Dapagliflozin Protects Cardiomyocytes against Doxorubicin-Induced Toxicity by Modulating Sirtuin 1/Sirtuin 3 and Ferroptosis Pathway

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  1. Dapagliflozin protects H9c2 cardiomyoblasts against doxorubicin-induced apoptosis, lipid peroxidation, and oxidative stress. H9c2 cells were treated with doxorubicin (Dox, 0.1 μM), dapagliflozin (DAPA, 10 μM), or pretreated with DAPA prior to Dox exposure. (A) Cell viability was assessed using the MTT assay. (B) Caspase-3/7 activity was measured to evaluate apoptotic response. (C) Flow cytometry using Annexin V-FITC/PI staining determined the proportions of viable, early apoptotic, late apoptotic, and necrotic cells. (D) Lipid peroxidation, a characteristic feature of ferroptosis, was measured using the C11-BODIPY probe. (E, F) Intracellular and mitochondrial ROS production was quantified using DCFH-DA and MitoSOX Red staining, respectively. Results are expressed as mean ± SD (n = 4). α p < 0.05 vs control, β p < 0.05 vs Dox. Scale bar: 50 μm.

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  2. Dapagliflozin improves mitochondrial function and modulates molecular markers of cellular metabolism and injury in doxorubicin-treated cells. H9c2 cells were treated as described in Figure . (A) Mitochondrial respiration parameters, including basal respiration, maximal respiration, ATP production, and spare respiratory capacity, were evaluated using the Seahorse XF Mito Stress Test. (B) Glycolytic function, including glycolysis and glycolytic capacity, was assessed using the Seahorse XF Glycolysis Stress Test. (C) Relative mRNA expression of genes involved in sirtuin signaling (SIRT1, SIRT3), ferroptosis (ACSL4, GPX4), apoptosis (BCL2, BAX), and mitochondrial dynamics/metabolism (DNM1, OPA1, PGC1α) were quantified using RT-qPCR. (D) Western blot analysis was used to evaluate protein expression of key regulators involved in these pathways. Results are expressed as mean ± SD (n = 4). α p < 0.05 vs control, β p < 0.05 vs Dox.

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  3. Pharmacological inhibition of SIRT1 or SIRT3 diminishes the protective effects of dapagliflozin against doxorubicin-induced cardiotoxicity. H9c2 cells were treated with dapagliflozin (DAPA, 10 μM) before doxorubicin (Dox, 0.1 μM) exposure, or pretreated with EX-527 (SIRT1 inhibitor, 1 μM) or 3-TYP (SIRT3 inhibitor, 5 μM), followed by DAPA before Dox exposure. (A) Cell viability was evaluated via MTT assay. (B) Apoptosis was assessed by measuring caspase-3/7 activity. (C) Flow cytometric analysis with Annexin V-FITC/PI staining distinguished stages of cell death. (D) Lipid peroxidation was quantitatively assessed using the C11-BODIPY fluorescent probe. (E, F) Levels of intracellular and mitochondrial ROS were detected using DCFH-DA and MitoSOX Red, respectively. Results are expressed as mean ± SD (n = 4). α p < 0.05 vs control, β p < 0.05 vs DAPA + Dox, γ p < 0.05 vs EX-527 + DAPA + Dox. Scale bar: 50 μm.

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  4. SIRT1 or SIRT3 inhibition impairs the ability of dapagliflozin to enhance mitochondrial bioenergetics and mitigate cellular injuries in doxorubicin-challenged cells. H9c2 cells were treated as mentioned in Figure . (A) Mitochondrial respiration metrics were assessed via Seahorse XF Mito Stress Test. (B) Glycolytic function was evaluated with the Seahorse XF Glycolysis Stress Test. (C) Transcript levels of key genes related to sirtuin signaling, ferroptosis, apoptosis, and mitochondrial function were determined by RT-qPCR. (D) Protein markers associated with these pathways were assessed by Western blotting. Results are expressed as mean ± SD (n = 4). α p < 0.05 vs control, β p < 0.05 vs DAPA + Dox, γ p < 0.05 vs EX-527 + DAPA + Dox.

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  5. Ferroptosis modulates dapagliflozin’s cardioprotective potentials in doxorubicin-treated cells. H9c2 cells were treated with dapagliflozin (DAPA, 10 μM) before doxorubicin (Dox, 0.1 μM) exposure, or pretreated with either Fer-1 (ferroptosis inhibitor, 1 μM) or erastin (ferroptosis inducer, 3 μM), followed by DAPA and then Dox. (A) Cell viability was assessed by MTT assay. (B) Caspase-3/7 activity was analyzed to measure apoptosis. (C) Apoptotic cell populations were evaluated using Annexin V-FITC/PI flow cytometry. (D) Lipid peroxidation was quantified with C11-BODIPY staining. (E, F) ROS levels were quantified using the fluorescent probes: DCFH-DA for the overall intracellular pool and MitoSOX Red specifically for the mitochondrial component. Results are expressed as mean ± SD (n = 4). α p < 0.05 vs control, β p < 0.05 vs DAPA + Dox, γ p < 0.05 vs Fer-1 + DAPA + Dox. Scale bar: 50 μm.

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  6. Ferroptosis inhibition enhances dapagliflozin-induced metabolic and molecular adaptations in doxorubicin-exposed cells. H9c2 cells were treated as indicated in Figure . (A) Mitochondrial respiratory profile was determined by Seahorse XF Mito Stress Test. (B) Glycolytic capacity was evaluated using Seahorse XF Glycolysis Stress Test. (C) mRNA expression of markers related to sirtuin activity, ferroptosis regulation, apoptosis, and mitochondrial dynamics was assessed by RT-qPCR. (D) Western blotting detected and quantified key proteins associated with these pathways. Results are expressed as mean ± SD (n = 4). α p < 0.05 vs control, β p < 0.05 vs DAPA + Dox, γ p < 0.05 vs Fer-1 + DAPA + Dox.

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  7. SIRT1 and SIRT3 are required for ferroptosis inhibition-mediated cardioprotective effects of dapagliflozin in doxorubicin-treated cells. H9c2 cells were treated with dapagliflozin (DAPA, 10 μM) before doxorubicin (Dox, 0.1 μM), or sequentially pretreated with EX-527 or 3-TYP, followed by Fer-1, then DAPA, and finally exposed to Dox. (A) Cell viability was determined by MTT assay. (B) Caspase-3/7 activity was measured to assess apoptosis. (C) Apoptotic populations were identified by flow cytometry with Annexin V-FITC/PI staining. (D) Lipid peroxidation was detected using C11-BODIPY fluorescence. (E, F) Intracellular and mitochondrial ROS levels were measured by DCFH-DA and MitoSOX Red staining, respectively. Results are expressed as mean ± SD (n = 4). α p < 0.05 vs control, β p < 0.05 vs DAPA + Dox, γ p < 0.05 vs EX-527 + Fer-1 + DAPA + Dox. Scale bar: 50 μm.

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  8. SIRT1 and SIRT3 are essential for dapagliflozin to enhance mitochondrial function and prevent ferroptosis-mediated cellular injury in doxorubicin-treated cells. H9c2 cells were treated as described in Figure . (A) Cellular bioenergetics in live cells were recorded using the Seahorse XF Mito Stress Test. (B) Glycolytic profile was measured using the Seahorse XF Glycolysis Stress Test. (C) Gene expression profiling of sirtuin, ferroptosis, apoptotic, and mitochondrial dynamics-related markers was conducted using RT-qPCR. (D) Western blotting examined protein expression changes of key regulators involved in these pathways. Results are expressed as mean ± SD (n = 4). α p < 0.05 vs control, β p < 0.05 vs DAPA + Dox, γ p < 0.05 vs EX-527 + Fer-1 + DAPA + Dox.

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  9. Cardioprotective mechanism of dapagliflozin in attenuating doxorubicin-induced cellular injuries through SIRT1/SIRT3 and ferroptosis regulation. Doxorubicin (Dox) induces cardiotoxicity by downregulating sirtuins, thereby promoting mitochondrial dysfunction, apoptosis, and ferroptosis, as evidenced by suppression of SIRT1/SIRT3, fusion (OPA1), biogenesis (PGC1α), antiapoptotic (BCL2), and antiferroptotic (GPX4) markers, along with activation of fission (DNM1), proapoptosis (BAX), and proferroptosis (ACSL4). Dapagliflozin (DAPA) mitigates Dox-induced cardiac injury through integrated activation of SIRT1/SIRT3 and inhibition of ferroptosis, ultimately restoring mitochondrial function, reducing oxidative and lipid damage, and enhancing cardiomyocyte survival. Interestingly, ferroptosis regulation reciprocally affects SIRT1/SIRT3 activity, suggesting potential feedback loops between sirtuin signaling and ferroptosis in DAPA-mediated cardiac defense. Green and red circles indicate gene up- and downregulation. Solid lines represent confirmed interactions, while dashed lines suggest potential feedback loops. Red arrows denote Dox-induced toxicity, and green arrows show DAPA-mediated…