Thesis (Ph.D.)--Boston University / Myostatin (mstn) belongs to the TGF-β family and is best known as a negative regulator of muscle growth. Hypermuscularity in mstn knockout mice has been widely recognized and numerous mstn inhibitors are in development to treat sarcopenia associated with aging and muscle wasting associated with chronic illness. To determine the health impact of long term mstn deficiency on physical performance, myostatin null (mstn-/-) mice were studied at three aging time points (5, 15, 22 months). Long term complete inhibition of myostatin was hypothesized to prevent the decline in physical performance expected with aging. The data disproves this hypothesis. While increased muscle was observed in the mstn-/- and the mice demonstrated an expected grip strength advantage at 5 months, by 15 and 22 months the overall grip strength was reduced with no significant difference compared to controls. Horizontal and vertical ambulatory activity at each aging time point showed a significantly decreased or a decreased trend when compared with age-matched wild type controls. Furthermore, the mstn-/- mice treadmill endurance was significantly less at 5 and 22 months and demonstrated a decreased trend at 15 months when compared to age-matched controls. Overall, long term myostatin deletion does not aid in the maintenance of overall physical performance as the animal ages. Since prior studies had also shown that myostatin affected both bone mass, cartilage development and skeletal tissue repair the effects of myostatin deletion were next examined relative to skeletal tissue structure and function. Micro Computerized Tomography (MicroCT) analysis showed increased size and ossification of the meniscus surrounding the knee, ectopic ossification surrounding the tarsal bones and extra ossification in joints between manus carpal bones in the myostatin null mice. These studies showed that this ectopic calcification increased with aging. Corresponding histology of 22 month old mstn-/- mice demonstrated increased fibrocartilage proliferation of the meniscus and loss of the tibial articular cartilage. Our current data suggests long term complete deletion of myostatin increases proliferation and differentiation of fibrocartilage and reduces articular cartilage maintenance leading to impaired joint function and an overall reduction in physical performance.

RNA interference (RNAi) is a means of regulating gene expression by targeting mRNA in a sequence-specific manner for degradation or translational inhibition. Short hairpin RNAs (shRNAs) and short interfering RNAs (siRNAs) have been extensively employed for manipulating gene expression in a wide range of species. The goal for this research was to produce transgenic livestock in which myostatin, a negative regulator of muscle growth, has been targeted for silencing by RNAi. This would demonstrate the utility of RNAi for reducing gene expression in large animal species. To successfully target the myostatin gene for reduction, siRNAs were designed to target the both the bovine and caprine myostatin mRNA sequence. These were then tested for effectiveness in vitro using both an HEK 293T cell line expressing caprine myostatin as well as adult bovine muscle cells. The most effective siRNA, GDF8-1026, was cloned into a lentiviral plasmid and used to transduce bovine fetal fibroblasts for somatic cell nuclear transfer cloning as well as perivitelline injection of in vitro produced bovine embryos. To date, seven pregnancies have been established using these two methods. Concern over the possibility of off-target effects associated with the expression of shRNAs in vivo prompted investigation into tissue-specific expression. Therefore, investigation into the use of a muscle-specific promoter to drive transgene expression was pursued. The bovine myogenin promoter and muscle creatine kinase (MCK) promoter were cloned into a lentiviral plasmid and evaluated in bovine fetal muscle cells and mouse C2C12 cells in vitro for their ability to drive GFP expression. Both promoters demonstrated an increase in GFP intensity at day nine of differentiation when compared to the nontransduced control. The retroviral basis of lentiviral plasmids has raised concern over the possible development of replication competent lentivirus (RCL). Therefore, analysis of tissues from recipients of lentivirus-treated embryos was performed to detect possible RCL. Tissues and blood serum were tested for RCL using p24 ELISA as well as qRT-PCR for the VSV-G gene. To date all tissues tested so far shown no evidence for RCL using these analyses. Analysis of offspring transgenic for an shRNA targeting myostatin will allow confirmation of RNAi as a useful tool for manipulating gene expression in large animal species.

En 1997, le caractère culard, un phénotype hypermusclé chez le bovin, est attribué à des mutations dans le gène de la myostatine (MSTN). Depuis, il a été confirmé qu’une baisse de l’activité de la MSTN conduisait à une augmentation de la masse musculaire chez d’autres espèces, y compris chez l’Homme. L’identification de ce facteur et des conséquences de son invalidation sur le développement musculaire ouvre de nombreuses perspectives en médecine humaine comme, par exemple, chez des personnes ayant eu une fonte musculaire importante suite à une immobilisation prolongée ou en conséquence du vieillissement ou d’une maladie chronique. L’objectif majeur de ce projet de recherche a consisté à évaluer de nouvelles stratégies permettant d’augmenter la masse musculaire chez la souris. Pour ce faire, nous nous sommes intéressés à une métalloprotéine de la matrice extracellulaire (MEC), la décorine (DCN), dont l’interaction avec la MSTN a été caractérisée comme étant zinc dépendante. Suite à l’injection de ce Small Leucine Rich Proteoglycan (SLRP) chez la souris dystrophique mdx et Gamma-sarcoglycan-/-, nous avons constaté une augmentation de la masse musculaire consécutive à un phénomène d’hypertrophie associé ou non à de l’hyperplasie. Des études de dose/cinétique ont montré que l’effet positif de la décorine sur la croissance musculaire était maximal 21 jours après administration. Nous avons également découvert qu’un fragment peptidique de 41 acides aminés du domaine N-terminal de la protéine DCN murine conservait une activité anti-myostatine et induisait une hypertrophie musculaire chez la souris dystrophique. Ce domaine, site de l’interaction directe entre la MSTN et la DCN, présente un motif CX3CXCX6C, caractéristique des SLRPs de classe I, dont le cluster de cystéines et son interaction avec le zinc ont été décrits comme indispensables à l’activité anti-MSTN de la DCN. Différentes études concernant les mécanismes induits lors de la séquestration de la MSTN par la DCN dans la MEC ont également été conduites afin d’expliquer les phénomènes observés chez la souris. Enfin, nous avons étudié le potentiel de la DCN pour favoriser la greffe de cellules myogéniques et développé différentes approches de thérapie génique. / In 1997, the double-muscling phenotype, a marked hypermuscularity in cattle, was related to mutations in the myostatin (MSTN) gene. Since, it was confirmed that a decrease of the myostatin’s activity drives an increase of the muscular mass in others species, including Human. The identification of this factor and the consequences of its invalidation on the muscular development open many perspectives in human medicine, as, for example, for people whom have an important muscular loss fallow up an extended immobilization or in consequence of old age or a chronic disease. The main purpose of this research project was to evaluate some new strategies permitting the increase of the muscular mass in mice. To achieve that, we investigated in detail the decorin (DCN), a metalloprotein of the extracellular matrix (ECM), interacting with MSTN in a zinc-dependent manner. After intramuscular injection of this Small Leucine Rich Proteoglycan (SLRP) in mdx and Gamma-Sarcoglycan-/- dystrophic mice, we observed a significant increase of the muscle mass conducted by hypertrophy associated or not with hyperplasia. Dose and cinetic studies showed that the positive effect of the decorin on muscular growth was maximal 21 days after administration. Furthermore, we showed that a peptide encompassing the 31-71 sequence retains full myostatin binding capacity and intramuscular injection of this peptide induces muscle hypertrophy in dystrophic mice. This direct interaction site between MSTN and DCN contains a conserved CX3CXCX6C pattern of class I SLRPs, whose cluster of cysteins and its interaction with zinc were shown to be crucial in the anti-MSTN activity of DCN. Various studies of the mechanism resulting of the sequestration of MSTN by DCN in ECM were conducted in order to explain the phenomenom observed in mice. Al last, we have studied the potential of DCN in the cellular transplantation and developped different anti-myostatin strategies of genetic therapy.

Myostatin, a TGF-β superfamily member, is a key negative regulator of embryonic and postnatal muscle growth. In order to further elucidate the role of myostatin during postnatal growth, several lines of investigation were undertaken in mice. Analysis of myostatin downstream target genes identified several known and unknown genes. From these, the regulation of an androgen receptor binding co-factor, ARA70, was selected for further investigation. Reverse Northern analysis on the differentially expressed cDNA library indicated an increased expression of ARA70 in myostatin-null muscles, which was later confirmed by Northern blot and semi-quantitative PCR analysis. In corroboration, treatment of myoblast cultures with exogenous myostatin resulted in the down-regulation of ARA70, confirming that myostatin is a negative regulator of ARA70 gene expression. The role of myostatin during sarcopenia, a progressive age-related loss of skeletal muscle mass and strength, was also investigated. The atrophy associated with sarcopenia is frequently correlated with insufficient muscle regeneration, resulting from an impaired propensity of satellite cells to activate and a subsequent decline in myogenesis. Myostatin is a known inhibitor of postnatal satellite cell activation and muscle regeneration, thus muscle mass and regeneration, and satellite cell behaviour were examined in young and aged myostatin-null mice. Myostatin-null mice had increased individual muscle weights, as a consequence of massive fibre hypertrophy and hyperplasia, and an increased proportion of type IIB fibres. Aging induced oxidative fibre type changes and atrophy in the wild-type muscle while no fibre type switching was observed in the myostatin-null muscle and atrophy was minimal. No decrease in satellite cell numbers was observed with aging in both genotypes; though a gradual decline in the number of activated satellite cells was noted during aging. However, both young and aged myostatin-null mice displayed increased satellite cells and activation compared to wild-type mice, suggesting a greater myogenic potential in the myostatin-null satellite cells. Consistent with this, aged myostatin-null myoblasts proliferated faster and displayed a higher fusion index during differentiation than the aged wild-type myoblasts, confirming that the reduced sarcopenia in the myostatin-null mice was due to a preserved increase in the myoblast myogenic activity. An increase in a Pax7-only myoblast population from myostatin-null muscle indicated an enhanced satellite cell self-renewal process, consistent with the increased satellite cell number observed on the myostatin-null muscle fibres. Additionally, muscle regeneration of aged myostatin-null muscle following notexin injury was accelerated, and fibre hypertrophy and type were recovered with regeneration, unlike the aged wild-type muscle. Testing the therapeutic value of a myostatin antagonist, Mstn-ant1, indicated that a short term blockade of myostatin by the antagonist significantly enhanced muscle regeneration in aged mice after injury and during sarcopenia. Antagonism of myostatin led to satellite cell activation, increased Pax7 and MyoD protein levels, and greater myoblast and macrophage cell migration culminating in enhanced muscle regeneration in the aged mice. In conclusion, the hypertrophic phenotype associated with myostatin-null mice may in part result from increased androgen receptor (AR) activity due to the up-regulation of ARA70, given that increased expression of the AR leads to hypertrophy. Additionally, the increased muscle mass in myostatin-null mice is likely to result from an augmented myogenic potential and self-renewal process. Overall, a prolonged absence of myostatin reduced sarcopenia and the associated loss of muscle regenerative capacity. Furthermore, the antagonism of myostatin displayed significant therapeutic potential in the alleviation of sarcopenia, through the restoration of the myogenic and inflammatory responses in the aged environment. Thus, the research work clearly demonstrates the role of myostatin in sarcopenia, and documents for the first time a valid therapeutic for alleviating sarcopenia.

Myostatin is a negative regulator of muscle growth. When it is not present or non-functional double-muscling occurs, the primary characteristic of this phenotype being an increase in muscle mass. Another characteristic of double-muscling is an increased proportion of type IIB muscle fibres, which rely on glycolysis as their primary energy source, as opposed to type IIA and type I fibres which rely on oxidative phosphorylation. This switch in muscle metabolism directly impacts on the mitochondria, as mitochondria from glycolytic muscle fibres have been shown to have differences in metabolic activity. The increased proportion of glycolytic muscle fibres present in myostatin knockout animals provides a unique model to investigate alterations in muscle fibre type metabolism. The mouse model of myostatin knockout utilised during this study was generated by genetic deletion of exon three of the myostatin gene. Verification of this knockout was attempted by western blot analysis, but only the latency associated protein (LAP) was detected. Interestingly, the LAP was barely detectable in the knockout muscle suggesting deletion of exon three affects binding of anti-myostatin antibodies to the LAP, as that part of the gene is not deleted. A comparison of the basal mitochondrial stress levels was made, also by western blot analysis. The knockout mitochondria showed no change in levels of heat shock protein 60 or superoxide dismutase 2, indicating that they are not being subjected to any increased stress due to the myostatin knockout phenotype. A comparative proteomics approach was used to detect changes in the mitochondrial proteome of myostatin knockout gastrocnemius muscle to gain clues to how mitochondria from glycolytic muscle fibres differ from those present in oxidative fibres. This was undertaken using two-dimensional electrophoresis (2-DE), in-gel tryptic digests and peptide mass fingerprinting by mass spectrometry. A 2-DE gel protein loading of 220 g was shown to give the best protein spot resolution and the most crucial step in the loading process was found to be the laying of the immobilized pH gradient, which had to be performed very carefully to obtain a consistent loading pattern. This study resolved only around 160 protein spots out of the estimated 1,000 to 2,000 proteins present in the mitochondria. Modulation of six proteins was seen at a plt0.1 level, but were unable to be identified using the current methodology. More abundant mitochondrial proteins were able to be identified, but showed no significant modulation. Malate dehydrogenase and 3-hydroxyacyl-CoA dehydrogenase, which were identified during this study, have been reported to have decreased activity in mitochondria from glycolytic muscle fibres. This study suggests that the change in activity observed by other researchers is due to inhibition of these enzymes in the glycolytic fibres or activation in the oxidative fibres.

This thesis proposal shows the efficiency of different methods of Myostatin inhibition by using CRISPR/CAS9. With the data cataloged by this thesis, researchers will have a better understanding of what methods are better to achieve their goals. The data was collected by reading multiple scientific articles involving the usage of CRISPR/CAS9 to inhibit the Myostatin protein. The data collected from all the different studies were analyzed in the same categories. The experiment that used CRIPSR/CAS9 on in vitro specimens had a superior Myostatin inhibition overall, therefore presenting higher muscle mass. The method using CRISPR/CAS9 to inhibit the Myostatin in vivo and in vitro depends on what the researcher is trying to accomplish. By reading this thesis researchers can understand that if they choose to use an in vitro treatment the results would be way more severe than using an in vivo application treatment.

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Orientador: Marcos Ferreira Minicucci / Resumo: Introdução: As doenças cardiovasculares são as principais causas de mortalidade em todo o mundo. O infarto agudo do miocárdio com supradesnivelamento de segmento ST (IAMCSST) vem apresentando redução da mortalidade após a introdução das terapias de reperfusão. Diversos fatores estão associados a pior prognóstico e foi evidenciado que massa e função muscular podem estar associadas a comorbidades como hipertensão arterial sistêmica, síndrome metabólica, diabetes mellitus, obesidade e morte precoce. A força e massa muscular são regulados por diversos fatores entre os quais podemos destacar a miostatina. A miostatina, conhecida classicamente como regulador negativo da musculatura tem apresentado papel controverso na literatura, sendo por vezes relacionada a perda de massa muscular. Até o presente momento não há estudos investigando o papel da miostatina nas síndromes coronarianas agudas. Objetivo: O objetivo do presente estudo é avaliar a associação dos valores séricos de miostatina, com a massa e função muscular, tempo de internação e mortalidade hospitalar de pacientes com IAMCSST admitidos no Hospital das Clínicas da Faculdade de Medicina de Botucatu. Materiais e métodos: Trata-se de um estudo prospectivo observacional com pacientes admitidos com diagnóstico de IAMCSST no Hospital das Clínicas da Faculdade de Medicina de Botucatu, no período de maio de 2018 a fevereiro de 2019. Foram incluídos pacientes com IAMCSST, que aceitaram participar e foram recrutados nas primeiras 48 ... (Resumo completo, clicar acesso eletrônico abaixo) / Mestre

O treinamento de força promove hipertrofia muscular esquelética e aumento da capacidade de gerar força. É preconizado que a ocorrência dessas adaptações depende da especificidade do estímulo de treinamento. De acordo com esse princípio, é esperado que as respostas adaptativas fossem específicas ao estímulo aplicado. Entretanto, tem sido observado, por exemplo, que os modelos de treinamento de força e treinamento de potência, relacionados especificamente a adaptações centrais, induzem semelhantes ganhos em força e hipertrofia. Diante dessas evidências, surgiram questionamentos sobre a validade desse princípio. Nesse sentido, o presente estudo teve como objetivo avaliar o efeito do treinamento de força e potência sobre a expressão do gene da miostatina (MSTN), associada ao controle do tamanho do músculo esquelético, e de genes relacionado a essa via de sinalização: FLST, FL3, GASP-1, ActIIB, SMAD-7 e FOXO-3A. Homens saudáveis, fisicamente ativos, foram randomicamente distribuídos, de forma balanceada, em três grupos: controle, força e potência. Os grupos treinados foram submetidos a oito semanas de intervenção (treinamento de força e treinamento de potência). Foram coletadas amostras de tecido muscular (vasto lateral) via biópsia percutânea nas condições pré e pós-treinamento. Essas amostras foram utilizadas para a análise da expressão de genes envolvidos na sinalização da MSTN por meio da PCR em tempo real. Não foi verificada alteração na expressão gênica de MSTN, ActIIB, GASP-1 e FOXO-3A após o treinamento de força e o treinamento de potência. Porém, foram observadas alterações no conteúdo de RNAm para FLST, FL3 e SMAD-7. Essas alterações foram semelhantes entre os distintos protocolos de treinamento. Os resultados do presente estudo sugerem que ambos os modelos de treinamento de força são capazes de induzir resposta similar sobre a expressão de genes envolvidos na sinalização da MSTN. A ausência de respostas específicas dentro do período investigado aponta para a necessidade de investigar o curso temporal das adaptações aos diferentes modelos de treinamento de força em longo prazo / Resistance training promotes skeletal muscle hypertrophy and increased ability to generate force. It is recommended that the occurrence of these adaptations depend on the specificity of the training stimulus. According to this principle, it is expected that the adaptive responses were specific to stimuli applied. However, it has been observed, for example, that models of strength training and power training, specifically related to central adaptations, induce similar gains in strength and hypertrophy. Given this evidence, questions arose about the validity of this principle. In this sense, the present study aimed to evaluate the effect of strength training and power on the expression of the myostatin gene (MSTN) associated with the control of skeletal muscle size and genes related to this signaling pathway: FLST, FL3, GASP-1, ActIIB, SMAD-7 and FOXO-7-3A. Men healthy, physically active, were randomly divided into three groups: control, strength and power. The trained groups underwent eight weeks of intervention (strength training and power training). Samples pre-and post-training were collected from muscle tissue by percutaneous biopsy of the vastus lateralis. These samples were used for analysis of the expression of genes involved in signaling MSTN by real time PCR. No change was observed in gene expression of MSTN, ActIIB, GASP-1 and FOXO-3A after strength training and power training. However, changes were observed in the content of mRNA for FLST, FL3 and SMAD-7. These changes were similar among the different training protocols. The results of this study suggest that both models of resistance training are able to induce a similar response on the expression of genes involved in signaling MSTN. The absence of specific responses within the investigated period points to the need to investigate the time course of adaptations to different models of resistance training in the long term

Estudos têm mostrado que o treinamento de força de baixa intensidade com oclusão vascular (TFOV) tem apresentado resultados similares nos ganhos de força e hipertrofia comparado ao treinamento de força (TF) de alta intensidade. O objetivo deste estudo foi comparar os efeitos de três diferentes programas de TF nos ganhos de força e hipertrofia musculares e na expressão da miostatina (MSTN) e seus antagonistas. Para isso, vinte e nove jovens do sexo masculino, sem experiência em TF, foram recrutados e divididos randomicamente nos grupos: treinamento de força de baixa intensidade sem oclusão (BI), treinamento de força de baixa intensidade com oclusão (BIO) e treinamento de força de alta intensidade sem oclusão (AI). Os grupos BIO e BI treinaram com intensidade de 20% 1RM, enquanto o grupo AI treinou com intensidade de 80% 1RM. A ANOVA one way foi utilizada para testar as diferenças percentuais nos ganhos de força (1RM) e na área de secção transversa (AST) do músculo quadríceps femoral. O modelo misto para análise das medidas repetidas foi utilizado para testar as diferenças nas variáveis miostatina (MSTN), folistatina-3 (FLST-3), SMAD-7 e GASP-1 nos grupos BI, BIO e AI nas condições pré e pós-treinamento. Os resultados mostraram que os aumentos de força e hipertrofia musculares nos grupos BIO e AI foram similares, entretanto superiores ao grupo BI. Esses resultados podem ser atribuídos a maior diminuição na expressão da MSTN nos grupos BIO (45%) e AI (41%) comparados com o grupo BI (16%) e o aumento na expressão dos genes que antagonizam sua atividade (SMAD-7, FLST-3 e GASP-1). Podemos concluir que a inibição na atividade da MSTN dos grupos BIO e AI podem responder em parte a similaridade nos ganhos de força e hipertrofia entre os grupos e a diferença para o grupo BI / It has been demonstrated that low intensity training associated to vascular occlusion (LIO) promotes similar gains in strength and muscle mass when compared to high intensity strength training (HI). The aim of the present study was to evaluate the effect of three different training programs on skeletal muscle hypertrophy and atrophy related gene expression. Twenty nine young male, with no previous experience in strength training were randomly allocated in three groups: low intensity strength training (i.e. 20% - 1-RM) (LI); low intensity strength training associated to vascular occlusion (i.e. 20% - 1-RM) (LIO); high intensity strength training (HI) (i.e. 80% - 1-RM). One-way ANOVA was used to assess differences in % delta change values of 1-RM and cross sectional area (CSA) of the quadriceps femoris. Mixed model analysis was used to compare myostatin (MSTN), folistatyn-3 (FLST-3), SMAD-7 e GASP-1 changes between groups pre and post training. Results demonstrated similar increases in strength and muscle hypertrophy for LIO and HI groups. Moreover, such increases were significantly greater when compared to LI. These results may be, at least in part, explained by a significant decrease in MSTN mRNA expression in LIO (45%) and HI (41%) when compared to LI (16%); additionally, SMAD-7; FLST-3 and GASP-1 mRNA expression were significantly increased. In conclusion, LIO training promotes similar gains than HI training. The results may be explained by changes in MSTN and related genes mRNA expression