Hansell H. Stedman

Duchenne muscular dystrophy (DMD) is a progressive primary myodegenerative disease caused by a genetic deficiency of the 427-kDa cytoskeletal protein dystrophin. Despite its single-gene etiology, DMD’s complex pathogenesis remains poorly understood, complicating the extrapolation from results of preclinical studies in genetic homologs to the design of informative clinical trials. Here we describe novel phenotypic assays which when applied to the mdx mouse resemble recently used primary end points for DMD clinical trials. By coupling force transduction, high-precision motion tracking, and respiratory measurements, we have achieved a suite of integrative physiological tests that provide novel insights regarding normal and pathological responses to muscular exertion. A common feature of these physiological assays is the precise tracking and analysis of volitional movement, thereby optimizing the relevance to clinical tests. Unexpectedly, the measurable biological distinction between dystrophic and control mice at early time points in the disease process is better resolved with these tests than with the majority of previously used, labor-intensive studies of individual muscle function performed ex vivo. For example, the dramatic loss of volitional movement following a novel, standardized grip test distinguishes control mice from mdx mice by a 17.4-fold difference of the means (3.5 ± 2.2 vs. 60.9 ± 12.1 units of activity, respectively; effect size 1.99). The findings have both mechanistic and translational implications of potential significance to the fields of basic myology and neuromuscular therapeutics. NEW & NOTEWORTHY This study uses novel phenotypic assays which when applied to the mdx mouse resemble recently used primary end points for DMD clinical trials. A measurable distinction between dystrophic and control mice was seen at early time points in vivo compared with invasive muscle studies performed ex vivo. These assays shed light on normal and pathological responses to muscular exertion and have significant mechanistic and translational implications for the fields of basic myology and neuromuscular therapeutics.

Ventilatory insufficiency remains the leading cause of death and late stage morbidity in Duchenne muscular dystrophy (DMD). To address critical gaps in our knowledge of the pathobiology of respiratory functional decline, we used an integrative approach to study respiratory mechanics in a translational model of DMD. In studies of individual dogs with the Golden Retriever muscular dystrophy (GRMD) mutation, we found evidence of rapidly progressive loss of ventilatory capacity in association with dramatic morphometric remodeling of the diaphragm. Within the first year of life, the mechanics of breathing at rest, and especially during pharmacological stimulation of respiratory control pathways in the carotid bodies, shift such that the primary role of the diaphragm becomes the passive elastic storage of energy transferred from abdominal wall muscles, thereby permitting the expiratory musculature to share in the generation of inspiratory pressure and flow. In the diaphragm, this physiological shift is associated with the loss of sarcomeres in series (∼60%) and an increase in muscle stiffness (∼900%) compared with those of the nondystrophic diaphragm, as studied during perfusion ex vivo. In addition to providing much needed endpoint measures for assessing the efficacy of therapeutics, we expect these findings to be a starting point for a more precise understanding of respiratory failure in DMD.

Background— The muscular dystrophies exemplify a class of systemic disorders for which widespread protein replacement in situ is essential for treatment of the underlying genetic disorder. Somatic gene therapy will require efficient, scale-independent transport of DNA-containing macromolecular complexes too large to cross the continuous endothelia under physiological conditions. Previous studies in large-animal models have revealed a trade-off between the efficiency of gene transfer and the inherent safety of the required surgical and pharmacological interventions to achieve this. Methods and Results— Rats and dogs underwent limb or hemibody isolation via atraumatic tourniquet placement or myocardial isolation via heterotopic transplantation. Recombinant adenovirus (10 ¹³ particles per kilogram) or recombinant adeno-associated virus (10 ¹⁴ genome copies/kg) encoding the lacZ transgene was delivered through pressurized venous infusion without pharmacological mediators. Muscle exhibited almost 100% myofiber transduction in rats and dogs by X-galactosidase staining and significantly higher β-galactosidase levels compared with nonpressurized delivery. No significant difference was seen in β-galactosidase levels between 100- or 400-mm Hg groups. The Conclusions— Uniform scale- and vector-independent skeletal and cardiac myofiber transduction is facilitated by pressurized venous infusion in anatomic domains isolated from the central circulation without pharmacological interference with cardiovascular homeostasis. We provide the first demonstration of uniform gene transfer to muscle fibers of an entire extremity in the dog, providing a firm foundation for further translational studies of efficacy in canine models for human diseases.

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Sarcomeric myosin heavy chain (MyHC) is the major contractile protein of striated muscle. Six tandemly linked skeletal MyHC genes on chromosome 17 and two cardiac MyHC genes on chromosome 14 have been previously described in the human genome. We report the identification of three novel human sarcomeric MyHC genes on chromosomes 3, 7, and 20, which are notable for their atypical size and intron-exon structure. Two of the encoded proteins are structurally most like the slow-beta MyHC, whereas the third one is closest to the adult fast IIb isoform. Data from pairwise comparisons of aligned coding sequences imply the existence of ancestral genomes with four sarcomeric genes before the emergence of a dedicated smooth muscle MyHC gene. To further address the evolutionary relationships of the distinct sarcomeric and nonsarcomeric rod sequences, we have identified and further annotated human genomic DNA sequences corresponding to 14 class-II MyHCs. An extensive analysis provides a timeline for intron gain and loss, gene contraction and expansion, and gene conversion among genes encoding class-II myosins. One of the novel human genes is found to have introns at positions shared only with the molluscan catchin/MyHC gene, providing evidence for the structure of a pre-Cambrian ancestral gene.