Analysis of the autophagic pathway in the dystrophic muscle

O músculo esquelético é um tecido que tem a capacidade de se regenerar após lesão, seja ela patológica ou induzida. Para tanto, células musculares progenitoras, presentes no músculo adulto, atuam fundindo-se entre si, ou com as fibras musculares danificadas, para formar novas fibras. A via da macroautofagia, implicada na degradação e reciclagem de proteínas e organelas danificadas via lisossomo, é essencial para a manutenção da massa muscular, mas já foi também implicada na diferenciação e funcionamento de células progenitoras do músculo. Além disso, essa via está desregulada em diversas doenças neuromusculares, o que destaca seu papel nesse tecido. Nesse estudo, a regulação da autofagia foi investigada em diferentes situações de formação e degradação do músculo. Para estudar o processo de diferenciação muscular in vitro utilizamos um modelo de células musculares imortalizadas normais, e de paciente com miopatia ligada ao X com autofagia excessiva (XMEA). A análise dos genes e proteínas p62, BNIP3, BECLIN1, VPS34, ATG12 e LC3, além de alvos de mTOR, mostrou um padrão similar de expressão em mioblastos indiferenciados e miotubos diferenciados a partir de células controle e nas derivadas de paciente XMEA. Estes resultados sugerem que a desregulação da via autofágica relacionada à doença provavelmente surge em estágios mais avançados, como se observa em doenças de acúmulo lisossomal. A investigação da diferenciação muscular nessas células mostrou um aumento na capacidade de fusão de mioblastos XMEA, que não foi relacionado a mudanças na expressão de genes envolvidos na miogênese. Isso indica que o defeito primário relacionado a XMEA, como a deficiência da ATPase vacuolar, pode interferir no processo de diferenciação muscular. Para estudar o músculo em condições patológicas, utilizamos modelos animais para distrofias musculares que possuem distintos graus de afecção do músculo, como o DMDmdx, modelo para distrofia muscular de Duchenne, o SJL/J, modelo para distrofia muscular de cinturas tipo 2B e o Largemyd, modelo para distrofia muscular congênita 1D. Observamos que não há alterações globais na expressão de genes e proteínas da autofagia. Adicionalmente, cada modelo murino teve alterações pontuais, destacando a ausência de correlação entre o grau de degeneração do músculo e as alterações observadas na via autofágica. Por outro lado, quando uma lesão muscular é induzida em músculo normal, houve uma diminuição da expressão de todos os genes estudados, Bnip3, Beclin1, Vps34, Atg12, Lc3 e Gabarapl1, com possível acúmulo das proteínas autofágicas p62 e Beclin1. Com a recuperação do músculo, após cinco dias da lesão, a maior parte dos genes estudados teve sua expressão normalizada. Tais resultados indicam que a lesão aguda se relaciona a uma resposta drástica e recuperação rápida na via da autofagia. Em conjunto, nossos resultados mostram que a via da autofagia é diferencialmente afetada a depender do estímulo dado ao músculo, seja ele de regeneração e formação de novas células musculares ou de degeneração. Dessa forma, este estudo pode ter implicações para o desenvolvimento de terapias que tenham como alvo a via autofágica, já que indica que o momento da intervenção terapêutica pode ser importante, assim como o estímulo que levou a alterações no tecido muscularThe skeletal muscle is a tissue that has the ability to regenerate upon lesion, whether it occurs pathologically or induced. Therefore, progenitor muscle cells, present in the adult muscle, act by fusing with each other or with damaged fibers in order to recover the tissue. The macroautophagy pathway, related to degradation and recycling of proteins and damaged organelles via lysosome, is essential for the maintenance of muscle mass, and it was also implicated in the differentiation and functioning of muscle progenitor cells. Besides that, this pathway is deregulated in several neuromuscular disorders, highlighting its important role in this tissue. In this study, the autophagic regulation was investigated in distinct contexts of muscle formation and degradation. To study the muscle differentiation process in vitro, we used a model of immortalized muscle cells from both a normal control and a patient with X-linked myopathy with excessive autophagy (XMEA). The genes and proteins p62, BNIP3, BECLIN1, VPS34, ATG12, LC3 and mTOR targets showed a similar pattern of expression in both undifferentiated myoblasts and differentiated myotubes, from both control cells and XMEA patient-derived cells. This fact suggests that autophagic deregulation might arise in later stages of the disease, in a pattern observed in disorders with protein accumulation. The investigation of muscle differentiation in the studied cells showed an enhancement of the myoblast fusion capacity in XMEA cells, which was not related to changes in the expression of myogenic genes. This observation indicates that the primary defect related to the XMEA pathology, as the deficiency of the vacuolar ATPase, might interfere in the process of muscle differentiation. In order to evaluate muscle in pathological conditions, we studied animal models for muscular dystrophies that have distinct patterns of muscle affection, such as the DMDmdx, model for the Duchenne muscular dystrophy, the SJL/J, model for the limb-girdle muscle dystrophy type 2B and the Largemyd, model for the congenital muscular dystrophy type 1D. We did not find any global alterations in the expression of autophagic genes and proteins. Additionally, each animal model had discrete changes, highlighting the absence of correlation between the pattern of muscle degeneration and alterations in the autophagy pathway. On the other hand, when a lesion is induced in normal muscle, there is a decrease in the expression of all studied genes, such as Bnip3, Beclin1, Vps34, Atg12, Lc3 and Gabarapl1, with a possible accumulation of the autophagic proteins p62 and Beclin1. With muscle recovery, five days after lesion, most of the studied genes had their expression returning to normal levels. These results indicate that the acute lesion is related to a drastic response and rapid recovery of the autophagic pathway. Together, our results show that autophagy is differentially affected depending on the stimulus given to the muscle, either of regeneration and formation of new muscle cells or degeneration. In that sense, this study may have implications for the development of therapies that target autophagy, since it indicates that the time point of therapeutic interventions may be important, as well as the stimulus that led to alterations in the skeletal muscle tissu

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

Primers used in relative quantification of gene expression.

<p>Primers used in relative quantification of gene expression.</p

List of DEGs exclusive of the mdx¹²⁹.

<p>List of DEGs exclusive of the mdx¹²⁹.</p

Comparative histological analysis.

<p>HE staining of gastrocnemius and diaphragm muscles, in mdx^C57BL and mdx¹²⁹ mice in the age of 6 months.</p

Quantification of <i>Spp1</i> transcript and OPN protein expression.

<p>(A) Fold changes in qPCR relative expression levels of osteopontin mRNA, as compared to the control group (129/Sv). (B) Western blotting analysis for OPN protein showing both the full length protein (66 kDa band) and one fragment of 32 kDa (cleaved product) in mdx^C57BL and mdx¹²⁹ as compared to normal control 129/Sv (con); M—myosin band. (C) Western blotting quantification showing the mean of each group for the band of 32 kDa and 66 kDa. An increase of 4,8 times of the 66 kDA band is observed in the mdx¹²⁹ group using myosin band as a protein loading control.</p

Originating the mdx¹²⁹ mouse.

<p>(A) Schematic representation of the cross-breeding. (B) Genotyping for the mdx mutation: acrylamide gel electrophoresis of the PCR competitive reaction showing the presence of the 134 pb band in two wild type normal DNA (N), a 117 pb band in the mdx (M) and both bands in two carrier females (H). (C) Dystrophin immunofluorescence analysis with DYS2 antibody showing the presence of dystrophin in the muscle membrane of normal control, and the absence of dystrophin in the mdx¹²⁹.</p

Graphical representation of comparative functional test in the mdx^C57Bl as compared to the three mdx¹²⁹ generations.

<p>Data of the fore limbs in the bar test and grip strength test are shown with the median expression and standard error range for each age/strain. The number of tested animals in each generation is also presented. * p<0,05.</p

Venn diagram showing genes in common and exclusive DEGs in each of compared lists.

<p>Venn diagram showing genes in common and exclusive DEGs in each of compared lists.</p

Quantification of the regeneration in the two mdx models.

<p>(A) Results of the qPCR expression of genes involved with the regeneration process (<i>Myf5</i>, <i>MyoD</i> and <i>Myog</i>). (B) Representation of the comparative immunohistochemical analysis of gastrocnemius for developmental myosin (red) in double reaction with laminin (green), showing the proportion of positive fibers in mdx^C57BL and mdx¹²⁹ mice in the age of 6 months. (C) Graphic representing the quantitative comparison between the two groups of mdx with normal control (con).</p