Severe cardiomyopathy in mice lacking dystrophin and MyoD

Abstract

The mdx mouse, a mouse model of Duchenne muscular dystrophy, carries a loss-of-function mutation in dystrophin, a component of the membrane-associated dystrophin-glycoprotein complex. Unlike humans, mdx mice rarely display cardiac abnormalities and exhibit dystrophic changes only in a small number of heavily used skeletal muscle groups. By contrast, mdx:MyoD-/- mice lacking dystrophin and the skeletal muscle-specific bHLH transcription factor MyoD display a severe skeletal myopathy leading to widespread dystrophic changes in skeletal muscle and premature death around 1 year of age. The severely increased phenotype of mdx:MyoD-/- muscle is a consequence of impaired muscle regeneration caused by enhanced satellite cell self-renewal. Here we report that mdx:MyoD-/- mice developed a severe cardiac myopathy with areas of necrosis associated with hypertrophied myocytes. Moreover, heart tissue from mdx:MyoD-/- mice exhibited constitutive activation of stress-activated signaling components, similar to in vitro models of cardiac myocyte adaptation. Taken together, these results support the hypothesis that the progression of skeletal muscle damage is a significant contributing factor leading to development of cardiomyopathy.

Figure 1

Cardiac hypertrophy in mdx mice lacking MyoD. Hearts from 5-month-old mdx:MyoD^−/− mice were compared with age-matched wt littermates. (A) Intact hearts of mdx:MyoD^−/− mice appeared significantly larger than hearts from wt mice. (B) Hematoxylin-eosin stained sections of hearts (cut transversely at midventricle) revealed an increased ventricular diameter in mdx:MyoD^−/− mice compared with wt. Although hearts were fixed ex vivo without diastole arrest, the apparent increase in ventricular diameter without concurrent changes in ventricular thickness suggests dilatation. We also observed marked cardiomyocyte hypertrophy and disarray in mdx:MyoD^−/− hearts not present in wt mice (C). A and B were photographed at identical magnifications. (C, ×40.)

Figure 2

Constitutive activation of SAPK effectors in ventricle protein lysates of 5-month-old wt, mdx, MyoD^−/−, and mdx:MyoD^−/− mice. Tyrosine phosphorylation of p38 (A) and JNK-1 (B) was assessed by immunoprecipitation with an antiphosphotyrosine antibody, followed by Western detection with anti-p38 or anti-JNK-1 (Upper). Lysates subjected to SDS/PAGE and Western blotting revealed equivalent amounts of p38 and JNK-1 within each genotype (Lower). Similar results were obtained in three independent experiments.

Figure 3

Advanced cardiac myopathy in older mdx:MyoD^−/− mice. >50% of 10-month-old mdx:MyoD^−/− mice exhibited visible fibrosis in the ventricular region (arrow in A and B). Masson trichrome-stained serial sections through mdx:MyoD^−/− hearts revealed the fibrosis to be confined primarily to epicardial portions of the left ventricle (compare C and D to E) and in regions encompassed by hypertrophied myocytes (arrowhead in B). (B ×40; C–F ×20.)

Figure 4

Constitutive activation of SAPK effectors in wt, early (hypertrophy), and late (damage) mdx:MyoD^−/− cardiomyopathy. Tyrosine phosphorylation of p38 (A) and JNK-1 (B) was assessed by immunoprecipitation with an antiphosphotyrosine antibody, followed by Western detection with anti-p38 or anti-JNK-1 (Top). p38 and JNK-1 were immunoprecipitated from these lysates and used in an in vitro kinase assay to test the ability of the proteins to phosphorylate an exogenous substrate (ATF2 for p38 and cJun for JNK-1) (Middle). Lysates subjected to SDS/PAGE followed by Western detection revealed equivalent amounts of p38 and JNK-1 within each genotype (Bottom). Similar results were obtained in independent experiments (three IP/Western blot analyses and two kinase assays). Note the IP:Western analysis in A revealing p38 phosphorylation was subjected to about a 10-fold shorter exposure than the experiment shown in Fig. 2A.