Multimodal three-dimensional characterization of murine skeletal muscle micro-scale elasticity, structure, and composition: Impact of dysferlinopathy, Duchenne muscular dystrophy, and age on three hind-limb muscles.

Skeletal muscle tissue function is governed by the mechanical properties and organization of its components, including myofibers, extracellular matrix, and adipose tissue, which can be modified by the onset and progression of many disorders. This study used a novel combination of quantitative micro-elastography and clearing-enhanced three-dimensional (3D) microscopy to assess 3D micro-scale elasticity and micro-architecture of muscles from two muscular dystrophies: dysferlinopathy and Duchenne muscular dystrophy, using male BLA/J and mdx mice, respectively, and their wild-type (WT) controls. We examined three muscles with varying proportions of slow- and fast-twitch myofibers: the soleus (predominantly slow), extensor digitorum longus (EDL; fast), and quadriceps (mixed), from BLA/J and WTBLA/J mice aged 3, 10, and 24 months, and mdx and WTmdx mice aged 10 months. Both dysferlin deficiency and age reduced the elasticity and variability of elasticity of the soleus and quadriceps, but not EDL. Overall, the BLA/J soleus was 20% softer than WT and less mechanically heterogeneous (-14% in standard deviation of elasticity). The BLA/J quadriceps at 24 months was 72% softer than WT and less mechanically heterogeneous (-59% in standard deviation), with substantial adipose tissue accumulation. While mdx muscles did not differ quantitatively from WT, regional heterogeneity was evident in micro-scale elasticity and micro-architecture of quadriceps (e.g., 11.2 kPa in a region with marked pathology vs 3.8 kPa in a less affected area). These results demonstrate differing biomechanical changes in hind-limb muscles of two distinct muscular dystrophies, emphasizing the potential for this novel multimodal technique to identify important differences between various myopathies.

Pilot investigations into the mechanistic basis for adverse effects of glucocorticoids in dysferlinopathy.

BACKGROUND: Dysferlinopathies are a clinically heterogeneous group of muscular dystrophies caused by gene mutations resulting in deficiency of the membrane-associated protein dysferlin. They manifest post-growth and are characterised by muscle wasting (primarily in the limb and limb-gridle muscles), inflammation, and replacement of myofibres with adipose tissue. The precise pathomechanism for dysferlinopathy is currently unclear; as such there are no treatments currently available. Glucocorticoids (GCs) are widely used to reduce inflammation and treat muscular dystrophies, but when administered to patients with dysferlinopathy, they have unexpected adverse effects, with accelerated loss of muscle strength. METHODS: To investigate the mechanistic basis for the adverse effects of GCs in dysferlinopathy, the potent GC dexamethasone (Dex) was administered for 4-5 weeks (0.5-0.75 µg/mL in drinking water) to dysferlin-deficient BLA/J and normal wild-type (WT) male mice, sampled at 5 (Study 1) or 10 months (Study 2) of age. A wide range of analyses were conducted. Metabolism- and immune-related gene expression was assessed in psoas muscles at both ages and in quadriceps at 10 months of age. For the 10-month-old mice, quadriceps and psoas muscle histology was assessed. Additionally, we investigated the impact of Dex on the predominantly slow and fast-twitch soleus and extensor digitorum longus (EDL) muscles (respectively) in terms of contractile function, myofibre-type composition, and levels of proteins related to contractile function and metabolism, plus glycogen. RESULTS: At both ages, many complement-related genes were highly expressed in BLA/J muscles, and WT mice were generally more responsive to Dex than BLA/J. The effects of Dex on BLA/J mice included (i) increased expression of inflammasome-related genes in muscles (at 5 months) and (ii) exacerbated histopathology of quadriceps and psoas muscles at 10 months. A novel observation was pronounced staining for glycogen in many myofibres of the damaged quadriceps muscles, with large pale vacuolated myofibres, suggesting possible myofibre death by oncosis. CONCLUSION: These pilot studies provide a new focus for further investigation into the adverse effects of GCs on dysferlinopathic muscles.

Considering the Promise of Vamorolone for Treating Duchenne Muscular Dystrophy.

This commentary provides an independent consideration of data related to the drug vamorolone (VBP15) as an alternative steroid proposed for treatment of Duchenne muscular dystrophy (DMD). Glucocorticoids such as prednisone and deflazacort have powerful anti-inflammatory benefits and are the standard of care for DMD, but their long-term use can result in severe adverse side effects; thus, vamorolone was designed as a unique dissociative steroidal anti-inflammatory drug, to retain efficacy and minimise these adverse effects. Extensive clinical trials (ongoing) have investigated the use of vamorolone for DMD, with two trials also for limb-girdle muscular dystrophies including dysferlinopathy (current), plus a variety of pre-clinical trials published. Vamorolone looks very promising, with similar efficacy and some reduced adverse effects (e.g., related to height) compared with other glucocorticoids, specifically prednisone/prednisolone, although it has not yet been directly compared with deflazacort. Of particular interest to clarify is the optimal clinical dose and other aspects of vamorolone that are proposed to provide additional benefits for membranes of dystrophic muscle: to stabilise and protect the sarcolemma from damage and enhance repair. The use of vamorolone (and other glucocorticoids) needs to be evaluated in terms of overall long-term efficacy and cost, and also in comparison with many candidate non-steroidal drugs with anti-inflammatory and other benefits for DMD.

Slow or fast: Implications of myofibre type and associated differences for manifestation of neuromuscular disorders.

Many neuromuscular disorders can have a differential impact on a specific myofibre type, forming the central premise of this review. The many different skeletal muscles in mammals contain a spectrum of slow- to fast-twitch myofibres with varying levels of protein isoforms that determine their distinctive contractile, metabolic, and other properties. The variations in functional properties across the range of classic 'slow' to 'fast' myofibres are outlined, combined with exemplars of the predominantly slow-twitch soleus and fast-twitch extensor digitorum longus muscles, species comparisons, and techniques used to study these properties. Other intrinsic and extrinsic differences are discussed in the context of slow and fast myofibres. These include inherent susceptibility to damage, myonecrosis, and regeneration, plus extrinsic nerves, extracellular matrix, and vasculature, examined in the context of growth, ageing, metabolic syndrome, and sexual dimorphism. These many differences emphasise the importance of carefully considering the influence of myofibre-type composition on manifestation of various neuromuscular disorders across the lifespan for both sexes. Equally, understanding the different responses of slow and fast myofibres due to intrinsic and extrinsic factors can provide deep insight into the precise molecular mechanisms that initiate and exacerbate various neuromuscular disorders. This focus on the influence of different myofibre types is of fundamental importance to enhance translation for clinical management and therapies for many skeletal muscle disorders.

Correction: Dysferlin-deficiency has greater impact on function of slow muscles, compared with fast, in aged BLAJ mice.

[This corrects the article DOI: 10.1371/journal.pone.0214908.].

Three-dimensional mechanical characterization of murine skeletal muscle using quantitative micro-elastography.

Skeletal muscle function is governed by both the mechanical and structural properties of its constituent tissues, which are both modified by disease. Characterizing the mechanical properties of skeletal muscle tissue at an intermediate scale, i.e., between that of cells and organs, can provide insight into diseases such as muscular dystrophies. In this study, we use quantitative micro-elastography (QME) to characterize the micro-scale elasticity of ex vivo murine skeletal muscle in three-dimensions in whole muscles. To address the challenge of achieving high QME image quality with samples featuring uneven surfaces and geometry, we encapsulate the muscles in transparent hydrogels with flat surfaces. Using this method, we study aging and disease in quadriceps tissue by comparing normal wild-type (C57BL/6J) mice with dysferlin-deficient BLAJ mice, a model for the muscular dystrophy dysferlinopathy, at 3, 10, and 24 months of age (sample size of three per group). We observe a 77% decrease in elasticity at 24 months in dysferlin-deficient quadriceps compared to wild-type quadriceps.

Dysferlin Deficiency Results in Myofiber-Type Specific Differences in Abundances of Calcium-Handling and Glycogen Metabolism Proteins.

Dysferlinopathies are a clinically heterogeneous group of muscular dystrophies caused by a genetic deficiency of the membrane-associated protein dysferlin, which usually manifest post-growth in young adults. The disease is characterized by progressive skeletal muscle wasting in the limb-girdle and limbs, inflammation, accumulation of lipid droplets in slow-twitch myofibers and, in later stages, replacement of muscles by adipose tissue. Previously we reported myofiber-type specific differences in muscle contractile function of 10-month-old dysferlin-deficient BLAJ mice that could not be fully accounted for by altered myofiber-type composition. In order to further investigate these findings, we examined the impact of dysferlin deficiency on the abundance of calcium (Ca2+) handling and glucose/glycogen metabolism-related proteins in predominantly slow-twitch, oxidative soleus and fast-twitch, glycolytic extensor digitorum longus (EDL) muscles of 10-month-old wild-type (WT) C57BL/6J and dysferlin-deficient BLAJ male mice. Additionally, we compared the Ca2+ activation properties of isolated slow- and fast-twitch myofibers from 3-month-old WT and BLAJ male mice. Differences were observed for some Ca2+ handling and glucose/glycogen metabolism-related protein levels between BLAJ soleus and EDL muscles (compared with WT) that may contribute to the previously reported differences in function in these BLAJ muscles. Dysferlin deficiency did not impact glycogen content of whole muscles nor Ca2+ activation of the myofilaments, although soleus muscle from 10-month-old BLAJ mice had more glycogen than EDL muscles. These results demonstrate a further impact of dysferlin deficiency on proteins associated with excitation-contraction coupling and glycogen metabolism in skeletal muscles, potentially contributing to altered contractile function in dysferlinopathy.

Mouse models for muscular dystrophies: an overview.

Muscular dystrophies (MDs) encompass a wide variety of inherited disorders that are characterized by loss of muscle tissue associated with a progressive reduction in muscle function. With a cure lacking for MDs, preclinical developments of therapeutic approaches depend on well-characterized animal models that recapitulate the specific pathology in patients. The mouse is the most widely and extensively used model for MDs, and it has played a key role in our understanding of the molecular mechanisms underlying MD pathogenesis. This has enabled the development of therapeutic strategies. Owing to advancements in genetic engineering, a wide variety of mouse models are available for the majority of MDs. Here, we summarize the characteristics of the most commonly used mouse models for a subset of highly studied MDs, collated into a table. Together with references to key publications describing these models, this brief but detailed overview would be useful for those interested in, or working with, mouse models of MD.

Dysferlin deficiency alters lipid metabolism and remodels the skeletal muscle lipidome in mice.

Defects in the gene coding for dysferlin, a membrane-associated protein, affect many tissues, including skeletal muscles, with a resultant myopathy called dysferlinopathy. Dysferlinopathy manifests postgrowth with a progressive loss of skeletal muscle function, early intramyocellular lipid accumulation, and a striking later replacement of selective muscles by adipocytes. To better understand the changes underpinning this disease, we assessed whole-body energy homeostasis, skeletal muscle fatty acid metabolism, lipolysis in adipose tissue, and the skeletal muscle lipidome using young adult dysferlin-deficient male BLAJ mice and age-matched C57Bl/6J WT mice. BLAJ mice had increased lean mass and reduced fat mass associated with increased physical activity and increased adipose tissue lipolysis. Skeletal muscle fatty acid metabolism was remodeled in BLAJ mice, characterized by a partitioning of fatty acids toward storage rather than oxidation. Lipidomic analysis identified marked changes in almost all lipid classes examined in the skeletal muscle of BLAJ mice, including sphingolipids, phospholipids, cholesterol, and most glycerolipids but, surprisingly, not triacylglycerol. These observations indicate that an early manifestation of dysferlin deficiency is the reprogramming of skeletal muscle and adipose tissue lipid metabolism, which is likely to contribute to the progressive adverse histopathology in dysferlinopathies.

Dysferlin-deficiency has greater impact on function of slow muscles, compared with fast, in aged BLAJ mice.

Dysferlinopathies are a form of muscular dystrophy caused by gene mutations resulting in deficiency of the protein dysferlin. Symptoms manifest later in life in a muscle specific manner, although the pathomechanism is not well understood. This study compared the impact of dysferlin-deficiency on in vivo and ex vivo muscle function, and myofibre type composition in slow (soleus) and fast type (extensor digitorum longus; EDL) muscles using male dysferlin-deficient (dysf-/-) BLAJ mice aged 10 months, compared with wild type (WT) C57Bl/6J mice. There was a striking increase in muscle mass of BLAJ soleus (+25%) (p<0.001), with no strain differences in EDL mass, compared with WT. In vivo measures of forelimb grip strength and wheel running capacity showed no strain differences. Ex vivo measures showed the BLAJ soleus had faster twitch contraction (-21%) and relaxation (-20%) times, and delayed post fatigue recovery (ps<0.05); whereas the BLAJ EDL had a slower relaxation time (+11%) and higher maximum rate of force production (+25%) (ps<0.05). Similar proportions of MHC isoforms were evident in the soleus muscles of both strains (ps>0.05); however, for the BLAJ EDL, there was an increased proportion of type IIx MHC isoform (+5.5%) and decreased type IIb isoform (-5.5%) (ps<0.01). This identification of novel differences in the impact of dysferlin-deficiency on slow and fast twitch muscles emphasises the importance of evaluating myofibre type specific effects to provide crucial insight into the mechanisms responsible for loss of function in dysferlinopathies; this is critical for the development of targeted future clinical therapies.