This thesis is a series of investigations into the regulation of skeletal muscle mitochondrial metabolism. Novel regulatory mechanisms regarding mitochondrial fatty acid oxidation are continually being identified and alterations in skeletal muscle mitochondrial metabolism have been implicated in the pathogenesis of type II diabetes (T2DM). Therefore, advancing our basic understanding of mitochondrial regulatory processes is required to provide insight into the progression of T2DM. In study one, the utilization of knockout mice for the putative mitochondrial fatty acid transport protein FAT/CD36, showed that mitochondrial FAT/CD36 plays a functional role in mitochondrial long chain fatty acid (LCFA) oxidation. Specifically, FAT/CD36 was found to be located on the outer mitochondrial membrane (OMM) upstream of acyl-CoA synthetase. In study two, it was observed that in rat muscle, malonyl-CoA (M-CoA) inhibition kinetics of carnitine palmitoyltransferase I (CPT-I) display a more physiological IC50 in permeabilzed muscle fibre bundles (PmFB) compared to isolated mitochondria. These data suggest that the cytoskeleton may have a role in regulating M-CoA inhibition. Additionally, a significant effect of LCFA-CoA on M-CoA inhibition kinetics was observed. These data indicate that M-CoA content does not need to decrease to promote an increase in CPT-I flux. Finally, in a model of T2DM (ZDF rat), submaximal ADP-stimulated respiration rates and the content of adenine nucleotide translocase 2 (ANT2) content were depressed compared to lean control animals. Resveratrol treatment in ZDF rats recovered these declines concomitantly with improving insulin-stimulated skeletal muscle glucose uptake and the cellular redox state. A number of novel findings are presented, specifically, 1) a functional role for mitochondrial FAT/CD36 in mitochondrial LCFA oxidation was confirmed and the topology of this protein along the OMM is expanded upon, 2) M-CoA inhibition kinetics of CPT-I were re-evaluated in PmFB and a regulatory role of LCFA-CoA on M-CoA inhibition kinetics is established, and 3) submaximal ADP-stimulated respiration rates and ANT2 content are depressed in the ZDF rat and resveratrol supplementation prevents these decrements.

This thesis investigated the effects of omega-3 supplementation (2.0 g/day EPA + 1.0 g/day DHA) for 12 weeks on human skeletal muscle sarcolemmal and mitochondrial membrane fatty acid (FA) composition and whole body energy expenditure in young healthy males. Supplementation resulted in significant incorporation of EPA and DHA into sarcolemmal and mitochondrial membranes, with an increase in total unsaturation of mitochondrial membranes. The incorporation profile of the sarcolemma and mitochondria differed, with the mitochondria mimicking changes in whole muscle. There were no changes in the protein content of mitochondrial and selected proteins involved in energy metabolism, except for a significant increase in the long form of UCP3. Despite changes in membrane FA compositions, there were no changes in whole body substrate oxidation at rest or during exercise. These data demonstrate that omega-3 supplementation for 12 weeks altered the FA composition of sarcolemmal and mitochondrial membranes in human skeletal muscle.

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No description available.

Skeletal satellite cells are adult stem cells located among muscle fibers. Proliferation, migration and subsequent differentiation of these cells are critical steps in the repair of muscle injury. We document in this study the roles and mechanisms through which the NAPDH oxidase complex regulates skeletal satellite cell proliferation. The NADPH oxidase subunits Nox2, Nox4, p22phox, p47phox and p67 phox were detected in primary human and murine skeletal muscle satellite cells. In human satellite cells, NADPH oxidase-fusion proteins were localized in the cytosolic and membrane compartments of the cell, except for p47 phox, which was detected in the nucleus. In proliferating subconfluent satellite cells, both Nox2 and Nox4 contributed to O2- production. However, Nox4 expression was significantly attenuated in confluent cells and in differentiated myotubes. Proliferation of satellite cells was significantly reduced by antioxidants (N-acetylcysteine and apocynin), inhibition of p22phox expression using siRNA oligonucleotides, and reduction of Nox4 and p47phox activities with dominant-negative vectors resulted in attenuation of activities of the Erk1/2, PI-3 kinase/AKT and NFkappaB pathways and significant reduction in cyclin D1 levels. We conclude that NADPH oxidase is expressed in skeletal satellite cells and that its activity plays an important role in promoting proliferation of these cells.

Cache Valley virus (CVV) induced malformations have been previously reproduced in ovine fetuses; however, no studies have established the CVV infection sequence of the cells targeted by the virus or the development of the antiviral response of the early, infected fetus that results in viral clearance before development of immunocompetency. To address these questions, ovine fetuses at 35 dg were inoculated in utero with CVV and euthanized at 7, 10, 14, 21 and 28 dpi. On postmortem examination arthrogryposis and oligohydramnios were observed in some infected fetuses. Morphologic studies showed necrosis in the central nervous system (CNS) and skeletal muscle of earlier infected fetuses and hydrocephalus, micromyelia and muscular loss in later infected fetuses. Using immunohistochemistry and in situ hybridization, intense CVV viral antigenic signal was detected in the brain, spinal cord, skeletal muscles and fetal membranes of infected fetuses. Viral signal decreased in targeted and infected tissues with the progression of the infection. To determine specific cell types targeted by CVV in the CNS, indirect immunofluorescence was applied to sections of the CNS using a double labeling technique with antibodies against CVV together with antibodies against neurons, astrocytes and microglia. CVV viral antigen was shown within the cytoplasm of neurons in the brain and spinal cord. No viral signal was observed in microglial cells; however, infected animals had marked microgliosis. The antiviral immune response in immature fetuses infected with CVV was evaluated. Gene expression associated with an innate, immune response was quantified by real-time, quantitative PCR. Upregulated genes in infected fetuses included ISG15, Mx1, Mx2, IL-1, IL-6, TNF-?, TLR-7 and TLR-8. The amount of Mx protein, an interferon stimulated GTPase capable of restricting growth of bunyaviruses, was elevated in the allantoic and amniotic fluid in infected fetuses. ISG15 protein expression was significantly increased in target tissues of infected animals. B lymphocytes and immunoglobulin-positive cells were detected in lymphoid tissues and in the meninges of infected animals. This demonstrated that the infected ovine fetus is able to stimulate an innate and adaptive immune response before immunocompetency that presumably contributes to viral clearance in infected animals.

The inflammatory process is a critical component of the repair and regeneration of skeletal muscle following injury. The influx of innate immune cells following injury is intricate, and temporal nature. Although required for proper repair and regeneration, the inflammatory process has been shown to exacerbate initial damage, prolonging the healing process. Complementary Alternative Medicine (CAM) treatments, such as massage therapy, are a promising substitute for pharmaceutical modulation of the inflammatory response, and recent studies into the efficacy of massage have begun to report the physiological benefits of massage application following injury. Nonetheless, there is a significant lack of sound mechanistic investigations into massage application and its effects on unperturbed tissue. To gain insight to its potential influences on healthy skeletal muscle, massage was applied at three different magnitudes of load in vivo. Using a custom fabricated device for cyclic compressive loading, Wistar rats receiving massage had an increased expression in genes associated with the immune response; a significant change in the macrophage populations within the muscle tissue; and demonstrated a systemic effect marked by the increase of immune cells in the non-massaged limb. Further elucidating the systemic and immunomodulatory effects of massage, Long Evans rats receiving non-constrained eccentric exercise followed by a single 30minute bout of massage, displayed a significant crossover effect just 6 hours post exercise through the modulation of inflammatory cells in the non-massaged limb. Together these investigations suggest that mechanotransductive properties of massage can promote modulation of the immune response absent of pharmaceuticals.

Background/Objectives: Individuals with a spinal cord injury (SCI) experience reductions in lower-extremity muscle mass and increased fatty-infiltration of skeletal muscle, predisposing them to an increased risk of specific secondary health conditions. To date, few investigations have prospectively examined changes in muscle in the chronic stage of SCI. Peripheral quantitative computed tomography (pQCT) is an imaging technique capable of measuring lower-extremity skeletal muscle cross-sectional area (CSA) and muscle density, the latter is a surrogate measure of muscle fatty infiltration. The purpose of this project was to a) determine the magnitude of muscle CSA and muscle density reduction in a chronic-SCI population with diverse impairments; b) identify demographic and injury characteristics associated with muscle CSA and density status; and c) determine if muscle CSA and muscle density change over a two-year period following chronic-paralysis and if so, what factors are associated with the changes. Materials and Methods: Seventy individuals [50/20 m/f, mean (± SD) age 48.9 ± 11.5 years; duration of injury 15.5 ± 10.0 years] with chronic (>2 years post-injury) SCI (C1-T12, AIS A-D) were enrolled in a two-year cohort study. Muscle CSA and muscle density values were calculated from pQCT scans of the 66%-site of the calf obtained at baseline and two follow-up visits separated by one year. Possible correlates of muscle CSA and density selected a priori included: gender, age, height, weight, waist circumference, age at injury, level of injury, injury duration, leg spasm frequency and severity scale score (SFSS), ISNCSCI calf-muscle lower-extremity motor score (cLEMS), wheelchair use, serum vitamin D level, and physical activity level. Dependent t-tests were used to compare muscle CSA and muscle density values of participants with complete and incomplete-SCI to age, gender, and height matched able-bodied controls. Multiple linear regression models were used to determine correlates of muscle CSA and muscle density. Repeated measures analysis of variance (rANOVA) were used to examine change in muscle CSA and density over the two-year study duration and multiple linear regression models were created to determine correlates of muscle CSA and density change from baseline. Results: Individuals with motor-complete SCI had a 45% reduction in muscle CSA and a 32% reduction in muscle density relative to controls. Participants with motor-incomplete SCI had a 17% reduction in muscle CSA and a 14% reduction in muscle density relative to controls. A reduced height, waist circumference, cLEMS, and wheelchair use were associated with a smaller muscle CSA in the best-fitting regression model (R2 = 0.66; p<0.0001). In the best-fitting regression model for muscle density, increased age, a lower cLEMS, reduced SFSS, fewer minutes of daily vigorous physical activity, and wheelchair use were associated with a lower muscle density (R2= 0.37; p<0.001). A high degree of individual variability in muscle CSA change (mean ± SD: -1.9 ± 6.2cm2; range: -22.6 to 8.5 cm2) and muscle density change (mean ± SD: -1.2 ± 3.28mg/cc; range: -8.6 to 6.4 mg/cc) was observed in those with both complete and incomplete SCI over the two-year study duration. rANOVA indicated a significant reduction in both muscle CSA and density after controlling for individual variability. A greater waist circumference at baseline was weakly associated with a reduction in muscle CSA (R2 = 0.14, p<0.05), and a lower weight and waist circumference at baseline were associated with a reduction in muscle density (R2 = 0.26, p < 0.001 and R2 = 0.20, p < 0.01, respectively). Conclusion: Age, completeness of injury, spasticity, physical activity participation, and ambulation ability were identified as potential clinical predictors of muscle status in individuals with chronic-SCI. Muscle CSA and density does not reach a “steady-state” after chronic-SCI. Further investigation is needed to determine the mechanisms responsible muscle CSA and density change in order to prevent continued reductions after chronic-SCI.

The muscle fibre cytoskeleton of skeletal and heart muscle cells is composed mainly of intermediate filaments (IFs), that surround the myofibrils and connect the peripheral myofibrils with the sarcolemma and the nuclear membrane. Desmin is the first muscle specific IF protein to be produced in developing muscles and is the main IF protein in mature muscles. In skeletal muscle, desmin is particularly abundant at myotendinous and neuromuscular junctions. In the heart an increased amount of desmin is found at intercalated discs and in Purkinje fibres of the conduction system. Interactions between the IFs themselves, and between IFs and other structures such as Z-discs and the sarcolemma, are mediated by intermediate filament associated proteins (IFAPs). A transgenic mice model, which lacks the desmin gene have been developed to study the function of desmin. In these mice, morphological abnormalities are observed in both heart and skeletal muscles. Similar defects have been observed in human myopathies, caused by different mutations in the desmin gene. In the present thesis, skeletal and heart muscles of both wild type and desmin knock-out (K/O) mice have been investigated. Furthermore the cytoskeletal organisation in skeletal muscles from human controls and from a patient with desmin myopathy was examined. In the desmin K/O mice, no morphological alterations were observed during embryogenesis. These mice postnatally developed a cardiomyopathy and a muscle dystrophy in highly used skeletal muscles. Ruptures of the sarcolemma appear to be the primary event leading to muscle degeneration and fibrosis both in cardiac and affected skeletal muscles. In the heart the muscle degeneration gave rise to calcifications, whereas in skeletal muscles regeneration of affected muscle was seen. In mature wild type mice, the IF proteins synemin and paranemin, and the IFAP plectin were present together with desmin at the myofibrillar Z-discs, the sarcolemma, the neuromuscular junctions and the myotendinous junctions. Nestin was only found in these junctional regions. In desmin K/O mice, all four proteins were detected at neuromuscular and myotendinous junctions. The normal network of synemin and paranemin were not observed, whereas the distribution of plectin was preserved. In normal human muscles, synemin, paranemin, plectin and αB-crystallin were colocalised with desmin in between the myofibrils, at the sarcolemma and at myotendinous and neuromuscular junctions. In the human desmin myopathy, the distribution of desmin varied considerably. A normal pattern was seen in some fibres areas, whereas other regions either contained large subsarcolemmal and intermyofibrillar accumulations of desmin or totally lacked desmin. Nestin, synemin, paranemin, plectin and αB-crystallin also exhibited an abnormal distribution. They were often aggregated in the areas that contained accumulations of desmin. In cultured satellite cells from the patient, a normal network of desmin was present in early passages, whereas aggragates of desmin occurred upon further culturing. In the latter, also the nestin network was disrupted, whereas vimentin showed a normal pattern. αB-crystallin was only present in cells with a disrupted desmin network. Plectin was present in a subset of cells, irrespective of whether desmin was aggregated or showed a normal network. From the present study it can be concluded that an intact desmin network is needed to maintain the integrity of muscle fibres. Desmin may be an important component in the assembly of proteins, which connect the extrasarcomeric cytoskeleton with the extracellular matrix.

Bcl-2 associated factor 1 (Bclaf1; previously known as Btf) is a nuclear protein that was originally identified as an interacting partner for the adenoviral anti-apoptotic Bcl-2 family member E1B-19K. Surprisingly, Bclaf1 does not share structural homology with the Bcl-2 family of proteins, but rather exhibits protein structure and subcellular distribution patterns reminiscent of proteins that regulate mRNA processing. In addition, Bclaf1 appears to be expressed at high levels in skeletal muscle and was recently shown to associate with emerin, a protein linked to muscular dystrophy. Despite these observations, roles for Bclaf1 in RNA processing and/or skeletal muscle differentiation remain to be elucidated. In an effort to identify new roles for Bclaf1 I conducted protein-protein interaction screens to identify candidate interacting proteins and pathways. I identified p32 and 9G8 as novel interacting partners for Bclaf1. Additional subsequent experiments demonstrated an interaction of Bclaf1 with tip associated protein (Tap) and association of Bclaf1 with ribonucleoprotein complexes. Given that all of these proteins have been linked to mRNA processing, a role for Bclaf1 in this pathway was investigated. Using several approaches, I demonstrated that Bclaf1 is able to associate with splicing complexes and mRNA species at various stages of processing. The function of Bclaf1 in the context of skeletal muscle differentiation was also explored using skeletal muscle cell lines and primary mouse myoblasts. Skeletal muscle differentiation led to a dramatic decrease in nuclear Bclaf1 steady-state protein, with the unexpected appearance of smaller Bclaf1 protein species that accumulated in the cytoplasm during differentiation due to cleavage by caspases. Furthermore, Bclaf1 depletion in a myoblast cell line led to increased myoblast fusion and myofiber dimensions during differentiation. Overall our findings indicate roles for Bclaf1 in the skeletal muscle differentiation program and in molecular events that regulate pre-mRNA splicing and related events.