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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online February 25, 2005 as doi:10.1096/fj.04-2511fje. |
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* Muscle Cell Biology Group, MRC Clinical Sciences Centre, Imperial College, London, UK;
Centre for Rheumatology, Royal Free Hospital School of Medicine, University College London, London, UK;
Research Centre for Genetic Medicine, Children’s National Medical Centre, Washington, DC, USA; and
Dubowitz Neuromuscular Unit, Department of Paediatrics, Hammersmith Hospital, Imperial College, London, UK
1 Correspondence: Muscle Cell Biology Group, MRC Clinical Sciences Centre, Imperial College, London W12 0NN, UK. E-mail: jmorgan{at}csc.mrc.ac.uk
SPECIFIC AIMS
Although glucocorticoid steroids are used as treatment for human Duchenne muscular dystrophy the precise mechanism(s) of action is not known. We have examined the influence of the glucocorticoid prednisolone on skeletal muscle gene expression profiles and muscle function in the dystrophin-deficient mdx mouse model of DMD.
PRINCIPAL FINDINGS
1. Steroid induces different gene expression profiles in mdx muscles after short- and long-term treatment
To investigate changes in skeletal muscle gene expression profiles in response to steroid, animals were treated with prednisolone for short (1 wk) and long-term (6 wk) periods, and global gene expression examined using Affymetrix gene chips. After 1 wk of treatment, 69 genes were up-regulated. These included genes associated with cell growth and proliferation, chemokines and cytokines, ion transport, metabolism and proteolysis, the muscle skeletal proteins myosin Va and delta sarcoglycan, and cell signaling including frizzled homologue 4 and FK506-binding protein 51 (FKBP51), a modulator of calcineurin. Twenty genes were down-regulated, including interleukin 17, microtubule associated protein tau, and homer2. After long-term treatment, 49 skeletal muscle genes were up-regulated, including two genes important in regulating muscle hypertrophy: p85
PI3 kinase regulatory subunit and insulin-like growth factor binding protein 6 (IGFBP6). Only nine genes were down-regulated at this time, including TNF receptor 21 (death receptor 6), a protein induced by TNF- and involved in inflammation. Gene expression changes after 1 and 6 wk of prednisolone treatment were almost entirely different. We focused on genes differentially regulated by prednisolone and involved in skeletal muscle structure, regeneration, and function (necrosis, fiber type, and size), and inflammation.
2. Steroid treatment induces changes in muscle protein expression in skeletal muscle
Using Western blot analysis, we confirmed the increased expression of delta sarcoglycan, a component of the dystroglycan complex (Fig. 1
a). IGFBP6, a marker for muscle regeneration, was significantly elevated after 6 wk treatment whereas the increase in FKBP51 message was maintained as protein throughout treatment (Fig. 2
a).
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3. Steroid induces changes in muscle regeneration and function
We examined defined markers of skeletal muscle regeneration in vivo, including the presence of centrally nucleated fibers, fibers containing the fetal isoform of myosin, and expression of myogenic regulatory factors MyoD and myogenin. No differences were found in the percentage of fibers containing centrally located nuclei, the number of fetal myosin-containing fibers, or the expression of MyoD and myogenin, between control and treated groups. We next examined the effects of prednisolone on muscle fiber type and hypertrophy. The percentage of muscle fibers in soleus muscles expressing slow (type 1) myosin was assessed and muscle fiber sizes were determined by measuring fiber diameter using image analysis. Although there was no difference in fiber size between control and prednisolone-treated mdx mice after 6 wk of treatment, there was a significant shift in skeletal muscle fiber type toward a faster muscle type, a significantly lower percentage of type 1 myosin expressing fibers in prednisolone-treated mdx but not C57Bl/10, soleus muscles (Fig. 2)
. This reverses the natural pathology seen in mdx soleus muscles, which exhibit a shift toward a slow fiber phenotype. To examine the effect of prednisolone on skeletal muscle necrosis, right legs of mice were irradiated with 18 Gy to prevent muscle growth and regeneration, thus separating these processes from the degenerative episodes that occur in mdx muscle. The loss of weight of irradiated mdx tibialis anterior muscles due to muscle necrosis and failure to grow and regenerate was not ameliorated by prednisolone treatment, indicating that steroid treatment did not reduce fiber necrosis. Despite its anti-inflammatory properties, macrophage numbers were not reduced in gastrocnemius muscles after 6 wk of prednisolone treatment (6.3 macrophages/muscle profile in control, untreated muscles, and 5.6 cells/muscle profile in prednisolone-treated mdx muscles).
4. Prednisolone did not significantly increase calcineurin activity or up-regulate utrophin in mdx soleus muscles
The significant change in skeletal muscle fiber type and the observation of the long-term up-regulation of FKBP51 led us to investigate the role of calcineurin in muscles from steroid-treated animals. We examined the activity of calcineurin and the expression of a major transcription factor involved in calcinuerin reponses, NFAT 2c, in muscles from prednisolone-treated and untreated mdx and normal C57Bl/10 mice. Although there was significant reduction in calcineurin activity in untreated mdx vs. untreated C57Bl/10 muscles (P<0.001, Student’s t test) (Fig. 2)
, the increase in calcineurin activity in prednisolone-treated vs. untreated mdx muscle was not statistically significant. There was no change in NFAT 2c protein in prednisolone-treated muscles (Fig. 2)
. Although utrophin, an autosomal paralogue of dystrophin, has been reported to be up-regulated by prednisone in human DMD muscle fibers in vitro and in mdx mice by calcineurin, we found no change in amounts of utrophin protein or its distribution in prednisolone-treated mdx muscles (Fig. 1a, b
).
CONCLUSIONS AND SIGNIFICANCE
Prednisolone is the only pharmacological treatment to have a beneficial effect on DMD. We used a novel strategy, gene expression profiling, coupled with protein analysis and assessments of skeletal muscle function, to examine the complex changes that occur in skeletal muscle after steroid treatment.
Our results show the complex effect of prednisolone on dystrophic mdx skeletal muscle, with almost entirely different genes being differentially regulated after 1 and 6 wk of treatment. This may be because the effect of the drug differs with time or the muscle itself has changed dramatically due to the ongoing pathological consequences of dystrophin deficiency.
Although glucocorticoids cause skeletal muscle atrophy and several genes (e.g., aldehyde dehydrogenase1 family 1, subfamily A1, proteasome subunit type 7, and homer 2) were differentially expressed in both prednisolone-treated mdx muscle and models of muscle atrophy, there was no alteration in mdx muscle fiber size after 6 wk of prednisolone treatment. It is possible that maintenance of muscle fiber size in treated muscles was achieved by up-regulation of the PI3 kinase regulatory subunit p85 , which inhibits PI3K signaling, and up-regulation of IGFBP-6, which prevents IGF-II mediated muscle hypertrophy, counteracting any hypertrophy caused by the slightly increased calcineurin activity in treated mdx muscles.
Prednisolone treatment significantly altered the fiber type composition of mdx soleus muscles, reversing the change toward a slow muscle phenotype previously observed in mdx. Mdx muscles undergo regeneration; inhibition of the RAS pathway has been shown to inhibit type 1 myosin gene transcription in regenerating muscle. C57Bl/10 muscles, which do not undergo degeneration and regeneration, did not undergo any fiber type change in response to prednisone. This suggests that the fiber type change in mdx muscle may occur via inhibition of the RAS pathway; the high levels of calcineurin in C57Bl/10 muscles may have maintained expression of the slow MHC 1 gene. Figure 3
shows our proposed model of the effect of prednisolone on mdx skeletal muscle.
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Prednisolone acts on dystrophin-deficient skeletal muscle in a complex manner, with effects on pathways involved in hypertrophy and fiber type. Its effect on gene expression differs depending on the time in the pathological process that it is administered. Our data show that prednisolone does not increase muscle degeneration, decrease muscle necrosis, or up-regulate utrophin in mdx muscles. However, prednisolone treatment does lead to a phenotypic improvement of mdx muscle.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2511fje;
This article has been cited by other articles:
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  K. A. Kleopa, A. Drousiotou, E. Mavrikiou, A. Ormiston, and T. Kyriakides Naturally occurring utrophin correlates with disease severity in Duchenne muscular dystrophy Hum. Mol. Genet., May 15, 2006; 15(10): 1623 - 1628. [Abstract] [Full Text] [PDF]
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 M. Yoshida, A. Yonetani, T. Shirasaki, and K. Wada Dietary NaCl supplementation prevents muscle necrosis in a mouse model of Duchenne muscular dystrophy Am J Physiol Regulatory Integrative Comp Physiol, February 1, 2006; 290(2): R449 - R455. [Abstract] [Full Text] [PDF]
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