Cisplatin induces calcium ion accumulation and hearing loss by causing functional alterations in calcium channels and exocytosis

Abstract

In recent years, molecular biology and biochemistry have been a focus of studies on the ototoxic side effects of cisplatin. In this paper, the application of cisplatin for 4 h and 72 h was studied from the perspective of electrophysiological function. Patch clamp experiments and immunofluorescence staining were performed on inner hair cells of the cochlea. The patch-clamp results showed that the calcium current amplitude decreased significantly at 4 h and 72 h after cisplatin treatment, the reversal potential was negatively polarized, and the activation time decreased. We suspected that intracellular calcium accumulation was responsible for this result and confirmed this hypothesis by using calpain to measure intracellular calcium concentrations. We tested membrane capacitive function, whose levels after cisplatin application were significantly lower than those in the control group, thus indicating dysfunctional cytoplasmic effervescent function. CtBP2 staining was used to verify this result and indicated a decrease in ribbon synapses. Simultaneously, we observed dysfunction of vesicle circulation after cisplatin application. We found that cisplatin induces the accumulation of calcium ions in inner hair cells by calpain staining and fluoresce intensity calculation, thus decreasing calcium current and synaptic vesicle release, and impairing vesicles cycling, all of which are important mechanisms of cisplatin-induced hearing loss.

Keywords: Cisplatin; calcium ions; exocytosis; inner hair cell; ototoxicity; ribbon synapse.

Figure 1

Establishment of a cisplatin-induced hearing loss mouse model. A. Representative auditory brainstem responses (ABRs) recorded from C57/BL6 control and cisplatin intraperitoneally injected mice (single dose (12 mg/kg) after 4 h and 72 h). B. Comparison of ABR threshold shifts after a single dose of intraperitoneal cisplatin. Two-way ANOVA followed by Tukey’s multiple comparisons test, *P<0.05, **P<0.01. C and D. Comparison of ABR wave I amplitude and latency at 8 KHz. Two-way ANOVA followed by Tukey’s multiple comparisons test, *P<0.05, **P<0.01, ***P<0.001. E. Loss of weight after cisplatin treatment. **P<0.01, one-way ANOVA. For all figures, data are depicted as mean ± SEM, N.S. means P>0.05, *P<0.05, **P<0.01 and ***P<0.001 as compared with the control group. (n, the number of mice used in this experiment).

Figure 2

Loss of outer hair cells after cisplatin treatment. A. Representative images show immunolabeling for MyosinVIIa examined 4 h and 72 h after cisplatin injection. Images comprise 40X Z-stack projections taken from the apical, middle and basal turns. Red: MyosinVIIa labeled IHCs; scale bar in bottom-right corner = 20 μm. B. Outer hair cell number changed significantly after 4 h and 72 h of cisplatin treatment. Data are shown as means ± SEM. Two-way ANOVA followed by Tukey’s multiple comparisons test, *P<0.05, **P<0.01, ***P<0.001. (Number of mice used in this experiment: 5 for each group).

Figure 3

Alterations in calcium currents in inner hair cells of control and CDDP treated mice. (A) Representative calcium currents recorded from three IHCs (control, 4 h and 72 h), induced by a voltage ramp from -70 to +60 mV under a voltage-clamp. (B) According to the Ca²⁺ current shown in (A), the conductance was calculated point by point and plotted against the membrane potential applied. The dashed lines in red depict Boltzmann fitting. (C-G) The Ca²⁺ current in IHCs from cisplatin treated mice has a smaller peak amplitude (C), a more negative reversal potential (D) and a steeper voltage dependency (E). No statistical significance was found in the half-activation voltage of the Ca²⁺ current (V_half) among the three groups (F). Normalize calcium currents were shown in (G). (Number of cells used in this experiment: control = 7, CDDP 4 h = 8, CDDP 3 d = 8). (H) Representative calcium currents recorded from three IHCs (control, 4 h and 72 h), induced by a voltage step clamped at -5 mV. The dashed lines in red were fitted with a single exponential equation. (I) A significant difference was found in the activation time constant (τ) of calcium channel from the CDDP 72 h group. *P<0.05, **P<0.01, ***P<0.001, one-way ANOVA.

Figure 4

Alterations in exocytosis in inner hair cells of control and CDDP treated mice. A. Representative Ca²⁺ currents and whole-cell capacitance measurements from IHCs (control, 4 h and 72 h). The stimulus protocol is shown on the top panel. Small sine waves were superposed on the holding potential before and after a step depolarization. The step depolarization induced a Ca²⁺ current (I_Ca) and triggered exocytosis, which can be quantified with capacitance increase (Δ_Cm). B. Both exocytosis (Δ_Cm, upper panel) and Ca²⁺ influx (Q_Ca, lower panel) are significantly lower in IHCs from cisplatin treated mice. *P<0.05; ***P<0.001. One-way ANOVA followed by Tukey’s multiple comparisons test.

Figure 5

Alterations in synaptic vesicle replenishment in IHCs. A. Representative current responses of three IHCs to double pulse stimulation (control, 4 h and 72 h). Both pulses (500 ms) depleted synaptic vesicles and induced notable I_Ca and Δ_Cm, and the ratio of Δ_Cm2/Δ_Cm1 can be calculated and used to quantify synaptic vesicle replenishment. B. Synaptic vesicle replenishment was significantly slower in IHCs from cisplatin treated 72 h mice. *P<0.05. One-way ANOVA followed by Tukey’s multiple comparisons test.

Figure 6

Cisplatin-induced loss of synaptic ribbons after 4 h and 72 h. A. Representative images revealing immunolabeling for CtBP2 examined 4 h and 72 h after cisplatin injection. Images comprise 120X Z-stack projections taken from the apical, middle and basal turn. Red: MyosinVIIa labeled IHCs, green: CtBP2-labeled synaptic ribbons and nuclei of IHCs, blue: DAPI labeled nuclei; scale bar = 5 μm. B. Quantification of CtBP2-immunolabeled ribbon particles in IHCs showed a significant reduction 4 h and 72 h after injection. n = 4 mice per group with one cochlea used per mouse. **P<0.01, ***P<0.001. (Number of mice used in this experiment: 5 for each group).

Figure 7

An increase in intracellular calcium concentration in IHCs induced by cisplatin. A. Representative images revealing immunolabeling for Calpain I. Images comprise 189X (63X oil objective and 3X zoom) Z-stack projections. Red: CtBP2-labeled synaptic ribbons and nuclei of IHCs, green: Calpain I labeled calpain proteins; scale bar = 5 μm. B. The CtBP2 region marked by the small white balls was used to calculate the surrounding fluorescence intensity of calpain in Imaris 9.3. C. Calpain I-immunolabeled calcium concentration in IHCs was normalize by the control group and showed a significant increase after 72 h cisplatin injection in all turns inside the cochlea. Significant difference was found only in the basal turn in the 4 h group. n = 4 mice per group with one cochlea used per mouse. *P<0.05, **P<0.01, ***P<0.001. (Number of mice used in this experiment: 5 for each group).