The Lymn Foundation made awards of $500 each to four new investigators for presenting posters that contributed new insights and understanding of biological or disease processes in skeletal muscle at two scientific conferences on recent research developments in muscle biology: New Directions in Biology and Diseases of Skeletal Muscle and Frontiers in Myogenesis. Committees consisting of leaders at each of the conferences recommended the investigators.

Awardees at New Directions in Biology and Diseases of Skeletal Muscle

Dallas, Texas, April 23-26, 2006

Kathryn E. Tifft

Regulation of emerin interactions by tyrosine phosphorylation

Cell Biology, Johns Hopkins School of Medicine, Baltimore, Maryland

Maura H. Parker

Overcoming Immune Rejection in Myoblast Transplant Therapy through Hematopoietic Cell Transplantation

Transplantation Biology, Fred Hutchinson Cancer Research Center, Seattle, Washington

Callaway Gardens, Pine Mountain, Georgia, April 27-May 2, 2006

Craig McFarlane

Myostatin signals through Pax7 to regulate post-natal myogenesis

AgResearch, Functional Muscle Genomics, Hamilton, New Zealand

Hugo C. Olguin

Satellite cell renewal: ups and downs on the road

University of Colorado, Boulder, Colorado

2006 ABSTRACTS

Regulation of emerin interactions by tyrosine phosphorylation

Tifft, Kathryn E, Wilson, Katherine L

Johns Hopkins School of Medicine, Baltimore, MD

Emery-Dreifuss muscular dystrophy (EDMD) is characterized by weakening of selected skeletal muscles, contractures of major tendons, and cardiac conduction defects. Loss of the inner nuclear membrane protein emerin causes X-linked EDMD. Emerin regulates gene expression during muscle development, potentially via direct binding to transcriptional regulators like BAF, GCL, Lmo7, Btf, and mRNA splicing factor YT521B. Emerin also binds structural proteins (lamin A, actin, and Nesprins) suggesting additional roles in nuclear structure. The binding sites in emerin for many partners overlap suggesting regulation of emerin binding partners and emerin functions. Five labs identified eleven tyrosine residues in emerin that are phosphorylated in vivo. We confirmed that emerin is tyrosine phosphorylated in HeLa cells and discovered that tyrosine phosphorylation decreases in the presence of Src inhibitors. We are testing the hypothesis that phosphorylation of specific tyrosine residues in emerin regulates specific partners using single missense mutations at known sites of tyrosine phosphorylation. Understanding how emerin is regulated by tyrosine phosphorylation may provide novel insight into emerin function and the mechanism of EDMD disease.

Overcoming Immune Rejection in Myoblast Transplant Therapy through Hematopoietic Cell Transplantation

Parker, Maura H., Storb, Rainer, Kuhr, Christian, Tapscott, Stephen

Clinical Research and Human Biology Divisions, Fred Hutchinson Cancer Research Center; Department of Surgery, University of Washington, Seattle, WA

Duchenne Muscular Dystrophy (DMD) is the most common and severe form of muscular dystrophy in humans. The goal of satellite cell-derived myoblast transplant therapy (MTT) for DMD is to increase dytrophin expression in existing fibers, and provide a stem cell source for future regeneration. The major limitation to the success of MTT has been immune rejection of transplanted myoblasts. We asked if generating a chimeric DMD-affected dog, in which the immune system is reconstituted with DLA-identical donor-derived hematopoietic cells, could provide a more effective platform for myoblast transplant therapy. DMD affected canines were subjected to myeloblative conditioning prior to hematopoietic cell transplant, generating a fully chimeric dog. The hematopoietic cells did not detectably contribute to either skeletal muscle or the satellite cell population. However, intramuscular injection of donor-derived satellite cell-derived myoblasts results in robust and stable expression of dystrophin in DMD affected skeletal muscle. This establishes chimeric recipients as a viable model for addressing myoblast transplantation in an immune tolerant, random-bred, large animal model of Duchenne muscular dystrophy.

Myostatin signals through Pax7 to regulate post-natal myogenesis

McFarlane, C., Hennebry, A., Thomas, M., Sharma, M., Kambadur, R.

AgResearch, Functional Muscle Genomics, East Street, Hamilton, New Zealand

The paired-box transcription factor Pax7 has been implicated in satellite cell specification, expansion of satellite cell populations and satellite cell self-renewal. Heterogeneity of Pax7 expression appears to exist in cultured myogenic cells, whereby high levels of Pax7 are associated with quiescent differentiation resistant cells. Indeed, delayed differentiation is observed in clonal C2C12 cell lines over-expressing Pax7, with higher proportions of quiescent reserve cells seen in Pax7 over-expressing cells. These reserve cells may form a population of cells analogous to self-renewed satellite cells. In support Pax7 expression is higher in reserve cell enriched populations of cultured C2C12 cells as compared to myotube populations. Myostatin, a Transforming Growth Factor-Beta (TGF-b) super-family member, has been well characterized as a negative regulator of post-natal myogenesis and in fact Myostatin has previously been shown to inhibit satellite cell activation and self-renewal. Here we show that treatment with Myostatin down-regulates Pax7 expression in proliferating satellite cell cultures. Furthermore, Pax7 expression is higher in primary cultures obtained from myostatin-null mice as compared to wild-type primary cultures. Interestingly, Pax7 expression persists through differentiation in cultured primary myoblasts from myostatin-null animals when compared to primary cultures from wild-type animals, thus Myostatin may regulate satellite cell activation, propagation and self-renewal through negatively regulating Pax7 expression.

Satellite cell renewal: ups and downs on the road

Olguin, Hugo C., Olwin, B. B.

University of Colorado Boulder, CO

The transcription factor Pax-7 appears to be crucial for the specification of the satellite cell (SC) lineage. Recent work also implicates Pax-7 in SC self-renewal in the adult skeletal muscle. However, the mechanisms regulating these activities remain unclear. Previously we showed that Pax-7 can block cell cycle progression and myogenesis in adult myoblasts and during the myogenic conversion of 10T1/2 cells. Our data indicates that the Pax-7-mediated block of differentiation affects MyoD function, prior to myogenin induction. Strikingly, independent experiments show that Pax-7 transcriptional activity may not be directly involved. Instead, Pax-7 affects MyoD protein stability, potentially in a cell cycle dependent manner. Supporting this, deletion analyses show that 1) protein domains thought to be involved in Pax-7 transcriptional activity, are not required for regulation of MyoD, and 2) deletion of an internal Pax-7 domain restores MyoD function and protein levels. Finally, myogenin but not MyoD appears to regulate Pax-7 function by affecting Pax-7 protein stability. Together, these results provide evidence for interdependent regulation between Pax-7 and MRFs. Increasing Pax-7 protein levels leads to enhanced degradation of MyoD, preventing MyoD function and the accumulation of myogenin. Lower levels of Pax-7 (and/or MyoD up-regulation) enhances MyoD function, inducing myogenin, which negatively feeds back to inhibit Pax-7 by decreasing Pax-7 protein stability. This represents a novel mechanism that may function early in the decision of an activated SC to either continue to proliferate, commit to terminal differentiation or to re-acquire a quiescent state, renewing the satellite cell pool. MDA to HO.