Hsp70 inhibits aminoglycoside-induced hearing loss and cochlear hair cell death

Abstract

Sensory hair cells of the inner ear are sensitive to death from aging, noise trauma, and ototoxic drugs. Ototoxic drugs include the aminoglycoside antibiotics and the antineoplastic agent cisplatin. Exposure to aminoglycosides results in hair cell death that is mediated by specific apoptotic proteins, including c-Jun N-terminal kinase (JNK) and caspases. Induction of heat shock proteins (Hsps) can inhibit JNK- and caspase-dependent apoptosis in a variety of systems. We have previously shown that heat shock results in robust upregulation of Hsps in the hair cells of the adult mouse utricle in vitro. In addition, heat shock results in significant inhibition of both cisplatin- and aminoglycoside-induced hair cell death. In this system, Hsp70 is the most strongly induced Hsp, which is upregulated over 250-fold at the level of mRNA 2 h after heat shock. Hsp70 overexpression inhibits aminoglycoside-induced hair cell death in vitro. In this study, we utilized Hsp70-overexpressing mice to determine whether Hsp70 is protective in vivo. Both Hsp70-overexpressing mice and their wild-type littermates were treated with systemic kanamycin (700 mg/kg body weight) twice daily for 14 days. While kanamycin treatment resulted in significant hearing loss and hair cell death in wild-type mice, Hsp70-overexpressing mice were significantly protected against aminoglycoside-induced hearing loss and hair cell death. These data indicate that Hsp70 is protective against aminoglycoside-induced ototoxicity in vivo.

Fig. 1

Hsp70 overexpression inhibits kanamycin-induced hearing loss. Hsp70-overexpressing mice and their wild-type littermates underwent hearing testing via auditory brainstem response measurement prior to receiving kanamycin or saline. Three weeks after the end of the treatment, hearing testing was repeated. Threshold shift is reported as the difference between pretest and posttest ABR thresholds. Saline-injected Hsp70-overexpressing mice (closed square) and their saline-injected wild-type littermates (closed triangle) had no significant hearing losses. Wild-type animals that received kanamycin (open triangle) had significant hearing losses at all frequencies tested, while Hsp70-overexpressing mice were protected against kanamycin-induced hearing loss (open square). Hsp70 OE Hsp70-overexpressing mice. Data points are means ± standard error of the mean for n = 5–6 animals per group. Asterisks indicate a significant difference between wild-type and Hsp70-overexpressing mice in the kanamycin-treated condition (two-way ANOVA p < 0.05)

Fig. 2

ABR thresholds following kanamycin treatment. Shown are representative posttest ABR recordings at 16 kHz recorded 3 weeks after the last day of injections. The recordings shown are from an individual mouse in each of the four treatment groups: a Wild-type mouse, saline-injected; b Hsp70-overexpressing mouse, saline-injected; c wild-type mouse, kanamycin-injected; and d Hsp70-overexpressing mouse, kanamycin-injected. At this frequency, ABRs of two (out of five) kanamycin-treated wild-type mice (c) were undetectable. In contrast, kanamycin-treated Hsp70-overexpressing mice (d) retained normal ABR waveforms. Arrowheads denote ABR threshold. NR denotes no response at output limits of equipment (“threshold” assigned = 105 dB SPL)

Fig. 3

Hsp70 overexpression inhibits kanamycin-induced cochlear hair cell death in vivo. After ABR posttesting, mice were euthanized, and their cochleae were prepared for immunochemistry using antimyosin VIIa to label hair cells. Saline-injected wild-type (a, e, i) and Hsp70-overexpressing mice (b, f, j) showed normal cochlear morphology with a single row of inner hair cells (IHCs) and three rows of outer hair cells (OHCs) intact in the apical (a, b), middle (e, f), and basal (i, j) cochlear turns. In kanamycin-injected wild-type mice, most outer hair cells in the basal turn were missing (k). Significant outer hair cell loss was also observed in middle turns of kanamycin-treated wild-type mice (g). Only occasional outer hair cells were missing in the apical turns of kanamycin-treated wild-type mice (c). Inner hair cells remained largely intact in all three turns of kanamycin-treated wild-type mice (c, g, k). Kanamycin-injected Hsp70-overexpressing mice (d, h, l) showed intact outer and inner hair cells, with only occasional outer hair cells missing. OHC1–3 outer hair cells rows 1–3, IHC inner hair cells, Hsp70 OE Hsp70-overexpressing mice. Scale bar in l = 25 μm and applies to all panels

Fig. 4

Kanamycin did not result in vestibular hair cell death in vivo. Control utricles were dissected from wild-type mice (a) and Hsp70-overexpressing littermates (c), immediately fixed and double-labeled for calmodulin and calbindin. Utricles from kanamycin-injected wild-type (b) and Hsp70-overexpressing (d) mice were collected 3 weeks after the last kanamycin injection. Extrastriolar and striolar hair cell densities (e) were not significantly different in any condition (one-way ANOVA p > 0.05). Scale bar in d represents 20 μm and applies to panels a–d. Bars represent mean hair cell densities ± standard error of the mean for n = 4–12 utricles per condition