Functional properties of skeletal muscle from transgenic animals with upregulated heat shock protein 70.

The influence of inducible heat stress proteins on protecting contracting skeletal muscle against fatigue-induced injury was investigated. A line of transgenic mice overexpressing the inducible form of the 72-kDa heat shock protein (HSP72) in skeletal muscles was used. We examined the relationship between muscle contractility and levels of the constitutive (HSC73) and inducible (HSP72) forms of the 72-kDa heat shock protein in intact, mouse extensor digitorum longus (EDL), soleus (SOL), and the diaphragm (DPH). In all transgenic muscles, HSP72 was expressed at higher levels compared with transgene-negative controls, where HSP72 was below the level of detection. At the same time, HSC73 levels were downregulated in all transgenic muscle types. Shipment-related stress caused an elevation in the levels of HSP72 in all muscles for 1 wk after arrival of the animals. We also found that, although no statistical differences in response to intermittent fatiguing stimulation in the contractile properties of intact transgene-positive muscles compared with their transgene-negative counterparts were observed, the response of intact transgene-positive EDL muscles to caffeine was enhanced. These findings demonstrate that elevated HSP72 does not protect EDL, SOL, or DPH muscles from the effects of intermittent fatiguing stimulation. However, HSP72 may influence the excitation-contraction coupling (ECC) process, either directly or indirectly, in EDL muscle. If the effects on ECC were indirect, then these results would suggest that manipulation of a specific gene might cause functional effects that seem independent of the manipulated gene/protein.

The role of stress hormones in exercise-induced suppression of alveolar macrophage antiviral function.

We hypothesized that a previously observed exercise-induced suppression of alveolar macrophage antiviral resistance results from increases in corticosterone and/or epinephrine. Mice (CD-1) were run to fatigue on a treadmill (exercise), or placed in Plexiglas lanes above the treadmill (control). The role of corticosterone was assessed by further dividing mice into groups receiving one of the following treatments; sham surgery, adrenalectomy, or adrenalectomy plus corticosterone replacement. Macrophage antiviral function was suppressed in the exercised mice compared to the control mice. However, macrophage antiviral function was not suppressed in the exercised mice that underwent adrenalectomy or adrenalectomy plus corticosterone replacement. We tested whether another adrenal factor (epinephrine) may be involved by dividing mice into exercise and control groups treated with either saline or propranolol. Macrophage antiviral function was again suppressed in the saline-treated exercised mice compared to saline-treated control mice, but no differences were found between the exercised mice receiving propranolol, control mice receiving propranolol, or saline-treated control mice. Isoproterenol, when added to alveolar macrophages in culture, also suppressed antiviral resistance. These findings suggest that decreased macrophage antiviral function following exercise may be due to increased release of adrenal catecholamines.

Delayed effects of exercise on the plasma leptin concentration.

Recent studies have concluded that a single exercise session has no immediate effect on the plasma concentration of leptin, a putative satiety factor. We tested the hypothesis that an increase in energy expenditure would decrease the leptin concentration but the effects would be manifest in a 48-hour period following exercise. Eleven active males completed two treadmill exercise sessions with different energy expenditure (800 or 1,500 kcal) at 70% maximal O2 consumption (Vo2max). Subjects maintained constant energy intake on the day before, the day of, and 2 days after exercise, as verified by dietary recall. Compared with preexercise in either exercise session, there were no differences in plasma leptin concentrations following exercise (0 and 24 hours postexercise) except at 48 hours postexercise, where an approximately 30% decrease (P < .05) was observed. With either duration of exercise, plasma glucose increased about 10% (P < .05), insulin decreased 35% to 46% (P < .05), and cortisol increased 41% to 50% (P < .05, 1,500 kcal only) immediately following exercise, but returned to preexercise values at 24 and 48 hours postexercise. A statistically significant correlation was observed between the changes in leptin and insulin (r = .49, P < .0001). Single exercise sessions of varying energy expenditure decreased the plasma leptin concentration after 48 hours in association with a preceding decrease in insulin.

Cytochrome oxidase in muscle of endurance-trained rats: subunit mRNA contents and heme synthesis.

Endurance training leads to an increase in the content of individual mitochondrial hemeproteins in skeletal muscle. To deduce the control mechanisms involved, cytochrome oxidase (COX) activity was compared with 1) the content of COX subunits III and IV and 2) 5-aminolevulinate synthase (ALAS) activity and mRNA content. In the plantaris muscle of female rats run daily for up to 28 days, ALAS activity was elevated 100% (P < 0.01) after 3 days and remained 150 and 125% higher (P < 0.001) after 7 and 28 days of running, respectively, than control. COX activity was also increased, but not until day 7 (40%; P < 0.05), and reached a maximal value 80% higher than control (P < 0.001) in the 28-day group. Compared with control, the content of COX subunit III and IV and ALAS mRNAs was not significantly changed by the training. The increased activities of COX and ALAS appear to be regulated by translational or posttranslational steps in the protein expression pathway. The induction of ALAS before COX suggests that the increased activity of COX may require increased synthesis of heme.

Lipase regulation of muscle triglyceride hydrolysis.

The cellular control of intramuscular triglyceride (TG) metabolism involves two major identified lipases: hormone-sensitive lipase (HSL) and lipoprotein lipase (LPL). Recently, the presence of HSL in muscle has been unequivocally demonstrated. However, although it is thought that HSL is responsible for intramuscular TG lipolysis, direct evidence for this is lacking. There is evidence to suggest that HSL and LPL are simultaneously activated under a variety of conditions. The two muscle lipases appear to be turned on by the same signal and function as a coordinated unit in meeting the energy demands of muscle. At a time when HSL is presumably hydrolyzing endogenous TG, LPL is sent to the capillary beds in search of substrate. TG uptake from circulation is highly related to muscle LPL activity. Exercise training increases LPL activity in plasma and in parenchymal cells in muscle. These results suggest that training may increase the capacity to clear TG from circulation and that LPL might have a role in replenishing muscle TG stores that have been decreased with exercise.

Effects of glucocorticoids and endurance training on cytochrome oxidase expression in skeletal muscle.

This investigation was undertaken to evaluate whether the mitochondrial disfunction associated with glucocorticoid treatment is expressed at the level of cytochrome-c oxidase (COX) and whether endurance training attenuates this response. Adult female rats were administered cortisol acetate (100 mg/kg body wt) or an equal volume of the vehicle solution for 11 days. Endurance training was performed by treadmill running up to 28 m/min (with intervals at 50 m/min for 2 min every 15 min), for 90 min/day, 6 days/wk, for 8-10 wk. During hormone treatments, the training animals ran every day. Exercise prevented 43-55% of the hormone-induced atrophy in various fast-twitch muscles or muscle groups. Cortisol acetate treatment produced no significant effects on COX enzyme activities or subunit mRNA content in deep red or superficial white quadriceps or mixed plantaris muscles. The levels of COX were increased as a result of training by 70-110% in plantaris and red quadriceps muscles, but no changes were seen in white quadriceps muscles. Both nuclear-encoded (COX IV) and mitochondrial-encoded (COX III) mRNAs were increased approximately twofold by the exercise program in these same muscles. These data indicate that the impaired mitochondrial functioning associated with glucocorticoids is not observed at the COX step of electron transport. The prolonged endurance-training regimen appears to induce relatively parallel increases in COX enzyme activity and mRNA expression with coordinate changes in nuclear and mitochondrial mRNAs.

Effects of four different single exercise sessions on lipids, lipoproteins, and lipoprotein lipase.

The purpose of this study was to determine the threshold of exercise energy expenditure necessary to change blood lipid and lipoprotein concentrations and lipoprotein lipase activity (LPLA) in healthy, trained men. On different days, 11 men (age, 26.7 +/- 6.1 yr; body fat, 11.0 +/- 1.5%) completed four separate, randomly assigned, submaximal treadmill sessions at 70% maximal O2 consumption. During each session 800, 1,100, 1,300, or 1,500 kcal were expended. Compared with immediately before exercise, high-density lipoprotein cholesterol (HDL-C) concentration was significantly elevated 24 h after exercise (P < 0.05) in the 1,100-, 1,300-, and 1,500-kcal sessions. HDL-C concentration was also elevated (P < 0.05) immediately after and 48 h after exercise in the 1,500-kcal session. Compared with values 24 h before exercise, LPLA was significantly greater (P < 0.05) 24 h after exercise in the 1,100-, 1,300-, and 1,500-kcal sessions and remained elevated 48 h after exercise in the 1,500-kcal session. These data indicate that, in healthy, trained men, 1,100 kcal of energy expenditure are necessary to elicit increased HDL-C concentrations. These HDL-C changes coincided with increased LPLA.

Exercise and tumor development in a mouse predisposed to multiple intestinal adenomas.

UNLABELLED: Epidemiological evidence suggests that physical activity may be protective against the development of colon cancer. Potential mechanisms remain largely unexplored due to the paucity of appropriate experimental models. PURPOSE: The purpose of this study was to examine the effect of exercise training on polyp development in an induced mutant mouse strain predisposed to multiple intestinal neoplasia (Min mouse). METHODS: Three-week-old male and female heterozygotes were randomly assigned to control (CON; 10 males, 6 females) or exercise (EX; 11 males, 11 females) groups. In the first week, EX mice were acclimated to treadmill running at 10-18 m x min(-1) for 15-60 min x d(-1). From 4-10 wk of age, mice ran at 18-21 m x min(-1) for 60 min. CON mice sat in Plexiglas lanes suspended above the treadmill for the same time periods. At 10 wk of age, the mice were sacrificed and the intestines removed, opened, and counted for polyps. RESULTS: Skeletal muscle oxidative capacity increased with training as shown by a 64% increase in citrate synthase activity in the gastrocnemius/soleus muscle of EX compared with CON (P = 0.009). There were no significant effects of exercise in the males and females combined on small intestine, colon, or total intestinal polyps (P > 0.05). When analyzed separately, however, there were fewer colon and total polyps in the EX than in the CON males, although the difference was not statistically significant (P = 0.06). CONCLUSIONS: These results suggest that seven weeks of exercise training do not affect the development of intestinal polyps in the Min mouse. Further studies are required to determine if a true sex difference exists or if variations on the current training protocol may affect tumor outcomes.

Muscle-specific regulation of the heme biosynthetic enzyme 5'-aminolevulinate synthase.

The induction of 5'-aminolevulinate synthase (ALV synthase) activity in adult muscle by overload occurs in the absence of proportional changes in its mRNA content. Complete interpretation of these findings is difficult because little is known of the basal regulation of ALV synthase expression in muscle. In three adult chicken muscle fiber types (n = 5 each), differences in ALV synthase activity were correlated (r greater than or equal to 0.89; P less than 0.05) to the activities of cytochrome oxidase (COX) and citrate synthase (CS) and to levels of the "liver" isoform of ALV synthase mRNA. During posthatch development, ALV synthase activity and mRNA levels (n = 3-6 per time point) also covaried with changes in COX and CS activity. The highest levels of ALV synthase mRNA in muscle are observed early in myogenesis prior to induction of COX activity. The regulation of ALV synthase is also tissue-specific because the higher basal levels of ALV synthase activity in liver mitochondria are associated with disproportionately less oxidative enzyme activity and less of the liver ALV synthase isoform mRNA than in muscle.

5'-Aminolevulinate synthase activity is decreased in skeletal muscle of anemic rats.

Expression of the rate-limiting heme biosynthetic enzyme 5'-aminolevulinate synthase (ALAS) was investigated in skeletal muscle of 3-wk-old rats fed an iron-deficient diet. After 14 days, ALAS activity had declined 70% relative to control (2.1 +/- 0.2 vs. 0.6 +/- 0.1 nmol.h-1.g-1; P less than 0.005). Similar decreases were observed for blood hemoglobin (11.4 +/- 0.2 vs. 3.9 +/- 0.3 g/dl; P less than 0.005) and muscle cytochrome c (14.5 +/- 1.3 vs. 7.1 +/- 0.6 nmol/g; P less than 0.005). An iron-deficient diet decreased body and skeletal muscle growth by 15 (P less than 0.005) and 10% (P less than 0.05), respectively, whereas concentrations of protein, RNA, ALAS mRNA, and citrate synthase activity in muscle were not different from control. One mechanism by which heme biosynthesis may be slowed in muscle of young anemic rats is a decrease in ALAS activity. At a time when enzyme activity was decreased, ALAS mRNA expression was not affected by an iron-deficient diet, suggesting that steps after transcription of the ALAS gene may regulate the decrease in activity.

Induction of HSP 32 gene in hypoxic cardiomyocytes is attenuated by treatment with N-acetyl-L-cysteine.

Increased synthesis of stress proteins may enhance myocardial viability during periods of low oxygen delivery. Our purpose was to determine if the oxidative stress protein heme oxygenase-1 [heat stress protein 32 (HSP 32)] was induced in hypoxic cardiomyocytes and whether this induction might be mediated by a redox-sensitive mechanism. Primary rat neonatal cardiomyocytes, cultured to express a tissuelike phenotype, responded to 12 h of hypoxia (< 0.5% ambient oxygen) with an approximately fivefold (range 3- to 7.5-fold; P < 0.05) increase in HSP 32 mRNA and a threefold (P < 0.05) increase in HSP 32 protein content. Exposure to 80 microM H2O2 for 3 h increased HSP 32 mRNA content to a similar extent. Expression of heme oxygenase-2 mRNA was unaffected by H2O2 or hypoxic treatments. Inclusion of 20 mM N-acetyl-L-cysteine in the medium during hypoxia reduced the increase in HSP 32 mRNA and protein expression by 25.50% compared with hypoxia alone. The data suggest that induction of HSP 32 protein may lead to an improved antioxidant defense in cardiomyocytes during hypoxia and that a redox-sensitive pathway mediates at least a portion of the hypoxic induction of the HSP 32 gene.

Muscle fatigue and induction of stress protein genes: a dual function of reactive oxygen species?

Definitive characterization of the mechanisms of skeletal muscle fatigue is still an area of active investigation. One emerging theory concerns a role for the reactive oxygen species (ROS) produced primarily as a consequence of elevated rates of mitochondrial respiration. It has been theorized that the long-lasting effects of low-frequency fatigue (LFF) can be attributed to disruption of some stage of the excitation contraction coupling (ECC) process. Recent evidence suggests that ROS likely denature one or more proteins directly associated with the sarcoplasmic reticulum (SR) Ca2+ release mechanism. Given the potential of ROS to damage intracellular proteins during subsequent bouts of muscle contractions, the capacity of preexisting antioxidant pathways may be complemented by the synthesis of inducible heat-stress proteins (HSPs). HSPs collectively function to maintain cellular protein conformation during stressful proteotoxic insults. The goal of this article is to illustrate how recent findings suggest a dual role of ROS generated during muscle contractions.

Regulation of 5'-aminolevulinate synthase activity in overloaded skeletal muscle.

The regulation of the mitochondrial enzyme 5'-aminolevulinate synthase (ALV synthase) activity during chronic weight-bearing activity (overload) in chicken skeletal muscle was investigated. Maximal enzyme activity was increased 2.5- and 4.0-fold after 3 and 7 days of overload. The content of ALV synthase mRNA (ng/mg total RNA) was not changed after 3 days but increased (20%; P less than 0.05) after 7 days of overload. Normalizing the content of ALV synthase mRNA relative to the increase in total RNA indicated that ALV synthase mRNA increased by 1.6- and 2.0-fold at 3 and 7 days, respectively. On this basis, the increase in enzyme activity per gram protein exceeded the increase in mRNA content per gram protein by 60-70%. During overload, the activity of cytochrome oxidase was unchanged after 3 days but increased by 1.5-fold (P less than 0.05) after 7 days of overload. The data indicate that 1) the initial rise in ALV synthase mRNA and activity due to overload occurs in the absence of a prior change in the level of cytochrome oxidase, an enzyme that requires heme for its assembly, and 2) induction of ALV synthase activity is regulated largely by processes at the translational or posttranslational steps.

Exercise training has a heparin-like effect on lipoprotein lipase activity in muscle.

Lipoprotein lipase (LPL) is anchored with high affinity to heparan sulphate proteoglycans on the luminal surface of the capillary endothelium. The levels of pre-heparin perfusate LPL activity increased from 16 +/- 1 to 145 +/- 6 U/hindlimb (nine-fold increase) in hindlimb muscle of exercise-trained rats measured immediately after the last bout of work. At the same time, post-heparin perfusate LPL activity decreased from 63 +/- 2 to 13 +/- 1 U/hindlimb (p less than 0.001). These results provide evidence that exercise-training has a heparin-like effect on capillary-bound LPL. The total amount of LPL (i.e., pre-heparin perfusate plus post-heparin perfusate) was twofold greater in the hindlimb of the trained animals versus the controls. The effect of exercise on muscle LPL activity appears to last for as long as 5 days after cessation of exercise. Serum triglycerides were reduced 38% and plasma free fatty acids increased fourfold. These results provide evidence that training increases the capacity to remove triglycerides from circulation.

Induction of heme oxygenase-1 (HSP32) mRNA in skeletal muscle following contractions.

The capacity of preexisting antioxidant pathways to handle oxidative stress during exercise may be complemented by the synthesis of inducible heat stress proteins (HSP). Our purpose was to determine if the amount of mRNA for HSP32, a major oxidative stress protein, was increased in muscle after repetitive contractions. Reverse transcriptase-polymerase chain reaction analysis showed that HSP32 mRNA (normalized to alpha-actin mRNA) was increased about seven- and about fourfold (P < 0.35) immediately after 1 h of exhaustive running and after 3 h of muscle contractions (10 Hz nerve stimulation), respectively. Northern blot analysis revealed that HSP70 mRNAs were 3.5- to 15.5-fold above control value (P < 0.05), whereas the content of another oxidative stress protein mRNA (macrophage stress protein 23) was unchanged 0 h after contractions. The relative increase in HSP32 mRNA was found to be dependent on active tension generation; passive tension did not increase the HSP32-to-actin mRNA ratio. Increases in HSP32 mRNA may underlie an inducible antioxidant pathway in muscle responsive to metabolic stresses associated with repeated muscle contractions.

Skeletal muscle protein synthesis and degradation in vitro: effects of temperature.

We compared the structure, function, protein synthesis, and degradation of 70- to 95-mg rat soleus muscles during 120 min of incubation at 20 and 37 degrees C. At 37 degrees C, muscles were characterized by a damaged central core region and a decline of isometric tension development during incubation. Protein synthesis in the core region at 37 degrees C was depressed relative to the peripheral region. At 20 degrees C, developed tension remained constant during incubation, and synthesis rates in the core region were not different from the peripheral region. Compared with fresh muscle, ATP concentration after incubation was not affected by temperature. After equilibration of phenylalanine specific activity between extracellular and intracellular spaces (60 min at 20 degrees C; 30 min at 37 degrees C), rates of protein synthesis at 20 [0.048 nmol tyrosine (Tyr) X mg wet mass-1 X 2 h-1] and 37 degrees C (0.160 nmol Tyr X mg wet mass-1 X 2 h-1) were linear up to 180 and 120 min, respectively. Rates of protein degradation at 20 (0.076 nmol Tyr X mg wet mass-1 X 2 h-1) and 37 degrees C (0.248 nmol Tyr X mg wet mass-1 X 2 h-1) measured after 60 min were linear up to 180 and 120 min, respectively. Incubation at 20 degrees C offers an approach to study 70- to 95-mg muscles in vitro without compromising structure and function.

Neurovascular-anastomosed muscle grafts in rabbits: functional deficits result from tendon repair.

In rabbits, 9 g rectus femoris (RFM) muscles were grafted with tendon repair and with (1) nerves and blood vessels intact, (2) nerves intact and blood vessels anastomosed, or (3) nerves and blood vessels anastomosed. The influences of tendon, nerve, and vascular repair on the functional capabilities of grafts were compared 15-120 days after grafting. Data were collected on the mass, total protein content, oxidative capacity, maximum force development, and fatigability of grafts and control RFM muscles. When stabilized 90-120 days after grafting, mean values for the three types of grafts were not significantly different. Compared with values for control RFM muscles, each type of graft had significantly lower mean values for mass, total protein content, and maximum force, but the grafts were more resistant to fatigue. In RFM grafts, nerve and vascular repair do not contribute significantly to the impairments. Consequently, tendon repair appears to be responsible for the major functional deficits.

delta-Aminolaevulinate synthase expression in muscle after contractions and recovery.

The synthesis of haem has been postulated to be a key regulatory step in muscle mitochondrial biogenesis. We examined the expression of delta-aminolaevulinate synthase (ALAs), the regulatory enzyme of haem metabolism, in 10 Hz electrically stimulated and non-stimulated control rat tibialis anterior (TA) muscle. ALAs activity and mRNA levels were measured at 0, 18 and 48 h of recovery after 3 h of acute stimulation, or after 7 days of stimulation (3 h/day). ALAs activity in control muscles averaged 7.8 +/- 0.8 nmol/h per g (n = 30). After 3 h of stimulation and during recovery, no change in ALAs activity occurred. ALAs mRNA during the same time was unchanged except at 48 h of recovery, when it increased 1.3-fold above control (P < 0.05). After 7 days of stimulation, ALAs activity was unchanged at 0 h, but increased at 18 and 48 h of recovery to 2.0- and 1.8-fold above control (P < 0.05). ALAs mRNA was also increased, but to a level averaging 1.6-fold above control (P < 0.05) at all times, indicating an increased mRNA stability or synthesis. No change in the haem-containing enzyme cytochrome c oxidase (CYTOX) activity occurred after 3 h of stimulation in the red section of the TA. After 7 days of stimulation, the increase in CYTOX activity averaged 1.7-fold above control (P < 0.05) at all times. Thus the induction of ALAs during recovery after 7 days was regulated by factors which not only change ALAs mRNA content, but which also affect ALAs mRNA at translational or post-translational steps. This induction occurred despite a 1.7-fold increase in CYTOX, implying that a precursor-product relationship does not always exist.

Characterization of serum-stimulated lipoprotein lipase from bovine heart.

1. A triglyceride (TG) lipase is present in whole homogenate and tissue extracts of beef myocardium with characteristics of lipoprotein lipase (LPL); i.e., activity is stimulated by serum, inhibited by NaCl and protamine sulfate, the protein binds to heparin-Sepharose, and the enzyme has an alkaline pH optimum. 2. This TG lipase, eluted from heparin-Sepharose at 0.9-1.0 M NaCl, has an apparent mol. wt of 64 K daltons. Its primary mRNA is 3.7 kb. 3. Expression of LPL mRNA and enzyme activities are in the ratio of approximately 20:8:1 for hearts of mouse, rat and beef, respectively and correlate with r = +0.99.

Expression of embryonic myosin heavy chain mRNA in stretched adult chicken skeletal muscle.

Chronic stretch of the chicken fast-twitch patagialis muscle increases the rate of growth and percentage of fast-twitch oxidative fibers. We have analyzed the effects of stretch on the expression of two previously identified "embryonic" myosin heavy chain (MHC) mRNAs (p251 and p110). Both MHC mRNAs were expressed in the patagialis at their highest levels in the embryo and 1 wk after hatching. During posthatch development (7-52 wk), the p110 mRNA was expressed in only trace quantities while the p251 mRNA was not detectable. After 2 wk of stretch of the patagialis in 7- or 38-wk-old birds, the p110 mRNA was increased to levels similar to that found in patagialis of newly hatched chicks, whereas expression of the p251 transcript was not affected. The existence of two other MHC mRNAs homologous to the p110 mRNA was suggested by the S1 mapping analysis, one of which was expressed at dramatically reduced levels in the stretched patagialis. It is concluded that stretch can cause selective alterations in the expression of developmentally regulated MHC isoforms in chicken fast-twitch muscle.