Heat shock inhibits both aminoglycoside- and cisplatin-induced sensory hair cell death

Abstract

Human hearing and balance impairments are often attributable to the death of sensory hair cells in the inner ear. These cells are hypersensitive to death induced by noise exposure, aging, and some therapeutic drugs. Two major classes of ototoxic drugs are the aminoglycoside antibiotics and the antineoplastic agent cisplatin. Exposure to these drugs leads to hair cell death that is mediated by the activation of specific apoptotic proteins, including caspases. The induction of heat shock proteins (HSPs) in response to cellular stress is a ubiquitous and highly conserved response that can significantly inhibit apoptosis in some systems by inhibiting apoptotic proteins. Induction of HSPs occurs in hair cells in response to a variety of stimuli. Given that HSPs can directly inhibit apoptosis, we hypothesized that heat shock may inhibit apoptosis in hair cells exposed to ototoxic drugs. To test this hypothesis, we developed a method for inducing HSP expression in the adult mouse utricle in vitro. In vitro heat shock reliably produces a robust up-regulation of HSP-70 mRNA and protein, as well as more modest up-regulation of HSP-90 and HSP-27. The heat shock does not result in death of hair cells. Heat shock has a significant protective effect against both aminoglycoside- and cisplatin-induced hair cell death in the utricle preparation in vitro. These data indicate that heat shock can inhibit ototoxic drug-induced hair cell death, and that the utricle preparation can be used to examine the molecular mechanism(s) underlying this protective effect.

Fig. 1

Cisplatin dose–response relationship. Utricles were cultured for 24 h in various concentrations of cisplatin. Hair cells were double-labeled and counted using calmodulin and calbindin immunofluorescence (see Methods). For both the striolar and extrastriolar regions, hair cell survival decreases as cisplatin concentration increases (one-way ANOVA, p < 0.05). For the extrastriolar region, Tukey's multiple comparison correction showed that the 25 μg/ml dose was statistically different from 0, 10, and 15 μg/ml at the 0.05 level of significance. Likewise, 20 μg/ml was statistically different from 0 μg/ml. For the striolar region, only the 25 μg/ml dose was different from 0 μg/ml at the 0.05 level of significance. Shown are mean hair cell numbers ± SEM for n = 5–7 utricles per concentration.

Fig. 2

Heat shock results in robust upregulation of HSP-70. (A) SYBR Green real-time RT-PCR results for HSP-70 mRNA in heat-shocked and control utricles. For each sample, C_t is defined as the number of thermocycles required for the SYBR Green fluorescence intensity to reach a threshold (0.1, indicated above by horizontal line) above background. HSP-70 mRNA levels are highest at 2 h after heat shock (C_t = 22.1 ± 0.06). HSP-70 mRNA levels remain high at 6 h after heat shock (C_t = 23.3 ± 0.01). C_t for control utricles is 30.1 ± 0.20. Assuming that the amount of PCR product doubles with every cycle, these data indicate a 256-fold difference in HSP-70 mRNA between control and heat-shocked utricles at 2 h after heat shock. Each experimental condition was run in duplicate, and data are shown for both of the duplicate samples. (B, C) HSP-70 immunoreactivity increases in hair cells after heat shock. Control and heat-shocked utricles were labeled by using phalloidin (red) and anti-HSP-70 (green). Control utricles show little HSP-70 immunoreactivity (B). HSP-70 immunoreactivity is significantly increased 6 h after heat shock (C).

Fig. 3

Heat shock results in upregulation of HSP-90. (A) Real-time RT-PCR for HSP-90 mRNA. HSP-90 mRNA levels increase 9.31-fold relative to controls by 2 h after heat shock [C_t (controls) = 28.6 cycles; C_t (2 h post-heat shock) = 25.4 cycles]. HSP-90 mRNA levels had further increased to 18.8-fold over controls by 6 h after heat shock [C_t (6 h) = 24.4 cycles]. (B, C) HSP-90 immunoreactivity increases in hair cells after heat shock. Control and heat-shocked utricles were labeled using phalloidin (red) and anti-HSP-90 (green). Control utricles show baseline levels of HSP-90 immunoreactivity (B). At 6 h after heat shock, HSP-90 immunoreactivity is increased in hair cells relative to controls (C).

Fig. 4

Heat shock results in up-regulation of HSP-27 mRNA. (A) Real-time RT-PCR for HSP-27 mRNA. Two hours after heat shock, HSP-27 mRNA levels are increased 7.7-fold relative to controls [C_t (controls) = 27.3 cycles; C_t (2 h after heat shock) = 24.3]. By 6 h after heat shock, HSP-27 levels have decreased again to 3.3-fold above controls [C_t (6 h post-heat shock) = 25.6 cycles]. (B, C) HSP-27 immunoreactivity in control and heat-shocked utricles. Utricles were labeled by using phalloidin (red) and anti-HSP-27 (green). HSP-27 immunofluorescence appears similar for control (B) and heat-shocked (C) utricles.

Fig. 5

Heat shock inhibits neomycin-induced hair cell death. Utricles were cultured at 37°C overnight. Two groups of utricles were then heat-shocked at 43°C for 30 min. Six hours after heat shock, one group of control utricles and one group of heat-shocked utricles were exposed to 1 mM neomycin for 24 h. Utricles were fixed and double-labeled for calmodulin and calbindin immunoreactivity, and hair cells were counted. (A) Control (no neomycin, no heat shock) utricles show normal numbers of striolar (red, calbindin +) and extrastriolar (green, calmodulin +) hair cells. (B) Heat-shocked (no neomycin) utricles appear similar to control utricles. (C) Neomycin-treated (no heat shock) utricles show significant hair cell loss, especially in the striolar region. (D) Heat-shocked, neomycin-treated utricles show decreased hair cell death relative to neomycin alone. (E) Quantification of hair cell densities in each condition. Heat shock treatment significantly inhibited neomycin-induced hair cell death in both the striolar and extrastriolar regions. Shown are mean ± SEM hair cell densities for n = 7–13 utricles per condition. Asterisks indicate significance (ANOVA, p < 0.05; Tukey's multiple comparison correction) relative to neomycin alone.

Fig. 6

Heat shock inhibits cisplatin-induced hair cell death. Six hours after heat shock, one group of control utricles and one group of heat-shocked utricles were exposed to 20 μg/ml cisplatin for 24 h. Heat shock treatment inhibited the hair cell death caused by exposure to cisplatin in the extrastriolar region. This protective effect of heat shock against cisplatin-induced hair cell death in the extrastriolar region is significant. There were no significant differences in the striolar region. Shown are mean ± SEM hair cell densities for n = 9–12 utricles per condition. Asterisk indicates significance (ANOVA, p < 0.001; Tukey's multiple comparison correction) relative to cisplatin alone.