Myogenin Regulates Exercise Capacity but Is Dispensable for Skeletal Muscle Regeneration in Adult mdx Mice

Duchenne muscular dystrophy (DMD) is the most prevalent inherited childhood muscle disorder in humans. mdx mice exhibit a similar pathophysiology to the human disorder allowing for an in-depth investigation of DMD. Myogenin, a myogenic regulatory factor, is best known for its role in embryonic myogenesis, but its role in adult muscle maintenance and regeneration is still poorly understood. Here, we generated an mdx:Myogflox/flox mouse harboring a tamoxifen-inducible Cre recombinase transgene, which was used to conditionally delete Myog during adult life. After tamoxifen treatment, three groups of mice were created to study the effects of Myog deletion: mdx:Myogflox/flox mice (mdx), Myogflox/flox mice (wild-type), and mdx:MyogfloxΔ/floxΔ:Cre-ER mice (mdx:Myog-deleted). mdx:Myog-deleted mice exhibited no adverse phenotype and behaved normally. When run to exhaustion, mdx:Myog-deleted mice demonstrated an enhanced capacity for exercise compared to mdx mice, running nearly as far as wild-type mice. Moreover, these mice showed the same signature characteristics of muscle regeneration as mdx mice. Unexpectedly, we found that myogenin was dispensable for muscle regeneration. Factors associated with muscle fatigue, metabolism, and proteolysis were significantly altered in mdx:Myog-deleted mice, and this might contribute to their increased exercise capacity. Our results reveal novel functions for myogenin in adult muscle and suggest that reducing Myog expression in other muscle disease models may partially restore muscle function

Quantitative evaluation of the beneficial effects in the mdx mouse of epigallocatechin gallate, an antioxidant polyphenol from green tea

In two separate previous studies, we reported that subcutaneous (sc) or oral administration of (−)-epigallocatechin-3-gallate (EGCG) limited the development of muscle degeneration of mdx mice, a mild phenotype model for Duchenne muscular dystrophy (DMD). However, it was not possible to conclude which was the more efficient route of EGCG administration because different strains of mdx mice, periods of treatment and methods of assessment were used. In this study, we investigated which administration routes and dosages of EGCG are the most effective for limiting the onset of dystrophic lesions in the same strain of mdx mice and applying the same methods of assessment. Three-week-old mdx mice were injected sc for 5 weeks with either saline or a daily average of 3 or 6 mg/kg EGCG. For comparison, age-matched mdx mice were fed for 5 weeks with either a diet containing 0.1% EGCG or a control diet. The effects of EGCG were assessed quantitatively by determining the activities of serum muscle-derived creatine kinase, isometric contractions of triceps surae muscles, integrated spontaneous locomotor activities, and oxidative stress and fibrosis in selected muscles. Oral administration of 180 mg/kg/day EGCG in the diet was found the most effective for significantly improving several parameters associated with muscular dystrophy. However, the improvements were slightly less than those observed previously for sc injection started immediately after birth. The efficacy of EGCG for limiting the development of dystrophic muscle lesions in mice suggests that EGCG may be of benefit for DMD patients

Muscular dystrophy in the mdx mouse is a severe myopathy compounded by hypotrophy, hypertrophy and hyperplasia

Background Preclinical testing of potential therapies for Duchenne muscular dystrophy (DMD) is conducted predominantly of the mdx mouse. But lack of a detailed quantitative description of the pathology of this animal limits our ability to evaluate the effectiveness of putative therapies or their relevance to DMD. Methods Accordingly, we have measured the main cellular components of muscle growth and regeneration over the period of postnatal growth and early pathology in mdx and wild-type (WT) mice; phalloidin binding is used as a measure of fibre size, myonuclear counts and BrdU labelling as records of myogenic activity. Results We confirm a two-phase postnatal growth pattern in WT muscle: first, increase in myonuclear number over weeks 1 to 3, then expansion of myonuclear domain. Mdx muscle growth lags behind that of WT prior to overt signs of pathology. Fibres are smaller, with fewer myonuclei and smaller myonuclear domains. Moreover, satellite cells are more readily detached from mdx than WT muscle fibres. At 3 weeks, mdx muscles enter a phase of florid myonecrosis, accompanied by concurrent regeneration of an intensity that results in complete replacement of pre-existing muscle over the succeeding 3 to 4 weeks. Both WT and mdx muscles attain maximum size by 12 to 14 weeks, mdx muscle fibres being up to 50% larger than those of WT as they become increasingly branched. Mdx muscle fibres also become hypernucleated, containing twice as many myonuclei per sarcoplasmic volume, as those of WT, the excess corresponding to the number of centrally placed myonuclei. Conclusions The best-known consequence of lack of dystrophin that is common to DMD and the mdx mouse is the conspicuous necrosis and regeneration of muscle fibres. We present protocols for measuring this in terms both of loss of muscle nuclei previously labelled with BrdU and of the intensity of myonuclear labelling with BrdU administered during the regeneration period. Both measurements can be used to assess the efficacy of putative antinecrotic agents. We also show that lack of dystrophin is associated with a number of previously unsuspected abnormalities of muscle fibre structure and function that do not appear to be directly associated with myonecrosis

Elimination of polyneuronal innervation in a fast muscle of normal and dystrophic mice.

The changes in the pattern of innervation of extensor digitorum longus (e.d.l.) during post-natal development was studied in normal and dystrophic mice. As in other mammals, individual muscle fibres of new-born mice are supplied by more than one axon. Up to 10 days after birth there was no difference in the extent of this polyneuronal innervation between normal and dystrophic muscle fibres. During post-natal development the polyneuronal innervation gradually disappeared. In normal e.d.l. muscles the rate of the elimination of polyneuronal innervation was faster during the first 10 post-natal days and then slowed down. By 16 days the final value of less than 10% of muscle fibres receiving more than one input was reached. In the dystrophic muscles the rate of elimination was similar to normal up to 10 days of age, but continued to decrease rapidly so that already by 11 days of age polyneuronal innervation was reduced to its final level of less than 10%. Thus the elimination of polyneuronal innervation was completed at least 3 days earlier in the dystrophic animals. It is suggested that the increased nerve activity said to be present in dystrophic mice could account for this finding

Trial of roxithromycin in subjects with asthma and serological evidence of infection with Chlamydia pneumoniae

Copyright © 2001 American Thoracic SocietyChlamydia pneumoniae and the severity of asthma, and uncontrolled observations have suggested that treatment with antibiotics active against C. pneumoniae leads to an improvement in asthma control. We studied the effect of roxithromycin in subjects with asthma and immunoglobulin G (IgG) antibodies to C. pneumoniae > 1:64 and/or IgA antibodies > 1:16. A total of 232 subjects, from Australia, New Zealand, Italy, or Argentina, were randomized to 6 wk of treatment with roxithromycin 150 mg twice a day or placebo. At the end of 6 wk, the increase from baseline in evening peak expiratory flow (PEF) was 15 L/min with roxithromycin and 3 L/min with placebo (p 0.02). With morning PEF, the increase was 14 L/min with roxithromycin and 8 L/min with placebo (NS). In the Australasian population, the increase in morning PEF was 18 L/min and 4 L/min, respectively (p = 0.04). At 3 mo and 6 mo after the end of treatment, differences between the two groups were smaller and not significant. Six weeks of treatment with roxithromycin led to improvements in asthma control but the benefit was not sustained. Further studies are necessary to determine whether the lack of sustained benefit is due to failure to eradicate C. pneumoniae.Peter N. Black, Francesco Blasi, Christine R. Jenkins, Raffaele Scicchitano, Graham D. Mills, Abraham R. Rubinfeld, Richard E. Ruffin, Peter R. Mullins, Josette Dangain, Bruce C. Cooper, Denise Bem David, And Luigi Allegr

Selective development of myogenic mesenchymal cells from human embryonic and induced pluripotent stem cells.

Human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells are promising sources for the cell therapy of muscle diseases and can serve as powerful experimental tools for skeletal muscle research, provided an effective method to induce skeletal muscle cells is established. However, the current methods for myogenic differentiation from human ES cells are still inefficient for clinical use, while myogenic differentiation from human iPS cells remains to be accomplished. Here, we aimed to establish a practical differentiation method to induce skeletal myogenesis from both human ES and iPS cells. To accomplish this goal, we developed a novel stepwise culture method for the selective expansion of mesenchymal cells from cell aggregations called embryoid bodies. These mesenchymal cells, which were obtained by dissociation and re-cultivation of embryoid bodies, uniformly expressed CD56 and the mesenchymal markers CD73, CD105, CD166, and CD29, and finally differentiated into mature myotubes in vitro. Furthermore, these myogenic mesenchymal cells exhibited stable long-term engraftment in injured muscles of immunodeficient mice in vivo and were reactivated upon subsequent muscle damage, increasing in number to reconstruct damaged muscles. Our simple differentiation system facilitates further utilization of ES and iPS cells in both developmental and pathological muscle research and in serving as a practical donor source for cell therapy of muscle diseases