Cisplatin exposure acutely disrupts mitochondrial bioenergetics in the zebrafish lateral-line organ

Abstract

Cisplatin is a commonly used chemotherapeutic agent that causes debilitating high-frequency hearing loss. No targeted therapies currently exist to treat cisplatin ototoxicity, partly because the underlying mechanisms of cisplatin-induced hair cell damage are not completely defined. Zebrafish may offer key insights to cisplatin ototoxicity because their lateral-line organ contains hair cells that are remarkably similar to those within the cochlea but are optically accessible, permitting observation of cisplatin injury in live intact hair cells. In this study, we used a combination of genetically encoded biosensors in zebrafish larvae and fluorescent indicators to characterize changes in mitochondrial bioenergetics in response to cisplatin. Following exposure to cisplatin, confocal imaging of live intact neuromasts demonstrated increased mitochondrial activity. Staining with fixable fluorescent dyes that accumulate in active mitochondria similarly showed hyperpolarized mitochondrial membrane potential. Zebrafish expressing a calcium indicator within their hair cells revealed elevated levels of mitochondrial calcium immediately following completion of cisplatin treatment. A fluorescent ROS indicator demonstrated that these changes in mitochondrial function were associated with increased oxidative stress. After a period of recovery, cisplatin-exposed zebrafish demonstrated caspase-3-mediated apoptosis. Altogether, these findings suggest that cisplatin acutely disrupts mitochondrial bioenergetics and may play a key role in initiating cisplatin ototoxicity.

Keywords: Cisplatin ototoxicity; Mitochondrial bioenergetics; Mitochondrial dysfunction; Zebrafish.

Fig. 1.

Hair cell mitochondrial activity increases following cisplatin exposure. Maximum-intensity projections of confocal images show TMRE-loaded Tg(TNKS1bp1:EGFP) zebrafish neuromasts with GFP-expressing supporting cells (SC) and GFP-negative hair cells after 2 h treatment with 0.1% DMSO (A-C) and 250 μM cisplatin (D – F). TMRE labeling is observed most prominently in hair cells (A-B; d-E). Mean hair cell TMRE intensity (normalized to control) was elevated in the cisplatin-exposed neuromasts (G, **p = 0.0021, unpaired t-test). n = 15 – 16 zebrafish. N = 4 experimental trials. Error bars = standard deviation.

Fig. 2.

The degree of cisplatin-induced mitochondrial hyperpolarization is dose dependent. Maximum-intensity projections of confocal images show neuromasts treated with 0.1% DMSO (A – C), 250 μM cisplatin (D – F), and 1 mM cisplatin (G – I). Hair cells were labeled with DAPI and mitochondria were stained with Mitotracker CMXRos and MitoTracker Deep Red. Mean intensities (normalized to control) increased in a dose-dependent manner across both MitoTracker CMXRos (J, p = 0.0058, one-way ANOVA) and MitoTracker Deep Red (K, p < 0.0001, one-way ANOVA) indicators. Post-hoc analysis with Tukey’s multiple comparisons test failed to detect significant changes in mean CMXRos intensities between DMSO and 250 μM cisplatin (J, ns = 0.23) and 250 μM cisplatin and 1 mM cisplatin (J, ns = 0.24). For MitoTracker Deep Red, significant differences were observed using Tukey’s multiple comparisons test between all treatment groups (K, **p = 0.0011, p < 0.0001). n = 135 neuromasts. N = 3 experimental trials. Error bars = standard deviation.

Fig. 3.

Cisplatin exposure causes acute increases in mitochondrial calcium levels and ROS production. Maximum-intensity projections of confocal images show mitoGCaMP3 and CellROX Deep Red fluorescence within neuromasts of Tg(myo6b:mitoGCaMP3) zebrafish treated with DMSO (A – C) and 250 μM cisplatin (D – F). Zebrafish treated with cisplatin demonstrate elevated levels of relative mitoGCaMP3 intensity (G, **p = 0.0074, Mann-Whitney U test, n = 32 – 34 zebrafish, N = 8 experimental trials) and increased CellROX Deep Red intensity (H, *p = 0.014, Mann-Whitney U test, n = 26 – 27 zebrafish, N = 7 experimental trials). Error bars = 95% CI.

Fig. 4.

Cisplatin activates caspase-3-mediated hair cell death. Maximum-intensity projections of confocal images showing HCS-1 staining of hair cell membranes and cleaved caspase-3 staining within the neuromast of 6 dpf larvae treated with 0.1% DMSO (A), 250 μM cisplatin (B), and 1 mM cisplatin (C) for 2 h, followed by recovery for 2 h. Median percentages of neuromasts with activated caspase-3 staining are greater in cisplatin-treated zebrafish compared to DMSO-treated zebrafish in a dose-dependent manner (D, **p = 0.003, ****p < 0.0001, Dunn’s multiple comparisons test). n = 90 neuromasts. N = 3 experimental trials. Error bars = 95% CI.