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.

Thesis (Ph. D.)--University of Missouri--Columbia, 2003. / "December 2003." Typescript. Vita. Includes bibliographical references (leaves 63-74). Also issued on the Internet.

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 105-111).

Uncoupling proteins (UCPs) are highly conserved inner mitochondrial membrane proteins that have been found in plants, nematodes, flies, and vertebrates. UCPs dissipate the proton gradient formed by the electron transport chain in an energy-expending process that generates heat. In mammals, the brown fat-specific UCP1 is thought to be the dominant, if not the only significant mediator of thermogenic responses. However, adult humans express only negligible amounts of brown fat and UCP1, yet still show significant non-shivering thermogenic responses (e.g. amphetamine-induced hyperthermia, diet induced thermogenesis, fever). Thus, the fact that human thermogenic mechanisms haven't been identified is a huge gap in our understanding of human thermoregulation. UCP3 is primarily expressed in skeletal muscle, an established thermogenic organ which is a major target of amphetamine-induced pathology. UCP3 knockout mice have a near complete loss (~80%) of amphetamine-induced thermogenesis and are completely protected from amphetamine-induced death over a range of lethal doses. With regard to mechanisms of UCP3 activation, we observed that norepinephrine and free fatty acids are elevated in the bloodstream prior to peak amphetamine-induced hyperthermia. However, little is known about the anatomic location of UCP3-dependent thermogenesis or the mechanisms by which fatty acids regulate UCP function. Thus, we sought to investigate the physiology and biochemical activation of UCP3 to establish the thermogenic potential of skeletal muscle uncoupling and elucidate the mechanisms of UCP3 function. The overall goal of this research was to identify the tissue target(s) and mechanisms involved in amphetamine-induced UCP3-dependent thermogenesis. Herein, we show that in addition to a deficit in induced thermogenesis, UCP3-null mice also lack responses to other physiologically-relevant stimuli (i.e. catecholamines and bacterial pathogens). Conversely, UCP3 knockout mice, engineered to express UCP3 only in skeletal muscle have an augmented thermogenic response to amphetamines. In order to explore UCP3's mechanism of activation, we performed a modified yeast two-hybrid analysis and identified [Delta][superscript 3,5][Delta][superscript 2,4]dienoyl-CoA isomerase (DCI) as a UCP3 binding partner. DCI, an auxiliary fatty acid oxidation enzyme, protects cells from the accumulation of toxic lipid metabolites. Using immunoprecipitation and fatty acid oxidation (FAO) assays, we determined that UCP3 and DCI directly bind in the mitochondrial matrix in order to augment lipid metabolism. These findings support a novel model in which skeletal muscle UCP3 is responsible for inducible thermogenesis through cooperation with binding partners such as DCI which enhance oxidation of fatty acids. Together, these studies shed light on thermogenic pathways in rodents that are likely to be relevant to humans. / text

Chromium has been reported to increase glucose clearance in insulin resistant and diabetic populations. Skeletal muscle is the tissue primarily responsible for glucose clearance. We therefore tested the effect of chromium chloride (CrCl3) on skeletal muscle glucose uptake both in the absence and presence of a submaximal level of insulin via the rat hindlimb perfusion technique. 0.096 μM CrCl3 was used with and without 200 μU/ml insulin. Our testing showed that insulin significantly increased [H3]-2 deoxyglucose (2-DG) uptake in both the gastrocnemius and quadriceps muscles. Additionally, the combination of CrCl3 and insulin (Cr-sIns) led to greater amounts of 2-DG uptake than insulin alone (sIns) in both the gastrocnemius (Cr-sIns 6.49±0.75 μmol/g/h, sIns 4.83±0.42 μmol/g/h) and quadriceps (Cr-sIns 6.74±0.62 μmol/g/h, sIns 4.54±0.43 μmol/g/h). However, CrCl3 without insulin (Cr) had no affect on 2-DG uptake above basal (Bas) in both the gastrocnemius (Cr 1.45±0.14 μmol/g/h, Bas 1.61±30 μmol/g/h) and the quadriceps (Cr 1.35±0.15 μmol/g/h, Bas 1.27±0.13 μmol/g/h). It has been speculated that chromium works to increase glucose uptake by increasing insulin signaling. To examine this, we used western blotting analysis to test both Akt and AS160 phosphorylation in the mixed gastrocnemius. We found that insulin increased Akt and AS160 phosphorylation, but chromium had no affect on Akt (Cr-sIns 25%±2%, sIns 22%±4%) or AS160 (Cr-sIns 35%±5%, sIns 36%±4%) phosphorylation in the absence or presence of insulin. Our results suggest that supplementation with CrCl3 can lead to an increase in glucose uptake in skeletal muscle, but only in the presence of insulin. However, this effect of CrCl3 does not appear to be a result of enhanced insulin signaling. / text

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), a small molecular weight, hydrophobic protein found in skeletal muscle, is a known regulator of sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) pumps. Earlier in vitro reconstitution experiments have shown that SLN uncouples ATP hydrolysis from Ca2+ transport by the SERCA pumps and increases the amount of heat released per mol of ATP hydrolyzed by inducing an increased rate of ???slippage??? during the reaction cycle of SERCA pumps. In order to determine whether SLN causes slippage of SERCA activity by uncoupling ATP hydrolysis from Ca2+ transport under more physiological conditions, comparisons were made between skeletal muscle Ca2+ ATPase activity and Ca2+ uptake in homogenates from soleus muscle of wild-type (WT) and Sln-null (KO) mice under conditions in which a Ca2+ gradient was preserved across the sarcoplasmic reticulum (SR) vesicles. Ca2+ ATPase activity, measured in the absence of the Ca2+ ionophore, A23187, was 15-25% lower in KO muscles, compared with WT, consistent with the proposal that SLN increases ???slippage??? and reduces the extent of back-inhibition of the Ca2+ ATPase. Ca2+ uptake, measured in homogenates without oxalate, was not different (p>0.05) in SR vesicles from WT and KO mice, indicating that the calculated Ca2+ transport efficiency (coupling ratio) in KO mice was increased by about 20% (P<0.04). The basal oxygen consumption (VO2) of soleus muscles isolated from WT and KO mice and the contribution of energy utilized by SERCA was also compared. Surprisingly, basal VO2 was not lower in the soleus of KO mice, but the contribution of energy utilized by SERCA pumps was about 7% lower (P<0.0001). It was also found that uncoupling protein 3 (UCP-3) was expressed at a higher (P<0.03) concentration in soleus muscle of KO compared to WT. Thus UCP-3 could, potentially, provide compensation, resulting in higher basal VO2 in KO mice than expected. These data show that at physiological SLN:SERCA ratios, SLN uncouples ATP hydrolysis from SR Ca2+ uptake in skeletal muscle, resulting in a lower contribution of Ca2+ handling to basal VO2. Thus, SLN is a key regulator of both ATP utilization in Ca2+ handling and of overall energy metabolism in skeletal muscle. To further examine the role of SLN in adaptive thermogenesis, obesity and glucose intolerance, KO and WT mice were placed on a high fat diet (HFD; 42% of kcal derived from fat) for an eight week period. Whole body metabolism, weight gain, glucose tolerance and insulin tolerance were measured before and after the HFD. Fat pads, liver, pancreas, hindlimb muscles and plasma samples were collected from standard chow fed control and HFD WT and KO mice. KO mice gained more weight (P<0.05) and became more obese (P<0.05) than WT mice after consuming the HFD. The comprehensive laboratory animal monitoring system (CLAMS) revealed no differences in whole body metabolic rate (ml O??2/kg/hr) between KO and WT mice pre diet; however, daily metabolic rate was lower (P<0.05) in KO mice compared with WT mice after the HFD which may explain the increased obesity in KO mice. Western blotting analyses revealed SLN protein content to be 3.8 fold higher (P<0.05) in WT soleus post HFD compared to control. Phospholamban (PLN), a homologue of SLN, was found to be 2.1 fold higher (P<0.05) in brown adipose tissue (BAT) in both WT and KO mice post HFD. Protein contents of other Ca2+ handling proteins (SERCA1a, SERCA2a, PLN and calsequestrin) within fast (white gastrocnemius) and slow (soleus) twitch muscle were not different between KO and WT mice following the HFD. Collectively, these results suggest that PLN and SLN could play a role in adaptive diet-induced thermogenesis. On the other hand, compared with chow fed control mice, the metabolic cost of Ca2+ handling in soleus muscle was significantly reduced post HFD in both WT and KO mice, although to a greater extent (P<0.05) in KO mice than WT mice. Moreover, there were no differences in resting energy expenditure of soleus muscles between WT and KO mice following the HFD. These observations can be accounted for by diet-induced increases in sympathetic nervous system activity in KO mice and other adaptive responses leading to increased energy expenditure of soleus in both WT and KO mice. Therefore, differences in whole body metabolic rate and obesity between high fat fed WT and KO mice do not appear to be due to adaptive thermogenesis mechanisms in skeletal muscle involving SLN. Interestingly, soleus and EDL muscle weights increased proportionately to body weight in high fat fed WT mice but not KO mice. Therefore, lower lean body tissue mass may explain the lower whole body metabolic rate and increased susceptibility to obesity in KO mice compared with WT mice. With increased obesity, KO mice became extremely glucose intolerant (P<0.05) post HFD compared to WT mice who also demonstrated glucose intolerance (P<0.05) compared to the pre-HFD values. Surprisingly, the insulin tolerance test responses were not different between KO and WT mice post HFD suggesting that KO mice did not develop greater whole body insulin resistance despite being more obese than WT mice. Blood serum analysis showed that non-esterified fatty acids (NEFA) and LDL cholesterol levels were also increased more (P<0.05) in KO mice compared to the WT mice post HFD. Overall, it is concluded that SLNs impact on Ca2+ handling influences not only ATP consumption by SERCA pumps in resting soleus muscle via uncoupling of ATP hydrolysis from SR Ca2+ uptake but also blunts the negative effect of high fat feeding by increasing resistance to diet-induced obesity and glucose intolerance in mice through mechanisms which are currently unidentified.

The disruption of the survival motor neuron (SMN1) gene leads to the children’s genetic disease spinal muscular atrophy (SMA). SMA is characterized by the degeneration of α-motor neurons and skeletal muscle atrophy. Although SMA is primarily considered a motor neuron disease, the involvement of muscle in its pathophysiology has not been ruled out. To gain a better understanding of the involvement of skeletal muscle pathophysiology in SMA, we have developed three aims: to identify cell-specific Smn-interacting proteins, to characterize postnatal skeletal muscle development in mouse models of SMA, and to assess the functional capacity of muscles from SMA model mice. We have used tandem affinity purification to discover Smn interacting partners in disease relevant cell types. We have identified novel cell-specific Smn interacting proteins of which we have validated myosin regulatory light chain as a muscle-specific Smn associated protein in vivo. We have taken advantage of two different mouse models of SMA, the severe Smn-/-;SMN2 mouse and the less severe Smn2B/- mouse, to study the postnatal development of skeletal muscle. Primary myoblasts from Smn2B/- mice demonstrate delayed myotube fusion and aberrant expression of the myogenic program. In addition, the expression of myogenic proteins was delayed in muscles from severe Smn-/-;SMN2 and less severe Smn2B/- SMA model mice. Muscle denervation and degeneration, however, are not the cause of the aberrant myogenic program. At the functional level, we demonstrate a significant decrease in force production in pre-symptomatic Smn-/-;SMN2 and Smn2B/- mice indicating that muscle weakness is an early event in these mice. Immunoblot analyses from hindlimb skeletal muscle samples revealed aberrant levels of developmentally regulated proteins important for muscle function, which may impact muscle force production in skeletal muscle of SMA model mice. The present study demonstrates early and profound intrinsic muscle weakness and aberrant expression of muscle proteins in mouse models of SMA, thus demonstrating how muscle defects can contribute to the disease phenotype independently of and in addition to that caused by motor neuron pathology.

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.