Role of Muscle-Specific Histone Methyltransferase (Smyd1) in Exercise-Induced Cardioprotection against Pathological Remodeling after Myocardial Infarction

Figures

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  Figure 1. Effects of exercise training on cardiac expression of Smyd1 and cardiac function in healthy normal rats without myocardial infarction (MI): (A) representative immunofluorescence staining images of rat heart sections from the healthy control (C) and exercise (EX) groups. Smyd1 was stained in red fluorescence, and the nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole) in blue color (scale bar = 100 µm); (B) Integrated optical density (IOD) of Smyd1 (as a ratio normalized with IOD of DAPI); (C,D) Smyd1 mRNA levels assessed with RT-qPCR (C) and Smyd1 protein expression assessed with Western blots (D) in the heart tissues; (E) ratio of phosphorylated AMP-activated protein kinase (p-AMPK) and total AMPK protein expression; (F) correlation analysis among Smyd1 expression and the ratio of p-AMPK/AMPK; and (G–J) effects of exercise training on cardiac function parameters. Data are expressed as mean ± Standard Deviation (SD), n = 5/group (B–E) or n = 10/group (F–J). Symbols indicate ** p < 0.01. Abbreviations: LVSP, left ventricular systolic pressure; LVEDP, left ventricular end-diastolic pressure; and ±dP/dtmax, the maximal rate of rise or decline of left ventricular pressure.

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  Figure 2. mRNA and protein expression of Smyd1 (SET and MYND domain containing 1), Trx1 (thioredoxin 1), MuRF1 (muscle RING-finger protein-1), and HSP90 (heat shock protein 90) and antioxidant capacity in the post-MI rat hearts: (A) Malondialdehyde (MDA) content in the infarcted myocardium; (B,C) mRNA level assessed by RT-qPCR (B) and protein level assessed by Western blot (C) of Trx1 in the infarcted myocardium; (D) immunofluorescent staining with Smyd1 (red color) and DAPI (blue color) in the sections from peri-infarcted area of heart (scale bar = 100 µm); (E) Integrated optical density (IOD) of Smyd1 (as a ratio normalized with IOD of DAPI); (F,G) Smyd1 mRNA levels assessed with RT-qPCR (F) and Smyd1 protein expression assessed with Western blots (G) in the heart; (H–K) mRNA assessed with RT-qPCR (H,J) and protein expression assessed with Western blots (I,K) of MuRF1 and HSP90 respectively in the heart tissues; (L) the ratio of phosphorylated AMPK (p-AMPK) and total AMPK protein expression; and (M) correlation analysis among Smyd1 expression and the ratio of p-AMPK/AMPK. Data are expressed as mean ± Standard Deviation (SD), n = 5/group (graphs A to L, except D) or n = 15/group (M).…

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  Figure 3. Effects of post-myocardial infarction (MI) exercise training on cardiomyocyte size in infarcted rat hearts: (A) Hematoxylin eosin (HE)-stained heart sections from the peri-infarcted area (scale bar = 100 µm); (B) quantification of the surviving cardiomyocyte cross-sectional area (CSA, n = 5/group); (C) Fluorescein isothiocyanate (FITC)-phalloidin-stained heart sections from the peri-infarcted area of myocardium (scale bar = 100 µm); (D) Integrated optical density (IOD) of F-actin in the peri-infarcted area of myocardium (n = 5/group); (E) correlation analysis among Smyd1 expression and CSA in cardiomyocytes (n = 15); and (F,G) Western blot analysis of cTnI (F) and α-actinin (G) in the infarcted myocardium (n = 5/group). Data are expressed as mean ± Standard Deviation (SD). Symbols indicate ** p < 0.01. Abbreviations: S, Sham group; MI, MI alone group; and MI + EX, post-MI exercise training group.

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  Figure 4. Effects of post-myocardial infarction (MI) exercise training on cardiac fibrosis and ventricular function: (A) Masson’s trichrome staining of cardiac tissue section for fibrosis evaluation. Images of cardiac muscle fibers (red color), collagen fibers (blue color), and nuclei (dark brown color) indicate the levels of fibrosis (scale bar = 100 µm); (B) quantitative analysis of collagen volume of fraction (CVF) in the post-MI rat hearts (n = 5/group); (C) Western blotting analysis of brain natriuretic peptide (BNP) expression in the infarcted myocardium (n = 5/group); and (D–G) effects of post-MI exercise training on cardiac function parameters (n = 10/group). Data are expressed as mean ± Standard Deviation (SD). Symbols indicate ** p < 0.01. Abbreviations: LVSP, left ventricular systolic pressure; LVEDP, left ventricular end-diastolic pressure; and ±dP/dtmax, maximal rate of rise or decline of left ventricular pressure. Abbreviations of groups: S, Sham group; MI, MI alone group; and MI + EX, post-MI exercise training group.

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  Figure 5. Effects of H2O2 on H9C2 cell size and Smyd1, Trx1, and F-actin expression: (A) cell morphology, volume, and survival rate following various concentrations of H2O2 in H9C2 cells (scale bar = 100 µm); (B) the level of intracellular reactive oxygen species (ROS) generation in H9C2 cells from control and H2O2 intervention groups was measured by 2′,7′-dichlorodihydrofluoresein diacetate (DCFH-DA) staining followed by quantification of fluorescence intensity; and (C–E) mRNA assessed with RT-qPCR and protein expression assessed with Western blots of α-actinin (C), Trx1 (D), and Smyd1 (E). Graph (F) shows immunofluorescence microscopy of cTnT (green color), Smyd1 (red color), and FITC-phalloidin (green color) in H9C2 cells, whereas the nuclei were stained by DAPI (blue color); scale bar = 200 µm. Data are expressed as mean ± Standard Deviation (SD), n = 5/group. Symbols indicate * p < 0.05 and ** p < 0.01. Abbreviations: C, Untreated control group and H20, H50, H100, H200, and H300, H9C2 cells treated with 20, 50, 100, 200, and 300 µmol/L H2O2, respectively.

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  Figure 6. Effects of H2O2 and AMP-activated protein kinase (AMPK) agonist (AICAR) on cell volume, F-actin and reactive oxygen species (ROS) in H9C2 rat cardiomyoblasts in vitro: (A,B) cell morphology and volume of H2O2, AICAR, or H2O2 + AICAR-treated H9C2 rat cardiomyoblasts (scale bar = 100 µm); (C,D) immunofluorescence staining of Smyd1 (red color, graph C) and integrated optical density (IOD, graph D). The nuclei of cells were stained by DAPI (blue color), scale bar = 200 µm; (E,F) immunofluorescence staining of FITC-phalloidin (green color, graph E) and IOD in H9C2 cells (graph F, scale bar = 400 µm); (G) representative images showing ROS generation levels under various treatment conditions; and (H) quantified fluorescence intensity. Data are expressed as mean ± Standard Deviation (SD), n = 5/group. Symbols indicate * p < 0.05 and ** p < 0.01. Abbreviations: C, Untreated control group; H, H2O2-treated group; A, AICAR-treated group; and HA, H2O2 + AICAR cotreated group.

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  Figure 7. Effects of H2O2 and AICAR on expression of Smyd1 and cytoskeleton-associated proteins in H9C2 rat cardiomyoblasts in vitro: Western blot results of Smyd1 (A), HSP90 (B), MuRF1 (C), cTnI (D), and α-actinin (E) expressions in H9C2 cells. Data are expressed as mean ± Standard Deviation (SD), n = 5/group. Symbols indicate * p < 0.05 and ** p < 0.01. Abbreviations: C, Untreated control group; H, H2O2-treated group; A, AICAR-treated group; and HA, H2O2 + AICAR cotreated group.

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  Figure 8. Illustrative descriptions of the experimental protocols, study methods, and investigated end-points: the in vivo experiments were performed in adult rats that underwent myocardial infarction (MI) with or without post-MI exercise training. The in vitro cellular experiments were conducted in cultured H9C2 rat cardiomyoblasts treated with or without H2O2 or exercise memetic—AICAR (5-amino-1-β-d-ribofuranosyl-imidazole-4-carboxamide). Please refer the main text for the meanings of additional abbreviations.