Activation of PPARα Ameliorates Cardiac Fibrosis in Dsg2-Deficient Arrhythmogenic Cardiomyopathy

Figures

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  Figure 1. Cardiac-specific Dsg2 knockout induced cardiac fibrosis. (A) Masson staining of heart sections in WT and CS-Dsg2−/− (−/−) mice. Arrow shows cardiac fibrosis. Collagen volume fraction in the hearts of WT and CS-Dsg2−/− mice was assessed. (B) Representative Western blots from mouse left ventricular (LV), interventricular septum (IVS), and right ventricle (RV). DSG2, PPARα, pSTAT3, pSMAD3, pAKT, α-SMA, and Collagen I were detected using specific antibodies. STAT3, SMAD3, AKT, and GAPDH were used as loading controls. (C) Results of quantitative PCR analysis of PPARα, TGF-β, α-SMA, and Collagen I mRNA levels in mouse LV and RV are expressed as fold change of control using β-actin as loading control. Results are expressed as mean values ± SEM. n = 6. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. WT.

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  Figure 2. Effects of Dsg2 siRNA and Stat3 siRNA on the expression levels of fibrotic markers in HL-1 cells. (A,B) HL-1 cells were transfected with control siRNA or Dsg2 siRNA. (A) Representative Western blots for DSG2, pSTAT3, α-SMA, and Collagen I were detected using specific antibodies. STAT3 and β-actin were used as loading controls. (B) Results of quantitative PCR analysis of Dsg2, α-SMA, and Collagen I mRNA levels in HL-1 cells treated with control or Dsg2 siRNA are expressed as fold change of control using β-actin as loading control. Results are expressed as mean values ± SEM. n = 3. * p < 0.05, ** p < 0.01 vs. control. (C,D) HL-1 cells were transfected with control siRNA or Stat3 siRNA. (C) Representative Western blots for STAT3 and α-SMA were detected using specific antibodies. β-actin were used as loading controls. (D) Results of quantitative PCR analysis of α-SMA and Collagen I mRNA levels in HL-1 cells treated with control or Stat3 siRNA are expressed as fold change of control using β-actin as loading control. Results are expressed as mean values ± SEM. n = 3. * p < 0.05, ** p < 0.01, vs. control.

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  Figure 3. Fenofibrate alleviated cardiac fibrosis in CS-Dsg2−/− mice. (A) Masson staining of heart sections in WT, CS-Dsg2−/− mice, and CS-Dsg2−/− mice treated with fenofibrate (Dsg2−/−F). Collagen volume fraction in the hearts of WT, CS-Dsg2−/−, and Dsg2−/−F mice were assessed. (B) Representative Western blots from ventricles of WT, CS-Dsg2−/−, and Dsg2−/−F mice. DSG2, PPARα, pSTAT3, pSMAD3, pAKT, α-SMA, and Collagen I were detected using specific antibodies. STAT3, SMAD3, AKT, and GAPDH were used as loading controls. (C) Results of quantitative PCR analysis of PPARα, TGF-β, α-SMA, and Collagen I mRNA levels in mouse ventricles are expressed as fold change of control using β-actin as loading control. Results are expressed as mean values ± SEM. n = 6. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control.

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  Figure 4. AAV9-Pparα alleviated cardiac fibrosis in CS-Dsg2−/− mice. (A) Masson staining of heart sections in CS-Dsg2−/− mice and CS-Dsg2−/− mice received AAV9-Pparα (Dsg2−/−P). Collagen volume fraction in the hearts of CS-Dsg2−/− and Dsg2−/−P mice were assessed. (B) Representative Western blots from ventricles of CS-Dsg2−/− and Dsg2−/−P mice. PPARα, pSTAT3, pSMAD3, pAKT, α-SMA, and Collagen I were detected using specific antibodies. STAT3, SMAD3, AKT, and GAPDH were used as loading controls. (C) Results of quantitative PCR analysis of PPARα, TGF-β, α-SMA, and Collagen I mRNA levels in mouse ventricles are expressed as fold change of control using β-actin as loading control. Results are expressed as mean values ± SEM. n = 6. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. CS-Dsg2−/−.