Background Preconditioning of the heart ameliorates doxorubicin (Dox)-induced cardiotoxicity. We tested whether pretreating cardiomyocytes by mitochondrial-targeted antioxidants, mitoquinone (MitoQ) or SKQ1, would provide better protection against Dox than co-treatment. Methods We investigated the dose-response relationship of MitoQ, SKQ1, and vitamin C on Dox-induced damage on H9c2 cardiomyoblasts when drugs were given concurrently with Dox (e.g., co-treatment) or 24 h prior to Dox (e.g., pretreatment). Moreover, their effects on intracellular and mitochondrial oxidative stress were evaluated by 2,7-dichlorofluorescin diacetate and MitoSOX, respectively. Results Dox (0.5–50 μM, n = 6) dose-dependently reduced cell viability. By contrast, co-treatment of MitoQ (0.05–10 μM, n = 6) and SKQ1 (0.05–10 μM, n = 6), but not vitamin C (1–2000 μM, n = 3), significantly improved cell viability only at intermediate doses (0.5–1 μM). MitoQ (1 μM) and SKQ1 (1 μM) significantly increased cell viability to 1.79 ± 0.12 and 1.59 ± 0.08 relative to Dox alone, respectively (both p < 0.05). Interestingly, when given as pretreatment, only higher doses of MitoQ (2.5 μM, n = 9) and SKQ1 (5 μM, n = 7) showed maximal protection and improved cell viability to 2.19 ± 0.13 and 1.65 ± 0.07 relative to Dox alone, respectively (both p < 0.01), which was better than that of co-treatment. Moreover, the protective effects were attributed to the significant reduction in Dox-induced intracellular and mitochondrial oxidative stress. Conclusion The data suggest that MitoQ and SKQ1, but not vitamin C, mitigated DOX-induced damage. Moreover, MitoQ pretreatment showed significantly higher cardioprotection than its co-treatment and SKQ1, which may be due to its better antioxidant effects.

Doxorubicin (DOX) is a widely used anti‐cancer drug but known to induce severe cardiotoxicity. Its adverse effect is caused by an overproduction of free radicals after its accumulation in mitochondria, particularly in heart which constitute about 30% mitochondria. Common antioxidant treatments have not shown significant protection, possibly due to its lack of accumulation within mitochondria. Mitochondria targeted antioxidants have been developed which can accumulate 100 times in mitochondria due to its lipophilic and positive charge properties. Mitoquinone (10‐(6'‐Ubiquinonyl) decyltriphenylphosphonium bromide; MitQ), a mitochondria targeted antioxidant, has shown protection against DOX induced cardiotoxicity in cells and animal studies. By contrast, SKQ1 (conjugate of decyl‐triphenylphosphonium cation (TPP+) with plastoquinone), another mitochondria targeted antioxidant, has shown better antioxidant capacity than MitQ in aqueous solution and lipid environment but no comparison has been made in cardiac tissue. The aim of this study is to compare the effects of MitQ and SKQ1, in their ability to attenuate DOX induced cell damage in H9c2 myoblast. Cell viability was analyzed using a cell counting kit which measured dehydrogenases activity that directly corresponds to cell viability. We found that DOX (500 nM – 50 μM; n=7) reduced cell viability in a dose dependent manner after 24 hour incubation. DOX (40 μM; n=7) reduced cell viability to 41 ± 3% of control. Cells cotreatment of SKQ1 (0.05 μM – 25 μM; n=7) or MitQ (0.05 μM – 10 μM; n=7) with DOX (40 μM) for 24 hours, showed similar dose dependent cellular protection. SKQ1 (5 μM; n=7) treated cells showed the highest cell protection having 55 ±19% increase in cell viability when compared to DOX. Similarly, MitQ (1μM; n=7) treated cells showed highest cell protection exhibiting 64 ± 7% increase in cell viability compared to DOX. Additionally, cells were also protected after pretreatment of SKQ1 (0.05 μM – 25 μM; n=7) or MitQ (0.05 μM – 10 μM; n=7) for 24 hours followed by washing out drug, then cells were treated with DOX (40μM; n=7). SKQ1 (5μM; n=7) pretreated cells showed the highest cell protection with 56 ± 16% increase in cell viability when compared to DOX. By contrast, MitQ (2.5 μM; n=7) pretreated cells showed highest cell protection with 121± 24% increase in cell viability when compared to DOX. Preliminary data suggest that MitQ and SKQ1 both showed protection against DOX induced cardiac cell damage. Moreover, MitQ is more efficacious than SKQ1 when given as pretreatment. MitQ is also more potent when compared to SKQ1. Further studies will be performed to investigate the mechanism of MitQ and SKQ1 protection against DOX. Support or Funding Information This research is supported by the Division of Research, Department of Bio‐Medical Sciences, and the Center for Chronic Disorders of Aging at Philadelphia College of Osteopathic Medicine.

Oxidative stress (e.g., increased hydrogen peroxide [H2O2] levels) and dicarbonyl stress (e.g. increased methylglyoxal [MG]) serves as an initiator for the pathogenesis of cardiovascular diseases and diabetic complications. Increased H2O2‐derived free radicals and MG can be highly reactive and attack cellular components resulting in cell damage and even cell death. Normally, endogenous antioxidant mechanisms and glyoxalase degrade H2O2 and MG, respectively. Additionally, autophagy facilitates the break‐down of damaged cellular components allowing the components to be recycled by the cells. Researchers are still unclear about whether autophagy exacerbates cell damage or protects cells under higher doses of H2O2 and MG. This study investigated cell damage and autophagy changes when rat cardiac H9c2 myoblasts were treated with H2O2 or MG. Moreover, autophagy enhancing or inhibiting drugs were tested to evaluate their effects on H2O2‐ or MG‐induced cell damage. We found that H2O2 (300–900 mM, n=4) dose‐dependently decreased cell viability via measuring live cell dehydrogenase activity. H2O2 (600 mM, n=4) decreased cell viability to 16 ± 1% of the control. Similarly, MG (400–1400 mM, n=5) decreased cell viability in a dose‐dependent manner. MG (1200 mM, n=5) decreased cell viability to 29 ± 6% of the control. These results were further confirmed with the use of double staining, which contained calcein‐AM for labeling live cells and propodium iodide for labeling dead cells. By using Cyto ID autophagy detection dye, we found that H9c2 cells exhibited higher autophagy fluorescence after incubation of H2O2 (300–600 mM, n=2) or MG (400–1200 mM, n=2). Furthermore, trehalose (0.1–100 mM, n=3), an autophagy enhancer, reduced MG (1200 mM) induced cell damage. Trehalose (100 mM, n=3) increased cell viability by 69 ± 27% when compared to MG (1200 mM). Our preliminary results suggest that higher doses of H2O2 and MG can cause cell damage accompanied with increased autophagy. The autophagy enhancer trehalose provided cell protection against MG. Further studies are needed to confirm the effects of autophagy modulation on H2O2 and MG‐induced cell damage by using different autophagy modulators. Support or Funding Information This research was supported by the Division of Research, Department of Biomedical Sciences, and the Center for Chronic Disorders of Aging at Philadelphia College of Osteopathic Medicine.