ABSTRACTS FROM 45TH ANNUAL MEETING OF THE AMERICAN SOCIETY FOR CELL BIOLOGY, DECEMBER 10-14, SAN FRANCISCO

Participation of CTGF in Skeletal Muscle Fibrosis
C. Vial, P. Cañón, E. Brandan; Cell & Mol. Biol., Catholic University of Chile, Santiago, Chile

Duchenne muscular dystrophy (DMD) is characterized by degeneration of muscle fibres, which leads to progressive muscular atrophy. Regeneration of these is dependent on the activation and proliferation of progenitor myoblasts, process that in DMD is insufficient to compensate for the continuous loss of myofibers and fibrotic scarring ensues. Connective tissue growth factor (CTGF) has been related to fibrotic processes for his ability of inducing connective tissue synthesis. CTGF is augmented in fibrotic disorders, but there is little information regarding its role in skeletal muscle disease, as well as its biological receptor. It has been reported that CTGF binds to the low-density lipoprotein receptor-related protein (LRP), and that it binds directly to integrins. The aim of our study is to elucidate CTGF’s role in skeletal muscle biology with relation to pathological fibrosis, and to study its receptor in myoblasts. Here we report that TGF-β and lysophosphatidic acid (LPA), two molecules involved in tissue scaring, induce CTGF expression in C2C12 mouse myoblast cell line in a dose dependent manner. In order to study the effect of CTGF in myoblasts we have generated a recombinant active CTGF (rCTGF). Here we report that incubation of myoblasts with rCTGF stimulates the production of fibronectin, collagen Iα2, and proteoglycans. In order to study CTGF´s receptor we have also use a siRNA to inhibit LRP and RGDS to inhibit integrins, resulting in an altered response to the cytokine. These results suggest that LRP might be an internalization receptor for CTGF and that in DMD not only are fibroblasts involved in the increase of connective tissue, but myoblasts can contribute to muscle fibrosis. (Supported by MDA3790, FONDAP, MIFAB)
 

Skeletal Muscle Fibrosis in Duchenne Muscular Dystrophy: Modulation by TGFβ and CTGF in mdx Mice
V. Mezzano, D. Cabrera, E. Brandan; Cell & Mol. Biol., Catholic University of Chile, Santiago, Chile

Duchenne muscular dystrophy (DMD) is a lethal, muscle wasting disease caused by a mutation in the dystrophin gene. The absence of the dystrophin protein leads to degeneration of muscle fibers and eventually fibrotic scarring ensues. The mdx mouse model of DMD also lacks dystrophin and its diaphragm reproduces the degenerative changes of DMD. The aim of our work is to understand the fibrotic process which parallels muscle degeneration and, particularly, the involvement of two potent regulators of extracellular matrix (ECM) synthesis: TGFβ and Connective Tissue Growth Factor (CTGF), which has been described as another profibrotic cytokine. We have isolated fibroblasts from mdx and control mouse diaphragm, studied their relationship to these factors both regarding their expression and synthesis, and their regulation of ECM molecules. Here we report that CTGF induces fibronectin in fibroblasts of control mice in a concentration dependent manner. Regarding mdx fibroblasts we observed that they express and synthezise more CTGF than controls. We, therefore, studied ECM molecules present in these cell cultures and observed an increase in fibronectin and chondroitin sulfate proteoglycans in mdx fibroblasts compared to control, increase which is not observed in skin isolated fibroblasts. Interestingly, regarding the ability of proteoglycans to influence the activity/bioavailability of TGFβ, we observed increased binding of TGFβ to TGFβ Receptor III, or betaglycan, a heparan sulfate proteoglycan. These results suggest a constitutive activation of mdx fibroblasts obtained from dystrophic muscle in spite of their being isolated from their regenerative/fibrotic niche offering new insights into the understanding of these disorders. (Supported by MDA 3790, FONDAP-MIFAB, PG53/04 School of Medicine PUC, CONICYT AT-24050106)
 

Functional Overlap between Dystrophin and the α7β1 Integrin
J. E. Rooney; Pharmacology, University of Nevada, Reno, Reno, NV

Duchenne muscular dystrophy (DMD) is the most common X-linked human disease, with a prevalence of 1 in every 3,500 live male births. DMD patients and mdx mice (the murine model for DMD) have mutations in the dystrophin gene that result in the absence of the dystrophin protein. A second laminin binding complex in skeletal muscle is the α7β1 integrin. The absence of the α7 integrin results in congenital myopathy in mice and humans. The transgenic over-expression of the α7 integrin chain in the skeletal muscle of severely dystrophic mice has been show to partially rescue the diseased phenotype. These correlations suggest that dystrophin and the α7β1 integrin may have complementary and overlapping functional and structural roles in maintaining skeletal muscle integrity. In order to test this hypothesis, mice deficient for both dystrophin and the α7 integrin (mdx/α7^-/-) were generated. These animals display severe signs of muscular dystrophy and die prematurely at 3-4 week of age. The muscle fibers of mdx/α7^-/- mice display extensive loss of membrane integrity, increased inflammatory cell infiltrate, fibrotic deposition and degeneration. These mice were also found to have increased muscle regeneration and changes in the expression patterns of laminin-2/4 and utrophin. Collectively, these results point to overlapping roles for dystrophin and the α7β1 integrin in skeletal muscle structure and function. The elucidation of these overlapping functional capacities may lead to the identification of novel therapies for muscular dystrophy through the exploitation and manipulation of cell signaling pathways or proteins shared by both complexes.
 

Telomere Shortening in Skeletal Muscle from MDX Mice
T. C. Lund,¹ S. A. Nelson,² R. W. Grange,³ D. A. Lowe²; ¹Department of Medicine, University of Minnesota, Minneapolis, MN, ²Department Physical Therapy and Rehabilitation, University of Minnesota, Minneapolis, MN, ³Dept. of Human Nutrition, Foods and Exercise, Virginia Polytechnic Institute and State University, Blacksburg, VA

Telomeres are the DNA sequences at the ends of chromosomes that keep tally of the number of cell divisions. Prior studies have shown that when telomeres become too short, chromosomes break and cell senescence or apoptosis occurs. Muscular dystrophy (MD) is a common X-linked neuromuscular disease in which the lack of the protein, dystrophin, causes muscle fiber damage. While previous studies have shown satellite cells are maintained in dystrophic muscle, they are reduced in number and this likely contributes to the pathogenesis of MD . We hypothesize that this is in part due to significant telomere shortening in these cells. Furthermore, our study stands out as the first to evaluate the diaphragm muscle for telomere dynamics. DNA was isolated from diaphragm and tibialis anterior muscles from mdx and wildtype mice aged 20, 40, 80, 100, 240, and 600 d (n=2-6 per age). Telomere length was determined by Southern blot. We found that telomere length was not different between muscles from 20, 40 or 80 d mdx and wildtype mice (P>0.05). Telomere length in diaphragm muscles from 100 and 600 d mdx mice was 84 +/- 3.8% and 59 +/- 1.4% of that from age-matched wildtype mice (P<0.05). In tibialis anterior muscles, telomere shortening was not evident in 100 d mdx mice (103 +/- 3.4% of wildtype) but in 600 d mice it was 68 +/- 2.3% of wildtype (P<0.05). These findings indicate that muscle satellite cells undergo telomere erosion and conceivably have a more finite life span in vivo than previously appreciated. Although considered to have stem cell characteristics, satellite cells in mdx muscle are perhaps under so much pressure to divide that they succumb to telomere erosion limiting their lifespan and their ability to repair damage muscle.
 

Isolation and Characterization of Myogenic Stem Cells from Adult Murine Skeletal Muscle and Bone Marrow
Z. Qu-Petersen; The Copenhagen Muscle Research Centre and Department of Neurology, Copenhagen National University Hospital, Copenhagen, Denmark

Skeletal muscle contains a stem cell pool of committed and pluripotent myogenic cells responsible for the growth and repair of postnatal muscle tissue. Investigations suggest that the pluripotent myogenic stem cells (pMySCs) can be of muscle and haematopoietic origins. To identify the pMySCs in different sources, we recently isolated distinct populations of MySCs from skeletal muscle and bone marrow of C57 BL/6 normal mice (5-7 weeks old) by preplate and clonal culture technique. Both populations are clonogenic, capable of extensive proliferation, and multi-lineage differentiation, demonstrating their property of stem cells. By flow cytometry, the muscle-derived MySCs (m-MySCs) were phenotypically characterized as Sca-1(+)CD34(-)CD24(-) while the bone marrow-derived MySCs (bm-MySCs) were characterized as Sca-1(+)CD34(low)CD24(+). Following initial differentiation in culture, both populations displayed a high degree of myogenicity, with 86% of m-MySCs and 64% of bm-MySCs capable of committing into myogenic lineage, but different myogenic pathways were identified. The m-MySCs generated three major progenies concerning specific neurons, glial cells and myoblasts, all of which contributed in vitro to multiple-myotube formation. The roles of the different myotubes (neuron/glia-reated or myoblast-related) in muscle development and regeneration should be investigated further. In contrast, only myoblast-related myotube formation was observed with bm-MySCs in culture. However, the capacity of bm-MySC’s clonal-proliferation and robust myofiber formation both in vitro and over transplantation suggest their potential use in muscular disease. In conclusion, the bm-MySCs represent a population of more committed myogenic stem cells in hematopoietic system, while the m-MySC represent a novel class of pluripotent neuromuscular progenitors/stem cells within skeletal muscle.