Skeletal muscle development and function is governed by a conserved set of Transcription Factors (TFs) that regulate gene expression. The TF gene regulation is stimulus driven and cell-type and time point specific. TBX5 is an essential dosage sensitive regulator of heart and limb development. In the skeletal system, TBX5 is expressed in early stages in the lateral plate mesoderm and gives rise to the forelimb. TBX5 is also involved in proliferation and differentiation and survival pathways in both heart and limb development. Mutations in TBX5 gene lead to HOS which is characterized by various types of cardiac and musculoskeletal defects. TBX5 mechanism of action and its spatiotemporal function in skeletal muscle development has yet to be fully understood. TBX5 regulation is controlled through various factors such as alternative splicing, protein-protein interactions, Post-Translational Modifications (PTMs) and microRNAs. To date, many TBX5 interactors have been identified in cardiac cells however TBX5 protein interactors and target genes in skeletal muscle cells have not been studied. Understanding the protein interactome of TBX5 in skeletal muscle will enhance the current understanding of its mechanism of action. In this study we have characterized TBX5 with focus on its regulation, expression and biochemical properties in cardiac and skeletal muscle cells and moreover its mechanism of action specifically in skeletal muscle proliferation and differentiation. Chapter 1 discusses TBX5 regulation through alternative splicing leading to the existence of 5 distinct TBX5 isoforms with variable transcriptional activity, cardiac and limb expression pattern, biochemical properties and function. We show the pro-proliferation role of TBX5a in myoblasts while TBX5c shows to be pro-differentiation leading to the formation of myotubes in skeletal muscle C2C12 myoblasts. This opposing role of the two TBX5 isoforms lead us to studying TBX5 mechanism of action in proliferation and differentiation of skeletal muscle cells. In this study using a mass spectrometry-based approach we have identified novel TBX5 interacting partners in skeletal muscle cells for the first time by using stably overexpressed 3xFlag TBX5 via retroviral transduction in C2C12 cell line. Nuclear protein extracts were immunoprecipitated and sent for HPLC-ESI-MS/MS to identify potential protein partners of TBX5 in skeletal muscle cells. Moreover, the same stable cell line was used to identify TBX5 downstream target genes in these cell types by sending RNA extracts for microarray analysis. Amongst the 200 protein interactors identified, MYBBP1a and TBX5 interaction was confirmed and studied. The microarray analysis identified over 1200 differentially expressed genes and potential downstream targets of TBX5a from which Myostatin (Mstn) and Cyclin D2 (CcnD2) were both significantly upregulated and further confirmed and studied in relation to proliferation and differentiation in skeletal muscle cells. Chapter 2 focuses on the cooperative interaction between TBX5a and MYBBP1a inhibiting muscle specific gene promoter, Myogenin (MyoG). TBX5a and TBX5c seem to both interact with MYBBP1a but result in variable transcriptional activity of both MyoG and Mstn gene promoters. We show that TBX5 is upstream of Mstn, it binds to the promoter on specific TBE sites, and is able to upregulate Mstn promoter activation. In vivo, we show that MDX mice limb skeletal muscle tissues show elevated levels of TBX5, MYBBP1a and MSTN expression which suggest that the TBX5 pathway is associated with and indicative of the onset of proliferation and regeneration in MDX skeletal muscle tissue. Chapter 3 discusses the role of TBX5 in proliferation and regeneration of skeletal muscle cells by identifying that TBX5 binds to CcnD2 promoter and upregulates its activation which is a known cell cycle gene critical in cell proliferation and survival. Moreover, we identify GATA4 as a TBX5a cofactor in myoblast proliferation and show synergistic activation of Ccnd2 promoter by cooperative TBX5a and GATA4 action. We further show that Tbx5 heterozygote mice exhibit decreased levels of CCND2 and other proliferation markers, as well as decreased expression of PAX7 (marker of satellite cells) compared to WT skeletal muscle tissues. We also show that the heterozygous loss of Tbx5 impairs the process of regeneration in a cardiotoxin-induced injury model in mouse limb tissues. Tbx5 heterozygote mice exhibit less proliferation and impaired regeneration 4 days after injury, followed by decreased formation of regenerated fibers by 7 days post-injury compared to the wildtype mice skeletal muscle tissues; suggesting that TBX5 function is important in maintaining adult muscle regenerative capacity. Together, this study has characterized TBX5 isoforms and identified novel TBX5 protein partners and targets in the skeletal muscle cells and sheds light on TBX5 regulatory mechanism in proliferation and differentiation of skeletal muscle cells and its potential implications in HOS and other muscular diseases.

A Distrofia Muscular de Duchenne (DMD) é provocada por mutações no gene distrofina. Este gene codifica a proteína distrofina, que exerce papel importante na manutenção da estabilidade da membrana da fibra muscular. Os objetivos do estudo consistiram em examinar o desempenho neuropsicológico de pacientes com DMD e verificar a influência de deleções downstream ao exon 45 sobre o mesmo. Foram avaliados os perfis de inteligência de 63 pacientes com DMD por meio das Escalas Wechsler de Inteligência ou o Teste de Raven. A faixa etária do grupo variou de 6 a 26 anos de idade e a escolaridade, 1 a 16 anos de estudo formal. Os pacientes com escores de inteligência normais (n=34) foram comparados com controles quanto às funções neuropsicológicas. O grupo controle foi composto por 34 jovens do sexo masculino pareados por idade. Os testes incluíram medidas de memória verbal (Teste de Aprendizado Verbal de Rey), habilidade viso-espacial (Teste de Organização Visual de Hooer), funções executivas (fluência verbal e Teste de Wisconsin de Classificação de Cartas). A avaliação também incluiu testes da bateria Cambridge Neuropsychological Test Automated Battery (CANTAB) para o exame de: tempo de reação simples (Simple Reaction Time); tempo de reação com dupla escolha (Choice Reaction Time), atenção visual sustentada (Rapid Visual Processing), amplitude atencional/memória operacional (Spatial Span, ordem direta e inversa), memória visual de curto e longo prazo (Pattern Recognition Memory), reconhecimento de estímulos complexos apresentados simultaneamente ou após intervalo (Delayed Matching to Sample), memória espacial (Spatial Recognition Memory) e tomada de decisão (Information Sampling Task). O Inventário de Depressão de Beck (BDI) foi administrado para exame de sintomas de depressão e o Inventário de Comportamentos da Infância e Adolescência (CBCL) foi utilizado como medida de alterações de comportamento. Utilizou-se a análise de variância (one-way ANOVA) na comparação entre grupos. As covariáveis foram controladas por meios estatísticos. Os pacientes com DMD obtiveram medias de QIs com 1 desvio padrão abaixo da media da população. Após controle para covariáveis, os pacientes com DMD mostraram desempenhos significantemente inferiores nos testes de Aritmética, Vocabulário, Compreensão, Dígitos e no Teste de Wisconsin (número de erros totais, erros perseverativos, respostas de nível conceitual e categorias completas). Também mostraram tempos de reação mais longos (Simple Reaction Time), menor amplitude atencional tanto na ordem direta, quanto inversa (Spatial Span) e menor número de acertos no teste de tomada de decisão (Information Sampling Task) (p<0,05). A proporção de pacientes com deleções no gene distrofina foi de 54% (34/63). Não foi encontrada associação entre os resultados de inteligência e dados genéticos. Comparados com controles, os 14 pacientes com deleção downstream ao exon 45 e QI&#8805;80 mostraram dificuldades mais específicas. O tempo de reação foi discutido conforme a complexidade da tarefa. Os participantes do grupo controle apresentaram escores mais altos no BDI e CBCL, indicando maior ocorrência de sintomas. Possíveis fatores relacionados foram discutidos. Há evidências de que as deleções downstream ao exon 45 (relacionadas à isoforma cerebral da distrofina Dp140) estão envolvidas com a menor eficiência de tomada de decisão dentre os pacientes com DMD. Os achados também sugerem a associação entre a DMD e disfunções frontais. / Duchenne Muscular Dystrophy (DMD) is caused by mutations in the dystrophin gene. This gene codes for dystrophin, a protein important for maintaining the stability of muscle-fiber membranes. The objectives of the study were to examine neuropsychological performance in patients with DMD and the influence of deletions in the dystrophin gene (the Dp140 regulatory region) on neuropsychological function. General intelligence was investigated in 63 DMD patients using the Wechsler Intelligence Scale or the Raven\'s Matrices Test. The age range for DMD participants was from 6 to 26 years, with a range of 1 to 16 years of formal education. The participants who had intellectual scores in the normal range (n=34) were compared to controls in terms of neuropsychological function. The control group was composed of 34 male age-matched subjects. Patients were divided into groups according to the region of mutation in the dystrophin gene and those with delection downstream of exon 45 were also compared to controls. The battery included the Wechsler scale subtests and measures of verbal memory (Rey Auditory Verbal Learning Test), viso-perceptual skills (Hooper Visual Organization Test), executive function (FAS, animals and Wisconsin Card Sorting Test). The assessment also included tests of the Cambridge Neuropsychological Test Automated Battery (CANTAB) to examine reaction time (Simple Reaction Time and Choice Reaction Time), sustained attention (Rapid Visual Processing), working memory (Spatial Span, forward and reverse), short and long term visual memory (Pattern Recognition Memory), recognition memory for complex stimuli presented simultaneously or after short interval (Delayed Matching to Sample), spatial memory (Spatial Recognition Memory), and decision making (Information Sampling Task). The Beck Depression Inventory (BDI) was administered for depression symptoms assessment and the Child Behavior Checklist (CBCL) was used as a measure of disruptive behavior. Analysis of variance (one-way ANOVA) was used. Covariates were controlled statistically. The DMD patients had mean IQs about one standard deviation lower than population means. Verbal IQ was significantly lower than Performance IQ. After controlling for covariates, significant difference (p<0.05) appeared between DMD patients and controls and DMD was associated to lower IQs (Full Scale, Verbal, and Performance) and worse performances on Arithmetic, Vocabulary, Comprehension, Digit Span, Wisconsin Test (total errors, perseverative errors, conceptual level responses, and categories completed), Spatial Span (forward and reverse recall), and on the number of correct trials on Information Sampling Task. They also showed slower simple reaction times (Simple Reaction Time). The proportion of patients with dystrophin gene deletions was 54% (34/63). No relationship was established between intelligence results and genetic data. Compared to controls, 14 patients with delection downstream of exon 45 and normal IQs showed more specific deficits. Reaction time was discussed in terms of complexity of the task. Controls showed significant higher BDI and CBCL scores than DMD patients. Possible related factors were discussed. There is evidence to indicate that delections downstream of exon 45 (related to cerebral dystrophin isoform Dp140) are involved in decision making impairment in patients with DMD. The findings suggest that DMD may be related to frontal dysfunction.

A Distrofia Muscular de Duchenne (DMD) é provocada por mutações no gene distrofina. Este gene codifica a proteína distrofina, que exerce papel importante na manutenção da estabilidade da membrana da fibra muscular. Os objetivos do estudo consistiram em examinar o desempenho neuropsicológico de pacientes com DMD e verificar a influência de deleções downstream ao exon 45 sobre o mesmo. Foram avaliados os perfis de inteligência de 63 pacientes com DMD por meio das Escalas Wechsler de Inteligência ou o Teste de Raven. A faixa etária do grupo variou de 6 a 26 anos de idade e a escolaridade, 1 a 16 anos de estudo formal. Os pacientes com escores de inteligência normais (n=34) foram comparados com controles quanto às funções neuropsicológicas. O grupo controle foi composto por 34 jovens do sexo masculino pareados por idade. Os testes incluíram medidas de memória verbal (Teste de Aprendizado Verbal de Rey), habilidade viso-espacial (Teste de Organização Visual de Hooer), funções executivas (fluência verbal e Teste de Wisconsin de Classificação de Cartas). A avaliação também incluiu testes da bateria Cambridge Neuropsychological Test Automated Battery (CANTAB) para o exame de: tempo de reação simples (Simple Reaction Time); tempo de reação com dupla escolha (Choice Reaction Time), atenção visual sustentada (Rapid Visual Processing), amplitude atencional/memória operacional (Spatial Span, ordem direta e inversa), memória visual de curto e longo prazo (Pattern Recognition Memory), reconhecimento de estímulos complexos apresentados simultaneamente ou após intervalo (Delayed Matching to Sample), memória espacial (Spatial Recognition Memory) e tomada de decisão (Information Sampling Task). O Inventário de Depressão de Beck (BDI) foi administrado para exame de sintomas de depressão e o Inventário de Comportamentos da Infância e Adolescência (CBCL) foi utilizado como medida de alterações de comportamento. Utilizou-se a análise de variância (one-way ANOVA) na comparação entre grupos. As covariáveis foram controladas por meios estatísticos. Os pacientes com DMD obtiveram medias de QIs com 1 desvio padrão abaixo da media da população. Após controle para covariáveis, os pacientes com DMD mostraram desempenhos significantemente inferiores nos testes de Aritmética, Vocabulário, Compreensão, Dígitos e no Teste de Wisconsin (número de erros totais, erros perseverativos, respostas de nível conceitual e categorias completas). Também mostraram tempos de reação mais longos (Simple Reaction Time), menor amplitude atencional tanto na ordem direta, quanto inversa (Spatial Span) e menor número de acertos no teste de tomada de decisão (Information Sampling Task) (p<0,05). A proporção de pacientes com deleções no gene distrofina foi de 54% (34/63). Não foi encontrada associação entre os resultados de inteligência e dados genéticos. Comparados com controles, os 14 pacientes com deleção downstream ao exon 45 e QI&#8805;80 mostraram dificuldades mais específicas. O tempo de reação foi discutido conforme a complexidade da tarefa. Os participantes do grupo controle apresentaram escores mais altos no BDI e CBCL, indicando maior ocorrência de sintomas. Possíveis fatores relacionados foram discutidos. Há evidências de que as deleções downstream ao exon 45 (relacionadas à isoforma cerebral da distrofina Dp140) estão envolvidas com a menor eficiência de tomada de decisão dentre os pacientes com DMD. Os achados também sugerem a associação entre a DMD e disfunções frontais. / Duchenne Muscular Dystrophy (DMD) is caused by mutations in the dystrophin gene. This gene codes for dystrophin, a protein important for maintaining the stability of muscle-fiber membranes. The objectives of the study were to examine neuropsychological performance in patients with DMD and the influence of deletions in the dystrophin gene (the Dp140 regulatory region) on neuropsychological function. General intelligence was investigated in 63 DMD patients using the Wechsler Intelligence Scale or the Raven\'s Matrices Test. The age range for DMD participants was from 6 to 26 years, with a range of 1 to 16 years of formal education. The participants who had intellectual scores in the normal range (n=34) were compared to controls in terms of neuropsychological function. The control group was composed of 34 male age-matched subjects. Patients were divided into groups according to the region of mutation in the dystrophin gene and those with delection downstream of exon 45 were also compared to controls. The battery included the Wechsler scale subtests and measures of verbal memory (Rey Auditory Verbal Learning Test), viso-perceptual skills (Hooper Visual Organization Test), executive function (FAS, animals and Wisconsin Card Sorting Test). The assessment also included tests of the Cambridge Neuropsychological Test Automated Battery (CANTAB) to examine reaction time (Simple Reaction Time and Choice Reaction Time), sustained attention (Rapid Visual Processing), working memory (Spatial Span, forward and reverse), short and long term visual memory (Pattern Recognition Memory), recognition memory for complex stimuli presented simultaneously or after short interval (Delayed Matching to Sample), spatial memory (Spatial Recognition Memory), and decision making (Information Sampling Task). The Beck Depression Inventory (BDI) was administered for depression symptoms assessment and the Child Behavior Checklist (CBCL) was used as a measure of disruptive behavior. Analysis of variance (one-way ANOVA) was used. Covariates were controlled statistically. The DMD patients had mean IQs about one standard deviation lower than population means. Verbal IQ was significantly lower than Performance IQ. After controlling for covariates, significant difference (p<0.05) appeared between DMD patients and controls and DMD was associated to lower IQs (Full Scale, Verbal, and Performance) and worse performances on Arithmetic, Vocabulary, Comprehension, Digit Span, Wisconsin Test (total errors, perseverative errors, conceptual level responses, and categories completed), Spatial Span (forward and reverse recall), and on the number of correct trials on Information Sampling Task. They also showed slower simple reaction times (Simple Reaction Time). The proportion of patients with dystrophin gene deletions was 54% (34/63). No relationship was established between intelligence results and genetic data. Compared to controls, 14 patients with delection downstream of exon 45 and normal IQs showed more specific deficits. Reaction time was discussed in terms of complexity of the task. Controls showed significant higher BDI and CBCL scores than DMD patients. Possible related factors were discussed. There is evidence to indicate that delections downstream of exon 45 (related to cerebral dystrophin isoform Dp140) are involved in decision making impairment in patients with DMD. The findings suggest that DMD may be related to frontal dysfunction.