Early Dapagliflozin and Melatonin Treatment Ameliorated LV Fibrosis via Suppressing TGF‐β1/Smads and Activating Nrf2‐ ARE Signaling in MI Rodent

Figures

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  FIGURE 1. Impact of DAPA on regulating the TGF‐β/Smad3 signaling in IR‐treated H9C2 cells. (A–G) Representative protein expression levels of transforming growth factor (TGF)‐β, phosphorylated Smad2 (p‐Smad2), p‐Smad3, matrix metalloproteinase (MMP)‐2, MMP‐9, Tissue Inhibitor of Metalloproteinases 1 (TIMP1), and TIMP2, respectively. Data are presented as mean ± SD. *P< 0.01, **P< 0.001 versus sham control (SC). Analysis was independently repeated three times (n = 3), and consistent trends were observed across experiments. DAPA = dapagliflozin; IR = ischemia–reperfusion. Note: Panel (A) displays the representative Western blot bands, whereas panel (B) illustrates the quantitative densitometric analysis presented as a bar graph.

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  FIGURE 2. Impact of melatonin (Mel) on Nrf2/ARE signaling in ischemia–reperfusion (IR)–treated H9C2 cells. (A–G) Representative protein expression levels of nuclear factor erythroid 2–related factor 2 (Nrf2), Kelch‐like ECH‐associated protein 1 (Keap‐1), Maf, heme oxygenase‐1 (HO‐1), NAD(P)H quinone dehydrogenase 1 (NQO1), glutathione peroxidase (GPx), and superoxide dismutase 1 (SOD1), respectively. Data are presented as mean ± SD. *P< 0.01, **P< 0.001 versus SC. Analysis was independently repeated three times (n = 3), and consistent trends were observed across experiments. Mel = melatonin; IR = ischemia–reperfusion. Note: Panel (A) displays the representative Western blot bands, whereas panel (B) illustrates the quantitative densitometric analysis presented as a bar graph.

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  FIGURE 3. Effects of double gene manipulation of TGF‐β and Nrf2 on fibrotic and Nrf2/ARE‐related protein expressions in ischemia–reperfusion (IR)–treated H9C2 cells. (A–G) Representative protein expression levels of TGF‐β, phosphorylated Smad3 (p‐Smad3), MMP‐9, Nrf2, Keap‐1, HO‐1, and NQO‐1, respectively. Data are presented as mean ± SD. *P< 0.01, **P< 0.001 versus SC. Analysis was independently repeated three times (n = 3), and consistent trends were observed across experiments. Note: Panel (A) displays the representative Western blot bands, whereas panel (B) illustrates the quantitative densitometric analysis presented as a bar graph.

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  FIGURE 4. Impact of melatonin (Mel) on mitochondrial cytochrome c retention and DNA damage markers in ischemia–reperfusion (IR)–treated H9C2 cells. (A–D) Representative immunofluorescence (IF) images (400×) showing mitochondrial cytochrome c localization, visualized by double staining for cytochrome c (green) and heat shock protein 60 (HSP60, red); merged images indicate mitochondrial cytochrome c retention. (E) Quantitative analysis of mitochondrial cytochrome c–positive signals. (F–I) Representative IF images (400×) showing γ‐H2AX expression (pink), a marker of DNA double‐strand breaks. (J) Quantitative analysis of γ‐H2AX–positive cells. (K–N) Representative IF images (400×) showing CD90+/53BP1+ cells, identified by double staining for CD90 (green) and 53BP1 (red); merged images indicate DNA damage–associated cells. (O) Quantitative analysis of CD90+/53BP1+ cells. Data are presented as mean ± SD. *P< 0.01, **P< 0.001, ***P< 0.0001 versus SC. Nuclei were counterstained with DAPI (blue). Scale bar = 20 μm.

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  FIGURE 5. Verification of effective Nrf2 knockdown and overexpression on Nrf2/ARE signaling and oxidative stress in H9C2 cells. (A–G) Representative protein expression levels of GPx, SOD1, HO‐1, NQO1, NOX‐1, and NOX‐2, respectively. Data are presented as mean ± SD. *P< 0.01, **P< 0.001, ***P< 0.0001 versus SC. Note: Panel (A) displays the representative Western blot bands, whereas panel (B) illustrates the quantitative densitometric analysis presented as a bar graph.

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  FIGURE 6. Time‐course transthoracic echocardiographic assessment of left ventricular function in AMI rats. (A–C) Baseline measurements of left ventricular ejection fraction (LVEF), left ventricular end‐diastolic diameter (LVEDd), and left ventricular end‐systolic diameter (LVESd), respectively, showing no significant differences among groups (P> 0.5). (D–F) Measurements of LVEF, LVEDd, and LVESd at day 7 post‐AMI.(G–I) Measurements of LVEF, LVEDd, and LVESd at day 28 post‐AMI. Data are presented as mean ± SD (n = 8 per group). *P< 0.01, **P< 0.001, ***P< 0.0001 versus SC. Statistical analysis was performed using one‐way ANOVA followed by Bonferroni post hoc test. AMI = acute myocardial infarction.

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  FIGURE 7. Histopathological assessment of left ventricular remodeling at day 28 after AMI. (A–E) Representative light microscopic images (200×) showing infarcted myocardium (gray area, yellow dotted outline). Scale bar = 50 μm. (F) Quantitative analysis of infarct area. (G–K) Representative Masson's trichrome–stained sections (200×) demonstrating myocardial fibrosis (blue area). Scale bar = 50 μm. (L) Quantitative analysis of fibrotic area. Data are presented as mean ± SD (n = 6 per group). *P< 0.01, **P< 0.001, ***P< 0.0001 versus SC. Statistical analysis was performed using one‐way ANOVA followed by Bonferroni post hoc test.

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  FIGURE 8. Cross‐sectional area of the mid‐left ventricle, cardiomyocyte size, and collagen deposition at day 28 after AMI induction. (A–E) Representative gross anatomical images of the mid‐ventricular cross‐section used to determine left ventricular cross‐sectional area. (F) Quantitative analysis of left ventricular cross‐sectional area. (G–K) Representative light microscopic images (400×) showing cardiomyocyte size in left ventricular myocardium. (L) Quantitative analysis of cardiomyocyte size. (M–Q) Representative microscopic images (400×) demonstrating collagen deposition (pink). (R) Quantitative analysis of collagen deposition area. Data are presented as mean ± SD. *P< 0.01, **P< 0.001, ***P< 0.0001 versus SC. Statistical analysis was performed using one‐way ANOVA followed by Bonferroni post hoc test (n = 6 per group). Scale bar = 20 μm.

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  FIGURE 9. Protein expressions of TGF‐β/Smad3 signaling–related molecules in left ventricular myocardium at day 28 after AMI induction. (A–H) Representative protein expression levels of TGF‐β, p‐Smad3, MMP‐2, MMP‐9, collagen I, TIMP1, and TIMP2, respectively. Data are presented as mean ± SD. *P< 0.01, **P< 0.001, ***P< 0.0001 versus SC. Statistical analysis was performed using one‐way ANOVA followed by Bonferroni post hoc test (n = 6 per group). Note: Panel (A) displays the representative Western blot bands, whereas panel (B) illustrates the quantitative densitometric analysis presented as a bar graph.

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  FIGURE 10. Protein expression levels of Nrf2/ARE signaling–related molecules in left ventricular myocardium at day 28 after AMI induction. (A–G) Representative protein expression levels of Nrf2, Keap‐1, Maf, HO‐1, NQO1, GPx and SOD1, respectively. Data are presented as mean ± SD. *P< 0.01, **P< 0.001, ***P< 0.0001 versus SC. Statistical analysis was performed using one‐way ANOVA followed by Bonferroni post hoc test (n = 6 per group). Note: Panel (A) displays the representative Western blot bands, whereas panel (B) illustrates the quantitative densitometric analysis presented as a bar graph.

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  FIGURE 11. Oxidative stress‐ and inflammation‐related protein expressions in left ventricular myocardium at day 28 after AMI induction. (A–D) Representative protein expression levels of NOX‐1, NOX‐2, NF‐κB, tumor necrosis factor‐α (TNF‐α), and oxidized proteins, respectively. For panel (A), (E) indicated the OxyBlot assay was conducted for the detection of oxidized protein; the left and right lanes represent the molecular weight marker and the control oxidized protein standard, respectively. M.W. = molecular weight; DNP = 1–3‐dinitrophenylhydrazone. Data are presented as mean ± SD. *P< 0.01, **P< 0.001, ***P< 0.0001 versus SC. Statistical analysis was performed using one‐way ANOVA followed by Bonferroni post hoc test (n = 6 per group). Note: Panel (A) displays the representative Western blot bands, whereas panel (B) illustrates the quantitative densitometric analysis presented as a bar graph.

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  FIGURE 12. Protein expressions of apoptosis‐ and mitochondrial/DNA damage–related biomarkers in left ventricular myocardium at day 28 after AMI induction. (A–E) Representative protein expression levels of cleaved caspase‐3 (c‐Casp3), cleaved poly (ADP‐ribose) polymerase (c‐PARP), γ‐H2AX, cytosolic cytochrome C (c‐CytoC), and phosphorylated dynamin‐related protein‐1 (p‐DRP1), respectively. Data are presented as mean ± SD. *P< 0.01, **P< 0.001, ***P< 0.0001 versus SC, as determined by one‐way ANOVA followed by Bonferroni post hoc test (n = 6 per group). Note: Panel (A) displays the representative Western blot bands, whereas panel (B) illustrates the quantitative densitometric analysis presented as a bar graph.