Increased circulating levels of glucocorticoids observed in many catabolic conditions play a major role in the induction of muscle atrophy. Indeed, inhibition of glucocorticoid action by glucocorticoid receptor antagonist attenuates and, in some cases, abolishes muscle atrophy. Circulating and tissue levels of IGF-I, a growth factor that stimulates the development of muscle mass, are frequently reduced in response to glucocorticoids. This decline could therefore trigger muscle atrophy in catabolic conditions. Indeed, systemic administration of IGF-I prevents glucocorticoid-induced muscle atrophy. However, use of systemic IGF-I administration is limited by its hypoglycemic and cardiac hypertrophic actions. Moreover, local IGF-I seems to play a more important role in the regulation of muscle mass than systemic IGF-I. Therefore, to limit loss of muscle mass observed in catabolic states, IGF-I administration must mimic as close as possible the autocrine production of IGF-I. The aim of this thesis was to investigate whether the restoration of IGF-I muscle content could reverse muscle atrophy induced by glucocorticoids. In this work we have tested the hypothesis that the local decrease in muscle IGF-I content might be responsible for the muscular atrophy induced by glucocorticoids. In our work, we have demonstrated that localized overexpression of IGF-I by gene electrotransfer prevents muscle atrophy in glucocorticoid-treated rats. High rate of fiber transfection and long term gene expression were obtained by combining multiple injection sites of DNA with electroporation. Human IGF-I gene electrotransfer using this optimised protocol resulted in increased muscle IGF-I mRNA and protein levels together with prevention of loss of skeletal muscle mass. Furthermore, alterations in the Akt/GSK-3â/â-catenin signaling pathway caused by glucocorticoids were prevented by local IGF-I gene overexpression. Finally, muscle overexpression of caAkt, dnGSK-3b and ÄNb-catenin was sufficient to mimic the anti-atrophic effect of IGF-I supporting the role of this signalling pathway in muscle atrophy caused by glucocorticoids. Taken together, our results show, for the first time in vivo, the role of the IGF-I/Akt/GSK-3b/b-catenin pathway in the skeletal muscle atrophy caused by glucocorticoids. In conclusion, our work highlights the crucial role of decreased muscle IGF-I in glucocorticoid-induced muscle atrophy. Indeed, the data presented in this thesis support the fact that the atrophic action of glucocorticoids is in part due to the downregulation of IGF-I, leading to the inhibition of its signalling pathways while restoration of muscle IGF-I levels is able to counteract totally muscle atrophy.

Recently, it has been postulated that fatigue is a mechanism to protect the muscle fiber from deleterious ATP depletion and cell death. The ATP-sensitive potassium (KATP) channel is believed to play a major role in this mechanism. Under metabolic stress, the channels open, reducing membrane excitability, Ca2+ release and force production. This alleviates energy demand within the fiber, as activation of the channel reduces ATP consumption from cellular ATPases. Loss of KATP channel activity during fatigue results in excessive intracellular Ca2+ ([Ca2+]i) levels, likely entering the fiber through L-type Ca2+ channels. It has been demonstrated that when mouse muscle lacking functional KATP channels are stimulated to fatigue, ATP levels become significantly lower than wild type levels. Thus, it was hypothesized that a lack of KATP channel activity impairs energy metabolism, resulting in insufficient ATP production. The focus of work for this M.Sc. project was to test this hypothesis. Fatigue was elicited in Kir6.2-/- FDB muscles for three min followed by 15 min recovery. After 60 sec, a 2.6-fold greater glycogen breakdown was observed in Kir6.2-/- FDB compared to wild type FDB. However, this effect disappeared thereafter, as there were no longer any differences between wild type and Kir6.2-/- FDB in glycogen breakdown by 180 sec. Glucose oxidation after 60 sec was also greater in Kir6.2-/- FDB compared to wild type FDB. However, levels of oxidation failed to increase in Kir6.2-/- FDB from 60 to 180 sec. Calculated ATP production during the fatigue period was 2.7-times greater in Kir6.2-/- FDB, yet measured ATP levels during fatigue are much lower in Kir6.2-/- FDB compared to wild type FDB. Taken together, it appears that muscle energy metabolism is impaired in the absence KATP channel activity.

Changes in intracellular Ca2+ ([Ca2+]f) and high-energy phosphates are known to induce adaptive changes in skeletal muscle during endurance exercising training, including mitochondrial biogenesis. Levels of [Ca2+]f are regulated by sarco(endo)plasmic reticulum Ca2+-ATPases (SERCAs) which are further regulated by sarcolipin (SLN), through a reduction in the apparent affinity of SERCAs for Ca2+. Furthermore, SLN reduces the efficiency of Ca2+ transport by SERCAs supporting a thermogenic role for SLN in skeletal muscle. Thus, it is possible SLN ablation could reduce Ca2+ and metabolic signaling during exercise training and attenuate increases in mitochondrial content. To investigate the potential role of SLN in the exercise-induced adaptive response of skeletal muscle, mice devoid of SLN (SLNKO) underwent endurance training for 8 weeks and were compared to WT controls. Maximal oxygen uptake (V̇O2 max) was measured with an exercise stress test while mitochondrial content was assessed through measurement of protein expression and maximal enzyme activities of several mitochondrial enzymes in soleus and extensor digitorum longus (EDL) muscles, which express high and low levels of SLN, respectively. All data were analyzed using a two-way analysis of variance (ANOVA) and student t-tests were conducted on enzyme data. V̇O2 max was found to not be significantly altered with exercise training in either genotype. Exercise training significantly increased the contents of adenine nucleotide translocase (ANT), cytochrome-c (cyt-c) and cytochrome-c oxidase subunit IV (COXIV) in soleus independent of genotype. Likewise, exercise training significantly increased cyt-c and COXIV expression (P<0.04), while increases in ANT expression were not significant (P=0.13) in the EDL. Two-way ANOVAs of mitochondrial enzymes in soleus revealed an interaction existed for succinate dehydrogenase (SDH) where its activity was increased only in the SLNKO mice (P<0.02). In comparison, exercise training significantly elevated activities of cytochrome c oxidase (COX) and citrate synthase (CS) activities (P<0.02) but not β-hydroxyacyl-CoA dehydrogenase (β-HAD; P=0.08), independent of genotype. Upon closer examination using student t-tests, it was determined that exercise training induced greater increases in COX and CS activity in SLNKO compared to WT controls (P<0.02), similar to and consistent with SDH data. In EDL, only SDH activity increased following exercise training, an effect that was independent of genotype. In conclusion, these data suggest that SLN ablation does not attenuate exercise-induced mitochondrial adaptations and may increase mitochondrial enzyme adaptations to exercise training in slow-twitch muscle. Further examination of the effects of SLN on Ca2+ and metabolic signaling may provide mechanisms explaining the results of this thesis.

Polyunsaturated fatty acids (PUFA) affect key cellular and physiological processes in the body ranging from cell signalling to inflammation. Compositional, dietary refinement and bioassay studies have shown strong associations between the PUFA composition of skeletal muscle with various contractile properties as well as the development of obesity and insulin resistance. The incorporation of PUFAs into rat soleus (slow-twitch oxidative), red gastrocnemius (fast-twitch oxidative), and white gastrocnemius (fast-twitch glycolytic) muscle were examined using stable isotope-labelled fatty acids. Two separate tracer studies were conducted. In the first study, four groups of rats were orally dosed with one of three isotopes of 18:2n-6; 13C18-18:2n-6 ethyl ester, 13C18- 18:2n-6 nonesterified fatty acid or 2H5-18:2n-6 ethyl ester and a control group received the vehicle only (olive oil). Animals were sacrificed 8 hours post dosing and soleus, red and white gastrocnemius muscles were collected for lipid analysis. In the second study, rats were orally administered a single dose of a mixture of 4 isotopes (13C18-18:2n-6, 2H5-18:3n-3, 13C16-16:0, and 2H2-18:1n-9) or vehicle only (olive oil) as a control. Groups of animals were sacrificed at 8, 24, and 48 h after dosing and four muscle types (heart, soleus, red and white gastrocnemius) were collected and analyzed for isotopic signal of these fatty acids and their corresponding desaturation and/or elongation products. Soleus accumulated significantly higher concentrations of labelled 18:2n-6, 18:3n-3 and most of n-6 fatty acids derived from 18:2n-6 followed by red gastrocnemius and white gastrocnemius. Heart muscle accumulated 20:5n-3 and 22:6n-3 increasingly over time while skeletal muscle accumulation was variable across muscle types. Labelled 20:5n-3 was detected in red and white gastrocnemius at 8 and 24 h with levels declining by 48 h while no 20:5n-3 was detected in soleus at anytime. Labelled 22:6n-3 was not detected in white gastrocnemius, but 22:6n-3 appeared to be increasing in red gastrocnemius over time. Soleus demonstrated a large accumulation of 22:6n-3 at 8 h with no detectable levels at 48 h. In conclusion we were able to demonstrate that the distribution and metabolism of various PUFAs differ in muscle types with distinct fibre type composition.

Polyunsaturated fatty acids (PUFA) affect key cellular and physiological processes in the body ranging from cell signalling to inflammation. Compositional, dietary refinement and bioassay studies have shown strong associations between the PUFA composition of skeletal muscle with various contractile properties as well as the development of obesity and insulin resistance. The incorporation of PUFAs into rat soleus (slow-twitch oxidative), red gastrocnemius (fast-twitch oxidative), and white gastrocnemius (fast-twitch glycolytic) muscle were examined using stable isotope-labelled fatty acids. Two separate tracer studies were conducted. In the first study, four groups of rats were orally dosed with one of three isotopes of 18:2n-6; 13C18-18:2n-6 ethyl ester, 13C18- 18:2n-6 nonesterified fatty acid or 2H5-18:2n-6 ethyl ester and a control group received the vehicle only (olive oil). Animals were sacrificed 8 hours post dosing and soleus, red and white gastrocnemius muscles were collected for lipid analysis. In the second study, rats were orally administered a single dose of a mixture of 4 isotopes (13C18-18:2n-6, 2H5-18:3n-3, 13C16-16:0, and 2H2-18:1n-9) or vehicle only (olive oil) as a control. Groups of animals were sacrificed at 8, 24, and 48 h after dosing and four muscle types (heart, soleus, red and white gastrocnemius) were collected and analyzed for isotopic signal of these fatty acids and their corresponding desaturation and/or elongation products. Soleus accumulated significantly higher concentrations of labelled 18:2n-6, 18:3n-3 and most of n-6 fatty acids derived from 18:2n-6 followed by red gastrocnemius and white gastrocnemius. Heart muscle accumulated 20:5n-3 and 22:6n-3 increasingly over time while skeletal muscle accumulation was variable across muscle types. Labelled 20:5n-3 was detected in red and white gastrocnemius at 8 and 24 h with levels declining by 48 h while no 20:5n-3 was detected in soleus at anytime. Labelled 22:6n-3 was not detected in white gastrocnemius, but 22:6n-3 appeared to be increasing in red gastrocnemius over time. Soleus demonstrated a large accumulation of 22:6n-3 at 8 h with no detectable levels at 48 h. In conclusion we were able to demonstrate that the distribution and metabolism of various PUFAs differ in muscle types with distinct fibre type composition.

There is substantial evidence that oxidative stress causes negative outcomes in many cell and tissue types. This is especially true of skeletal muscle, as it is continually subjected to various sources of reactive oxygen species (ROS). Oxidative stress in muscle has been linked to several disease states as well as to the normal aging process. Oxidative stress has also been associated with increased apoptotic signalling. Furthermore, elevated apoptosis is consistently observed in aged skeletal muscle and is thought to be one of the mechanisms of age-related muscle atrophy. Due to its post-mitotic nature, skeletal muscle may be more susceptible to the harmful effects of oxidative stress in light of its limited regenerative capacity. As a protective measure, a sophisticated antioxidant system exists in muscle consisting of both enzymatic (superoxide dismutases (SOD’s), catalase, glutathione peroxidase) and non-enzymatic elements (glutathione: GSH). GSH is a ubiquitously expressed tripeptide essential to maintenance of the redox status of the cell. Its role in skeletal muscle apoptosis, especially in different muscle types, is currently unclear. To elucidate the potential role of GSH in skeletal muscle apoptosis and oxidative stress, L-buthionine-[S,R]-sulfoximine (BSO) was used to deplete GSH in young (34.85 ± 0.68 wks) and old (69.11 ± 3.61 wks) male Sprague-Dawley rats. Thiol levels (GSH, GSSG), ROS production, 4-hydroxy-2-nonenal (4HNE) levels, DNA fragmentation and apoptosis-related protein expression were examined in soleus (SOL) and white gastrocnemius (WG) muscle. BSO led to significant GSH depletion (89% in SOL, 96% in WG) compared to age-matched controls. Catalase upregulation, in the absence of change in SOD levels, was evident as a result of BSO treatment and advancing age in both muscle tissues. BSO treatment also resulted in increased DNA fragmentation in WG and SOL, with elevated ROS production in SOL only; both of these effects were independent of age. Advancing age resulted in elevated caspase activity and Hsp70 protein content, with a concomitant decrease in anti-apoptotic ARC in SOL but not WG. Additionally, ROS production, 4HNE content, DNA fragmentation and ARC levels were all significantly elevated in SOL compared to WG. These data indicate that SOL may be subjected to a state of elevated cellular stress. There is also some evidence that GSH depletion increases DNA fragmentation while age contributes to a degradative loss of glycolytic muscle.

Sarcolipin (SLN) is a small sarcoplasmic reticulum (SR) integral membrane protein that regulates the SR Ca2+-ATPase (SERCA). Previous studies indicate that the functional interaction between SLN and SERCA is thermogenic in nature. Recently, SLN knock-out (SLNKO) mice have been shown to develop excessive obesity and glucose intolerance when placed on a high-fat diet (HFD; 42% kcal derived from fat) relative to wild-type (WT) littermates, implicating SLN in diet-induced obesity. The purpose of this thesis was two-fold: 1) to determine whether an excessively obese phenotype persists when SLNKO mice are given access to voluntary exercise, and 2) to determine if SLN ablation results in a deficit in skeletal muscle oxidative capacity, given the integral role cellular Ca2+ plays in mitochondrial metabolism. Mice were fed either standard chow or a HFD for 8 weeks, and remained sedentary or given access to voluntary running wheels during this period. Glucose tolerance was assessed pre- and post-diet, along with weight gain and adiposity. Skeletal muscle succinate dehydrogenase (SDH), citrate synthase (CS), cytochrome c oxidase (COX), and 3-hydroxyacyl CoA dehydrogenase (ß-HAD) activities were measured in the soleus (SOL) and extensor digitorum longus (EDL) of both chow- and high-fat fed sedentary mice. Both average daily running distance and total exercise volume were not different between WT and SLNKO mice given voluntary running wheels. As before, sedentary SLNKO mice gained more mass following the HFD relative to WT counterparts (P < 0.05); however, no difference in mass gain existed between genotype for voluntary exercising mice on a HFD. Despite this, SLNKO animals were more obese and glucose intolerant following high-fat feeding, regardless of activity status (P < 0.05). Under chow-fed conditions COX activity was higher in the EDL of SLNKO mice (P < 0.05), while no differences in SDH, CS, or ß-HAD existed between genotype in either muscle group. Following the HFD, no changes in mitochondrial enzyme activities within the SOL existed. COX activity in the EDL remained elevated in SLNKO mice post-HFD (P < 0.001), while ß-HAD increased in both WT and SLNKO animals relative to chow-fed controls (P < 0.05). These findings suggest that increasing energy expenditure through voluntary activity cannot compensate for increased basal SERCA Ca2+-pumping efficiency during caloric excess. Additionally, ablation of SLN does not result in a metabolic deficit within skeletal muscle, nor does it limit the adaptive enzymatic response of SLNKO mice to high-fat feeding. Thus, the findings of this study provide further support of the view that SLN’s thermogenic role is the primary mechanism of diet-induced obesity in SLNKO mice.

Skeletal muscle function is important to the human body for daily activities. Mechanical signals are critical to the maintenance of that function. Muscle diseases, such as the muscular dystrophies, in which the force transmission apparatus is compromised, have devastating effects on muscle function and quality of life. Mechanical signals activate intracellular signaling to maintain function. ERK2 has been shown to be quickly and strongly upregulated following stretch, leading to cell proliferation. Stretch has been shown to cause deformation of caveolae, invaginations of the plasma membrane that inhibit ERK signaling. This leads to the hypothesis that stretch induced deformation of caveolae may initiate mechanotransduction by activating ERK2. Reducing caveolin-3 expression via siRNA knockdown eradicated the stretch-induced effect on ERK2 activation, indicating that caveolin is required for the stretch response. Stabilizing caveolae structure by temperature reduction or destabilizing caveolae by cholesterol depletion resulted in changes consistent with the hypothesis that proper caveolae structure plays an important role in inhibition of signaling molecules and that deformation mediates mechanotransduction, resulting in changes in activation of ERK2.

An acute bout of resistance exercise (RE) and dietary protein consumption stimulate muscle protein synthesis (MPS). This anabolic effect is believed to be attenuated with resistance exercise training (RET), however, the mechanism for this plateau" is unknown. In addition, the ideal timing for protein consumption to optimize MPS is not well characterized. The central hypothesis of this research is that RE stimulates cumulative (measured over 24-36 h) MPS in rats and humans. Study one determined whether an acute bout of RE in rats enhances MPS when assessed with the traditional flooding dose (~ 25 min) and 2H2O (4 and 24 h measurements); thus a comparison of the two methodologies was made. An acute session of RE did not result in an elevation in MPS when quantified by either the flooding dose or 2H2O over 4 and 24 h (methods compared qualitatively). Therefore, an acute bout of RE in rats does not appear to be anabolic and adaptation resulting from multiple bouts is likely necessary. Study two determined if RET in rats results in attenuation in MPS (plateau effect) 16 h following the final RE session (peak anabolic window) and if it is due to an increase in 4E-BP1 (a key regulator of mRNA translation initiation) activity; or if the timing in anabolism changes, which could be detected with a cumulative assessment (2H2O). MPS at 16 h was unchanged following RE training. Consistent with this finding, there were no differences in 4E-BP1 activity. Conversely, cumulative MPS was significantly increased with RET, suggesting a temporal shift in anabolism. Study three determined if dietary protein consumed immediately following RE augments cumulative (24 h) MPS in young adult human males when energy and macronutrients are controlled. RE and post-RE protein had no effect on mixed MPS; however, myofibrillar MPS was significantly increased with RE suggesting specific changes within a heterogeneous protein pool. Collectively, these are the first studies to assess changes in cumulative MPS with RE in rats and humans. The long term goals of this research are to understand muscle protein anabolism in "free-living" mammals and the mechanisms that regulate this process.

Flying fish has specialized pectoral fins. When they are activated, they will rush out of the water, expand their pectoral fins and flap their caudal fin to glide. The pectoral fins are controlled by two groups of muscles in which the external appearance is pink. No histological investigations have been made on their muscles to verify whether they are red muscles. The purposes of this study were to compare the pectoral fin muscle, trunk white muscle and trunk red muscle by histological and proteome methods so as to understand if the pectoral fin muscles is red muscles and to infer their function. Cyselurus poecilopteins was used for this study, Result show that the sizes for the cross section of the pectoral-fin-muscle-fibers were between the white and red muscles, and a large amount of connective tissue and fat tissues are present in the space among the muscle cells. It is interpreted the pectoral fin muscles of flying fish might not belong to white muscle and red muscle, and they probably utilize lipid metabolism to provide enough energy for the gliding activates. The proteomic pages for the three muscle types were compared and differences were found in the muscle proteins: actin, myosin regulatory light chain, myosin light polypeptide; enzymes: isocitrate dehydrogenase, malate synthase, queuosine biosynthesis protein¡Fstress proteins: heat shock protein (HSP70 and HSP60). Expressions of these proteins were high in the pectoral-fin muscles than in the white and red muscles. These results suggest that the flying fish¡¦ pectoral-fin muscles may involve in the oxidative and glycolysis pathways, and the muscle fibers type maybe belong to an intermediate type of muscle fiber.