David S. Criswell

High intensity training-induced changes in skeletal muscle antioxidant enzyme activity

These experiments tested the hypothesis that high intensity (interval) training is superior to moderate intensity (continuous) exercise training in the upregulation of antioxidant enzyme activity in skeletal muscle. To test this postulate, we examined changes in oxidative and antioxidant enzyme activities in rat skeletal muscle following 12 wk of either interval (6 x approximately 5-min intervals at approximately 80-95% VO2max) or continuous (45 min at approximately 70% VO2max) exercise training. Both continuous and interval training resulted in significantly elevated (P < 0.05) succinate dehydrogenase (SDH) and 3-hydroxyacyl-CoA-dehydrogenase (HADH) activities in the gastrocnemius (G) and soleus (S) muscles compared with controls. SDH and HADH activities in the G and S muscles did not differ between the two exercise groups. Glutathione peroxidase (GPX) activity exceeded controls (P < 0.05) in only the interval trained S muscle. Soleus superoxide dismutase (SOD) activity was higher (P < 0.05) in both exercise groups compared with controls. No differences in SOD activity existed between interval and continuous trained animals. We conclude that when matched for oxygen cost, interval and continuous exercise training result in similar increases in SOD activity. However, high intensity interval exercise is superior to moderate intensity continuous exercise in the promotion of GPX activity in the S.

Nitric oxide and AMPK cooperatively regulate PGC-1α in skeletal muscle cells

Nitric oxide (NO) induces mitochondrial biogenesis in skeletal muscle cells via upregulation of the peroxisome proliferator‐activated receptor‐γ coactivator 1α (PGC‐1α). Further, we have shown that nitric oxide interacts with the metabolic sensor enzyme, AMPK. Therefore, we tested the hypothesis that nitric oxide and AMPK act synergistically to upregulate PGC‐1α mRNA expression and stimulate mitochondrial biogenesis in culture. L6 myotubes treated with nitric oxide donors, S‐nitroso‐N‐penicillamine (SNAP, 25 μm) or diethylenetriamine‐NONO (DETA‐NO, 50 μm), exhibited elevated AMPK phosphorylation, PGC‐1α mRNA and protein, and basal and uncoupled mitochondrial respiration (P < 0.05). Pre‐treatment of cultures with the AMPK inhibitor, Compound C, prevented these effects. Knockdown of AMPKα1 in L6 myotubes using siRNA reduced AMPKα protein content and prevented upregulation of PGC‐1α mRNA by DETA‐NO. Meanwhile, siRNA knockdown of AMPKα2 had no effect on total AMPKα protein content or PGC‐1α mRNA. These results suggest that NO effects on PGC‐1α expression are mediated by AMPKα1. Paradoxically, we found that the AMPK‐activating compound, AICAR, induced NO release from L6 myotubes, and that AICAR‐induced upregulation of PGC‐1α mRNA was prevented by inhibition of NOS with NG‐nitro‐l‐arginine methyl ester (l‐NAME, 1 mm). Additionally, incubation of isolated mouse extensor digitorum longus (EDL) muscles with 2 mm AICAR for 20 min or electrical stimulation (10 Hz, 13 V) for 10 min induced phosphorylation of AMPKα (P < 0.05), which was completely prevented by pre‐treatment with the NOS inhibitor, l‐NG‐monomethyl arginine (l‐NMMA, 1 mm). These data identify the AMPKα1 isoform as the mediator of NO‐induced effects in skeletal muscle cells. Further, this study supports a proposed model of synergistic interaction between AMPK and NOS that is critical for maintenance of metabolic function in skeletal muscle cells.

Antioxidant administration attenuates mechanical ventilation-induced rat diaphragm muscle atrophy independent of protein kinase B (PKB Akt) signalling.

Oxidative stress promotes controlled mechanical ventilation (MV)‐induced diaphragmatic atrophy. Nonetheless, the signalling pathways responsible for oxidative stress‐induced muscle atrophy remain unknown. We tested the hypothesis that oxidative stress down‐regulates insulin‐like growth factor‐1–phosphotidylinositol 3‐kinase–protein kinase B serine threonine kinase (IGF‐1–PI3K–Akt) signalling and activates the forkhead box O (FoxO) class of transcription factors in diaphragm fibres during MV‐induced diaphragm inactivity. Sprague–Dawley rats were randomly assigned to one of five experimental groups: (1) control (Con), (2) 6 h of MV, (3) 6 h of MV with infusion of the antioxidant Trolox, (4) 18 h of MV, (5) 18 h of MV with Trolox. Following 6 h and 18 h of MV, diaphragmatic Akt activation decreased in parallel with increased nuclear localization and transcriptional activation of FoxO1 and decreased nuclear localization of FoxO3 and FoxO4, culminating in increased expression of the muscle‐specific ubiquitin ligases, muscle atrophy factor (MAFbx) and muscle ring finger‐1 (MuRF‐1). Interestingly, following 18 h of MV, antioxidant administration was associated with attenuation of MV‐induced atrophy in type I, type IIa and type IIb/IIx myofibres. Collectively, these data reveal that the antioxidant Trolox attenuates MV‐induced diaphragmatic atrophy independent of alterations in Akt regulation of FoxO transcription factors and expression of MAFbx or MuRF‐1. Further, these results also indicate that differential regulation of diaphragmatic IGF‐1–PI3K–Akt signalling exists during the early and late stages of MV.

Mechanistic Links Between Oxidative Stress and Disuse Muscle Atrophy

Long periods of skeletal muscle inactivity promote a loss of muscle protein resulting in fiber atrophy. This disuse-induced muscle atrophy results from decreased protein synthesis and increased protein degradation. Recent studies have increased our insight into this complicated process, and evidence indicates that disturbed redox signaling is an important regulator of cell signaling pathways that control both protein synthesis and proteolysis in skeletal muscle. The objective of this review is to outline the role that reactive oxygen species play in the regulation of inactivity-induced skeletal muscle atrophy. Specifically, this report will provide an overview of experimental models used to investigate disuse muscle atrophy and will also highlight the intracellular sources of reactive oxygen species and reactive nitrogen species in inactive skeletal muscle. We then will provide a detailed discussion of the evidence that links oxidants to the cell signaling pathways that control both protein synthesis and degradation. Finally, by presenting unresolved issues related to oxidative stress and muscle atrophy, we hope that this review will serve as a stimulus for new research in this exciting field.

Effects of clenbuterol on contractile and biochemical properties of skeletal muscle

We investigated the effects of clenbuterol on the muscle mass, contractile properties, myosin phenotype, and bioenergetic enzyme activity in the gastrocnemius (GS)-plantaris (PL)-soleus (SO) muscle complex. Rats were sham-injected or treated with clenbuterol (2 mg.kg-1, subcutaneously) for 14 d. Clenbuterol increased (P < 0.05) body weight and muscle complex weight. Also, clenbuterol treatment resulted in an increase in total muscle force production and maximal shortening velocity (P < 0.05). No difference (P > 0.05) in relative force production (force.g-1 muscle) existed between experimental groups. However, muscle fatigue increased with clenbuterol treatment. Myosin heavy chain (MHC) composition was not altered in the GS or PL muscles, but shifted toward the fast Type II MHC in the SO. Myosin light chain (MLC) composition was not altered in any of the muscles. Clenbuterol caused a decrease in oxidative and glycolytic enzyme activity in the GS and PL, but not the SO. These data suggest that the clenbuterol-induced increase in muscle mass and maximal force generation is due to hypertrophy of both fast and slow fibers. Furthermore, these findings support the notion that beta-agonists may be beneficial in combating conditions that result in muscle wasting and dysfunction.

Regional training-induced alterations in diaphragmatic oxidative and antioxidant enzymes

We examined the relationship between the intensity and duration of exercise training and the up-regulation of diaphragmatic oxidative and antioxidant enzyme activities. Nine groups of rats exercised for 10 weeks (4 days/week). Groups of animals exercised at three intensities (low, moderate, and high); at each exercise intensity, a group of animals ran at one of three exercise durations (30, 60, and 90 min/day). Sedentary animals served as controls. Muscle oxidative capacity was assessed by citrate synthase (CS) activity while antioxidant capacity was evaluated by total superoxide dismutase (SOD) and total glutathione peroxidase (GPX) activities. All intensities and durations of exercise training promoted significant (P < 0.05) increases in costal diaphragmatic CS, SOD, and GPX activities. Increases in costal CS, SOD, and GPX activity were independent of the exercise intensity and duration. High and moderate intensity exercise of 90 min duration significantly elevated (P < 0.05) crural diaphragm CS activity. Further, high and moderate intensity exercise of durations > or = 60 min promoted significant (P < 0.05) increases in crural diaphragm SOD activities. Exercise did not influence (P > 0.05) crural diaphragm GPX activity. We conclude that the training threshold for up-regulation of oxidative and antioxidant enzyme activities differs between the costal and crural diaphragm.

Exercise-induced hypoxemia in athletes: role of inadequate hyperventilation

SummaryThese experiments examined the exercise-induced changes in pulmonary gas exchange in elite endurance athletes and tested the hypothesis that an inadequate hyperventilatory response might explain the large intersubject variability in arterial partial pressure of oxygen (Pa02) during heavy exercise in this population. Twelve highly trained endurance cyclists [maximum oxygen consumption (VO2max) range = 65-77 ml·kg−1·min−1] performed a normoxic graded exercise test on a cycle ergometer toVO2max at sea level. During incremental exercise atVO2max 5 of the 12 subjects had ideal alveolar to arterial P02 gradients (PA-aO2) of above 5 kPa (range 5-5.7) and a decline from restingPaO2 (ΔPaO2) 2.4 kPa or above (range 2.4-2.7). In contrast, 4 subjects had a maximal exercise (PA-aO2) of 4.0-4.3 kPa with ΔPaO2 of 0.4-1.3 kPa while the remaining 3 subjects hadPA-aO2 of 4.3-5 kPa with ΔPaO2 between 1.7 and 2.0 kPa. The correlation between PAO2 andPaO2 atVO2max was 0.17. Further, the correlation between the ratio of ventilation to oxygen consumption VSPaO2 and arterial partial pressure of carbon dioxide VSPaO2 atVO2max was 0.17 and 0.34, respectively. These experiments demonstrate that heavy exercise results in significantly compromised pulmonary gas exchange in approximately 40% of the elite endurance athletes studied. These data do not support the hypothesis that the principal mechanism to explain this gas exchange failure is an inadequate hyperventilatory response.

Adaptive strategies of respiratory muscles in response to endurance exercise.

The purpose of this review is to discuss the adaptive strategies of mammalian respiratory muscles in response to whole-body endurance exercise training. It is now clear that endurance training results in small (i.e., 20-30%) but significant increases in mitochondrial enzyme activity and the activities of key antioxidant enzymes (i.e., superoxide dismutase and glutathione peroxidase) within the rodent diaphragm. Interestingly, the magnitude of this training-induced increase in costal diaphragmatic oxidative and antioxidant enzyme activity is relatively independent of the exercise duration and intensity. Although the crural diaphragm of rodents is also capable of increasing its oxidative and antioxidant capacity in response to endurance training, high-to moderate-intensity exercise of long duration is required to promote these changes. Endurance training also increases the oxidative capacity of other key rodent inspiratory muscles, such as the parasternal intercostals and external intercostals. Furthermore, endurance training results in small (approximately 10%) increased in the oxidative capacity of key abdominal (expiratory) muscles. Whether the improvement in oxidative capacity of respiratory muscles is of significant magnitude to result in improvement in respiratory muscle performance remains an unanswered question.

Mechanism of specific force deficit in the senescent rat diaphragm

Aging is associated with a decline in the maximal in vitro specific force in the rat costal diaphragm. The purpose of this study was to determine if this force deficit is associated with a decrease in the concentration of myofibrillar protein in diaphragm fibers of senescent rats. Isometric twitch and tetanic contractile properties were measured on diaphragm strips from young adult (9-month-old: n = 12) and senescent (26-month-old: n = 13) male specific pathogen free-barrier protected Fischer 344 rats. Maximal tetanic force (Po) normalized to the cross-sectional area (CSA) of the in vitro diaphragm strips was 16.4% lower in the senescent diaphragms (21.03 +/- 0.4 N/cm2) compared to the young adult (25.16 +/- 0.5 N/cm2) (p < 0.001). Diaphragm water content was significantly higher in the senescent group (75.9% of total wet mass) compared to the young adult (72.1% of total wet mass, p < 0.05). Subtracting the contribution of water from the CSA of the diaphragm strips significantly reduced (p < 0.05) the senescent specific Po deficit (from -16.4 to -6.4%). Further, correcting Po for the contribution of myofibrillar protein to CSA resulted in no age group differences in specific force. These data indicate that the age-related decline in diaphragm in vitro maximal specific Po can be explained by an age-related increase in the water content of the diaphragm muscle. Future experiments are necessary to determine the mechanism(s) responsible for this observation.

Diaphragmatic fiber type specific adaptation to endurance exercise

Recent evidence suggests that exercise training results in a significant improvement in the oxidative capacity of the mammalian diaphragm; however, limited data exist concerning which diaphragmatic fiber types are metabolically altered due to training. To test the hypothesis that exercise training increases the oxidative capacity of diaphragmatic type I and IIa fibers only, we examined the effects of endurance training on the fiber type specific changes in oxidative capacity, cross-sectional area, and capillarity of the costal diaphragm. Female Fischer-344 rats (age ca 180 days) were divided into either sedentary control group (n = 6) or an exercise training group (n = 6). The trained animals exercised for 10 wks on a motor-driven treadmill (60 min.day-1; 5 days.wk-1) at a work rate equal to ca 55-65% VO2max. Capillaries were identified histologically and fiber types determined using ATPase histochemistry. Fiber cross-sectional area (CSA) and succinate dehydrogenase (SDH) activity in individual fibers were measured using a computerized image analysis system. Compared to control animals, training did not increase the capillary to fiber ratio in any diaphragm fiber type (P greater than 0.05); however, training increased capillary density (capillary No./CSA) in type IIa fibers due to a reduction in cell CSA (P less than 0.05). Further, training resulted in significant (P less than 0.05) increases in total diaphragmatic SDH activity (delta increase = 17.5%) and an increase in SDH activity in both type I (delta increase = 14%) and IIa fibers (delta increase = 17.4%). In contrast, training did not alter (P greater than 0.05) SDH activity in type IIb fibers.(ABSTRACT TRUNCATED AT 250 WORDS)