Mitochondrial-Targeted SS-31 Attenuates the Doxorubicin-Induced Cardiomyoblast H9C2 Cell Senescence

Figure 1. Low-concentration doxorubicin (DOX) induces a senescent phenotype in H9C2 cells. (A) Schematic overview of the experiment protocol. H9C2 cells were plated on Day −1. On Day 0, cells were treated with DOX (5–100 nM) with or without SS-31 for 3 h. Following DOX exposure, the medium was replaced with fresh DOX-free medium (with or without SS-31), and cells were maintained in culture for an additional 3 days. With this treatment regimen, SS-31 was added both at hour 0 with DOX and after DOX washout. (B) Representative images of senescence-associated β-galactosidase (SA-β-gal) staining. The senesecent cells were stained blue color for positive SA-β-gal activity. Scale bar: 500 μm in the upper panel, and 100 μm in the lower enlarged panel. (C) DOX concentrations below 100 nM induced SA-β-gal activity in a dose-dependent manner. N = 4–10. (D) Impact of low-dose DOX on cell proliferation. DOX treatment resulted in significant, dose-dependent cell growth retardation. In (C,D), each black dot represents a single independent experiment. Open bar, no treatment control; Grey bar, DOX added as indicated concentration. N = 3–14. All the data are mean ± SEM, * p < 0.01 vs. control.

Figure 2. SS-31 partially mitigates the DOX-induced senescence in H9C2 cells. (A) Representative images of SA-β-gal staining in DOX-exposed H9C2 cells with or without SS-31 treatment. Scale bar: 500 μm. (B) Quantification of SA-β-gal positive cells following treatment with 50 nM DOX and varying concentrations of SS-31 (0.1, 1, and 3 µM), N = 3–5. (C) Effects of SS-31 on DOX-induced cell growth inhibition. SS-31 treatment did not significantly reverse the DOX-induced growth inhibition, N = 4–14. (D) Quantification of cell area. 50 nM DOX treatment significantly induced cell hypertrophy, which was markedly attenuated by SS-31. N = 42–59 cells from three independent experiments. In (B,C), each black dot represents a single independent experiment. Open box, control; Grey box, SS-31 only; Blue box, DOX only; Purple box, DOX plus SS-31 at the concentration indicated. All the data are mean ± SEM; ** p < 0.01 vs. control, # p < 0.05 vs. DOX, and ## p < 0.01 vs. DOX.

Figure 3. SS-31 attenuates the expression of molecular senescence markers and SASP components in DOX-treated H9C2 cells. H9C2 cells were exposed to 50 nM DOX for 3 h, followed by treatment with 1 µM SS-31 for 48 or 72 h. Representative Western blot images and corresponding quantifications are shown for: (A) p16, (B) p21, (C) TNF-α, (D) IL-6, (E) IL-1β, and (F) MMP3. (G) Summary quantification of protein expression levels normalized to total protein. In (G), each black dot represents a single independent experiment. Open box, control; Grey box, SS-31 only; Blue box, DOX only; Purple box, DOX plus SS-31. N = 3–8. All the data are mean ± SEM, * p < 0.05 vs. control, # p < 0.05 vs. DOX.

Figure 4. SS-31 reduces DOX-induced upregulation of γ-H2AX but does not rescue the DOX-induced downregulation of loss of Lamin B1. (A,C,E) Western blot image showing the expression levels of γ-H2AX, H2AX and Lamin B1 in H9C2 cells following treatment with 50 nM DOX in the presence or absence of 1 μM SS-31. (B,D,F) Quantification of γ-H2AX, H2AX, and Lamin B1 protein levels. Each black dot represents a single independent experiment. Open box, control; Grey box, SS-31 only; Blue box, DOX only; Purple box, DOX plus SS-31. N = 3–4 in each group. All the data are mean ± SEM, ** p < 0.01 vs. control, # p < 0.05 vs. DOX.

Figure 5. SS-31 does not reverse the cell cycle arrest induced by 50 nM DOX. Quantitative analysis of the percentage of cells in the (A) G0/G1 phase, (B) S phase, and (C) G2/M phase. Each black dot represents a single independent experiment. Open box, control; Grey box, SS-31 only; Blue box, DOX only; Purple box, DOX plus SS-31. N = 4. All the data are mean ± SEM, ** p < 0.01 vs. control.

Figure 6. SS-31 mitigates DOX-induced mitochondrial superoxide production. (A) Representative confocal microscopy images of H9C2 cells stained for mitochondrial superoxide. Upper panel, MitoSOX; middle panels, MitoTracker Green; lower panel, merge of MitoSOX and MitoTracker Green. Cells were treated with 50 nM DOX in the presence or absence of 1 μM SS-31. Scale bar: 50 μm. (B) Quantification of mitochondrial superoxide levels, expressed as the fluorescence intensity ratio of MitoSOX to MitoTracker Green. Open box, control; Grey box, SS-31 only; Blue box, DOX only; Purple box, DOX plus SS-31. N = 36–55 cells from three independent preparations. All the data are mean ± SEM, ** p < 0.01 vs. control, ## p < 0.01 vs. DOX.

Figure 7. SS-31 attenuates the DOX-induced elevation of mitochondrial respiration. (A) Average traces of oxygen consumption rate (OCR) in intact H9C2 cells using Seahorse XF96 mitochondrial stress assay. The vertical lines indicate the sequential injection of oligomycin A, (OA, 5 µM), carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP, 10 µM), and a combination of antimycin A (AA, 5 µM) and rotenone (Rot, 5 µM). (B–F) Quantification of mitochondrial respiration parameters, including (B) basal respiration, (C) maximal respiration, (D) proton leak-related respiration, (E) ATP production-related respiration, and (F) respiration control ratio (RCR), calculated as the ratio of maximal to basal OCR. N = 6 independent experiments. (G) Quantification of total cellular ATP levels. Data were normalized to control cells to represent relative changes in ATP content. N = 13 measurements from 3 independent preparations. Each black dot represents a single independent experiment. Open box, control; Grey box, SS-31 only; Blue box, DOX only; Purple box, DOX plus SS-31. All the data are mean ± SEM, ** p < 0.01 vs. control, # p < 0.05 vs. DOX.

Figure 8. SS-31 suppresses the DOX-induced increase in citrate synthase expression and mitochondrial area. (A) Western blot image of citrate synthase in H9C2 cells following exposure to 50 nM DOX with or without 1 μM SS-31. (B) Quantification of citrate synthase protein levels. N = 3–4. All the data are mean ± SEM. (C) SS-31 suppresses the DOX-induced increase in mitochondrial area. N = 103–107 cells from 5 independent preparations. The DOX-induced elevation of citrate synthase and mitochondrial area, indicative of increased mitochondrial mass, was significantly attenuated by SS-31 treatment. Open box, control; Grey box, SS-31 only; Blue box, DOX only; Purple box, DOX plus SS-31. ** p < 0.01 vs. control, # p < 0.05 vs. DOX.