Fiber composition and oxidative capacity of hamster skeletal muscle.

The hamster is a valuable biological model for physiological investigation. Despite the obvious importance of the integration of cardiorespiratory and muscular system function, little information is available regarding hamster muscle fiber type and oxidative capacity, both of which are key determinants of muscle function. The purpose of this investigation was to measure immunohistochemically the relative composition and size of muscle fibers composed of types I, IIA, IIX, and IIB fibers in hamster skeletal muscle. The oxidative capacity of each muscle was also assessed by measuring citrate synthase activity. Twenty-eight hindlimb, respiratory, and facial muscles or muscle parts from adult (144-147 g bw) male Syrian golden hamsters (n=3) were dissected bilaterally, weighed, and frozen for immunohistochemical and biochemical analysis. Combining data from all 28 muscles analyzed, type I fibers made up 5% of the muscle mass, type IIA fibers 16%, type IIX fibers 39%, and type IIB fibers 40%. Mean fiber cross-sectional area across muscles was 1665 +/- 328 microm(2) for type I fibers, 1900 +/- 417 microm(2) for type IIA fibers, 3230 +/- 784 microm(2) for type IIX fibers, and 4171 +/- 864 microm(2) for type IIB fibers. Citrate synthase activity was most closely related to the population of type IIA fibers (r=0.68, p<0.0001) and was in the rank order of type IIA > I > IIX > IIB. These data demonstrate that hamster skeletal muscle is predominantly composed of type IIB and IIX fibers.     

Metabolic capacity of individual muscle fibers from different anatomic locations.

We studied muscle fibers by quantitative biochemistry to determine whether metabolic capacity varied among fibers of a given type as a function of their anatomic location. Muscles were selected from both contiguous and diverse anatomic regions within the rats studied. The individual fibers, classified into myosin ATPase fiber types by histochemical means, were assessed for fiber diameters and analyzed for the activities of enzymes representing major energy pathways: malate dehydrogenase (MDH, oxidative), lactate dehydrogenase (LDH, glycolytic), and adenylokinase (AK, high-energy phosphate metabolism). We found that neither the average activities of each of the three enzymes nor the fiber diameters varied in Type I or Type IIa fibers selected from superficial to deep portions of the triceps surae of the hindlimb. However, the IIb fibers in the deep region of this muscle group had significantly greater oxidative capacity, less glycolytic capacity, and smaller diameters than the superficially situated IIb fibers. Type IIa fibers in lateral gastrocnemius, extensor digitorum longus, psoas, diaphragm, biceps brachii, superficial masseter, and superior rectus muscles were highly variable in both diameter and enzyme profiles, with a correlation between MDH activity and fiber diameter. Therefore, our results show that both intermuscular and intramuscular metabolic variations exist in muscle fibers of a given type.     

Metabolic adaptations to training precede changes in muscle mitochondrial capacity.

To determine whether increases in muscle mitochondrial capacity are necessary for the characteristic lower exercise glycogen loss and lactate concentration observed during exercise in the trained state, we have employed a short-term training model involving 2 h of cycling per day at 67% maximal O2 uptake (VO2max) for 5-7 consecutive days. Before and after training, biopsies were extracted from the vastus lateralis of nine male subjects during a continuous exercise challenge consisting of 30 min of work at 67% VO2max followed by 30 min at 76% VO2max. Analysis of samples at 0, 15, 20, and 60 min indicated a pronounced reduction (P less than 0.05) in glycogen utilization after training. Reductions in glycogen utilization were accompanied by reductions (P less than 0.05) in muscle lactate concentration (mmol/kg dry wt) at 15 min [37.4 +/- 9.3 (SE) vs. 20.2 +/- 5.3], 30 min (30.5 +/- 6.9 vs. 17.6 +/- 3.8), and 60 min (26.5 +/- 5.8 vs. 17.8 +/- 3.5) of exercise. Maximal aerobic power, VO2max (l/min) was unaffected by the training (3.99 +/- 0.21 vs. 4.05 +/- 0.26). Measurements of maximal activities of enzymes representative of the citric acid cycle (succinic dehydrogenase and citrate synthase) were similar before and after the training. It is concluded that, in the voluntary exercising human, altered metabolic events are an early adaptive response to training and need not be accompanied by changes in muscle mitochondrial capacity.     

Structural basis of muscle O(2) diffusing capacity: evidence from muscle function in situ.

Although evidence for muscle O(2) diffusion limitation of maximal O(2) uptake has been found in the intact organism and isolated muscle, its relationship to diffusion distance has not been examined. Thus we studied six sets of three purpose-bred littermate dogs (aged 10-12 mo), with 1 dog per litter allocated to each of three groups: control (C), exercise trained for 8 wk (T), or left leg immobilized for 3 wk (I). The left gastrocnemius muscle from each animal was surgically isolated, pump-perfused, and electrically stimulated to peak O(2) uptake at three randomly applied levels of arterial oxygenation [normoxia, arterial PO(2) (Pa(O(2))) 77 +/- 2 (SE) Torr; moderate hypoxia, Pa(O(2)): 33 +/- 1 Torr; and severe hypoxia, Pa(O(2)): 22 +/- 1 Torr]. O(2) delivery (ml. min(-1). 100 g(-1)) was kept constant among groups for each level of oxygenation, with O(2) delivery decreasing with decreasing Pa(O(2)). O(2) extraction (%) was lower in I than T or C for each condition, but calculated muscle O(2) diffusing capacity (Dmus(O(2))) per 100 grams of muscle was not different among groups. After the experiment, the muscle was perfusion fixed in situ, and a sample from the midbelly was processed for microscopy. Immobilized muscle showed a 45% reduction of muscle fiber cross-sectional area (P < 0.05), and a resulting 59% increase in capillary density (P < 0.05) but minimal reduction in capillary-to-fiber ratio (not significant). In contrast, capillarity was not significantly different in T vs. C muscle. The results show that a dramatically increased capillary density (and reduced diffusion distance) after short-term immobilization does not improve Dmus(O(2)) in heavily working skeletal muscle.     

Increased antioxidant capacity does not attenuate muscle atrophy caused by unweighting.

Previous studies have increased antioxidant capacity in skeletal muscle to attenuate oxidative stress and muscle atrophy during limb immobilization (Appell HJ, Duarte JAR, and Soares JMC. Int J Sports Med 18: 157-160, 1997; Kondo H, Miura M, Nakagaki I, Sasaki S, and Itokawa Y. Am J Physiol Endocrinol Metab 262: E583-E590, 1992). The purpose of this study was to determine the level of oxidative stress in muscle during hindlimb unweighting (HLU) and whether antioxidant supplementation can attenuate the atrophy and changes in contractile properties resulting from 14 days of unweighting. Muscle unweighting caused a 44% decrease in soleus (Sol) and a 30% decrease in gastrocnemius (GS) mass, a 7% decrease in body weight, and 28% decrease in tetanic force in the GS. Protein carbonyls increased by 44% in the Sol with HLU. Antioxidant supplementation did not attenuate the GS or Sol atrophy or the decrease in GS force generation during HLU. Sol and GS protein concentration was not different between groups. The GS was also subjected to three different oxidative challenges to determine whether the supplement increased the antioxidant capacity of the muscle. In all cases, muscles exhibited an increased antioxidant capacity. These data indicate that antioxidant supplementation was not an effective countermeasure to the atrophy associated with HLU.     

Age is independently related to muscle metabolic capacity in premenopausal women.

The purpose of this study was to determine whether muscle metabolic capacity was inversely related to age after adjusting for physical activity in sedentary premenopausal women. Eighty-three women (ages 23-47 yr) had their free-living, activity-related energy expenditure evaluated with doubly labeled water procedures, and room calorimeter determined sleeping energy expenditure. Maximum O(2) uptake and strength were evaluated in all subjects, whereas 31P-magnetic resonance spectroscopy determined metabolic economy during maximal exercise, and muscle biopsy maximal enzyme activity was evaluated in subsets of the sample (48 and 18 subjects, respectively). Age was significantly related to whole body treadmill endurance time (r = -0.32), plantar flexion strength (r = -0.29), maximum O(2) uptake (r = -0.27), (31)P-magnetic resonance spectroscopy ADP recovery rate (r = -0.44), and anaerobic glycolytic capacity (r = -0.37), and muscle biopsy citrate synthase activity (r = -0.48), glyceraldehyde-3-phosphate dehydrogenase (r = -0.54), phosphofructokinase (r = -0.62), and phosphorylase (r = -0.58) activity even after adjusting for activity-related energy expenditure. These data suggest that, in sedentary premenopausal women, both oxidative and glycolytic muscle capacity decrease with age even when physical activity is taken into account.     

Skeletal muscle oxidative capacity in young and older women and men.

It has been suggested that a decline in skeletal muscle oxidative capacity is a general consequence of aging in humans. However, previous studies have not always controlled for the effects of varying levels of physical activity on muscle oxidative capacity. To test the hypothesis that, when matched for comparable habitual physical activity levels, there would be no age-related decline in the oxidative capacity of a locomotor muscle, the postexercise recovery time of phosphocreatine was compared in the tibialis anterior muscle of young [n = 19; 33.8 +/- 4.8 (SD) yr] and older [n = 18; 75.5 +/- 4.5 yr] healthy women and men of similar, relatively low, activity levels. The intramuscular metabolic measurements were accomplished by using phosphorus magnetic resonance spectroscopy. The results indicate that there was no age effect on the postexercise recovery time of phosphocreatine recovery, thus supporting the stated hypothesis. These data suggest that there is no requisite decline in skeletal muscle oxidative capacity with aging in humans, at least through the seventh decade.     

Whole body and muscle respiratory capacity with dobutamine and hindlimb suspension.

The effects of repeated injections of dobutamine, a synthetic catecholamine, were studied in control and tail-suspended rats to determine whether this drug could improve the metabolic response to unweighting. Dobutamine prevented the decrease in maximal oxygen uptake (VO2max) induced by hindlimb suspension. Furthermore, VO2max was 12% greater in dobutamine-treated animals than in saline-treated control animals. Soleus muscle weight and mean fiber cross-sectional area were decreased by 60 and 75%, respectively, in saline- and dobutamine-treated suspended rats. Total capillary length was unaffected by unweighting and increased 21% in all animals receiving dobutamine. The drug prevented the increase in total mitochondrial volume density (+30%) induced by unweighting but did not change total mitochondrial volume. Our results suggest that 1) dobutamine is useful to prevent the decrease of total aerobic capacity during hindlimb suspension, 2) dobutamine increases VO2max in control rats, and 3) total capillary length in soleus muscle is increased by the drug in all groups, although no beneficial effects on mitochondria can be detected.     

Exercise-induced increase in the capacity of rat skeletal muscle to oxidize ketones.

During and after strenuous prolonged exercise, sedentary individuals develop high blood levels of acetoacetate and beta-hydroxybutyrate whereas exercise-trained animals and human subjects do not. We have investigated the possibility that exercise training can increase the capacity of skeletal muscle to oxidize ketones. In this study we measured rates of D-beta[3-14-C]-hydroxybutyrate and [3-14-C]acetoacetate oxidation, and the levels of activity of the enzymes involved in the oxidation of ketones in homogenates of gastrocnemius muscles of exercise-trained and of untrained male rats. The trained animals had markedly lower blood ketone levels immediately and 60 min after a 90 min long bout of exercise than did the sedentary animals. The rates of D-beta-[13-14C]hydroxybutryate and [3-14-C]acetoacetate oxidation were twice as high in homogenates of muscles from the trained as compared to the sedentary rats. The increases in levels of activity in gastrocnemius muscle in response to the exercise program were: beta-hydroxybutyrate dehydrogenase threefold; 3-ketoacid CoA-transferase twofold; and acetoacetyl-CoA thiolase 55%. This exercise-induced increase in the capacity of skeletal muscle to oxidize ketones could play a role in preventing development of ketosis in the physically trained animal during and following prolonged strenuous exercise.     

Metabolic capacity of muscle fibers from high-altitude natives

We evaluate the effects of chronic hypoxia on the metabolic phenotype of the muscle fiber types of humans. The subjects were three Quechua natives residing in the Peruvian Andes at an altitude greater than 3300 m, and three lowlanders from below 700 m. Biopsy specimens were obtained from the vastus lateralis muscles of volunteers. Muscle fibers were identified histochemically as type 1 (oxidative), 2a (oxidativeglycolytic) or 2b (glycolytic). The relative contribution of each fiber type to the total cross-sectional area of each biopsy sample was determined. In individual fibers, the activities of malate dehydrogenase (MDH, citric acid cycle), lactate dehydrogenase (LDH, glycolysis) and adenylokinase (high-energy phosphate) were quantified. The cross-sectional area of the muscle occupied by each fiber type is comparable between Quechuas and lowlanders. Type 1 fibers are the only fiber type to demonstrate statistically significant (P 50.05) differences in enzyme activities between Quechuas and lowlanders. MDH activity is, on average, 19.6% less (P 0.0001) and LDH activity 28.1% more (P 0.0001) in the type 1 fibers of the Quechuas. Chronic hypoxia appears to produce a shift from oxidative to glycolytic metabolism in those fibers which are typically the, most aerobic in human muscle.     

Buffering capacity of deproteinized human vastus lateralis muscle

The in vitro deproteinized vastus lateralis muscle buffer capacity, carnosine, and histidine levels were examined in 20 men from 4 distinct populations (5 sprinters, 800-m runners; 5 rowers; 5 marathoners; 5 untrained). Needle biopsies were obtained at rest from the vastus lateralis muscle. The buffer capacity was determined in deproteinized homogenates by repeatedly titrating supernatant extracts over the pH range of 7.0-6.0 with 0.01 N HCl. Carnosine and histidine levels were determined on an amino acid AutoAnalyzer. Fast-twitch fiber percentage was determined by staining intensity of myosin adenosinetriphosphatase. High-intensity running performance was assessed on an inclined treadmill run to fatigue (20% incline; 3.5 m X s-1). Significantly (P less than 0.01) elevated buffer capacities, carnosine levels, and high-intensity running performances were demonstrated by the sprinters and rowers, but no significant differences existed between these variables for the marathoners vs. untrained subjects. Low but significant (P less than 0.05) interrelationships were demonstrated between buffer capacity, carnosine levels, and fast-twitch fiber composition. These findings indicate that the sprinters and rowers possess elevated buffering capabilities and carnosine levels compared with marathon runners and untrained subjects.     

Aerobic capacity of frog skeletal muscle during hibernation

Frogs submerged at 3 degrees C in hypoxic water (Po2=60 mmHg) depress their metabolic rate to 25% of that seen in control animals with access to air. The hypometabolic state of the skeletal muscle in such cold-submerged frogs is thought to be the most important contributor to the overall metabolic depression. The aim of this study was to determine whether the aerobic capacity of frog skeletal muscle became altered during 1-4 mo of hibernation to match the reduction in adenosine triphosphate (ATP) demand. To this end, the activities of key mitochondrial enzymes were measured in the skeletal muscle and in isolated mitochondria of frogs at different stages during hibernation. We also measured the activity of lactate dehydrogenase (LDH) as an indicator of glycolytic capacity. The activities of cytochrome c oxidase, citrate synthase, and LDH were significantly lower in frog skeletal muscle after 4 mo of hibernation compared with control conditions. The reduction in skeletal muscle aerobic capacity is apparently due to changes in the intrinsic properties of the mitochondria. Overall, these results indicate an important reorganisation of ATP-producing pathways during long-term metabolic depression to match the lowered ATP demand.     

Effects of exogenous cytochrome c on respiratory capacity of heart and skeletal muscle.

Cytochrome c is easily extracted from mitochondria by salt buffers. Yet studies of mitochondrial respiratory capacity, which routinely use KCl solutions for preparation of muscle, have failed to include the enzyme in the assay medium. The present study demonstrated that exogenous cytochrome c stimulated the oxidation of pyruvate + malate by 120, 66, 65, and 35% for the mitochondrial fraction of heart, fast-twitch red, mixed and fast-twitch white rodent muscle. Lesser increases were obtained for whole homogenate preparations. These data suggest that previous studies may have underestimated the true respiratory capacity of these tissues.