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Adult skeletal muscle is highly plastic and responds readily to environmental stimuli. One of the most commonly utilized methods to study skeletal muscle adaptations is immunofluorescence microscopy. By analyzing images of adult muscle cells, also known as myofibers, one can quantify changes in skeletal muscle structure and function (e.g. hypertrophy and fiber type). Skeletal muscle samples are typically cut in transverse or cross sections, and antibodies against sarcolemmal or basal lamina proteins are used to label the myofiber boundaries. The quantification of hundreds to thousands of myofibers per sample is accomplished either manually or semi-automatically using generalized pathology software, and such approaches become exceedingly tedious. In the first study, I developed MyoVision, a robust, fully automated software that is dedicated to skeletal muscle immunohistological image analysis. The software has been made freely available to muscle biologists to alleviate the burden of routine image analyses. To date, more than 60 technicians, students, postdoctoral fellows, faculty members, and others have requested this software. Using MyoVision, I was able to accurately quantify the effects of β-catenin knockout on myofiber hypertrophy. In the second study, I tested the hypothesis that myofiber hypertrophy requires β-catenin to activate c-myc transcription and promote ribosome biogenesis. Recent evidence in both mice and human suggests a close association between ribosome biogenesis and skeletal muscle hypertrophy. Using an inducible mouse model of skeletal myofiber-specific genetic knockout, I obtained evidence that β-catenin is important for myofiber hypertrophy, although its role in ribosome biogenesis appears to be dispensable for mechanical overload induced muscle growth. Instead, β-catenin may be necessary for promoting the translation of growth related genes through activation of ribosomal protein S6. Unexpectedly, we detected a novel, enhancing effect of myofiber β-catenin knockout on the resident muscle stem cells, or satellite cells. In the absence of myofiber β-catenin, satellite cells activate and proliferate earlier in response to mechanical overload. Consistent with the role of satellite cells in muscle repair, the enhanced recruitment of satellite cells led to a significantly improved regeneration response after chemical injury. The novelty of these findings resides in the fact that the genetic perturbation was extrinsic to the satellite cells, and this is even more surprising because the current literature focuses heavily on intrinsic mechanisms within satellite cells. As such, this model of myofiber β-catenin knockout may significantly contribute to better understanding of the mechanisms of satellite cell priming, with implications for regenerative medicine.

Vitamin D has been connected with increased intramyocellular lipid (IMCL) mitochondrial function in skeletal muscle. It is also shown to prevent lipotoxicity in several tissues, but this has not yet been examined in skeletal muscle. Perilipin 2 (PLIN2), a lipid droplet protein upregulated with vitamin D treatment, is integral to managing IMCL capacity and lipid oxidation in skeletal muscle. Increased lipid storage and oxidation is associated with increased tolerance to a hyperlipidic environment and resistance to lipotoxicity. Therefore, I hypothesized that vitamin D increases β-oxidation and lipid turnover though a PLIN2 mediated mechanism, thereby preventing lipotoxicity. This hypothesis was divided into two specific aims: 1) Characterize the effect of vitamin D and PLIN2 on lipid turnover and β-oxidation in mature myotubes, and 2) Determine the role of vitamin D and PLIN2 in regulating key markers of lipotoxicity. To address these aims, cells were treated with or without vitamin D, palmitate, and PLIN2 siRNA in an eight group, 2x2x2 design. Key experiments included quantitative real time polymerase chain reaction for markers of lipid accumulation, lipolysis, and lipotoxicity; Seahorse oxygen consumption assay; 14C-palmitate oxidation assay; and analyses of lipid accumulation and profile. Failure of the palmitate treatment to produce a reliable model for lipotoxicity resulted in negative data for Aim 2 of this dissertation and a focus on vitamin D and PLIN2 knockdown treatments as a four group, 2x2 model. Aim 1 showed that vitamin D reliably increases markers of lipolysis and lipid accumulation. Most of these markers were in turn decreased after PLIN2 knockdown, and DGAT2 exhibited an interaction effect between the two treatments. Contrary to our hypothesis and some published research, PLIN2 knockdown did not prevent lipid accumulation. Vitamin D increased oxygen consumption, especially consumption driven by mitochondrial complex II. PLIN2 knockdown decreased oxygen consumption and demonstrated an interaction effect specific to mitochondrial complex II. Data in this dissertation show that vitamin D increases mitochondrial function, and these effects are at least in part accomplished through a PLIN2 mediated mechanism. However, this work lacks the data required to make specific claims regarding β-oxidation and lipid turnover. This research is some of the first to show that PLIN2 knockdown carries negative impacts for skeletal muscle mitochondria and makes valuable contributions to general knowledge of how vitamin D and lipid storage impact muscle health and function. This ultimately provides additional evidence to advocate for vitamin D supplementation as a means of improving musculoskeletal health and function. Future research should investigate how vitamin D and PLIN2 impact markers of lipotoxicity in skeletal muscle.

The purpose of this dissertation was to explore the physical and psychological responses to the combination of major trauma (Injury Severity Score [ISS] > 15) and variable periods of immobility. Specific aims were to: 1) develop a conceptual model that illustrates physiological and psychological alterations that occur after injury and subsequent immobility, and their association with skeletal muscle responses and recovery; 2) evaluate daily measures of skeletal muscle strength (bicep and quadricep) using dynamometry and skeletal muscle (rectus femoris and biceps brachii) muscle thickness measured with ultrasound in patients after major trauma; and 3) assess the predictive ability of anxiety and depressive symptoms after traumatic injury on delayed ambulation (> 48 hours) following hospital admission. Specific Aim 1 was addressed by development of a conceptual model to describe the association between injury responses, immobility and skeletal muscle after trauma based on a comprehensive review of the state of the science. This model guided the research reported in Aims 2 and 3. The second specific aim was addressed with the conduct of an observational study in which we evaluated daily skeletal muscle strength with dynamometry and muscle thickness with ultrasound to evaluate the impact of trauma and immobility on skeletal muscle in patients after major trauma (n = 19). Participants with delayed ambulation after trauma (more than 48 hours immobility) demonstrated significantly less muscle strength compared with those who had early ambulation (bicep: delayed ambulation 12.9 ± 3.8, early ambulation 17.7 ± 4.7, p = 0.004; quadriceps: delayed ambulation 9.9 ± 3.1, early ambulation 17.1 ± 4.6, p = 0.001). Muscle thickness was unchanged over time in those with delayed ambulation; however, in those who ambulated early, muscle thickness significantly increased by 0.17 cm (p = 0.008) from baseline to day 5. The third specific aim was addressed with data collected during the same observational study of patients after trauma (n = 19). Participants provided measures of anxiety and depressive symptoms at baseline. Anxiety was not a predictor of delayed ambulation; however, depressive symptoms increased the likelihood of delayed ambulation by 67% (Odds Ratio [OR]: 1.67, 95% CI: 1.02 – 2.72, p = 0.041). Early ambulation was associated with significantly greater muscle strength and thickness as determined by dynamometry and muscle ultrasound, and depressive symptoms significantly increased the likelihood of delayed ambulation. Systematic evaluation of the association between trauma injury, immobility, skeletal muscle function and structure, and psychological state will provide an opportunity for the appropriate evaluation after injury and development of effective, tailored interventions to improve short- and long-term physiological and psychological recovery.

Au sein du muscle squelettique, le réticulum sarcoplasmique occupe une place essentielle dans la régulation de l’homéostasie calcique et de la contraction musculaire. En particulier, le transporteur calcique SERCA, situé à la membrane du réticulum endoplasmique permet de reconstituer le contenu calcique réticulaire suite à une contraction musculaire. Dans le muscle squelettique, l’activité de SERCA est contrôlée par un peptide inhibiteur spécifique appelé la myoréguline. Nous nous intéressons au rôle du récepteur nucléaire Rev-erb-α, un répresseur de transcription connu pour favoriser la fonction musculaire et dont l’activité peut être modulée par des ligands pharmacologiques. Nos résultats montrent que Rev-erb-α réprime l’expression de la myoréguline en se fixant sur son promoteur, ce qui a pour conséquence l’augmentation de l’activité de SERCA et la hausse du contenu calcique réticulaire. Un traitement avec un agoniste de Rev-erb-α, le SR9009, améliore l’homéostasie calcique et la contractilité musculaire de souris mdx/utr+/-, un modèle de la myopathie de Duchenne. Par ailleurs, le réticulum endoplasmique est le siège de la conformation des protéines de la voie sécrétoire. Des altérations de la conformation protéique provoquent un stress réticulaire et le déclenchement de la réponse aux protéines mal-conformées qui peut conduire jusqu’à l’apoptose. Il est décrit que le stress réticulaire est un phénomène impliqué dans l’activation de la cellule satellite musculaire suite à une blessure. Nous avons établi que Rev-erb-α, en augmentant l’interaction entre le réticulum endoplasmique et la mitochondrie accroit l’activation de la réponse aux protéines mal-conformées et l’apoptose de cellules satellites activées, ce qui pourrait impacter le potentiel de régénération musculaire. En conclusion, nous avons identifié Rev-erb-α comme un modulateur de la fonction du réticulum endoplasmique dans le muscle squelettique. Dans le futur, des thérapies ciblant spécifiquement Rev-erb-α pourraient être développées dans le cadre de pathologies musculaires chez l’Homme. / Within skeletal muscle, the sarcoplasmic reticulum plays an essential role in the regulation of calcium homeostasis and muscle contraction. In particular, the SERCA transporter, located at the membrane of the endoplasmic reticulum, by pumping calcium from cytosol from reticular lumen, allows the reticular calcium content to be reconstituted following muscle contraction. In skeletal muscle, SERCA activity is controlled by a specific inhibitory peptide called myoregulin. We are interested in the role of the nuclear receptor Rev-erb-α, a transcription repressor known to promote muscle function and whose activity can be modulated by pharmacological ligands. Our results show that Rev-erb-α represses the expression of myoregulin by binding to its promoter, which results in an increase in SERCA activity and an increase in reticular calcium content. Treatment with a Rev-erb-α agonist, SR9009, improves calcium homeostasis and muscle contractility in mdx/utr+/- mice, a model of Duchenne myopathy. In addition, the endoplasmic reticulum is the site of protein conformation of the secretory pathway. Alteration in protein conformation causes reticular stress and triggers the unfolded protein response that can lead to apoptosis. It is described that reticular stress is a phenomenon involved in the activation of skeletal muscle satellite cell following an injury. We have established that Rev-erb-α, by increasing the interaction between endoplasmic reticulum and mitochondria enhances the activation of unfolded protein response and apoptosis of activated satellite cells, which could impact the muscle regeneration capacity. In conclusion, we have identified Rev-erb-α as a modulator of endoplasmic reticulum function in skeletal muscle. In the future, specific Rev-erb-α targeting therapies may be developed for human muscle diseases.

Action potentials of striated muscle are created through movement of ions through membrane ion channels. ATP-sensitive potassium (KATP) channels are the only known channels that are gated by the intracellular energetic level ([ATP]/[ADP] ratio). KATP channels are both effectors and indicators of cellular metabolism as part of a negative feedback system. Decreased intracellular energetic level alters the gating of KATP channels, which is reflected in alterations of the action potential morphology. These changes protect the cell from exhaustion or injury by altering energy-consuming processes that are driven by membrane potential. Assessing the effects of KATP channel activation on resting membrane potential and action potential morphology, and the relationship to cellular stress is important to the understanding of normal cellular function. To better understand how muscle cells adapt to energetic stress, the monophasic action potential (MAP) electrode and floating microelectrode were used to record action potentials in intact hearts and skeletal muscles, respectively. Intact organs provide a more physiological environment for the study of energetics and membrane electrical phenomena. Utilizing these techniques, a stress on the intracellular energetic state resulted in greater and faster shortening of the duration of cardiac action potentials, and hyperpolarization of the membrane of skeletal muscle in a KATP channel dependent manner. Motion artifacts are a limitation to studying transmembrane action potentials, but the MAP and floating microelectrode techniques uniquely allow for reading of action potential morphology uncoupled from motion artifacts. The use of the floating microelectrode in skeletal muscles is a novel approach that provides previously unavailable data on skeletal muscle membrane potentials in situ.

Skeletal muscle atrophy is a common and often debilitating complication of diverse stresses including muscle disuse, fasting, aging, critical illness and many chronic illnesses. However, the pathogenesis of muscle atrophy is still poorly understood. The thesis herein describes my studies investigating the molecular mechanisms of skeletal muscle atrophy. Using mouse skeletal muscle and cultured skeletal myotubes as experimental systems, I discovered a novel stress-induced pathway in skeletal muscle that causes muscle atrophy. The pathway begins with stress-induced expression of ATF4, a basic leucine zipper (bZIP) transcription factor with an evolutionarily ancient role in cellular stress responses. I found that diverse stresses including fasting and muscle disuse increase expression of ATF4 in skeletal muscle. ATF4 then activates the growth arrest and DNA damage-inducible 45a (Gadd45a) gene, leading to increased expression of Gadd45a protein, an essential and inducible subunit of DNA demethylase complexes. Gadd45a localizes to skeletal myonuclei where it interacts with and stimulates demethylation of a specific region in the promoter of the cyclin dependent kinase inhibitor 1a (Cdkn1a) gene. By demethylating the Cdkn1a promoter, Gadd45a activates the Cdkn1a gene, leading to increased expression of Cdkn1a protein, also known as p21WAF1/CIP1. Cdkn1a stimulates protein breakdown (a critical pro-atrophy process) and inhibits anabolic signaling, protein synthesis and PGC-1α expression (processes that maintain healthy skeletal muscle and protect against atrophy). As a result, Cdkn1a causes skeletal muscle fibers to undergo atrophy. Importantly, interventions that reduce any one component of this pathway (ATF4, Gadd45a or Cdkn1a) reduce skeletal muscle atrophy during fasting, muscle disuse, and perhaps other skeletal muscle stresses such as illness and aging. Conversely, forced expression of any one component of this pathway is sufficient to cause skeletal muscle fiber atrophy in the absence of upstream stress. These data suggest the ATF4/Gadd45a/Cdkn1a pathway as a potential therapeutic target. Collectively, my studies demonstrate that the sequential, stress-induced expression of ATF4, Gadd45a and Cdkn1a is a critical process in the pathogenesis of skeletal muscle atrophy. This significantly advances our understanding of how muscle atrophy occurs and it opens up new avenues of investigation into the causes and treatment of muscle atrophy.

Skeletal muscle atrophy is a debilitating condition that commonly occurs as a secondary consequence of many acute and chronic medical conditions, including muscle disuse, heart and renal failure, starvation, cancer, HIV/AIDS, and aging. Though it leads to weakness, falls, and fractures, and reduces independence and quality of life for millions of Americans annually, no effective pharmacologic therapies for muscle atrophy exist. This is largely due to a poor understanding of the pathogenesis of skeletal muscle atrophy at a molecular level. In this thesis, I describe my studies into the molecular pathogenesis of skeletal muscle atrophy. Using mouse models, I showed that the gene encoding the pro-atrophy nuclear protein Gadd45a is regulated by distinct pathways after muscle denervation and fasting, and also identified a novel protein regulating skeletal muscle fiber size. First, we demonstrated that denervation-induced muscle atrophy, unlike atrophy mediated by fasting, does not require the bZIP transcription factor ATF4. However, the lysine deacetylase HDAC4 is sufficient to induce Gadd45a mRNA and necessary for Gadd45a mRNA induction after denervation, but not after fasting. Taken together, these data show that Gadd45a is a central convergence point for muscle atrophy caused by several stimuli, and also demonstrate that distinct pathways mediate Gadd45a induction in different models of skeletal muscle atrophy. Second, we identified spermine oxidase as a critical regulator of muscle fiber size. We observed that spermine oxidase mRNA and spermine oxidase protein were reduced by several distinct causes of muscle atrophy (i.e. immobilization, denervation, fasting, and aging). Furthermore, spermine oxidase overexpression increased muscle fiber size, while spermine oxidase knockdown caused muscle fiber atrophy. Restoring spermine oxidase expression significantly attenuated muscle atrophy after limb immobilization, denervation, and fasting. Finally, we identified p21 as a key upstream regulator of spermine oxidase expression, and spermine oxidase as a required mediator of p21-mediated skeletal muscle fiber atrophy. Collectively, these findings greatly advance our understanding of the molecular pathogenesis of skeletal muscle atrophy. These data demonstrate that Gadd45a is a convergence point for multiple pro-atrophy pathways and identify spermine oxidase as a novel therapeutic target for the treatment of skeletal muscle atrophy. These discoveries suggest several important new areas for future research, and further our understanding of this common, debilitating condition.

It has long been known that in addition to disruptions in glucose homeostasis, individuals with insulin resistance have a breakdown in lipid dynamics, often manifested by elevated levels of circulating fatty acids (FA) together with accumulation of lipids in insulin-sensitive tissues, including skeletal muscle. However, little is known about how common therapies used to treat insulin resistant individuals (such as Rosiglitazone and exercise training) improve skeletal muscle lipid and glucose metabolism. Thus, the primary aim of the studies undertaken for this thesis was to enhance our understanding of the mechanisms by which Rosiglitazone and exercise training improve skeletal muscle lipid metabolism and insulin sensitivity in two distinct models of insulin resistance. The first investigation determined the effect of chronic Rosiglitazone treatment on the accumulation of lipid metabolites and enzymatic regulators of lipid metabolism in the skeletal muscle of obese Zucker rats. The observation that Rosiglitazone treatment exacerbated the accumulation of muscle ceramide and diacylglycerol in skeletal muscle, while improving glucose tolerance led to the conclusion that this insulin sensitising drug improves insulin sensitivity by mechanisms other than reduction of fatty acid metabolites in this tissue. Accordingly, the second investigation sought to identify an alternative mechanism by which Rosiglitazone treatment may improve skeletal muscle insulin sensitivity. It was found that Rosiglitazone restored AMP-activated protein kinase (AMPK) á2 activity in the skeletal muscle of obese Zucker rats, providing a potential peroxisome proliferator activated receptor (PPAR) ã-independent mechanism by which this drug may mediate its insulinsensitising actions. The final experiment undertaken for this thesis determined the independent and interactive effects on Rosiglitazone and exercise training on various aspects of skeletal muscle glucose and lipid metabolism in a model of diet-induced insulin resistance, the high-fat fed rat. Exercise training, but not Rosiglitazone treatment restored skeletal muscle insulin sensitivity in high-fat fed rats. Improvements in insulin sensitivity with exercise training were associated with increased FA oxidation, increased AMPK activity and a normalisation of the expression of the Akt substrate, AS160. In contrast, Rosiglitazone treatment was associated with increased FA uptake and decreased insulin-stimulated glucose uptake in skeletal muscle. Exercise prevented the accumulation of skeletal muscle lipids in Rosiglitazone-treated animals when the two treatments were combined. In summary, the results from the studies undertaken for this thesis provide novel information regarding the mechanisms by which two insulinsensitising therapies, exercise training and Rosiglitazone treatment, act to improve glucose and lipid metabolism in skeletal muscle.It has long been known that in addition to disruptions in glucose homeostasis, individuals with insulin resistance have a breakdown in lipid dynamics, often manifested by elevated levels of circulating fatty acids (FA) together with accumulation of lipids in insulin-sensitive tissues, including skeletal muscle. However, little is known about how common therapies used to treat insulin resistant individuals (such as Rosiglitazone and exercise training) improve skeletal muscle lipid and glucose metabolism. Thus, the primary aim of the studies undertaken for this thesis was to enhance our understanding of the mechanisms by which Rosiglitazone and exercise training improve skeletal muscle lipid metabolism and insulin sensitivity in two distinct models of insulin resistance. The first investigation determined the effect of chronic Rosiglitazone treatment on the accumulation of lipid metabolites and enzymatic regulators of lipid metabolism in the skeletal muscle of obese Zucker rats. The observation that Rosiglitazone treatment exacerbated the accumulation of muscle ceramide and diacylglycerol in skeletal muscle, while improving glucose tolerance led to the conclusion that this insulin sensitising drug improves insulin sensitivity by mechanisms other than reduction of fatty acid metabolites in this tissue. Accordingly, the second investigation sought to identify an alternative mechanism by which Rosiglitazone treatment may improve skeletal muscle insulin sensitivity. It was found that Rosiglitazone restored AMP-activated protein kinase (AMPK) á2 activity in the skeletal muscle of obese Zucker rats, providing a potential peroxisome proliferator activated receptor (PPAR) ã-independent mechanism by which this drug may mediate its insulinsensitising actions. The final experiment undertaken for this thesis determined the independent and interactive effects on Rosiglitazone and exercise training on various aspects of skeletal muscle glucose and lipid metabolism in a model of diet-induced insulin resistance, the high-fat fed rat. Exercise training, but not Rosiglitazone treatment restored skeletal muscle insulin sensitivity in high-fat fed rats. Improvements in insulin sensitivity with exercise training were associated with increased FA oxidation, increased AMPK activity and a normalisation of the expression of the Akt substrate, AS160. In contrast, Rosiglitazone treatment was associated with increased FA uptake and decreased insulin-stimulated glucose uptake in skeletal muscle. Exercise prevented the accumulation of skeletal muscle lipids in Rosiglitazone-treated animals when the two treatments were combined. In summary, the results from the studies undertaken for this thesis provide novel information regarding the mechanisms by which two insulinsensitising therapies, exercise training and Rosiglitazone treatment, act to improve glucose and lipid metabolism in skeletal muscle.

<p>Human skeletal muscles possess thixotropic, i.e. history-dependent mechanical properties. This means that the degree of passive muscle stiffness and resting tension is dependent on the immediately preceding history of contractions and length changes. Athletes, for instance, reduce passive muscle stiffness by various types of ‘limbering-up’ procedures, whereas muscle stiffness gradually increases during inactivity.</p><p>Passive resistance of antagonistic muscles may significantly add to the total load during voluntary muscle contractions. This resistance may vary from one moment to another, depending on immediately preceding events. This research was conducted to determine whether history-dependent variations in passive muscular forces influence motor control of voluntary joint movements and steady maintenance of joint positions in healthy subjects. </p><p>In study I, the EMG signal revealed motor compensations for history-dependent variations in passive stiffness of the antagonists during slow voluntary wrist joint movements. Studies II and III demonstrated that the voluntary muscle activity required to maintain a certain wrist joint position was highly influenced by previous changes in forearm muscle length and contractions. Study IV showed that rapid voluntary movements varied in speed and onset time depending on the prevailing degree of muscle resistance, and in addition that the central nervous reaction time required to execute rapid movements was highly influenced by immediately preceding muscle-conditioning procedures.</p><p>History-dependent variations in passive muscular forces seem to be effectively compensated by the motor control system. Presumably, voluntary motor commands to the muscles are automatically adjusted in strength to history-dependent changes in passive muscular forces. Such adjustments occur within the central nervous system, which receives information about the mechanical state of the muscles. Several issues in connection with muscle thixotropy remain unaddressed. For instance, do alterations in the normal thixotropic mechanical behaviour of the muscles impose a particular problem in patients with certain neuromuscular diseases? </p>