Ryanodine receptors (RyRs) are intracellular homotetrameric Ca2+-release channels constituting a family of three different isoforms, named RyRl, RyR2 and RyR3. RyRl and RyR2 are highly expressed in skeletal and cardiac muscles respectively, where they localize in the terminal cisternae of the sarcoplasmic reticulum (SR). Although RyRl and RyR2 have been found to be expressed in several other tissues at much lower level than in striated muscles, their major functional role is related to Ca2+-release from the SR following electrical depolarization of the plasma membrane, a process referred to as excitation-contraction (e-c) coupling and known to regulate striated muscle contraction. The third isoform, RyR3, is characterized by a wide pattern of expression, without any specific association to a tissue or a cell-type. The finding that RyR3 is also expressed in mammalian skeletal muscles parallels the presence of two distinct isoforms, o- and P-RyR, in non-mammalian vertebrate skeletal muscles, and suggests that two functionally distinct RyRs may be involved in the regulation of skeletal muscle contraction. The expression of RyR3 was analyzed in murine skeletal muscle from late foetal stages to adult, throughout neonatal phases of development. RyR3 was expressed widely during skeletal muscle post-natal development, disappearing in all muscles analyzed except diaphragm and soleus. RyR3 knockout mice were generated, and contractile properties of skeletal muscles were analyzed. Skeletal muscle contraction in RyR3-/- mice was impaired during the neonatal phase of development. In skeletal muscles isolated from RyR3-1- mice, the twitch elicited by electrical stimulation was strongly depressed. A significant reduction of the contractile activity was also elicited after stimulation with caffeine, an activator of Ca2+-release through RyRs. In the adults, no differences were detected between wild-type and mutant mice. These results are the first demonstrations of a physiological role of RyR3 in excitation-contraction coupling mechanisms of skeletal muscle, and support the model of a two-channel system regulating skeletal muscle contraction. In order to further characterize the RyR3-1- mouse, [3H]ryanodine binding experiments were performed on diaphragm and total hindlimb skeletal muscles from RyR3+/+ and RyR3-1- mice. Preliminary results will be presented and discussed.

Cachexia is a highly complex syndrome identified by metabolic, hormonal and cytokine-related abnormalities, but can be shortly characterized as accelerated skeletal muscle and adipose tissue loss in the context of a chronic inflammatory response. Cachexia is a debilitating complication of several diseases such as AIDS, sepsis, diabetes, renal failure, burn injury and cancer. Cachexia is responsible for 25-30% of cancer patient deaths. One of the most obvious outcomes of cancer cachexia is the redistribution of the total protein content, namely the depletion of skeletal muscle protein levels and increase in the acute phase response protein levels as a response to tissue injury. Although the plasticity of muscle mass and utility of skeletal muscle as a protein source are known facts, there have not been many studies concerning the mechanism of conversion of skeletal muscle proteins to other protein forms, for which the organism has greater need. IL-6 and activation of the acute phase response have been linked to cancer cachexia. However, IL-6 is generally not thought to signal directly on skeletal muscle and to date no studies have manipulated the STAT3 pathway for regulating skeletal muscle mass. Our data demonstrate direct action of IL-6 on activation of the STAT3 and acute phase response pathway at the skeletal muscle. In addition, our observations that STAT3 is broadly activated in cachexia and that STAT3 activation is sufficient and necesssary to induce muscle wasting are also novel. Thus, these studies define a new pathway leading to muscle wasting, which can be a potential target for reversing muscle wasting in cancer cachexia. Successful inhibition of skeletal muscle wasting and other metabolic derangements of cachexia will increase quality of life and survival of a significant fraction of cancer patients.

The purpose of this thesis was to investigate the role of lactate in the regulatory volume response of mammalian skeletal muscle to hypertonic challenge-induced cell shrinkage. It was demonstrated that adult mice skeletal muscle single fibres responded to increased extracellular osmolarity in a dose-dependent manner when exposed to NaCl or sucrose challenge. This regulatory response to sucrose and NaCl however was abolished when cells were pre-treated with bumetanide, a specific sodium-potassium-chloride cotransport (NKCC) inhibitor, demonstrating that the NKCC is primarily responsible for eliciting a regulatory volume increase (RVI). When cells were exposed to NaLac treatment, bumetanide incubation did not significantly diminish the ability of the cells to recover volume. Furthermore, these cells lost less volume compared to NaCl or sucrose control. Inhibiting the single muscle fibres with either monocarboxylate transport (MCT) inhibitor phloretin or pCMBS resulted in significantly greater volume loss and impaired volume recovery. Combined MCT inhibition of phloretin or pCMBS with NKCC inhibition (bumetanide) led to unexpected findings, whereby the cells lost very little volume. These data suggest that while skeletal muscle fibres may utilize the NKCC to regulate volume, the ability for these cells to employ the most efficient means of volume regulation involves the inclusion of lactate as well via MCT uptake. / NSERC

Fatty acid transport across the plasma membrane is an important site of regulation in skeletal muscle lipid metabolism, and is governed by a number of fatty acid transport proteins including, CD36, FABPpm, and FATP1 and 4. While each transporter is capable of independently stimulating fatty acid transport, less is known about their specific functions under various metabolic conditions, although CD36 appears to be key. The purpose of this thesis was to examine skeletal muscle fatty acid metabolism in several rodent models where CD36 has been altered, particularly via whole body deletion, by muscle specific overexpression, or in the face of permanent redistribution of CD36 to the plasma membrane. Using these models, this thesis sought to answer the following questions: 1) Is caffeine-stimulated fatty acid oxidation CD36-dependent? 2) Does CD36 function in tandem with FABPpm, and does this enhance fatty acid uptake at the plasma membrane and/or influence the metabolic fate of incoming fatty acids? 3) Is intramuscular lipid distribution altered in a rodent model of obesity, in which CD36-mediated fatty acid uptake is increased? Specific novel findings include the following: 1) Caffeine-stimulated calcium release can elicit the translocation of a number of fatty acid transporters in skeletal muscle, but CD36 is essential for caffeine-induced increases in fatty acid uptake and oxidation. 2) In spite of difficulties associated with protein co-overexpression, it appears that simultaneous overexpression of CD36 and FABPpm enhances fatty acid transport across the plasma membrane, and that these transporters may collaborate to increase insulin-induced fatty acid esterification and AICAR-induced oxidation. 3) Finally, in the obese Zucker rat model, augmented CD36-dependent fatty acid transport into muscle in combination with elevated lipid supply, results in lipid accretion within the IMF region of muscle, an effect that could not be explained by compartment-specific changes in selected glycerolipid synthesizing enzymes. Taken together, these studies emphasize the importance of CD36 in the regulation of plasmalemmal fatty acid transport, and further elucidate the metabolic implication of CD36 alteration on overall skeletal muscle metabolism.

No description available.

Insulin resistance is a major metabolic defect associated with obesity and type 2 diabetes. The incidences of both are increasing at an alarming rate. Excessive consumption of nutrient rich foods have been implicated in the pathogenesis of insulin resistance. However, the mechanisms involved in the onset of insulin resistance in skeletal muscle caused by acute nutrient oversupply in vivo have not been fully elucidated. The broad aim of this thesis was to examine the mechanisms associated with the onset of skeletal muscle insulin resistance in models of acute nutrient oversupply. The effect of glucose oversupply was investigated in the first study, which resulted in insulin resistance at the whole body and skeletal muscle level following 5h of glucose infusion, but not after 3h. There was no change in markers of oxidative stress over the same time course during which insulin resistance developed. Furthermore, co-infusion of the antioxidant taurine had no effect on the decreased glucose uptake in skeletal muscle from glucose infused animals. There was no evidence of activation of inflammatory/stress signalling pathways or defects in the phosphorylation state of multiple insulin signalling intermediates over the same time course. In isolated soleus strips taken from control, 1h, or 5h glucose infused animals, insulin stimulated 2-deoxyglucose transport was similar. Although, insulin-stimulated glycogen synthesis was significantly reduced after 5h of glucose infusion, in the presence of significantly increased glycogen content. The reduced flux through the glycogen synthesis pathway and a reduced content of glucose-6-phosphate suggests in this model that the rate limiting step has shifted from glucose transport to glucose phosphorylation by hexokinase (HK). In an acute lipid and insulin infusion model, the onset of insulin resistance was similar to that observed in the glucose infusion model. The mechanisms for the insulin resistance in skeletal muscle in this model was not associated with defects in the phosphorylation of key insulin signalling intermediates or activation of inflammatory/stress signalling pathways. Furthermore, there was no change in markers of oxidative stress and the co-infusion of taurine had no effect on the onset of insulin resistance. There was an increased exposure of long chain acyl-CoA (LCACoA), although there was no change in the content of other lipid intermediates such as DAG or ceramides. Interestingly, muscle pyruvate dehydrogenase (PDH) kinase 4 (PDHK4) protein content was significantly decreased in hyperinsulinaemic glycerol infused rats after 3 and 5h, and this decrease was blunted in muscle from hyperinsulinaemic 3 and 5h lipid infused rats. These findings suggest that lipid infusion may reduce glucose metabolism by inhibition of the glucose phosphorylation due to LCACoA inhibition of HK and mitochondrial substrate competition regulated by increased PDHK4. In conclusion, the current studies demonstrate that the insulin resistance associated with nutrient oversupply was not associated with significant changes in phosphorylation of key insulin signalling intermediates, activation of inflammatory and stress signalling pathways, or a change in markers of oxidative stress. Overall, the studies in this thesis suggest that the initial onset of insulin resistance due to glucose and lipid oversupply (in the presence of high insulin) is associated with metabolic feedback regulation, which is likely to be a protective mechanism of the skeletal muscle to limit any further insult by the excess nutrients.

Obesity and type 2 diabetes are the most prevalent metabolic diseases in the western world and affect over 50% of the world’s population. During obesity non-adipose tissues such as the liver and skeletal muscle take up and store excess fatty acids (FA) as lipids such as triacylglycerols (TAG) and diacylglycerols (DAG). Excessive lipid storage in non-adipose tissues can result in the dysfunction of cellular processes and lead to programmed cell death (apoptosis). Lipid-induced apoptosis was investigated in the key glucoregulatory tissues, the liver and skeletal muscle. Lipid-induced apoptosis was detected in vitro in both hepatocytes and myotubes but was not detected in the livers or skeletal muscles of genetically obese mice or high-fat fed mice. Further investigation discovered despite exacerbated TAG accumulation, endoplasmic reticulum stress (ER) was not activated in the liver and pathways of cellular remodelling (proteolysis and autophagy) were not initiated in skeletal muscle. These studies demonstrated that the liver and skeletal muscle are adaptable to increased lipid storage in physiological models but not isolated cell culture systems. In vitro experiments demonstrated unsaturated FAs could protect hepatocytes from lipoapoptosis and it has been suggested this is due to driving FA accumulation into TAG lipid droplets. Adipose triglyceride lipase (ATGL) is one of the primary TAG lipases. To explore TAG metabolism in the liver, primary hepatocytes were derived from ATGL null mice and ATGL was over-expressed in the livers of chronically obese mice. / It was found that cellular FA uptake and TAG esterification was increased and TAG lipolysis and FA oxidation were decreased in the ATGL null hepatocytes. This resulted in exacerbated TAG and diacylglycerol (DAG) storage. The gene expression of metabolic regulators such as cytochrome c oxidase subunit 2 (COX2), medium chain acyl Co-A dehydrogenase (MCAD), peroxisome proliferators-activated receptor co-activator 1! (PGC1!), nuclear respiratory factor 1 (NRF1) and FA translocase/cluster of differentiation 36 (FAT/CD36) were increased in ATGL null hepatocytes compared with wild type hepatocytes, suggesting that the reduction in FA oxidation in the ATGL null hepatocytes was probably due to limited FA substrate availability. Interestingly, despite increased TAG and DAG, the hepatocytes remained insulin sensitive. To investigate hepatic ATGL over-expression an adenovirus containing an ATGL insert was injected into chronic high fat fed mice. Hepatic ATGL over-expression in the iii chronically obese mice reduced TAG, DAG and ceramide content in the liver. This resulted in improved hepatic insulin signalling and whole body insulin sensitivity. In summary, studies from this thesis suggested the use of in vitro systems are not a substitute for in vivo models when assessing the toxic effects of lipid oversupply, TAG accumulation may be a protective mechanism against cellular remodelling and programmed cell death, and increased ATGL expression in the liver can reduce hepatic steatosis and enhance whole body insulin sensitivity. Therefore, increasing hepatic ATGL expression could be a therapeutic approach to treat obesity and type 2 diabetes.

Duchenne Muscular Dystrophy (DMD) is a fatal X-linked disorder affecting 1 out of every 3500 male births. The underlying cause of DMD is mutations within the dystrophin gene resulting in loss of protein expression, which leads to myofiber instability and damage. The constant damage of skeletal muscle causes sustained immune infiltration, marked by increased levels of cytokines, such as TGF-beta. Interestingly, TGF-beta can decrease the myogenic potential of satellite cells, thus preventing muscle regeneration. Previously, our lab has shown that knockdown of the Ste20 Like Kinase, SLK, in normal mammary epithelial cells was sufficient to delay TGF-beta induced epithelial to mesenchymal transition. Therefore, we speculated that decreasing SLK levels would be sufficient to decrease the anti-myogenic effects of TGF-beta both in cultured myoblasts and in a mouse model of muscular dystrophy. In the first section of this study, we explored the effect of muscle specific deletion of SLK on muscle development and regeneration. Skeletal muscle specific deletion of SLK did not impair muscle development, but caused a myopathy in older mice. Additionally, muscle regeneration was delayed, but not inhibited by SLK deletion. These findings indicated that SLK has beneficial roles in skeletal muscle, but was not absolutely required for optimal muscle development and regeneration. In the second section, we investigated the potential for SLK knockdown to mitigate the anti-myogenic effects of TGF-beta in vitro. Decreasing levels of SLK restored myoblast differentiation in the presence of TGF-beta in a p38 dependent manner. In the final section, we determined that SLK levels are elevated in dystrophic muscle and that subsequent deletion of SLK in the mdx mouse enhances terminal differentiation of myoblasts without further exacerbating the pathology of the disease. Collectively, this work demonstrates that SLK inhibition can provide a protective effect against the anti-myogenic effects of TGF-beta via upregulation of p38 activity.

The importance of activity during the development of central components of the nervous system such as the visual system has long been recognized (Wiesel & Hubel 1963) and it is beginning to be understood that sensory experience and motor behavior are equally important for neuromuscular development (Brumley et al. 2015; Sharp & Bekoff 2015). The chick embryo model has proven to be especially useful in studying the relationships among motor behavior, sensory experience, and neuromuscular development (Oppenheim et al. 1978; Sharp & Bekoff 2001) due to its accessibility and early onset of movement behavior. Traditionally, neuromuscular blockers have been used to broadly study the role of neural activity and muscle activity during development (Oppenheim et al. 1978; Ding et al. 1983). In order to noninvasively alter neural activity in specific populations of cells, the Sharp lab has developed an optogenetic approach that allows the expression of ChIEF, a variant of channelrhodopsin-2, in the spinal cord of living chick embryos (Sharp & Fromherz 2011). In order to better understand the unique role that muscle activity plays in neuromuscular development, it would be advantageous to directly and noninvasively control muscle activity through light-activation of ChIEF expressed in muscle fibers. Therefore, the primary objective of this thesis research was to achieve ChIEF expression in the plasma membrane of myotubes in living chick embryos. Initial attempts to express ChIEF in chick muscle resulted in low success rates. The CAG promoter in pPB-ChIEF-Tom, the plasmid vector that encodes ChIEF, was likely hindering expression of ChIEF in muscle tissue. Therefore, standard molecular cloning techniques were used to replace the CAG promoter with the myosin light chain promoter which was known to drive transgene expression in chick muscle (Wang et al. 2011). The new DNA construct that resulted from modifying pPB-ChIEF-Tom was identified as pPB-MLC-ChIEF-Tom (mChIEF). ChIEF was successfully expressed in hindlimb muscles of chick embryos via somite electroporation of mChIEF and observed between E7 and E18. Expression patterns corresponded with the current understanding of muscle progenitor contributions of somites to hindlimb muscles (Rees et al. 2003). ChIEF was located in the outer membrane of muscle fibers on E9, E14, and E18 when tissue was histologically examined in conjunction with myosin heavy chain immunofluorescence. Importantly, light-activation of ChIEF in the hindlimb muscle of living chick embryos resulted in muscle contraction and light-evoked hindlimb movements. In addition to demonstrating the functionality of ChIEF expression, an effort was made to characterize the effects of altered parameters of light stimuli on light-evoked movement and determine whether light-evoked muscle contraction could be used to imitate normal, neuronal muscle control. Light intensity was directly related to amplitude and rate of light-evoked movement. Light duration was directly related to amplitude and latency of peak movement. Unfused and fused tetanus were observed when bursts of short duration light pulses with varying interpulse intervals were used to activate ChIEF. This thesis research strongly suggests that light-activation of ChIEF expressed in living, chick embryo hindlimb muscle results in muscle contractions in manner similar to normal, neurally-driven muscle contraction.

The role of glycogen in endurance performance has been accepted in theory. It has been shown that higher resting muscle glycogen levels prolong endurance performance. On the other hand, low glycogen levels have been associated with fatigue. Ultimately, a person's muscle glycogen level dictates the duration in which an activity can be maintained at a maximal effort, after which time, performance will decrease. As of yet, there has been no evidence as to what happens to the fatigued muscle. Force production in skeletal muscle is dictated by the release and uptake of Ca²⁺ from the sarcoplasmic reticulum (SR). Force production is proportional to [Ca²⁺], as [Ca²⁺] increases so does force. At the point of fatigue, there is a decrease in force production. Since fatigue has been associated with glycogen depletion, it is likely that SR function has been altered causing this decrease in force. The purpose of this study was to determine the effect of glycogen depletion on the SR. Twenty male Sprague-Dawley (Harlan Sprague-Dawley, Indianapolis, IN) rats weighing, 345 ± 70 gm were housed two per cage in the Virginia Tech Lab Animal Resources facility. They were fed ad libitum (Purina Rodent Laboratory Chow and water) until time of experiment. Ten of the rats were used as control animals and the other ten were assigned to the experimental group. Rats were allowed a minimum of 5 days to acclimate to their housing. On the morning of the day of testing, rats were selected in pairs according to the housing cage in an effort to decrease variations in food consumption. To reduce muscle glycogen levels, experimental rats were given an initial injection of either epinephrine (1mg/g: ip) while control rats were injected with saline (equal volume) at 0 hr. Thirty minutes later they received another injection of epinephrine or saline (0.5 mg/g: ip). At the end of the hour the rats were anesthetized with pentobarbital sodium (60 mg/kg:ip) for tissue harvesting. Upon reaching a surgical plane of anesthesia one gastrocnemious muscle was extracted for the muscle glycogen assay and the other removed for SR vesicle preparation. Rats were then euthanized with an overdose of pentobarbital sodium. The tissue was assayed for glycogen and glucose levels as well as for Ca²⁺ uptake and release and ATPase activity. It was found that epinephrine animals had 23% less glycogen than did the control animals and almost twice the amount of glucose (control — 2.9 nmol/g and epinephrine — 5.9 nmol/g). Ca²⁺ uptake rates in epinephrine animals were significantly decreased by 19.7% (p < .05). Control animals had a release rate of 77.15 ± 1.26 nmol/mg/min and epinephrine animals had a release rate of 75.01 ± 1.86 nmol/mg/min. Ca²⁺ release rates were decreased but not significantly. Ca²⁺ stimulated ATPase activity was significantly decreased by 17.7% in epinephrine animals (p < .05). This is one of the first studies that demonstrate that glycogen reduction in a rested muscle causes altered SR function similar to those caused by exercise. This study shows that low glycogen levels are associated with decreased SR function, which is the primary reason for causing the loss of force in muscle. Ultimately, this study suggests that glycogen loading will enhance endurance performance. / Master of Science