Exercise Training Alleviates Cardiac Fibrosis through Increasing Fibroblast Growth Factor 21 and Regulating TGF-β1-Smad2/3-MMP2/9 Signaling in Mice with Myocardial Infarction

Abstract

Exercise training has been reported to alleviate cardiac fibrosis and ameliorate heart dysfunction after myocardial infarction (MI), but the molecular mechanism is still not fully clarified. Fibroblast growth factor 21 (FGF21) exerts a protective effect on the infarcted heart. This study investigates whether exercise training could increase FGF21 protein expression and regulate the transforming growth factor-β1 (TGF-β1)-Smad2/3-MMP2/9 signaling pathway to alleviate cardiac fibrosis following MI. Male wild type (WT) C57BL/6J mice and Fgf21 knockout (Fgf21 KO) mice were used to establish the MI model and subjected to five weeks of different types of exercise training. Both aerobic exercise training (AET) and resistance exercise training (RET) significantly alleviated cardiac dysfunction and fibrosis, up-regulated FGF21 protein expression, inhibited the activation of TGF-β1-Smad2/3-MMP2/9 signaling pathway and collagen production, and meanwhile, enhanced antioxidant capacity and reduced cell apoptosis in the infarcted heart. In contrast, knockout of Fgf21 weakened the cardioprotective effects of AET after MI. In vitro, cardiac fibroblasts (CFs) were isolated from neonatal mice hearts and treated with H₂O₂ (100 μM, 6 h). Recombinant human FGF21 (rhFGF21, 100 ng/mL, 15 h) and/or 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR, 1 mM, 15 h) inhibited H₂O₂-induced activation of the TGF-β1-Smad2/3-MMP2/9 signaling pathway, promoted CFs apoptosis and reduced collagen production. In conclusion, exercise training increases FGF21 protein expression, inactivates the TGF-β1-Smad2/3-MMP2/9 signaling pathway, alleviates cardiac fibrosis, oxidative stress, and cell apoptosis, and finally improves cardiac function in mice with MI. FGF21 plays an important role in the anti-fibrosis effect of exercise training.

Keywords: cardiac fibrosis; exercise training; fibroblast growth factor 21; myocardial infarction.

Figure 1

Schematic diagram of experimental design.

Figure 2

Knockout of Fgf21 attenuated exercise-induced inhibition of cardiac fibrosis and improvement of heart function following MI. (A) Masson staining, cardiomyocytes (red), collagen fibers (blue) and nuclei (brown) are shown, scale bar = 200 µm; (B) Sirius red staining, cardiomyocytes (yellow), collagen fibers (red) and nuclei (brown) are shown, scale bar = 200 µm; (C) Statistical analysis of CVF, the level of cardiac fibrosis in the infarcted heart was evaluated by the Image Pro Plus analysis software. CVF was calculated as collagen area/total area of myocardial tissue × 100%; (D–F) Protein expressions of COL-I, COL-III and FGF21 in the myocardium; (G–K) Statistical analysis of echocardiographic results and cardiac function in mice. Data are expressed as mean ± Standard Deviation (SD), n = 8 in S, MI, MAE and MRE groups and n = 4 in KME group. Statistical method: one-way analysis of variance (ANOVA). ** p < 0.01. CVF, collagen volume fraction; COL- I, collagen type I; COL-III, collagen type III; LVIDd, left ventricular internal diameter at end diastole; LVIDs, left ventricular internal diameter at end systole; EF, left ventricular ejection fraction; FS, left ventricular fractional shortening; S, Sham group; MI, MI-sedentary group; MAE, post-MI aerobic exercise training group; MRE, post-MI resistance exercise training group; and KME, the Fgf21 KO mice with aerobic exercise training group.

Figure 3

rhFGF21 and/or AICAR inhibited the activation of the TGF-β1-Smad2/3-MMP2/9 signaling pathway in CFs with H₂O₂ treatment. (A) The protein expressions of TGF-β1, Smad2/3, MMP2, and MMP9 in CFs. (B) Statistical analysis. Data are expressed as mean ± Standard Deviation (SD), n = 6. Statistical method: one-way analysis of variance (ANOVA). ** p < 0.01. rhFGF21, Recombinant human FGF21; AICAR, 5-Aminoimidazole-4-carboxamide ribonucleotide; CFs, cardiac fibroblasts; C, Untreated control group.

Figure 4

rhFGF21 and/or AICAR induced CFs apoptosis and reduced oxidative stress and fibrosis levels in CFs with H₂O₂ treatment. (A) TUNEL staining; the positive apoptotic particles showed red fluorescence, the nuclei showed blue, scale bar = 200 px; (B) statistical analysis of TUNEL staining; (C) MDA content of CFs; (D) the protein expressions of SOD2, COL- I and COL- III in CFs. (E) Statistical analysis. Data are expressed as mean ± Standard Deviation (SD), n = 6. Statistical method: one-way analysis of variance (ANOVA). ** p < 0.01. MDA, Malondialdehyde; C, Untreated control group; AICAR, 5-Aminoimidazole-4-carboxamide ribonucleotide; rhFGF21, Recombinant human FGF21.

Figure 5

Exercise training inactivated the TGF-β1-Smad2/3-MMP2/9 signaling pathway via FGF21. (A) The protein expressions of TGF-β1, Smad2/3, MMP2 and MMP9. (B) Statistical analysis. Data are expressed as mean ± Standard Deviation (SD), n = 8 in S, MI, MAE and MRE groups and n = 4 in KME group. Statistical method: one-way analysis of variance (ANOVA). ** p < 0.01. S, Sham group; MI, MI-sedentary group; MAE, post-MI aerobic exercise training group; MRE, post-MI resistance exercise training group; and KME, the Fgf21 KO mice with aerobic exercise training group.

Figure 6

Exercise inhibited cell apoptosis and oxidative stress in the infarcted heart via FGF21. (A) TUNEL staining; the positive apoptotic particles showed red fluorescence, the nuclei showed blue, scale bar = 200 px; (B) statistical analysis of TUNEL staining; (C) the protein expression of SOD2; (D,E) MDA content and CAT activity. Data are expressed as mean ± Standard Deviation (SD), n = 8 in S, MI, MAE and MRE groups and n = 4 in KME group. Statistical method: one-way analysis of variance (ANOVA). * p < 0.05 and ** p < 0.01. S, Sham group; MI, MI-sedentary group; MAE, post-MI aerobic exercise training group; MRE, post-MI resistance exercise training group; and KME, the Fgf21 KO mice with aerobic exercise training group.

Figure 7

Aerobic and resistance exercise training up-regulated FGF21 inhibits the activation of TGF-β1-Smad2/3-MMP2/9 pathway and alleviates cardiac fibrosis following MI.