Assessment of systemic AAV-microdystrophin gene therapy in the GRMD model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disease caused by the absence of dystrophin, a membrane-stabilizing protein encoded by the DMD gene. Although mouse models of DMD provide insight into the potential of a corrective therapy, data from genetically homologous large animals, such as the dystrophin-deficient golden retriever muscular dystrophy (GRMD) model, may more readily translate to humans. To evaluate the clinical translatability of an adeno-associated virus serotype 9 vector (AAV9)-microdystrophin (µDys5) construct, we performed a blinded, placebo-controlled study in which 12 GRMD dogs were divided among four dose groups [control, 1 × 1013 vector genomes per kilogram (vg/kg), 1 × 1014 vg/kg, and 2 × 1014 vg/kg; n = 3 each], treated intravenously at 3 months of age with a canine codon-optimized microdystrophin construct, rAAV9-CK8e-c-µDys5, and followed for 90 days after dosing. All dogs received prednisone (1 milligram/kilogram) for a total of 5 weeks from day -7 through day 28. We observed dose-dependent increases in tissue vector genome copy numbers; µDys5 protein in multiple appendicular muscles, the diaphragm, and heart; limb and respiratory muscle functional improvement; and reduction of histopathologic lesions. As expected, given that a truncated dystrophin protein was generated, phenotypic test results and histopathologic lesions did not fully normalize. All administrations were well tolerated, and adverse events were not seen. These data suggest that systemically administered AAV-microdystrophin may be dosed safely and could provide therapeutic benefit for patients with DMD.

A Low-Cost Pulse Generator for Exacerbating Muscle Fiber Detachment Phenotypes in Zebrafish.

Muscle fiber detachment from myoseptal boundaries is a common finding in zebrafish models of muscular dystrophies. In some instances, there is a weakening of the interaction between muscle fiber and myosepta, which is yet to manifest as a fiber detachment phenotype. Therefore, to push the fiber detachment of muscle, mutant fish but not their wild-type siblings, beyond their binding threshold, a series of small electrical pulses can be applied to the larvae to create a maximal force contraction and ultimately fiber detachment. To do this, we built a digital pulse generator which delivers four 8 ms 30 V pulses in quick succession, and it has the advantage over older analog approaches to pulse generation because it improves accuracy and is appreciably less expensive. Our pulse generator significantly increases fiber detachment in the laminin-α2 deficient, congenital muscular dystrophy type 1a (MDC1a) model lama2-/- fish when compared with controls.

Contractile efficiency of dystrophic mdx mouse muscle: in vivo and ex vivo assessment of adaptation to exercise of functional end points.

Progressive weakness is a typical feature of Duchenne muscular dystrophy (DMD) patients and is exacerbated in the benign mdx mouse model by in vivo treadmill exercise. We hypothesized a different threshold for functional adaptation of mdx muscles in response to the duration of the exercise protocol. In vivo weakness was confirmed by grip strength after 4, 8, and 12 wk of exercise in mdx mice. Torque measurements revealed that exercise-related weakness in mdx mice correlated with the duration of the protocol, while wild-type (WT) mice were stronger. Twitch and tetanic forces of isolated diaphragm and extensor digitorum longus (EDL) muscles were lower in mdx compared with WT mice. In mdx, both muscle types exhibited greater weakness after a single exercise bout, but only in EDL after a long exercise protocol. As opposite to WT muscles, mdx EDL ones did not show any exercise-induced adaptations against eccentric contraction force drop. qRT-PCR analysis confirmed the maladaptation of genes involved in metabolic and structural remodeling, while damage-related genes remained significantly upregulated and angiogenesis impaired. Phosphorylated AMP kinase level increased only in exercised WT muscle. The severe histopathology and the high levels of muscular TGF-ß1 and of plasma matrix metalloproteinase-9 confirmed the persistence of muscle damage in mdx mice. Therefore, dystrophic muscles showed a partial degree of functional adaptation to chronic exercise, although not sufficient to overcome weakness nor signs of damage. The improved understanding of the complex mechanisms underlying maladaptation of dystrophic muscle paves the way to a better managment of DMD patients.NEW & NOTEWORTHY We focused on the adaptation/maladaptation of dystrophic mdx mouse muscles to a standard protocol of exercise to provide guidance in the development of more effective drug and physical therapies in Duchenne muscular dystrophy. The mdx muscles showed a modest functional adaptation to chronic exercise, but it was not sufficient to overcome the progressive in vivo weakness, nor to counter signs of muscle damage. Therefore, a complex involvement of multiple systems underlies the maladaptive response of dystrophic muscle.

A Simple and Low-cost Assay for Measuring Ambulation in Mouse Models of Muscular Dystrophy.

Measuring functional outcomes in the treatment of muscular dystrophy is an essential aspect of preclinical testing. The assessment of voluntary ambulation in mouse models is a non-invasive and reproducible activity assay that is directly analogous to measures of patient ambulation such as the 6-minute walk test and related mobility scores. Many common methods for testing mouse ambulation speed and distance are based on the open field test, where an animal's free movement within an arena is measured over time. One major downside to this approach is that commercial software and equipment for high-resolution motion tracking is expensive and may require transferring mice to specialized facilities for testing. Here, we describe a low-cost, video-based system for measuring mouse ambulation that utilizes free and open-source software. Using this protocol, we demonstrate that voluntary ambulation in the dystrophin-null mdx mouse model for Duchenne muscular dystrophy (DMD) is decreased relative to wild-type mouse activity. In mdx mice expressing the utrophin transgene, these activity deficits are not observed and the total distance traveled is indistinguishable from wild-type mice. This method is effective for measuring changes in voluntary ambulation associated with dystrophic pathology, and provides a versatile platform that can be readily adapted to diverse research settings.

Effect of intraperitoneal injection of glucose on glucose oxidation and energy expenditure in the mdx mouse model of duchenne muscular dystrophy.

Previous studies suggesting that glucose metabolism could be impaired in the skeletal muscles of the mdx mouse led us to study the metabolic response to i.p. injection of either glucose or glucose and insulin in the free-moving mdx mouse. In the first study, changes in blood glucose and plasma insulin levels were measured in mice with chronic venous cannulae. In the second study, the thermogenic response to glucose and the changes induced on glucose oxidation (Gox) and lipid oxidation (Lox) were assessed by indirect calorimetry. The experiments showed that insulin response, as well as whole body glucose uptake, were normal in mdx mice. Addition of exogenous insulin abolished the increase in blood glucose level similarly in mdx and control mice. The thermogenic response to glucose was identical in mdx and control mice but, when insulin was injected with glucose, it increased significantly in mdx mice. Exogenous glucose increased Gox less and decreased Lox less in mdx than in control mice. Addition of exogenous insulin reduced the difference between mdx and control mice but affected Gox and Lox less in mdx than in control mice. Key words Duchenne muscular dystrophy middle dot mdx Mouse middle dot Glucose tolerance test middle dot Respiratory quotient middle dot Energy metabolism middle dot Indirect calorimetry

Components of energy expenditure in the mdx mouse model of Duchenne muscular dystrophy.

Previous observations showing that basal heat production rates and glucose metabolism were reduced in mdx mouse skeletal muscles incubated in vitro led us to study the components of total energy expenditure by open-circuit indirect calorimetry in the intact, free-moving mdx mouse. Our purpose was to verify if the mdx mouse exhibited whole-body alterations in energy metabolism. The results revealed that total and basal energy expenditure, as well as spontaneous activity, energetic cost of activity, and, therefore, energy expended in relation to activity were not significantly different in C57B1/10 (control) and in dystrophic (mdx) mice. In contrast, the thermic effect of food was 32% larger in mdx than in control mice and was accompanied by significant differences in post-prandial glucose and lipid oxidation. The present in vivo study could not show a direct demonstration that impaired glucose metabolism by skeletal muscles participated in this phenomenon. However, since post-prandial glucose metabolism by skeletal muscles contributes a significant part of the thermic effect of food, the present data are in line with previous studies in vitro that show that mdx mouse skeletal muscles probably suffer an impaired control of their energy metabolism.