Codon-optimized human Smad7 gene therapy enhances skeletal muscle mass and function in a murine model of Duchenne muscular dystrophy.

not available

The objective of this investigation was to determine whether short-term heavy resistance training (RT) in healthy older men could eliminate deficits in muscle mass and strength (ST) compared with healthy younger men. Seventeen older men (60-71 yr) performed supervised RT for 22 weeks. Before and after RT, measurements were made for lean tissue mass (LTM), muscle thickness (MT), and ST (leg and bench press 1 repetition maximum) and were compared with values of younger men (n = 22-60 for the different measures, 18-31 yr). Before training, older men had significantly lower (p < 0.05) LTM (58.4 ± 7.0 kg), MT (3.4 ± 0.7 cm), and ST (leg press = 168 ± 33 kg; bench press = 75 ± 18 kg) compared with younger men (LTM 64.3 ± 7.1 kg; MT 4.0 ± 0.8 cm; leg press = 231 ± 54 kg; bench press = 121 ± 31 kg). All deficits were eliminated after 22 weeks of RT (LTM = 60.5 ± 7.6 kg; MT = 4.0 ± 0.7 cm; leg press = 222 ± 48 kg; bench press = 107 ± 19 kg). Short-term, heavy RT in healthy older men is sufficient to overcome deficits in muscle mass and ST when compared with healthy younger men. The practical application from this research is that healthy older men can be prescribed a whole-body heavy RT program to substantially increase muscle mass and ST to levels similar to young, active individuals.

Delivery of therapeutic transgenes with adeno-associated viral (AAV) vectors for treatment of myopathies has yielded encouraging results in animal models and early clinical studies. Although certain AAV serotypes efficiently target muscle fibers, transduction of the muscle stem cells, also known as satellite cells, is less studied. Here, we used a Pax7nGFP;Ai9 dual reporter mouse to quantify AAV transduction events in satellite cells. We assessed a panel of AAV serotypes for satellite cell tropism in the mdx mouse model of Duchenne muscular dystrophy and observed the highest satellite cell labeling with AAV9 following local or systemic administration. Subsequently, we used AAV9 to interrogate CRISPR/Cas9-mediated gene editing of satellite cells in the Pax7nGFP;mdx mouse. We quantified the level of gene editing using a Tn5 transposon-based method for unbiased sequencing of editing outcomes at the Dmd locus. We also found that muscle-specific promoters can drive transgene expression and gene editing in satellite cells. Lastly, to demonstrate the functionality of satellite cells edited at the Dmd locus by CRISPR in vivo, we performed a transplantation experiment and observed increased dystrophin-positive fibers in the recipient mouse. Collectively, our results confirm that satellite cells are transduced by AAV and can undergo gene editing to restore the dystrophin reading frame in the mdx mouse.

A Single Intravenous Injection of Adeno-associated Virus Serotype-9 Leads to Whole Body Skeletal Muscle Transduction in Dogs

Background: Cystinosis is associated with growth failure, myopathy and multiple risk factors for impaired bone development. The objectives of this study were to quantify muscle mass, muscle strength, and bone mineral density (BMD) in children and adults with cystinosis. Methods: This cross-sectional study assessed DXA regional lean mass, spine and hip BMD, handgrip and leg strength in 38 participants with cystinosis (ages 5-37 years) and 289 healthy controls. All BMD, muscle mass and muscle strength measures were expressed as sex-specific Z-scores relative to age and adjusted for height Z-score or limb length. Linear regression models were used to assess muscle strength relative to muscle mass (muscle specific force) and determine the impact of adjusting BMD results for muscle status. Results: Among adults, arm and leg lean mass (p &lt; 0.001), handgrip strength (p &lt; 0.001), proximal and distal leg strength (p &lt; 0.001), muscle specific force (p &lt; 0.01) and femoral neck and total hip BMD (p&lt;0.001) were markedly low, compared with controls. On average, muscle strength was more than 2 SD below normal, due to both low muscle mass and poor muscle quality. Among the children and adolescents, upper extremity lean mass and grip strength were preserved. However, leg lean mass (p &lt; 0.01), strength (p &lt; 0.001), muscle specific force (p &lt; 0.001) and femoral neck and total hip BMD (p &lt; 0.01) were reduced, compared with controls, approaching deficits seen in adults. Adjustment for lean mass and muscle strength markedly attenuated the BMD deficits. Conclusions: Cystinosis is associated with deficits in muscle mass and strength that far exceed those observed in CKD alone and is associated with low proximal femur BMD. Future studies are needed to determine if physical activity or other interventions to address sarcopenia in cystinosis will improve physical function and bone strength.

Mitochondrial dysfunction has been theorized to contribute to age-associated muscle atrophy. Deficits in muscle mass are thought to be due to a degree of anabolic resistance to growth-promoting stimuli; however, it is not known whether mitochondrial dysfunction contributes to an impaired synthetic response following resistance exercise. While long-term resistance training has been shown to influence mitochondrial transcripts, the acute response has not been well characterized. PURPOSE: To determine the ability of a single resistance-training bout to alter mitochondrial-related transcript abundance and whether such a response is influenced by age. METHODS: 19 younger (YNG; n=10, 21±3 y) and older (OLD; n=9, 70±4 y) sedentary males completed a single unilateral resistance-training bout for the knee extensors (four sets of 10 repetitions at 75%1RM for leg extension and leg press exercises). Muscle biopsies were taken from the unexercised leg to establish baseline measures and from the exercised leg at three, 24 and 48 hours post exercise along with blood from the antecubital vein. RESULTS: Both groups had similar lean body mass (66.0±10.8 kg YNG; 62.9±6.4 kg OLD); however OLD had reduced strength (leg press: 228±94 lbs YNG; 161±50 lbs OLD; leg extension: 117±47 lbs YNG; 82±23 lbs OLD; p<0.05). Serum creatine kinase was elevated over baseline to a similar extent for both groups at 24 and 48 h post-exercise (289±153% at 24h, 267±161% 48 h; p<0.001). PGC-1α mRNA increased three h post-exercise (441±274%, p<0.01), while Tfam mRNA was elevated at 24 h (123±29.5%, p<0.05). In contrast, ND1 and ND4 mRNA were repressed 48 h after exercise (82±60% and 63±26% of baseline, p<0.05). No effects of age were noted except for ND4 mRNA, where expression in YNG was elevated relative to OLD 24 h post-exercise (154±57%, p<0.05), before dropping to a repressed level similar to the older group at 48 h. CONCLUSION: Age does not appear to influence the dynamics of the PGC-1α, Tfam and ND1 mRNA response to a single resistance-training bout. Further work is required to clarify the significance of the signaling pathways responsible for the induction of PGC-1α, and whether this results in the co-activation of proteins responsible for the induction of mitochondrial adaptations to exercise.

Erythropoietin promotes myoblast proliferation and inhibits fibrosis and thus it could impede the pathogenesis of muscle degenerative diseases. However, its stimulation of erythropoiesis limits its use as a therapeutic agent. An erythropoietin analog, carbamylated erythropoietin (C-EPO), retains these protective actions, yet it does not interact with the erythropoietin receptor. To determine whether treatment with C-EPO alleviates the signs of muscular dystrophy in an animal model of Duchenne muscular dystrophy, we treated mdx mice with intraperitoneal injections of 50 μg/kg and 100 μg/kg C-EPO for 4 and 12 weeks, and we monitored weight, serum creatine kinase levels, and changes in muscle histology. Moderate histological improvement was observed at 4 weeks, which did not translate into a significantly decreased level of serum creatine kinase. At the doses tested, C-EPO is not an effective therapeutic for the treatment of a mouse model of Duchenne muscular dystrophy.

Three-dimensional optical coherence tomography (3D-OCT) was used to image the structure and pathology of skeletal muscle tissue from the treadmill-exercised mdx mouse model of human Duchenne muscular dystrophy. Optical coherence tomography (OCT) images of excised muscle samples were compared with co-registered hematoxylin and eosin-stained and Evans blue dye fluorescence histology. We show, for the first time, structural 3D-OCT images of skeletal muscle dystropathology well correlated with co-located histology. OCT could identify morphological features of interest and necrotic lesions within the muscle tissue samples based on intrinsic optical contrast. These findings demonstrate the utility of 3D-OCT for the evaluation of small-animal skeletal muscle morphology and pathology, particularly for studies of mouse models of muscular dystrophy.

CRISPR-Cas9 gene-editing technology has revolutionised the creation of precise and permanent modifications to DNA, enabling the generation of diverse animal models for investigating potential treatments. Here, we provide a protocol for the use of CRISPR-Cas9 to create murine models of Duchenne Muscular Dystrophy (DMD) along with a step-by-step guide for their phenotypic and molecular characterisation. The experimental procedures include CRISPR microinjection of embryos, molecular testing at the DNA, RNA, and protein levels, forelimb grip strength testing, immunostaining and serum creatine kinase (CK) testing. We further provide suggestions for analysis and interpretation of the generated data, as well as the limitations of our approach. These protocols are designed for researchers who intend to generate and use mouse models to study DMD as well as those seeking a detailed framework of phenotyping to contribute to the broader landscape of genetic disorder investigations.

Computed Tomography Measures of Nutrition in Patients With End-Stage Liver Disease Provide a Novel Approach to Characterize Deficits

Full-length dystrophin restoration via targeted exon integration by AAV-CRISPR in a humanized mouse model of Duchenne muscular dystrophy

Murine models of Duchenne muscular dystrophy: is there a best model?

The mdx mouse is the most widely-used animal model of the human disease Duchenne muscular dystrophy, and quantitative PCR analysis of gene expression in the muscles of this animal plays a key role in the study of pathogenesis and disease progression and in evaluation of potential therapeutic interventions. Normalization to appropriate stably-expressed reference genes is essential for accurate quantitative measurement, but determination of such genes is challenging: healthy and dystrophic muscles present very different transcriptional environments, further altering with disease progression and muscle use, raising the possibility that no single gene or combination of genes may be stable under all experimental comparative scenarios. Despite the pedigree of this animal model, this problem remains unaddressed. The aim of this work was therefore to comprehensively assess reference gene suitability in the muscles of healthy and dystrophic mice, identifying reference genes appropriate for specific experimental comparisons, and determining whether an essentially universally-applicable set of genes exists. Using a large sample collection comprising multiple muscles (including the tibialis anterior, diaphragm and heart muscles) taken from healthy and mdx mice at three disease-relevant ages, and a panel of sixteen candidate reference genes (FBXO38, FBXW2, MON2, ZFP91, HTATSF1, GAPDH, ACTB, 18S, CDC40, SDHA, RPL13a, CSNK2A2, AP3D1, PAK1IP1, B2M and HPRT1), we used the geNorm, BestKeeper and Normfinder algorithms to identify genes that were stable under multiple possible comparative scenarios. We reveal that no single gene is stable under all conditions, but a normalization factor derived from multiple genes (RPL13a, CSNK2A2, AP3D1 and the widely-used ACTB) appears suitable for normalizing gene expression in both healthy and dystrophic mouse muscle regardless of muscle type or animal age. We further show that other popular reference genes, including GAPDH, are markedly disease- or muscle-type correlated. This study demonstrates the importance of empirical reference gene identification, and should serve as a valuable resource for investigators wishing to study gene expression in mdx mice.

Duchenne muscular dystrophy (DMD) is a severe hereditary myopathy. Standard treatment by glucocorticosteroids is limited because of numerous side effects. The aim of this study was to test immunomodulation by human immunoglobulin G (IgG) as treatment in the experimental mouse model (mdx) of DMD. 2g/kg human IgG compared to human albumin was injected intraperitoneally in mdx mice at the age of 3 and 7weeks. Advanced voluntary wheel running parameters were recorded continuously. At the age of 11 weeks, animals were killed so that blood, diaphragm, and lower limb muscles could be removed for quantitative PCR, histological analysis and exvivo muscle contraction tests. IgG compared to albumin significantly improved the voluntary running performance and reduced muscle fatigability in an exvivo muscle contraction test. Upon IgG treatment, serum creatine kinase values were diminished and mRNA expression levels of relevant inflammatory markers were reduced in the diaphragm and limb muscles. Macrophage infiltration and myopathic damage were significantly ameliorated in the quadriceps muscle. Collectively, this study demonstrates that, in the early disease course of mdx mice, human IgG improves the running performance and diminishes myopathic damage and inflammation in the muscle. Therefore, IgG may be a promising approach for treatment of DMD. Two monthly intraperitoneal injections of human immunoglobulin G (IgG) improved the early 11-week disease phase of mdx mice. Voluntary running was improved and serum levels of creatine kinase were diminished. In the skeletal muscle, myopathic damage was ameliorated and key inflammatory markers such as mRNA expression of SPP1 and infiltration by macrophages were reduced. The study suggests that IgG could be explored as a potential treatment option for Duchenne muscular dystrophy and that pre-clinical long-term studies should be helpful.

Aging reduces skeletal muscle mass and strength, but the underlying molecular mechanisms remain elusive. Here, we used mouse models to investigate molecular mechanisms of age-related skeletal muscle weakness and atrophy as well as new potential interventions for these conditions. We identified two small molecules that significantly reduce age-related deficits in skeletal muscle strength, quality, and mass: ursolic acid (a pentacyclic triterpenoid found in apples) and tomatidine (a steroidal alkaloid derived from green tomatoes). Because small molecule inhibitors can sometimes provide mechanistic insight into disease processes, we used ursolic acid and tomatidine to investigate the pathogenesis of age-related muscle weakness and atrophy. We found that ursolic acid and tomatidine generate hundreds of small positive and negative changes in mRNA levels in aged skeletal muscle, and the mRNA expression signatures of the two compounds are remarkably similar. Interestingly, a subset of the mRNAs repressed by ursolic acid and tomatidine in aged muscle are positively regulated by activating transcription factor 4 (ATF4). Based on this finding, we investigated ATF4 as a potential mediator of age-related muscle weakness and atrophy. We found that a targeted reduction in skeletal muscle ATF4 expression reduces age-related deficits in skeletal muscle strength, quality, and mass, similar to ursolic acid and tomatidine. These results elucidate ATF4 as a critical mediator of age-related muscle weakness and atrophy. In addition, these results identify ursolic acid and tomatidine as potential agents and/or lead compounds for reducing ATF4 activity, weakness, and atrophy in aged skeletal muscle.