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Dystrophin- and MLP-deficient mouse hearts: marked differences in morphology and function, but similar accumulation of cytoskeletal proteins

James R. Wilding^*, Jürgen E. Schneider, A. Elizabeth Sang^*, Kay E. Davies, Stefan Neubauer and Kieran Clarke^*,1

^* Departments of Physiology,
Cardiovascular Medicine, and
Human Anatomy and Genetics, University of Oxford, Oxford, England, UK

¹Correspondence: Department of Physiology, University of Oxford, Parks Road, Oxford OX1 3PT, England, UK. E-mail: kieran.clarke{at}physiol.ox.ac.uk

SPECIFIC AIMS

Mutation of the genes encoding cytoskeletal proteins such as dystrophin and muscle LIM protein (MLP) causes dilated cardiomyopathy in humans. Dystrophin-deficient (mdx) mice are a commonly used model of human muscle disease, yet their cardiac morphology and function in vivo is unknown. We therefore used cine magnetic resonance imaging to measure cardiac parameters in 2- to 3-month-old mdx mice and compared these with the MLP null mouse model of dilated cardiomyopathy. We tested the hypothesis that dystrophin and MLP loss would perturb levels of other cytoskeletal proteins.

PRINCIPAL FINDINGS

1. Markers of dystrophic and cardiac pathology
Mdx mice had 14-fold higher serum creatine kinase activity than their C57BL/10 controls, consistent with sarcolemmal instability and muscular dystrophy, but had normal cardiac levels of ß-myosin heavy chain protein. In contrast, MLP null mice had normal serum creatine kinase activity but elevated ß-myosin heavy chain levels, compared with Sv129/B6 controls, suggesting activation of the hypertrophic gene program.

2. Cardiac parameters in vivo
Cardiac morphology and function were characterized in vivo in mdx and MLP null mice, and their respective control strains, using magnetic resonance imaging with a spatial resolution of 50 x 50 µm (Fig. 1 ). In both models, heart rate was similar to controls. Dystrophin deficiency did not significantly alter left ventricle/body weight ratio, end-diastolic volume, end-systolic volume, or stroke volume, and compared with controls, mdx mouse hearts had similar ejection fractions (55±3 vs. 61±2%) and cardiac output (15±1 vs. 16±1 mL/min). MLP deficiency, by contrast, significantly increased left ventricle/body weight ratio, end-diastolic and end-systolic volumes, and decreased stroke volumes relative to controls. MLP null mouse left ventricular ejection fractions were also lower than controls (25±5 vs. 66±1%, P<0.01), as was cardiac output (9±1 vs. 17±1 mL/min, P<0.01). Dystrophin deficiency did not affect mouse heart morphology or function, whereas MLP deficiency caused dilated cardiomyopathy with heart failure.

View larger version (63K): [in this window] [in a new window]   Figure 1. In vivo cine MRI images of an mdx and C57BL/10 control mouse heart (A), and an MLP-null and Sv129/B6 control mouse heart (B). Top row shows long-axis views, middle row shows the short axis of a mid-ventricular slice at end diastole, and bottom row shows the short axis at end systole.

3. End-diastolic pressure-volume relationships
In isolated, perfused hearts from mdx and MLP null mice, end-diastolic pressure-volume relationships and passive left ventricular stiffness were normal, compared with controls, over the end-diastolic pressure range 0–30 mmHg.

4. Cytoskeletal protein levels
In mdx and MLP null mouse hearts, levels of ß-tubulin and desmin were significantly higher than in the respective controls (Fig. 2 ). Dystrophin-deficient mouse hearts had higher MLP levels and MLP null hearts had higher dystrophin levels. mdx mouse hearts accumulated utrophin and MLP null mouse hearts accumulated syncoilin. In mdx diaphragm muscle, levels of syncoilin, ß-tubulin, desmin, and MLP were significantly higher than in controls, whereas cytoskeletal protein levels were normal in MLP null diaphragm.

View larger version (73K): [in this window] [in a new window]   Figure 2. Cytoskeletal proteins accumulated in mdx and MLP null mouse heart. Left: representative immunoblots of cytoskeletal proteins in mdx and C57BL/10 control mouse heart. Right: representative immunoblots of cytoskeletal proteins in MLP null and Sv129/B6 control mouse heart.

CONCLUSIONS AND SIGNIFICANCE

The cytoskeleton plays a role in various forms of heart disease: mutation of genes encoding cytoskeletal proteins can cause cardiomyopathy; post-translational modification of dystrophin may promote cardiomyopathy; and the disordered accumulation of microtubules and desmin may lead to contractile dysfunction. The mechanisms that link cytoskeletal gene mutations with cardiomyopathy are unclear, but a defective response to mechanical stress may be involved, with dystrophin mutations leading to sarcolemmal weakness and aberrant signaling and MLP mutations leading to the loss of a putative stretch-sensing complex.

In mice, dystrophin-deficiency reproduces the sarcolemmal weakness, and MLP-loss the T-cap/telethonin mislocalization, that characterizes human dilated cardiomyopathy caused by mutation of the same genes, yet only murine MLP-loss reproduces the chamber dilation and heart failure. Other studies have provided evidence of mild cardiomyopathy in the mdx mouse, consisting of ECG abnormalities, depressed isolated working heart function, histological changes, and activation of hypertrophic signaling pathways. No studies have determined this model’s cardiac morphology and function in vivo before. We used high-resolution cine-MRI to characterize mouse hearts because this technique provides accurate, volumetric information on cardiac structure in vivo and can detect subtle alterations in heart morphology and function. We found that dystrophin deficiency in the mouse heart did not affect left ventricular mass, cavity volume, ejection fraction, or cardiac output. Histological changes and ECG abnormalities may have been present in the mdx mouse hearts, and functional impairment may have occurred under conditions of increased workload, but disease did not manifest as morphological or functional changes under the conditions studied here. Left ventricular diastolic stiffness was also normal in both mdx and MLP null mouse hearts. In the absence of morphological changes, this may suggest that mdx mouse myocardial stiffness was unaffected by the loss of dystrophin. Mass-to-volume ratio was greatly reduced in the MLP null mouse left ventricle, which would decrease ventricular stiffness, but this may have been offset by the collagen deposition that reportedly occurs in this model.

Loss of dystrophin or MLP in the mouse heart and in dystrophic diaphragm muscle resulted in increased levels of cytoskeletal proteins that were associated with the dystrophin/utrophin-associated protein complex (dystrophin, utrophin, and syncoilin), the intermediate filament network (desmin and syncoilin), microtubules (ß-tubulin) or the interface between the Z-disk and the extrasarcomeric cytoskeleton/intercalated disk (desmin and MLP). The absence of such changes in MLP null mouse diaphragm may have reflected the relatively low expression of MLP in this muscle in controls with a consequent lack of muscle remodeling due to MLP loss. In the heart, accumulation of ß-tubulin and desmin is associated with cardiac failure caused by pressure overload or cardiomyopathy. Our data indicate that these proteins can accumulate in the absence of such pathological changes, because the mdx mouse left ventricle had normal morphology, function, and passive properties. In contrast, ß-tubulin and desmin accumulation in MLP null mouse hearts was associated with heart failure due to dilated cardiomyopathy. It is surprising that similar cytoskeletal proteins accumulated in mdx and MLP null mouse hearts, given their different phenotypes. There is evidence of structural compensation for dystrophin deficiency by utrophin, talin and vinculin in mdx skeletal muscle, and by utrophin in mdx cardiac muscle. Utrophin is a homologue of dystrophin, and its accumulation may be particularly effective at compensating for the loss of dystrophin. Our data suggest that ß-tubulin, desmin, and MLP also help structurally compensate for dystrophin deficiency in cardiac and diaphragm muscle, perhaps strengthening the association of myofibrils with the sarcolemma (Fig. 3 ). Similarly, cytoskeletal protein accumulation may help compensate for MLP deficiency in mouse hearts lacking this protein, but this was not sufficient to prevent morphological and functional remodeling.

View larger version (27K): [in this window] [in a new window]   Figure 3. Diagram showing effects of dystrophin or MLP loss on cardiac remodeling. The absence of dystrophin (black cross) or MLP (gray cross) increases the susceptibility of muscle to mechanical stress, which is a potent stimulus for remodeling. Mechanical stress may in turn cause compensatory increases in cytoskeletal protein levels (black and gray arrows for dystrophin and MLP-deficient hearts, respectively), which help maintain the link between myofibrils and the sarcolemma, thereby retarding the progression of cardiac disease. In the case of murine MLP deficiency, cytoskeletal protein accumulation was not able to prevent morphological and functional remodeling.

We used 2- to 3-month-old mice because this age is commonly used for mdx mouse heart studies. Since there is evidence that cardiac disease is more severe in older animals, it would be important to determine the morphology and function of older mdx mouse hearts. The absence of morphological or functional changes in hearts from 2- to 3-month-old mdx mice casts doubt on the value of this model in the development of therapies aimed at treating cardiac disease associated with dystrophin deficiency in patients, whereas the MLP null mouse provides an excellent model of human heart failure caused by dilated cardiomyopathy.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-1731fje;

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