Neurotrophic factors decrease the release of creatine kinase and prostaglandin E2 from metabolically stressed muscle.

We have used an in vitro model of oxidative stress by exposing rat muscle to 2:4 dinitrophenol. This causes an efflux of creatine kinase (CK) and prostaglandin E2 (PGE2) commonly used as indicators of muscle cell damage. We then investigated compounds with a putative cell protective effect in the system. Ciliary neurotrophic factor, brain derived neurotrophic factor and insulin like growth factor 1 all prevent the release of PGE2 and CK. To the extent that these indicators may reflect cell damage, the results might support the investigation of the therapeutic potential of these compounds in muscle disease.

Evidence of a temperature-sensitive step in the release of prostaglandin E2 in calcium ionophore-stimulated rat muscle.

Recent studies have shown that mild hypothermia (32-35 degrees C) confers striking protection against ischemic muscle and neuronal injuries, although the mechanisms are unknown. We previously demonstrated that the release of prostaglandin E2 (PGE2) from metabolically stressed muscles was dependent on calcium and was abolished at or below 35 degrees C. In this study, we examined the temperature response of the release of arachidonic acid (AA) and its cyclooxygenase metabolites, PGE2 and prostaglandin F2 alpha (PGF2 alpha) from rat skeletal muscle in the presence of calcium ionophore A23187, an agent that directly elevates intracellular calcium. Calcium ionophore markedly stimulated the release of AA, PGE2 and PGF2 alpha at 37 degrees C, as expected. Reducing the temperature to 35 degrees C and below sharply decreased PGE2 and PGF2 alpha release but not AA release. The activity of phospholipase A2 stimulated by calcium ionophore was unaffected when temperature of incubation was lowered from 37 to 32 degrees C. The results suggest that reducing temperature from 37 degrees C to 35 degrees C or below inhibits the conversion from free arachidonate to PGs in calcium ionophore-stimulated muscle.

Evidence of two mechanisms of prostaglandin release in an in vitro model of muscle damage. Possible therapeutic implications.

In spite of recent progress, treatment of muscle disease based on specific gene therapy is not yet available. An alternative approach is to develop treatment which affords non-specific protection against general factors involved in cell damage. This approach is used effectively to prevent neuronal damage in experimental brain ischemia in animals and has been proposed for human trials. The most effective intervention is the use of mild (35 degrees C) hypothermia. An in vitro model to study muscle cell damage employs the rat epitrochlearis muscle exposed to low concentrations of 2:4-dinitrophenol, an uncoupler of oxidative phosphorylation. The efflux of prostaglandin E2 (PGE2) from the muscle is used as an indicator of muscle damage. We now show that there are two types of PGE2 release. "Basal" efflux gradually declines with decreasing temperatures and is not affected by removal of calcium from the medium. The efflux of PGE2 in response to metabolic stress is dependent on the presence of calcium and is abolished by mild hypothermia of 35 degrees C. The latter effect suggests that cell death is muscle and neurons have features in common and that muscle may be a useful tissue in which to investigate this phenomenon further.

Effects of calcium channel antagonists on the release of prostaglandin E2 from metabolically stressed muscle.

Calcium influx plays a critical role in the activation of the arachidonic cascade in muscle damage. We examined the effects of L-type calcium channel antagonists on the release of prostaglandin E2 (PGE2), a bioactive metabolite of arachidonic acid metabolism, from skeletal muscle. The basal release of PGE2 was not affected by calcium channel inhibitors, such as nifedipine and verapamil. The release of PGE2 induced by dinitrophenol, an uncoupler of oxidative phosphorylation, was abolished by nifedipine and verapamil at 50 and 150 microM, respectively. It was not necessary to include the calcium channel blockers in the medium before or at the time of dinitrophenol stimulation to produce the effect on PGE2 release. The release of PGE2 was prevented for as long as calcium channel blockers were present in the medium after the dinitrophenol stress.

Mild hypothermia preserves contractile function and inhibits prostaglandin E2 release from metabolically stressed skeletal muscle.

An in vitro model of muscle damage was used to investigate the protective effect of mild hypothermia in muscle injury. Rat epitrochlearis muscles were dissected in their entirety and suspended in Krebs-Ringer solution and DNP, a mitochondrial uncoupler, was added. PGE2 and lactate release and the contractile response to stimulation were measured and compared to untreated controls. Experiments were done at 37, 35, 33 and 27 degrees C. At 37 degrees C, DNP stimulated muscle releases large amounts of PGE2 and lactate and is unable to contract. As the temperature is reduced, there is progressive preservation of contractile force, although high lactate levels at the lowest temperatures indicate that the metabolic stress is still present. In contrast, DNP stimulated PGE2 release is completely inhibited at or below 35 degrees C and may be related to a similar protective phenomenon seen in experimental ischemic neuronal death.

Relationship of oxypurine release to contractile failure in dinitrophenol-treated rat skeletal muscle.

The efflux of hypoxanthine and uric acid from skeletal muscle has been noted to follow exercise and metabolic stress both in vivo and in vitro. Since the action of xanthine oxidase and hypoxanthine generates free radicals with potential damaging effect on the muscle membranes, an in vitro model was used to study the relationship of metabolic stress, oxypurine release and muscle contraction. When rat epitrochlearis muscle was exposed to the mitochondrial uncoupler dinitrophenol at 37 degrees C, lactate release was pronounced and hypoxanthine and uric acid appeared in the incubating medium. The twitch tension, in response to supramaximal stimulation, was reduced to less than 5% of the initial value. When the same experiment was repeated at 27 degrees C, hypoxanthine and uric acid formation was inhibited, although lactate release indicated that metabolic stress was still present. Twitch tension was relatively preserved (57% of the initial value). The lower temperature did not alter the decrease in ATP and phosphocreatine levels in the muscle which is produced by dinitrophenol. There was an inverse relationship between oxypurine release and twitch tension in individual muscles (r = 0.80, P < 0.01 for hypoxanthine and r = 0.95, P < 0.0002 for uric acid). Xanthine dehydrogenase/xanthine oxidase was detected in muscle and between 16 and 22% of the activity was in the oxidase form.

Duchenne dystrophy: randomized, controlled trial of prednisone (18 months) and azathioprine (12 months)

Prednisone has been shown to improve strength in Duchenne dystrophy. Azathioprine often benefits corticosteroid-responsive diseases and can reduce the dose of prednisone needed. The present study reports a randomized, controlled trial of prednisone and azathioprine designed to assess the longer-term effects of prednisone and to determine whether azathioprine alone, or in combination with prednisone, improves strength. Ninety-nine boys (aged five to 15 years) with Duchenne dystrophy were randomized to one of three groups: (I) placebo; (II) prednisone 0.3 mg/kg/d; or (III) prednisone 0.75 mg/kg/d. After 6 months, azathioprine 2 to 2.5 mg/kg/d was added in groups I and II and placebo added in group III. The study showed that the beneficial effect of prednisone (0.75 mg/kg/d) is maintained for at least 18 months and is associated with a 36% increase in muscle mass. There was weight gain, growth retardation, and other side effects. Azathioprine did not have a beneficial effect. This study suggests that prednisone's beneficial effect is not due to immunosuppression.

Mononuclear cell analysis of muscle biopsies in prednisone- and azathioprine-treated Duchenne muscular dystrophy.

Prednisone improves strength and function in patients with Duchenne dystrophy. Although the mechanism of this effect is uncertain, prior studies suggested that the benefit might result from immunosuppressive effects on T lymphocytes invading muscle. A recent randomized, double-blind, controlled trial of prednisone and azathioprine demonstrated that azathioprine had no effect in Duchenne dystrophy, raising questions about the role of immunosuppression in mediating clinical improvement. The goal of this current study was to compare the effects of prednisone and azathioprine on mononuclear infiltrates from biopsies performed at the end of the controlled clinical trial (reported separately in the article by Griggs et al on page 520). We studied 14 patients from the prednisone group (0.75 mg/kg/d), 10 from the combination therapy group (prednisone 0.3 mg/kg/d and azathioprine 2.5 mg/kg/d), and 13 from the azathioprine group (2.5 mg/kg/d), and used monoclonal antibodies for cell typing. There were no significant differences between the groups for total T cells, T-cell subsets, B cells, natural killer cells, total mononuclear cells, necrotic muscle fibers, or fibers focally invaded by mononuclear cells. These data indicate that azathioprine decreases mononuclear subsets infiltrating muscle to a similar degree as does prednisone, although azathioprine-treated patients do not show a clinical improvement. This implies that immunosuppressive actions on cellular infiltrates in muscle are probably not the primary mechanism of prednisone-induced clinical improvement.

Long-term benefit from prednisone therapy in Duchenne muscular dystrophy.

Two successive, 6-month, randomized, double-blind, controlled trials of prednisone showed that 0.75 mg/kg/d was the optimal dose to improve strength in boys with Duchenne muscular dystrophy (DMD). We attempted to maintain 93 boys on that dose for an additional 2 years. During the 3 years of observation, the decline in average muscle strength scores of all boys taking prednisone was 0.072 units/yr, as compared with an expected decline of 0.341 units/yr from natural history controls. The occurrence of side effects in some boys prevented maintenance of the full dose, which may have lessened the response. At the time of last visit, dosages ranged from 0.15 mg/kg to 0.75 mg/kg. In addition to maintaining their strength, several of the boys actually improved their performance in lifting kilogram weights and in some timed function tests. Treatment of DMD with prednisone significantly slows the progression of weakness and loss of function for at least 3 years.

Glutathione depletion during experimental damage to rat skeletal muscle and its relevance to Duchenne muscular dystrophy.

1. The release of glutathione has been studied in comparison with the release of creatine kinase from isolated rat soleus muscles subjected to certain forms of experimental damage. 2. Excessive electrically stimulated contractile activity or treatment of muscles with the mitochondrial inhibitor, 2,4-dinitrophenol, induced a substantial release of both creatine kinase and glutathione and a reduction in the total glutathione content of the muscle. The time course of this release and depletion indicates that the efflux of the two molecules is not directly related and that a reduction in muscle glutathione content does not occur before cytosolic enzyme release. 3. 2,4-Dinitrophenol-stimulated release of creatine kinase was significantly reduced by the omission of external calcium from the incubation media, but glutathione release and depletion was relatively unaffected by this. Deliberate elevation of the muscle intracellular calcium content with the calcium ionophore, A23187, induced a substantial loss of creatine kinase, but had no significant effect on the release of glutathione. 4. Muscle biopsies from patients with Duchenne muscular dystrophy were found to have an elevated content of glutathione and an equivalent protein-thiol content compared with control subjects. 5. We conclude that, although release of glutathione from skeletal muscle occurs after excessive contractile activity or inhibition of mitochondrial metabolism, this is not a key step in the damaging processes leading to cytosolic enzyme release, neither is it relevant to the ongoing damage to skeletal muscle which occurs in patients with Duchenne muscular dystrophy.

A comparison of daily and alternate-day prednisone therapy in the treatment of Duchenne muscular dystrophy.

We previously reported the results of a randomized, double-blind 6-month trial of prednisone therapy in which 102 boys aged 5 to 15 years with Duchenne muscular dystrophy received daily doses of 1.5 and 0.75 mg/kg per day and were compared with those receiving placebo. The strength and function in both prednisone-treated groups improved equally and were significantly better than in the placebo group. To compare alternate-day and daily dosing of prednisone with respect to benefits and adverse side effects, the placebo group was started on alternate-day prednisone therapy, and the treatment group regimens were changed to equivalent doses of alternate-day prednisone without breaking the double-blind nature. At the end of 6 months, the group that was changed from daily to alternate-day therapy had declined in strength back to levels observed 12 months previously, at the start of daily therapy. The group in which alternate-day therapy was started showed a significant improvement in strength at 3 months, similar in magnitude to the response of boys treated with daily therapy. However, their strength declined significantly in the subsequent 3 months compared with boys who received daily therapy. The frequency of side effects was not significantly different for alternate-day therapy compared with daily therapy. We conclude that alternate-day prednisone therapy effectively increases strength but does not sustain the improvement to the same extent as daily therapy or mitigate side effects.

Prednisone in Duchenne dystrophy. A randomized, controlled trial defining the time course and dose response. Clinical Investigation of Duchenne Dystrophy Group.

A randomized, controlled trial of daily prednisone was conducted in 99 boys (aged 5 to 15 years) with Duchenne dystrophy to define the time course of improvement and the dose response to treatment. Prednisone at 0.3 mg/kg (n = 33), prednisone at 0.75 mg/kg (n = 34), and placebo (n = 32) were administered for 6 months. Patients were examined using manual muscle and myometry testing, timed functional testing, pulmonary function testing, and laboratory measurements at 10 days, 1 month, 2 months, 3 months, and 6 months of treatment. Boys treated with prednisone had stronger average muscle strength scores, than did boys treated with placebo as early as 10 days after starting therapy. At the 3-month visit, the boys in the group given 0.75 mg/kg of prednisone were significantly stronger than those in the group given 0.3 mg/kg of prednisone, indicating a dose response. At 6 months, significant side effects occurred in the group treated with 0.75 mg/kg of prednisone, including weight gain, cushingoid appearance, and excessive hari growth. Only weight gain was observed in the group taking prednisone at a dose of 0.3 mg/kg. Importantly, no side effects were evident at 10 days or 1 month of treatment, despite improvement in muscle strength and function. We conclude that prednisone produces a rapid increase in muscle strength in patients with Duchenne dystrophy and that this improvement is maximal at a prednisone dosage of 0.75 mg/kg or less.

Creatine kinase and prostaglandin E2 release from isolated Duchenne muscle.

We studied the release of creatine kinase (CK) activity and prostaglandin E2 (PGE2) from isolated strips of biceps muscle from patients with Duchenne muscular dystrophy and nondystrophic control patients. CK release was significantly higher from the dystrophic samples than controls during the initial period of incubation, but this difference reduced with time of incubation. Immediate immersal of muscle strips into calcium-free bathing medium reduced the initial difference between the efflux from dystrophic and nondystrophic samples, whereas treatment with the calcium ionophore maintained the difference between the groups throughout the period of incubation (150 minutes). These results support the hypothesis that the lack of dystrophin in Duchenne muscle leads to damage to the tissue via a failure of calcium homeostasis. PGE2 release from the muscle strips followed a similar pattern to CK activity, supporting the possibility that, at least partly, the calcium-mediated damage involves activation of phospholipid hydrolysis.

Randomized, double-blind trial of mazindol in Duchenne dystrophy.

There is evidence that growth hormone may be related to the progression of weakness in Duchenne dystrophy. We conducted a 12-month controlled trial of mazindol, a putative growth hormone secretion inhibitor, in 83 boys with Duchenne dystrophy. Muscle strength, contractures, functional ability and pulmonary function were tested at baseline, and 6 and 12 months after treatment with mazindol (3 mg/d) or placebo. The study was designed to have a power of greater than 0.90 to detect a slowing to 25% of the expected rate of progression of weakness at P less than 0.05. Mazindol did not benefit strength at any point in the study. Side effects attributable to mazindol included decreased appetite (36%), dry mouth (10%), behavioral change (22%), and gastrointestinal symptoms (18%); mazindol dosage was reduced in 43% of patients. The effect of mazindol on GH secretion was estimated indirectly by comparing the postabsorptive IGF-I levels obtained following 3, 6, 9, and 12 months in the mazindol treated to those in the placebo groups. Although mazindol-treated patients gained less weight and height than placebo-treated patients, no significant effect on IGF-I levels was observed. Mazindol doses not slow the progression of weakness in Duchenne dystrophy.