No description available.

Skeletal muscle is a complicated and heterogeneous striated muscle tissue that serves critical mechanical and metabolic functions in the organism. The process of generating skeletal muscle, myogenesis, is elaborately coordinated by members of the protein kinase family, which transmit diverse signals initiated by extracellular stimuli to myogenic transcriptional hierarchy in muscle cells. Mitogen-activated protein kinases (MAPKs) including p38 MAPK, c-Jun N terminal kinase (JNK) and extracellular signal-regulated protein kinase (ERK) are components of serine/threonine protein kinase cascades that play important roles in skeletal muscle differentiation. The exploration of MAPK upstream kinases identified mitogen activated protein kinase kinase kinase kinase 4 (MAP4K4), a serine/threonine protein kinase that modulates p38 MAPK, JNK and ERK activities in multiple cell lines. Our lab further discovered that Map4k4 regulates peroxisome proliferator-activated receptor γ (PPARγ) translation in cultured adipocytes through inactivating mammalian target of rapamycin (mTOR), which controls skeletal muscle differentiation and hypotrophy in kinase-dependent and -independent manners. These findings suggest potential involvement of Map4k4 in skeletal myogenesis. Therefore, for the first part of my thesis, I characterize the role of Map4k4 in skeletal muscle differentiation in cultured muscle cells. Here I show that Map4k4 functions as a myogenic suppressor mainly at the early stage of skeletal myogenesis with a moderate effect on myoblast fusion during late-stage muscle differentiation. In agreement, Map4k4 expression and protein kinase activity are declined with myogenic differentiation. The inhibitory effect of Map4k4 on skeletal myogenesis requires its kinase activity. Surprisingly, none of the identified Map4k4 downstream effectors including p38 MAPK, JNK and ERK is involved in the Map4k4-mediated myogenic differentiation. Instead, expression of myogenic regulatory factor Myf5, a positive mediator of skeletal muscle differentiation is transiently regulated by Map4k4 to partially control skeletal myogenesis. Mechanisms by which Map4k4 modulates Myf5 amount have yet to be determined. In the second part of my thesis, I assess the relationship between Map4k4 and IGF-mediated signaling pathways. Although siRNA-mediated silencing of Map4k4 results in markedly enhanced myotube formation that is identical to the IGF-induced muscle hypertrophic phenotype, and Map4k4 regulates IGF/Akt signaling downstream effector mTOR in cultured adipocytes, Map4k4 appears not to be involved in the IGF-mediated ERK1/2 signaling axis and the IGF-mediated Akt signaling axis in C2C12 myoblasts. Furthermore, Map4k4 does not affect endogenous Akt signaling or mTOR activity during C2C12 myogenic differentiation. The results presented here not only identify Map4k4 as a novel suppressor of skeletal muscle differentiation, but also add to our knowledge of Map4k4 action on multiple signaling pathways in muscle cells during skeletal myogenesis. The effects that Map4k4 exerts on myoblast differentiation, fusion and Myf5 expression implicate Map4k4 as a potential drug target for muscle mass growth, skeletal muscle regeneration and muscular dystrophy.

Fundação de Amparo a Pesquisa do Estado de Minas Gerais / Skeletal muscle consists of muscle fiber types that have different physiological and biochemical characteristics. Basically, the muscle fiber can be classified into type I and type II, presenting, among other features, contraction speed and sensitivity to fatigue different for each type of muscle fiber. These fibers coexist in the skeletal muscles and their relative proportions are modulated according to the muscle functionality and the stimulus that is submitted. To identify the different proportions of fiber types in the muscle composition, many studies use biopsy as standard procedure. As the surface electromyography (EMGs) allows to extract information about the recruitment of different motor units, this study is based on the assumption that it is possible to use the EMG to identify different proportions of fiber types in a muscle. The goal of this study was to identify the characteristics of the EMG signals which are able to distinguish, more precisely, different proportions of fiber types. Also was investigated the combination of characteristics using appropriate mathematical models. To achieve the proposed objective, simulated signals were developed with different proportions of motor units recruited and with different signal-to-noise ratios. Thirteen characteristics in function of time and the frequency were extracted from emulated signals. The results for each extracted feature of the signals were submitted to the clustering algorithm k-means to separate the different proportions of motor units recruited on the emulated signals. Mathematical techniques (confusion matrix and analysis of capability) were implemented to select the characteristics able to identify different proportions of muscle fiber types. As a result, the average frequency and median frequency were selected as able to distinguish, with more precision, the proportions of different muscle fiber types. Posteriorly, the features considered most able were analyzed in an associated way through principal component analysis. Were found two principal components of the signals emulated without noise (CP1 and CP2) and two principal components of the noisy signals (CP1 and CP2 ). The first principal components (CP1 and CP1 ) were identified as being able to distinguish different proportions of muscle fiber types. The selected characteristics (median frequency, mean frequency, CP1 and CP1 ) were used to analyze real EMGs signals, comparing sedentary people with physically active people who practice strength training (weight training). The results obtained with the different groups of volunteers show that the physically active people obtained higher values of mean frequency, median frequency and principal components compared with the sedentary people. Moreover, these values decreased with increasing power level for both groups, however, the decline was more accented for the group of physically active people. Based on these results, it is assumed that the volunteers of the physically active group have higher proportions of type II fibers than sedentary people. Finally, based on these results, we can conclude that the selected characteristics were able to distinguish different proportions of muscle fiber types, both for the emulated signals as to the real signals. These characteristics can be used in several studies, for example, to evaluate the progress of people with myopathy and neuromyopathy due to the physiotherapy, and also to analyze the development of athletes to improve their muscle capacity according to their sport. In both cases, the extraction of these characteristics from the surface electromyography signals provides a feedback to the physiotherapist and the coach physical, who can analyze the increase in the proportion of a given type of fiber, as desired in each case. / A musculatura esquelética é constituída por tipos de fibras musculares que possuem características fisiológicas e bioquímicas distintas. Basicamente, elas podem ser classificadas em fibras do tipo I e fibras do tipo II, apresentando, dentre outras características, velocidade de contração e sensibilidade à fadiga diferentes para cada tipo de fibra muscular. Estas fibras coexistem na musculatura esquelética e suas proporções relativas são moduladas de acordo com a funcionalidade do músculo e com o estímulo a que é submetido. Para identificar as diferentes proporções de tipos de fibra na composição muscular, muitos estudos utilizam a biópsia como procedimento padrão. Como a eletromiografia de superfície (EMGs) nos permite extrair informações sobre o recrutamento de diferentes unidades motoras, este estudo parte da hipótese de que seja possível utilizar a EMGs para identificar diferentes proporções de tipos de fibras em uma musculatura. O objetivo deste estudo foi identificar as características dos sinais EMGs que sejam capazes de distinguir, com maior precisão, diferentes proporções de tipos de fibras. Também foi investigado a combinação de características por meio de modelos matemáticos apropriados. Para alcançar o objetivo proposto, sinais emulados foram desenvolvidos com diferentes proporções de unidades motoras recrutadas e diferentes razões sinal-ruído. Treze características no domínio do tempo e da frequência foram extraídas do sinais emulados. Os resultados de cada característica extraída dos sinais emulados foram submetidos ao algorítimo de agrupamento k-means para separar as diferentes proporções de unidades motoras recrutadas nos sinais emulados. Técnicas matemáticas (matriz confusão e técnica de capabilidade) foram implementadas para selecionar as características capazes de identificar diferentes proporções de tipos de fibras musculares. Como resultado, a frequência média e a frequência mediana foram selecionadas como capazes de distinguir com maior precisão as diferentes proporções de tipos de fibras musculares. Posteriormente, as características consideradas mais capazes foram analisadas de forma associada por meio da análise de componentes principais. Foram encontradas duas componentes principais para os sinais emulados sem ruído (CP1 e CP2) e duas componentes principais para os sinais com ruído (CP1 e CP2 ), sendo as primeiras componentes principais (CP1 e CP1 ) identificadas como capazes de distinguirem diferentes proporções de fibras. As características selecionadas (frequência mediana, frequência média, CP1 e CP1 ) foram utilizadas para analisar sinais EMGs reais, comparando pessoas sedentárias com pessoas fisicamente ativas praticantes de treinamentos físicos de força (musculação). Os resultados obtidos com os diferentes grupos de voluntários mostram que as pessoas fisicamente ativas obtiveram valores mais elevados de frequência média, frequência mediana e componentes principais em comparação com as pessoas sedentárias. Além disto, estes valores decaíram com o aumento do nível de força para ambos os grupo, entretanto, o decaimento foi mais acentuado para o grupo de pessoas fisicamente ativas. Com base nestes resultados, presume-se que os voluntários do grupo fisicamente ativo apresentam maiores proporções de fibras do tipo II, se comparado com as pessoas sedentárias. Por fim, com base nos resultados obtidos, pode-se concluir que as características selecionadas foram capazes de distinguir diferentes proporções de tipos de fibras musculares, tanto para os sinais emulados quanto para os sinais reais. Estas características podem ser utilizadas em vários estudos, como por exemplo, para avaliar a evolução de pessoas com miopatias e neuromiopatia em decorrência da reabilitação fisioterápica, e também para analisar o desenvolvimento de atletas que visam melhorar sua capacidade muscular de acordo com sua modalidade esportiva. Em ambos os casos, a extração destas características dos sinais de eletromiografia de superfície proporciona um feedback ao fisioterapeuta e ao treinador físico, que podem analisar o aumento na proporção de determinado tipo de fibra, conforme desejado em cada caso. / Mestre em Ciências

La Myotubularine (MTM1) est une 3-phosphatase à phosphoinositides (PI) mutée dans la myopathie centronucléaire liée au chromosome X (XLCNM), caractérisée par une faiblesse musculaire et un positionnement anormal des noyaux dans les fibres musculaires. MTM1 définit une grande famille de phosphatases, exprimées dans tous les tissus, et qui englobent des phosphatases catalytiquement actives et inactives. Les myotubularines actives dephosphorylent le phosphatidylinositol 3 monophosphate [PtdIns3P] et le 3,5-bisphosphate [PtdIns(3,5)P2] en PtdIns et PtdIns5P, respectivement. Le rôle de MTM1 et son activité phosphatase à lipide dans le muscle restaient peu connus. L’étude approfondie de la protéine a révélé une association de MTM1 au réticulum sarcoplasmique des triades, un sous-compartiment impliqué dans la régulation calcique. La caractérisation de la souris Mtm1 KO, qui reproduit la XLCNM, a témoigné d’une anomalie de l’organisation et de la forme du réticulum sarcoplasmique. Afin d’explorer l’implicationde l’activité phosphatase de MTM1 dans l’organisation de réticulum sarcoplasmique, j’ai utilisé une approche in vivo avec des virus adéno-associé (AAV) pour moduler l’activité phosphatase en sur-exprimant MTM1 et sa forme phosphatase-inactive (MTM1-C375S) dans un muscle sauvage. L’observation des muscles transduits a dévoilé une implication de MTM1 dans le remodelage du réticulum sarcoplasmique et un rôle potentiel de PtdIns3P avec MTM1 dans la courbure des membranes du réticulum sarcoplasmique. Afin de comprendre l’importance de l’activité phosphatase dans le maintien du phénotype XLCNM, les muscles de souris Mtm1 KO ont été injectés avec ces AAVs contenant la forme active et inactive de MTM1 au moment de l’apparition des premiers signes de XLCNM. Étonnamment, la forme phosphatase-inactive(MTM1-C375S) a sauvé le phénotype de la souris Mtm1 KO de la même façon que la forme active, suggérant que l'activité de phosphatase de MTM1 n’est pas nécessaire pour le maintien de la structure intracellulaire des fibres du muscle adulte. Ces données suggèrent que MTM1 exerce une fonction phosphatase-indépendante dans le maintien de la structure musculaire, certainement via des interactions protéine-protéine, et une fonction phosphatase-dépendente dans le remodelage de la forme du réticulum sarcoplasmique dans le muscle squelettique. / Myotubularin (MTM1) is a phosphoinositide (PI) 3-phosphatase mutated in X-linked centronuclear myopathy (XLCNM), a rare congenital myopathy characterized by muscle weakness and abnormal positioning of nuclei in muscle fibers. MTM1 defines a large family of ubiquitously expressed catalytically active and inactive phosphatases. Active myotubularins dephosphorylate both phosphatidylinositol 3-phosphate [PtdIns3P] and 3,5-bisphosphate [PtdIns(3,5)P2] to PtdIns andPtdIns5P, respectively. The specific role of MTM1 and its PI phosphatase activity in muscle remains unknown. Comprehensive analysis of the protein unveiled the association of MTM1 with the sarcoplasmic reticulum (SR) at the triads. Characterization of Mtm1-KO mouse, which reproduce the XLCNM phenotype, revealed a defect of SR organization and shape. In order to gain insight into the involvement of MTM1 phosphatase activity on SR shape and organization, we employed an in vivo approach using Adeno-Associated Virus (AAV) to modulate the phosphatase activity by overexpressingMTM1 and its phosphatase inactive mutant in wild type muscle. The analysis of transduced muscle revealed the involvement of MTM1 in the SR remodeling and its potential role together with PtdIns3P in modulating membrane curvature. In order to understand the importance of the phosphatase activity in the generation of the XLCNM phenotype, Mtm1 KO mice were injected with AAV expressing the active form and the phosphatase inactive form. Surprisingly, both, the phosphatase active and the phosphatase inactive mutant corrected the Mtm1-KO mouse phenotype to a similar extent, thus suggesting that the PI-phosphatase activity of MTM1 is not essential for adult skeletal muscle maintenance. Our data indicates that MTM1 has a phosphatase-independent function in adult muscle structure maintenance and a phosphatase-dependent function in sarcoplasmic reticulum remodeling and shape in skeletal muscle.

A doença de Pompe (GSDII), autossômica recessiva, é causada pela deficiência da enzima lisossomal que degrada o glicogênio, -glucosidase ácida (GAA). O quadro clínico varia de acordo com a idade de início da doença, grau de progressão e envolvimento dos tecidos: predominantemente cardíaco e muscular esquelético na forma de início-precoce (FIP) e mais restrito no músculo esquelético na forma de início-tardio (FIT). A sobrevida média na FIP é de 9-12 meses. Com avanço dos métodos histológicos, histoquímicos e imunoistoquímicos intensificou-se a análise estrutural e funcional dos tipos de fibras musculares. O estudo da vascularização também é de importância pelo aporte nutricional e funcional das fibras. O objetivo do presente trabalho é analisar a correlação da distribuição do tipo de fibras com a forma de apresentação clínica da doença de Pompe, seu genótipo correspondente e a quantidade residual da enzima GAA. Analisou-se 10 biópsias musculares de pacientes FIP e 09 de FIT comparados com o grupo controle, pareados por idade e gênero. Os pacientes foram selecionados segundo dados clínicos e laboratoriais, sendo feito o seqüenciamento de toda parte codificante do gene e Western Blotting (WB) com anticorpo monoclonal 15362-157, cedido pela Genzyme (primário 1:200 e secundário 1:10.000). A confirmação do diagnóstico foi feita através da medida da atividade residual de GAA em papel filtro, da presença de miopatia vacuolar com grânulos PAS e fosfatase ácida positivos em biópsia muscular e pela presença de mutação no gene GAA. A reação de imunoistoquímica foi realizada para fibras tipo I (lenta), tipo II (rápida) e densidade capilar (ulex), utilizando anticorpos monoclonais, respectivamente: antimiosina lenta (1:80), anti-miosina rápida (1:40) da Novocastra e ulex da Vector (1:800). A contagem das fibras foi realizada por 2 observadores em todo fragmento do corte transversal da biópsia com auxílio de um programa semi-automatizado. Observou-se predomínio de fibras tipo II em ambos os gêneros na FIP e predomínio de fibras tipo I em mulheres e tipo II em homens, na FIT. Aumento da densidade capilar, em comparação com os controles, foi notada em ambas as formas IP e IT. Verificou-se em média 90% de fibras vacuoladas nos casos FIP com completa distorção da arquitetura, enquanto na FIT, a porcentagem de fibras vacuoladas foi variável (0-88%). Como alguns genes constitutivos influenciam na distribuição das fibras musculares, como o gene ACE, o polimorfismo deste gene foi analisado quanto aos genótipos I/I, D/D e I/D. Observou-se ausência de concordância entre o genótipo do ACE e a distribuição de fibras em 60% dos casos da FIP e FIT, atribuindo-se o resultado da distribuição do tipo de fibras como parte da patologia da doença de Pompe. A gravidade da doença variou inversamente com a quantidade de enzima residual, sendo compatível com o quadro clínico do paciente. A presença de mutação deletéria em ambos os alelos foi observada em 3/10 casos de IP, sendo que todos os 3 casos apresentaram ausência total de enzima no WB. Há maior envolvimento de fibras tipo II em GSDII, sem depleção da microcirculação muscular. Estudos demonstram que a remoção do depósito de glicogênio ocorre diferencialmente nos tipos de fibra, sendo menos eficiente nas fibras tipo II. O achado do presente estudo poderá ter implicações na resposta à recente terapêutica proposta por reposição enzimática. / The glycogen storage disease type II (GSDII), autosomal recessive disorder, is caused by the deficiency of GAA (acid -glucosidase) a lysossomal enzyme that degrades the glycogen. The clinical findings are in accordance to great variability of age onset, degree of disease progression and extent of tissue involvement: predominantly cardiac and skeletal muscle in the infantile form (I) and more restricted to the skeletal muscle in the late-onset form (LO). The average survival time of the infantile form is 9-12 months. With advances of the histological, histochemical and imunohistochemical methods structural and functional analysis of muscle fiber types were intensified. The study of the capillary density is also important for nutritional and functional aspects. The objective of the present work is to analyze the correlations of the fiber type distribution to clinical presentation, genotype and residual GAA enzymatic activity. We analyzed 10 muscle biopsies of infantile and 09 of late-onset patients and compared to age and gender matched controls. The patients were selected according to clinical and laboratorial data, molecular diagnosis by full gene sequencing, and Western Blotting (WB) with monoclonal antibody 15362-157, courtesy Genzyme Science Group (primary 1:200 and secondary 1:10.000). Diagnostic confirmation was made by GAA enzymatic measurement in DBS, presence of vacuolar myopathy in muscle biopsy, and presence of mutation in GAA gene. The imunohistochemical study was carried out by detection of type I (slow), type II (fast) fibers and capillaries, using monoclonal antibodies, respectively: anti-slow myosin (1:80), anti-fast myosin (1:40) (Novocastra) and ulex (1:800) (Vector). Morphometry was performed by 2 observers using a half-automatized program. Type II fiber predominance was observed in both gender in the infantile form, type I fiber predominance in women and type II predominance in men with LO. Increase of the capillary density, in comparison to controls was noticed in both forms. 90% of vacuolated fibers with complete distortion of fiber architecture were demonstrated in I cases, while in LO, the percentage of vacuolated fibers ranged from 0 to 88%. As some constitutive gene, like ACE, influence muscle fiber distribution, its polymorphisms I/I, D/D and I/D gene were analyzed. Absence of agreement was observed between ACE genotype and fiber type distribution in 60% of I and LO cases, which was attributed as consequence of Pompe disease pathology itself. The disease severity varied inversely to the amount of residual GAA enzymatic activity, being compatible with the patient clinical findings. The presence of deleterious mutation in both alleles was observed in 3/10 infantile cases, and all 3 presented total enzyme absence at WB. A greater fiber type II involvement was observed in GSDII, without decrease in muscle capillary density. Recent studies demonstrated that glycogen deposit removal occurs distinctively in different fiber types, being less efficient in type II fibers. The present findings might have implications in the reply to the recent proposed enzyme replacement therapy.

A doença de Pompe (GSDII), autossômica recessiva, é causada pela deficiência da enzima lisossomal que degrada o glicogênio, -glucosidase ácida (GAA). O quadro clínico varia de acordo com a idade de início da doença, grau de progressão e envolvimento dos tecidos: predominantemente cardíaco e muscular esquelético na forma de início-precoce (FIP) e mais restrito no músculo esquelético na forma de início-tardio (FIT). A sobrevida média na FIP é de 9-12 meses. Com avanço dos métodos histológicos, histoquímicos e imunoistoquímicos intensificou-se a análise estrutural e funcional dos tipos de fibras musculares. O estudo da vascularização também é de importância pelo aporte nutricional e funcional das fibras. O objetivo do presente trabalho é analisar a correlação da distribuição do tipo de fibras com a forma de apresentação clínica da doença de Pompe, seu genótipo correspondente e a quantidade residual da enzima GAA. Analisou-se 10 biópsias musculares de pacientes FIP e 09 de FIT comparados com o grupo controle, pareados por idade e gênero. Os pacientes foram selecionados segundo dados clínicos e laboratoriais, sendo feito o seqüenciamento de toda parte codificante do gene e Western Blotting (WB) com anticorpo monoclonal 15362-157, cedido pela Genzyme (primário 1:200 e secundário 1:10.000). A confirmação do diagnóstico foi feita através da medida da atividade residual de GAA em papel filtro, da presença de miopatia vacuolar com grânulos PAS e fosfatase ácida positivos em biópsia muscular e pela presença de mutação no gene GAA. A reação de imunoistoquímica foi realizada para fibras tipo I (lenta), tipo II (rápida) e densidade capilar (ulex), utilizando anticorpos monoclonais, respectivamente: antimiosina lenta (1:80), anti-miosina rápida (1:40) da Novocastra e ulex da Vector (1:800). A contagem das fibras foi realizada por 2 observadores em todo fragmento do corte transversal da biópsia com auxílio de um programa semi-automatizado. Observou-se predomínio de fibras tipo II em ambos os gêneros na FIP e predomínio de fibras tipo I em mulheres e tipo II em homens, na FIT. Aumento da densidade capilar, em comparação com os controles, foi notada em ambas as formas IP e IT. Verificou-se em média 90% de fibras vacuoladas nos casos FIP com completa distorção da arquitetura, enquanto na FIT, a porcentagem de fibras vacuoladas foi variável (0-88%). Como alguns genes constitutivos influenciam na distribuição das fibras musculares, como o gene ACE, o polimorfismo deste gene foi analisado quanto aos genótipos I/I, D/D e I/D. Observou-se ausência de concordância entre o genótipo do ACE e a distribuição de fibras em 60% dos casos da FIP e FIT, atribuindo-se o resultado da distribuição do tipo de fibras como parte da patologia da doença de Pompe. A gravidade da doença variou inversamente com a quantidade de enzima residual, sendo compatível com o quadro clínico do paciente. A presença de mutação deletéria em ambos os alelos foi observada em 3/10 casos de IP, sendo que todos os 3 casos apresentaram ausência total de enzima no WB. Há maior envolvimento de fibras tipo II em GSDII, sem depleção da microcirculação muscular. Estudos demonstram que a remoção do depósito de glicogênio ocorre diferencialmente nos tipos de fibra, sendo menos eficiente nas fibras tipo II. O achado do presente estudo poderá ter implicações na resposta à recente terapêutica proposta por reposição enzimática. / The glycogen storage disease type II (GSDII), autosomal recessive disorder, is caused by the deficiency of GAA (acid -glucosidase) a lysossomal enzyme that degrades the glycogen. The clinical findings are in accordance to great variability of age onset, degree of disease progression and extent of tissue involvement: predominantly cardiac and skeletal muscle in the infantile form (I) and more restricted to the skeletal muscle in the late-onset form (LO). The average survival time of the infantile form is 9-12 months. With advances of the histological, histochemical and imunohistochemical methods structural and functional analysis of muscle fiber types were intensified. The study of the capillary density is also important for nutritional and functional aspects. The objective of the present work is to analyze the correlations of the fiber type distribution to clinical presentation, genotype and residual GAA enzymatic activity. We analyzed 10 muscle biopsies of infantile and 09 of late-onset patients and compared to age and gender matched controls. The patients were selected according to clinical and laboratorial data, molecular diagnosis by full gene sequencing, and Western Blotting (WB) with monoclonal antibody 15362-157, courtesy Genzyme Science Group (primary 1:200 and secondary 1:10.000). Diagnostic confirmation was made by GAA enzymatic measurement in DBS, presence of vacuolar myopathy in muscle biopsy, and presence of mutation in GAA gene. The imunohistochemical study was carried out by detection of type I (slow), type II (fast) fibers and capillaries, using monoclonal antibodies, respectively: anti-slow myosin (1:80), anti-fast myosin (1:40) (Novocastra) and ulex (1:800) (Vector). Morphometry was performed by 2 observers using a half-automatized program. Type II fiber predominance was observed in both gender in the infantile form, type I fiber predominance in women and type II predominance in men with LO. Increase of the capillary density, in comparison to controls was noticed in both forms. 90% of vacuolated fibers with complete distortion of fiber architecture were demonstrated in I cases, while in LO, the percentage of vacuolated fibers ranged from 0 to 88%. As some constitutive gene, like ACE, influence muscle fiber distribution, its polymorphisms I/I, D/D and I/D gene were analyzed. Absence of agreement was observed between ACE genotype and fiber type distribution in 60% of I and LO cases, which was attributed as consequence of Pompe disease pathology itself. The disease severity varied inversely to the amount of residual GAA enzymatic activity, being compatible with the patient clinical findings. The presence of deleterious mutation in both alleles was observed in 3/10 infantile cases, and all 3 presented total enzyme absence at WB. A greater fiber type II involvement was observed in GSDII, without decrease in muscle capillary density. Recent studies demonstrated that glycogen deposit removal occurs distinctively in different fiber types, being less efficient in type II fibers. The present findings might have implications in the reply to the recent proposed enzyme replacement therapy.