Morphological and biochemical changes in old rat muscles: effect of increased use

Male Wistar rats were strength and swim trained during a substantial period of old age to determine the influence of aging and activity on the histochemical and metabolic characteristics of a predominantly slow (soleus) and a predominantly fast (plantaris) skeletal muscle. Strength training counteracted the age-related atrophy of the fibers and the age-induced changes in fiber-type distribution of both muscles. Swim training, on the other hand, was without any effect on these parameters. The activity of both mitochondrial and cytoplasmic enzymes became lower with aging in the soleus muscle, whereas only the activity of the cytoplasmic enzymes became lower in the plantaris. Strength training reduced the aerobic capacity of both muscles, whereas swim training had the opposite effect. Aging induced a lower glycogen concentration of the lateral gastrocnemius muscle. This was avoided by swim training. The phosphocreatine and adenosine 5'-triphosphate concentrations were unchanged with aging but became higher with strength training. The activity pattern, therefore, seems to have a considerable influence on the age-related modification of the histochemical and metabolic characteristics of skeletal muscles of the rat. The effect, however, is related to the recruitment pattern of the fiber populations and the form of activity.     

Metabolic adaptations in skeletal muscle of streptozotocin-diabetic rats following exercise training

Compensatory metabolic adaptations induced in streptozotocin-diabetic rat skeletal muscle by submaximal endurance training have been investigated. The gastrocnemius muscles of sedentary streptozotocin-diabetic rats were found to have a lower than normal myoglobin content, succinate dehydrogenase activity, and capacity to oxidize pyruvate and palmitate-1-[¹⁴C]. The values of these parameters were significantly increased in the diabetic skeletal muscle by the training program, obtaining levels similar to those of normal sedentary animals.     

Some hormonal factors (hypophysectomy, castration and testosterone administration) modifying the course of "compensatory" muscle hypertrophy in the rat.

1. Maximum compensatory hypertrophy of the soleus and plantaris muscle in male rats is attained seven days after tenotomy of the gastrocnemius muscle (39% and 9% respectively). When tenotomy of the gastrocnemius was performed seven days ater hypophysectomy, hypertrophy in these two muscles was aproximately half that found in control animals. 2. After 81-day castration of young male rats the weight of the saleus and plantaris was reduced and hypertrophy following tenotomy of the gastrocneumius muscle did not develop. 3. Chronically castrated rats received testosterone two weeks prior to tenotomy of the gastrocnemius and a week during the muscle hypertrophy phase. Hypertrophy of the soleus in castrated rats which had received testosterone seven days after tenotomy of the gastrocnemius was 25% as compared with muscles of castrated animals. The corresponding value in the plantaris muscle was 10%. 4. These results indicate that even calf muscles of the rat, namely the soleus and plantaris muscles, are significantly affected by testosterone under these conditions, although it is not, as yet, clear whether its action is direct or indirect.     

Anatomic capillarization is maintained in relative excess of fiber oxidative capacity in some skeletal muscles of late middle-aged rats.

The anatomic size of the capillary-to-fiber (C/F) interface plays an important role in O(2) flux from blood to tissue by determining the surface area available for diffusion and is maintained in relative proportion to fiber mitochondrial volume across a wide range of muscle aerobic capacity. In the present study, we examined an estimate of the anatomic size of the C/F interface [the quotient of the individual C/F ratio and fiber perimeter, C/F perimeter exchange (CFPE) index] and fiber oxidative capacity in different skeletal muscles, or muscle regions, to test the hypothesis that capillarization would be maintained in relative excess of reduced fiber oxidative capacity in aged muscles. The right gastrocnemius, plantaris, and soleus muscles from young adult (8 mo old) and late middle-aged (28-30 mo old) Fischer 344 x Brown Norway F1 hybrid rats were excised for evaluation of flux through electron transport chain complexes I-III and/or morphometric estimation of capillarization. Muscle mass was lower in the gastrocnemius muscles of the older animals (2,076 +/- 32 vs. 1,825 +/- 47 mg in young adult vs. late middle-aged, respectively; mean +/- SE) but not the plantaris or soleus muscles. Fibers were smaller in the white region of gastrocnemius muscles but larger in the red region of gastrocnemius muscles of the older animals. There was no difference in the number of capillaries around a fiber, the individual C/F ratio, or the CFPE index between groups for any muscle/region, whereas flux through complexes I-III was reduced by 29-43% in late middle-aged animals. Thus the greater quotient of indexes of anatomic capillarity (individual C/F ratio or CFPE index) and fiber oxidative capacity in soleus and the white region of gastrocnemius muscles, but not in the red region of gastrocnemius muscles of the older animals, shows that anatomic capillarity is maintained in relative excess of oxidative capacity in some muscle regions in late middle-aged rats.     

Heat stress inhibits skeletal muscle hypertrophy

Heat shock proteins (Hsps) are molecular chaperones that aid in protein synthesis and trafficking and have been shown to protect cells/tissues from various protein damaging stressors. To determine the extent to which a single heat stress and the concurrent accumulation of Hsps influences the early events of skeletal muscle hypertrophy, Sprague-Dawley rats were heat stressed (42 degrees C, 15 minutes) 24 hours prior to overloading 1 plantaris muscle by surgical removal of the gastrocnemius muscle. The contralateral plantaris muscles served as controls. Heat-stressed and/or overloaded plantaris muscles were assessed for muscle mass, total muscle protein, muscle protein concentration, Type I myosin heavy chain (Type I MHC) content, as well as Hsp72 and Hsp25 content over the course of 7 days following removal of the gastrocnemius muscle. As expected, in non-heat-stressed animals, muscle mass, total muscle protein and MHC I content were significantly increased (P < 0.05) following overload. In addition, Hsp25 and Hsp72 increased significantly after 2 and 3 days of overload, respectively. A prior heat stress-elevated Hsp25 content to levels similar to those measured following overload alone, but heat stress-induced Hsp72 content was increased significantly greater than was elicited by overload alone. Moreover, overloaded muscles from animals that experienced a prior heat stress showed a lower muscle mass increase at 5 and 7 days; a reduced total muscle protein elevation at 3, 5, and 7 days; reduced protein concentration; and a diminished Type I MHC content accumulation at 3, 5, and 7 days relative to nonheat-stressed animals. These data suggest that a prior heat stress and/or the consequent accumulation of Hsps may inhibit increases in muscle mass, total muscle protein content, and Type I MHC in muscles undergoing hypertrophy.     

A comparative study of the effects of the diamine oxidase inhibitor aminoguanidine, with or without dietary restriction, on the nucleic acid and protein composition of cardiac and type I and type II skeletal muscles of the rat.

It has been proposed that the diamine oxidase inhibitor aminoguanidine may be a potential therapeutically important anabolic agent. An investigation was therefore made into the effects of aminoguanidine treatment with or without nutritional restriction, on cardiac and skeletal muscles containing mainly of either Type I (i.e. soleus) or Type II fibres (i.e. plantaris) or a mixture of Type I and II fibres (i.e. gastrocnemius). After 3 weeks, dietary restrictions reduced cardiac weight, protein, RNA and DNA contents by between 31 per cent and 36 per cent. Similar, but smaller, reductions were observed in the soleus (18-31 per cent), plantaris (22-34 per cent) and gastrocnemius (22-34 per cent). Aminoguanidine had no effect on the heart of the rats fed ad libitum, nor did it alter the response to dietary restriction. Treatment with aminoguanidine had no overt anabolic effect on skeletal muscle, but a reduction in DNA content was observed. It was concluded that cardiac protein and nucleic acid contents are more sensitive to dietary deprivation than either anaerobic or aerobic skeletal muscles. Furthermore, aminoguanidine does not appear to promote growth or reduce catabolism as previous studies have suggested.     

Endurance training decreases the alkaline proteolytic activity in mouse skeletal muscles

Alkaline and myofibrillar protease activities of rectus femoris, soleus, and tibialis anterior muscles and the pooled sample of gastrocnemius and plantaris muscles were analyzed in male NMRI-mice during a running-training program of 3, 10, or 20 daily 1-h sessions. The activity of citrate synthase increased during the endurance training, reflecting the increased oxidative capacity of skeletal muscles. The activities of alkaline and myofibrillar proteases continually decreased in the course of the training program in all muscles studied. Instead, the activity of beta-glucuronidase (a marker of lysosomal hydrolases) increased in all muscles. The highest activities were observed at the beginning of the training program. Present results, together with our earlier observations, show that the type of training, running as opposed to swimming, modulates the training responses in alkaline protease activities. Further, diverse adaptations in the activities of alkaline proteases and a lysosomal hydrolase suggest difference in the function of different proteolytic systems.     

Angiotensin-converting enzyme inhibition attenuates myonuclear addition in overloaded slow-twitch skeletal muscle

Because optimal overload-induced skeletal muscle hypertrophy requires ANG II, we aimed to determine the effects of blocking ANG II production [via angiotensin-converting enzyme (ACE) inhibition] on potential mediators of hypertrophy in overloaded skeletal muscle, namely, myonuclear addition and fibroblast content. In a 2 x 2 design, adult (200-225 g) female Sprague-Dawley rats were placed into one of four groups (n = 8/group): 7-day skeletal muscle overload, sham operation, 7-day skeletal muscle overload with ACE inhibition, or sham operation with ACE inhibition. Functional overloads of the plantaris and soleus muscles were produced via bilateral surgical ablation of the synergistic gastrocnemius muscle, and ACE inhibition was accomplished by the addition of the ACE inhibitor enalapril maleate to the animals' daily drinking water (0.3 mg/ml). Myonuclear addition and extrasarcolemmal nuclear proliferation, as measured by in vivo 5-bromo-2'-deoxyuridine labeling, were significantly (P < or = 0.05) increased by overload in both the slow-twitch soleus and fast-twitch plantaris muscles. Furthermore, ACE inhibition attenuated these overload-induced increases in the soleus muscle but not in the plantaris muscle. However, the effect of ACE inhibition on soleus extrasarcolemmal nuclei was not likely due to differences in fibroblast content because overload elicited significant increases in vimentin-positive areas in soleus and plantaris muscles, and these areas were unaffected by ACE inhibition in either muscle. There was no effect of ACE inhibition on any measure in sham-operated muscles. Collectively, these data indicate that ANG II may mediate the satellite cell response to overload in slow-twitch soleus but not in fast-twitch plantaris muscles and that this effect may occur independently of changes in fibroblast content.     

Interactive effect of exercise training and growth hormone administration on glucose tolerance and muscle GLUT4 protein expression in rats

The insulin-resistance effect of growth hormone (GH) administration has been frequently reported. The present study investigated the effect of GH administration on glucose tolerance and muscle GLUT4 protein expression in exercise-trained and untrained rats. Forty-eight rats were weight-matched and assigned to the following 4 groups: control, GH, exercise training, and exercise training + GH groups. After 2 weeks of GH injections (65 µg/kg/day) and exercise training, the glucose tolerance and insulin response were measured in these rats. The GLUT4 protein level, glycogen storage, and citrate synthase activity were determined in red gastrocnemius and plantaris muscles. Daily GH administration elevated the curves of the oral glucose tolerance test and insulin response compared with those of saline-injected control rats. Furthermore, exercise training completely eliminated this GH-induced insulin resistance as determined 18 h after the last bout of exercise training. Additionally, exercise training significantly increased muscle glycogen storage and GLUT4 protein levels. GH administration did not affect the GLUT4 protein and glycogen storage increases induced by exercise training, but the citrate synthase activity in the plantaris muscle was further elevated by GH administration to a level above that induced by training. In conclusion, this is the first study that demonstrates that regular exercise training prevents GH-induced insulin-resistance side effect in rats.     

Biochemical adaptation in the skeletal muscle of rats depleted of creatine with the substrate analogue beta-guanidinopropionic acid.

Rats were fed on a diet containing 1% beta-guanidinopropionic acid (GPA), a creatine substrate analogue, for 6-10 weeks to deplete their muscle of creatine. This manipulation was previously shown to give a 90% decrease in [phosphocreatine] in skeletal and cardiac muscle and a 50% decrease in [ATP] in skeletal muscle only. Maximal activities of creatine kinase and of representative enzymes of aerobic and anaerobic energy metabolism were measured in the superficial white, medial and deep red portions of the gastrocnemius muscle, in the soleus and plantaris muscle and in the heart. Fast-twitch muscles were smaller in GPA-fed animals than in controls, but the size of the soleus muscle was unchanged. The activities of aerobic enzymes increased by 30-40% in all fast-twitch muscle regions except the superficial gastrocnemius, but were unchanged in the soleus muscle. The activities of creatine kinase and phosphofructokinase decreased by 20-50% in all skeletal-muscle regions except the deep gastrocnemius, and the activity of glycogen phosphorylase generally paralleled these changes. There were no significant changes in the activities of any of the enzymes measured in the heart. The glycogen content of the gastrocnemius-plantaris complex was increased by 185% in GPA-fed rats. The proportion of Type I fibres in the soleus muscle increased from 81% in control rats to 100% in GPA-fed rats, consistent with a previous report of altered isometric twitch characteristics and a decrease in the maximum velocity of shortening in this muscle [Petrofsky & Fitch (1980) Pflugers Arch. 384, 123-129]. We conclude that fast-twitch muscles adapt by a combination of decreasing diffusion distances, increasing aerobic capacity and decreasing glycolytic potential. Slow-twitch muscles decrease glycolytic potential and become slower, thus decreasing energy demand. These results suggest that persistent changes in the [phosphocreatine] and [ATP] are alone sufficient to alter the expression of enzyme proteins and proteins of the contractile apparatus, and that fibre-type-specific thresholds exist for the transformation response.     

Metabolic alterations in skeletal muscle of chronically streptozotocin-diabetic rats

This study was accomplished to determine the effects of chronic streptozotocin diabetes and insulin treatment on selected enzymes and substrates used in energy transduction in muscles composed of different muscle fiber types. Triglyceride concentration in all the muscles of diabetic rats was significantly elevated. Glycogen and protein concentrations were unchanged. The enzyme activities of hexokinase and alanine aminotransferase were significantly reduced and 3-hydroxyacyl-CoA dehydrogenase increased in all the muscles. Declines in phosphofructokinase, lactate dehydrogenase, citrate synthase, and succinate dehydrogenase activities were found in the red gastrocnemius and plantaris. Glycerol-3-phosphate dehydrogenase activity was lower than normal in the red gastrocnemius. Insulin treatment to the diabetic rats returned the altered triglyceride content and enzyme activities to normal, with exception of the lower alanine aminotransferase activity in the red gastrocnemius and plantaris. However, this enzyme was significantly ameliorated when compared with the untreated diabetic rats. The findings show that hypoinsulinism has a differential effect on the enzymatic profile of the different skeletal muscle fiber types, with those of the red gastrocnemius being most severely affected. Insulin treatment returned the enzymatic profile of the fiber types in diabetic rats to essentially normal.     

Effect of prolonged intermittent hypoxia and exercise training on glucose tolerance and muscle GLUT4 protein expression in rats

We compared the chronic effect of intermittent hypoxia and endurance training on the glucose tolerance and GLUT4 protein expression in rat skeletal muscle. Thirty-two Sprague-Dawley rats were matched for weight and assigned to one of the following four groups: control, endurance training, hypoxia, or hypoxia followed by endurance training. Hypoxic treatment consisted of breathing 14% O₂ for 12 h/day under normobaric conditions, and the training protocol consisted of making animals swim 2 times for 3 h/day. At the end of the 3rd week, an oral glucose tolerance test (OGTT) was performed 16 h after treatments. At the end of the 4th week, GLUT4 protein, mRNA, and glycogen storage in skeletal muscle were determined. Endurance training significantly improved OGTT results. Glycogen content and GLUT4 protein expression in the plantaris and red gastrocnemius, but not in the soleus or white gastrocnemius muscles, were also elevated. Chronic intermittent hypoxia also improved OGTT results, but did not alter GLUT4 protein expression. Additionally, hypoxia followed by exercise training produced significant increases in GLUT4 protein and mRNA in a greater number of muscles compared to endurance training alone. Both exercise training and hypoxia significantly reduced body mass, and an additive effect of both treatments was found. In conclusion, chronic intermittent hypoxia improved glucose tolerance in the absence of increased GLUT4 protein expression. This treatment facilitated the exercise training effect on muscle GLUT4 expression and glycogen storage. These new findings open the possibility of utilizing intermittent hypoxia, with or without exercise training, for the prevention and clinical treatment of type 2 diabetes or insulin resistance.     

Strategies of adaptation of small arteries in diaphragm and gastrocnemius muscle to aerobic exercise training

Aerobic exercise training is associated with adaptive changes in skeletal muscles and their vascular bed; such changes in individual muscles may vary depending on their characteristics and recruitment. This study was aimed at comparing the effects of eight-week treadmill training on the locomotor and respiratory muscles in rats. The training course increased the aerobic performance in rats, which was evidenced by an increase in maximum O₂ consumption and a decrease in the blood lactate concentration in ramp test. The succinate dehydrogenase activity was increased in the red portion of the gastrocnemius muscle, but not in the diaphragm of trained rats. Arterial segments were isolated from feed arteries and studied by wire myography. The relaxation in response to acetylcholine in gastrocnemius arteries in trained animals was higher as compared with controls (due to higher NO production), while contractile responses to noradrenaline (in the presence of propranolol) were not changed. On the contrary, the endothelial function of diaphragm arteries was not affected by training, but contractile responses to activation of α-adrenoceptors were markedly increased. Thus, aerobic training may increase the blood supply rate to both locomotor and respiratory muscles, but the underlying regulatory mechanisms are different. The results obtained allow us to reveal the physiological mechanisms that determine the physical performance of the body under conditions of compromised functioning of the respiratory system.     

Effects of hindlimb suspension and androgen treatment on testosterone receptors in rat skeletal muscles

This study tested the specific and combined effects of testosterone treatment and hindlimb suspension (HS) on the properties of steroid receptors in skeletal muscle. Male rats were either administered weekly high doses of testosterone heptylate (10 mg x kg(-1)) or olive oil placebo, and were either tail-suspended or acted as controls. After 3 weeks of treatment, three muscles were excised from each animal, soleus (SOL), extensor digitorum longus (EDL), and plantaris. The results showed that the testosterone treatment was unable to minimise the HS-induced atrophy of skeletal muscle. As expected, HS altered the fibre-type composition of SOL muscles (-33% of type I, +188% and +161% of type IIa and intermediate fibres respectively, P < 0.01). No overall effect of treatment was detected on the fibre-type composition of either slow or fast-twitch muscles. Binding capacity determined by a radiocompetition technique was increased by HS, especially in SOL and EDL muscles (P < 0.01), while HS or steroid treatment decreased the affinity of the steroid receptors. The combination of HS and testosterone administration resulted in a decrease in binding capacity and affinity of steroid receptors in skeletal muscles. Steroid receptors in fast-twitch muscles exhibited a higher affinity than those in slow-twitch muscles, and it is suggested that it is likely that testosterone treatment is more effective in fast-twitch than in slow-twitch muscles. It was concluded that the lack of preventive effect of testosterone treatment on HS-induced SOL muscle atrophy could be explained by both a decrease in steroid sensitivity and the removal of mechanical factors.     

Blood flow and glycogen use in hypertrophied rat muscles during exercise

Previous findings suggest that skeletal muscle that has enlarged as a result of removal of synergistic muscles has a similar metabolic capacity and improved resistance to fatigue compared with normal muscle. The purpose of the present study was to follow blood flow and glycogen loss patterns in hypertrophied rat plantaris plantaris and soleus muscles during treadmill exercise to provide information on the adequacy of perfusion of the muscles during in vivo exercise. Thirty days following surgical removal of gastrocnemius muscle, blood flows (determined with radiolabeled microspheres) and glycogen concentrations were determined in all of the ankle extensor muscles of experimental and sham-operated control rats during preexercise and after 5-6 min of treadmill exercise at 15 m/min. There were no differences (P greater than 0.05) in blood flows per unit mass or glycogen concentrations between control and hypertrophied plantaris or soleus muscles at either time, although both muscles were larger (P less than 0.05) in the experimental group (plantaris: 95%; soleus: 40%). None of the other secondary ankle extensor muscles (tibialis posterior, flexor digitorum longus or flexor hallicus longus) hypertrophied in response to removal of gastrocnemius. These results provide indirect evidence that O2 delivery in the enlarged muscles is not compromised during low-intensity treadmill exercise due to limited perfusion.     

Evidence for increased peroxidative activity in muscles from streptozotocin-diabetic rats

The ability of cardiac and skeletal muscles from diabetic rats to metabolize superoxide and hydrogen peroxide was determined by the activities of superoxide dismutase (SOD) and catalase, respectively. Male and female Sprague-Dawley rats, 43 days old, were made diabetic with a single intravenous injection of streptozotocin (70 mg/kg body weight). On the 80th day after injection the blood glucose concentration of these rats was increased fourfold, and the plasma insulin concentration was decreased four- to fivefold compared to controls. Body weights of male diabetic rats were 61% and those of female diabetic rats were 66% of their ad libitum-fed controls. The seven different skeletal muscles examined weighed less in the diabetic rats than in controls of the same age and body weight. The hearts of the diabetic rats weighed more than those of controls of the same age and body weight. Comparison to the body weight controls allowed the distinction of specific effects due to lack of insulin from effects due to retardation in muscle growth. Increased catalase activity in all muscles examined from diabetic rats (plantaris, gastrocnemius, and heart) suggested a response in catalase activity similar to that of starved rats. SOD activity was not altered in the diabetic rat skeletal muscles and erythrocytes, but was somewhat decreased in the heart.     

Chronic activation of 5'-AMP-activated protein kinase increases GLUT-4, hexokinase, and glycogen in muscle.

This study was designed to determine whether chronic chemical activation of AMP-activated protein kinase (AMPK) would increase glucose transporter GLUT-4 and hexokinase in muscles similarly to periodic elevation of AMPK that accompanies endurance exercise training. The adenosine analog, 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), has previously been shown to be taken up by cells and phosphorylated to form a compound (5-aminoimidazole-4-carboxamide ribonucleotide) that mimics the effect of AMP on AMPK. A single injection of AICAR resulted in a marked increase in AMPK in epitrochlearis and gastrocnemius/plantaris muscles 60 min later. When rats were injected with AICAR (1 mg/g body wt) for 5 days in succession and were killed 1 day after the last injection, GLUT-4 was increased by 100% in epitrochlearis muscle and by 60% in gastrocnemius muscle in response to AICAR. Hexokinase was also increased approximately 2. 5-fold in the gastrocnemius/plantaris. Gastrocnemius glycogen content was twofold higher in AICAR-treated rats than in controls. Chronic chemical activation of AMPK, therefore, results in increases in GLUT-4 protein, hexokinase activity, and glycogen, similarly to those induced by endurance training.     

Effect of endurance training on the phospholipid content of skeletal muscles in the rat

Only few data are available on the effect of training on phospholipid metabolism in skeletal muscles. The aim of the present study was to examine the effect of 6 weeks of endurance training on the content of particular phospholipid fractions and on the incorporation of blood-borne [14C]-palmitic acid into the phospholipids in different skeletal muscles (white and red sections of the gastrocnemius, the soleus and the diaphragm) of the rat. Lipids were extracted from the muscles and separated using thin-layer chromatography into the following fractions: sphingomyelin, phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, cardiolipin and neutral lipids (this fraction being composed mostly of triacylglycerols). It was found that training did not affect the content of any phospholipid fraction in soleus muscle. It increased the content of sphingomyelin in white gastrocnemius muscle, cardiolipin and phosphatidylethanolamine in red gastrocnemius muscle and phosphatidylinositol in white gastrocnemius muscle and diaphragm. The total phospholipid content in red gastrocnemius muscle of the trained group was higher than in the control group. Training reduced the specific activity of sphingomyelin and cardiolipin in all muscles, phosphatidylcholine in soleus, red, and white gastrocnemius muscles, phosphatidylserine in all muscles, phosphatidylinositol in all except the soleus muscle, and phosphatidylethanolamine in hindleg muscles, but not in the diaphragm compared to the corresponding values in the sedentary group. It was concluded that endurance training affects skeletal muscle phospholipid content and the rate of incorporation of the blood-borne [14C]palmitic acid into the phospholipid moieties.