Myosin heavy chain composition in the rat diaphragm: effect of age and exercise training.

Increases in aerobic capacity in both young and senescent rats consequent to endurance exercise training are now known to occur not only in locomotor skeletal muscle but also in diaphragm. In the current study the effects of aging and exercise training on the myosin heavy chain (MHC) composition were determined in both the costal and crural diaphragm regions of female Fischer 344 rats. Exercise training [treadmill running at 75% maximal oxygen consumption (1 h/day, 5 day/wk, x 10 wk)] resulted in similar increases in plantaris muscle citrate synthase activity in both young (5 mo) and old (23 mo) trained animals (P < 0.05). Computerized densitometric image analysis of fast and slow MHC bands revealed the ratio of fast to slow MHC to be significantly higher (P < 0.005) in the crural compared with costal diaphragm region in both age groups. In addition, a significant age-related increase (P < 0.05) in percentage of slow MHC was observed in both diaphragm regions. However, exercise training failed to change the relative proportion of slow MHC in either the costal or crural region.     

Diaphragm contractile dysfunction in MyoD gene-inactivated mice

MyoD is one of four myogenic regulatory factors found exclusively in skeletal muscle. In an effort to better understand the role that MyoD plays in determining muscle contractile properties, we examined the effects of MyoD deletion on both diaphragmatic contractile properties and myosin heavy chain (MHC) phenotype. Regions of the costal diaphragm from wild-type and MyoD knockout [MyoD (-/-)] adult male BALB/c mice (n = 8/group) were removed, and in vitro diaphragmatic contractile properties were measured. Diaphragmatic contractile measurements revealed that MyoD (-/-) animals exhibited a significant (P < 0.05) downward shift in the force-frequency relationship, a decrement in maximal specific tension (P(o); -33%), a decline in maximal shortening velocity (V(max); -37%), and concomitant decrease in peak power output (-47%). Determination of MHC isoforms in the diaphragm via gel electrophoresis revealed that MyoD elimination resulted in a fast-to-slow shift (P < 0.05) in the MHC phenotype toward MHC types IIA and IIX in MyoD (-/-) animals. These data indicate that MyoD deletion results in a decrease in diaphragmatic submaximal force generation and P(o), along with decrements in both V(max) and peak power output. Hence, MyoD plays an important role in determining diaphragmatic contractile properties.     

Training-induced alterations in young and senescent rat diaphragm muscle.

The current study sought to examine the effects of chronic endurance treadmill running on oxidative capacity and capillary density in specific diaphragm muscle fiber types in young (5 mo) and senescent (greater than or equal to 23 mo) female Fischer 344 rats. Both young and senescent animals trained at approximately 75% of maximal O2 consumption for 1 h/day 5 days/wk for 10 wk. Plantaris citrate synthase activity was significantly increased (P less than 0.01) in both young and old trained groups. Densitometric analysis of succinate dehydrogenase (SDH) activity in diaphragm type I, IIa, and IIb muscle fibers was done using a computerized image-processing system. There were no age-related differences in SDH activity between the young and old groups for any of the fiber types. In addition, SDH activity was found to be significantly increased (P less than 0.05) in all three fiber types in both the young and senescent trained animals compared with their sedentary counterparts. Fiber size and capillary density did not differ between young and senescent rats, nor did exercise affect this measure. Each fiber, irrespective of type, had an average of approximately four capillaries in contact with it. However, type IIb fibers had a significantly lower capillary density per unit area than type I or IIa muscle fibers. The results indicate that the senescent costal diaphragm maintains its ability to adapt to an increased metabolic demand brought about by locomotor exercise. Of further interest is the finding that training adaptations occurred in all three fiber types, suggesting that increased work of breathing from moderate exercise leads to recruitment of all three fiber types.(ABSTRACT TRUNCATED AT 250 WORDS)     

Aging and respiratory muscle metabolic plasticity: effects of endurance training.

We examined the oxidative and antioxidant enzyme activities in respiratory and locomotor muscles in response to endurance training in young and aging rats. Young adult (4-mo-old) and old (24-mo-old) female Fischer 344 rats were divided into four groups: 1) young trained (n = 12), 2) young untrained (n = 12), 3) old trained (n = 10), and 4) old untrained (n = 6). Both young and old endurance-trained animals performed the same training protocol during 10 wk of continuous treadmill exercise (60 min/day, 5 days/wk). Compared with young untrained animals, the young trained group had significantly elevated (P less than 0.05) activities of 3-hydroxyacyl-CoA dehydrogenase (HADH), glutathione peroxidase (GPX), and citrate synthase (CS) in both the costal diaphragm and the plantaris muscle. In contrast, training had no influence (P greater than 0.05) on the activity of lactate dehydrogenase within the costal diaphragm in young animals. In the aging animals, training did not alter (P greater than 0.05) activities of CS, HADH, GPX, or lactate dehydrogenase in the costal diaphragm but significantly (P less than 0.05) increased CS, HADH, and GPX activities in the plantaris muscle. Furthermore, training resulted in higher activities of CS and HADH in the intercostal muscles in the old trained than in the old untrained animals. Finally, activities of CS, HADH, and GPX were significantly (P less than 0.05) lower in the plantaris in the old untrained than in the young untrained animals; however, CS, HADH, and GPX activities were greater (P less than 0.05) in the costal diaphragm in the old sedentary than in the young untrained animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alterations in diaphragmatic oxidative and antioxidant enzymes in the senescent Fischer 344 rat.

We investigated age-related changes in antioxidant, glycolytic, beta-oxidation, and tricarboxylic acid cycle enzyme activity in the diaphragm and plantaris muscle of female Fischer 344 rats. Tissue samples from the costal and crural diaphragm and plantaris muscle were obtained from 30 animals in the following age groups: 1) 6 mo old (n = 10), 2) 26 mo old (n = 10), and 3) 30 mo old (n = 10). Aging had no effect (P greater than 0.05) on the activities of citrate synthase (CS) and 3-hydroxyacyl-CoA dehydrogenase (HADH) in the costal or crural diaphragm. Similarly, no age-related differences existed (P greater than 0.05) in the crural diaphragm in lactate dehydrogenase (LDH) or glutathione peroxidase (GPX) activity. In contrast, the activities of LDH and GPX were significantly (P less than 0.05) higher in the costal diaphragm in the 30- than in the 6-mo old animals. In addition, the ratio of LDH to CS activity increased (P less than 0.05) as a function of age in the costal diaphragm. Conversely, the ratio of CS to GPX activity in the costal diaphragm was lower (P less than 0.05) in the 30- than in the 6-mo old animals. No significant (P greater than 0.05) age-related differences existed in LDH-to-CS or CS-to-GPX activity ratios in the crural diaphragm. Finally, aging resulted in a significant decrease (P less than 0.05) in the activities of LDH, CS, and HADH in the plantaris muscle. These data demonstrate that, unlike many hindlimb locomotor muscles, the oxidative capacity of the Fischer 344 rat diaphragm does not decrease in old age.     

A physiological level of clenbuterol does not prevent atrophy or loss of force in skeletal muscle of old rats.

Supraphysiological levels of clenbuterol (CL) reduce muscle degradation in both young and old animals; however, these pharmacological levels induce side effects that are unacceptable in the elderly. In this study, we tested the hypothesis that a "physiological" dose of CL (10 microg. kg(-1). day(-1)) would attenuate the loss of in situ isometric force and mass in muscles of senescent rats during hindlimb suspension (HS). Adult (3 mo) and senescent (38 mo) Fischer 344 x Brown Norway rats received CL or a placebo during 21 days of normal-weight-bearing or HS conditions (8 rats/age group). HS reduced soleus muscle weight-to-body weight ratio by 31%, muscle cross-sectional area by 37%, and maximal isometric tetanic force (P(o)) by 76% in senescent rats. CL attenuated the loss of P(o) and muscle weight by 17 and 8%, respectively, in the soleus of senescent rats relative to HS+placebo conditions, but it did not improve muscle weight normalized for body weight. CL did not reduce the decrease in soleus P(o) or mass after HS in adult rats. CL failed to reduce the loss of plantaris weight (-20%) and P(o) (-46%) in senescent rats after HS. Our data support the conclusion that physiological levels of CL do not improve fast muscle atrophy and only modestly reduce slow muscle atrophy, and, therefore, it is largely an ineffective countermeasure for preventing muscle wasting from HS in senescent rats.     

Regional metabolic differences in the rat diaphragm

This study characterized the biochemical properties of the rat diaphragm by measuring the activities of selected citric acid cycle and glycolytic enzymes. The diaphragm was removed from 10 female Sprague-Dawley rats (180 days old) and dissected into five discrete anatomic regions: crural (region 1), left posterior costal (region 2), left anterior costal (region 3), right anterior costal (region 4), and right posterior costal (region 5). Sections were assayed for total protein concentration and the activities of succinate dehydrogenase (SDH) and lactate dehydrogenase (LDH). The SDH activity in the crural region was approximately 18% lower (P less than 0.05) than that in any costal region. Furthermore, protein concentration was significantly lower (P less than 0.05) in the crural region compared with all costal regions. In contrast, costal regions 2-5 did not significantly differ from each other in protein concentration or SDH activity. LDH activity did not differ significantly (P greater than 0.05) between regions. Finally, the LDH-to-SDH activity ratio was significantly higher (P less than 0.05) in the crural diaphragm compared with all costal regions. We conclude that the crural region of the rat diaphragm is significantly lower in oxidative capacity than all the costal regions. Investigators who use a rodent model to study diaphragmatic function and plasticity should consider the oxidative heterogeneity of the diaphragm when designing experiments.     

Senescence alters blood flow responses to acute heat stress

Renal and splanchnic sympathetic nerve discharge (SND) responses to heating are significantly reduced in senescent compared with young Fischer-344 (F344) rats (Kenney MJ and Fels RJ. Am J Physiol Regul Integr Comp Physiol 283: R513-R520, 2002). However, the functional significance of this finding is not known. We tested the hypothesis that blood flow distribution profiles to heating are altered in senescent (24 mo old) compared with mature (12 mo old) and young (3 mo old) F344 rats. Visceral organ, skeletal muscle, and tail blood flows were determined with the radionuclide-tagged microsphere technique before (control, 38 degrees C) and during heating that increased body temperature to 41 degrees C in anesthetized F344 rats. Vascular conductance in the kidney, stomach, large intestine, pancreas, spleen, and tail was significantly reduced during control before heating in senescent compared with young F344 rats. Heating significantly decreased kidney, stomach, small and large intestine, and pancreas vascular conductance in young and mature but not senescent F344 rats. Vascular conductance at 41 degrees C in the kidney and small intestine was significantly lower and in the stomach tended to be lower in young compared with senescent rats. Splenic conductance increased during heating in young and senescent rats but was highest in young rats. Tail conductance during heating was significantly increased in young rats but remained unchanged in mature and senescent rats. These results demonstrate a marked attenuation in heating-induced vascular conductance changes in senescent rats, suggesting an important functional consequence for the attenuated SND responses to heating in aged rats.     

Exercise training protects against contraction-induced lipid peroxidation in the diaphragm

Endurance exercise training promotes a small but significant increase in antioxidant enzyme activity in the costal diaphragm (DIA) of rodents. It is unclear if these training-induced improvements in muscle antioxidant capacity are large enough to reduce oxidative stress during prolonged contractile activity. To test the hypothesis that training-related increases in DIA antioxidant capacity reduces contraction-induced lipid peroxidation, we exercise trained adult female Sprague-Dawley (n = 7) rats on a motor-driven treadmill for 12 weeks at approximately 75% maximal O2 consumption (90 min/day). Control animals (n = 8) remained sedentary during the same 12-week period. After training, DIA strips from animals in both experimental groups were excised and subjected to an in vitro fatigue contractile protocol in which the muscle was stimulated for 60 min at a frequency of 30 Hz, every 2 s, with a train duration of 330 m. Compared to the controls, endurance training resulted in an increase (P < 0.05) in diaphragmatic non-protein thiols and in the activity of the antioxidant enzyme superoxide dismutase. Following the contractile protocol, lipid peroxidation was significantly lower (P < 0.05) in the trained DIA compared to the controls. These data support the hypothesis that endurance exercise training-induced increases in DIA antioxidant capacity protect the muscle against contractile-related oxidative stress.     

Collagen gene expression in rat left ventricle: interactive effect of age and exercise training.

Whether or not exercise training of sufficient intensity and duration to produce left ventricle (LV) hypertrophy also regulates deposition of interstitial collagen and cross-linking at the pretranslational level is unknown. Therefore, the effects of exercise training on gene expression for the two principal fibrillar collagens in LV, types I and III, were assessed in young adult (5 mo), middle-aged (15 mo), and old (26 mo) rats. We also evaluated the potential interaction of changes in mRNA for these procollagens with alterations in LV extracellular matrix characteristics by simultaneously measuring collagen concentration (hydroxyproline) and extent of mature collagen cross-linking (hydroxylysylpyridinoline, HP). Ten weeks of treadmill running resulted in LV hypertrophy and an increased maximal oxygen uptake in all three age groups of trained rats compared with sedentary controls. Percent collagen in rat LV almost doubled (P < 0.0001) from 5 to 26 mo of age, an increase unaffected by exercise training. With aging, a significant decline in expression of mRNAs for both collagen type I (P < 0.005) and type III (P < 0.001) was observed in LV free wall (LVF) but not septum (LVS). Training prevented this decline in LVF mRNAs for the two principal fibrillar collagens in middle-aged rats whereas it attenuated the decline in senescent animals. HP concentration increased significantly with aging in both LVF (P < 0.005) and LVS (P < 0.01). Training modulated this effect, but again only in LVF, so that HP was significantly lower (P < 0.05) in this region of the LV in old trained rats compared with sedentary counterparts. We conclude that exercise training modulates the effects of aging on collagen gene mRNAs and HP cross-linking regionally within the LV.     

Growth hormone restores aged diaphragm myosin composition and performance after chronic undernutrition.

The effects of growth hormone (GH) on diaphragm muscle myosin heavy chain (MHC) composition and mechanical performance were investigated in Fischer 344 male rats aged to senescence (24.5 mo of age). Chronic undernutrition (UN), refeeding (RF), and RF+GH were compared with ad libitum feeding by using a model of UN that produced a 50% decrease in body weight over a 12-mo period. The effect of aging was assessed by comparing MHC composition of ad libitum-fed rats at 12 and 24.5 mo of age. At senescence, significant decreases in slow type I (-23%) and fast type IIA (-31%) MHC had occurred with aging. Conversely, UN over this aging period increased types I (32-73%) and IIA (22-23%) MHC and decreased fast types IIB (32-54%) and IIX (30-31%) MHC. RF and RF+GH reversed these shifts back toward control values. At senescence, maximal specific force, maximal velocity, and specific power capacity were not different across treatment groups. During repetitive isotonic contraction trials, the diaphragms of UN rats maintained power production over time (54% of initial power at 60 s), whereas the power production of diaphragms of ad libitum-fed rats fell to 0% (P < 0.05). In comparison with UN rats, the diaphragms of RF and RF+GH rats produced 23 (not significant) and 11% (P < 0.05) of initial power, respectively, suggesting that RF+GH treatment restored performance characteristics after UN. We conclude that RF+GH can reverse alterations in MHC composition and mechanical performance produced by chronic UN in the aged rat diaphragm.     

Diaphragmatic perfusion heterogeneity during exercise with inspiratory resistive breathing

Regional distribution of diaphragmatic blood flow (Q; 15-microns-diam radionuclide-labeled microspheres) was studied in normal (n = 7) and laryngeal hemiplegic (LH; n = 7) ponies to determine whether the added stress of inspiratory resistive breathing during maximal exercise may cause 1) redistribution of diaphragmatic Q and 2) crural diaphragmatic Q to exceed that in maximally exercising normal ponies. LH-induced augmentation of already high exertional work of breathing resulted in diminished locomotor exercise capacity so that maximal exercise in LH ponies occurred at 25 km/h compared with 32 km/h for normal ponies. The costal and crural regions received similar Q in both groups at rest. However, exercise-induced increments in perfusion were significantly greater in the costal region of the diaphragm. At 25 km/h, costal diaphragmatic perfusion was 154 and 143% of the crural diaphragmatic Q in normal and LH ponies. At 32 km/h, Q in costal diaphragm of normal ponies was 136% of that in the crural region. Costal and crural diaphragmatic Q in LH ponies exercised at 25 km/h exceeded that for normal ponies but was similar to the latter during exercise at 32 km/h. Perfusion pressure for the three conditions was also similar. It is concluded that diaphragmatic perfusion heterogeneity in exercising ponies was preserved during the added stress of inspiratory resistive breathing. It was also demonstrated that vascular resistance in the crural and costal regions of the diaphragm in maximally exercised LH ponies remained similar to that in maximally exercising normal ponies.     

Aging alters the contribution of nitric oxide to regional muscle hemodynamic control at rest and during exercise in rats

Advanced age is associated with altered skeletal muscle hemodynamic control during the transition from rest to exercise. This study investigated the effects of aging on the functional role of nitric oxide (NO) in regulating total, inter-, and intramuscular hindlimb hemodynamic control at rest and during submaximal whole body exercise. We tested the hypothesis that NO synthase inhibition (N(G)-nitro-l-arginine methyl ester, l-NAME; 10 mg/kg) would result in attenuated reductions in vascular conductance (VC) primarily in oxidative muscles in old compared with young rats. Total and regional hindlimb muscle VCs were determined via radiolabeled microspheres at rest and during treadmill running (20 m/min, 5% grade) in nine young (6-8 mo) and seven old (27-29 mo) male Fisher 344 × Brown Norway rats. At rest, l-NAME increased mean arterial pressure (MAP) significantly by ～17% and 21% in young and old rats, respectively. During exercise, l-NAME increased MAP significantly by ～13% and 19% in young and old rats, respectively. Compared with young rats, l-NAME administration in old rats evoked attenuated reductions in 1) total hindlimb VC during exercise (i.e., down by ～23% in old vs. 43% in young rats; P < 0.05), and 2) VC in predominantly oxidative muscles both at rest and during exercise (P < 0.05). Our results indicate that the dependency of highly oxidative muscles on NO-mediated vasodilation is markedly diminished, and therefore mechanisms other than NO-mediated vasodilation control the bulk of the increase in skeletal muscle VC during the transition from rest to exercise in old rats. Reduced NO contribution to vasomotor control with advanced age is associated with blood flow redistribution from highly oxidative to glycolytic muscles during exercise.     

Sympathetic nerve regulation to heating is altered in senescent rats

Renal and splanchnic sympathetic nerve discharge (SND) responses to increased (38-41 degrees C) internal temperature were determined in anesthetized young (3-6 mo old), mature (12 mo old), and senescent (24 mo old) Fischer 344 (F344) rats. We hypothesized that SND responses would be altered in senescent and mature rats as demonstrated by attenuated sympathoexcitatory responses to heating and by the absence of hyperthermia-induced SND pattern changes. The following observations were made. 1) Renal and splanchnic SND responses were significantly increased during heating in young and mature but not in senescent rats. 2) At 41 degrees C, renal and splanchnic SND responses were higher in young compared with senescent rats, and renal SND was higher in mature than in senescent rats. 3) Heating changed the SND bursting pattern in young, but not in mature or senescent, rats. 4) SND responses to heating did not differ between baroreceptor-innervated (BRI) and sinoaortic-denervated (SAD) senescent rats but were higher in SAD compared with BRI young rats. These results demonstrate an attenuated responsiveness of sympathetic neural circuits to heating in senescent F344 rats.     

Endurance-training-induced cellular adaptations in respiratory muscles

Controversy exists concerning the adaptability of mammalian respiratory muscles in response to endurance training. We examined the effects of 8 wk of progressive treadmill exercise (45 min/day 5 days/wk) on the biochemical adaptations of rat diaphragm and intercostal muscles. Female Sprague-Dawley rats were randomly assigned to a sedentary control (n = 10) or an exercise-training group (n = 10). Endurance training resulted in an enhanced oxidative capacity in the anterior costal diaphragm as evidenced by a 29% increase (P less than 0.05) in the activity of succinate dehydrogenase (SDH) in trained animals compared with controls (4.15 +/- 0.13 vs. 3.21 +/- 0.17 mumol.g-1.min-1). Similarly, SDH activity in the intercostal muscles was 32% greater (P less than 0.05) in the trained animals than in the untrained animals (1.72 +/- 0.11 vs. 1.30 +/- 0.06 mumol.g-1.min-1). In contrast, the crural region of the diaphragm showed no significant increase (P greater than 0.05) in oxidative capacity as a result of the training program (3.28 +/- 0.12 vs. 3.13 +/- 0.18). Furthermore, training did not alter (P less than 0.05) lactate dehydrogenase activity in the intercostals or in the crural or the costal diaphragm. These data demonstrate that the oxidative capacity of the costal diaphragm and the intercostal muscles can be enhanced by increasing respiratory loads via regular endurance exercise. We speculate that the lack of metabolic adaptation in the crural region of the diaphragm was not due to limited plasticity of the fibers in this area but to failure to the exercise-training program to provide the appropriate stimulus for cellular adaptation.     

Age-related differences in apoptosis with disuse atrophy in soleus muscle

Muscle atrophy is associated with a loss of muscle fiber nuclei, most likely through apoptosis. We investigated age-related differences in the extent of apoptosis in soleus muscle of young (6 mo) and old (32 mo) male Fischer 344 x Brown Norway rats subjected to acute disuse atrophy induced by 14 days of hindlimb suspension (HS). HS-induced atrophy (reduction in muscle weight and cross-sectional area) was associated with loss of myofiber nuclei in soleus muscle of young, but not old, rats. This resulted in a significant decrease in the myonuclear domain (cross-sectional area per nucleus) in young and old rats, with changes being more pronounced in old animals. Levels of apoptosis (TdT-mediated dUTP nick end labeling and DNA fragmentation) were higher in soleus muscles of old control rats than young animals. Levels were significantly increased with HS in young and old rats, with the greatest changes in old animals. Caspase-3 activity in soleus muscle tended to be increased with age, but changes were not statistically significant (P=0.052). However, with HS, caspase-3 activity significantly increased in young, but not old, rats. Immunohistochemistry showed that the proapoptotic endonuclease G (EndoG, a mitochondrion-specific nuclease) was localized in the subsarcolemmal mitochondria in control muscles, and translocation to the nucleus occurred in old, but not young, control animals. There was no difference between EndoG total protein content in young and old control rats, but EndoG increased almost fivefold in soleus muscle of old, but not young, rats after HS. These results show that deregulation of myonuclear number occurs in old skeletal muscle and that the pathways involved in apoptosis are distinct in young and old muscles. Apoptosis in skeletal muscle is partly mediated by the subsarcolemmal mitochondria through EndoG translocation to the nucleus in response to HS.     

Regeneration and transplantation of muscles in old rats and between young and old rats.

In order to compare the regenerative ability of skeletal muscle between young (5 month) and old (26 month) rats, sliced or intact extensor digitorum longus muscles were freely autografted into young and old rats and also reciprocally grafted from young to old inbred animals and vice versa. Sixty days after grafting, the transplants were analyzed for contractile and histochemical properties. There was a relative similarity between the contraction times of both normal control muscles and of all groups of transplants, although the contraction time tended to be prolonged and histochemical fiber pattern was more often found to be uniform in grafts of senescent animals. All groups of transplants possessed histochemically heterogeneous fiber types at 60 days. The experiments demonstrate that skeletal muscle in old rats possesses a substantial degree of regenerative ability and that the free tranpllantation of entire muscles in old animals is feasible.     

Role of nitric oxide on diaphragmatic contractile failure in Escherichia coli endotoxemic rats

Contractile dysfunction of the respiratory muscles plays an important role in the genesis of respiratory failure during sepsis. Nitric oxide (NO), a free radical that is cytotoxic and negatively inotropic in the heart and skeletal muscle, is produced in large amounts during sepsis by a NO synthase inducible (iNOS) by LPS and/or cytokines. The aim of this study was to investigate whether iNOS was induced in the diaphragm of Escherichia coli endotoxemic rats and whether inhibition of iNOS induction or of NOS synthesis attenuated diaphragmatic contractile dysfunction. Rats were inoculated intravenously (IV) with 10 mg/kg of E. coli endotoxin (LPS animals) or saline (C animals). Six hours after LPS inoculation animals showed a significant increase in diaphragmatic NOS activity (L-citrulline production, P < 0.005). Inducible NOS protein was detected by Western-Blot in the diaphragms of LPS animals, while it was absent in C animals. LPS animals had a significant decrease in diaphragmatic force (P < 0.0001) measured in vitro. In LPS animals, inhibition of iNOS induction with dexamethasone (4 mg/kg IV 45 min before LPS) or inhibition of NOS activity with N(G)-methyl-L-arginine (8 mg/kg IV 90 min after LPS) prevented LPS-induced diaphragmatic contractile dysfunction. We conclude that increased NOS activity due to iNOS was involved in the genesis of diaphragmatic dysfunction observed in E. coli endotoxemic rats.     

Aging and acute exercise enhance free radical generation in rat skeletal muscle.

Reactive oxygen species (ROS) are implicated in the mechanism of biological aging and exercise-induced oxidative damage. The present study examined the effect of an acute bout of exercise on intracellular ROS production, lipid and protein peroxidation, and GSH status in the skeletal muscle of young adult (8 mo, n = 24) and old (24 mo, n = 24) female Fischer 344 rats. Young rats ran on a treadmill at 25 m/min and 5% grade until exhaustion (55.4 +/- 2.7 min), whereas old rats ran at 15 m/min and 5% grade until exhaustion (58.0 +/- 2.7 min). Rate of dichlorofluorescin (DCFH) oxidation, an indication of ROS and other intracellular oxidants production in the homogenate of deep vastus lateralis, was 77% (P < 0.01) higher in rested old vs. young rats. Exercise increased DCFH oxidation by 38% (P < 0.09) and 50% (P < 0.01) in the young and old rats, respectively. DCFH oxidation in isolated deep vastus lateralis mitochondria with site 1 substrates was elevated by 57% (P < 0.01) in old vs. young rats but was unaltered with exercise. Significantly higher DCFH oxidation rate was also found in aged-muscle mitochondria (P < 0.01), but not in homogenates, when ADP, NADPH, and Fe(3+) were included in the assay medium without substrates. Lipid peroxidation in muscle measured by malondialdehyde content showed no age effect, but was increased by 20% (P < 0.05) with exercise in both young and old rats. Muscle protein carbonyl formation was unaffected by either age or exercise. Mitochondrial GSH/ GSSG ratio was significantly higher in aged vs. young rats (P < 0.05), whereas exercise increased GSSG content and decreased GSH/GSSG in both age groups (P < 0.05). These data provided direct evidence that oxidant production in skeletal muscle is increased in old age and during prolonged exercise, with both mitochondrial respiratory chain and NADPH oxidase as potential sources. The alterations of muscle lipid peroxidation and mitochondrial GSH status were consistent with these conclusions.     

Respiratory muscle blood flows during physiological and chemical hyperpnea in the rat.

Whether the diaphragm retains a vasodilator reserve at maximal exercise is controversial. To address this issue, we measured respiratory and hindlimb muscle blood flows and vascular conductances using radiolabeled microspheres in rats running at their maximal attainable treadmill speed (96 +/- 5 m/min; range 71-116 m/min) and at rest while breathing either room air or 10% O(2)-8% CO(2) (balance N(2)). All hindlimb and respiratory muscle blood flows measured increased during exercise (P < 0.001), whereas increases in blood flow while breathing 10% O(2)-8% CO(2) were restricted to the diaphragm only. During exercise, muscle blood flow increased up to 18-fold above rest values, with the greatest mass specific flows (in ml. min(-1). 100 g(-1)) found in the vastus intermedius (680 +/- 44), red vastus lateralis (536 +/- 18), red gastrocnemius (565 +/- 47), and red tibialis anterior (602 +/- 44). During exercise, blood flow was higher (P < 0.05) in the costal diaphragm (395 +/- 31 ml. min(-1). 100 g(-1)) than in the crural diaphragm (286 +/- 17 ml. min(-1). 100 g(-1)). During hypoxia+hypercapnia, blood flows in both the costal and crural diaphragms (550 +/- 70 and 423 +/- 53 ml. min(-1). 100 g(-1), respectively) were elevated (P < 0.05) above those found during maximal exercise. These data demonstrate that there is a substantial functional vasodilator reserve in the rat diaphragm at maximal exercise and that hypoxia + hypercapnia-induced hyperpnea is necessary to elevate diaphragm blood flow to a level commensurate with its high oxidative capacity.