P.C.M. Martins

Animal Models for Genetic Neuromuscular Diseases

The neuromuscular disorders are a heterogeneous group of genetic diseases, caused by mutations in genes coding sarcolemmal, sarcomeric, and citosolic muscle proteins. Deficiencies or loss of function of these proteins leads to variable degree of progressive loss of motor ability. Several animal models, manifesting phenotypes observed in neuromuscular diseases, have been identified in nature or generated in laboratory. These models generally present physiological alterations observed in human patients and can be used as important tools for genetic, clinic, and histopathological studies. The mdx mouse is the most widely used animal model for Duchenne muscular dystrophy (DMD). Although it is a good genetic and biochemical model, presenting total deficiency of the protein dystrophin in the muscle, this mouse is not useful for clinical trials because of its very mild phenotype. The canine golden retriever MD model represents a more clinically similar model of DMD due to its larger size and significant muscle weakness. Autosomal recessive limb-girdle MD forms models include the SJL/J mice, which develop a spontaneous myopathy resulting from a mutation in the Dysferlin gene, being a model for LGMD2B. For the human sarcoglycanopahties (SG), the BIO14.6 hamster is the spontaneous animal model for δ-SG deficiency, whereas some canine models with deficiency of SG proteins have also been identified. More recently, using the homologous recombination technique in embryonic stem cell, several mouse models have been developed with null mutations in each one of the four SG genes. All sarcoglycan-null animals display a progressive muscular dystrophy of variable severity and share the property of a significant secondary reduction in the expression of the other members of the sarcoglycan subcomplex and other components of the Dystrophin-glycoprotein complex. Mouse models for congenital MD include the dy/dy (dystrophia-muscularis) mouse and the allelic mutant dy2J/dy2J mouse, both presenting significant reduction of α2-laminin in the muscle and a severe phenotype. The myodystrophy mouse (Largemyd) harbors a mutation in the glycosyltransferase Large, which leads to altered glycosylation of α-DG, and also a severe phenotype. Other informative models for muscle proteins include the knockout mouse for myostatin, which demonstrated that this protein is a negative regulator of muscle growth. Additionally, the stress syndrome in pigs, caused by mutations in the porcine RYR1 gene, helped to localize the gene causing malignant hypertermia and Central Core myopathy in humans. The study of animal models for genetic diseases, in spite of the existence of differences in some phenotypes, can provide important clues to the understanding of the pathogenesis of these disorders and are also very valuable for testing strategies for therapeutic approaches.

Dmdmdx/Largemyd: a new mouse model of neuromuscular diseases useful for studying physiopathological mechanisms and testing therapies

SUMMARY Although muscular dystrophies are among the most common human genetic disorders, there are few treatment options available. Animal models have become increasingly important for testing new therapies prior to entering human clinical trials. The Dmdmdx mouse is the most widely used animal model for Duchenne muscular dystrophy (DMD), presenting the same molecular and protein defect as seen in humans with the disease. However, this mouse is not useful for clinical trials because of its very mild phenotype. The mouse model for congenital myodystrophy type 1D, Largemyd, harbors a mutation in the glycosyltransferase Large gene and displays a severe phenotype. To help elucidate the role of the proteins dystrophin and LARGE in the organization of the dystrophin-glycoprotein complex in muscle sarcolemma, we generated double-mutant mice for the dystrophin and LARGE proteins. The new Dmdmdx/Largemyd mouse model is viable and shows a severe phenotype that is associated with the lack of dystrophin in muscle. We tested the usefulness of our new mouse model for cell therapy by systemically injecting them with normal murine mesenchymal adipose stem cells (mASCs). We verified that the mASCs were hosted in the dystrophic muscle. The new mouse model has proven to be very useful for the study of several other therapies, because injected cells can be screened both through DNA and protein analysis. Study of its substantial muscle weakness will also be very informative in the evaluation of functional benefits of these therapies.

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.

Mitochondrial Cardioencephalomyopathy Due to a Novel SCO2 Mutation in a Brazilian Patient: Case Report and Literature Review

OBJECTIVES To review all patients with SCO2 mutations and to describe a Brazilian patient with cardioencephalomyopathy carrying compound heterozygous mutations in SCO2, one being the known pathogenic p.E140K mutation and the other a novel 12-base pair (bp) deletion at nucleotides 1519 through 1530 (c.1519_1530del). DESIGN Case report and literature review. SETTING University hospital. PATIENT Infant girl presenting with an encephalomyopathy, inspiratory stridor, ventilator failure, progressive hypotonia, and weakness, leading to death. MAIN OUTCOME MEASURES Clinical features, neuroimaging findings, muscle biopsy with histochemical analysis, and genetic studies. RESULTS This infant girl was the first child of healthy, nonconsanguineous parents. She developed progressive muscular hypotonia and ventilatory failure. At the end of the first month of life, she developed cardiomegaly and signs of cardiac failure. Routine blood tests showed lactic acidosis and mild elevation of the creatine kinase level. Brain magnetic resonance imaging showed increased T2 and fluid-attenuated inversion recovery signals in the putamen bilaterally. Nerve conduction studies showed severe axonal sensorimotor neuropathy. Muscle biopsy revealed a neurogenic pattern with mitochondrial proliferation and total absence of cytochrome- c oxidase histochemical stain. Sequencing of SCO2 showed that the patient had compound heterozygote SCO2 mutations: the previously described c.1541G>A (p.E140K) mutation and a novel 12-bp deletion at nucleotides 1519 through 1530 (c.1519_1530del). The patient died at age 45 days. CONCLUSIONS Our findings and the literature review indicate that it is important to consider the diagnosis of mitochondrial disease in newborns with hypotonia and cardiomyopathy. In our case, the accurate diagnosis of SCO2 mutations is particularly important for genetic counseling.

Kinin and Purine Signaling Contributes to Neuroblastoma Metastasis

Bone marrow metastasis occurs in approximately 350,000 patients that annually die in the U.S. alone. In view of the importance of tumor cell migration into the bone marrow, we have here investigated effects of various concentrations of stromal cell-derived factor-1 (SDF-1), bradykinin- and ATP on bone marrow metastasis. We show for first time that bradykinin augmented chemotactic responsiveness of neuroblastoma cells to SDF-1 and ATP concentrations, encountered under physiological conditions. Bradykinin upregulated VEGF expression, increased metalloproteinase activity and induced adhesion of neuroblastoma cells. Bradykinin augmented SDF-1-induced intracellular Ca2+ mobilization as well as resensitization and expression of ATP-sensing P2X7 receptors. Bradykinin treatment resulted in higher gene expression levels of the truncated P2X7B receptor compared to those of the P2X7A full-length isoform. Bradykinin as pro-metastatic factor induced tumor proliferation that was significantly decreased by P2X7 receptor antagonists; however, the peptide did not enhance cell death nor P2X7A receptor-related pore activity, promoting neuroblastoma growth. Furthermore, immunodeficient nude/nude mice transplanted with bradykinin-pretreated neuroblastoma cells revealed significantly higher metastasis rates compared to animals injected with untreated cells. In contrast, animals receiving Brilliant Blue G, a P2X7 receptor antagonist, did not show any specific dissemination of neuroblastoma cells to the bone marrow and liver, and metastasis rates were drastically reduced. Our data suggests correlated actions of kinins and purines in neuroblastoma dissemination, providing novel avenues for clinic research in preventing metastasis.

Kinin-B2 Receptor Activity in Skeletal Muscle Regeneration and Myoblast Differentiation

The bioactive peptide bradykinin obtained from cleavage of precursor kininogens activates the kinin-B2 receptor functioning in induction of inflammation and vasodilatation. In addition, bradykinin participates in kidney and cardiovascular development and neuronal and muscle differentiation. Here we show that kinin-B2 receptors are expressed throughout differentiation of murine C2C12 myoblasts into myotubes. An autocrine loop between receptor activation and bradykinin secretion is suggested, since bradykinin secretion is significantly reduced in the presence of the kinin-B2 receptor antagonist HOE-140 during differentiation. Expression of skeletal muscle markers and regenerative capacity were decreased after pharmacological inhibition or genetic ablation of the B2 receptor, while its antagonism increased the number of myoblasts in culture. In summary, the present work reveals to date no functions described for the B2 receptor in muscle regeneration due to the control of proliferation and differentiation of muscle precursor cells.

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.

T.P.38 Effects of steroid hormones on myostatin expression and on genes of muscle regeneration pathway

Abstract Myostatin is an important negative regulator of skeletal muscle growth, while decanoato de nadrolone, an anabolic steroid, is a strong positive effector. Inhibition of myostatin has been tested as an approach for treatment neuromuscular diseases. In order to investigate the possible interaction between myostatin and anabolic steroids, as a therapeutic strategy, we studied myostatin expression in the quadriceps femoris of normal mice treated with Decadurabolin® (D), flutamide (F), an antagonist of the androgen receptor, and Decadurabolin administration, post flutamide treatment (FD), as compared to controls, treated with saline (S). We also studied the relative expression of the genes, myogenin, MyoD and Myf5, involved in the pathway of muscle regeneration. We observed significant increase in the body mass in the (D) and (FD) groups, and a decrease in the group (F), when compared to groups (S). Real-time PCR quantitative analysis for myostatin expression showed no statistically significant differences between the studied groups. On the other hand, the groups (D) and (FD) showed a significant decrease in the expression of myogenin, MyoD and Myf5, while animals of the group (F) showed a significant increase in the expression of these genes. We conclude that administration of anabolic steroid, or its inhibition did not alter the expression of the myostatin gene, despite the increase or decrease in the body mass observed in group (D), (FD) and (F). However, the blockade of androgen receptor by flutamide, clearly stimulate the regeneration cascade, by increasing the expression of genes related to proliferation (MyoD and Myf5) and cell differentiation (myogenin). Additional studies will elucidate the possible role of other pathways in this stimulus for regeneration. Financial support: FAPESP-CEPID, CNPq-INCT, FINEP, ABDIM.