Awardees at New Directions in Biology and Diseases of Skeletal Muscle

April 27-30, 2008 in New Orleans, Louisiana

Douglas P. Millay

Calcium as the central mediator of degeneration in muscular dystrophy

Molecular Cardiovascular Biology, Cincinnati Children's Hospital,
Cincinnati, Ohio

Kevin Sonnemann

TAT-Utrophin crosses cell barriers to combat dystrophin deficiency

Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota

Awardees at the 37th European Muscle Congress

September 13 - September 16, 2008 in Oxford, England

Christiane Look

Adipocyte-derived factors suppress heart contraction

Department of Internal Medicine III, TU Dresden; Max-Delbrck-Center for Molecular Medicine, Berlin-Buch, Germany

Zacharias Orfanos

The dynamics of sarcomere assembly in Drosophila flight muscle

Department of Biology, University of York, Heslington, York, YO10 5DD, United Kingdom

2008 ABSTRACTS

Calcium as the central mediator of degeneration in muscular dystrophy

Millay,Douglas P; Goonasekera, Sanjeewa; Molkentin, Jeffery D

Molecular Cardiovascular Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH

Muscular dystrophy (MD) is a muscle wasting disease, which typically results in death by the second or third decade of life. Unfortunately, there are no cures for the disease and current therapeutic treatments available only extend lifespan by a few years. Many forms of muscular dystrophy are caused by a mutation in proteins that reside within the dystrophin-glycoprotein complex (DGC). The DGC serves as a structural attachment apparatus that spans the underlying contractile units to the basal lamina outside the cell, ultimately providing support for the cell membrane. Without a completely functional DGC the integrity of the muscle cell membrane is compromised, allowing for contraction-induced membrane tears. Ca2+ entry via tears in the membrane, along with dysregulation of Ca2+ channels, results in an increase in cytosolic Ca2+ levels in dystrophic myotubes. Increased cytosolic Ca2+ can cause degeneration through a number of downstream effectors, including its effect on degradative enzymes and cell death proteins. Thus, we hypothesize that Ca2+ is the central mediator of skeletal muscle degeneration in MD. To date this hypothesis has not been rigorously tested in vivo and results of these studies could result in novel therapeutic approaches. To investigate the role of Ca2+ in skeletal muscle degeneration we generated three independent skeletal muscle specific transgenic mice that overexpress membrane calcium/cation channels. One transgenic (total of 3 lines) expresses the sodium/calcium exchanger (NCX1), which typically transports Ca2+ out and Na+ into the cell, although our preliminary studies determined that NCX TG fibers have increased baseline Ca2+ levels and Ca2+ transients. Remarkably, the medium and high expressing lines exhibited skeletal muscle disease as judged by histological analysis (central nuclei, fiber area, fibrosis), while the low expressing line did not display overt disease. Furthermore, the quadriceps muscle of 15 month old NCX TG (high line) mice had a large amount of fat replacement, indicating loss of regenerative capacity. We have crossed the low and high NCX TG with a model of MD, which lacks delta-sarcoglycan (Scgd-/-, a component of the DGC), and, preliminarily, no benefit was observed at 6 weeks or 6 months of age from the low line cross. As another model of altered Ca2+ levels we have also generated mice overexpressing transient receptor potential channel of the canonical subclass (TRPC3, one line). Similarly, these mice also exhibit disease at 2 months of age as muscle weights are reduced compared to NTG and gross histology is abnormal. Compared to the NCX TG mice, TRPC3 TGs have more skeletal muscle disease (3.8% of quadriceps myocytes contain central nuclei in high line of NCX TG and 26.6% in the same muscle of the TRPC3 TGs) possibly because the pathway involving TRPC3 is a true influx pathway. We are currently also crossing the TRPC3 TGs with the d-sarcoglycan MD model. Finally, Plasma Membrane Calcium ATPase (PMCA4b). Young animals did not display skeletal muscle disease, however there was some disease at 6 months of age and we are currently crossing these mice with our MD model to test whether MD pathogenesis can be reduced through greater calcium clearance. Overall, our preliminary results indicate altered Ca2+ handling can cause disease without a mutation in the DGC and our future results should allow for analysis of whether enhanced removal of intracellular Ca2+ benefits MD pathogenesis or even enhances it by altering trans-sarcolemmal flux.we have generated one line of TG mice overexpressing

TAT-Utrophin crosses cell barriers to combat dystrophin deficiency

Sonnemann, Kevin J; Heun, Hanke; Baltgalvis, Kristen; Lowe, Dawn; Ervasti, James M

Deptartment of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN

Upregulation of the dystrophin homolog utrophin by gene delivery or pharmacological means is thought to provide one possible mechanism to restore membrane integrity and combat the phenotypic effects of dystrophin deficiency. However, despite significant effort, no therapeutic interventions are currently available. Here we identify a novel protein-delivery therapy and show that repeated intraperitoneal injections of cell-penetrating recombinant TAT-utrophin protein into the dystrophin-deficient mdx mouse elevated utrophin levels in all tissues examined, partially restored muscle membrane integrity, reduced the prevalence of muscle fibrosis and degeneration, and improved physiological performance in a dose-dependent manner. These results triple the known size capacity for effective TAT-mediated cell transduction and establish the feasibility of TAT-utrophin as a novel protein-based therapy for the treatment of skeletal muscle and cardiac diseases caused by loss of dystrophin.

Adipocyte-derived factors suppress heart contraction

Christiane Look, Valeria Lamounier-Zepter, Stefan R. Bornstein, Monika Ehrhart-Bornstein, Ingo Morano

Department of Internal Medicine, TU Dresden; Max-Delbruck-Center for Molecular Medicine, Berlin-Buch, Germany

Obesity is a major risk factor for metabolic syndrome and cardiovascular disorders. Obesity-related heart disease is the most serious complication of human obesity. Despite of several investigations the pathophysiological mechanisms involved remain unclear. Latest studies have emphasized the importance of adipose tissue as a highly endocrine organ which releases a wide variety of biological active substances. In this context we have recently showed that adipose tissue exerts highly potent cardiodepressant activity with an acute effect directly on cardiomyocytes contraction. In the present study we investigated whether the adipocyte-derived factors also affect the whole cardiac function. We isolated adipocytes from human subcutaneous fat tissue and treated isolated perfused rat hearts (Langendorff mode) with the secretory products of the adipocytes. We recorded changes in coronary flow (CF) and developed isovolumetric left ventricular pressure (LVP). From the LVP we calculated maximal rate of isovolumetric pressure increase (+dP/dtmax) and maximal rate of isovolumetric pressure decrease (-dP/dtmax) as contractility parameters. We could observe a significant and reversible decrease of all four parameters within a few seconds after incubation with the adipocyte-factors. This cardiodepressant effect could not be blocked by the perfusion of the hearts with a solution containing the non-selective COX-inhibitor indomethacin. These data indicate that human adipocytes secrete factors with a strong acute depressant effect on cardiac force generation as well as coronary flow due to contraction of the coronary vessels, thus suggesting a direct role of adipose tissue in the pathogenesis of cardiac dysfunction.

The dynamics of sarcomere assembly in Drosophila/ flight muscle

Z Orfanos, JC Sparrow

Department of Biology, University of York, Heslington, York, YO10 5DD, United Kingdom

Differentiation of striated muscle involves the organization of proteins, expressed after myoblast fusion, into the highly ordered myofibrils. These are serial assemblies of the fundamental functional contractile unit known as the sarcomere. Sarcomeric assembly is most commonly studied in cells in culture. I am using an in vivo molecular genetic approach in the Drosophila flight muscle, one of the most regular muscles found in nature. The process starts with the appearance of linear arrays of electron-dense "Z-bodies", precursors of Z-discs that define the sarcomere borders. Using a GFP-tagged version of the Z-disc protein sallimus, I am following assembly dynamics with confocal microscopy. Here I show that assembly is synchronous throughout the muscle, and I pinpoint the start of the lateral extension from Z-bodies to mature Z-discs in the overall assembly process. Furthermore, using a GFP-tagged version of myosin heavy chain, I show that different isoforms of the protein are used before and after the beginning of the lateral Z-disc extension phase. Finally, I attempt to pinpoint the first appearance of various sarcomeric proteins (such as projectin and flightin) during development and relate their functions to the above processes. My results contribute towards the establishment of a model for sarcomere assembly in the Drosophila flight muscle.