Nitro-Oleic Acid (NO2-OA) Improves Systolic Function in Dilated Cardiomyopathy by Attenuating Myocardial Fibrosis

Figures

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  Figure 1. Effect of NO2-OA on left ventricular systolic function in DCM. (A) Experimental design of murine model treatment. (B) Heart-weight-to-body-weight ratio is reduced after 4 weeks of treatment with NO2-OA. Baseline echocardiographic assessment of (C) LVEF, (F) CO, and (I) GLS shows reduced baseline parameters in 12-week-old Mlp−/− animals compared to WT and (D,G,J) improved parameters in 16-week-old animals after 4 weeks of treatment with NO2-OA. Absolute change of LVEF, CO, and GLS from weeks 12–16 is shown in (E,H,K). Representative M-mode echocardiography tracings of (L) WT, (M) Mlp−/−, and (N) Mlp−/− + NO2-OA at 16 weeks. Data are expressed as mean ± SD, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, one-way ANOVA with Tukey multiple comparison test, n = 15–17 as indicated.

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  Figure 2. Effect of NO2-OA on interstitial myocardial fibrosis in DCM. (A–C) Representative images of whole heart sections and magnified areas from WT, Mlp−/−, and Mlp−/− + NO2-OA stained with picrosirius red for collagen detection. (D) Analysis of fibrotic area shows significantly reduced interstitial fibrosis in mice treated with NO2-OA. (E) Biochemical analysis of total collagen content in LV tissue homogenates reveals a reduction of collagen by NO2-OA. Data are expressed as mean ± SD, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, one-way ANOVA with Tukey multiple comparison test, n = 9–10 as indicated, scalebar as indicated.

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  Figure 3. TGFβ signaling Mlp−/− hearts. (A) qPCR reveals increased mRNA expression levels of Tgfβ in LV of Mlp−/− mice, whereas (B) expression of Tgfβ R1 is unaffected. Representative immunoblot and quantification demonstrate increased protein expression levels of TGFβ (C,F) and TGFβ latency-associated protein (LAP) (D,G) in Mlp−/− mice, whereas protein expression levels of TGFβ receptor 1 (R1) (E,H) is not influenced by the DCM phenotype. Data are expressed as mean ± SD, * p < 0.05, one-way ANOVA with Tukey multiple comparison test, n = 5–7 as indicated.

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  Figure 4. Effect of NO2-OA on TGFβ signaling in isolated primary cardiac fibroblasts. Representative (A) immunoblots, (B) quantification for expression of ⍺SMA, and (C) representative immunofluorescence show an increased ⍺SMA expression in cardiac fibroblasts upon treatment with TGFβ, which is significantly attenuated by concomitant treatment with NO2-OA. Protein expression analysis of the phosphorylation ratio of Smad2 (D,G), Stat3 (E,H), and Erk1/2 (F,I) revealed attenuated phosphorylation by NO2-OA. Data are expressed as mean ± SD, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, one-way ANOVA with Tukey multiple comparison test, n = 6 as indicated, scalebar as indicated.