PRMTs and exercise. / Protein arginine methyltransferases (PRMTs) play an important role in muscle. Using three unique but complementary approaches across human and mouse models, we examined PRMT biology during conditions of exercise-induced skeletal muscle plasticity. In response to acute and chronic cues for muscle plasticity in human muscle, an array of PRMT-specific increases and reductions in expression and activity were observed. Following this we generated coactivator-associated arginine methyltransferase 1 (CARM1) skeletal muscle-specific knockout (mKO) mice to further examine the role of this enzyme. We discovered that the rate of arginine methylation is equivalent to that of phosphorylation and ubiquitination in healthy muscle. CARM1 mKO displayed altered transcriptome and arginine methylproteomic signatures, confirming remodelled muscle contractile and neuromuscular junction characteristics, which foreshadowed the animal’s decreased acute exercise tolerance. Removal of CARM1 reduced voluntary wheel running (VWR) performance in a sex-dependent manner and eliminated the strong, positive correlation between VWR distance and mitochondrial number observed in WT mice. While CARM1 was shown to regulate AMPK-PGC-1α signaling during acute conditions of activity-induced muscle plasticity, molecular measures of PRMT biology were mostly unaffected by VWR and the removal of this enzyme. In conclusion, these results indicate that changes to expression and activity are PRMT-specific and reveal the broad impact of CARM1 in the maintenance and remodelling of skeletal muscle biology. / Thesis / Doctor of Philosophy (PhD) / Skeletal muscle is a malleable tissue that can adapt to an array of physiological demands. Past research suggests that protein arginine methyltransferases (PRMTs) regulate skeletal muscle remodelling. However, their role in exercise-induced skeletal muscle plasticity is unknown. Therefore, the purpose of this work was to investigate PRMT biology during acute and chronic exercise. Our data demonstrate that in human muscle a variety of PRMT-specific alterations in expression and activity occur in response to cues for muscle plasticity. Using mice lacking coactivator-associated methyltransferase 1 (CARM1) in skeletal muscle, we studied the impact removal of CARM1 has on the acute and chronic muscle adaptations to training. Our data demonstrate that in addition to changing molecular signals and physiological function at rest, the deletion of CARM1 decreased acute exercise ability and altered chronic training performance in a sex-dependent manner. Altogether, these findings expand our knowledge of PRMTs in skeletal muscle biology.

No / Objectives Ischaemia modified albumin (IMA) has been used as a marker of myocardial ischaemia but little is known about its production during ischaemia of other tissues. The clinical models of patients with intermittent claudication and major arterial surgery were used to investigate IMA production from ischaemic skeletal muscle. Materials and methods IMA was measured pre-operatively, at end ischaemia, and 5min, 4, 24, 48, 72 and 144h post-surgery in patients undergoing (a) revascularisation for intermittent claudication (IC, n=15), (b) abdominal aortic aneurysm repair (AAA, n=12) and controls (n=16). Results The median pre-operative IMA concentration in IC patients was significantly higher than the AAA group (88.3 versus 83.5U/ml, p=0.036) and controls (88.3 versus 80.3U/ml, p=0.031). IMA concentrations increased significantly during arterial clamping in both IC and AAA groups (88.3 versus 120.0U/ml, p=0.001; 83.5 versus 118.8U/ml, p=0.002, respectively) consistent with increased skeletal muscle ischaemia. In contrast, there was only a mild perioperative increase in the controls (80.3 versus 91.6U/ml, p=0.012). Conclusions Patients with intermittent claudication have significantly elevated IMA and skeletal muscle ischaemia during arterial surgery results in significantly increased circulating IMA. When IMA is used to detect myocardial ischaemia, ischaemic skeletal muscle must be excluded.

Skeletal muscle disease (myopathy) carries an enormous psychological, social and economic impact on the lives of the patients and their caregivers. There is also an appreciable amount of economic burden on the healthcare system and our society especially when most patients are in their childhood/adolescent lives. It is not a surprising fact that a percentage of myopathies are of undetermined cause, which makes the need to identify new genes that play a critical role in muscle health of paramount importance. The Xin gene is designated as an indispensable component for the normal development and morphogenesis of striated muscle; however, the exact roles of Xin in skeletal muscle are still undefined. Studies to date have demonstrated that Xin is expressed in activated satellite cells and newly formed myofibers following tissue injury. When using in vitro reduction of Xin expression in skeletal muscle cells, the outcome was impairments in satellite cell function and muscle response to injury. That said, there is still a significant lack of knowledge in the literature regarding the in vivo effects of the absence of Xin on skeletal muscle structure and function. Therefore, the aim of our studies was to characterize skeletal muscles in the absence of Xin and identify the potential roles of Xin in maintaining muscle integrity and health. In our studies, we uncover a new form of muscle disease resulting from the lack of Xin in a mouse model (Xin-/- mice). Xin-/- skeletal muscles show a mild form of myopathy at the light microscopy level that is associated with ultrastructural and functional defects peculiar to the Ca2+ handling. In addition, Xin-/- muscle demonstrated features of mitochondrial dysfunction that are likely secondary to the Ca2+ defects caused by the absence of Xin. Our studies provide a platform to investigate the potential manifestation of “loss of function” mutation of Xin in skeletal muscle and its possibly related pathology in human diseases. / Thesis / Doctor of Philosophy (PhD) / Skeletal muscle covers the bones, produces movements and stabilizes joints. Diseases of skeletal muscle are common. Specifically, inherited diseases of skeletal muscle can cause an inability to eat, walk, run, work and even breathe. By starting at an early age, these conditions may result in the patient being bed-ridden with severe psychological, social and financial burdens for them and their caregivers. When skeletal muscles that control breathing are involved, death can occur at a young age. A considerable proportion of skeletal muscle disease are of unknown cause, and therefore, there is a constant need to discover more factors that influence muscle health to provide treatment plans for these patients. This dissertation uncovers the role of a new factor, Xin, involved in keeping skeletal muscle healthy. The achieved results of our studies will help physicians use this knowledge into identifying new types of muscle disease and providing targeted treatments for patients.

The purpose of this investigation was to examine the effects of selective and non-selective β-blockade on muscle excitability and fatiguability during exercise. Ten healthy males (x̄ age= 21.9 ± 7.1 yrs) participated in all phases of the study. The first stage was designed to determine equipotent doses of the selective (metoprolol) and non-selective (propranolol) β-blocking agents within each subject. Symptom limited, maximal graded exercise tests were performed on an electrically braked cycle ergometer during a control condition and after the administration of 100 mg of metoprolol. Following this, exercise tests were performed to establish a dose of propranolol equipotent to that of 100 mg of metoprolol within each subject. In 8 of the subjects, 80 mg of propranolol produced a heart rate attenuation equal to that of 100 mg of metoprolol. In contrast, 1 subject required 60 mg of propranolol while another required 100 mg of propranolol to match the effects of the metoprolol treatment. Significant reductions in the submaximal and maximal oxygen uptakes were observed during the metoprolol (9% ↓ ± 7%; 10% ↓ ± 4%, respectively) and the propranolol (9% ↓ ± 7%; 19% ↓ ± 4%, respectively) treatments. Similarly, the time to exhaustion was reduced significantly by 13% (± 8%) and 19% (± 8%) following the administration of metoprolol and propranolol, respectively. The reductions in the maximal oxygen uptake and the time to exhaustion elicited by the β-blocking agents were significantly greater following the non -selective versus the selective drug treatments. It was hypothesized that part of the impairment in exercise performance with β-blockade could be the result of an inhibition in the activity of the adrenergically cor,trolled Na⁺-K⁺ ATPase with a subsequent failure in muscle excitability during exercise. Thus, in the second stage of this investigation, a double blind design was utilized to investigate the effects of metoprolol and propranolol on muscle excitability and fatiguability. Subjects performed a 4 minute fatigue protocol consisting of intermittent, isometric voluntary contractions of the knee extensor muscles in one leg. The protocols were performed on three separate occasions following the administration of either placebo, 100 mg of metoprolol or an equipotent dose of propranolol. Surface electrodes were used to record the voluntary EMG activity and M-waves from the vastus medialis in the active and inactive legs throughout each of the drug trials. During the control trial, significant declines in the evoked twitch torque (77% ↓ ± 15%) and the voluntary torque (55% ↓ ± 11%) were observed but these recovered completely within 15 minutes following the exercise. In contrast, both the voluntary EMG activity and the M-waves recorded from the active and inactive legs were maintained throughout fatigue and recovery in the control state. Neither the evoked contractile properties for the voluntary muscle strength of the knee extensors were affected by the administration of metoprolol or propranolol. The fatiguability of the quadriceps was also unaffected by the β-blocking agents. Similarly, the β-blockade treatments did not alter the EMG activity or the M-waves measured from either of the legs at rest and over the course of fatigue and recovery. The results of this investigation suggest that although β-blocking agents do impair dynamic exercise performance, there is no effect of these agents on peripheral skeletal muscle function during single limb exercise. These observations have been explained in relation to the possible central and hemodynamic effects of β-blockade. / Thesis / Master of Science (MSc)

Many have been searching for and contemplating the origins of syphilis. By understanding its emergence as a human pathogen we will be better able to elucidate its evolution through time and space as well as shed light on its current state. Ancient DNA techniques used to isolate Treponema pallidum subspecies pallidum DNA from archaeological human specimens provides direct evidence of its existence in the past. To date, only Kolman et al. (1999) have been successful in this endeavour. Along with this protocol, two other published protocols and novel allele specific techniques this thesis aims to add new cases of venereal syphilis identification from historic human remains. To accomplish this, sixteen skeletal samples from different time periods and geographic locations were collected for this project. Of importance are those dating from the Civil War time period from the United States as medical documents state these individuals suffered and/or died from the complications of syphilis. Samples from the United Kingdom are also critical to this analysis as they have confirmed pre-Columbian dates. Along with attempts to isolate bacterial DNA, endogenous DNA (mitochondrial and amelogenin) is analyzed to provide an idea of the different levels of molecular preservation. General preservation as well as the identification of syphilis are also performed using microscopic techniques. By using a tiered approach (macroscopic to microscopic to molecular), a better idea of both preservation and disease presence can be ascertained. Results indicate that although syphilis could be identified at both the macroscopic and microscopic levels for some individuals and endogenous DNA was present, treponemal DNA failed to amplify. Many different reasons are suggested, for example poor conservation methods, misdiagnosis and diagenesis, but the most important possibility is the lack of bacterial DNA in bone at later stages of syphilis which was confirmed using the rabbit model. As a result, the present techniques may not be conducive for treponemal DNA isolation from ancient human remains. / Thesis / Doctor of Philosophy (PhD)

Obesity-related metabolic derangements have been linked to toll-like receptor 4 (TLR4), an innate immune system receptor, due to its role in proinflammatory pathways. Lipopolysaccharide (LPS), a gram-negative bacteria cell wall component, is the ligand for TLR4, and has been shown to be elevated in states of metabolic disease. Heightened levels of circulating endotoxin is termed metabolic endotoxemia and has been linked to systemic inflammation which is associated with obesity, type 2 diabetes mellitus (T2DM), and cardiovascular disease (CVD). Immune cells exhibit a protective ability to develop endotoxin tolerance. The objective of this study was to determine if endotoxin tolerance exists in skeletal muscle cells, and if a condition that mimics a state of over nutrition, such as elevated levels of fatty acids, affect this tolerance. To this end, L6 skeletal muscle cells were treated with low (50 pg/mL)- and high (500 ng/mL)-doses of LPS, with and without the presence of free fatty acids (FFAs). Tolerance was assessed by measuring: 1) changes in mRNA expression of interleukin-6 (IL-6) and monocyte chemoattractant-1 (MCP-1) as markers of a pro-inflammatory response; and 2) mRNA levels of peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC1-°) and mitochondrial oxidative capacity via an XF24 Flux Analyzer (Seahorse Bioscience) as measures of the metabolic response. Tolerance to LPS was observed in response to low- and high-doses with MCP-1 mRNA transcription but not IL-6. Changes in PGC1-° and mitochondrial OCR exhibited a tolerant effect in response to the high dose of LPS but not the low dose. The addition of free fatty acids to LPS treatments did not prevent the tolerant effects under any conditions. In conclusion, LPS tolerance exists in skeletal muscle cells but appears to differ depending on pro-inflammatory target and LPS concentration. Additionally, fatty acids, in the current model, have no effect on LPS tolerance. / Master of Science

The vast majority of horses engage in some form of exercise, whether it be for leisure or competition. Despite almost half of the approximately 7.2 million horses engaging in structured athletic work, very little is known about one of the most critical facets of recovery: satellite cells (SCs). Satellite cells lie adjacent to the myofiber of skeletal muscle, poised to enter the myogenic program and fuse to the nearby muscle after a damaging event. Hepatocyte growth factor (HGF) and insulin-like growth factor-1 (IGF-1) transcript abundance increased after an exhaustive bout of endurance exercise in concert with myogenic regulator factors and preceding increased SC abundance in a previous study. This suggests that SCs may participate in repair of exercise-induced muscle damage. To assess the role of HGF in this process, equine SCs (eqSCs) were isolated from the gluteus medius of mature thoroughbred geldings for activation, proliferation and differentiation assays. Activation was not accelerated by 1, 5, 10, or 25 ng/mL HGF. Instead, 25 ng/mL HGF increased the proliferation rate of eqSC via protein kinase C δ and decreased differentiation. The influence of dietary L-citrulline, an amino acid that has the potential to influence SC activity and nutrient availability by its metabolism to L-arginine, was assessed during recovery from exercise in unfit adult horses. To model submaximal exercise, horses were exercised for 1 h at an average heart rate of 116 bpm, suggested to be typical of a heavy exercise session by the National Research Council. L-citrulline decreased myogenin mRNA abundance compared to controls while exercise increased peroxisome proliferator-activated receptor gamma coactivator 1- α (PGC1α) mRNA abundance, a master regulator of energy metabolism, at 1 d post-exercise. Although SCs were not activated in response to a single bout of submaximal exercise, metabolic regulators increased in the early period of recovery. Through these studies eqSC dynamics during exercise are better defined. / Doctor of Philosophy / The horse is well-known as an athletic creature and is often used in amateur and professional athletic events. Despite its popularity as a pastime in low and high-stakes competition, certain facets directly related to performance during exercise remain relatively unstudied. One crucial component of recovery from exercise is the intrinsic ability of skeletal muscle to repair exercise-induced muscle damage. This is accomplished largely through the incorporation of new nuclei, which originate from a position orbiting the muscle, hence the name satellite cells. This cell is essential to muscle regeneration from injury as often demonstrated in rodent models, but the role of satellite cells in recovery from exercise remains elusive in all species, but particularly so in horses. For instance, whether satellite cells only contribute nuclei after exercise to stimulate gains in muscle mass or whether they may also play a role in the process of adaptation to exercise is not clearly understood. The purpose of my work was to define the response of satellite cells to hepatocyte growth factor, a factor present in skeletal muscle during exercise that is already well-studied in rodent models. Additionally, to determine whether the addition of the non-essential amino acid, citrulline, would influence satellite cells and nutrient reserves after a session of submaximal exercise. I found that hepatocyte growth factor does not influence satellite cells isolated from horses in the same way it influences those from rodents, nor through the same mechanisms. Additionally, I found that satellite cells were not stimulated after a session of submaximal exercise, but a factor involved in regulation of genetic expression that is associated with satellite cells and skeletal muscle was downregulated with the addition of citrulline. Together, these results suggest that satellite cells may behave like other species in some ways, such as some responses to hepatocyte growth factor and the lack of response to a submaximal bout of exercise, but that there is still much to be learned in order to begin to influence management and training decisions as regards skeletal muscle recovery.

Skeletal muscle functions as an endocrine organ. Exosomes, small vesicles containing mRNAs, miRNAs, and proteins, are secreted from muscle cells and facilitate cell-to-cell communication. Our recent work found greater exosome release from oxidative compared to glycolytic muscle. Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) is a key driver of mitochondrial biogenesis, a characteristic of oxidative muscle. It was hypothesized that PGC1α regulates exosome biogenesis and secretion in skeletal muscle. The purpose of this study is to determine if PGC-1α regulates skeletal muscle exosome biogenesis and secretion. On day 4 of differentiation, human primary myotubes from vastus lateralis biopsies from lean donors (BMI < 25.0 kg/m2) were exposed to adenovirus encoding human PGC-1α or GFP control. On day 6 of differentiation, culture media was replaced with exosome-free media. On day 8, cells were collected for mRNA and protein analysis, and culture media was collected for exosome isolation. Overexpression of PGC-1α increases regulators of exosome biogenesis in the endosomal sorting complexes required for transport (ESCRT) pathway: Alix (CON: 1.0 ± 0.2 vs. PGC-1α: 7.6 ± 3.8), TSG-101 (CON: 1.0 ± 0.1 vs. PGC-1α: 7.3 ± 2.1), CD63 (CON: 1.0 ± 0.17 vs. PGC-1α: 3.7 ± 0.4), Clathrin (CON: 1.0 ± 0.2 vs. PGC-1α: 11.6 ± 2.5), and the secretion pathway: Rab27b (CON: 1.0 ± 0.3 vs. PGC-1α: 3.2 ± 0.3), STAM (CON: 1.0 ± 0.3 vs. PGC-1α: 7.3 ± 0.6), and VTA1 (CON: 1.0 ± 0.1 vs. PGC-1α: 7.3 ± 2.4). Exosome count and total extracellular vesicle count were not significantly different from control. Overexpression of PGC-1α increases gene 9 expression of regulators of exosome biogenesis and secretion in human primary myotubes. In the future, in vitro studies assessing exosomal content from PGC-1 OE cells as well as in vivo effects of PGC-1 OE on exosome production and release should be investigated to further understand the role PGC-1 plays in exosome secretion.

Thesis (Ph. D.)--University of Virginia, 2008. / Includes bibliographical references. Also available in electronic form as viewed 2/16/2009.

A traumatic skeletal muscle injury that involves the loss of a substantial portion of tissue will not regenerate on its own. Little is understood about the ability of the muscle to recover function after such a defect injury, and few research models exist to further elucidate the repair and regeneration processes of defected skeletal muscle. In the current research, a model of muscle injury was developed in the lateral gastrocnemius (LGAS) of the rat. In this model, the muscle gradually remodels but functional recovery does not occur over 42 days. Repair of the defect with muscle-derived extracellular matrix (ECM), improves the morphology of the LGAS. Blood vessels and myofibers grow into the ECM implant in vivo, but functional recovery does not occur. Addition of bone marrow-derived mesenchymal stem cells (MSCs) to the implanted ECM in the LGAS increases the number of blood vessels and regenerating myofibers within the ECM. Following 42 days of recovery, the cell-seeded ECM implanted LGAS produces significantly higher isometric force than the non-repaired and non-cell seeded ECM muscles. These results suggest that the LGAS muscle defect is a suitable model for the study of traumatic skeletal muscle injury with tissue loss. Additionally, MSCs seeded on an implanted ECM lead to functional restoration of the defected LGAS. / text