Oestrogen is believed to be a potent antioxidant, with potential membrane stabilising and gene regulatory effects. Oestrogen has been shown to be an effective cardioprotectant, with for example a lower incidence of atherosclerosis in pre menopausal females compared to age-matched males and in post menopausal females on hormone replacement therapy compared to age-matched males. What has yet to be determined is the extent to which oestrogen can protect skeletal muscle. It has been shown that certain markers of exercise-induced muscle damage (EIMD) are lower in females in both animal and human studies, but as yet no conclusive evidence from human studies has shown that oestrogen provides a protective mechanism against ERvID or whether susceptibility to EIKID varies across the normal menstrual cycle, where oestrogen fluctuations are high. Furthermore, if oestrogen provides a protective mechanism against EIlVID, it is unknown at which phase during the muscle damage and repair cycle this occurs. It is also debatable if the potential inhibitory effects that oestrogen has on the inflammatory response, with regard to repair and regeneration of the skeletal muscle are positive or negative. The thesis is comprised of a critical review of the nature of EIMD and the potential effects that oestrogen has on the muscle damage and repair cycle. This is followed by three empirical studies which were designed to explore this question. These are outlined below: Study 1 Study one was in two parts. The first part of this study aimed at determining if the phase of the menstrual cycle, could in anyway affect eumennorheic (normally 2 menstruating) females in their susceptibility to exercise-induced muscle damage. An eccentric exercise procedure (elbow flexor muscle group) was performed on a randomly assigned arm during either the menses or ovulatory phase of the menstrual cycle. The contra-lateral limb underwent the same procedure during the alternate phase (random assignment determined in which phase the participant was first damaged). Simple markers of EDM were assessed at baseline and every 24 h up to three days post exercise, during both phases. No significant differences were seen in any markers of BIMD across phases of the menstrual cycle. The second part of this study investigated whether prolonged ingestion of exogenous oestrogen, in the form of the combined oral contraceptive pill attenuated any of the symptoms associated with EIMD. The only symptom to show a significant interaction between groups was perceived soreness, with the pill users reporting significantly (P<0.01) less soreness than the eumennorheic females in the days following the exercise protocol. This suggested that oestrogen may modulate the pain associated with EIMD. Study 2 The second study focussed on gender differences in exercise-induced muscle damage, with particular focus on the secondary symptoms and events which occur following ERVID. Male and female participants performed a bout of eccentrically biased exercise. Markers of both EIMD and inflammation were taken prior to the eccentric exercise and across a 7-day follow up period. Gender differences in the response to EIMD were seen in creatine kinase activity and mid-thigh circumference, with males showing a larger response on both variables. In addition to this, males reported significantly less soreness than females following the exercise protocol. 3 Interestingly, with the exception of neutrophil elastase release, there were no differences in other markers of inflammation between men and women. Total elastase concentration, a marker of neutrophil activation, did not differ between genders. However, elastase release per neutrophil was significantly lower in females, which may be indicative of gender differences in the inflammatory response associated with EDvID. Study 3 With the recognition that oestrogen could potentially reduce or inhibit the inflammatory response the third and final study investigated whether female skeletal muscle was more susceptible to exercise-induced muscle damage after a second bout of eccentric exercise, due to poor regeneration and repair following the initial bout. Males and females performed a bout of eccentrically biased exercise. Markers of EEVM included creatine kinase, soreness, isometric strength and isokinetic strength assessment. The procedure was then repeated two weeks later to determine if gender differences existed in terms of the repeated bout effect associated with EIlvID. Only one variable showed a gender x time x bout interaction (P<0.05), that was the fatigue index. It was shown that following the initial bout of damage, males and females responded very differently, with female muscle being less fatiguable in the 48 h following damage compared to the males, but with both groups responding very similarly in the repeated bout. This may be due to differences in gender and their response to EIlVID, or due to differences in fibre type between genders.

No description available.

Introduction. Fused Positron Emission Tomography and Computed Tomography (PET/CT) is an emerging investigative tool in cardiovascular disease that provides an imaging-based quantification of pathophysiological processes of interest. The purpose of this thesis was to study the application of PET to identify fundamental pathophysiological processes driving 3 forms of cardiovascular disease: aortic stenosis, myocardial infarction, and atherosclerosis. Methods. Aortic Stenosis. Patients with a spectrum of calcific aortic valve disease (n=121) who underwent PET-CT imaging for the identification of valvular calcification (18Ffluoride) and inflammation (18F-fluorodeoxyglucose, 18F-FDG) underwent serial imaging and clinical follow-up over 2 years. Baseline imaging findings were compared with echocardiographic and CT markers of disease progression and clinical outcome. Myocardial Infarction. Patients underwent PET-CT imaging with 18F-fluciclatide (a novel αvβ3-selective radiotracer highlighting active angiogenesis, inflammation and fibrosis) after ST-segment elevation MI (n=21), alongside stable patients with chronic total occlusion (CTO) of a major coronary vessel (n=7), and healthy volunteers (n=9). Myocardial radiotracer uptake was compared with clinical and cardiac magnetic resonance imaging (CMR) markers of infarction and remodeling. Atherosclerosis. Patients with a spectrum of atherosclerotic disease categorized as stable or unstable (recent MI) underwent PET/CT imaging with 18F-fluciclatide (n=46). Thoracic aortic 18F-fluciclatide uptake was compared with aortic atherosclerotic burden quantified by CT plaque thickness, plaque volume and calcium scoring. Histological validation. Tissue from the aortic valve, myocardium and carotid arteries of study subjects was acquired and examined ex vivo using histology and autoradiography. Results. Aortic Stenosis. Baseline valvular 18F-fluoride uptake correlated strongly with the rate of progression in AVC (r=0.80, p < 0.001) and with haemodynamic progression (mean aortic valve gradient r=0.32, p=0.001). It emerged as independently associated with clinical outcome after age and sex-adjustment (HR 1.55 [1.33-1.81], p < 0.001). 18F-FDG demonstrated moderate correlations with disease progression as assessed by CT (r=0.43, p=0.001) and echocardiography (18F-FDG r=0.30, p=0.001), and was associated with clinical outcomes independent of age and sex (HR 1.35 [1.16-1.58], p < 0.001). Valvular 18F-fluoride uptake correlated with immunohistochemical markers of calcification activity. There was no correlation between 18F-FDG uptake and inflammation. Myocardial Infarction. 18F-Fluciclatide binding was demonstrated in ex vivo peri-infarct myocardium and uptake was increased in vivo at sites of acute infarction compared to remote myocardium (tissue-to-background ratio (TBRmean) 1.34±0.22 vs 0.85±0.17 respectively, p < 0.001) and myocardium of healthy volunteers (TBRmean 1.34±0.22 vs 0.70±0.03; p < 0.001). There was no 18F-fluciclatide uptake at sites of established prior infarction in patients with CTO, with myocardial activity similar to healthy volunteers (TBRmean 0.71±0.06 vs. 0.70±0.03,p=0.83). 18F-Fluciclatide uptake occurred at sites of regional wall hypokinesia (wall motion index ≥1 vs 0; TBRmean 0.93±0.31 vs 0.80±0.26 respectively, p < 0.001), was increased in segments displaying functional recovery (TBRmean 0.95±0.33 vs 0.81±0.27, p=0.002) and associated with increase in probability of regional recovery. Atherosclerosis. 18F-Fluciclatide vascular binding ex vivo co-localised with regions of increased αvβ3 integrin expression, and markers of inflammation and angiogenesis. 18F-Fluciclatide uptake in vivo correlated with measures of aortic atherosclerotic burden: plaque thickness (r=0.57, p=0.001), total plaque volume (r=0.56, p=0.001) and the CT aortic calcium score (r=0.37, p=0.01). Patients with recent MI had greater aortic 18F-fluciclatide uptake than those with stable disease (TBRmax 1.33 vs 1.21, p=0.01). Conclusions. In a range of cardiovascular diseases, PET-CT can provide insights into key pathophysiological processes, guide patient risk stratification and prognosis, and identify important biomarkers of disease activity that can be used for the development of future therapeutic interventions.

Óxido nítrico (NO) é uma molécula que exerce uma multiplicidade de funções fisiológicas importantes que vêm sendo estudadas em diversos tecidos. No entanto, o papel do NO em processos fisiológicos e patológicos no músculo estriado é pouco conhecido, apesar de algumas evidências apontarem para uma função de regulação redox e interação com células satélites progenitoras. Neste estudo avaliamos a participação do NO na regeneração muscular em um modelo de inflamação aguda in vivo. O trauma muscular foi induzido por um aparelho similar a uma prensa com impacto direto sobre o gastrocnêmio. Foram utilizados 40 ratos, Wistar, divididos em quatro grupos: (i) controle (CO); (ii) falso (sham) trauma; (iii) trauma; (iv) trauma com exposição ao nitro-L-arginina metil éster (L-NAME), um inibidor da sintase do óxido nítrico, em dois tempos experimentais: 24 horas e 7 dias após lesão. Vinte e quatro horas após o trauma, o músculo lesionado apresentava intensa vasodilatação e reação inflamatória na análise histológica, lipoperoxidação tecidual e estresse nitrosativo, aumento do mRNA das citocinas próinflamatórias (IL-1β, IL-6, iNOS), metaloproteinase-2, expressão proteica de iNOS e MMP-2, ativação do NF-κB e intensa atividade proliferativa. O tratamento com LNAME diminuiu significativamente os achados das alterações histológicas e moleculares em 24 horas após a lesão e, sete dias após o trauma, houve um aumento na expressão do TGF-β, na deposição de colágeno e proliferação celular. Entretanto, o grupo tratado com L-NAME apresentou uma maior expressão de TGF-β e de deposição de colágeno quando comparado ao grupo trauma e proliferação celular semelhante. Estes resultados indicam que a lesão muscular é associada com a ativação do sistema NO, o qual parece estar envolvido no equilíbrio entre os processos de regeneração e fibrose durante o reparo. / Nitric oxide (NO) is a molecule that carries a variety of important physiological functions that have been studied in various tissues. However, the role of NO in physiological and pathological processes in striated muscle is poorly known, although some evidence suggests a role of redox regulation and interaction with progenitor satellite cells. We evaluated the role of NO in muscle regeneration in a model of acute inflammation in vivo. The muscle trauma was induced by a device similar to a press with a direct impact on the gastrocnemius. A total of 40 rats were divided into four groups: (i) control (CO), (ii) sham trauma, (iii) trauma, (iv) trauma + L-NAME, an inhibitor of nitric oxide synthase in two experimental phases: 24 hours and 7 days after injury. Twenty-four hours after the trauma, the injured muscle showed intense vasodilatation and inflammation in the histological analysis, tissue lipid peroxidation and nitrosative stress, activation of NF-κB, increased mRNA of pro-inflammatory, (IL- 1β, IL-6, iNOS), metalloproteinase-2 and HGF, and increased total cell proliferation. The administration of the L-NAME significantly reduced iNOS, MMP-2 and activation of NF-κB reduced the histological and molecular findings 24 hours after injury, and 7 days after injury, there was an increase in the expression of TGF-β, collagen deposition, and total cell proliferation. However, the L-NAME group showed increased expression and collagen deposition when compared to the trauma and similar total cell proliferation. These results indicate that muscle injury is associated with activation of the NO, which seems to be involved in the balance between the processes of regeneration and fibrosis during the repair.

Óxido nítrico (NO) é uma molécula que exerce uma multiplicidade de funções fisiológicas importantes que vêm sendo estudadas em diversos tecidos. No entanto, o papel do NO em processos fisiológicos e patológicos no músculo estriado é pouco conhecido, apesar de algumas evidências apontarem para uma função de regulação redox e interação com células satélites progenitoras. Neste estudo avaliamos a participação do NO na regeneração muscular em um modelo de inflamação aguda in vivo. O trauma muscular foi induzido por um aparelho similar a uma prensa com impacto direto sobre o gastrocnêmio. Foram utilizados 40 ratos, Wistar, divididos em quatro grupos: (i) controle (CO); (ii) falso (sham) trauma; (iii) trauma; (iv) trauma com exposição ao nitro-L-arginina metil éster (L-NAME), um inibidor da sintase do óxido nítrico, em dois tempos experimentais: 24 horas e 7 dias após lesão. Vinte e quatro horas após o trauma, o músculo lesionado apresentava intensa vasodilatação e reação inflamatória na análise histológica, lipoperoxidação tecidual e estresse nitrosativo, aumento do mRNA das citocinas próinflamatórias (IL-1β, IL-6, iNOS), metaloproteinase-2, expressão proteica de iNOS e MMP-2, ativação do NF-κB e intensa atividade proliferativa. O tratamento com LNAME diminuiu significativamente os achados das alterações histológicas e moleculares em 24 horas após a lesão e, sete dias após o trauma, houve um aumento na expressão do TGF-β, na deposição de colágeno e proliferação celular. Entretanto, o grupo tratado com L-NAME apresentou uma maior expressão de TGF-β e de deposição de colágeno quando comparado ao grupo trauma e proliferação celular semelhante. Estes resultados indicam que a lesão muscular é associada com a ativação do sistema NO, o qual parece estar envolvido no equilíbrio entre os processos de regeneração e fibrose durante o reparo. / Nitric oxide (NO) is a molecule that carries a variety of important physiological functions that have been studied in various tissues. However, the role of NO in physiological and pathological processes in striated muscle is poorly known, although some evidence suggests a role of redox regulation and interaction with progenitor satellite cells. We evaluated the role of NO in muscle regeneration in a model of acute inflammation in vivo. The muscle trauma was induced by a device similar to a press with a direct impact on the gastrocnemius. A total of 40 rats were divided into four groups: (i) control (CO), (ii) sham trauma, (iii) trauma, (iv) trauma + L-NAME, an inhibitor of nitric oxide synthase in two experimental phases: 24 hours and 7 days after injury. Twenty-four hours after the trauma, the injured muscle showed intense vasodilatation and inflammation in the histological analysis, tissue lipid peroxidation and nitrosative stress, activation of NF-κB, increased mRNA of pro-inflammatory, (IL- 1β, IL-6, iNOS), metalloproteinase-2 and HGF, and increased total cell proliferation. The administration of the L-NAME significantly reduced iNOS, MMP-2 and activation of NF-κB reduced the histological and molecular findings 24 hours after injury, and 7 days after injury, there was an increase in the expression of TGF-β, collagen deposition, and total cell proliferation. However, the L-NAME group showed increased expression and collagen deposition when compared to the trauma and similar total cell proliferation. These results indicate that muscle injury is associated with activation of the NO, which seems to be involved in the balance between the processes of regeneration and fibrosis during the repair.

Óxido nítrico (NO) é uma molécula que exerce uma multiplicidade de funções fisiológicas importantes que vêm sendo estudadas em diversos tecidos. No entanto, o papel do NO em processos fisiológicos e patológicos no músculo estriado é pouco conhecido, apesar de algumas evidências apontarem para uma função de regulação redox e interação com células satélites progenitoras. Neste estudo avaliamos a participação do NO na regeneração muscular em um modelo de inflamação aguda in vivo. O trauma muscular foi induzido por um aparelho similar a uma prensa com impacto direto sobre o gastrocnêmio. Foram utilizados 40 ratos, Wistar, divididos em quatro grupos: (i) controle (CO); (ii) falso (sham) trauma; (iii) trauma; (iv) trauma com exposição ao nitro-L-arginina metil éster (L-NAME), um inibidor da sintase do óxido nítrico, em dois tempos experimentais: 24 horas e 7 dias após lesão. Vinte e quatro horas após o trauma, o músculo lesionado apresentava intensa vasodilatação e reação inflamatória na análise histológica, lipoperoxidação tecidual e estresse nitrosativo, aumento do mRNA das citocinas próinflamatórias (IL-1β, IL-6, iNOS), metaloproteinase-2, expressão proteica de iNOS e MMP-2, ativação do NF-κB e intensa atividade proliferativa. O tratamento com LNAME diminuiu significativamente os achados das alterações histológicas e moleculares em 24 horas após a lesão e, sete dias após o trauma, houve um aumento na expressão do TGF-β, na deposição de colágeno e proliferação celular. Entretanto, o grupo tratado com L-NAME apresentou uma maior expressão de TGF-β e de deposição de colágeno quando comparado ao grupo trauma e proliferação celular semelhante. Estes resultados indicam que a lesão muscular é associada com a ativação do sistema NO, o qual parece estar envolvido no equilíbrio entre os processos de regeneração e fibrose durante o reparo. / Nitric oxide (NO) is a molecule that carries a variety of important physiological functions that have been studied in various tissues. However, the role of NO in physiological and pathological processes in striated muscle is poorly known, although some evidence suggests a role of redox regulation and interaction with progenitor satellite cells. We evaluated the role of NO in muscle regeneration in a model of acute inflammation in vivo. The muscle trauma was induced by a device similar to a press with a direct impact on the gastrocnemius. A total of 40 rats were divided into four groups: (i) control (CO), (ii) sham trauma, (iii) trauma, (iv) trauma + L-NAME, an inhibitor of nitric oxide synthase in two experimental phases: 24 hours and 7 days after injury. Twenty-four hours after the trauma, the injured muscle showed intense vasodilatation and inflammation in the histological analysis, tissue lipid peroxidation and nitrosative stress, activation of NF-κB, increased mRNA of pro-inflammatory, (IL- 1β, IL-6, iNOS), metalloproteinase-2 and HGF, and increased total cell proliferation. The administration of the L-NAME significantly reduced iNOS, MMP-2 and activation of NF-κB reduced the histological and molecular findings 24 hours after injury, and 7 days after injury, there was an increase in the expression of TGF-β, collagen deposition, and total cell proliferation. However, the L-NAME group showed increased expression and collagen deposition when compared to the trauma and similar total cell proliferation. These results indicate that muscle injury is associated with activation of the NO, which seems to be involved in the balance between the processes of regeneration and fibrosis during the repair.

Submitted by Nadir Basilio (nadirsb@uninove.br) on 2018-06-19T13:32:21Z No. of bitstreams: 1 Evaldo Moreira da Silva Junior.pdf: 1681061 bytes, checksum: 3c8938d092baff83e502672b6d6a1b69 (MD5) / Made available in DSpace on 2018-06-19T13:32:21Z (GMT). No. of bitstreams: 1 Evaldo Moreira da Silva Junior.pdf: 1681061 bytes, checksum: 3c8938d092baff83e502672b6d6a1b69 (MD5) Previous issue date: 2015-12-10 / Muscle lesions although more common in athletes also affects sedentary people, leading to partial or total removal of the individual from their daily activities. The muscle repair process comprises interdependent phases orchestrated by immune cells that invade the tissue behind the injury occurrence. Delays or enhancements at different stages can lead to chronic inflammation cases or fibrosis, reinforcing the search for an alternative treatment. The therapeutic ultrasound (US) is widely used to treat muscle injuries, but lack scientific evidence to explain its mechanism of action. This study aimed aims to elucidate the role of US in the inflammatory infiltrate cells presentes on the muscle tissue behind acute injury. For this purpose, 45 Wistar rats will were used, separated into 3 groups (5 animals in the control group, 20 animals suffer injury and will not receive treatment and 20 animals suffer injury and will be treat with US). The adopted procedure is the cryoinjury lesion consisting of two bat applications of cooled in liquid nitrogen directly into the in tibialis anterior (TA) muscle. The US treatment and these groups will be evaluate after 1, 2, 3 and 7 days. The US treatment (stationary mode, pulse 1: 4, with a frequency of 1 MHz and intensity of 0.4 W / cm2) was performed daily for 3 minutes. At the end of the study, the animals were euthanized with an anesthetic overdosis and the TA muscles were removed for neutrophil infiltration analysis and the different phenotypes of macrophages during muscle remodeling by immunostaining (positive for CD68, CD80, CD163, CD206 and elastase). The images were analyzed and quantified using the Image J software (National Institute of Health - NIH, USA). Statistical analysis of data was performed according to evaluating the distribution of the same by & Kolmogorov Smirnov test. Treatment with US generated in the damaged areas of the treated muscles, reduction in the number of neutrophils (elastase +) during the periods 1 and 2 days, an increase of M1 macrophages (CD80 +) in one day period and reducing them in periods of 2, 3 and 7 days. It was also noted when comparing the injured muscles untreated, the US generated reduced profile macrophages M2a (CD206 +) on day 2 and increased this phenotype cells present on day 3. The labeling of total macrophages (CD68 +) uS-treated animals was lower than that present in untreated animals on days 2, 3 and 7. There was no difference in other temporal comparisons. As continuity and the intensity of cells presence with pro or anti inflammatory action can leading to chronic inflammation or fibrosis cases, attesting the US's ability can modulate the occurrence of these cells is an important step in understanding the therapeutic potential of this resource in the treatment of muscle injuries. / As lesões musculares embora mais comuns em atletas, atingem também os não praticantes de atividades física, levando o indivíduo ao afastamento de suas atividades diárias. O reparo muscular compreende fases interdependentes orquestradas pelas células imunes que invadem o tecido logo após a ocorrência da lesão. O ultrassom (US) terapêutico é vastamente utilizado no tratamento de lesões musculares, porém faltam evidências científicas que expliquem seu mecanismo de ação. Este trabalho visou elucidar o papel do US sobre as células componentes do infiltrado inflamatório presente no tecido muscular após lesão aguda. Para tanto, foram utilizados 45 ratos Wistar, separados em 3 grupos (5 animais no grupo controle, 20 animais sofreram lesão e não receberam tratamento e 20 animais sofreram lesão e foram tratados com US). O procedimento de lesão adotado foi a criolesão com duas aplicações de bastão resfriado em nitrogênio líquido diretamente no músculo tibial anterior (TA). O US (modo estacionário, pulsado 1:4, com frequência de 1 MHz e intensidade de 0,4 W/cm2) foi aplicado diariamente por 3 minutos no respectivo músculo. Ao término dos períodos experimentais (1, 2, 3 e 7 dias), os músculos TA foram analisados quanto a presença de neutrófilos e dos diferentes fenótipos de macrófagos por meio de imunomarcação (positividade para elastase, CD68, CD80 e CD206). As imagens foram analisadas e quantificadas por meio do software Image J (NIH, EUA). A análise estatística dos dados foi realizada de acordo com a avaliação da distribuição dos mesmos pelo teste de Kolmogorov & Smirnov. O tratamento com US gerou, nas áreas lesionadas dos músculos tratados, redução no número de neutrófilos (elastase+) nos períodos de 1 e 2 dias, aumento de macrófagos M1 (CD80+) no período de 1 dia e redução destes nos períodos de 2, 3 e 7 dias. Também foi possível observar quando da comparação com os músculos lesionados não tratados, que o US gerou diminuição de macrófagos de perfil M2a (CD206+) no dia 2 e aumento da presença de células deste fenótipo no dia 3. A marcação de macrófagos totais (CD68+) nos animais tratados com US foi menor do que a presente nos animais não tratados nos dias 2, 3 e 7. Não houve diferença nas demais comparações temporais. Como continuidade e a intensidade da presença de células com ação pró ou anti inflamatória pode levar a quadros de inflamação crônica ou de fibrose, a comprovação da capacidade do US em modular a ocorrência destas células representa uma importante passo para a compreensão do potencial terapêutico deste recurso no tratamento de lesões musculares.

Glucocorticoids are the most widely prescribed medications due primarily to their anti-inflammatory and immunosuppressive actions, however, their use is not without side effects. Among these, glucocorticoids cause profound muscle atrophy, yet paradoxically are used as the first line of treatment for muscle wasting disorders such as Duchenne Muscular Dystrophy (DMD) and inflammatory myopathies. In DMD patients, glucocorticoid treatment can improve muscle strength during the first 6 months of treatment and can delay loss of muscle function by up to three years. While recent advancements have been made to understand the effect of glucocorticoids (GCs) on the myofiber, the impact of GCs on skeletal muscle stem cells (MuSCs), the adult stem cells responsible for muscle regeneration, and their role in myogenic differentiation, are relatively unknown. To study the role of glucocorticoid signalling during muscle repair, I developed a conditional null mouse (GRMuSC-/-) model in which glucocorticoid receptor (GR) expression is knocked out specifically in MuSCs (GRMuSC-/-). One-week following acute muscle injury, WT and GRMuSC-/- mice both underwent robust repair assessed by myofiber cross-sectional area (CSA) analysis. However, the GR-/- MuSCs failed to return to quiescence following repair resulting in a significant increase in average myofiber CSA at 28- and 42- days post-injury, as compared to controls. Loss of the GR led to a significant increase in the percentage of PAX7+Ki67+ cycling cells in GRMuSC-/- mice (as compared to controls) at 42 days post injury. In the uninjured contralateral limb, I observed significantly fewer MuSCs in GRMuSC-/- mice with a concomitant increase in fibers with centrally located nuclei, indicating that these PAX7+ MuSCs progressed to differentiation in the absence of direct injury. In an uninjured model, two weeks following loss of GR expression there was an increase in the percentage of BrdU+ and Ki67+ cycling cells in resting GRMuSC-/- tibialis anterior muscles as compared to WT, suggesting that the GR acts to maintain MuSC quiescence. Consistent with this, immunostaining of single EDL myofiber fibers at T2h post-dissociation revealed that loss of GR in MuSCs lead to precocious activation and subsequent proliferation of MuSCs as compared to controls. Bulk RNA-sequencing from in situ fixed MuSCs in resting muscle revealed that the gene signature of GR-/- MuSCs was consistent with cells that have exited from the quiescent state and are activated for differentiation. Despite precocious activation, GR-/- myoblasts differentiate and fuse normally, however the myotubes produced had abnormal morphology and aberrant myonuclear placement in regenerated muscle fibers in vivo.

Skeletal muscle possesses a remarkable plasticity, able to repair, remodel and adapt to various stressors. A population of resident muscle stem cells, commonly referred to as satellite cells (SC), are largely responsible for skeletal muscle plasticity. The loss of muscle mass and plasticity typically observed in aging has been attributed to the deterioration of SC function. SC reside in a quiescent state, but following stimuli they become active, proliferate and eventually differentiate, fusing to existing muscle fibres. The progression of SC through this process, termed the myogenic program, is orchestrated by a complex network of transcription factors, termed myogenic regulatory factors. SC function is regulated by various growth factors and/or cytokines. The delivery of these signalling factors to SC is, in part, dependent on their proximity and exposure to local microvascular blood flow. The purpose of this thesis was to examine the relationship between skeletal muscle capillaries and muscle SC. We examined the effect of age on the spatial relationship between SC and muscle fiber capillaries, and observed that type II muscle fiber SC were located at a greater distance from the nearest capillary in older men as compared to their younger counterparts. We then examined the changes in SC activation status following a single bout of resistance exercise, prior to and following a 16wk progressive resistance training (RT) program. We observed that following RT, there was an enhanced SC activation in response to a single bout of resistance exercise. This enhanced response was accompanied by an increase in muscle capillarization following training. Furthermore, we investigated the impact of muscle fiber capillarization on the expansion and activation status of SC in acute response to muscle damaging exercise in healthy young men. We observed that muscle capillarization was positively related to SC pool activation and expansion. Taken together, we demonstrate that muscle capillarization may be related to the SC response following acute resistance exercise or exercise-induced injury, and may be implicated in adaptation to RT. Furthermore, the spatial relationship between muscle capillaries and SC is negatively altered by aging. / Thesis / Doctor of Philosophy (PhD) / Skeletal muscle health is, in part, maintained by a population of stem cells associated with individual muscle fibres. When muscle is damaged or stressed, these cells become activated, aid in muscle repair, and help drive adaptations to exercise. The central purpose of this thesis was to examine the relationship between muscle capillaries and muscle stem cells, and determine how that relationship impacts muscle stem cell function. We demonstrated that muscle stem cells and capillaries exist in close proximity to each other in skeletal muscle. We observed that a greater muscle capillarization is linked to improved muscle stem cell function during muscle repair. However, we also report that the distance between muscle capillaries and muscle stem cells becomes greater in aging, and may be a root cause of impaired muscle stem cell function in aging.

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