No description available.

Perisotin-like-factor (PLF) is a member of the Fasciclin family of proteins, which are characterized by the presence of 150 amino acid long conserved Fasciclin domains. Members of this family have been implicated in numerous cellular processes like adhesion, migration, axonal guidance and growth cone extension. PLF plays an important role during embryonic development, however very low levels are present in adults. PLF is up regulated in adult tissues when they are exposed to stress or insult. We have previously shown that PLF is up regulated in heart when they are mechanically overloaded and in lung when exposed to cigarette smoke. Similarly in bone, PLF is highly up regulated in fractured bone calli, albeit not present in normal adult bone. Furthermore PLF leads to increased bone formation in vivo in the bone marrow cavity when over expressed adenovirally. These results suggest that PLF promotes tissue repair and healing after injury. Additionally, PLF expression was up regulated in musculoskeletal tissues in our innovative model of Upper extremity-Work Related Musculoskeletal Disorder (WMSD). In this model, which draws parallels between the exposure to risk factors and development of associated disorders in human PLF was expressed in bone, skeletal muscle, tendon and nerves. Coincidentally, these are the same tissues in which PLF is expressed during embryonic development. Additionally, PLF was detected in inflammatory cells like macrophages suggesting a role for PLF in inflammation. Molecules like PLF which play important roles during development, reappear when the tissues require them, promoting their remodeling and recovery can be targeted as therapeutic agents. Such molecules can be targeted after injury or disease. In this study we focus our attention on the role of PLF in musculoskeletal (muscle and bone) remodeling in our animal model of WMSD. / Anatomy

Many factors influence the efficiency of muscle growth including genetics, nutrition, and environment. The neonatal period is characterized as a time of rapid growth. Growth rate is reduced during neonatal nutrient restriction possibly due to altered satellite cell activity which can permanently alter growth potential. Therefore, optimal nutrition is important for maximizing the growth potential of the animal. Heat stress leads to changes in digestion and metabolism, thus alters nutrient availability to muscle. Heat stress is a prevalent problem in the agriculture industry resulting in great economic losses due to reduced growth, fertility, and increased morbidity. The use of functional feed additives is a potential strategy to mitigate these negative effects. The objective of this dissertation was to investigate nutritional strategies to improve growth in pigs during key malleable periods. Three nutritional studies were conducted to discern the optimal inclusion levels of calcium phosphate, energy, and protein in the diet to maximize neonatal muscle growth. Adequate dietary calcium phosphate was most efficient for satellite cell function which may be mediated by micro-RNA. Differentiation promoting miR-206 and correspondingly the fusion rate was highest in adequate calcium phosphate diets. Excess protein diets enhanced body and muscle growth, while deficient protein was detrimental to growth. Dietary protein treatments altered energy metabolism genes, and genes regulating protein degradation were upregulated in deficient protein diets. Dietary energy levels did not influence body weight, however feed efficiency improved with energy balance. Excess energy diets had the lowest fusion rates and the lowest differentiation promoting miR-1 expression. These data suggest that nutrient inclusion levels are important for satellite cell function and may mediate satellite cell activity through the expression of micro-RNAs. The final study sought to discern the ability of supplementation of an artificial high-intensity sweetener and capsicum oleoresin to mitigate the negative effects of heat stress on pig performance. Heat stress leads to increased body temperature and respiration and was detrimental to metabolic flexibility. Supplementation helped improve feed efficiency and maintain metabolic flexibility. These data indicate that supplementation may be an efficient strategy to mitigate heat stress. / Doctor of Philosophy / Muscle is an important tissue to consider when optimizing growing conditions in feed animals due to its function as a consumer good. Many factors influence the efficiency of muscle growth including genetics, nutrition, and environment. Fractional growth rates are highest during the neonatal period and animals require adequate nutrients to facilitate this growth. Nutrient restriction reduces growth rate and can lead to permanent changes the animals' body size and composition later in life. Therefore, optimal nutrition is important for maximizing the growth potential of the animal. While the nutrients in feed can be controlled to improve growth, other factors are more difficult to regulate. Heat stress is a prevalent problem in the agriculture industry resulting in great economic losses due to reduced growth, fertility, and increased morbidity. The use of functional feed additives is a potential strategy to alleviate these negative effects. The objective of this dissertation was to investigate nutritional strategies to improve growth in pigs during key malleable periods. Three nutritional studies were conducted to determine the optimal inclusion levels of calcium phosphate, energy, and protein in the diet to maximize neonatal muscle growth. Satellite cells are muscle-specific stem cells that help facilitate the growth of muscle. Altering the ability of satellite cells to proliferate and fuse impairs the ability of muscle to grow and repair. Adequate dietary calcium phosphate was most efficient for satellite cell function. Excess protein diets enhanced body and muscle growth, while deficient protein was detrimental to growth. Dietary protein treatments altered energy metabolism genes, and genes regulating protein degradation were upregulated in deficient protein diets. Dietary energy levels did not influence body weight, however, feed efficiency improved with energy balance. Satellite cells from excess energy diets had the lowest fusion rates. These data suggest that nutrient inclusion levels are important for satellite cell function and growth. The final study sought to discern the ability of the supplementation of an artificial high-intensity sweetener and capsicum oleoresin to mitigate the negative effects of heat stress on pig performance. Heat stress leads to increased body temperature and respiration and was detrimental to metabolic flexibility. Supplementation helped improve feed efficiency and maintain metabolic flexibility. These data indicate that supplementation may be an efficient strategy to mitigate heat stress.

Skeletal muscle is a heterogeneous, multinucleated, post-mitotic tissue that contains many functionally diverse fibre types that are capable of adjusting their phenotypic properties in response to altered contractile demands. This plasticity, or adaptability of skeletal muscle is largely dictated by variations in motoneuron firing patterns. For example, in response to increased tonic firing of slow motoneurons, which occurs during bouts of endurance training or chronic low-frequency stimulation (CLFS), skeletal muscle adapts by transforming from a faster to a slower phenotypic profile. CLFS is an animal model of endurance training that induces fast-to-slow fibre type transformations in the absence of fibre injury in the rat. The underlying signaling mechanisms regulating this fast-to-slow fibre type transformation, however, remain to be fully elucidated. It has been suggested that myogenic stem cells, termed satellite cells, may regulate and/or facilitate this transformational process. Therefore, the signaling mechanisms involved in CLFS-induced satellite cell activation as well as the role satellite cells may play in CLFS-induced skeletal muscle adaptation were investigated in rat. A pharmacological inhibitor of nitric oxide (NO) synthase, Nω-nitro-L-arginine methyl ester, was used to investigate CLFS-induced satellite cell activation in the absence of endogenous NO production. Results suggest that NO is required for early CLFS-induced satellite cell activation, but a yet-to-be defined pathway exists that is able to fully compensate in the absence of prolonged NO production. A novel method of satellite cell ablation (i.e. weekly focal γ-irradiation application) was used to investigate CLFS-induced skeletal muscle adaptation in the absence of a viable satellite cell population. Myosin heavy chain (MHC), an important structural and regulatory protein component of the contractile apparatus, was used as a cellular marker of the adaptive response to CLFS. Findings suggest that satellite cell activity may be required for early fast-to-slow MHC-based transformations to occur at the protein level without delay in the fast fibre population, and may also play an obligatory role in the final transformation from fast type IIA to slow type I fibres. Interestingly, additional results show that NO appears to be a key mediator of MHC isoform gene expression during CLFS-induced fast-to-slow fibre type transformations.

No description available.

Le muscle squelettique adulte est un tissu possédant la capacité fondamentale d’adapter sa taille à la demande fonctionnelle : il peut s’atrophier ou s’hypertrophier en réponse à une variation de la charge mécanique qui lui est appliquée. A l’heure actuelle, les facteurs impliqués dans la plasticité musculaire demeurent méconnus. D’une part, grâce à différents modèles d’atrophie musculaire, nous démontrons que le facteur de transcription Srf joue le rôle de médiateur de la mécano-transduction par la voie actine/Mrtfs/Srf. L’arrêt de l’activité mécanique provoque une accumulation nucléaire d’actine monomérique, une délocalisation de Mrtf-A, coactivateur de Srf, et une diminution de l’activité de Srf, se traduisant notamment par une baisse de la transcription Srf-dépendante. Les gènes cibles de Srf comptant un grand nombre de protéines sarcomériques, telles que l’α-actine squelettique, la réduction de leur expression pourrait participer à l’atrophie musculaire. De plus, nos travaux suggèrent que la diminution de l’activité de Srf pourrait influencer l’organisation du réseau mitochondrial et le flux autophagique par des mécanismes qui restent à élucider. D’autre part, en tirant parti d’un modèle d’invalidation conditionnelle et inductible de Srf dans les cellules satellites, nous montrons que le phénomène d’hypertrophie compensatoire requiert l’expression de Srf par les cellules satellites. L’absence de Srf n’altère ni la prolifération ni l’entrée en différenciation des myoblastes, néanmoins elle provoque un défaut de fusion des myoblastes aux fibres au cours de l’hypertrophie induite par surcharge. Ainsi, nos travaux démontrent que Srf est un acteur majeur de la plasticité musculaire, à la fois en tant que médiateur de la mécano-transduction par la voie actine/Mrtfs/Srf et par son implication dans la fusion des cellules satellites aux fibres musculaires, nécessaire à l’hypertrophie compensatoire. / Adult skeletal muscle is able to adapt its size to functional demand. It can undergo atrophy or hypertrophy according to mechanical load. To date, the molecules that mediate muscle plasticity remain unclear.Using different models inducing muscle atrophy, we show that the transcription factor Srf is a mediator of mechanotransduction through the actin/Mrtfs/Srf pathway. Mechanical load abolition leads to G-actin nuclear accumulation, delocalization of Mrtf-A, an Srf coactivator, and Srf activity downregulation. This results in a decrease in Srf-dependent transcription. Many Srf target genes encode sarcomeric proteins such as α-skeletal actin, thus a downregulation of Srf-dependent transcription could participate to muscle atrophy. In addition, our results suggest that Srf activity decrease could affect mitochondrial network organization and autophagic flux in a way that remains to be determined. Besides, using a satellite cell-specific conditional and inducible Srf knockout, we show that overload hypertrophy requires Srf expression by satellite cells. Myoblasts proliferation and early differentiation are not altered by Srf loss. However, mutant myoblasts are unable to fuse with myofibers during overload hypertrophy. Altogether, our results demonstrate that Srf is an important player in skeletal muscle plasticity: it is a mediator of mechanotransduction via the actin/Mrtfs/Srf pathway and its expression by satellite cells is required for myoblasts to fuse with myofibers during overload hypertrophy.

Muscle stem cells self-renew to maintain the long-term capacity for skeletal muscles to regenerate. However, the homeostatic regulation of muscle stem cell self-renewal is poorly understood. By utilizing high-throughput screening and transcriptomic approaches, we identify the critical function of dystrophin, the epidermal growth factor receptor (EGFR), and fibronectin in the establishment of cell polarity and in determining symmetric and asymmetric modes of muscle stem cell self-renewal. These findings reveal an orchestrated network of paracrine signaling that regulate muscle stem cell homeostasis during regeneration and have profound implications for the pathogenesis and development of therapies for Duchenne muscular dystrophy.

No description available.

<p>Prediabetes can lead to Type II Diabetes Mellitus, yet Prediabetes is a disease in its own right with its own physiological complications. Despite the pervasiveness of Prediabetes in our society and the negative impact on current and future health the extent of myopathy, short of muscle insulin resistance, and the mechanisms behind development of muscle insulin resistance remains unclear. Animal models of diet-induced obesity (DIO) have been employed to assess development of muscle insulin resistance and changes to muscle health. However there is a lack of clarity as to the molecular mechanisms leading to muscle insulin resistance. The goal of the studies presented here was to elucidate changes to muscle health and potential mechanisms contributing to muscle insulin resistance in response to DIO. Since the ability to perform exercise is to date one of the best therapies for Prediabetes and exercise contributes to a healthy muscle mass, the ability of muscle to undergo proper regeneration was also assessed following DIO. The results presented in this work demonstrate that skeletal muscle tissue adapts to increased dietary lipid by an early increase in functional lipid oxidation, mitigating IMCL deposition, despite glucose intolerance. Unfortunately this adaptive response is reversed with prolonged dietary fat intake and the development of muscle insulin resistance. Of note was the stronger link between IMCLs and muscle insulin resistance, compared to inflammation. DIO also led to decrements in satellite cell functionality which, along with physiological changes to HGF content and signaling, likely resulted in the observed impairment in regenerative ability. The results reported here improve our understanding of changes to muscle health and the mechanisms behind development of muscle insulin resistance with DIO. These findings have implications for therapies and treatments for Prediabetes.</p> / Doctor of Philosophy (Medical Science)

Skeletal muscle satellite cells located between the plasma membrane and the basal lamina of muscle fibres, could for many years, only be studied in situ by electron microscopy. The introduction of immunohistochemistry and the discovery of molecular markers of satellite cells then made them accessible for light microscopic studies and a wealth of information is today available. Satellite cells are myogenic stem cells that can be activated from a quiescent state to proliferate for self-renewal or differentiate into myogenic cells. The satellite cells are involved in muscle growth during fetal and postnatal development and play a key role in repair and regeneration of damaged muscle fibres. The satellite cells are also essential for muscle fibre hypertrophy and maintenance of muscle mass in the adult. When the present thesis was initiated, studies on satellite cells in human skeletal muscle relied on the neuronal cell adhesion molecule (NCAM) as a marker for satellite cell identification. The results from different studies varied markedly. Therefore the aims of the present thesis were i) to develop a highly reliable method using light microscopy for satellite cell identification and quantification in biopsies of human skeletal muscle in normal and pathological conditions. A molecular marker for the myofibre basal lamina or plasma membrane to enhance the reliability of myonuclei and satellite cell identification were to be included. Furthermore unbiased morphometric methods should be used in the quantification process. ii) to evaluate which molecular markers which had been described for satellite cell and stem cell identification in different cell states (quiescence, activated or differentiated) are the most useful for studies on human skeletal muscle. iii) to further explore the function and heterogeneity of satellite cells with respect to different markers in human skeletal muscle by studying the effects of strength-training, intake of anabolic substances and pathological conditions. A new immunofluorescence method was developed where in the same tissue section, two satellite cell markers, the basal lamina and nuclei were monitored. From the evaluation of different markers it was found that both NCAM and Pax7 identified the majority of satellite cells but that both markers were needed for reliable identification. The members of the myogenic regulatory family were evaluated and by using the new method MyoD and myogenin were found to be useful markers to identify activated and differentiated satellite cells. Upon re-examination of biopsies from power-lifters, power-lifters using anabolic substances and untrained subjects it was observed that the new results on satellite cell frequency were significantly different from those obtained when using staining for NCAM and nuclei alone. In addition three subtypes of satellite cells (94.4% NCAM+/Pax7+, 4.2% NCAM+/Pax7– and 1.4% NCAM–/Pax7+) were observed. Thus the multiple marker method gave more information about satellite cells heterogeneity in human muscle and we propose that this is more reliable than previous methods. Low numbers of MyoD or myogenin stained satellite cells were observed in both untrained and strength trained subjects. Other markers such as DLK1/FA1, a member of the EGF-like family and c-Met, the receptor for hepatocyte growth factor showed that satellite cell heterogeneity in human muscle is far greater than previously shown. Furthermore, new evidence is presented for so called fibre splitting observed in hypertrophic muscle fibres to be due to defect regeneration of partially damaged fibres.