Deficits in skeletal muscle mitochondrial metabolism are associated with a wide variety of chronic skeletal muscle and metabolic-related diseases, including diabetes and sarcopenia. Even in patients with advanced skeletal muscle-related diseases, exercise is a well-established method to improve skeletal muscle mitochondrial metabolism, culminating in enhanced whole-body metabolism and decreased disease severity. In response to exercise, there is an increase in reactive oxygen species (ROS) production. Historically, ROS were solely considered to drive disease development. However, ROS are also required for physiological adaptation and many questions still remain regarding their downstream pathways. One significant producer of skeletal muscle ROS with exercise is Nadph oxidase 4 (Nox4). Nox4 is unique compared to other Nox members as it predominantly produces hydrogen peroxide (H2O2), an effective signaling molecule. Here we demonstrate an essential role for Nox4 in mediating the beneficial effects of exercise. This work will contribute to our understanding of physiological ROS and their downstream targets by identifying a novel role for Nox4 in exercise adaptation. Further defining the molecular events that promote exercise adaptation will be essential for formulating new treatment strategies for patients with chronic metabolic diseases. / Doctor of Philosophy / Exercise is a widely effective tool for both preventing and reversing disease. Even patients with advanced skeletal muscle and metabolic-related diseases can benefit from continual and repeated exercise training. While decades of work have supported the effectiveness of exercise as a therapeutic intervention, the mechanistic understanding of what occurs at the cellular level remains incomplete. Here, we elucidate a novel pathway mediating important metabolic adaptations to exercise. In response to exercise stress, reactive oxygen species (ROS) are produced in skeletal muscle. ROS facilitate metabolic adaptations to meet the body's need for increased energy. One significant source of ROS comes from Nadph oxidase 4 (Nox4) which plays an essential role in metabolic regulation. The skeletal muscle metabolic response to stress is largely dependent on adaptations that include changes in gene expression, substrate oxidation, and mitochondrial metabolic adaptations. These mitochondrial adaptations include mitochondrial recycling after exercise in skeletal muscle (referred to as mitophagy). We have shown that Nox4 increases the expression of a subset of metabolic genes, is required for substrate oxidation after exercise, and is important for exercise-induced mitophagy.

The purpose of this study was to examine the relationship between metabolic adaptive response to 5 days of high SFA feeding, independent of positive energy balance, and diet-induced agonism of pro-inflammatory pathways. A secondary aim was to determine if the metabolic adaptive response in skeletal muscle to a single, high fat meal was altered by 5 days of high saturated fat feeding. Twelve college-age, non-obese males were studied and skeletal muscle samples were obtained prior to and concluding the consumption of a high SFA diet. In a subset of volunteers (N=6), we fed participants a high fat meal after the initial skeletal muscle biopsy and measured changes in postprandial endotoxin concentrations for four hours following the meal challenge. A second biopsy was obtained four hours after the meal challenge. Skeletal muscle samples were used to measure fatty acid oxidation, glucose oxidation, oxidative enzyme activities, mRNA expression of metabolic targets, and phosphorylation and total content of inflammatory proteins. In response to five days of high SFA feeding, skeletal muscle glucose and complete palmitate oxidation were significantly reduced as was the ratio of complete to incomplete fatty acid oxidation. Five days of high SFA feeding also attenuated the meal challenge-induced up-regulation of oxidative genes while augmenting postprandial increases in plasma endotoxin concentrations. To assess the relationship between metabolic adaptability and diet-induced inflammatory response we categorized volunteers by the diet induced percent change in fatty acid oxidation. Volunteers who were the least capable to adapt to high SFA feeding displayed the most robust increases in phosphorylation of inflammatory proteins. Lastly, we measured the correlation between the meal challenge associated percent change in oxidative and inflammatory markers in samples obtained prior to and following five days of high SFA feeding. We observed positive associations between the percent change in oxidative and inflammatory markers in samples obtained prior to the high SFA diet that were not observed following five days of high SFA feeding. These findings suggest that diet induced inflammatory response is involved in the regulation of adaptive response to high SFA feeding and that this relationship becomes dysregulated with chronic high SFA intake. / Ph. D.

The main goal for my research was to challenge the long held belief that the mechanical properties of maturing dystrophic compared to control skeletal muscle membranes are weaker, leading to onset of Duchenne muscular dystrophy (DMD). We built on a previous report from our lab that suggested sarcolemmal membranes from dystrophic mice are not more susceptible to damage early in maturation (i.e., age 9-12 days) and determined if and when muscle mechanical properties change as the mice mature. Across four studies, I have helped define the role of dystrophin-deficient skeletal muscle membranes in the onset of DMD. A linear viscoelastic muscle model was used to determine passive stiffness and damping in control and dystrophic muscles from maturing mice aged 14-35 days. Results confirmed my hypothesis that there are no differences in passive mechanical properties between normal and dystrophic mice. Recognizing the limitations of the linear model, a nonlinear model was developed to determine the stiffness and damping of active and passive dystrophic muscles from maturing mice aged 21 and 35 days. The nonlinear model achieved a significantly better fit to experimental data than the linear model when muscles were stretched to 15% strain beyond resting length. Active and passive mechanical properties of dystrophic mice were not different than control at 14 and 28 days of age. The previously developed nonlinear model was used to determine a more complete time-course (14-100 days of age) of dystrophic muscle mechanical properties. There was no difference in passive stiffness between mdx and control muscles at each age. However, the mdx:utrn-/- muscles showed increased stiffness compared to control and mdx muscles at 21 and 28 days, suggesting a temporary change within the muscle that only occurs with a lack of both utrophin and dystrophin. Fast-twitch and slow-twitch muscle mechanical properties were compared in control and dystrophic mice aged 3, 5, and 9 weeks of age. Dystrophic and control slow-twitch muscles did not have different mechanical properties, suggesting that a lack of dystrophin does not affect slow-twitch muscles during maturation (3-5 weeks) or well after maturation (9 weeks). / Ph. D.

Toll-like receptor 4 (TLR4) is a transmembrane receptor, which upon activation by lipopolysaccharide (LPS) from Gram-negative bacteria, plays an important role in the induction of the innate immune response. Our lab has previously demonstrated that activation of TLR4 in skeletal muscle results in the preferential oxidation of glucose for ATP production over that of fatty acids. Currently, the exact mechanism(s) for TLR4-induced modulation of metabolism are not known. The purpose of this project was to test the hypothesis that activation of TLR4 pathway causes increased ROS production, which contributes to deceased fatty acid oxidation and altered mitochondrial respiration in skeletal muscle. To this end, skeletal muscle cells were studied following acute and chronic treatments with LPS, and a mouse model with muscle-specific over expression of TLR4 (mTLR4) was studied under chow fed conditions and following 16 weeks of high fat feeding. Acute LPS treatment of C2C12 cells resulted in mitochondrial uncoupling as evidenced by higher levels of state IV respiration, reduced maximally simulated respiration, and a robust induction of uncoupling protein 3. These observations occurred in conjunction with increased pyruvate dehydrogenase activity. The LPS-induced changes in substrate preferences and maximally-stimulated mitochondrial respiration were prevented in the presence of the antioxidants, N-acetyl-L-cyteine (NAC) and catalase. Using isolated flexor digitorum brevis (FDB) muscle fibers from C57BL/6J mice, we showed that LPS treatment results in significant increases in ROS production that are evident at 15 min and still increasing at 45 min following the addition of LPS to incubation media. Hyperpolarization of mitochondrial membrane potential was also evident at 15 min post LPS treatment in FDB fibers. Fatty acid oxidation measured in skeletal muscle whole homogenates from the mTLR4 mice was significantly reduced compared to wild-type littermates on a standard chow diet. Following a 16 week high fat diet, the mTLR4, compared to wild-type mice, gained more weight and fat mass, were glucose intolerant, and displayed elevated production of mitochondrial-derived reactive oxygen species (ROS) from complex III. In conclusion, these data show that TLR4 activation elicits a change in mitochondrial substrate preference in that acetyl-CoA derived from pyruvate oxidation is the preferred substrate for the TCA cycle over that derived from β-oxidation of fatty acids. These data also lend strong support to the idea that the TLR4-mediated change in substrate preference is dependent on the production of ROS. / Ph. D.

Duchene muscular dystrophy (DMD) is a lethal X-linked disease that afflicts approximately 1 in 3500 newborn males. Boys with DMD will become progressively weaker causing wheelchair dependence by their early teens and death by their mid to late twenties. Currently there is no cure for DMD, the exact mechanism of disease action remains elusive, and treatments to improve quality of life are limited. Two areas of DMD research that could begin to fill this void and provide simple, cost effective therapy aimed to improve quality of life are neutriceutical and exercise therapies. We hypothesized that leucine, a branched chain amino acid (BCAA) with anabolic properties, given to sedentary and exercised x-linked dystrophic mice (mdx) over 4 weeks would improve skeletal muscle function and decrease markers of skeletal muscle degradation. In sedentary mdx mice, leucine improved tetanic extensor digitorum longus (EDL) stress (p < 0.05), gastrocnemius mammalian target or rapamycin (mTOR) phosphorylation (p < 0.05), while decreasing the rate of real-time calpain activity in flexor digitorum brevis (FDB) fibers (p < 0.05) compared to sedentary mice given no leucine. In exercised mdx mice, leucine improved total running distance over the 4 week testing period by 40% (p < 0.02) and increased EDL stress at every frequency recorded (p < 0.05). Our data lead us to the conclusion that the BCAA leucine can increase EDL muscle stress in dystrophic animals, and that the effects of leucine treatment are enhanced when leucine supplementation is combined with exercise. Leucine supplementation should be explored further and in higher order species of muscular dystrophy to determine if its use could provide clinical improvements in DMD patients. / Ph. D.

Keratins are a family of proteins found within human hair, skin and nails, as well as a broad variety of animal tissue. Prior research suggests hydrogel constructs of keratin and keratin derivatives exhibit several mechanical and biological properties that support their use for tissue engineering and regenerative medicine applications. Microparticle formulations of these hydrogels are an intriguing delivery vehicle for drugs and cellular payloads for tissue engineering purposes due to the ability to exploit size, surface area, loading potential and importantly, non-invasive delivery (i.e. injection) of cells and biologics. Here we examine the water-in-oil emulsion synthesis procedure to produce keratin microparticles using an oxidized keratin derivative, keratose (KOS). Analyses of particle size, microstructure, and other characterization techniques were performed. Drug loading characteristics, release kinetics, and feasibility of use in two different microparticles was subsequently investigated, first using a model-drug and later testing an antibiotic payload on bacterial cultures to validate antibacterial applications. A suspension culture technique was developed to load bone marrow-derived mesenchymyal stromal cells (BM-MSCs), testing the capacity to maintain viability and express key protein-based factors in cell growth and development. Finally, we tested the in vitro effects of cell-loaded microparticles on the L6 skeletal muscle cell line to determine potentially beneficial outcomes for skeletal muscle tissue regeneration. Largely spherical particles with a porous internal structure were obtained, displaying hydrogel properties and forming viscoelastic gels with small differences between synthesis components (solvents, crosslinkers), generating tailorable properties. The uniquely fibrous microstructure of KOS particles may lend them to applications in rapid drug release or other payload delivery wherein a high level of biocompatibility is desired. Data showed an ability to inhibit bacterial growth in the emulsion-generated system, and thereby demonstrated the potential for a keratin-based microparticle construct to be used in wound healing applications. Dense cell populations were loaded onto particles. Particles maintained cell viability, even after freeze-thaw cycling, and provided a material substrate that supported cell attachment through the formation of focal adhesions. Finally, in vitro studies show that both KOS and BM-MSCs support varying aspects of skeletal muscle development, with combinatorial treatments of cell-loaded particles conferring the greatest growth responses. / Doctor of Philosophy / Keratins and keratin hydrogels may exhibit several properties that support their use for tissue engineering and regenerative medicine applications. Microparticle formulations of these hydrogels are an intriguing delivery vehicle for payloads for tissue engineering purposes. Here we examine the water-in-oil emulsion synthesis procedure to produce keratin microparticles that were analyzed based on drug loading characteristics. A suspension culture technique was developed to load bone marrow-derived mesenchymyal stromal cells (BM-MSCs). Finally, we tested these products to determine potentially beneficial outcomes for skeletal muscle tissue regeneration. Particles with a porous structure were obtained. The microstructure of these particles may lend them to applications in drug release or other payload delivery. Data showed an ability to load and unload specific drug payloads. Dense cell populations were loaded onto particles. Finally, studies show that both keratin and BM-MSCs support skeletal muscle development, with combinatorial treatments of cell-loaded particles conferring the greatest growth responses.

The STAC3 gene is a functionally undefined gene predicted to encode a protein containing two SH3 domains and one cysteine-rich domain. In this study, we determined the potential role of the STAC3 gene in proliferation and differentiation of bovine satellite cells. We isolated satellite cells from skeletal muscle of adult cattle and transfected them with STAC3 small interfering RNA (siRNA) or scrambled siRNA. Cell proliferation assays revealed that STAC3 knockdown had no effect on the proliferation rate of bovine satellite cells. We assessed the differentiation status of bovine satellite cells by quantifying the expression levels of myogenin and myosin heavy chain genes, and by quantifying fusion index. STAC3 knockdown stimulated mRNA and protein expression of myogenin, and myosin heavy chain 3 and 7, and increased fusion index of bovine satellite cells. These data together suggest that STAC3 inhibits differentiation of bovine satellite cells into myotubes. To determine the underlying mechanism, we identified and validated AP1?1 as a STAC3-interacting protein by yeast two-hybrid screening and co-immunoprecipitation. In C2C12 cells, STAC3 knockdown decreased the expression level of AP1?1 protein. In bovine satellite cells, STAC3 knockdown increased the membrane localization of glucose transporter 4 (GLUT4) and glucose uptake. These data together suggest the following mechanism by which STAC3 inhibits differentiation of bovine satellite cells: STAC3 increases AP1?1 stability in cells; a high level of AP1?1 keeps GLUT4 from translocating to the plasma membrane; reduced membrane localization of GLUT4 impedes glucose uptake; and restricted glucose uptake inhibits differentiation of satellite cells into myotubes. / Master of Science

Once an animal is harvested for meat, skeletal muscle attempts to maintain ATP at or near antemortem levels. To maintain ATP levels postmortem, stored glycogen is catabolized to produce ATP through glycolysis and possibly oxidative metabolism. Hydrolysis of the produced ATP acidifies muscle until an ultimate pH is reached. The ultimate pH of meat directly impacts the quality characteristics of color, texture, and water holding capacity. Therefore, our research intends to describe the contributions glycolysis and oxidative metabolism play in determining ultimate pH and fresh meat quality. Traditionally, glycogen content at death was thought to be responsible for dictating ultimate pH. This was especially true in oxidative muscle with limited glycogen stores. Yet, our research indicated that in the presence of excess glycogen, oxidative muscle maintains a high ultimate pH. Rather, pH inactivation of phosphofructokinase is responsible for terminating postmortem glycolysis and brackets ultimate pH between 5.9 – 5.5. Meat with a pH below this range is uncommon. However, AMPK γ3R200Q mutant pigs produce meat with an ultimate pH near 5.3. Due to lower AMP deaminase abundance in their muscle, AMP levels are elevated late postmortem. Because AMP is a potent activator of phosphofructokinase, the aberrant meat quality from AMPK γ3R200Q mutant pigs is caused by extended postmortem glycolysis. Combined, these data further our understanding of the factors that contribute to the formation of fresh meat quality. We also characterized AMPK γ3R200Q muscle by investigating antemortem skeletal muscle lactate transport. Lactate is transported in or out of tissues by proton-linked iii monocarboxylate transporters (MCTs). Previous reports indicated that acute activation of AMPK increased monocarboxylate transporter expression in skeletal muscle of other species. Yet, it was unknown the impact chronic activation of AMPK will have on MCT1, MCT2, and MCT4 expression in pigs. Compared to wild-type pigs, the longissimus lumborum of AMPK γ3R200Q pigs increased both MCT2 and MCT4 protein expression. Our data suggest glycolytic skeletal muscle from the AMPK γ3R200Q pigs has increased capacity for antemortem lactate export from muscle and possibly increased pyruvate transport into the mitochondria. / Ph. D.

High-fat diets and obesity have been linked to unfavorable changes in gut bacteria and increased leakage of bacterially-derived lipopolysaccharide (endotoxin) from the intestinal tract into circulation, which is associated with low-grade inflammation, metabolic dysregulation and degradation of tight-junction proteins between intestinal cells. Probiotic supplementation is the practice of ingesting live strains of bacteria that are proposed to have a beneficial effect on the host by enriching the intestine with healthy bacteria. The purpose of this project was to determine if probiotic supplementation would prevent increased inflammatory tone, decreased oxidative capacity, and decreased tight-junction protein expression associated with high-fat feeding and elevated endogenous endotoxin. Male C57BL/6J mice were fed either a control (CD, 10% fat) or high-fat (HFD, 60% fat) diet for 4 weeks while receiving a daily oral gavage of water (C-VSL#3, HF-VSL#3) or probiotics (C+VSL#3, HF+VSL#3) equivalent to 1.2 billion live cultures. Changes in body weight, body composition, respiratory exchange ratio, energy expenditure, and glucose and insulin tolerance were measured in live mice. Markers of metabolic function were measured in whole muscle homgenates and mitochondria isolated from red and white skeletal muscle. Plasma endotoxin was measured in blood collected from fasted mice at the time of euthanization. The large and small intestines were collected and mRNA levels of tight-junction proteins and markers of nutrient sensing were measured. To determine a possible protective effect against endogenous LPS, a second cohort of mice were given an intraperitoneal injection of 0.1µg/kg LPS or saline to induce endotoxemia after four weeks of the aforementioned feeding protocol. Markers of metabolic function and inflammation were measured in mitochondria, skeletal muscle and liver. VSL#3 supplementation improved glucose homeostasis and markers of inflammation while enhancing nutrient sensing in the gut. / Ph. D.

Postnatal skeletal muscle growth occurs through myonuclear accretion and high protein turnover rate. While fiber type composition of the muscle could affect protein turnover rate, less is known about how fiber type influences the regulation of protein synthesis and degradation signaling pathways. Thus, the hypothesis of this work was that variation in fiber type composition will differentially affect the regulation of signaling pathways related to protein turnover in skeletal muscle hypertrophy in growing pigs. Downregulated protein synthesis signaling and reduced expression of type II MyHC isoforms have been reported in skeletal muscles of low birth weight (LBWT) neonatal pigs. Therefore, we sought to determine whether these changes are sustained until weaning and would explain the reduction in LBWT pig growth compared to their normal birth weight (NBWT) sibling at weaning. Another objective was to determine whether the regulation of protein turnover signaling pathways are correlated to fiber type differences in skeletal muscles. Our data suggest that the longissimus dorsi (LD, glycolytic) muscle of LBWT pigs experienced compensatory growth while the soleus (oxidative) remained proportionally smaller. Growth of the LD was accompanied by upregulation of translation initiation. Additionally, there was no difference in expression of MyHC isoforms between NBWT and LBWT pigs. These data suggest the rapid growth of the LD of LBWT pigs may be attributed to an upregulation of protein synthesis signaling and occurred only in glycolytic muscles. A caveat in LBWT pig model is that the reduction in type II MyHC at birth is not the only factor that could influence muscle growth, and that other factors may have confounded our results. This is why we aimed to use β-adrenergic agonist as a means to induce a shift fiber type in muscles to a more glycolytic phenotype. Our objective was to determine the influence of the β-adrenergic agonist Ractopamine (RAC) induced slow-to-fast fiber type transformation on the regulation of protein synthesis and degradation pathways. Although supplementation improved translational capacity, enhanced S6K1 phosphorylation, and reduced the abundance of calcium-dependent proteases, RAC feeding had no effect on body or muscle weights. These results suggest that a fiber type transformation without other physiological influences does not alter protein turnover signaling in favor of hypertrophy in growing pigs. / Master of Science / Skeletal muscles grow by increasing the amount of protein contained within them. The amount of protein deposited is determined by the net balance between the rates at which proteins are synthesized and degraded. However, not all skeletal muscles grow at the same rate. One factor that is thought to influence protein synthesis and degradation rates is the types of muscle fibers that are present within a muscle. These fibers can display a range of contractile and metabolic characteristics, from slow-twitch oxidative fibers to fast-twitch glycolytic fibers. In the presented studies, we sought to determine whether changes in fiber type composition result in difference to the signaling pathways the regulate protein synthesis and degradation, ultimately leading to differences in the muscle growth of young pigs. We have previously shown reduced activation of the protein synthesis pathway in the skeletal muscle of low birth weight (LBWT) newborn pigs. These pigs also had lower expression of glycolytic fibers. In experiment 1, we aimed to compare the signaling pathways regulating protein synthesis and degradation in LBWT and normal birth weight (NBWT) pigs at weaning. We also sought to determine if the regulation of these signaling pathways changed between muscles with differing fiber type compositions. The glycolytic longissimus dorsi (LD) muscle of LBWT pigs grew rapidly between birth and weaning whereas the highly oxidative soleus did not. In addition, the LD of LBWT pigs had greater protein synthesis signaling and similar expression of muscle fibers compared with NBWT pigs, suggesting the improvement in protein synthesis signaling of LBWT pigs between birth and weaning may be related to a shift in fiber type. In experiment 2, we used a compound called ractopamine hydrochloride (RAC) to promote a slow-to-fast fiber type switch in the muscle of young pigs. With this study, we sought to determine the effect of this fiber type transformation, without the influence of birth weight, on the regulation of protein synthesis and degradation pathways. Although RAC-fed pigs showed some minor changes that could improve protein synthesis and decrease protein degradation, RAC feeding had no observable effect on body weight or muscle growth. These results suggest that a fiber type transformation alone is not enough to promote muscle growth in growing pigs.