Chronic stimulation of mammalian muscle: changes in metabolite concentrations in individual fibers.

Single fibers were analyzed from rabbit fast-twitch tibialis anterior muscles freeze-clamped during continuous stimulation at 10 Hz for up to 8 wk. ATP declined after 1 wk to a stable level approximately 30% below controls. Phosphocreatine decreased earlier and to a greater extent (approximately 50%). Glycogen varied considerably among stimulated fibers and decreased on average approximately 75% by 8 wk. Glucose, lactate, citrate, and malate had changed little in the first 30 h and then increased four-, two-, four-, and sevenfold, respectively, over the next 5 wk. Glucose 6-phosphate showed the most unexpected behavior: with an overall upward trend, it descended to extremely low values (10% of control) after approximately 1 wk of stimulation. As long as high- and low-oxidative fibers were present, the former showed slightly higher levels of ATP, lactate, and malate; other metabolites did not differ in a consistent way. These unexpected observations, which differ strikingly from data for acute stimulation, shed light on adaptations that enable a chronically stimulated muscle to sustain a continuous high level of ATP utilization.

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.

Progressive metabolite changes in individual human muscle fibers with increasing work rates.

Muscle biopsies were obtained from vastus lateralis muscles of four volunteers exercising at increasing work rates on a bicycle ergometer. Samples were taken at rest (t1), after a work load 23% below the blood lactate threshold (t2), 23% above this threshold (t3), and at exhaustion (t4). Individual muscle fibers were typed by their lactate dehydrogenase and adenylokinase levels and assayed for lactate, glucose-6-phosphate, and malate, (which preliminary data indicated to be the most responsive to increased activity) as well as ATP and phosphocreatine. The results in three of the four cases indicated that by the time of the t2 sample, almost all fibers, regardless of type, had been recruited. Additionally, there were no major differences in lactate concentration between type 1 and 2 fibers from muscle samples taken at t1, t2, and t3. It is concluded that in a muscle with fast-twitch glycolytic and slow-twitch oxidative fibers, all fibers share in the contraction to a substantial degree, even at moderate work loads, and that both the type 1 and 2 fibers contribute significantly to the initial rise in blood lactate during a graded exercise task. Metabolite responses in type 2 fibers differed in certain respects among the four participants. This is attributed to differences in their training backgrounds and consequent differences in type 2 fiber oxidative enzyme levels.

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.

Chronic stimulation of mammalian muscle: enzyme changes in individual fibers.

Single fibers of rabbit fast-twitch tibialis anterior (TA) muscles were analyzed after continuous low-frequency stimulation for up to 8 wk. After 2-5 wk, every fiber showed higher levels of citrate synthase, hexokinase, and 3-oxoacid CoA-transferase than any control fiber; in some cases these levels were 2-10 times higher (well above any found even in the control soleus, a slow-twitch muscle). Average levels of malate dehydrogenase and alanine transaminase also rose dramatically, but peak single fiber levels were not much above the highest in controls. These differential effects confirm at the single fiber level that chronic stimulation can alter mitochondrial composition. Lactate dehydrogenase, fructose-bisphosphatase, and adenylate kinase declined to levels far below those of any control TA fiber, and, in the case of fructose-bisphosphatase, to within the activity range of control soleus fibers. According to their staining reaction for myofibrillar ATPase, TA fibers were initially 23% type IIA, and 74% type IIB, but by 5 wk these had been converted to a mixture of type I, IIA, and IIC fibers. At 5 wk, levels of lactate dehydrogenase, adenylate kinase, and malate dehydrogenase were characteristic of their (new) ATPase type, but 3-oxoacid CoA transferase had increased to levels 6-15 times higher than in control fibers of the same type.

A method for branched-chain amino acid aminotransferase activity in microgram and nanogram tissue samples.

A method for branched-chain amino acid aminotransferase is described which is based on running the reaction in the reverse of the usual direction with glutamate and alpha-ketoisocaproate as substrates. The alpha-ketoglutarate generated is reduced with glutamate dehydrogenase and NADH. For sensitivity in the nanomole range, the NAD+ generated is measured directly by converting to the highly fluorescent strong alkali product. For smaller samples, down to the 0.2- to 2-pmol range, the NAD+ is amplified by enzymatic cycling.

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.

Heterogeneity in regard to enzymes and metabolites within individual muscle fibers.

The possibility of variability along individual rat and human muscle fibers was assessed for four enzymes and four substances responsive to stimulation. Heterogeneity was determined by the differences between ends of fiber segments several millimeters long and by fluctuations in successive samples along the entire length of such segments. Among the enzymes, a cytosolic enzyme, lactate dehydrogenase, varied the least: average coefficient of variation (CV) of 5%. This is only a little greater than the analytic error. On the other hand, the CV for a mitochondrial enzyme, fumarase, was 13%. A mixed cytosolic-mitochondrial enzyme, malate dehydrogenase, was intermediate (CV of 9%). The CV for glycogen phosphorylase, which is normally bound to glycogen particles, was 34% along one fiber segment. Among the nonenzyme components, average CVs in stimulated fibers were 34, 20, 7, and 7%, respectively, for glucose 6-phosphate, phosphocreatine, ATP, and malate. Major differences were not random but developed gradually over distances of 0.5-2 mm along the fibers, and in some cases significant correlations between enzyme and metabolite levels were demonstrable.

Effects of detraining on enzymes of energy metabolism in individual human muscle fibers.

Muscle biopsies were obtained from three cyclists and four runners at the end of 10-24 mo of intensive training and after intervals of detraining up to 12 wk. Control samples came from four untrained persons and four former athletes. Macro mixed fiber samples were assayed for lactate dehydrogenase, adenylate kinase, glycogen phosphorylase, citrate synthase, malate dehydrogenase, beta-hydroxyacyl-CoA dehydrogenase, succinate dehydrogenase, beta-hydroxybutyrate dehydrogenase, creatine kinase, hexokinase, 1-phosphofructokinase, fructosebisphosphatase, protein, and total creatine. In the case of three trained persons and two controls, the first six of the enzymes were also measured in individual fibers. Before detraining, enzymes of oxidative metabolism were substantially higher than in controls, and differences in levels between type I and type II fibers were smaller. During detraining, oxidative enzymes were decreased in both fiber types but the type II fibers did not fall to control levels even after 12 wk. Phosphorylase increased with detraining in both fiber types. The same is true for lactate dehydrogenase and adenylate kinase, except in the case of the type I fibers of one individual. Among the other six enzymes (measured in mixed fiber samples), only hexokinase was consistently affected (decreased) by detraining.

Correcting a potential defect in an enzymatic cycle for NADP.

An enzymatic cycle for NADP which uses as one of its enzymes glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides has occasionally caused trouble due to failure to completely heat-kill this enzyme before the indicator step. It was found that a very small increase in pH was the cause of this. It was also found that the other two proteins present in the reagent greatly increase heat inactivation of the enzyme. The inactivation problem is completely overcome by keeping the pH below 7.2.

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.

Malar mass due to Actinomyces odontolyticus.

Actinomyces odontolyticus was isolated from a patient with a soft tissue mass in the malar region. The organism was identified on the basis of morphological, cultural, and biochemical characteristics. On histological examination, the tissue mass contained several granulomatous foci with small, basophilic staining areas resembling microscopic sulfur granules. This is believed to be the first reported case of actinomycosis due to A. odontolyticus.