Dystrophin-deficient cardiomyopathy in mouse: expression of Nox4 and Lox are associated with fibrosis and altered functional parameters in the heart

Abstract

Duchenne muscular dystrophy (DMD; dystrophin-deficiency) causes dilated cardiomyopathy in the second decade of life in affected males. We studied the dystrophin-deficient mouse heart (mdx) using high-frequency echocardiography, histomorphometry, and gene expression profiling. Heart dysfunction was prominent at 9-10months of age and showed significantly increased LV internal diameter (end systole) and decreased posterior wall thickness. This cardiomyopathy was associated with a 30% decrease in shortening fraction. Histologically, there was a 10-fold increase in connective tissue volume (fibrosis). mRNA profiling with RT-PCR validation showed activation of key pro-fibrotic genes, including Nox4 and Lox. The Nox gene family expression differed in mdx heart and skeletal muscle, where Nox2 was specifically induced in skeletal muscle while Nox4 was specifically induced in heart. This is the first report of an altered profibrotic gene expression profile in cardiac tissue of dystrophic mice showing echocardiographic evidence of cardiomyopathy.

Figure 1. Dystrophin-deficient mdx mice shows decreased cardiac function using high frequency echocardiography

(A) Long axis view of the left ventricle with the end-diastolic volume measured using Simpson’s method. (B) Enlarged view of M-mode tracing of left ventricle showing measurements used to calculate shortening fraction.

Figure 2. Left ventricle of mdx mice shows increased fibrosis area by histomorphometry

(A) Graphic depiction of ratio of connective tissue area to normal myocardium in 9-10 month old mdx mice and wild type mice. Significantly elevated tissue area of fibrosis was seen in the mdx left ventricle (p=0.0001). (B) H&E stained left ventricle in wild type mice (10X). (C) H&E stained left ventricle in mdx mice showing patchy areas of fibrosis (10X). (D) H&E stained left ventricle myocardium in wild type mice showing normal cellular morphology (40X). (E) H&E stained left ventricle myocardium in mdx mice showing focal cardiomyocyte degeneration and increased fibroblast infiltration (40X). (F) Corresponding tri-chrome stained tissue to plate B (10X). (G) Corresponding tri-chrome stained tissue to plate C showing increased areas of collagen (10X). (H) Corresponding tri-chrome stained tissue to plate D showing normal cellular morphology (40X). (I) Corresponding tri-chrome stained tissue to plate E showing extensive collagen infiltration of myocardium.

Figure 3. Increased Nox4 mRNA expression in left ventricle of mdx mice with cardiomyopathy

Real time PCR results for Nox1, Nox2, Nox4 and Lox in cardiac (A) and skeletal muscle (B) tissue of 9-10 month old mdx and wild type mice. Significant increases were seen in Nox4 and Lox in mdx mice in cardiac muscle and in Nox2 and Lox in skeletal muscle. P values are for mdx vs. wild type for each respective gene.

Figure 4. Increased Nppb mRNA expression in left ventricle of mdx mice showing cardiomyopathy

Real time PCR results for Igf-1, Irs-1, Pdpk1, Nppb and Sln in cardiac tissue of 9-10 month old mdx and wild type mice. A significant increase in Nppb was seen in mdx mice (p=0.009).

Figure 5. Increased Nox4 protein found in the nuclei of left ventricular cardiomyocytes in 9-10 month old mdx mice

Immunofluorescence using primary Nox4 antibodies demonstrates increased binding in cardiomyocytes found in the left ventricles of mdx (top panel) mice compared to wild type (wt) mice (bottom panel). Counterstaining with DAPI confirms localization to the nuclei.