Follistatin Attenuates Myocardial Fibrosis in Diabetic Cardiomyopathy via the TGF-β–Smad3 Pathway

Figures

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  FIGURE 1. Relationship between FST and diabetic cardiomyopathy. Serum levels of FST in healthy controls and patients with diabetic cardiomyopathy [(A), n≥13]. Levels of FST in serum [(B), n = 5] and heart tissues of controls and db/db mice [(C, D), n≥5]. Protein levels of FST in control and db/db mice [(E), n = 3)]. Expression of FST in AC16 cells with or without HG + PA treatment [(F), n = 5]. *p< 0.05; **p< 0.01; ***p<0.001.

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  FIGURE 2. Overexpression of FST improves heart functions. Serum FST levels in controls and db/db mice [(C), n≥4]. FST mRNA levels in mouse heart in each group [(A), n≥5]. ELISA results of heart FST levels in each group [(B), n≥4]. Western blot results of heart FST levels in each group [(D), n = 6]. The ratio of E/A and echocardiograph images in each group [(E), n = 6]. Hemodynamic analysis of db/db mice and controls [(F,G), n≥4]. *p< 0.05; **p< 0.01; ***p<0.001.

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  FIGURE 3. Overexpression of FST is associated with preserved cardiac hypertrophy. The ratio of heart weight/tibia length (HW/TL) of each group [(A), n = 6]. Representative images of H&E staining and quantitative analysis (magnification: 400× [(B), n≥5]). LVPWd was measured by echocardiography [(C), n≥5]. Quantitative real-time PCR analysis of mRNA transcription of ANP, MYH7/MYH6 [(D, E), n≥4], and myostatin [(F), n≥4] in the myocardial tissues. Protein levels of p-AKT and AKT and quantitative analysis in heart tissues [(G), n = 4]. *p< 0.05; **p< 0.01; ***p<0.001.

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  FIGURE 4. Overexpression of FST abrogates cardiac fibrosis. Representative images of Sirius red staining and quantitative analysis of myocardial interstitial collagen (magnification: 200×[(A), n = 4]). Quantitative real-time PCR analysis of mRNA transcription of fibrosis markers COL1 and CTGF [(B, C), n≥4]. Western blotting and quantitative analysis of COL1 and CTGF [(F, G), n = 4]. Quantitative real-time PCR analysis of mRNA transcription of MMP9 and MMP2 [(D, E), n≥4] and activity measured by zymography [(H), n = 4] in each group. *p< 0.05; **p< 0.01; ***p<0.001.

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  FIGURE 5. Overexpression of FST reduces cardiac fibrosis by inhibiting the activation of the TGF-β–Smad3 pathway in vivo and in vitro. Quantitative real-time PCR analysis of TGF-β1, activin A, and TGF-β3 mRNA transcription [(A–C), n≥4] and p-Smad3 and Smad3 protein expression in each group [(D), n = 4]. In vitro, NIH3T3 cells were treated with or without high glucose (30 mM), recombinant FST protein (10 ng/ml), and TGF-β1 (20 μM). Protein expression of COL1, COL3, p-Smad3, and Smad3 with different treatments [(E–G), n = 3]. *p< 0.05; **p< 0.01; ***p<0.001.

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  FIGURE 6. Overexpression of FST decreases cardiac oxidative stress in db/db mice. Representative images of DHE staining and quantitative analysis of myocardial ROS levels in each group (magnification: 400×[(A), n = 4]). The activity of MnSOD, the content of MDA, and the quantitative real-time PCR analysis of mRNA transcription of NOX4 and NOX2 in heart tissues (B–E, n≥4).*p< 0.05; **p< 0.01; ***p<0.001.

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  FIGURE 7. Schematic diagram of the mechanism of FST in improving diabetic cardiomyopathy. Overexpression of FST in the heart attenuate diabetic cardiomyopathy through the following ways: causing compensatory hypertrophy by the Myostatin/Akt pathway, reducing fibrosis through the TGF-β/Smad3 pathway, and decreasing oxidative stress by increasing the scavenging capacity of reactive oxygen species and reducing the production of reactive oxygen species.