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Cardiomyopathy is independent of skeletal muscle disease in muscular dystrophy ¹

XIAOLEI ZHU^*, MATTHEW T. WHEELER, MICHELE HADHAZY^*, MAN-YEE J. LAM^* and ELIZABETH M. MCNALLY^*,2

^* Department of Medicine, Section of Cardiology,
Department of Molecular Genetics and Cell Biology, and
Department of Human Genetics, The University of Chicago, Chicago, Illinois, USA

²Correspondence: The University of Chicago, Section of Cardiology, 5841 S. Maryland, MC 6088, Chicago, IL 60637, USA. E-mail: emcnally{at}medicine.bsd.uchicago.edu

SPECIFIC AIMS

Dystrophin and its associated proteins, the sarcoglycans, are normally expressed in heart and skeletal muscle, and mutations that alter the expression of these membrane-associated proteins lead to muscular dystrophy and cardiomyopathy in humans. Cardiomyopathy commonly accompanies muscular dystrophy, and it has been suggested that extrinsic parameters may contribute to the development of cardiomyopathy in muscular dystrophies. To test whether severe skeletal muscle dystrophy affecting the respiratory musculature contributes to cardiomyopathy, we rescued skeletal muscle -sarcoglycan in -sarcoglycan null mice.

PRINCIPAL FINDINGS

1. Skeletal muscle expression of -sarcoglycan
We expressed full-length murine -sarcoglycan under control of the myosin light chain (MLC)1/3 promoter since this promoter has been shown to be specific to skeletal muscle. Several lines of mice carrying the transgene encoding -sarcoglycan under control of the MLC1/3 sequence (LCg) were generated; one was selected for study since it expressed -sarcoglycan at levels comparable to wild-type levels. LCg⁺ mice were bred to gsg^-/- mice to generate mice that carried the transgene on the -sarcoglycan null background. Northern blot and immunoblot analysis of LCg⁺/gsg^-/- cardiac and skeletal muscle preparations demonstrated that -sarcoglycan was not found in cardiac muscle but was expressed in fast skeletal muscle fibers, consistent with the known specificity of the MLC1/3 promoter.

2. Restoration of the skeletal muscle sarcoglycan complex in LCg⁺/gsg^-/- mice
Immunostaining of LCg⁺/gsg^-/- skeletal muscle revealed normal expression of -sarcoglycan at the periphery of skeletal muscle myofibers. -Sarcoglycan is part of the dystrophin glycoprotein complex (DGC) (Fig. 1 ). Mutations in any single sarcoglycan gene lead to instability of the sarcoglycan complex at the plasma membrane of skeletal and cardiac muscle. Instability of sarcoglycan is seen when dystrophin is absent, as it is in Duchenne muscular dystrophy (DMD). The presence of the LCg transgene leads not only to the normal expression of -sarcoglycan, but also to the restoration of the -, ß-, and -sarcoglycan subunits.

View larger version (47K): [in this window] [in a new window]   Figure 1. Shown is a schematic of the dystrophin glycoprotein complex (DGC). Dystrophin, the protein product of the Duchenne muscular dystrophy gene, is associated with transmembrane proteins including sarcoglycan, dystroglycan, and sarcospan. Intracellular components of the DGC include dystrobrevin and the syntrophins. Mutations in the genes encoding sarcoglycan subunits or dystrophin lead to destabilization of the sarcoglycan complex, muscle membrane instability, cardiomyopathy, and muscular dystrophy.

3. Correction of skeletal muscle dystrophy
Muscles from LCg⁺/gsg^-/- mice were examined and found to exhibit a normal histological pattern. Muscle from gsg^-/- mice has marked fiber size variation, an increase in centrally placed nuclei, focal necrosis, calcification, and replacement by connective and adipose tissue. Skeletal muscle from LCg⁺/gsg^-/- mice showed no evidence of these characteristics of muscular dystrophy. Because the MLC1/3 promoter expresses in fast-type skeletal muscle fibers and most murine muscle fibers have a significant component of fast fibers, nearly all muscle groups were corrected by the presence of the LCg transgene.

4. Correction of membrane permeability defects in LCg⁺/gsg^-/- mice
Evans blue dye (EBD) is a small molecule that can be used as a vital tracer. In muscular dystrophies where sarcoglycan is reduced or absent from the plasma membrane, muscle becomes abnormally permeable to EBD. To determine whether the LCg transgene corrected the membrane permeability defects seen in gsg^-/-, we treated LCg⁺/gsg^-/- mice with EBD. As shown in Fig. 2 , stripes of blue can be seen grossly in gsg^-/- muscle but not in LCg⁺/gsg^-/- muscle, consistent with correction of membrane permeability defects in skeletal muscle by the LCg transgene. We evaluated the diaphragm muscle since this is the major respiratory muscle affected by the dystrophic process. Only rare EBD-positive fibers were seen (shown as red fibers on fluorescence microscopy) in LCg⁺/gsg^-/- mice whereas abundant EBD uptake was seen in gsg^-/- diaphragm muscle.

View larger version (100K): [in this window] [in a new window]   Figure 2. Membrane permeability defects are corrected by the LCg transgene. EBD uptake is indicative of membrane permeability defects as blue (grossly) and red staining on fluorescence microscopy. EBD uptake in abdominal wall (A) and limb (C) muscle from gsg-/- mice is seen as blue stripes. There is a lack of EBD uptake in LCg+,gsg-/- mice in the abdominal wall (B) and the limb (D). The soleus muscle remains permeable to EBD (arrowhead in panel E) in LCg+,gsg-/- muscle since it is composed of mainly slow muscle fibers, and the MLC1/3 promoter is fast fiber specific. F, G) Double staining with anti--sarcoglycan (orange) and anti-slow fiber antibody (green) on sections from gastrocnemius muscle from LCg+,gsg-/- mice. F) In the gastrocnemius muscle of LCg+,gsg-/- mice, the only fiber that is not expressing -sarcoglycan (*) expresses slow myosin heavy chain (seen as green staining). G) In soleus muscle from LCg+,gsg-/- mice, many fewer fibers are expressing -sarcoglycan (orange) and these slow fibers express slow myosin (green). H–L) Microscopic EBD uptake (red) in gastrocnemius (H, I) and diaphragm (J–L) muscle sections. I, K) The absence of EBD uptake in LCg+,gsg-/- muscle; H, J) abundant EBD uptake in gsg-/-. An antibody to -sarcoglycan (green) was used to show -sarcoglycan expression in the same sections (H–L). Counterstaining with DAPI shown as blue color for nuclei was included in the mounting medium. Normal diaphragm muscle is shown in panel L.

5. Persistent cardiomyopathy despite skeletal muscle rescue in LCg⁺/gsg^-/- mice
We studied the hearts of LCg⁺/gsg^-/- mice and compared them to age-matched littermate gsg^-/- mice. We confirmed that -sarcoglycan was not expressed in the hearts of gsg^-/- and LCg⁺/gsg^-/- mice. Focal necrosis and fibrosis was corrected in LCg⁺/gsg^-/- skeletal muscle but not in LCg⁺/gsg^-/- cardiac muscle (Fig. 3 ). Fibrosis (blue) was frequently noted in the right ventricles of gsg^-/- and LCg⁺/gsg^-/- mice. We found frequent EBD-positive cardiomyocytes consistent with membrane instability. Like areas of fibrosis, EBD-positive cardiomyocytes were seen in the right ventricle. These findings suggest that the right ventricle may be especially susceptible to the pathological mechanisms that arise from the loss of sarcoglycan. Both gsg^-/- and LCg⁺/gsg^-/- hearts had up-regulation of ß-myosin heavy chain, a molecular correlate of cardiac failure.

View larger version (108K): [in this window] [in a new window]   Figure 3. Focal necrosis and membrane permeability defects are present in the hearts of gsg-/- and LCg+,gsg-/- mice. A–C) Trichrome-stained, short-axis cross sections of hearts. Regions of necrosis are seen in gsg-/- and LCg+,gsg-/- hearts stained blue and highlighted in the boxed regions. D–F) Focal necrosis in the right ventricle of gsg-/- and LCg+,gsg-/- mice that is absent in normal tissue. G–I) Staining with a polyclonal -sarcoglycan antibody indicating that -sarcoglycan is not expressed in the hearts of gsg-/- and LCg+,gsg-/- mice. Counterstaining with DAPI (blue) shows nuclei. J–L) Regions of EBD uptake in the right ventricle of gsg-/- and LCg+,gsg-/- hearts, and EBD uptake is not seen in normal hearts. J–L) Counterstaining with a carboxyl-terminal dystrophin antibody (Dys2) is shown indicating that dystrophin expression is normal in these cardiomyopathic hearts.

CONCLUSIONS

Dystrophin and the sarcoglycan subunits are found in cardiac muscle at similar levels to their expression in skeletal muscle. Studies with vital dyes demonstrate abnormal uptake within cardiac myocytes, yet several lines of evidence have suggested that cardiac-extrinsic processes may contribute to the development of cardiomyopathy in DMD and the limb girdle muscular dystrophies (LGMDs). Lordosis and scoliosis arising from muscular dystrophy in postural musculature frequently limit respiratory performance. This, coupled with intrinsic respiratory muscle disease affecting the diaphragm and intercostal muscles leads to restrictive lung disease, hypoxemia, and pulmonary hypertension, which in turn may produce a secondary cardiomyopathy. Although the development of cardiac degeneration in these disorders may originate from cardiac-intrinsic loss of dystrophin or its associated sarcoglycans, these clinicopathologic findings indicate that severe skeletal muscle disease itself may contribute to cardiovascular morbidity in the muscular dystrophies.

The mdx mouse is a model for DMD since it lacks full-length dystrophin. mdx mice have histopathologic changes similar to those in DMD patients, including focal degeneration and regeneration. Megeney and colleagues showed that skeletal muscle damage is a crucial determinant in the progression of cardiomyopathy in the mdx mouse by generating mice deficient for both dystrophin and the skeletal muscle-specific transcription factor MyoD (mdx:MyoD^-/-). mdx:MyoD^-/- mice develop a severe cardiomyopathy. Since MyoD is expressed exclusively in skeletal and not cardiac muscle, cardiomyopathy in these mice develops as a secondary consequence of severe skeletal muscle disease. These findings are relevant since many cell and gene therapies being contemplated for these disorders are aimed at correcting only the skeletal muscle defects.

We used a mouse model for LGMD2C (gsg^-/-) to determine genetically whether severe skeletal muscle disease contributes to cardiomyopathy. We selected gsg^-/- mice since this model has findings similar to those in human muscular dystrophy patients including prominent skeletal muscle weakness and cardiomyopathy. In the present study, we generated transgenic mice (LCg⁺) expressing -sarcoglycan under the control of skeletal muscle-specific MLC1/3 promoter and enhancer. These transgene ‘rescued’ mice show dramatic improvement of skeletal muscle dystrophy with correction of membrane permeability defects, lack of centrally placed nuclei, and complete absence of fibrosis. Despite correction of the skeletal muscle disease, degeneration and membrane permeability defects persist in cardiac muscle. Since the LCg transgene is not expressed in cardiac muscle, these data demonstrate that cardiomyopathy in these disorders is independent of skeletal muscle disease.

Despite normal skeletal muscle in LCg⁺,gsg^-/- mice, focal necrosis remained a prominent finding within the right ventricles of LCg⁺,gsg^-/- mice. It has been noted that the right ventricle is preferentially affected in both DMD and LGMD patients and this was thought to relate to respiratory muscle involvement and scoliosis. Our findings imply that the right ventricle is sensitive to cardiomyopathic defects in dystrophin and sarcoglycan-related disorders. These findings argue that therapy in these patients should target both cardiac and skeletal muscle to prevent sudden death arising from cardiomyopathy.

Our results differ from those seen in mdx:MyoD^-/- mice. The MyoD mutation causes a markedly abnormal skeletal muscle regeneration, yet in DMD patients, regeneration is an important feature. Accordingly, the severe cardiomyopathy in mdx:MyoD^-/- mice may not reflect the physiological defects found in DMD patients. Alternatively, there may be a fundamental difference between dystrophin-mediated and sarcoglycan-mediated cardiomyopathy. Contraction-induced injury plays a major role in mdx muscular dystrophy; the addition of signal transduction impairment may be involved in sarcoglycan disorders.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0954fje; to cite this article, use FASEB J. (May 8, 2002) 10.1096/fj.01-0954fje.

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