C.F. Almeida

Systemic Delivery of Human Mesenchymal Stromal Cells Combined with IGF-1 Enhances Muscle Functional Recovery in LAMA2 dy/2j Dystrophic Mice

The combination of cell therapy with growth factors could be a useful approach to treat progressive muscular dystrophies. Here, we demonstrate, for the first time, that IGF-1 considerably enhances the myogenesis of human umbilical cord (UC) mesenchymal stromal cells (MSCs) in vitro and that IGF-1 enhances interaction and restoration of dystrophin expression in co-cultures of MSCs and muscle cells from Duchenne patients. In vivo studies showed that human MSCs were able to reach the skeletal muscle of LAMA2dy/2j dystrophic mice, through systemic delivery, without immunosuppression. Moreover, we showed, for the first time, that IGF-1 injected systemically together with MSCs markedly reduced muscle inflammation and fibrosis, and significantly improved muscle strength in dystrophic mice. Our results suggest that a combined treatment with IGF-1 and MSCs enhances efficiency of muscle repair and, therefore, should be further considered as a potential therapeutic approach in muscular dystrophies.

The mdx Mutation in the 129/Sv Background Results in a Milder Phenotype: Transcriptome Comparative Analysis Searching for the Protective Factors.

The mdx mouse is a good genetic and molecular murine model for Duchenne Muscular Dystrophy (DMD), a progressive and devastating muscle disease. However, this model is inappropriate for testing new therapies due to its mild phenotype. Here, we transferred the mdx mutation to the 129/Sv strain with the aim to create a more severe model for DMD. Unexpectedly, functional analysis of the first three generations of mdx129 showed a progressive amelioration of the phenotype, associated to less connective tissue replacement, and more regeneration than the original mdxC57BL. Transcriptome comparative analysis was performed to identify what is protecting this new model from the dystrophic characteristics. The mdxC57BL presents three times more differentially expressed genes (DEGs) than the mdx129 (371 and 137 DEGs respectively). However, both models present more overexpressed genes than underexpressed, indicating that the dystrophic and regenerative alterations are associated with the activation rather than repression of genes. As to functional categories, the DEGs of both mdx models showed a predominance of immune system genes. Excluding this category, the mdx129 model showed a decreased participation of the endo/exocytic pathway and homeostasis categories, and an increased participation of the extracellular matrix and enzymatic activity categories. Spp1 gene overexpression was the most significant DEG exclusively expressed in the mdx129 strain. This was confirmed through relative mRNA analysis and osteopontin protein quantification. The amount of the 66 kDa band of the protein, representing the post-translational product of the gene, was about 4,8 times higher on western blotting. Spp1 is a known DMD prognostic biomarker, and our data indicate that its upregulation can benefit phenotype. Modeling the expression of the DEGs involved in the mdx mutation with a benign course should be tested as a possible therapeutic target for the dystrophic process.

Quantitative T2 Combined with Texture Analysis of Nuclear Magnetic Resonance Images Identify Different Degrees of Muscle Involvement in Three Mouse Models of Muscle Dystrophy: mdx, Largemyd and mdx/Largemyd

Quantitative nuclear magnetic resonance imaging (MRI) has been considered a promising non-invasive tool for monitoring therapeutic essays in small size mouse models of muscular dystrophies. Here, we combined MRI (anatomical images and transverse relaxation time constant—T2—measurements) to texture analyses in the study of four mouse strains covering a wide range of dystrophic phenotypes. Two still unexplored mouse models of muscular dystrophies were analyzed: The severely affected Largemyd mouse and the recently generated and worst double mutant mdx/Largemyd mouse, as compared to the mildly affected mdx and normal mice. The results were compared to histopathological findings. MRI showed increased intermuscular fat and higher muscle T2 in the three dystrophic mouse models when compared to the wild-type mice (T2: mdx/Largemyd: 37.6±2.8 ms; mdx: 35.2±4.5 ms; Largemyd: 36.6±4.0 ms; wild-type: 29.1±1.8 ms, p<0.05), in addition to higher muscle T2 in the mdx/Largemyd mice when compared to mdx (p<0.05). The areas with increased muscle T2 in the MRI correlated spatially with the identified histopathological alterations such as necrosis, inflammation, degeneration and regeneration foci. Nevertheless, muscle T2 values were not correlated with the severity of the phenotype in the 3 dystrophic mouse strains, since the severely affected Largemyd showed similar values than both the mild mdx and worst mdx/Largemyd lineages. On the other hand, all studied mouse strains could be unambiguously identified with texture analysis, which reflected the observed differences in the distribution of signals in muscle MRI. Thus, combined T2 intensity maps and texture analysis is a powerful approach for the characterization and differentiation of dystrophic muscles with diverse genotypes and phenotypes. These new findings provide important noninvasive tools in the evaluation of the efficacy of new therapies, and most importantly, can be directly applied in human translational research.

G.P.40 LGMD2G with clinical presentation of congenital muscular dystrophy: A rare phenotype

Abstract LGMD2G is a relatively rare and mild autosomal recessive form of progressive neuromuscular disorder with a wide spectrum of inter and intrafamilial clinical variability. The age at onset ranges from 9 to 15 years old and loss of ambulation is uncommon, or in the third or fourth decade of life. LGMD2G is caused by mutations in the TCAP gene, causing the deficiency of the sarcomeric protein telethonin in muscle of affected patients. Up to now, only a few families have been described, mainly in Brazil. In a recent screening for mutation in the TCAP gene, including 200 additional LGMD adult patients, we identified six new cases, confirming the rarity of the disease also in Brasil. All these patients carried the same c.157C>T (Q53X) homozygous missense mutation, identified in the first families, suggesting a common origin of this mutation. In addition,we studied a five years-old patient with clinical diagnosis of congenital muscular dystrophy. Immunohistochemical analysis for muscle protein excluded the deficiency of dystrophin, the four sarcoglycans, α 2-laminin, dysferlin and calpain3 proteins. Surprisingly, telethonin analysis showed a total deficiency, both through immune fluorescence and western blot analyses. Sequencing of the TCAP gene identified the common c.157C>T mutation in a homozygous state in the patient and in heterozygosity in both non-consanguineous parents. This called our attention to the fact that the rarity of the disease could be due to the screening of the improper patients. Telethonin deficiency could be, in fact, the cause of more severe forms of muscular dystrophies. To test this hypothesis, we analyzed telethonin deficiency in the muscle of 118 patients with clinical diagnosis of congenital MD, from which we selected sixteen patients for gene sequencing. However, no alteration was found in any of them. Therefore, mutation in the TCAP gene is not a common cause of severe forms of MD. FAPESP-CEPID, CNPq-INCT, FINEP, ABDIM.

Muscle Satellite Cells: Exploring the Basic Biology to Rule Them

Adult skeletal muscle is a postmitotic tissue with an enormous capacity to regenerate upon injury. This is accomplished by resident stem cells, named satellite cells, which were identified more than 50 years ago. Since their discovery, many researchers have been concentrating efforts to answer questions about their origin and role in muscle development, the way they contribute to muscle regeneration, and their potential to cell-based therapies. Satellite cells are maintained in a quiescent state and upon requirement are activated, proliferating, and fusing with other cells to form or repair myofibers. In addition, they are able to self-renew and replenish the stem pool. Every phase of satellite cell activity is highly regulated and orchestrated by many molecules and signaling pathways; the elucidation of players and mechanisms involved in satellite cell biology is of extreme importance, being the first step to expose the crucial points that could be modulated to extract the optimal response from these cells in therapeutic strategies. Here, we review the basic aspects about satellite cells biology and briefly discuss recent findings about therapeutic attempts, trying to raise questions about how basic biology could provide a solid scaffold to more successful use of these cells in clinics.

A.P.14: A new in/del in the critical splicing region of the VMA21 gene causing X-linked myopathy with excessive autophagy (XMEA)

X-linked myopathy with excessive autophagy (XMEA) is an inherited, slowly progressive myopathy, characterized by membrane-bound sarcoplasmic vacuoles in muscle fibers. Proximal muscle weakness in early childhood is observed, but with no cardiac, nor cognitive impairment. Recent findings identified mutations in the vacuolar membrane ATPase activity 21 (VMA21), as causative of XMEA. Among Six different single-nucleotide substitutions in VMA21 (in 14 XMEA families), four were intronic, and in two of them, the IVS1–27A base is involved. These mutations result in a reduction in VMA21 mRNA, and protein, and a consequent elevated lysosomal pH with partial block the final degradation stage of autophagy. Only a few XMEA families have been worldwide identified. Here we describe the first XMEA Brazilian family carrying a small in/del in the VMA21 gene. The 5-year-old propositus presented a characteristic dystrophic phenotype. He walked at the age of 2 and showed difficulties for running, climbing stairs, and raising from the floor. No calf hypertrophy nor joint contractures were observed. CK level was 1330 U/l, and ECG showed altered conduction in the right branch. Muscle biopsy showed a dystrophic pattern and autophagic vacuoles. Emerin was normal. Family history revealed a recessive X-linked inheritance, with 5 affected males linked through asymptomatic females. The affected maternal grandfather, aged 48, was wheelchair bound since the age of 30, presenting also cardiac alterations and joint contractures in the upper limbs. Exome sequencing identified a small insertion-deletion, including the IVS1-27A base previously described. This new family/mutation reinforces the importance of this splice site branchpoint for the appropriate transcription/translation of VMA21, and normal lysosome function. Additionally, it expands the clinical variability, including cardiac involvement and joint contractures to the XMEA phenotype.

G.P.210

Muscle satellite cells have been widely studied, especially to understand their mechanism of action in muscle regeneration and correspondent implications in the different dystrophic processes. Two mice models for muscular dystrophies, Large myd and Lama2 dy2j / J , have a pattern of an intense and very similar degeneration, but with differences in the expression of genes involved in the regeneration cascade, as we shown in our recent work. Therefore, they are interesting models to study possible differences in the mechanism of activation and action of satellite cells in the dystrophic muscle. The main objective of this work was to evaluate gene expression profile of the satellite cells from both dystrophic mouse models, as compared to normal murine muscle, to try to explain the difference observed in the respective muscles. For this evaluation, we harvested muscle derived cells after enzyme dissociation of muscle tissue. The cells were then pre-plated in culture flasks (PP1) and re-plated after 24h (PP2). The different populations were than characterized by flow cytometry markers and analyzed using a murine gene expression microarray panel of more than 26,000 genes. We observed 383 differentially expressed genes in Large myd and 110 in Lama2 dy2j / J . The glycosilation alteration, exhibited by Large myd alters the expression of many genes, especially those involved with myogenesis and activation of cell differentiation. The altered genes of Lama2 dy2j / J are more related with cell membrane or extracellular matrix. These observations are corroborating our previous gene expression results, suggesting that the mutation present in Large myd mouse leads to defects in the regeneration potential of satellite cells, what does not occur in the Lama2 dy2j / J model.

Myogenic Differentiation of ES Cells for Therapies in Neuromuscular Diseases: Progress to Date

The neuromuscular disorders are a heterogeneous group of genetic diseases characterized by progressive degeneration and impaired regeneration of skeletal muscle, resulting in weakness. The mobility of patients is very reduced, leading, depending on the disease severity, to wheelchair dependency and reduced life expectancy and quality. Currently there are no proven treatments for these diseases, except for palliative measures to improve a patient’s quality of life. Nevertheless, cell therapy using embryonic or somatic stem cells is considered to offer the best potential for success, and many projects are now being undertaken to evaluate the therapeutic possibilities of this approach. Theoretically, due to their pluripotency, embryonic stem cells can give rise to any type of tissue, and raises the possibility of successfully treating many diseases. However, a simple injection of ES cells into various body locations in model organisms often leads to formation of undesirable teratomas and not to healthy new tissues. Accordingly, ES cells must be partially differentiated and selected prior to injection to increase the likelihood of implantation and growth of tissue exhibiting differentiation of the desired type. Although some modest advances have been achieved, to date the use of ES cells for therapy of neuromuscular disorders still remains a distant goal. In this review we mainly consider the role of embryonic stem cells in neuromuscular therapeutic approaches.

Myogenic Potential of Murine Embryonic Stem Cells in the Dmdmdx Mouse Model for Duchenne Muscular Dystrophy

Danielle Ayub-Guerrieri1, Poliana C. M. Martins-Machado1, Paula C.G. Onofre-Oliveira1, Lygia V. Pereira2, Camila F. Almeida1, Vanessa F. Lopes1 and Mariz Vainzof1 1Laboratory of Muscle Proteins and Comparative Histopathology, Human Genome Research Center, Biosciences Institute, University of Sao Paulo 2Dept of Genetics and Evolutionary Biology, Biosciences Institute, University of Sao Paulo Brazil