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Abstract P2069: In Vivo Investigation Of The Mechanisms Regulating Truncated Dystrophin Protein Turnover In The Heart

Numerous inherited and acquired cardiac diseases are directly linked to a deficiency in the protein dystrophin, such as the development of severe cardiomyopathy in Duchenne Muscular Dystrophy (DMD) patients. Dilated cardiomyopathy, arrhythmias, and congestive heart failure represent the most important life-limiting condition in 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 an innovative and valuable system to determine the direct physiological significance of dystrophin protein deficiency including determining the half-life of truncated dystrophin constructs. This system provides a novel means to investigate mechanistically how deletions in dystrophin affect therapeutically shortened dystrophin turnover in cardiac muscle in vivo. We used a floxed allele approach together with a cardiac directed (αMHC Mer-Cre-Mer) inducible Cre for precise control of full-length dystrophin or therapeutic 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 tissues were then 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 reported a fast turnover rate for micro-dystrophin in the heart, with calculated half-life of between 5-7 days. In marked contrast, full-length dystrophin was highly stable with dystrophin protein content (~ 40%) at 6 months, after dystrophin gene excision. Studies of in vivo full-length and micro-dystrophin protein half-life in skeletal muscle, are ongoing. This work will provide key information required for long-term success of ongoing and future DMD therapies featuring gene therapy with shortened dystrophins.