Combined therapy with dapagliflozin and entresto offers an additional benefit on improving the heart function in rat after ischemia-reperfusion injury

Figures

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  Fig. 1. Sacubitril (Sac)/valsartan (Val) and dapagliflozin therapy suppressed the expressions of ROS, MPTP and apoptosis in H9C2 cells. A) Illustrating the flow cytometric analysis of total intracellular reactive oxygen species (ROS). B) Analytical result of mean fluorescent intensity (MIF) of DCFDA, ∗ vs. other groups with different symbols (†, ‡), p < 0.0001. C) Illustrating the cytometric analysis of mitochondrial ROS. D) Analytical result of MIF of MitoSOX, ∗ vs. other groups with different symbols (†, ‡), p < 0.0001. E) Illustrating the flow cytometric analysis of mitochondrial permeability transitional pore (MPTP). F) Analytical result of MFI of MPTP (i.e., by calcein-AM assay), ∗ vs. other groups with different symbols (†, ‡), p < 0.0001. G) Flow cytometric analysis of early apoptosis (annexin V+/PI-), ∗ vs. other groups with different symbols (†, ‡), p < 0.001. H) Flow cytometric analysis of late apoptosis (annexin V+/PI+), ∗ vs. other groups with different symbols (†, ‡), p < 0.001. All statistical analyses were performed by one-way ANOVA, followed by Bonferroni multiple comparison post hoc test (n = 6). Symbols (∗, †, ‡) indicate significance (at 0.05 level). Group A = H9C2…

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  Fig. 2. Sac/Val and dapagliflozin therapy downregulated the oxidative stress and DNA-damaged biomarkers and upregulated the antioxidants in H9C2 cells. A) Protein expression of NOX-1, ∗ vs. other groups with different symbols (†, ‡), p < 0.001. B) Protein expression of NOX-2, ∗ vs. other groups with different symbols (†, ‡), p < 0.001. C) Protein expression of γ-H2AX, ∗ vs. other groups with different symbols (†, ‡), p < 0.001. D) The oxidized protein expression, ∗ vs. other groups with different symbols (†, ‡), p < 0.001 (Note: the left and right lanes shown on the upper panel represent protein molecular weight marker and control oxidized molecular protein standard, respectively). M.W. = molecular weight; DNP = 1–3 dinitrophenylhydrazone. E) Protein expression of SIRT1, ∗ vs. other groups with different symbols (†, ‡), p < 0.001. F) Protein expression of SIRT3, ∗ vs. other groups with different symbols (†, ‡), p < 0.001. G) Protein expression of superoxide dismutase (SOD), ∗ vs. other groups with different symbols (†, ‡), p < 0.001. H) Protein expression of catalase (CAT), ∗ vs. other groups with different symbols (†, ‡), p < 0.001. I) Protein expression of glutathione peroxidase (GPX),…

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  Fig. 3. Entresto and dapagliflozin treatment effectively preserved heart function and attenuated infarct size in LV myocardium by day 3 after acute IR induction. A) The baseline of left ventricular ejection fraction (LVEF), p > 0.5. By day 3 after ischemia-reperfusion (IR) induction, the analytical result of LVEF, ∗ vs. other groups with different symbols (†, ‡, §), p < 0.0001. B to F) Illustrating the H.E. stain for identification of infarction area (yellow dotted line). G) Analytical result of infarction area, ∗ vs. other groups with different symbols (†, ‡, §), p < 0.0001. All statistical analyses were performed by one-way ANOVA, followed by Bonferroni multiple comparison post hoc test (n = 8 for LVEF; n = 6 for H.E. stain). Symbols (∗, †, ‡, §) indicate significance (at 0.05 level). Abbreviations: LV: left ventricular; SC: sham-operated control; IR: ischemia reperfusion; DAPA: dapagliflozin; En: entresto.

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  Fig. 4. Entresto and dapagliflozin therapy suppressed the fibrosis and DNA-damaged marker in LV myocardium by day 3 after acute IR induction. A to E) Illustrating the microscopic finding (100x) for identification of fibrosis in LV myocardium (blue color). All scale bars in lower right corner represent 100 μm. F) Analytical result of fibrotic area, ∗ vs. other groups with different symbols (†, ‡, §), p < 0.0001. G to K) Illustrating immunofluorescent microscopic finding (400x) for identification of γ-H2AX + cells (pink color). All scale bars in lower right corner represent 20 μm. L) Analytical result of number of positively stained γ-H2AX cells, ∗ vs. other groups with different symbols (†, ‡, §), p < 0.0001. All statistical analyses were performed by one-way ANOVA, followed by Bonferroni multiple comparison post hoc test (n = 6). Symbols (∗, †, ‡, §) indicate significance (at 0.05 level). Abbreviations: LV: left ventricular; SC: sham-operated control; IR: ischemia reperfusion; DAPA: dapagliflozin; En: entresto.

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  Fig. 5. Entresto and dapagliflozin therapy ameliorated inflammatory cell infiltration in LV myocardium by day 3 after acute IR induction. A to E) Illustrating the immunohistochemical microscopic finding (400x) for identification of myeloperoxidase (MPO)+ cells (gray color). F) Analytical result of number of positively stained MPO cells, ∗ vs. other groups with different symbols (†, ‡, §), p < 0.0001. G to K) Illustrating the immunofluorescent microscopic finding for identification of CD68+ cells (pink color). L) Analytical result of number of positively stained CD68 cells, ∗ vs. other groups with different symbols (†, ‡, §), p < 0.0001. All scale bars in lower right corner represent 20 μm. All statistical analyses were performed by one-way ANOVA, followed by Bonferroni multiple comparison post hoc test (n = 6). Symbols (∗, †, ‡, §) indicate significance (at 0.05 level). Abbreviations: LV: left ventricular; SC: sham-operated control; IR: ischemia reperfusion; DAPA: dapagliflozin; En: entresto.

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  Fig. 6. Entresto and dapagliflozin therapy ameliorated the expression of BNP and preserved connexin43 expression in LV myocardium by day 3 after acute IR induction. A to E) Illustrating the immunohistochemical microscopic finding (400x) for identification of cellular expression of brain natriuretic peptide (BNP) (gray color). F) Analytical result of the expression of BNP, ∗ vs. other groups with different symbols (†, ‡, §), p < 0.0001. G to K) Illustrating the cellular expression of connexin43 (Cx43) (green strip-like band) (red arrows). L) Analytical result of Cx43 expression, ∗ vs. other groups with different symbols (†, ‡, §), p < 0.0001. All scale bars in lower right corner represent 20 μm. All statistical analyses were performed by one-way ANOVA, followed by Bonferroni multiple comparison post hoc test (n = 6). Symbols (∗, †, ‡, §) indicate significance (at 0.05 level). Abbreviations: LV: left ventricular; SC: sham-operated control; IR: ischemia reperfusion; DAPA: dapagliflozin; En: entresto.

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  Fig. 7. Entresto and dapagliflozin therapy inhibited oxidative stress and enhanced antioxidants in LV myocardium by day 3 after acute IR induction. A) Protein expression of NADPH-oxidase-1 (NOX-1), ∗ vs. other groups with different symbols (†, ‡, §), p < 0.0001. B) Protein expression of NOX-2, ∗ vs. other groups with different symbols (†, ‡, §), p < 0.0001. C) Oxidized protein expression, ∗ vs. other groups with different symbols (†, ‡), p < 0.001 (Note: the left and right lanes shown on the upper panel represent protein molecular weight marker and control oxidized molecular protein standard, respectively). M.W. = molecular weight; DNP = 1–3 dinitrophenylhydrazone. D) Protein expression of SIRT1, ∗ vs. other groups with different symbols (†, ‡, §), p < 0.0001. E) Protein expression of SIRT3, ∗ vs. other groups with different symbols (†, ‡, §), p < 0.0001. All statistical analyses were performed by one-way ANOVA, followed by Bonferroni multiple comparison post hoc test (n = 6). Symbols (∗, †, ‡, §) indicate significance (at 0.05 level). Abbreviations: LV: left ventricular; SC: sham-operated control; IR: ischemia reperfusion; DAPA: dapagliflozin; En: entresto.

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  Fig. 8. Entresto and dapagliflozin therapy inhibited apoptosis, fibrosis and autophagy in LV myocardium by day 3 after acute IR induction. A) Protein expression of mitochondrial (mito)-Bax, ∗ vs. other groups with different symbols (†, ‡, §), p < 0.0001. B) Protein expression of cleaved caspase 3 (c-Casp3), ∗ vs. other groups with different symbols (†, ‡, §), p < 0.0001. C) Protein expression of cleaved Poly (ADP-ribose) polymerase (c-PARP), ∗ vs. other groups with different symbols (†, ‡, §), p < 0.0001. D) Protein expression of phosphorylated (p)-Smad3, ∗ vs. other groups with different symbols (†, ‡, §), p < 0.0001. E) Protein expression of transforming growth factor (TGF)-β, ∗ vs. other groups with different symbols (†, ‡, §), p < 0.0001. F) Protein ratio of CL3B-II to CL3B-I, ∗ vs. other groups with different symbols (†, ‡, §), p < 0.0001. All statistical analyses were performed by one-way ANOVA, followed by Bonferroni multiple comparison post hoc test (n = 6). Symbols (∗, †, ‡, §) indicate significance (at 0.05 level). Abbreviations: LV: left ventricular; SC: sham-operated control; IR: ischemia reperfusion; DAPA: dapagliflozin; En: entresto.

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  Fig. 9. Entresto and dapagliflozin therapy preserved the mitochondrial integrity and attenuated myocardial hypertrophy and DNA damage in LV myocardium by day 3 after acute IR induction. A) Protein expression of cytosolic cytochrome c (cyt-CytoC), ∗ vs. other groups with different symbols (†, ‡, §), p < 0.0001. B) Protein expression of mitochondrial cytochrome c (mit-CytoC), ∗ vs. other groups with different symbols (†, ‡, §), p < 0.0001. C) Protein expression of β-myosin heavy chain (β-MHC), ∗ vs. other groups with different symbols (†, ‡, §), p < 0.0001. D) Protein expression of α-MHC, ∗ vs. other groups with different symbols (†, ‡, §), p < 0.0001. All statistical analyses were performed by one-way ANOVA, followed by Bonferroni multiple comparison post hoc test (n = 6). Symbols (∗, †, ‡, §) indicate significance (at 0.05 level). Abbreviations: LV: left ventricular; SC: sham-operated control; IR: ischemia reperfusion; DAPA: dapagliflozin; En: entresto.

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  Fig. 10. Entresto and dapagliflozin therapy suppressed the upstream and downstream inflammatory signalings. A) Protein expression of high-mobility group protein 1 (HMG-1), ∗ vs. other groups with different symbols (†, ‡, §), p < 0.0001. B) Protein expression of toll-like receptor (TLR)-4, ∗ vs. other groups with different symbols (†, ‡, §), p < 0.0001. C) Protein expression of Myeloid differentiation primary response 88 (MYD88), ∗ vs. other groups with different symbols (†, ‡, §), p < 0.0001. D) Protein expression of phosphorylated nuclear factor (p-NF)-κB, ∗ vs. other groups with different symbols (†, ‡, §), p < 0.0001. E) Protein expression of tumor necrosis factor (TNF)-α, ∗ vs. other groups with different symbols (†, ‡, §), p < 0.0001. F) Protein expression of interleukin (IL)-1β, ∗ vs. other groups with different symbols (†, ‡, §), p < 0.0001. G) Protein expression of IL-6, ∗ vs. other groups with different symbols (†, ‡, §), p < 0.0001. H) Protein expression of IKB-α, ∗ vs. other groups with different symbols (†, ‡, §), p < 0.0001. I) Protein expression of IKB-β, ∗ vs. other groups with different symbols (†, ‡, §), p < 0.0001. All statistical analyses were performed by one-way…

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  Fig. 11. Illustrating the underlying mechanisms of entresto-dapagliflozin treatment for preserving the heart function and integrity of cardiac structure. Abbreviations: LVEF: left ventricular ejection fraction; IR: ischemia-reperfusion; DAPA: dapagliflozin; cyt-CytoC: cytosolic cytochrome c; mit-CytoC: cytosolic cytochrome c.