In vivo investigation of the mechanisms regulating truncated dystrophin protein turnover in intact cardiac and skeletal muscle



Poster Number: 181


Addeli B.B Angulski Spies, PhD, University of Minnesota, Nora Ahmed, MD, PhD, Medical school, University of Minnesota, John Bauer, University of Minnesota, Joseph Metzger, PhD, University of Minnesota

The Muscular Dystrophy Association (MDA) mission highlights a major goal of finding a cure for Duchenne Muscular Dystrophy (DMD). Currently, there is great excitement in the muscular dystrophy community as multiple ongoing gene-based therapies are advancing through clinical trials. These therapies utilize miniaturized dystrophin constructs, and these modified dystrophins are the focus of this study. We implemented a gene regulated system with high temporal and spatial specificity to determine the direct physiological significance of dystrophin protein deficiency including determining the half-life of truncated dystrophin in vivo. This system provides a robust means to investigate mechanistically how deletions in dystrophin affect therapeutically shortened dystrophin turnover in cardiac and skeletal muscle in vivo. We used a floxed allele approach together with a cardiac (αMHC) and skeletal (HSA) directed inducible Cre for precise control of dystrophin and micro-dystrophin gene excision. We examined the time course of full-length and micro-dystrophin mRNA as a biologically relevant surrogate for intact gene excision efficiency. Our data showed evidence of significant full-length dystrophin cDNA and micro-dystrophin gene excision, with a complete loss of micro-dystrophin mRNA and a gene excision efficiency of 80% for full-length dystrophin. Heart and skeletal tissues were extracted for protein quantitative analysis of micro-dystrophin (5, 10 and 30 days) and of full-length dystrophin (1, 3 and 6 months) post tamoxifen administration. Results demonstrate a fast turnover rate for micro-dystrophin in the heart and skeletal muscles, with calculated half-life of between 5-7 days in vivo. In marked contrast, full-length dystrophin was highly stable with dystrophin protein content (~ 40%) at 6 months, after dystrophin gene excision. Murf1 and Atrogin 1 were upregulated at the message level with Murf1 being also upregulated at the protein level in micro-dystrophin-derived skeletal muscle tissues compared to full length dystrophin, suggesting a possible involvement of Murf1 in the micro-dystrophin polyubiquitination and protein degradation pathway. Results from these studies will be highly informative in guiding the long-term success of ongoing and future DMD therapies featuring gene therapy with shortened dystrophins.