Myostatin Mutation Promotes Glycolysis by Increasing Phosphorylation of Phosphofructokinase via Activation of PDE5A-cGMP-PKG in Cattle Heart

Figures

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  FIGURE 1. Myostatin mutation decreased SMAD2/3 expression but had no effect on cardiac anatomy of cattle. (A) Whole heart images of MT and WT cattle. (B,C) Statistical results of heart weight (HW)/tibial length (TL) and HW/body weight (BW) in MT and WT cattle. (D) Expression of MSTN mRNA in heart of MT and WT cattle. (E,F) Expression of MSTN protein in heart of MT and WT cattle. (G) Representative expression of MSTN immunofluorescence images of heart (green, MSTN; blue, nuclei; scale bar, 25 μm). (H) Expression of MSTN signaling pathway factor mRNA in heart of MT and WT cattle. (I, J) Expression of MSTN signaling pathway factor protein in heart of MT and WT cattle. All data are presented as mean ± SD. Compared with the WT group, *p < 0.05, **p < 0.01, ns: no significant; t-tests were used to calculate the p-values.

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  FIGURE 2. Myostatin mutation did not cause cardiac histology change. (A) Hematoxylin and eosin (HE) staining of heart tissue samples (400×). (B) Calculated cross-sectional area of heart samples (n > 100 cells for each group). (C) mRNA levels of the hypertrophic markers. (D,E) Expression of MYH7 protein in heart of MT and WT cattle. (F) Representative expression of MYH7 immunofluorescence images of heart (green, MYH7; blue, nuclei; scale bar, 25 μm). (G) Representative images of Sirius Red-stained sections and polarized light microscopy from MT and WT cattle. (H,I) Analysis of cardiac fibrosis, and in polarized light collagen type I (red-yellow) and collagen type III (green) in MT and WT cattle. (J) mRNA levels of the fibrosis markers. All data are presented as mean ± SD. Compared with the WT group, *p < 0.05, **p < 0.01, ns: no significant; t-tests were used to calculate the p-values.

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  FIGURE 3. Myostatin mutation mutant effect on heart glycolysis of cattle. (A–C) Activity for key enzymes involved in glycolysis pathway. (D–G) Expression of protein for key enzymes involved in glycolysis pathway in MT and WT cattle heart. (H–K) Expression of phosphorylation of protein for key enzyme protein involved in glycolysis pathway by immunoprecipitation in MT and WT cattle heart. Western blots obtained by immunoblotting antibodies directed against HK2, PFK, PK, and phosphorylation. Abbreviations: PFK, phosphofructokinase; HK2, hexokinase; PK, pyruvate kinase. All data are presented as mean ± SD. Compared with the WT group, *p < 0.05, **p < 0.01, ns: no significant; t-tests were used to calculate the p-values.

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  FIGURE 4. MSTN regulates PDE5A-cGMP-PKG pathway expression via SMAD2/SMAD3. (A) Volcano plot of differential gene expression analysis. (B) Clustering heat map of differential gene expression. (C) KEGG analysis of DEGs between WT and MT groups. (D–F) Expression of PDE5A and PKG protein in heart of MT and WT cattle. (G) cGMP content of MT and WT cattle heart. (H) Expression of PDE5A mRNA in heart of MT and WT cattle. (I) Schematic representation of SMAD2/SMAD3 binds the PDE5A promoter region. (J) ChIP-qPCR detected the binding of SMAD2/SMAD3 to the PDE5A promoter region in cattle hearts with MT. (K) Promoter activity of PDE5A was determined by a luciferase assay. All data are presented as mean ± SD. *p < 0.05, **p < 0.01, ns: no significant; t-tests were used to calculate the p-values.

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  FIGURE 5. Activation of cGMP-PKG promotes glycolysis via increasing phosphorylation of PFK. (A) PKG interacts with PFK by co-immunoprecipitation and immunoblotting analyses in cattle heart. (B,C) The expression of PKG at the protein level after the activation of 8-Br-cGMP in the H9C2 cells. (D,E) Phosphorylation level of PFK protein by immunoprecipitation after the activation of 8-Br-cGMP in the H9C2 cells. (F) Activity for PFK enzymes after the activation of 8-Br-cGMP in the H9C2 cells. (G,H) Content for key metabolites in glycolysis pathway after the 8-Br-cGMP treatment. FDP, Fructose-1,6 diphosphate; G6P, Glucose-6-phosphate. All data are presented as mean ± SD. Compared with the control group, *p < 0.05, **p < 0.01, ns: no significant; t-tests were used to calculate the p-values.

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  FIGURE 6. Knockdown of PKG inhibited glycolysis via decreasing phosphorylation of PFK. (A–C) The expressions of PKG at the mRNA and protein levels after knockdown of PKG in the H9C2 cells. (D,E) Phosphorylation level of PFK protein by immunoprecipitation of the PKG knockdown H9C2 cells. (F) Enzyme activity of PFK assay in PKG knockdown H9C2 cells. (G,H) FDP and G6P, the key metabolites in glycolysis, assayed respectively. Abbreviations: PFK, phosphofructokinase; FDP, Fructose-1,6 diphosphate; G6P, Glucose-6-phosphate. All data are presented as mean ± SD. Compared with the control group, *p < 0.05, **p < 0.01, ns: no significant; t-tests were used to calculate the p-values.

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  FIGURE 7. Schematic of mechanisms for the activation of PDE5A-cGMP-PKG signaling and the promotion of glycolysis by MSTN. MSTN activate the pathway via combining with ActRIIB, which then binds to the type I receptor to form a receptor complex. After the receptor complex is activated by the signal, SMAD2/SMAD3 is activated via phosphorylation. Then, SMAD2/SMAD3 will detach from receptor complex and bind to SMAD4. Then, the heteromeric complex enters the nucleus, binds to the promoter region, and alters transcription of PDE5A gene. The left one is the wild-type cattle and the right one is the MSTN mutant cattle. The MSTN mutant decreased SMAD2/3 expression, which led to transcription downregulation of PDE5A and thereby promoted the cGMP/PKG pathway, increased the phosphorylation level of PFK, and subsequently enhanced glycolysis. The red arrow indicates upregulation and the green arrow indicates downregulation.