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Persistent and improved functional gain in mdx dystrophic mice after treatment with L-arginine and deflazacort

Department of Human Anatomy and Cell Science, Faculty of Medicine, University of Manitoba, Winnipeg, Canada

¹Correspondence: Faculty of Medicine, University of Manitoba, 730 William Ave., Winnipeg, MB R3E 0W3, Canada. E-mail: janders{at}ms.umanitoba.ca

SPECIFIC AIMS

Experiments were designed to test the hypothesis that combined treatment with deflazacort and L-arginine would mitigate dystrophic fiber injury induced by 24 h of voluntary exercise, increase myogenic cell proliferation, and change the progressive loss of function in mdx mouse dystrophy.

PRINCIPAL FINDINGS

Remarkably, in addition to promoting proliferation and preventing exercise-induced damage and regeneration in the short term, functional gain at the end of treatment not only persisted, but was increased 3 months after treatment.

We used a physiologic muscle injury, 24 h of voluntary exercise, to induce a repair response in mdx mouse dystrophic muscle after 17 days of a double-blind treatment protocol [placebo, deflazacort, deflazacort plus L-arginine (NOS substrate) or deflazacort plus L-NAME (NOS inhibitor)].

1. Acute effects on muscle injury
In a pilot study on placebo-treated mice (measured as acute changes in proportion of muscle fibers permeable to Evans Blue Dye, EBD), 24 h of voluntary exercise increased the proportion of EBD+ fibers in four muscles, as expected. This was especially noted in quadriceps, the muscle selected for experiments on treatment effects.

In the study of treatment effects, quadriceps muscle in mdx mice without exercise exhibited a significantly smaller proportion of EBD+ fibers after treatment with deflazacort plus L-arginine. After exercise, muscle from two groups (those treated with placebo and those treated with deflazacort plus L-arginine) displayed increased proportions of EBD+ fibers. In comparison, exercise did not increase the proportion of acute permeability in fibers after treatment with deflazacort alone.

2. Short-term effects on muscle regeneration
Muscle regeneration was measured in mdx mouse quadriceps muscle (as expression of the muscle-specific regulatory gene, myf5 relative to total RNA), and served as an index of the extent of the regenerative response induced by 1) the fiber damage from ongoing dystrophy; and additionally from 2) the period of exercise activity 4 days earlier. Daily treatment was maintained in each group from days 17–21 after exercise. Results of this experiment are presented in Fig. 1 . In groups without exercise, myf5 expression was reduced by deflazacort, especially in combination with L-arginine (roughly halved), consistent with a lower susceptibility to the contraction-induced fiber injury from normal activity (compared with muscle in placebo-treated mice) after these treatments. Four days after 24 h of voluntary exercise, myf5 expression increased in placebo-treated mdx mice as expected. The rise in myf5 expression induced by exercise-related damage was smaller in the group treated with deflazacort. Treatment with deflazacort plus L-arginine completely prevented the induction of muscle regeneration by fiber damage caused by voluntary exercise.

View larger version (14K): [in this window] [in a new window]   Figure 1. Short-term regeneration in quadriceps muscle. Histogram (mean ±SE) showing myf5 mRNA expression (relative to total RNA), an indicator of regeneration in quadriceps muscles from mdx mice treated for 21 days with placebo (Pl), deflazacort (D), or deflazacort with either L-arginine (D+LA) or L-NAME (D+LN), 4 days after exercise (black bars) or not (white bars). In mdx muscle without exercise, myf5 expression was reduced by deflazacort treatment. Exercise induced a significant increase in regeneration (following exercise damage) in muscle of mice treated with placebo or D+LN. Exercise-induced injury and regeneration were reduced by deflazacort treatment compared with that in the placebo group. D+LA mice showed no exercise-induced myf5 expression. *Difference from same treatment group without exercise; #difference from respective placebo group; &difference from placebo and deflazacort groups without exercise.

These findings demonstrate that deflazacort treatment, especially when combined with L-arginine, spared dystrophic muscle from a susceptibility to exercise-induced injury that normally in dystrophic muscle, would have had sufficient impact to induce a regenerative response. Recalling that fiber membrane permeability to EBD was increased after exercise in the deflazacort plus L-arginine group, the results suggest additional treatment effects. For example, the previously reported increased concentrations of utrophin, a structural homologue to dystrophin, and CAPON, an anchor protein for neuronal NOS (nNOS), may stabilize the dystrophin-deficient muscle sarcolemma enough to preclude severe membrane injury that would induce myogenic regeneration.

3. Long-term effects on function
The functional outcome of treatment (measured grossly as the total distance run during 24 h of voluntary exercise) was compared in the short-term (after 21 days treatment) and longer-term (3 months after the end of treatment), as presented in Fig. 2 . Mdx mice treated with placebo showed a reduction in distance run over time after treatment, consistent with the characteristic progression of mdx mouse muscular dystrophy.

View larger version (12K): [in this window] [in a new window]   Figure 2. Long-term persistence of treatment effects on function. A histogram (mean ±SE) showing the distance run over a 24 h period of voluntary exercise (normalized to body weight) in groups treated with placebo (Pl), deflazacort (D) and deflazacort plus L-arginine (D+LA), either immediately after treatment (short-term, white bars) or 3 months later (long-term, black bars). After placebo treatment, mice showed a decline in the distance run 3 months later, consistent with progression of mdx mouse muscular dystrophy. mdx mice in the long-term deflazacort-treated group maintained voluntary distance run at the level demonstrated by animals immediately after 3 wk treatment. Notably, mdx mice treated for 3 wk with D+LA showed an increase in the voluntary distance run, 3 months after the end of treatment, indicating a long term functional benefit of D+LA treatment. *Difference from long-term animals in the placebo group; &significant difference from the short-term placebo group; #significant difference from short-term D+LA group.

Deflazacort treatment improved function in the short term, and that level of function was maintained over the longer term, 3 months after treatment. For animals that received deflazacort plus L-arginine, the short-term benefits to function were the same as for deflazacort alone: distance run was higher at the end of treatment, compared with the placebo group. In the longer term, overall function (distance run over 24 h) was further increased, 3 months after treatment with deflazacort plus L-arginine. Therefore, whereas deflazacort treatment alone prevented a functional decline in mdx mice by this measure, the persistent functional gain produced from treatment with deflazacort alone was markedly improved in the longer term after combined treatment with L-arginine.

CONCLUSIONS AND SIGNIFICANCE

These studies demonstrated significant persistence and potentiation of a functional gain following a combined drug treatment for muscular dystrophy in mdx mice, a model of human Duchenne muscular dystrophy (DMD). Results demonstrated that 1) deflazacort, especially with L-arginine, reduced the extent of muscle necrosis that was induced by exercise in dystrophic muscle compared with placebo treatment, despite an increase in fiber membrane permeability observed immediately after exercise; 2)deflazacort alone prevented progressive loss of function; 3) combined L-arginine and deflazacort treatment spared dystrophic muscle from damage-induced regeneration caused by short-term voluntary exercise; and 4)combination of L-arginine and deflazacort treatments potentiated the long-term the functional gain of deflazacort alone. These results encourage the development of new pharmacologic interventions for muscular dystrophy.

Recent, exciting explorations of potential genetic and cell-based, myoblast transplantation therapies are aimed to restore dystrophin or utrophin in DMD or otherwise obviate the impact of a gene mutation, and provide a real cure for DMD (and other neuromuscular diseases). Such important aims are directed as medium- and long-term goals. In the interim, drug treatments are required to belay the lethal progression of muscular dystrophy in affected individuals at all stages of disease progression. While glucocorticoid treatment is generally accepted as a "gold standard" of care in many countries, there are significant caveats to steroid treatment, due to side effects and other nonspecific and immunological mechanisms of action. Therefore, findings that short-term treatment through a combination of deflazacort (a glucocorticoid) with L-arginine (a NOS substrate), provides significant functional improvement in the short-term are notable. Evidence that functional gains from treatment not only persist in the longer term, but also substantially increase overall function is important.

Dystrophin deficiency from the cytoskeleton of skeletal muscle fibers makes fibers susceptible to exercise-induced injury (Fig. 3 ). Major advances in the field of muscle cell and molecular biology have developed through studies of the mdx mouse model of DMD. The present findings are consistent with an elegant series of investigations by Tidball and colleagues on double mutant mdx mice overexpressing nNOS, in which muscle damage was reduced in both skeletal and cardiac muscle compared with control mdx muscle. Results from other experiments in this laboratory have shown NO regulates satellite cell activation and apparently also regulates satellite cell quiescence in normal muscle (Wozniak and Anderson, unpublished), where an intact dystrophin-glycoprotein complex (DGC) stabilizes nNOS in the cytoskeleton. Addition of L-arginine to the gains from a protective treatment with deflazacort may stabilize the up-regulated utrophin within the DGC; promote the increased expression of nNOS and CAPON as we previously reported; and help to restore the imbalance of quiescence and activation for satellite cells in mdx muscle (Fig. 3) . Molecular biochemistry studies of the DGC-nNOS interplay in a range of dystrophic, mini-dystrophin and nNOS mutants are needed to address the mechanisms that are the basis of the treatment gains recorded here. The potentiation of a functional gain in dystrophic muscle may arise from effects on the fiber DGC (alleviating deficiencies in cytoskeletal integrity). Alternatively, the effect may arise by partly normalizing the regulation of quiescence and activation in the highly activated satellite cells in dystrophic muscle. Whether or not such effects can be distinguished experimentally, both would augment the effectiveness of muscle regeneration.

View larger version (37K): [in this window] [in a new window]   Figure 3. The pathophysiology of muscular dystrophy and treatment effects. Key elements in the pathophysiology of dystrophin-deficient myopathy, exhibited in mdx mice and human Duchenne muscular dystrophy (DMD). A cascade of events develops from the primary loss of dystrophin, through interactions with the DGC (e.g., with nNOS down-regulation and satellite cell hyperactivation), and inflammation and fibrosis that occur in response to ongoing fiber damage. Events lead to progressive decline in function. Curved arrows indicate processes affected by treatment with deflazacort plus L-arginine that reduce or partly reverse the pathophysiology. One arrow, at ‘Utrophin and CAPON up-regulation," is indicated as positive (+), meaning the treatment increases (rather than reverses) this effect of the primary mutation.

These in vivo experiments integrated cell and molecular biology studies with a functional assay of mdx mouse muscular dystrophy. Since nitric oxide mediates the activation of skeletal muscle precursors (satellite cells) and certain aspects of early muscle regeneration, these studies explored the potential for an NO-based therapy for muscular dystrophy. Results suggested a strong, new option for treatment, one which provided a persistent functional gain and long-term improvement in mdx mouse muscular dystrophy. These findings suggest a potential to improve the quality of life of boys with DMD using pharmacologic therapies, pending ongoing studies of cell and gene therapies which may take 5–10 years to reach fruition.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4821fje;

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