Forced exercise increases muscle mass in EAE despite early onset of disability.

We aimed to determine whether 10 days of treadmill exercise can increase skeletal muscle mass and intramuscular concentrations of brain derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in experimental autoimmune encephalomyelitis (EAE). Forty female Lewis rats were randomly assigned to either EAE sedentary (EAE-Sed), EAE exercise (EAE-Ex), Control sedentary (Con-Sed) and Control exercise (Con-Ex). Exercising animals completed a 10 day forced exercising training program. Hind limb skeletal muscles were excised and weighed with soleus muscle used for BDNF and NGF quantification. Statistical analysis was done using a one-way analysis of variance. Disability was more pronounced in the EAE-Ex group than in the EAE-Sed group. Exercising animals (EAE-Ex and Con-Ex) had significantly greater bilateral EDL, plantaris and gastrocnemius muscle mass compared to their sedentary animals (p=0.01). The EAE-Ex group had significantly higher NGF concentrations (1.98+/-0.3 pg/mg) compared to Con-Ex (0.96+/-0.07 pg/mg, p=0.003) and Con-Sed (1.2+/-0.2 pg/mg, p=0.04) groups. The main effect of exercise represented a significantly lower BDNF concentrations in the soleus of exercising animals compared to sedentary animals (p=0.03). Our study provides preliminary evidence that exercise increases skeletal muscle mass despite the early onset of disability in EAE animals.

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.

Effect of oral contraceptives on peripheral blood flow in untrained women at rest and during exercise.

BACKGROUND: Endothelium-derived nitric oxide is an important mediator of exercise-induced changes in skeletal muscle blood flow. Given the recently documented effects of estrogens on nitric oxide synthase, it is hypothesized that oral contraceptives (OC) containing estrogen would increase nitric oxide production at rest and after endurance exercise. Further, we postulated that OC use would augment skeletal muscle blood flow at rest and during exercise. METHODS: Fourteen women (non-smokers) were divided into two groups: control (CON; sedentary, normal menstrual-cycling women who have not used oral contraceptives for > or = 12 mons; 18-38 yrs old; n = 7), and oral contraceptive users (OC; sedentary women who have been using low-dose estrogen/progestin oral contraceptives for > or = 12 mons; 19-38 yrs old; n = 7). Measurements of forearm blood flow were obtained from each group, using strain gauge plethysmography, at rest and during an exercise protocol in which intermittent handgrip exercise was performed at 15%, 30%, and 45% of maximum voluntary contraction (MVC). Additionally, venous blood samples were taken before and after a 90 min treadmill walk for measurement of serum nitrate/nitrite, an indirect assessment of steady-state nitric oxide production. RESULTS: There was no difference in forearm blood flow (ml/min/ 100 cc tissue) at rest (CON = 2.7; OC = 2.8); however, the hyperemic response to handgrip exercise was significantly (p < 0.05) lower in the OC group at 30% (9.0 vs CON = 14.2) and 45% (12.0 vs CON=17.0) of MVC. Serum nitrate values at rest and following 90 min of treadmill walking did not differ between groups (p > 0.05). CONCLUSIONS: Contrary to our hypotheses, these data indicate a compromised hyperemic response in the forearm of OC users. Further, chronic OC use may not affect nitric oxide production during low intensity treadmill exercise.

Overexpression of IGF-I in skeletal muscle of transgenic mice does not prevent unloading-induced atrophy.

This study examined the association between local insulin-like growth factor I (IGF-I) overexpression and atrophy in skeletal muscle. We hypothesized that endogenous skeletal muscle IGF-I mRNA expression would decrease with hindlimb unloading (HU) in mice, and that transgenic mice overexpressing human IGF-I (hIGF-I) specifically in skeletal muscle would exhibit less atrophy after HU. Male transgenic mice and nontransgenic mice from the parent strain (FVB) were divided into four groups (n = 10/group): 1) transgenic, weight-bearing (IGF-I/WB); 2) transgenic, hindlimb unloaded (IGF-I/HU); 3) nontransgenic, weight-bearing (FVB/WB); and 4) nontransgenic, hindlimb unloaded (FVB/HU). HU groups were hindlimb unloaded for 14 days. Body mass was reduced (P < 0.05) after HU in both IGF-I (-9%) and FVB mice (-13%). Contrary to our hypothesis, we found that the relative abundance of mRNA for the endogenous rodent IGF-I (rIGF-I) was unaltered by HU in the gastrocnemius (GAST) muscle of wild-type FVB mice. High-level expression of hIGF-I peptide and mRNA was confirmed in the GAST and tibialis anterior (TA) muscles of the transgenic mice. Nevertheless, masses of the GAST and TA muscles were reduced (P < 0.05) in both FVB/HU and IGF-I/HU groups compared with FVB/WB and IGF-I/WB groups, respectively, and the percent atrophy in mass of these muscles did not differ between FVB and IGF-I mice. Therefore, skeletal muscle atrophy may not be associated with a reduction of endogenous rIGF-I mRNA level in 14-day HU mice. We conclude that high local expression of hIGF-I mRNA and peptide in skeletal muscle alone cannot attenuate unloading-induced atrophy of fast-twitch muscle in mice.

Nerve-responsive troponin I slow promoter does not respond to unloading.

We examined the regulation of the troponin I slow (TnIs) promoter during skeletal muscle unloading-induced protein isoform transition, by using a transgenic mouse line harboring the -4,200 to +12 base pairs region of the human TnIs promoter. Eighteen female transgenic mice ( approximately 30 g body mass) were randomly divided into two groups: weight-bearing (WB) controls (n = 9) and hindlimb unloaded (HU; n = 9). The HU mice were tail suspended for 7 days. Body mass was unchanged in the WB group but was reduced (-6%; P < 0.05) after the HU treatment. Absolute soleus muscle mass (-25%) and soleus mass relative to body mass (-16%) were both lower (P < 0.05) in the HU group compared with the WB mice. Northern blot analyses indicate that 7 days of HU result in a 64% decrease (P < 0.05) in the abundance of endogenous TnIs mRNA (microg/mg muscle) in the mouse soleus. Furthermore, there is a trend for the abundance of the fast troponin I mRNA to be increased (+34%). Analysis of transgenic chloramphenicol acetyltransferase activity in the soleus muscle revealed no difference (P > 0.05) between WB and HU groups. We conclude that additional elements are necessary for the TnIs gene to respond to an unloading-induced, slow-to-fast isoform transition stimulus.

Molecular events underlying skeletal muscle atrophy and the development of effective countermeasures.

Skeletal muscle adapts to loading; atrophying when exposed to unloading on Earth or in spaceflight. Significant atrophy (decreases in muscle fiber cross-section of 11-24%) in humans has been noted after only 5 days in space. Since muscle strength is determined both by muscle cross-section and synchronization of motor unit recruitment, a loss in muscle size weakens astronauts, which would increase risks to their safety if an emergency required maximal muscle force. Numerous countermeasures have been tested to prevent atrophy. Resistant exercise together with growth hormone and IGF-I are effective countermeasures to unloading as most atrophy is prevented in animal models. The loss of muscle protein is due to an early decrease in protein synthesis rate and a later increase in protein degradation. The initial decrease in protein synthesis is a result of decreased protein translation, caused by a prolongation in the elongation rate. A decrease in HSP70 by a sight increase in ATP may be the factors prolonging elongation rate. Increases in the activities of proteolytic enzymes and in ubiquitin contribute to the increased protein degradation rate in unloaded muscle. Numerous mRNA concentrations have been shown to be altered in unloaded muscles. Decreases in mRNAs for contractile proteins usually occur after the initial fall in protein synthesis rates. Much additional research is needed to determine the mechanism by which muscle senses the absence of gravity with an adaptive atrophy. The development of effective countermeasures to unloading atrophy will require more research.

Myogenic regulatory factors during regeneration of skeletal muscle in young, adult, and old rats.

Myogenic factor mRNA expression was examined during muscle regeneration after bupivacaine injection in Fischer 344/Brown Norway F1 rats aged 3, 18, and 31 mo of age (young, adult, and old, respectively). Mass of the tibialis anterior muscle in the young rats had recovered to control values by 21 days postbupivacaine injection but in adult and old rats remained 40% less than that of contralateral controls at 21 and 28 days of recovery. During muscle regeneration, myogenin mRNA was significantly increased in muscles of young, adult, and old rats 5 days after bupivacaine injection. Subsequently, myogenin mRNA levels in young rat muscle decreased to postinjection control values by day 21 but did not return to control values in 28-day regenerating muscles of adult and old rats. The expression of MyoD mRNA was also increased in muscles at day 5 of regeneration in young, adult, and old rats, decreased to control levels by day 14 in young and adult rats, and remained elevated in the old rats for 28 days. In summary, either a diminished ability to downregulate myogenin and MyoD mRNAs in regenerating muscle occurs in old rat muscles, or the continuing myogenic effort includes elevated expression of these mRNAs.

Association of insulin-like growth factor mRNA expressions with muscle regeneration in young, adult, and old rats.

The purpose of this study was to determine whether impaired regeneration of skeletal muscle in old rats can be attributed to diminished expression of insulin-like growth factor (IGF) mRNAs. Fischer 344 male rats aged 2 (young), 12 (adult), and 24 mo (old) were given an injection of the myotoxic anesthetic, bupivacaine, into the left tibialis anterior muscle. Muscle mass and protein concentration recovered to contralateral control values by 28 days in young, but not adult or old rats. The temporal and maximal expressions of IGF-I mRNA were similar during recovery from bupivacaine on days 5 and 10 in young, adult, and old rat muscles. IGF-I mRNA levels were reduced toward control levels in young rats by 15 days, but remained elevated in adult and old rats. IGF-I receptor mRNA in bupivacaine-injected muscle of old rats was elevated significantly higher than injected muscle of young and adult rats at recovery day 5. Five days after bupivacaine injection, IGF-II mRNA was increased 46-fold in young rats but was only increased fourfold in adult rats. Thereafter, IGF-II mRNA expression was similar for young, adult, and old rats at 10 and 15 days of recovery. In summary, we demonstrate that impaired regeneration of the tibialis anterior muscle in adult or old rats after bupivacaine-induced damage is associated with a prolonged elevation of IGF-I mRNA expression and/or diminished initial IGF-II mRNA expression.

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.

Regulation of contractile protein gene expression in unloaded mouse skeletal muscle.

NASA: Hindlimb unloading was performed on mice in an effort to study the regulation of contractile protein genes. In particular, the regulation of myosin heavy chain IIb was examined. During unloading, muscle fibers undergo a type conversion. Preliminary data from this study does not support the hypothesis that the fiber type conversion is due to an increase in promoter activity of fast isoform genes, such as myosin heavy chain IIb. The consequences of this finding are examined, with particular focus on other factors controlling gene regulation.^ieng

Alterations in phenotypic and contractile properties of the rat diaphragm: influence of hypothyroidism.

This study examined the influence of experimental hypothyroidism on myosin isoform distribution and contractile function of the costal diaphragm. Adult female Sprague-Dawley rats were randomly assigned to control (n = 12) or hypothyroid groups (n = 13) over a 6-wk treatment period. In comparison to the control group, in the hypothyroid group the relative distribution of type I myosin heavy chain (MHC) was increased 35% (P < 0.05), whereas type IIb MHC decreased 63% (P < 0.05). Similarly, Ca(2+)-activated myosin adenosinetriphosphatase activity (nmol Pi.mg-1.min-1) in the hypothyroid group was reduced 30% compared with the control group (P < 0.05). Furthermore, significant reductions in diaphragmatic maximal tetanic specific tension (Po; N.cm-2; -21%) and maximal shortening velocity (Vmax; muscle length/s; -25%) were observed in the hypothyroid group. These data provide the first evidence that hypothyroid produces a fast-(type IIb) to-slow (type I) shift in costal diaphragmatic MHC isoform profile that is highly correlated to the observed decrease in Vmax. Finally, the present findings indicate that hypothyroidism does not alter myofibrillar content or noncontractile elements of the diaphragm, thereby suggesting an alternative mechanism(s) to explain the reduction in specific Po.

Clenbuterol-induced fiber type transition in the soleus of adult rats.

This study examined the effects of 6 weeks of treatment with the beta(2)-adrenoceptor agonist, clenbuterol, on the soleus muscle of adult female Sprague-Dawley rats. Animals (4 months old) were divided into two groups: clenbuterol treated (CL, n = 7) (2 mg.kg-1 body mass injected subcutaneously every other day), and control (CON, n = 7) (injected with isotonic saline). Post-treatment body weights were approximately 5% greater in the CL group compared to CON (P < 0.05). Polyacrylamide gel electrophoresis (SDS-PAGE) of soleus myofibrillar protein indicated a clenbuterol-induced decrease (P < 0.05) in the relative percentage of type I myosin heavy chain (MHC) with a concomitant increase (P < 0.05) in type IIdx MHC, while the proportion of type IIa MHC was unaffected. ATPase fiber typing revealed increases (P < 0.05) in the proportion of type II fibers expressed both as a percentage of total fiber number and total cross-sectional area (CSA). Finally, mean type II fiber CSA was approximately 25% greater (P < 0.05) in the CL groups as compared to the CON group. These data indicate that clenbuterol treatment results in alterations in the MHC phenotype and an increased proportion of type II fiber CSA in the soleus of adult rats. These observations were due to an increase in the total number of type II fibers, as well as hypertrophy of these fibers. Thus, the relative increase in the number of histochemically determined type II fibers and the emergence of the normally unexpressed type IIdx MHC isoform in the soleus suggest a clenbuterol-induced transition of muscle fiber phenotype as well as selective hypertrophy of the type II fibers.

No effect of aging on skeletal muscle insulin-like growth factor mRNAs.

This study examined the hypothesis that during aging insulin-like growth factor (IGF) mRNAs are reduced in skeletal muscle. IGF-I, IGF-II, and IGF-binding protein-5 (IGFBP-5) mRNAs were measured with a ribonuclease protection assay in the gastrocnemius of specific pathogen-free Fischer-344 rats. We hypothesized that IGF-I, IGF-II, and IGFBP-5 mRNA concentration (normalized to 18S RNA) in the gastrocnemius muscle of growing animals (3 mo) would be downregulated in a coordinated manner with muscle size during aging-associated atrophy. As indicated by muscle wet weight and total protein content, the gastrocnemius muscle was growing in the 3-mo group (P < 0.01 smaller compared with 12 mo), fully developed at 12 mo, and was atrophied at 24 mo of age (P < 0.05 compared with 12 mo). IGF-I mRNA concentration in the gastrocnemius of 12- and 24-mo-old rats was 39-49% less than in 3-mo-old rats (P < 0.05). Contrary to our hypothesis, there was not a significant skeletal muscle IGF-I mRNA difference between middle age (12 mo) and senescence (24 mo). Thus IGF-I mRNA changed during maturation (3-12 mo) but not during aging (12-24 mo). Skeletal muscle IGF-II mRNA concentration was not different among 3-, 12-, and 24-mo-old animals. Furthermore, animal age did not have an effect on IGFBP-5 mRNA concentration. We conclude that the aging-associated atrophy of skeletal muscle is not caused by altered pretranslational regulation of IGF-I, IGF-II, or IGFBP-5 in skeletal muscle.

Myosin heavy chain composition in young and old rat skeletal muscle: effects of endurance exercise.

The objective of this study was to determine the effects of age and exercise on the myosin heavy chain (MHC) composition of skeletal muscle. Young (3 mo) and old (22 mo) female specific pathogen-free barrier-reared Fischer 344 rats were randomly assigned to young untrained or young trained and old untrained or old trained groups, respectively. Young trained and old trained animals performed endurance exercise training on a motorized treadmill for 8 wk. Succinate dehydrogenase activity and MHC isoforms were measured in the plantaris (Plan), lateral and medial gastrocnemius (Gast), and soleus (Sol) muscles. In sedentary animals, aging resulted in a decrease (P < 0.05) in type IIb MHC and an increase (P < 0.05) in type IIa MHC in both the Gast and Plan muscles. Also, aging resulted in a small but significant increase (approximately 4%; P < 0.05) in type I MHC in the Sol. Exercise training resulted in significant (P < 0.05) increases in Gast, Plan, and Sol succinate dehydrogenase activity in both young and old animals. Furthermore, exercise training resulted in a decrease (P < 0.05) in the percentage of type IIb MHC and an increase (P < 0.05) in the percentage of type IIa MHC in the Plan in both young and old animals. These data suggest that there is an age-related shift in locomotor muscle MHC isoforms from a faster to a slower isoform.

Gender differences in diaphragmatic metabolic properties of the adult Sprague-Dawley rat.

Adult male Sprague-Dawley rats (> or = 180 days old) develop an obesity-exacerbated insulin resistance in contrast with female animals of the same strain. Given the fact the maintenance of muscle mass requires an adequate supply of insulin and active insulin receptors, we postulated that gender differences might exist in both protein content and metabolic properties of skeletal and cardiac muscle in adult Sprague-Dawley rats. Therefore, to test this hypothesis, we examined activities of bioenergetic enzymes and total protein content in the diaphragm, the heart and the plantaris muscle in 12-month-old male and female animals. Mean (+/- SD) body weights of male animals were significantly (P < 0.05) greater than female animals (598 +/- 8 vs. 362 +/- 19 g) and the diaphragm weight/body weight ratio was significantly lower in males compared to females (2.36 +/- 0.05 vs. 3.02 +/- 0.13 mg/g). The activities of isocitrate dehydrogenase (NADP-specific) and succinate dehydrogenase were significantly lower (P < 0.05) in male animals compared to females in both the crural and costal regions of the diaphragm, the heart, and the plantaris muscle. In contrast, no gender differences (P > 0.05) existed in lactate dehydrogenase activity in any of the muscles studied. Finally, muscle protein concentration was significantly higher in female animals when compared to males (P < 0.05) in all muscles studied except the heart. These data support the hypothesis that gender differences exist for adult Sprague-Dawley rats in general and specific protein content of the diaphragm, locomotor muscles, and the heart.

Inducibility of NADP-specific isocitrate dehydrogenase with endurance training in skeletal muscle.

NADP(+)-specific isocitrate dehydrogenase (ICDH-NADP: EC 1 x 1 x 1 x 42) is primarily a mitochondrial matrix enzyme in both mammalian skeletal muscle and heart and has markedly higher activity levels than the NAD(+)-specific isozyme. To date, it is unknown whether ICDH-NADP activity is inducible with in vivo exercise training in locomotor or respiratory skeletal muscle. Therefore, the purpose of this investigation was to quantify alterations in ICDH-NADP activity in respiratory muscles (costal and crural diaphragm) and locomotor muscles (medial gastrocnemius, plantaris and soleus) following 8 weeks of treadmill endurance training. Ten of the animals had been assigned randomly to an exercise group (TR) and had completed 8 weeks of progressive (5 days week-1: 45 min day-1) treadmill endurance training while the remaining 10 animals comprised a sedentary control (C). Mean ICDH-NADP activities in Tr were significantly higher (P < 0.05) when compared with C in the medial gastrocnemius (61.3%), plantaris (42.9%) soleus (21.4%). Mean costal diaphragm ICDH-NADP activity noted in trained animals when compared to the sedentary control group was not significantly higher (10.8% greater for TR; P = 0.14). No mean differences (P = 0.58) were noted in the crural diaphragm. The results indicate that ICDH-NADP is inducible with endurance training in locomotor skeletal muscle. A coefficient of determination of 0.624 (i.e. 62.4% of the variance could be explained) for ICDH-NADP was calculated, with the oxidative enzyme marker succinate dehydrogenase (P < 0.05) indicating a positive, moderate relationship.(ABSTRACT TRUNCATED AT 250 WORDS)