Beneficial Effects of Mineralocorticoid Receptor Antagonism on Myocardial Fibrosis in an Experimental Model of the Myxomatous Degeneration of the Mitral Valve

Figures

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  Figure 1. Effects of Spironolactone on cardiac fibrosis in a mouse model of myxomatous valve disease. Representative microphotographs of mouse myocardial sections with Sirius red staining for interstitial (A) and perivascular (C) fibrosis. Individual datapoints show the quantification of interstitial fibrosis (* p = 0.0452 Nordexfenfluramine (NDF) vs. control; $ p = 0.0001; * p = 0.0166 NDF + Spiro vs. control) (B) and perivascular fibrosis (* p = 0.0307; $ p = 0.035) (D). Quantification of collagen type I (* p = 0.0329; $ p = 0.0007) (E). The box plots show the individual datapoints and the horizontal bars indicate the mean and SEM in arbitrary units versus the control group. Magnifications 40× (Scale bar 50 µm). The results were analyzed using one-way analysis of variance (ANOVA), followed by Tukey’s multiple comparisons tests. * vs. control, $ vs. NDF.

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  Figure 2. Effect of Spironolactone on profibrotic molecules and myofibroblast activation markers in a mouse model of myxomatous mitral valve disease. Quantification of Gal-3 (* p = 0.0338; $ p = 0.0217) and ST2 (* p = 0.0289) (A) protein levels in myocardium from controls, NDF-treated mice and NDF + Spiro-treated mice. Quantification of TGF-β (Transforming growth factor beta) and CT-1 (Cardiotrophin-1) (* p = 0.0014; $ p = 0.0016) (B) mRNA levels from controls, NDF-treated mice and NDF + Spiro-treated mice. Representative microphotographs of α-smooth muscle actin (α-SMA) immunostaining in myocardium from controls, NDF-treated mice and NDF + Spiro-treated mice (C). Quantification of vimentin (* p = 0.0033; $ p = 0.0051) and α-SMA protein levels in myocardium from controls, NDF-treated mice and NDF +Spiro-treated mice (D). The box plots show the individual datapoints and the horizontal bars indicate the mean and SEM in arbitrary units versus the control group. Magnifications 40× (Scale bar 50 µm). The results were analyzed using one-way ANOVA, followed by Tukey’s multiple comparisons tests. * vs. control, $ vs. NDF.

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  Figure 3. Effect of spironolactone treatment on the expression of non-fibrillar proteins in a mouse model of mitral valve fibromyxomatous degeneration. Representative microphotographs of fibronectin, aggrecan, decorin and lumican immunostainings are shown (A,C,E,G). The protein expressions of fibronectin (* p = 0.0005 Control vs. NDF; * p = 0.0013 Control vs. NDF+Spiro), aggrecan (* p = 0.0008; $ p = 0.0072), decorin (* p = 0.0209; $ p = 0.0459), lumican (* p = 0.0017; $ p = 0.0072), syndecan-1 and syndecan-4 (* p = 0.032; $ p = 0.0009) were measured by ELISA (Enzyme-Linked Immuno Sorbent Assay) in myocardium from controls, NDF-treated mice and NDF +Spiro-treated mice (B,D,F,H–J). The box plots show the individual datapoints and the horizontal bars indicate the mean and SEM in arbitrary units versus the control group. Magnifications 40× (Scale bar 50 µm). The results were analyzed one-way ANOVA, followed by Tukey’s multiple comparisons tests. * vs. control, $ vs. NDF.

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  Figure 4. NDF induces the expression of activation and fibrosis markers in adult human cardiac fibroblast. NDF effects on the activation marker α-SMA protein expression in adult human cardiac fibroblasts (* p = 1.0 × 10−7; $ p = 1.6 × 10−5 Control + NDF vs. NDF + Spiro) (A). Quantification of collagen type I (* p = 0.0410; $ p = 0.0208 (B) and the non-fibrillar protein fibronectin (* p = 0.0172)) (C) in human cardiac fibroblasts treated with NDF and NDF+Spiro. Quantification of proteoglycans (aggrecan (* p = 6 × 10−6; $ p = 1.25 × 10−4 Control+NDF vs. NDF+Spiro) (D), hyaluronan (E), decorin (F), glypican (G), syndecan-1 (H) and syndecan-4 (I) in human cardiac fibroblasts treated with NDF and NDF + Spiro. The box plots show the individual datapoints and the horizontal bars indicate the mean and SEM in arbitrary units versus the control group. The results were analyzed using one-way ANOVA, followed by Tukey’s multiple comparisons tests. * vs. control, $ vs. control +NDF.

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  Figure 5. Refining the mineralocorticoid receptor antagonist (MRA) as a new pharmacological approach to treat myocardial fibrosis associated with myxomatous mitral valve disease.