The D2.mdx mouse as a preclinical model of the skeletal muscle pathology associated with Duchenne muscular dystrophy

Abstract

Duchenne muscular dystrophy (DMD) is an X-linked, lethal muscle degenerative disease caused by loss of dystrophin protein. DMD has no cure and few treatment options. Preclinical efforts to identify potential DMD therapeutics have been hampered by lack of a small animal model that recapitulates key features of the human disease. While the dystrophin-deficient mdx mouse on the C57BL/10 genetic background (B10.mdx) is mildly affected, a more severe muscle disease is observed when the mdx mutation is crossed onto the DBA/2J genetic background (D2.mdx). In this study, the functional and histological progression of the D2.mdx skeletal muscle pathology was evaluated to determine the distinguishing features of disease. Data herein details the muscular weakness and wasting exhibited by D2.mdx skeletal muscle, as well as severe histopathological features, which include the rapid progression of fibrosis and calcifications in the diaphragm and progressive fibrosis accumulation in limb muscles. Furthermore, a timeline of D2.mdx progression is provided that details distinct stages of disease progression. These data support the D2.mdx as a superior small animal model for DMD, as compared to the B10.mdx model. The insights provided in this report should facilitate the design of preclinical evaluations for potential DMD therapeutics. Effective preclinical evaluation of potential therapeutics for human diseases requires thorough understanding of the disease phenotype exhibited by animal models utilized during such studies. Duchenne muscular dystrophy (DMD) is a fatal X-linked pediatric muscle disease with no cure and limited treatment options for affected patients. DMD is caused by mutations in the DMD gene resulting in complete loss of the gene's protein product, dystrophin 1 , a molecule that provides stabilization of the sarcolemma during muscular contraction by linking the muscle cytoskeleton and extracellular matrix 2,3. Preclinical work to study DMD has largely utilized the mdx mouse harboring a nonsense mutation in exon 23 of Dmd, which has been primarily maintained on the C57BL/10 genetic background (referred to as B10.mdx). This genetic homolog of DMD, however, exhibits a markedly mild disease phenotype compared to the human disease, limiting its potential to accurately model possible DMD therapeutics. Recent efforts to identify murine models of DMD that more accurately recapitulate the severity of the human condition than the B10.mdx mouse have led to the discovery that crossing the mdx mutation onto the DBA/2J genetic background (referred to D2.mdx) results in a more severe disease phenotype. This model exhibits greater muscle damage, impaired muscle regeneration, muscle wasting, and exacerbated progression of intramuscular fibrosis than age-matched B10.mdx mice 4-7. The heightened severity of the D2.mdx mouse has been linked to a hyper-fibrotic polymorphism in latent TGFβ binding protein (LTBP) 4 8 , which has also been identified as a genetic modifier affecting disease progression within DMD patient populations 9. For...

Indices of Defective Autophagy in Whole Muscle and Lysosome Enriched Fractions From Aged D2-mdx Mice

“…Maintenance of autophagosome degradation in limb muscle is associated with attenuated functional decline in limb muscles (soleus and EDL) compared to diaphragms all from these animals (Spaulding et al, 2019b(Spaulding et al, , 2020b. The mechanism underlying this fundamental difference is beyond the scope of this investigation, though speculatively may be influenced by the different activation patterns of these muscles, exposure to different forces, their differing fiber types, and/or differing metabolic profiles (Silverman and Atwood, 1980;Parry and Parslow, 1981;Petrof et al, 1993a;Zardini and Parry, 1994;Augusto et al, 2004), compounded by different rates of disease progression (Hammers et al, 2020). While muscle-dependent differences in autophagy have been previously reported, to our knowledge, the mechanism underlying these differences has not been fully elucidated (Mizushima et al, 2004;Raben et al, 2008;Ogata et al, 2010;Mofarrahi et al, 2013;Fernando et al, 2020).…”

“…In support, cardioprotective effects were observed by treating mdx mice with rapamycin nanoparticles, and suggested amelioration of defective autophagy in dystrophic hearts (Bibee et al, 2014). Since the mdx model displays a mild disease phenotype, others have argued the more severe D2-mdx model may better recapitulate disease (Coley et al, 2016;van Putten et al, 2019) and, therefore, may more accurately predict therapeutic success in clinical trials (Hammers et al, 2020). However, how dystrophin deficiency alters autophagy in the D2-mdx model is not well-understood.…”

“…Mutations in the latent TGFβ binding protein gene (LTBP) cause increased fibrosis and in the Anxa6 gene result in decreased muscle repair in the DBA background, in addition to the nonsense mutation in exon 23 of dystrophin gene, were attributed for the severity of this model compared to the mdx model (Coley et al, 2016;van Putten et al, 2019). Given the potential importance of this new model for therapeutic development (Hammers et al, 2020), the purpose of this study was to perform an initial exploration of autophagosome degradation in the D2-mdx model. We hypothesized that, similar to mdx mice, dystrophin deficiency in D2-mdx mice would cause decreased lysosomal abundance and accumulation of autophagosomes.…”

Duchenne Muscular Dystrophy Animal Models

“…In contrast to original mdx mice D2-mdx mice developed cardiomyopathy at an earlier age, moreover, more fibrous tissue was observed in the hearts of D2-mdx mice [37]. The more pronounced dystrophic phenotype and faster progression of the disease in D2-mdx mice compared to mdx mice on C57BL/10 background makes D2-mdx mouse strain more suitable for evaluation of treatment efficacy in preclinical trials [42]. Immune-deficient mdx mice are used to test cell therapies as one of the approaches to treating muscular dystrophy.…”

The pan HDAC inhibitor Givinostat improves muscle function and histological parameters in two Duchenne muscular dystrophy murine models expressing different haplotypes of the LTBP4 gene

“…Recently, in the search of genetic determinants of DMD severity, the LTBP4 gene emerged as an important predictor of functional outcome trajectories. The LTBP4 gene encodes the latent transforming growth factor-β (TGF-β)-binding protein (Ltbp) 4 that binds TGF-β in the extracellular matrix, sequestering this cytokine [ 16 – 18 ]. During inflammatory processes, TGF-β is released from the Ltbp4 complex by proteolysis of the proline-rich hinge domain.…”