Rostrocaudal variation of fiber type composition in rat intercostal muscles.

We used the histochemical stain for ATPase to compare the fiber-type composition of rat internal and external intercostal muscles from thoracic (T) segments 2-5, 8, and 11. At each level, type II fibers were more numerous than type I fibers, type II B fibers were more numerous than II A fibers, and type I fibers were more numerous in external than in internal intercostals. However, fiber type composition varied from segment to segment. For example, the proportion of type II A fibers increased in a rostrocaudal gradient in internal but not external intercostals, and type I fibers were more prevalent at rostral and caudal than at intermediate levels in both internal and external intercostals. These results provide a basis for interpreting previous physiological and molecular studies which have compared intercostal muscles from different segmental levels.

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.

Clinical investigation in Duchenne muscular dystrophy: penicillamine and vitamin E.

A double-blind controlled trial of penicillamine and vitamin E against placebo was conducted in 106 boys with Duchenne muscular dystrophy. Nine dropped out of the study after being randomized. Patients were evaluated using an established protocol that measures muscle strength, joint contractures, functional grade, and pulmonary function. The trial lasted for 18 months. The power of the study to detect a 75% slowing of the disease was 0.95 at a P less than 0.05. No therapeutic effect was observed in the patients taking penicillamine and vitamin E as compared with those taking placebo.

2,4-Dinitrophenol, muscle biopsy, and McArdle's disease.

Exercise is simulated in muscle biopsy preparations by using low concentrations of 2,4-dinitrophenol (DNP), which do not produce contracture or anatomic damage. The validity of this simulation is supported by (1) the biochemical effects of simultaneous muscle contraction and DNP are not additive, suggesting that exercise and DNP stress the same pathways; (2) the effects of increasing concentrations of DNP and increasing levels of stimulation are similar with an early drop in phosphocreatine, increasing lactate and inosine monophosphate (IMP), and a late fall in ATP levels; and (3) DNP provocation in a patient with McArdle's disease demonstrated an absence of lactate and high levels of IMP correlating with clinical findings. DNP provocation may be a simple way of studying metabolic pathways in neuromuscular diseases.

Clinical investigation of Duchenne muscular dystrophy. Interesting results in a trial of prednisone.

We investigated the effect of high-dose prednisone therapy in 33 boys with Duchenne muscular dystrophy. The drug was given daily in doses of 1.5 mg/kg of body weight (to a maximum of 80 mg) for six months. Muscle strength, joint contractures, timed functional tests, functional ability, and pulmonary function were measured at the beginning and end of the treatment period. The trial was designed using natural history controls, and the power of the study was 0.80 to detect a slowing of 50% in the rate of progression. During the period of the trial, muscle strength, functional grades, timed functional tests, and pulmonary function improved. Contractures followed the expected natural history of the illness.

Effect of Duchenne muscular dystrophy on enzymes of energy metabolism in individual muscle fibers.

Individual muscle fibers from patients with Duchenne muscular dystrophy at an early stage in their disease, and from apparently normal boys of similar age, were analyzed for 13 enzymes of energy metabolism. This approach avoided the serious problems with muscle homogenate assays from increases in nonparenchymal components and permitted assessment of disease changes in different fiber types. Some enzymes of glycogenolysis (phosphorylase, phosphoglucomutase, and pyruvate kinase) were decreased in dystrophic fibers of all types. Phosphofructokinase was decreased in presumptive type II fibers. Lactate dehydrogenase was increased in type I fibers and essentially unchanged in type II. Phosphoglucoisomerase was near normal. Two enzymes of glucose metabolism not involved in glycogenolysis, hexokinase and glycogen synthase, were near normal, but a third, fructose bisphosphatase, was sharply reduced. Two enzymes of oxidative metabolism, citrate synthase, and beta-hydroxyacyl CoA dehydrogenase, were unchanged or increased. Two enzymes of high energy phosphate transfer, creatine kinase and adenylokinase, were only marginally affected. The net result is to leave the type II fibers, which normally exert the greatest force, with a severe deficit in the glycogenolytic enzyme machinery to maintain that force.

Clinical investigation of Duchenne muscular dystrophy. A methodology for therapeutic trials based on natural history controls.

Between 1979 and 1987 we documented the natural history of Duchenne muscular dystrophy in 170 patients, aged from 3 to 23 years, by making serial measurements in over 5000 individual evaluations. This database makes it possible to design and conduct therapeutic trials using natural history controls. Such trials do not replace the need for randomized placebo-controlled trials of promising agents but they do require fewer patients, are cost-effective, and permit the use of high-risk therapy where toxicity monitoring may be important. Natural history-controlled trials, therefore, may serve as a screening method for new therapeutic agents. Drugs showing a significant benefit can then be evaluated in a randomized controlled trial.

McArdle's disease with myoadenylate deaminase deficiency: observations in a combined enzyme deficiency.

Exercise and work potential of a patient with coexistent myophosphorylase and myoadenylate deaminase (AMPDA) deficiency was compared with that of three patients with myophosphorylase deficiency alone. The patient with the combined defect failed to produce an abnormal rise in serum ammonia or hypoxanthine as seen in the other patients after forearm exercise. Maximum oxygen consumption and work rates during cycle ergometer testing were similar in all patients, but well below controls. The occurrence of two defects involving short-term energy metabolism in muscle presents an opportunity to define further the metabolic role of AMPDA.

Acetazolamide-responsive myotonia congenita.

We have studied 14 patients from a kindred with an autosomal dominant form of myotonia, with features differing from most cases of autosomal dominant or recessive myotonia congenita. All patients had painful muscle stiffness that was provoked by fasting and oral potassium administration and was relieved by carbohydrate-containing foods. Muscle biopsies showed the presence of type 1, 2A, and 2B fibers, as opposed to the absence of type 2B fibers seen in some patients with myotonia congenita. Acetazolamide was dramatically effective in alleviating myotonia in all patients and was more effective than other antimyotonic agents.

Chronic stimulation of mammalian muscle: changes in enzymes of six metabolic pathways.

Twenty-one enzymes of different metabolic systems were measured in the rabbit fast-twitch tibialis anterior (TA) muscle after electrical stimulation (10 Hz, 24 h/day) for 1 day to 10 wk. Nine analytical methods are either new, (3-oxoacid CoA-transferase, branched-chain-amino-acid aminotransferase, carnitine acetyltransferase, thiolase), improved (glutamate dehydrogenase, glycogen synthase, adenylic acid deaminase), or specially adapted (hexokinase, phosphoglucomutase). The activities (based on protein) of 12 mitochondrial or partly mitochondrial enzymes were lower in control TA than in control (slow) soleus (30-84% of soleus level). After 2 wk, 11 of these had surpassed the control soleus level. Maximal increases (3- to 14-fold) occurred after 2-5 wk, and thereafter six of the enzymes declined, whereas the other five maintained or increased their levels. Five glycolytic and two high-energy phosphate transfer enzymes, originally much higher in control TA than in control soleus, decreased gradually to levels at 8-10 wk only 27-123% higher than in soleus. Noncollagen protein concentration dropped 46%, explained largely by a sixfold increase in extracellular (chloride) space and a modest increase in collagen. The data constitute strong evidence for coordinate regulation of (mainly cytosolic) enzymes of glycolysis, glycogenolysis, gluconeogenesis, and high-energy phosphate transfer. Changes in the (mainly mitochondrial) enzymes of oxidative metabolism were more divergent, partly because of a hitherto undescribed secondary phase in the metabolic response. This phase may reflect a lower energy consumption in muscles adapted to continuous activity.

Inosine monophosphate production is proportional to muscle force in vitro.

An in vitro system of muscle stimulation has been developed that combines physiologic measurements with biochemical evaluation. In the rat epitrochlearis muscle, 2 minutes of stimulation at 4 Hz results in a marked elevation of inosine monophosphate (IMP) levels. The amount of IMP generated is proportional to the amount of force developed. Depriving the muscle of oxygen results in an increased amount of IMP generation. This model provides direct confirmation of similar findings in the human forearm exercise test.

Controlled trial of thyrotropin releasing hormone in amyotrophic lateral sclerosis.

A double-blind controlled trial of thyrotropin releasing hormone (TRH) 150 mg IM daily in 30 patients with amyotrophic lateral sclerosis is reported. The drug/placebo was administered for 2 months, followed by a 2-month "wash-out". Evaluation of strength, functional ability, and respiratory functions was performed. A temporary increase in the strength of some muscles was detected following the administration of TRH, but no change in functional performance was noted. Neither the patients nor the investigators believed the effects were of any marked clinical significance. The course of the illness was not altered.

Comparison of muscle fiber typing by quantitative enzyme assays and by myosin ATPase staining.

Fibers in cross sections of human and rat muscle were typed by using histochemical ATPase stains, and the results were compared with those of quantitative enzyme assays of fragments of the same fibers dissected from serial freeze-dried sections. Two enzymes previously used to assess the metabolic type were measured in each case: lactate dehydrogenase and either adenylokinase (human fibers) or malate dehydrogenase (rat fibers). With human fibers there was essentially complete agreement between ATPase staining and the metabolic enzyme assays in distinguishing types I and II fibers. The agreement was less consistent with regard to type IIA and IIB fibers. A number of ATPase type IIC fibers were identified in one human muscle, and were found to fall between ATPase types I and IIA on the basis of metabolic enzyme assay results. Rat-fiber ATPase types I, IIA, and IIB from the plantaris muscle were rather well segregated on a two-dimensional lactate dehydrogenase-malate dehydrogenase grid. In the rat soleus muscle, ATPase types I and IIA fibers were shifted to lower lactate dehydrogenase levels, with IIC fibers interposed between them.

Exercising muscle does not produce hypoxanthine in adenylate deaminase deficiency.

The failure of forearm exercise to increase plasma hypoxanthine in subjects with adenylate deaminase deficiency confirms this enzyme's role in hypoxanthine production by normal forearm exercise. The conversion of adenosine monophosphate (AMP) to hypoxanthine may reflect an alternative method of adenosine triphosphate (ATP) regeneration in working muscle.

Hypoxanthine and Mcardle disease: a clue to metabolic stress in the working forearm.

Forearm exercise in a patient with myophosphorylase deficiency resulted in abnormally high levels of hypoxanthine in the venous blood. The post-exercise hypoxanthine response may reflect ATP depletion in the muscle and provides a useful screening test in muscle diseases with abnormal energy metabolism.

Forearm exercise increases plasma hypoxanthine.

Plasma hypoxanthine was measured in three normal subjects during aerobic forearm exercise. The comparative increase of hypoxanthine greatly exceeded that of ammonia or lactate. It is proposed that hypoxanthine production reflects ATP breakdown in muscle. The test may prove useful in the investigation of patients with metabolic muscle disease.

Metabolite changes in individual rat muscle fibers during stimulation.

Rat plantaris and soleus muscles were stimulated intensely in vivo for 1 and 15 min, freeze-clamped, and freeze-dried, and individual fibers were dissected free. Fibers, assigned to four groups on the basis of lactate dehydrogenase and malate dehydrogenase, were each separately analyzed for ATP, P-creatine, glycogen, glucose, glucose-6-phosphate (glucose-6-P), lactate, citrate, and malate. Some fibers were also analyzed for fructose 1,6-phosphate, total adenylate and total creatine. Although each group as a whole showed significant and often large differences in control composition and response to stimulation, individual fibers varied enough to create an almost continuous spectrum of metabolite levels from one extreme to the other. The data suggest that the slowest twitch fibers were the most active in the control state. Stimulation for 1 min caused a small increase in ATP in all groups with a large decrease in P-creatine in "fast white" fibers and a modest decrease in the rest. After 15-min stimulation, fast white fibers had lost 60% of initial ATP and 97% of initial P-creatine, whereas in other fiber types these compounds underwent little further change. Metabolite changes with stimulation were also greatest in fast white fibers. Glucose-6-P rose 15-fold in 1 min, then fell to below control by 15 min when glycogen had been exhausted; lactate rose two to six times more than in other types. Glucose rose in all groups to levels at 15 min, compatible with equilibrium with blood plasma.

Enzyme levels in individual rat muscle fibers.

Individual muscle fibers from the rat anterior tibialis and soleus muscles were each analyzed in duplicate for lactate dehydrogenase (LDH, EC 1.1.1.27), malate dehydrogenase (MDH, EC 1.1.1.37), 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35), fumarate hydrotase (EC 4.2.1.2), glycogen phosphorylase (EC 2.4.1.1), 6-phosphofructokinase (EC 2.7.1.11), pyruvate kinase (EC 2.7.1.40), fructose-bisphosphatase (EC 3.1.3.11), and creatine kinase (EC 2.7.3.2). A few fibers were also analyzed for adenylate kinase (EC 2.7.4.3). In general, there was a wide and almost continuous spectrum of coordinated enzyme activities. In the tibialis muscle, two fiber groups could be clearly distinguished on the basis of MDH activity. The high MDH group had on the average lower LDH activity, but there was a great deal of overlap in LDH between the two groups. Less overlap was observed for phosphorylase and fructose-bisphosphatase, both inversely related to MDH. Only one main group of fibers (presumably slow twitch) was found in the soleus muscle, although enzyme activities also covered a wide range. These soleus fibers were clearly distinguished from the high MDH tibialis group by much lower activities of LDH, pyruvate kinase, and fructose-bisphosphatase.