Combined Therapy with SS31 and Mitochondria Mitigates Myocardial Ischemia-Reperfusion Injury in Rats

Figures

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  Figure 1. SS31 inhibits menadione-induced oxidative stress in H9C2 cells. (A) Protein expression of 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 < 0.0001. (Note: left and right lanes shown on the upper panel represent a protein molecular weight marker and control oxidized molecular protein standard, respectively). M.W = molecular weight. DNP = 1-3 dinitrophenylhydrazone. (D) Protein expression of mitochondrial SIRT3 * vs. other groups with different symbols (†, ‡, §, ¶), p < 0.0001. (E) Protein expression of mitochondrial SIRT1 * vs. other groups with different symbols (†, ‡, §, ¶), p < 0.0001. All statistical analyses were performed by one-way ANOVA and followed by the Bonferroni multiple comparison post hoc test (n = 6 for each group). Symbols (*, †, ‡, §, ¶) indicate significance at the 0.05 level.

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  Figure 2. SS31 protects mitochondria from oxidative damage. (A–E) Illustrating the IF microscopic finding (400×) of cytosolic cytochrome C (cyt-Cyto C) staining (red color) (i.e., an indicator of mitochondrial damage). The ultrastructural cytosolic cytochrome C in H9C2 cells was notably increased by menadione treatment when compared to the normal control that was notably preserved after SS31 treatment. Blue color indicates nuclei stained by DAPI. (F) The analytical result of number of cyt-Cyto C+ cells * vs. other groups with different symbols (†, ‡, §, ¶), p < 0.0001. (G–K) Illustrating IF microscopic finding (400×) of mitotracker-positively stained cells (i.e., endogenous mitochondria) (green color) (red arrows). (L) The analytic result of number of mitotracker+ cells * vs. other groups with different symbols (†, ‡, §, ¶), p < 0.0001. (M) ATP concentration (i.e., measured by ELISA kit) of H9C2 cells * vs. other groups with different symbols (†, ‡, §, ¶), p < 0.0001. All statistical analyses were performed by one-way ANOVA and was followed by a Bonferroni multiple comparison post hoc test (n = 6 for each group). Symbols (*, †, ‡, §, ¶) indicate significance at the 0.05 level.

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  Figure 3. Refreshment of mitochondria in H9C2 cells and SS31 cyto-protective effect abolished by SIRT3 suppression. (A) Activity (OCR) of isolated mitochondria (determined by the Mito stress test kit and the XFe24 Analyzer) (n = 4). (B) Immunofluorescent (IF) (400×) staining showing nuclear DAPI stain (B-1,B-5), endogenous mitochondria (B-2,B-6), exogenous mitochondria (red color) being transferred into H9C2 cells (B-7), and (B-3) indicating that no exogenous mitochondria are transfused into H9C2 cells. (B-4) indicating merged of (B-1–B-3), and (B-8) indicating merged pictures of (B-5–B-7) with a yellow-green color indicating the fusion of exogenous and endogenous mitochondria in H9C2 cells. (C) Protein expression of NOX-1, * vs. other groups with different symbols (†, ‡), p < 0.0001. (D) Protein expression of NOX-2 * vs. other groups with different symbols (†, ‡), p < 0.0001. (E) Protein expression of mitochondrial Bax * vs. other groups with different symbols (†, ‡), p < 0.0001. (F) Protein expression cytosolic cytochrome C (cyt-Cyto C) * vs. other groups with different symbols (†, ‡), p < 0.0001. (G) Protein expression of mitochondrial cytochrome C (mit-Cyto C) * vs. other groups with…

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  Figure 4. SIRT1 suppression inhibits SS31-mediated cyto-protective effect. (A) Protein expression of nuclear factor (NF)-κB, * vs. other groups with different symbols (†, ‡), p < 0.0001. (B) Protein expression of tumor necrosis factor (TNF)-α * vs. other groups with different symbols (†, ‡), p < 0.0001. (C) Protein expression of interleukin (IL)-6 * vs. other groups with different symbols (†, ‡), p < 0.0001. (D–G) Illustrating the immunofluorescent microscopic finding (400×) of TUNEL assay for identification of apoptotic nuclei (green color) in H9C2 cells (green color). (H) Analytical result of the number of apoptotic nuclei * vs. other groups with different symbols (†, ‡), p < 0.0001. All statistical analyses were performed by one-way ANOVA, which was followed by a Bonferroni multiple comparison post hoc test (n = 6 for each group). Symbols (*, †, ‡) indicate significance at the 0.05 level. Mena = menadione.

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  Figure 5. Transthoracic echocardiographic findings after the IR procedure. (A) At baseline, the left ventricular end-diastolic dimension (LVEDd), p > 0.5. (B) At baseline, left ventricular end-systolic dimension (LVESd), p > 0.5. (C) At baseline, left ventricular ejection fraction (LVEF), p > 0.5. (D) Upon 72 h after myocardial IR procedure, LVEDd, p > 0.3. (E) Upon 72 h after myocardial IR procedure, LVESd * vs. other groups with different symbols (†, ‡, §), p < 0.001. (F) Upon 72 h after myocardial IR procedure, LVEF, * vs. other groups with different symbols (†, ‡, §), p < 0.001. All statistical analyses were performed by one-way ANOVA, which was followed by a Bonferroni multiple comparison post hoc test (n = 8 for each group). Symbols (*, †, ‡, §) indicate significance at the 0.05 level. SC = sham control. IR = ischemia-reperfusion. Mito = mitochondria.

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  Figure 6. Pro-inflammatory, anti-inflammatory, apoptotic, and anti-apoptotic biomarkers in LV myocardium IR procedure. (A) Protein expression of matrix metalloproteinase (MMP)-9 * vs. other groups with different symbols (†, ‡, §, ¶), p < 0.0001. (B) Protein expression of tumor necrosis factor (TNF)-α * vs. other groups with different symbols (†, ‡, §, ¶), p < 0.0001. (C) Protein expression of interleukin (IL)-1ß * vs. other groups with different symbols (†, ‡, §, ¶), p < 0.0001. (D) Protein expression of IL-10 * vs. other groups with different symbols (†, ‡, §, ¶), p < 0.0001. (E) Protein expression of mitochondrial (mito)-Bax * vs. other groups with different symbols (†, ‡, §, ¶), p < 0.0001. (F) Protein expression of cleaved caspase 3 (c-Casp 3) * vs. other groups with different symbols (†, ‡, §, ¶), p < 0.0001. (G) Protein expression of cleaved poly (ADP-ribose) polymerase (c-PARP) * vs. other groups with different symbols (†, ‡, §, ¶), p < 0.0001. (H) Protein expression of Bcl-2 * vs. other groups with different symbols (†, ‡, §, ¶), p < 0.0001. All statistical analyses were performed by one-way ANOVA, which was followed by the Bonferroni multiple comparison post hoc test (n = 8 for…

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  Figure 7. Oxidative-stress biomarkers in LV myocardium after the IR procedure. (A) Protein expression of 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 < 0.0001. (Note: 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 phosphorylated (p)-Smad3 * vs. other groups with different symbols (†, ‡, §), p < 0.0001. Protein expression of total Smad3 (i.e., gray-colored bar chart) did not differ among the five groups (p > 0.5). (E) Protein expression of transforming the growth factor (TGF)-ß * vs. other groups with different symbols (†, ‡, §, ¶), p < 0.0001; (F) Protein expression of p-Smad1/5, * vs. other groups with different symbols (†, ‡, §, ¶), p < 0.0001. (G) Protein expression of bone morphogenetic protein (BMP-2) * vs. other groups with different symbols (†, ‡, §, ¶), p < 0.0001. All statistical…

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  Figure 8. Energy integrity and pressure/volume overload biomarkers in LV myocardium after IR procedure. (A) Protein expression of Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) * vs. other groups with different symbols (†, ‡, §), p < 0.0001. (B) Protein expression of mitochondrial cytochrome C (mit-Cyto C) * vs. other groups with different symbols (†, ‡, §), p < 0.0001. (C) Protein expression of cytosolic cytochrome C (cyt-Cyto C) * vs. other groups with different symbols (†, ‡, §), p < 0.0001. (D) Protein expressions of brain natriuretic peptide (BNP) * vs. other groups with different symbols (†, ‡, §), p < 0.0001. (E) Protein expression of ß-myosin heavy chain (β-MHC) * vs. other groups with different symbols (†, ‡, §), p < 0.0001. (F) Protein expression of α-MHC * vs. other groups with different symbols (†, ‡, §), p < 0.0001. All statistical analyses were performed by one-way ANOVA and was followed by the Bonferroni multiple comparison post hoc test (n = 8 for each group). Symbols (*, †, ‡, §) indicate significance at the 0.05 level. SC = sham control, IR = ischemia-reperfusion, and Mito = mitochondria.

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  Figure 9. Infarct and collagen-deposition areas in the LV myocardium after the IR procedure. (A–E) Showing the light microscopic finding (400×) of H.E., stain in order to identify the infarct area (white-blue color). Scale bars in the right lower corner represents 20 µm. (F) Analytical result of the infarct area * vs. other groups with different symbols (†, ‡, §, ¶), p < 0.0001. (G–K) Illustrating the microscopic finding (200×) of Sirius red satin for the identification of collagen deposition area (pink color). (L) Analytical result of the collagen deposition area * vs. other groups with different symbols (†, ‡, §, ¶), p < 0.0001. Scale bars in the right lower corner represent 50 µm. All statistical analyses were performed by one-way ANOVA, which was followed by the Bonferroni multiple comparison post hoc test (n = 8 for each group). Symbols (*, †, ‡, §, ¶) indicate significance at the 0.05 level. SC = sham control, IR = ischemia-reperfusion, and Mito = mitochondria.

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  Figure 10. Inflammation in LV myocardium after the IR procedure. (A–E) Showing immunofluorescent (IF) microscopic finding (400×) of cellular expression of CD68 (green color). (F) Analytical result of number of CD68+ cells * vs. other groups with different symbols (†, ‡, §, ¶), p < 0.0001. (G–K) Illustration of IF microscopic finding (400×) of cellular expression of CD11 (green color). (L) Analytical result of number of CD11+ cells * vs. other groups with different symbols (†, ‡, §, ¶), p < 0.0001. Scale bars in the right lower corner represent 20 µm. All statistical analyses were performed by one-way ANOVA, which was followed by the Bonferroni multiple comparison post hoc test (n = 8 for each group). Symbols (*, †, ‡, §, ¶) indicate significance at the 0.05 level. SC = sham control, IR = ischemia-reperfusion, and Mito = mitochondria.

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  Figure 11. DNA-damage and Gap junction biomarkers in LV myocardium after the IR procedure. (A–E) Immunofluorescent (IF) microscopic finding (400×) of cellular expression of γ-H2AX (green). (F) Analytical result of number of γ-H2AX + cells * vs. other groups with different symbols (†, ‡, §, ¶), p < 0.0001. (G–K) IF microscopic finding (400×) of cellular expression of connexin43 (Cx43). (L) Analytical result of positively-stained Cx43 * vs. other groups with different symbols (†, ‡, §, ¶), p < 0.0001. Scale bars in the right lower corner represent 20 µm. All statistical analyses were performed by one-way ANOVA, which was followed by the Bonferroni multiple comparison post hoc test (n = 8 for each group). Symbols (*, †, ‡, §, ¶) indicate significance at the 0.05 level. SC = sham control. IR = ischemia-reperfusion. Mito = mitochondria.

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  Figure 12. Actin-phalloidin staining (A–E) for measuring the sarcomere length of cardiomyocytes in LV myocardium after the IR procedure. (A–E) Immuno-fluorescent microscopic (1000×) finding for identifying the sarcomere length. (a–e) Illustrating the manifestations of distance of sarcomere length (SL) in five groups (i.e., (A–E)). (F) Results of statistical analysis of the changes in length of sarcomere identified by troponin-I staining * vs. other groups with different symbols (†, ‡, §, ¶), p < 0.0001. The scale bars in the right lower corner represent 10 µm. (G–K) Illustrating the Masson’s trichrome stain (200×) for identification of the fibrotic area (blue color). (L) Results of statistical analysis of the fibrotic area * vs. other groups with different symbols (†, ‡, §, ¶), p < 0.0001. All statistical analyses were performed by one-way ANOVA, which was followed by the Bonferroni multiple comparison post hoc test (n = 8 for each group). Symbols (*, †, ‡, §, ¶) indicate significance at the 0.05 level. SC = sham control, IR = ischemia-reperfusion, and Mito = mitochondria.