in humans.

Fig. 1. Hearing loss in Myh1^−/− mice due to outer hair cell dysfunction.

a Overall appearance of Myh1^+/+, Myh1^+/−, and Myh1^−/− mice at postnatal Day 30. b Body weights of the Myh1^+/+, Myh1^+/−, and Myh1^−/− mice. c Tone-pip ABR thresholds, d click ABR thresholds, and e ABR I/O functions of the Myh1^+/+, Myh1^+/−, and Myh1^−/− mice at postnatal Day 30. f DPOAE thresholds and g DPOAE I/O functions at 6 kHz and h 30 kHz. b–h Myh1^+/+ (black, n = 5), Myh1^+/− (blue, n = 7), and Myh1^−/− (red, n = 7) mice. The data are presented as the means ± SEMs. Statistical comparisons were performed using two-way ANOVA with Bonferroni correction for multiple comparisons in (b–h). **P < 0.01 and ***P < 0.001.

Fig. 2. Myh1 expression in the organ of Corti.

a Cross section of the inner ear from C57BL6/N mice at postnatal Day 30. MYH1, red; MYO7A, green. b Whole-mount images of the inner ears of C57BL6/N mice at postnatal Day 30. MYH1, red; phalloidin, green.

Fig. 3. Impaired prestin activity in Myh1^−/− mouse outer hair cells.

a Input voltage-clamp pulse protocol used for stimulating prestin in the outer hair cells. A 1 kHz sine wave with a 10 mV amplitude was simultaneously applied at −150 to 100 mV for a 250 ms duration. b, c Representative traces of Myh1^+/+ and Myh1^−/− mouse outer hair cell recordings at postnatal Day 21. d Voltage-normalized nonlinear capacitance (NLC) relationship of Myh1^+/+ (n = 6) and Myh1^−/− (n = 8) mouse outer hair cells. The bell-shaped curve was fit to the derivative of the Boltzmann equation. e V_h values calculated from the outer hair cells of Myh1^+/+ and Myh1^−/− mice. f Normalized charge transfer density calculated from the outer hair cells of Myh1^+/+ and Myh1^−/− mice. Data are presented as the means ± SEMs. Statistical comparisons were performed using two-sample independent t-tests in (e) and (f). *P < 0.05 and **P < 0.01.

Fig. 4. Simulation of Myh1^+/+ and Myh1^−/− outer hair cells in response to sound stimulus.

a Illustration of the outer hair cell (OHC) circuit. g_MET conductance of the mechanoelectrical transducer (MET) channel, g_K,s conductance of the small conductance K⁺ channel, g_K,n conductance of I_K,n, corresponding to the KCNQ4 channel, C_m cell membrane capacitance, EP endocochlear potential, E_K K⁺ reversal potential, V_m basolateral membrane potential. b Stimulation of stereocilia at 1000 Hz for 200 ms. c Membrane potential oscillation by 1000 Hz stereocilia stimulation. d MET channel gating induced by 1000 Hz stereocilia stimulation. e K⁺ channel gating induced by 1000 Hz stereocilia stimulation. f Membrane capacitance oscillation induced by 1000 Hz stereocilia stimulation. g OHC electromotility power at 1000 Hz stereocilia stimulation. h OHC electromotility power across different frequencies of stereocilia stimulation. i Normalized electromotility power of Myh1^−/− OHCs compared to Myh1^+/+ OHCs across different frequencies. In (b–h), Myh1^+/+ OHCs are indicated by black lines, and Myh1^−/− OHCs are indicated by red lines.

Fig. 5. Molecular dynamics simulation of identified MYH1 variants.

a Location of the MYH1 variants in terms of the domain structure and amino acid conservation across other vertebrates. b The overall architecture of MYH1 (residues 1–843) predicted using AlphaFold2. The variant residues are indicated. c Root mean square deviation (RMSD) analysis of a 100 ns MD simulation of the wild-type and mutant MYH1 proteins. d Root mean square fluctuation (RMSF) analysis of the last 50 ns. e Principal component analysis (PCA) of MD simulation trajectories.

Fig. 6. Prestin activity in wild-type or mutant MYH1-expressing cells.

a Voltage-normalized nonlinear capacitance (NLC) relationship of overexpressed prestin with or without MYH1 in HEK293T cells. b V_h values measured in cells overexpressing prestin with or without MYH1. c Normalized charge transfer density measured in cells overexpressing prestin with or without MYH1. d Immunoprecipitation (IP) and coimmunoprecipitation (co-IP) of prestin- and MYH1-transfected HEK293T cells. The input lanes represent 1/10 of the immunoprecipitated cell lysate. e NLC relationship between overexpressed prestin and mutant MYH1. f V_h values and normalized charge transfer density measured from cells overexpressing prestin and mutant MYH1. g Normalized charge transfer density measured from cells overexpressing prestin and mutant MYH1. The data are presented as the means ± SEMs. Statistical comparisons were performed using two-sample independent t-tests in (b) and (c). *P < 0.05 and ****P < 0.0001.

Fig. 7. Traction stress maps of wild-type or mutant MYH1-expressing cells.

a Representative phase contrast (top panel) and traction field images (bottom panel) of COS-7 cells adhered to an elastic polyacrylamide gel coated with fibronectin (Young’s modulus of 20 kPa with a Poisson’s ratio of 0.48). Scale bar = 50 µm. b Projected cell area measured in cells overexpressing wild-type or mutant MYH1. c Root mean square (RMS) traction measured in cells overexpressing wild-type or mutant MYH1. The data are presented as the means ± SEMs. Statistical comparisons were performed using two-way ANOVA with Bonferroni correction for multiple comparisons in (b) and (c). ns not significant; *P < 0.05; **P < 0.01.