Skeletal Muscle Enzyme Research Articles

UPREGULATION OF ANTIOXIDANT CAPACITY BY ENDURANCE TRAINING IN PIGS FOLLOWING CHRONIC CORONARY ARTERY OCCLUSION

Growing evidence indicates that oxidative stress is associated with muscle fatigue and weakness in skeletal muscle of heart disease patients. Previously, our laboratory and others demonstrated that reactive oxygen species (ROS) and reactive nitrogen species (RNS) have the ability to inhibit key antioxidant enzymes in skeletal muscle, thus increasing susceptibility to oxidative stress. Endurance training has been shown to elevate skeletal muscle mantioxidant enzyme activities in healthy animal models. Chronic coronary artery occlusion in pigs was used to model heart disease and to test the following hypothesis: twelve weeks of treadmill endurance training will increase the antioxidant capacity in skeletal muscle in pigs with chronic coronary occlusion. Miniature Yucatan swine were divided into three groups: sedentary animals (n = 6) with coronary artery occlusion (SO); endurance trained animals (n = 6) with coronary occlusion (EO); and sedentary (n = 3) without coronary occlusion (CON). Skeletal muscle samples (triceps brachii) were extracted after a 12 week training period, and frozen in liquid nitrogen until analysis. Superoxide dismutase (total SOD, Mn-SOD isoform, and Cu, Zn-SOD isoform), glutathione peroxidase (GPX), and catalase (CAT) activities were measured. The quenching of ABTS+ radical was used as a marker of non-enzymatic antioxidant scavenging capacity (TASC). Total SOD, Mn-SOD, and Cu, Zn-SOD activities were significantly higher in EO than SO by 82.6%, 72.0%, and 112.0%, respectively. GPX activity was 176.6% greater in EO than in SO. CAT trended higher (P = 0.059) in EO when compared to SO. TASC was also significantly improved (12.7%) by exercise training over SO. All antioxidant measurements for EO were not different from CON, while SO values were lower than CON. These data indicate that endurance exercise training is critical for the protection of antioxidant capacity in pigs with chronic coronary occlusion. Grant support by NIH PO1-HL52490 and HL64931.

Activation of AMP-activated protein kinase increases mitochondrial enzymes in skeletal muscle.

Muscle contraction causes an increase in activity of 5'-AMP-activated protein kinase (AMPK). This study was designed to determine whether chronic chemical activation of AMPK will increase mitochondrial enzymes, GLUT-4, and hexokinase in different types of skeletal muscle of resting rats. In acute studies, rats were subcutaneously injected with either 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR; 1 mg/g body wt) in 0.9% NaCl or with 0.9% NaCl alone and were then anesthetized for collection and freezing of tissues. AMPK activity increased in the superficial, white region of the quadriceps and in soleus muscles but not in the deep, red region of the quadriceps muscle. Acetyl-CoA carboxylase (ACC) activity, a target for AMPK, decreased in all three muscle types in response to AICAR injection but was lowest in the white quadriceps. In rats given daily, 1 mg/g body wt, subcutaneous injections of AICAR for 4 wk, activities of citrate synthase, succinate dehydrogenase, and malate dehydrogenase were increased in white quadriceps and soleus but not in red quadriceps. Cytochrome c and delta-aminolevulinic acid synthase levels were increased in white, but not red, quadriceps. Carnitine palmitoyl-transferase and hydroxy-acyl-CoA dehydrogenase were not significantly increased. Hexokinase was markedly increased in all three muscles, and GLUT-4 was increased in red and white quadriceps. These results suggest that chronic AMPK activation may mediate the effects of muscle contraction on some, but not all, biochemical adaptations of muscle to endurance exercise training.

Activities of phospholipases A and lysophospholipases in glycolytic and oxidative skeletal muscles in the rabbit

Oxidative muscles contain more free fatty acids than glycolytic muscles, which could explain in part their higher sensitivity to oxidation. These fatty acids are partly the result of phospholipid hydrolysis catalysed by phospholipases A and lysophospholipases. Up to now, very little is known on the activities of these enzymes in skeletal muscles. This study deals with the activities of phospholipases A and lysophospholipases in five rabbit muscles covering a large range of metabolic types (oxidative Soleus and Semimembranosus proprius muscles, glycolytic Psoas major and Longissimus lumborum muscles and intermediate Gastrocnemius laterale muscle). The results showed that (a) phospholipases A and lysophospholipases had maximal activity at pH 8-9; (b) phospholipases A and lysophospholipases retained more than 50% of their maximal activity at pH 5.5-6, the ultimate pH of muscles; (c) lysophospholipases exhibited a higher activity than phospholipases A (4-7-fold higher in the oxidative muscles, 11-fold higher in the intermediate muscle and 18-23-fold higher in the glycolytic muscles); and (d) phospholipase A and lysophospholipase activities were higher in oxidative muscles than in glycolytic muscles (10-25-fold higher for phospholipases A and 4-5-fold higher for lysophospholipases). Thus oxidative muscles have a higher potential activity for post-mortem hydrolysis of phospholipids. © 2000 Society of Chemical Industry.

Turkey cytochrome c oxidase contains subunit VIa of the liver type associated with low efficiency of energy transduction.

Cytochrome c oxidase was isolated from turkey liver, heart and breast skeletal muscle and separated by SDS/PAGE. The N-terminal amino-acid sequence of subunit VIa from all tissues and internal sequences from the skeletal muscle enzyme show homology to the mammalian liver-type subunit VIaL, which was verified by isolation and sequencing of the cDNA of turkey subunit VIa. No cDNA corresponding to subunit VIaH (mammalian heart-type) could be found by RACE-PCR with mRNA from all turkey tissues. Measurement of proton translocation with the reconstituted enzymes from turkey liver and heart revealed H+/e- ratios below 0.5 that were independent of the intraliposomal ATP/ADP ratio, as previously found with the bovine liver enzyme. Under identical conditions, the bovine heart enzyme revealed H+/e- ratios of 0.85 at low and 0.48 at high intraliposomal ATP/ADP ratios. The results suggest that in birds the lower H+/e-ratio of cytochrome c oxidase participates in elevated resting metabolic rate and thermogenesis.

Evaluation of Methods for the Determination of Mitochondrial Respiratory Chain Enzyme Activities in Human Skeletal Muscle Samples

The quantification of mitochondrial enzyme activities in skeletal muscle samples of patients suspected of having mitochondrial myopathies is problematic. Therefore, we have evaluated different methods for the determination of activities cytochrome c oxidase and NADH:CoQ oxidoreductase in human skeletal muscle samples. The measurement of cytochrome c oxidase activity in the presence of 200 μM ferrocytochrome c and the detection of NADH:CoQ oxidoreductase as rotenone-sensitive NADH:CoQ1 reductase resulted in comparable citrate synthase-normalized respiratory chain enzyme activities of both isolated mitochondria and homogenates from control human skeletal muscle samples. These methods allowed the precise detection of deficiencies of respiratory chain enzymes in skeletal muscle of two patients harboring only 20 and 27% of deleted mitochondrial DNA, respectively. Therefore, citrate synthase-normalized respiratory chain activities can serve as stable reference values for the determination of a putative mitochondrial defect in human skeletal muscle.

Muscle fibres: applications for the study of the metabolic consequences of enzyme deficiencies in skeletal muscle

Mitochondrial function in saponin-permeabilized muscle fibres can be studied by high-resolution respirometry, laser-excited fluorescence spectroscopy and fluorescence microscopy. We applied these techniques to study metabolic effects of changes in the pattern of mitochondrial enzymes in skeletal muscle of patients with chronic progressive external ophthalmoplegia or Kearns-Sayre syndrome harbouring large-scale deletions of mitchondrial DNA (mtDNA). In all patients combined deficiencies of respiratory chain enzymes containing mitochondrially encoded subunits were observed. The citrate synthase-normalized activity ratios of these enzymes decreased linearly with increasing mtDNA heteroplasmy. This indicates the absence of any well-defined mutation thresholds for mitochondrial enzyme activities in the entire skeletal muscle. We applied metabolic control analysis to perform a quantitative estimation of the metabolic influence of the observed enzyme deficiencies. For patients with degrees of mtDNA heteroplasmy below about 60% we observed at almost normal maximal rates of respiration an increase in flux control coefficients of complexes I and IV. Permeabilized skeletal-muscle fibres of patients with higher degrees of mtDNA heteroplasmy and severe enzyme deficiencies exhibited additionally decreased maximal rates of respiration. This finding indicates the presence of a 'metabolic threshold' which can be assessed by functional studies of muscle fibres providing the link to the phenotypic expression of the mtDNA mutation in skeletal muscle.

Activity of antioxidant enzymes in rat skeletal muscle and brown fat: effect of cold and propranolol

1. Effects of acute (6 h) and chronic (21 day) cold (6°C) exposure, as well as propranolol (15 mg/kg) on the activities of CuZnSOD, MnSOD and catalase in the rat skeletal muscle (SM) and brown adipose tissue (BAT), which are important sites of cold-induced thermogenesis, were investigated. 2. The changes in the activity of antioxidant enzymes were tissue specific and dependent on the duration of cold exposure. Thus, in the SM of acutely cold exposed rats, the activity of all antioxidant enzymes studied was elevated, whereas in the BAT the activity of both SOD s decreased and that of catalase remained unchanged. In cold acclimated rats, the activity of all the three enzymes was increased in the BAT whereas in the SM, CuZnSOD activity was enhanced, MnSOD activity decreased and catalase activity returned to the control level. 3. Propranolol also differently altered the antioxidant enzyme activity in SM and BAT, alterations being dependent on the acclimation temperature. Thus, in room acclimated rats propranolol decreased the activity of all antioxidant enzymes in SM but did not affect those in BAT. However, in the SM propranolol prevented the elevation of MnSOD and catalase activities, induced by acute cold. In cold acclimated rats propranolol inhibited CuZnSOD activity in both SM and BAT but increased that of MnSOD.

Kinetics of thyroid hormone induced changes in liver and skeletal muscle enzymes of Clarias batrachus.

Kinetics of triiodothyronine (T3) induced changes were studied in cytoplasmic malate dehydrogenase (cMDH), mitochondrial malate dehydrogenase (mMDH) and lactate dehydrogenase (LDH) of the liver and skeletal muscle of a catfish, Clarias batrachus. The rates of gradual inductions in the activities of all the three metabolic enzymes were faster in skeletal muscle than those of the liver. These time-dependent and tissue-specific inductions may be due to the possible differences in the rates of different enzymic syntheses. The maximum inductions in the activities of cMDH, mMDH and LDH were recorded around 19 hr after T3 treatment. Thereafter, the activities of all the enzymes gradually declined to their half levels within the next 12 hr which reflected the physiological half-life of these metabolic enzymes in the freshwater catfish.

EFFECT OF TREADMILL TRAINING AND CLENBUTEROL TREATMENT ON BODY FAT ACCUMULATION AND MUSCLE ENZYME ACTIVITIES OF RATS.

1392 The effects of exercise training, chronic administration of clenbuterol(β&2-adrenergic agonist), and the combination of these two treatment on body fat content and muscle enzyme activities were investigated. Forty male Sprague-Dawley rats were randomly assigned to one of four treatment groups: sedentary control(CON), clenbuterol(CL), exercise trained(TR), or exercise trained/clenbuterol(TR+CL). The training protocol consisted of running on motor-driven treadmill up an 8% grade, 5 days/wk for 8 weeks. Rats receiving clenbuterol were intubated with 0.8 mg/kg body wt/day dissolved in deionized water. Exercise training and clenbuterol administration significantly not only reduced the body fat content as evidenced by the decreasing retroperitoneal fat weight, but also increased the skeletal muscle mass without the changes of growth rate. The combined effect of these two treatment in increasing the muscle mass was additive, but in reducing the fat content, it was not additive. Chronic clenbuterol treatment significantly reduced the hexokinase and citrate synthase activities in skeletal muscle. These decreased enzyme activities significantly reduced endurance performance. Thus, the results of the current study indicate that the effects of clenbuterol treatment are the same as the effects of exercise training in body composition, but each of clenbuterol treatment and training inversely affects the changes in skeletal muscle enzyme activities of normal rats. Supported by Korea Research Foundation.

Increased mRNAs for procollagens and key regulating enzymes in rat skeletal muscle following downhill running.

The purpose of the study was to investigate pre-translational regulation of collagen expression after a single bout of exercise. We analysed steady-state messenger ribonucleic acid (mRNA) levels for collagen types I, III and IV, alpha- and beta-subunits of prolyl 4-hydroxylase and lysyl oxidase (enzymes modifying procollagen chains), and enzyme activity of prolyl 4-hydroxylase from rat soleus muscle (MS) and the red parts of quadriceps femoris muscle (MQF) after 12 h and after 1, 2, 4, 7 and 14 days of downhill (-13.5 degrees ) treadmill running at a speed of 17 m.min-1 for 130 min. Histological and biochemical assays revealed exercise-induced muscle damage in MQF but not MS. Steady-state mRNA levels for the alpha- and beta-subunits of prolyl 4-hydroxylase in MQF, lysyl oxidase in MS and MQF were increased 12 h after running, whereas prolyl 4-hydroxylase activity did not increase until 2 days after exercise. The mRNA levels for the fibrillar collagens (I and III) and basement membrane type IV collagen significantly increased 1 day and 12 h after exertion, respectively. Peak mRNA levels were observed 2-4 days after running, the increases being more pronounced in MQF than in MS. No significant changes were observed in types I or III collagen at the protein level. Strenuous downhill running thus causes an increase in gene expression for collagen types I and III and their post-translational modifying enzymes in skeletal muscle in a co-ordinated manner. These changes, together with the increased gene expression of type IV collagen, may represent the regenerative response of muscle extracellular matrix to exercise-induced injury and an adaptive response to running exertion.

Changes in skeletal and cardiac muscle enzymes during the Scottish Coast to Coast Triathlon.

While skeletal muscle injury is common after prolonged exercise, evidence in the literature supporting cardiac muscle injury is conflicting. Creatine kinase and cardiac troponin-I were measured, in 31 amateur athletes (25 male) before, and 12-24 hours after, a 300 km cycling/running/canoe triathlon event. A short questionnaire was used to assess level of fitness, training and previous experience. Creatine kinase levels were greater after the 45 km cross-country run compared with after a 155 km road cycle (60.5 +/- 62.8 iu/L/kg vs 19.3 +/- 9.6 iu/kg, P = 0.03). Individuals performing running and cycling events consecutively had creatine kinase similar to those observed after running alone (50.2 +/- 53.8 iu/L/kg vs 60.5 +/- 62.8 iu/L/kg, P = 0.55). Cardiac troponin-I was elevated above the normal range (0.1 ng/L) in six athletes (four in running and cycling events, one in the running and one in the cycling event). We conclude that running produces significantly more skeletal muscle injury than cycling and that strenuous endurance exercise involving running and cycling in amateur trained athletes is associated with release of cardiac specific enzymes. The functional and longer term consequences of this require further study.

Induced mutant mouse lines that express lipoprotein lipase in cardiac muscle, but not in skeletal muscle and adipose tissue, have normal plasma triglyceride and high-density lipoprotein-cholesterol levels.

The tissue-specific expression of lipoprotein lipase (LPL) in adipose tissue (AT), skeletal muscle (SM), and cardiac muscle (CM) is rate-limiting for the uptake of triglyceride (TG)-derived free fatty acids and decisive in the regulation of energy balance and lipoprotein metabolism. To investigate the tissue-specific metabolic effects of LPL, three independent transgenic mouse lines were established that expressed a human LPL (hLPL) minigene predominantly in CM. Through cross-breeding with heterozygous LPL knockout mice, animals were generated that produced hLPL mRNA and enzyme activity in CM but lacked the enzyme in SM and AT because of the absence of the endogenous mouse LPL gene (L0-hLPL). LPL activity in CM and postheparin plasma of L0-hLPL mice was reduced by 34% and 60%, respectively, compared with control mice. This reduced LPL expression was sufficient to rescue LPL knockout mice from neonatal death. L0-hLPL animals developed normally with regard to body weight and body-mass composition. Plasma TG levels in L0-hLPL animals were increased up to 10-fold during the suckling period but normalized after weaning and decreased in adult animals. L0-hLPL mice had normal plasma high-density lipoprotein (HDL)-cholesterol levels, indicating that LPL expression in CM alone was sufficient to allow for normal HDL production. The absence of LPL in SM and AT did not cause detectable morphological or histopathological changes in these tissues. However, the lipid composition in AT and SM exhibited a marked decrease in polyunsaturated fatty acids. From this genetic model of LPL deficiency in SM and AT, it can be concluded that CM-specific LPL expression is a major determinant in the regulation of plasma TG and HDL-cholesterol levels.

Chemotaxin-dependent translocation of immunoreactive ADP-ribosyltransferase-1 to the surface of human neutrophil polymorphs.

mRNA from human polymorphonuclear neutrophil leucocytes (PMNs) was probed with cDNA encoding human skeletal muscle arginine-specific ADP-ribosyltransferase (ART1). A single 2.6-kb transcript was identified, which was similar in size to that observed in human skeletal muscle RNA. An 872-bp cDNA fragment, corresponding to the amino acid sequence of the processed human skeletal muscle enzyme, was generated by reverse transcription-PCR amplification of RNA from human PMNs, and was found to be identical to the ART1 cDNA derived from human skeletal muscle. ART1 was expressed as a fusion protein with glutathione S-transferase (GST) in insect cells, and antibodies were raised against the fusion protein in a rabbit. Following removal of GST immunoreactivity by immunoprecipitation, these antibodies were used to measure the abundance of immunoreactive ART1 on the surface of PMNs. Exposure of PMNs to formyl-Met-Leu-Phe (FMLP) was followed by a rapid increase in the abundance of cell surface ART1 (T1/2 = 1.9 min), and the concentration of FMLP for half-maximum response was 28.6 nM. Similar responses were observed after exposure of the cells to platelet-activating factor or interleukin-8, and we conclude that some of the effects of these chemotaxins are mediated by translocation of an intracellular pool of ART1 to its site of catalytic activity on the outer aspect of the plasma membrane.

Novel Aspects of Tetramer Assembly and N-terminal Domain Structure and Function Are Revealed by Recombinant Expression of Human AMP Deaminase Isoforms

AMP deaminase isoforms purified from endogenous sources display smaller than predicted subunit molecular masses, whereas baculoviral expression of human AMPD1 (isoform M) and AMPD3 (isoform E) cDNAs produces full-sized recombinant enzymes. However, nearly 100 N-terminal amino acid residues are cleaved from each recombinant polypeptide during storage at 4 degreesC. Expression of N-truncated cDNAs (DeltaL96AMPD1 and DeltaM90AMPD3) produces stable recombinant enzymes exhibiting subunit molecular masses and kinetic properties that are similar to those reported for purified isoforms M and E. Conversely, wild type recombinant isoforms display significantly higher Km(app) values in the absence of ATP. Gel filtration analysis demonstrates native tetrameric structures for all recombinant proteins, except the wild type AMPD1 enzyme, which forms aggregates of tetramers that disperse upon cleavage of N-terminal residues at 4 degreesC. These data: 1) confirm that available literature on AMP deaminase is likely derived from N-truncated enzymes and 2) are inconsistent with a new model proposing native trimeric structure of an N-truncated rabbit skeletal muscle AMP deaminase (Ranieri-Raggi, M., Montali, U., Ronca, F., Sabbatini, A., Brown, P. E., Moir, A. J. G., and Raggi, A. (1997) Biochem. J. 326, 641-648). N-terminal residues also influence actomyosin-binding properties of the enzyme, which are enhanced and suppressed by AMPD1 and AMPD3 sequences, respectively. Finally, co-expression of AMPD1 and AMPD3 recombinant polypeptides produces tetrameric enzymes with either isoform-specific or mixed subunits, and also reveals that tetramer assembly is driven by relative polypeptide abundance with no apparent preference for like subunits.

Nitric oxide synthase I (NOS I) is a costameric enzyme in rat skeletal muscle

Previously, we and others have reported association of nitric oxide (NO)-generating nitric oxide synthase I (NOS I) with dystrophin in the subsarcolemmal cytoskeleton of striated muscle fibers. Since this structure shows a costameric organization the detailed distribution of NOS I and other molecules inside and outside the subsarcolemmal cytoskeleton was investigated. Using catalytic histochemistry and immunohistochemistry on rat skeletal muscle NOS I was colocalized in the costameres together with dystrophin, beta-dystroglycan, alpha-, beta- and gamma-sarcoglycan, beta 1-integrin, vinculin, paxillin and caveolin-3. Additionally, only NOS appeared in uncharacterized subsarcolemmal wave-like structures. These data show 1) a growing family of proteins assembled in the costameres including NOS I as described here for the first time, 2) expanded distribution patterns for NOS I, and 3) therefore, presumably uneven NO concentrations within the skeletal muscle fibers which may have implications for NO function.

Drug-Induced Rhabdomyolysis

The syndrome of rhabdomyolysis is the result of skeletalmuscle injury that alters the integrity of the sarcolemmaand leads to the eventual release of intracellular contentsinto the plasma. The causes are diverse, and includemuscle trauma (for instance, from vigorous exercise, crushinjuries, battering, or seizures), inadequate bloodperfusion, heat stroke, electrolyte imbalance, hereditaryenzyme deficiencies, infections and ingestion of drugs andtoxins. Various serious medical disorders, particularlythose resulting in states of disordered energy production,are also associated with rhabdomyolysis.In general, drug toxicity involves organs, such as thekidney, liver, gastrointestinal tract and the central nervoussystem, with skeletal muscle being usually less readilyaffected. Although drug-induced rhabdomyolysis wasuncommon in the past, it is no longer rare, due to theintroduction of more and increasingly potent drugs intoclinical practice.The incidence of drug-induced rhabdomyolysis isuncertain, largely because most of it is unreported.Similarly, the mortality rates are unknown. However, ithas been suggested that rhabdomyolysis from all causesleads to 5%-25% of cases of acute renal failure.

Influence of acclimation temperature on mitochondrial DNA, RNA, and enzymes in skeletal muscle.

Skeletal muscle fibers typically undergo modifications in their mitochondrial content, concomitant with alterations in oxidative metabolism that occur during the development of muscle fiber and in response to physiological stimuli. We examined how cold acclimation affects the mitochondrial properties of two fish skeletal muscle fiber types and how the regulators of mitochondrial content differed between tissues. After 2 mo of acclimation to either 4 or 18 degrees C, mitochondrial enzyme activities in both red and white muscle were higher in cold-acclimated fish. No significant differences were detected between acclimation temperatures in the abundance of steady-state mitochondrial mRNA (cytochrome-c oxidase 1, subunit 6 of F0F1-ATPase), rRNA (16S), or DNA copy number. Steady-state mRNA for nuclear-encoded respiratory (adenine nucleotide translocase 1) and glycolytic genes showed high interindividual variability, particularly in the cold-acclimated fish. Although mitochondrial enzymes were 10-fold different between the two muscle types, mitochondrial DNA copy number differed only 4-fold. The relative abundance of mitochondrial mRNA and nuclear mRNA in red and white muscle reflected the differences in copy number of their respective genes. These data suggest that the response to physiological stimuli and determination of tissue-specific mitochondrial properties likely result from the regulation of nuclear-encoded genes.

Associations between skeletal muscle properties, physical fitness, physical activity and coronary heart disease risk factors in men

High physical fitness and physical activity are associated with favourable lipid levels, especially a high level of high density lipoprotein cholesterol (HDL-C). A person's skeletal muscle properties, metabolism and percentage of different muscle fibres (ST-%), which may modify coronary heart disease (CHD) risk factors, such as serum insulin, obesity and serum sex hormones may also influence his fitness level and leisure-time physical activity. We studied the associations of physical fitness, physical activity and ST-% with serum lipids and lipoproteins in 72 healthy men. Their parameters were compared with those of 20 men with defined CHD. Significant interrelationships between ST-%, fitness and leisure-time physical activity index (LTPAI) were observed. Multiple regression analysis showed that ST-%, fitness and leisure-time physical activity explained about 32% of the variation in HDL-C in the healthy men. In healthy men ST-% correlated positively with fitness ( r s=0.62, P<0.001) and with LTPAI ( r s=0.62, P<0.001). Fitness level also correlated significantly with LTPAI ( r s=0.81, P<0.001). Serum insulin showed negative associations with ST-% ( r s=−0.63, P<0.001) and fitness ( r s=−0.54, P<0.001) and LTPAI ( r s=−0.62, P<0.001). Free fraction of testosterone correlated negatively with serum HDL-C level ( r s=−0.34, P<0.01), with fitness ( r s=−0.41, P<0.001) and with LTPAI ( r s=−0.54, P<0.001). In sedentary men with the lowest fitness and physical activity the mean of ST-% (45%) was similar to that in CHD patients (44%). However, ST-% in men in the highest tertile of physical activity and fitness (68%) was significantly higher than in CHD patients and in men in the lowest tertile of physical activity and fitness. Skeletal muscle enzyme activity in lipid metabolism was significantly lower in both CHD patients and in sedentary and low-fit men than that in fitter and physically active men. The present data imply that skeletal muscle properties are important determinants of risk profiles, such as physical activity, fitness and serum lipid and lipoprotein patterns. Although fitness is a graded, independent predictor of mortality from CHD, a relatively high fitness level is not enough. This was clearly observed in the clustering analysis, in which the healthy men, according to their ST-%, fitness, leisure-time physical activity and serum sex hormone binding globulin (SHBG), fell into three natural groups: (i) Inactive men with lowest ST-% (mean 42%), lowest fitness (10.7 METs) and lowest HDL-C (1.36 mm/l); (ii) Fit men with high ST-% (66%), high fitness (14.5 METs) and moderately high HDL-C (1.54 mol/l); (iii) Active men with high ST-% (66%), highest fitness (14.9 METs) and highest serum HDL (1.83 mmol/l). The results support the idea that both fitness and physical activity give further protection against CHD by modifying risk factors. Our findings also suggest that skeletal muscle properties should be considered in the studies which assess CHD risk factors and their modifications especially in the field of health-related fitness.

Effect of microgravity on the expression of mitochondrial enzymes in rat cardiac and skeletal muscles.

The purpose of this study was to examine the expression of nuclear and mitochondrial genes in cardiac and skeletal muscle (triceps brachii) in response to short-duration microgravity exposure. Six adult male rats were exposed to microgravity for 6 days and were compared with six ground-based control animals. We observed a significant 32% increase in heart malate dehydrogenase (MDH) enzyme activity, which was accompanied by a 62% elevation in heart MDH mRNA levels after microgravity exposure. Despite modest elevations in the mRNAs encoding subunits III, IV, and VIc as well as a 2.2-fold higher subunit IV protein content after exposure to microgravity, heart cytochrome c oxidase (CytOx) enzyme activity remained unchanged. In skeletal muscle, MDH expression was unaffected by microgravity, but CytOx activity was significantly reduced 41% by microgravity, whereas subunit III, IV, and VIc mRNA levels and subunit IV protein levels were unaltered. Thus tissue-specific (i.e., heart vs. skeletal muscle) differences exist in the regulation of nuclear-encoded mitochondrial proteins in response to microgravity. In addition, the expression of nuclear-encoded proteins such as CytOx subunit IV and expression of MDH are differentially regulated within a tissue. Our data also illustrate that the heart undergoes previously unidentified mitochondrial adaptations in response to short-term microgravity conditions more dramatic than those evident in skeletal muscle. Further studies evaluating the functional consequences of these adaptations in the heart, as well as those designed to measure protein turnover, are warranted in response to microgravity.

Redox regulation of antioxidant enzymes in skeletal muscle

Redox regulation of antioxidant enzymes in skeletal muscle