<p>The extent of skeletal muscle hypertrophy in response to resistance training is highly variable in humans. To explain the nature of this variability, we focused on the myogenic stem cell population, the satellite cell (SC) as a potential mediator of hypertrophy. Twenty-three males (aged 18-35yrs) underwent 16wk of progressive, whole body resistance training, resulting in changes of 7.9%±1.6 (range of -1.9 – 24.7%) and 21.0%±4.0 (range of -7.0 to 51.7%) in quadriceps volume and myofibre cross-sectional area (CSA) respectively. The SC response to a single bout of resistance exercise (80% 1RM), resulted in an expansion in type one fibre associated SC (MHCI-SC) content of 43.7%±10.4 24h post-exercise pre-training, that shifted, post-training, to an increase in type two fibre associated SC (MHCII-SC) content of 47.6%±21.2 72h post-exercise. Analysis of individual SC responses revealed a correlation between the relative change in MHCII-SC content between 24-72h pre-training and the percentage increase in quadriceps lean tissue mass assessed by MRI (r=0.663, p=0.001). The proportion of SC co-localized with MSTN decreased progressively in the acute time-course following exercise and correlated with SC expansion between Pre-24h (r=0.563, p=0.012) and Pre-72h (r=0.454, p=0.045) in the pre- and post-training time-courses. In conclusion, the SC response to exercise appears to become more specific with training; while individual capacity to invoke the SC response is predictive of training induced muscular hypertrophy and may be limited by the degree of MSTN co-localization.</p> / Master of Science (MSc)

Myostatin is a secreted growth factor of the transforming growth-factor $\beta$ (TGF$\beta$) superfamily, and a powerful negative regulator of muscle mass in vertebrates. As such, there is considerable interest in developing pharmacological agents which inhibit myostatin signalling in order to stimulate muscle growth in the context of pathological muscle wasting. Like other TGF$\beta$ family proteins, myostatin is biosynthesised as an inactive (latent) precursor protein which requires proteolytic processing to liberate the mature bioactive growth factor. To examine the molecular basis of pro-myostatin latency and the mechanism by which it is activated in the extracellular space, I have determined the crystal structure of unprocessed human pro-myostatin and studied the properties of the protein at various stages of activation. Crystallographic analysis of pro-myostatin reveals a unique domain-swapped dimeric structure, with an open V-shaped conformation distinct from the prototypical family member, TGF$\beta$1. Following cleavage of the prodomains by furin, pro-myostatin persists as a stable non-covalent complex which is resistant to the natural inhibitor follistatin and exhibits significantly weaker bioactivity than the mature growth factor. A number of distinct structural features combine to stabilise the interaction between pro and mature domains and in doing so confer latency to the pro-complex. This facilitates a controlled, step-wise process of activation in the extracellular space and contributes to a complex network of regulatory control. The results presented here provide a structural basis for understanding the effect of natural polymorphisms on myostatin function and a starting point for structure-guided development of next generation myostatin inhibitors. As a proof-of-concept, I present preliminary data on prodomain derived stapled peptides as inhibitors of myostatin signalling.

Made available in DSpace on 2016-06-02T20:19:16Z (GMT). No. of bitstreams: 1 3721.pdf: 561806 bytes, checksum: df650c263a05271fbcab53bdbda89a04 (MD5) Previous issue date: 2011-05-11 / Universidade Federal de Minas Gerais / Introduction: Myostatin (MSTN), a member of TGF-&#946; family, is characterized as a negative regulator of muscle mass and is present in small quantities in adipose tissue. Accumulating evidences suggest its participation in energetic metabolism. Follistatin (FS) is a non-related protein which inhibits MSTN action. Objective: The objective of this study was to determine the influence of exercise in the expression of MSTN, its receptors (ActRIIB and ALK4), and FS in muscle (white gastrocnemius) and fat (epidydimal, mesenteric, subcutaneous and brow adipose tissue BAT) in streptozotocin-induced diabetic rats. Material and Methods: Control and diabetic animals were randomly assigned to a swimming training group (EC and ED) or a sedentary group (SC and SD). Exercising animals swam for 4 weeks. MSTN, ActRIIB, ALK4 and FS mRNA was quantified by real time RT-PCR and protein expression by Western blotting. Results: Blood glucose was significantly lower in ED than in SD. MSTN and FS mRNA expression increased in SD compared to SC in muscle and subcutaneous fat. The expression of ActRIIB mRNA increased in SD compared to SC in muscle, mesenteric fat and BAT. Expression of ALK4 mRNA increased in SD compared to SC only in BAT. The protein expression of MSTN increased in muscle of the DS when compared to CS. The expression of N-terminal propeptide from the MSTN increased in DS compared to CS in mesenteric fat and TAM. After training, MSTN and ActRIIB were lower in EC compared to SC in BAT. MSTN increased in mesenteric fat and FS mRNA expression decreased in muscle, mesenteric, subcutaneous fat and BAT in ED when compared with SD. ALK4 mRNA expression was lower in BAT when compared to ED with SD. The expression of LAP of MSTN decreased in muscle and mesenteric fat f DE compared to DS. Conclusion: The results indicate that MSTN, its receptors and FS expression changes in both muscle and adipose tissues in diabetic rats and that their expression can be modulated by exercise in Diabetes Mellitus. / Introdução: Miostatina (MSTN), é um membro da família TGF-&#946;, é caracterizada como um regulador negativo da massa muscular e está presente em pequenas quantidades no tecido adiposo. Evidências sugerem sua participação no metabolismo energético. Folistatina (FS) é uma proteína que se liga a MSTN e inibe sua ação. Objetivo: O objetivo deste estudo foi determinar a influência do exercício na expressão da MSTN, seus receptores (ActRIIB e ALK4) e da FS no músculo (gastrocnêmio branco) e tecidos adiposos (gordura mesentérica, epididimal, subcutânea e tecido adiposo marrom TAM) em ratos com diabetes induzido por STZ. Material e métodos: Animais controle e diabéticos eram randomicamente distribuídos no grupo natação (CE e DE) ou grupo sedentário (CS e DS). Os animais realizaram exercício por 4 semanas. O RNAm da MSTN, ActRIIB, ALK4 e FS foram quantificados por RT-PCR e expressão protéica por Western Blotting. Foi utilizado teste ANOVA two-way (posthoc de Tukey). Resultados: A concentração glicêmica no sangue do DE foi menor quando comparado ao DS. A expressão de MSTN e FS aumentaram no DS comparado ao CS no músculo e na gordura subcutânea. A expressão do ActRIIB aumentou no DS comparado ao CS no músculo, gordura mesentérica e TAM. A expressão de ALK4 aumentou no DS comparado com CS somente no TAM. A expressão protéica da MSTN aumentou no músculo do DS comparado ao CS. A expressão do pró-peptídeo da MSTN aumentou no DS comparado ao CS na gordura mesentérica e TAM. Após o treinamento de natação a expressão da MSTN e ActRIIB foram menores no CE comparado ao CS no TAM. A expressão da MSTN aumentou na gordura mesentérica e a expressão da FS diminuiu no músculo, gordura mesentérica e subcutânea e TAM nos animais DE quando comparados ao DS. A expressão de ALK4 foi menor no TAM quando comparado DE com DS. A expressão do LAP da MSTN diminuiu no músculo e na gordura mesentérica do DE comparado ao DS. Conclusão: Os resultados indicam que as expressões da MSTN, de seus receptores e da FS mudaram tanto no músculo quanto no tecido adiposo de ratos diabéticos e que essa expressão pode ser modulada pelo exercício no Diabetes Mellitus.

Thesis (MSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: The aim of this study was to investigate the powerful muscle regulator, myostatin, and its regulators in response to an acute bout of plyometric training. The participants were recruited and screened by characterization by means of isometric force production tests, baseline blood creatine kinase levels and VO2 max results. The selected individuals (n=15) were subjected to a baseline muscle biopsy for comparative purposes. The study made use of plyometric jumping, as source of eccentric exercise, to serve as an exercise intervention after which muscle biopsies (4 hours post and 24 hours post) and blood draw (4 hours post, 24 hours post and 48 hours post) samples were taken. Maximal voluntary isometric contractions of the knee extensors were also measured immediately after the exercise protocol and after 1 week recovery. Creatine kinase (CK) analysis on the serum samples was used to conclude muscle damage. The muscle biopsy samples were used for protein quantification (Western blot) and gene expression assessment (semi-quantitative and real-time PCR). The results showed decreased force production immediately after eccentric exercise (p < 0.05), while returning back to baseline values at 1 week post exercise and CK results showed a significant increases at 4 hours (p<0.05), 24 hours (p<0.001) and 48 hours (p<0.01) after exercise. There were no significant differences in myostatin precursor protein (43 kDa), phosphorylated Smad2,3, Smad7 or activin receptor IIb in response to eccentric exercise. However, the follistatin protein was increased at both 4 hours and 24 hours after exercise (p<0.01). RNA analysis of the extracellular matrix (ECM) protein, decorin, revealed the existence of the splice variants A1 and A2 in human skeletal muscle. The RT-PCR analysis (n=4) of these variants showed no significant difference when comparing pre- to post-exercise. The decorin core protein was also investigated by means of antibody probing and results revealed the need for ABC chondroitinase enzyme treatment before immunoblotting of human skeletal muscle samples. The results concerning knee extensor force reduction and circulating creatine kinase showed the effectiveness of plyometric jumping in producing skeletal muscle damage in the lower limbs of unfit individuals, unaccustomed to eccentric exercise. In conclusion, myostatin, and its associated signalling cascade, are not activated in early muscle regeneration, but follistatin is increased during this phase possibly aiding and initiating the muscle repair process. Future studies: Variants of decorin are expressed in human skeletal muscle, increasing the complexity that should be taken into account in studies concerning the regulation of decorin in a human model. Investigation into myostatin protein at different post-translational levels needs more clarification. Published methods and materials used in different laboratories are not consistent and investigators should attempt to standardise protocols in order to compare results between studies more effectively. Of importance, these results show that the myostatin at protein level report different results compared to mRNA analysis and that more investigation into myostatin regulatory factors, with special reference to follistatin and decorin, is needed in future human models. / AFRIKAANSE OPSOMMING: Die doel van hierdie studie was om die kragtige spiere reguleerder, miostatin, en sy reguleerders in reaksie op 'n akute aanval van pliometriese spronge te ondersoek. Die deelnemers is gewerf en gekeur deur karakterisering deur middel van isometriese krag produksie toetse, basislyn bloed kreatien kinase vlakke en VO2maks resultate. Die geselekteerde individue (N = 15) is onderhewig aan 'n basislyn spierbiopsie vir vergelykende doeleindes. Die studie het gebruik gemaak van pliometriese spronge (essentriese spier aksie) as die oefening intervensie waarna spierbiopsie (4 uur na en 24 uur na) en bloed (4 uur na, 24 uur na en 48 uur na) monsters geneem is. Isometriese kontraksies van die knieverlengers is ook gemeet onmiddellik na die oefening protokol en na 1 week se herstel. Kreatine kinase (KK) ontleding van die serum monsters is gebruik om spierskade aftelei. Die spierbiopsie monsters was gebruik vir proteïen kwantifisering (Western klad) en die assessering van geen uitdrukking (semi-kwantitatiewe en real-time PCR). Die resultate het gewys dat krag produksie afgeneem het onmiddellik na essentriese oefening (p <0.05), terwyl dit terugkeer na die oorspronklike waardes 1 week na oefening en KK resultate toon 'n beduidende toename by 4 uur (p <0,05), 24 uur (p <0,001) en 48 uur (p <0,01) na oefening. Daar was geen betekenisvolle verskille in Miostatien voorloper proteïen (43 kDa), gefosforileerde Smad2,3, Smad7 of Activin reseptoor IIb in reaksie op essentriese oefening. Dit is egter die follistatien proteïen wat verhoog by beide 4 uur en 24 uur na oefening (p <0,01). RNS ontleding van die ekstrasellulêre matriks (ESM) proteïen, decorin, het die bestaan van die splitsing variante A1 en A2 in menslike skeletspier, aan die lig gebring. Die RT-PCR analise (n = 4) van hierdie variante het geen betekenisvolle verskille getoon wanneer voor met na-oefening vergelyk is. Die decorin kern proteïen is ook ondersoek deur middel van teenliggaam afhanklike metodes en resultate het die behoefte aan ABC chondroitinase ensiem behandeling voor immunokladding van menslike skeletspier monsters gesteun. Die resultate aangaande knieverlenger krag vermindering en sirkuleerende kreatien kinase het die doeltreffendheid van pliometriese spronge in die vervaardiging van skeletspier skade in die onderste ledemate van individue ongewoond aan essentriese oefening verseker. Ten slotte, Miostatien, en sy verwante sein kaskade, is nie geaktiveer vroeg in spier herstelling, maar follistatien is tydens hierdie fase verhoog en help moontlik met die aanvang van die spier herstel. Toekomstige studies: variante van decorin word uitgedruk in menslike skeletspier, wat die kompleksiteit aangaande decorin verhoog en dit is iets wat in ag geneem moet word in studies wat handel oor die regulering van decorin in mens modelle. Ondersoek na miostatien proteïen op verskillende na-translasie vlakke moet meer duidelikheid verkry. Gepubliseer metodes en materiaal wat gebruik word in verskillende laboratoriums is nie konsekwent en ondersoekbeamptes moet probeer om protokolle te standaardiseer sodat resultate van studies meer effektief kan vergelyk word. Van belang is, die resultate wys dat miostatien op proteïen vlak verskillende resultate vertoon in vergelyking met boodskapper-RNS ontleding en dat meer ondersoek na miostatien regulerende faktore, met spesiale verwysing na follistatien en decorin, nodig is in toekomstige menslike modelle.

Submitted by Angela Maria de Oliveira (amolivei@uepg.br) on 2018-11-20T17:29:47Z No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Amanda de Paula Lima.pdf: 1103885 bytes, checksum: c825dfd59f4730fd1cd0b63b5796cb44 (MD5) / Made available in DSpace on 2018-11-20T17:29:47Z (GMT). No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Amanda de Paula Lima.pdf: 1103885 bytes, checksum: c825dfd59f4730fd1cd0b63b5796cb44 (MD5) Previous issue date: 2018-09-10 / Fundação Araucária de Apoio ao Desenvolvimento Científico e Tecnológico do Paraná / A presente pesquisa foi realizada com o objetivo avaliar os efeitos da suplementação combinada de lisina e metionina sobre o desempenho produtivo e expressão de genes relacionados com o crescimento muscular em alevinos de tilápia do Nilo revertidos sexualmente. Trezentos e trinta e seis alevinos de tilápia do Nilo (peso inicial 0,90 ± 0,01 g) foram distribuídos em 24 aquários de 70L com sistema contínuo de fluxo de água (2,0 L/min), em um delineamento inteiramente casualizado com quatro tratamentos e seis repetições. Foram elaboradas quatro dietas isoproteicas (~330,50 g/kg de proteína bruta) e isocalóricas (~18,59 MJ/kg) sem suplementação de lisina e metionina (Controle), suplementada com lisina (Lys), suplementada com metionina (Met) e suplementada com lisina e metionina (Lys+Met) durante oito semanas. Os peixes foram alimentados manualmente, quatro vezes por dia até saciedade aparente. Peixes tratados com as dietas Lys e Met apresentaram maior peso corporal e taxa de crescimento específico em relação aos peixes mantidos com as demais dietas. Peixes tratados com dieta Lys apresentaram maior taxa de eficiência proteica em comparação aos peixes mantidos com as outras dietas. O índice hepatostomático e a gordura corporal foram menores nos peixes alimentados com as dietas Met e Lys+Met em comparação aos peixes tratados com a dieta controle. O consumo, sobrevivência, umidade, proteína bruta, cinzas corporais e a expressão do mRNA da miostatina não foram influenciados pelas dietas. Peixes que receberam dieta Lys+Met apresentaram maior nível de expressão de mRNA da MyoD em comparação aos peixes que receberam a dieta controle, mas nenhum efeito da suplementação isolada de lisina e metionina foi observada. Em conclusão, a suplementação combinada de lisina e metionina melhora o desempenho produtivo e aumenta a expressão de mRNA de MyoD e miogenina e reduz conteúdo de gordura corporal de alevinos de tilápias do Nilo. / This work was carried out with the objective of evaluating the effects of the combination of lysine and methionine on the performance of growth and expression of genes related to muscle growth in sexually reversed Nile tilapia fingerlings. Fish (n = 336, initial weight 0.90 ± 0.01 g) were randomly distributed into 24 70 L aquaria with a continuous water flow system in an entirely randomized design with four treatments and six replicates. Four isoproteic (~330.50 g/kg crude protein) and isocaloric (~ 18.59 MJ / kg) diets without lysine or methionine supplementation (Control), or supplemented with lysine (Lys), methionine (Met) and lysine and methionine (Lys + Met) were elaborated. Fish were hand fed until apparent satiety. Fish fed diets Lys+Met and Met showed higher final body weight and specific growth ratio compared to fish fed other diets. The protein efficiency ratio was higher in fish diet Lys compared to fish fed other diets. Fish fed Met and Lys+Met diets showed lower hepatosomatic index and whole-body fat compared to fish fed the control diet. Feed intake, survival and whole-body moisture, crude protein, ash and mRNA expression of myostatin of fish were not affected by diets. Fish fed diet Lys+Met demonstrated higher mRNA expression level of MyoD compared to those fed the control diet. In conclusion, Nile tilapia fingerlings fed combined lysine and methionine demonstrates improved growth performance in line to higher mRNA expression of MyoD and myogenin, and also reduced body fat contents

HMb, metabólito da leucina, utilizado para aumentar a síntese protéica. Investigamos o efeito do HMb sobre o eixo somatotrófico, bem como o mRNA de IGF-I e miostatina muscular. Ratos tratados com HMb (320 mg/Kg de peso corporal /mL de salina-0,9%), ou salina (controle), gavagem, 4 semanas, decapitados, sangue para avaliação sérica: insulina (RIE), glicose (colorimetria) e IGF-I (RIE). Extração de RNA total, para avaliação do mRNA de IGF-I e miostatina (Fígado, músculo extensor digital longo, Sóleo), avaliação da expressão do mRNA do GH, por Northern Blot, e expressão do GH ,Western blotting (hipófise). Dados analisados pelo teste-T de Student (P<0,05). Tratamento aumentou o conteúdo de mRNA de GH (> 60%), da proteína GH (>20%), do mRNA do IGF-I (~24%), da concentração sérica de IGF-I (p<0,05), indicando uma ativação do eixo somatotrófico pelo HMb, sem alterações no mRNA de miostatina e IGF-I muscular, ainda um aumento da insulina (~2x), sem alterações na glicose sérica, resultado do efeito hiperglicemiante do GH, ou um efeito direto do HMb na secreção de insulina. / HMb, metabolite of leucine, used to increase protein synthesis. Evaluate the effect HMb on the somatotrophic axis activity, as well as muscle mRNA IGF-I and myostatin. Rats treated with HMb (320 mg / kg body weight / mL of saline-0, 9%) or saline (control), gavage, 4 weeks, decapitated, blood for evaluation of serum: insulin (RIA), glucose (colorimetric) and IGF-I (RIA). Extraction of RNA total, for evaluation the mRNA IGF-I and myostatin (liver, muscle extensor digitalis longus (EDL) and soleus), evaluation of the GH mRNA expression of by Northern blot, and GH content, western blotting (pituitaries). Data analyzed by Student t-test (P <0.05). HMb treatment increased the content of GH mRNA (> 60%), GH (> 20%), IGF-I mRNA (~ 24%), IGF-I (p <0.05), indicates that the somatotrophic axis activity is increased by the HMb, without changes in mRNA of myostatin and muscle IGF-I, insulin also increased (~ 2x), without changes in serum glucose, hyperglycemiant result of the effect of GH or a direct effect of HMb in the secretion of insulin.

Nosso objetivo foi investigar o efeito da suplementação de leucina na via intracelular acionada por miostatina, uma proteína reguladora negativa da massa muscular, em um modelo de atrofia gerada por imobilização em ratos. Nossa hipótese inicial contemplava que a expressão de miostatina se elevaria com a imobilização e que a leucina poderia atenuar esse aumento. Nossos resultados, entretanto, mostraram uma regulação gênica no sentido de suprimir a via da miostatina. A suplementação com leucina, associada à imobilização, também provocou queda da expressão gênica de miostatina. Em ambos os grupos a expressão proteica permaneceu inalterada. Em contrapartida, a folistatina, inibidor endógeno da miostatina, apresentou acentuado aumento de expressão no grupo imobilizado e imobilizado/suplementados com leucina. Nossos resultados mostraram que o efeito protetor da leucina no modelo atrófico utilizado não se dá através da sinalização da miostatina. Os dados também sugerem uma resposta biológica coordenada entre o ligante, miostatina, e seu inibidor endógeno, a folistatina. / Our purpose was to investigate the effect of leucine supplementation upon the intracellular pathway triggered by myostatin, a negative regulator of muscle mass, in an atrophic model driven by immobilization in rats. Our initial hypothesis contemplated that myostatin expression would be elevated under immobilization and that leucine supplementation would be able to attenuate this raise. However our results showed a strong downregulation of myostatin gene expression in atrophy induced by cast. The immobilization associated with leucine supplementation also caused mRNA myostatin downregulation. Protein levels were unchanged. On the other hand, follistatin, a myostatin endogenous inhibitor, was markedly upregulated in both immobilized and immobilized/supplemented groups. Our results revealed that leucine protective effect in this atrophic model is not through myostatin signailing. Furthermore, these data suggest a coordinated biological response between ligand, myostatin, and its endogenous inhibitor, follistatin.

Antagonism of the activin receptor signaling pathway represents a promising potential therapy for the muscular dystrophies and other muscle wasting disorders (i.e., cachexia or sarcopenia). Previous research has shown that antagonism of activin signaling promotes muscle growth, attenuates muscle wasting, and restores function in both wild type and diseased animals. Our laboratory has recently developed a novel small molecule (SGI-1252) that inhibits activin downstream (i.e., Smad2/3 phosphorylation) signaling. Purpose: In this study we determined how eight weeks of orally administered SGI-1252 affected TGF-ß signaling, whole body mass, individual limb muscle mass, and muscle fiber cross sectional area (CSA). Methods: Wild-type (WT) mice were treated with SGI-1252 or a vehicle control (VC) via oral gavage (400 mg/kg 3 times per week) for 8 weeks. Body mass was measured twice per week during the 8-week treatment period. At the end of the treatment period, gastrocnemius and tibialis anterior (TA) muscles were excised, weighed, and prepared for histological and biochemical analyses. Results: Following 8 weeks of treatment, there was no difference in weight gain between SGI-1252 (24.8 ± 1.8g) and VC treated mice (23.2 ± 1.5g) (p = 0.06). Gastrocnemius whole muscle mass was significantly greater in the SGI-1252 treated group relative to the VC treated mice (139.6 ± 12.8 mg vs 128.8 ± 14.9 mg) (p = 0.04), although when normalized with body mass there was no difference in gastrocnemius mass. For the TA muscle, there were no significant differences in whole muscle mass between SGI-1252 and VC groups, yet TA muscles in the SGI-1252 treated group had a reduced muscle fiber CSA compared to controls (621 ± 44 µm2 vs 749 ± 36 µm2) (p = 0.0005). There was a statistical trend of decreasing Smad2 phosphorylation in the SGI-1252 treated TA muscles (mean SGI-1252 = 0.668 vs VC = 0.848) (p = 0.06), and no significant differences in Smad2 phosphorylation in the gastrocnemius. Conclusions: Contrary to our hypothesis, 8 weeks of orally administered SGI-1252 was not effective in promoting increases in whole body mass, limb whole muscle mass, or myofiber cross sectional area. This may be due to the inability of SGI-1252, at the administered dose, to effectively decrease signaling downstream of the activin receptor. Clearly, studies using a wider range of doses and delivery methods will be needed to ascertain the efficacy of SGI-1252 as a potential therapeutic.

Acute quadriplegic myopathy (AQM), or critical illness myopathy, is a common debilitating acquired disorder in critically ill intensive care unit (ICU) patients characterized by generalized muscle wasting and weakness of limb and trunk muscles. A preferential loss of the thick filament protein myosin is considered pathognomonic of this disorder, but the myosin loss is observed relatively late during the disease progression. In attempt to explore the potential role of factors considered triggering AQM in sedated mechanically ventilated (MV) ICU patients, we have studied the early effects, prior to the myosin loss, of neuromuscular blockade (NMB), corticosteroids (CS) and sepsis separate or in combination in a porcine experimental ICU model. Specific interest has been focused on skeletal muscle gene/protein expression and regulation of muscle contraction at the muscle fiber level. This project aims at improving our understanding of the molecular mechanisms underlying muscle specific differences in response to the ICU intervention and the role played by the different triggering factors. The sparing of masticatory muscle fiber function was coupled to an up-regulation of heat shock protein genes and down-regulation of myostatin are suggested to be key factors in the relative sparing of masticatory muscles. Up-regulation of chemokine activity genes and down-regulation of heat shock protein genes play a significant role in the limb muscle dysfunction associated with sepsis. The effects of corticosteroids in the development of limb muscle weakness reveals up-regulation of kinase activity and transcriptional regulation genes and the down-regulation of heat shock protein, sarcomeric, cytoskeletal and oxidative stress responsive genes. In contrast to limb and craniofacial muscles, the respiratory diaphragm muscle responded differently to the different triggering factors. MV itself appears to play a major role for the diaphragm muscle dysfunction. By targeting these genes, future experiments can give an insight into the development of innovative treatments expected at protecting muscle mass and function in critically ill ICU patients.

Myostatin is a major player in the regulation of mammalian muscle growth and development, maintaining the balance between proliferation and differentiation prenatally and the quiescence of satellite cells in adults. An absence or overexpression of myostatin results in double-muscling and cachexia respectively, placing myostatin as a promising target in the treatment of muscle wastage diseases. As a transforming growth factor-β superfamily member, myostatin is produced as a precursor protein, consisting of a propeptide region N-terminal to the growth factor domain. Cleavage of the precursor between the domains forms the myostatin latent complex, an inhibitory structure which is exported from the cell where a second cleavage event releases the active myostatin growth factor. The precursor protein, propeptide, and latent complex play important roles in the regulation of myostatin. However, their structure and function are poorly understood, and a possible role for the myostatin precursor protein in the muscle wastage disease sporadic inclusion body myositis, suggests that pre-growth factor forms of myostatin may be additional important therapeutic targets. This thesis presents an investigation into the structure and function of the myostatin precursor protein, the latent complex, and the propeptide region within these, with comparisons to a mutant form of myostatin responsible for the naturally-occurring double-muscled phenotype of the Piedmontese cattle breed. Results suggest that the diverse functions of the propeptide region are facilitated by regions of intrinsic disorder within a primarily structured domain, and that conformational alterations accompany the precursor to latent complex transition, resulting in a tighter inhibitory structure. Comparative analyses between the wild-type and mutant proteins suggest that the Piedmontese phenotype is due to a reduced capacity for covalent dimerisation and significant structural alterations within the type I receptor-binding domain. Investigation into misfolded myostatin precursor protein found that the precursor is able to form cytotoxic amyloid aggregates and mature fibrils under partially denaturing conditions, suggesting a possible mechanism for the role of the myostatin precursor in sporadic inclusion body myositis. Together, these novel results contribute important information towards an understanding of myostatin structure, function and regulation in both normal and disease scenarios.