Duchenne muscular dystrophy (DMD) is a lethal genetic muscle disorder caused by recessive mutations in dystrophin gene, affecting 1/3000 males. Gene therapy has been proven to ameliorate dystrophic pathology. To investigate therapeutic benefits from long-term effect of human mini-dystrophin and functional outcomes, transgenic mdx mice (Tg-mdx) containing a single copy of human mini-dystrophin (∆hDys3849) gene, five rods (Rods1-2, Rods22-24), and two hinges (H1 and H4) driven by a truncated creatine-kinase promoter (dMCK) in a recombinant adeno-associated viral vector (rAAV) backbone, were generated and used to determine gene expression and improvement of muscle function. Human mini-dystrophin gene expression was found in a majority of the skeletal muscles, but no expression in cardiac muscle. Dystrophin-associated glycoproteins (DAGs) such as sarcoglycans and nNOS were restored at the sarcolemma and coincided with human mini-dystrophin gene expression at the ages of 6, 10, and 20 months; Morphology of dystrophic muscle expressing the human mini-dystrophin gene was improved and central nuclei were reduced. Myofiber membrane integrity was improved by Evans blue dye test. Improvement in treadmill running and grip force was observed in transgenic mice at 6 months. Tetanic force and specific force of tibialis anterior (TA) muscle were significantly increased at the ages of 6, 10, and 20 months. Pseudohypertrophy was not found in TA muscle at 10 and 20 months when compared with wild-type C57 (WT) group. This study demonstrated that the long-term effects of human mini-dystrophin effectively ameliorated pathology and improved the functions of the dystrophic muscles in the transgenic DMD mouse model.

Duchenne Muscular Dystrophy (DMD) patients often suffer from both muscle wasting and osteoporosis. Our previous studies have revealed reduced regeneration potential in skeletal muscle and bone, concomitant with ectopic calcification of soft tissues in double knockout (dKO, dystrophin-/-; utrophin-/-) mice, a severe murine model for DMD. We found significant involvement of RhoA/ROCK (Rho-Associated Protein Kinase) signaling in mediating ectopic calcification of muscles in dKO mice. However, the cellular identity of these RhoA+ cells, and the role that RhoA plays in the chronic inflammation-associated pathologies has not been elucidated. Here, we report that CD68+ macrophages are highly prevalent at the sites of ectopic calcification of dKO mice, and that these macrophages highly express RhoA. Macrophages from dKO mice feature a shift towards a more pro-inflammatory M1 polarization and an increased expression of various senescence-associated secretory phenotype (SASP) factors that was reduced with the RhoA/ROCK inhibitor Y-27632. Further, systemic inhibition of RhoA activity in dKO mice led to reduced number of RhoA+/CD68+ cells, as well as a reduction in fibrosis and ectopic calcification. Together, these data revealed that RhoA signaling may be a key regulator of imbalanced mineralization in the dystrophic musculoskeletal system and consequently a therapeutic target for the treatment of DMD or other related muscle dystrophies.

Duchenne muscular dystrophy (DMD) is a devastating genetic disorder that leads to compromised cellular membranes, caused by the absence of membrane-bound dystrophin protein. Muscle membrane leakage results in disrupted intracellular homeostasis, protein degradation, and muscle wasting. Improving muscle membrane integrity may delay disease progression and extend the lifespan of DMD patients. Here, we demonstrate that exosomes, membranous extracellular vesicles, can elicit functional improvements in dystrophic mice by improving muscle membrane integrity. Systemic administration of exosomes from different sources induced phenotypic rescue and mitigated pathological progression in dystrophic mice without detectable toxicity. Improved membrane integrity conferred by exosomes inhibited intracellular calcium influx and calcium-dependent activation of calpain proteases, preventing the degradation of the destabilized dystrophin-associated protein complex. We show that exosomes, particularly myotube-derived exosomes, induced functional improvements and alleviated muscle deterioration by stabilizing damaged muscle membrane in dystrophic mice. Our findings suggest that exosomes may have therapeutic implications for DMD and other diseases with compromised membranes.

Duchenne muscular dystrophy (DMD) is a devastating disorder caused by loss of functional dystrophin protein, resulting in muscle wasting. Enhancing muscle growth by inhibiting myostatin, a growth factor negatively regulating skeletal muscle mass, is a promising approach to slow disease progression. Direct administration of myostatin propeptide, a natural inhibitor of mature myostatin, has shown limited efficacy probably due to low serum stability. Here, we demonstrate that serum stability, delivery efficiency and efficacy of propeptide can be significantly enhanced by anchoring propeptide to the surface of exosomes by fusing the inhibitory domain of myostatin propeptide into the second extracellular loop of CD63 (EXOpro). Repeated administrations of EXOpro accelerated muscle regeneration and growth, resulting in significantly increased muscle mass and functional rescue without any detectable toxicity in mdx mice. Importantly, EXOpro partially rehabilitated bone structure and promoted bone regeneration in mdx mice. Our findings demonstrate that anchoring to exosomes increased delivery and serum stability of propeptide and augmented the inhibitory efficacy of myostatin propeptide and thus provide a delivery platform for propeptide-based intervention in DMD.

Limb girdle muscular dystrophy type 2L (LGMD2L) and Miyoshi myopathy type 3 (MMD3) are autosomal recessive muscular dystrophy caused by mutations in the gene encoding anoctamin-5 (ANO5), which belongs to the anoctamin protein family. Two independent lines of mice with complete disruption of ANO5 transcripts did not exhibit overt muscular dystrophy phenotypes; instead, one of these mice was observed to present with some abnormality in sperm motility. In contrast, a third line of ANO5-knockout (KO) mice with residual expression of truncated ANO5 expression was reported to display defective membrane repair and very mild muscle pathology. Many of the ANO5-related patients carry point mutations or small insertions/deletions (indels) in the ANO5 gene. To more closely mimic the human ANO5 mutations, we engineered mutant ANO5 rabbits via co-injection of Cas9 mRNA and sgRNA into the zygotes. CRISPR-mediated small indels in the exon 12 and/or 13 in the mutant rabbits lead to the development of typical signs of muscular dystrophy with increased serum creatine kinase (CK), muscle necrosis, regeneration, fatty replacement and fibrosis. This novel ANO5 mutant rabbit model would be useful in studying the disease pathogenesis and therapeutic treatments for ANO5-deficient muscular dystrophy.

Fukutin-related protein (FKRP) mutations are the most common cause of dystroglycanopathies known to cause both limb girdle and congenital muscular dystrophy. The P448Lneo- mouse model has a knock-in mutation in the FKRP gene and develops skeletal, respiratory, and cardiac muscle disease.
We studied the natural history of the P448Lneo- mouse model over 9 months and the effects of twice weekly treadmill running. Forelimb and hindlimb grip strength (Columbus Instruments) and overall activity (Omnitech Electronics) assessed skeletal muscle function. Echocardiography was performed using VisualSonics Vevo 770 (FujiFilm VisualSonics). Plethysmography was performed using whole body system (ADInstruments). Histological evaluations included quantification of inflammation, fibrosis, central nucleation, and fiber size variation.
P448Lneo- mice had significantly increased normalized tissue weights compared to controls at 9 months of age for the heart, gastrocnemius, soleus, tibialis anterior, quadriceps, and triceps. There were no significant differences seen in forelimb or hindlimb grip strength or activity monitoring in P448Lneo- mice with or without exercise compared to controls. Skeletal muscles demonstrated increased inflammation, fibrosis, central nucleation, and variation in fiber size compared to controls (p < 0.05) and worsened with exercise. Plethysmography showed significant differences in respiratory rates and decreased tidal and minute volumes in P448Lneo- mice (p < 0.01). There was increased fibrosis in the diaphragm compared to controls (p < 0.01). Echocardiography demonstrated decreased systolic function in 9-month-old mutant mice (p < 0.01). There was increased myocardial wall thickness and mass (p < 0.001) with increased fibrosis in 9-month-old P448Lneo- mice compared to controls (p < 0.05). mRNA expression for natriuretic peptide type A (Nppa) was significantly increased in P448Lneo- mice compared to controls at 6 months (p < 0.05) and for natriuretic peptide type B (Nppb) at 6 and 9 months of age (p < 0.05).
FKRP-deficient P448Lneo- mice demonstrate significant deficits in cardiac and respiratory functions compared to control mice, and this is associated with increased inflammation and fibrosis. This study provides new functional outcome measures for preclinical trials of FKRP-related muscular dystrophies.

This study aimed to evaluate the role of echocardiography-based strain analysis in the early diagnosis and guidance for management of dilated cardiomyopathy (DCM). Muscular dystrophy mice (which spontaneously develop DCM) and control (C57 BL/6 J) mice were sequentially evaluated by ultrasound biomicroscopy, conventional left ventricle (LV) measurement, two-dimensional (2-D) strain analysis and myocardial histologic analysis for 12 consecutive months. Significant alternation of LV remodeling and dysfunction could be detected by conventional echocardiography after 9 mo, by strain analysis after 5 mo and by histologic analysis after 4 mo. The global longitudinal systolic peak strain (PK) was the most sensitive strain marker for early detection of myocardial structural abnormality in the subclinical stage. Moreover, losartan administration before the PK decrease was associated with significantly preserved LV function. These results suggest that myocardial strain analysis (particularly longitudinal PK) is sensitive for the early detection of LV dysfunction in mice with dilated cardiomyopathy.

Acetoacetate (AA) is a ketone body and acts as a fuel to supply energy for cellular activity of various tissues. Here, we uncovered a novel function of AA in promoting muscle cell proliferation. Notably, the functional role of AA in regulating muscle cell function is further evidenced by its capability to accelerate muscle regeneration in normal mice, and it ameliorates muscular dystrophy in mdx mice. Mechanistically, our data from multiparameter analyses consistently support the notion that AA plays a non-metabolic role in regulating muscle cell function. Finally, we show that AA exerts its function through activation of the MEK1-ERK1/2-cyclin D1 pathway, revealing a novel mechanism in which AA serves as a signaling metabolite in mediating muscle cell function. Our findings highlight the profound functions of a small metabolite as signaling molecule in mammalian cells.

OBJECTIVE: Mesenchymal stem cells (MSCs) represent a powerful tool in regenerative medicine because of their differentiation and migration capacities. We aimed to investigate the possibility of Flk-1(+)Sca-1(-) mesenchymal stem cells (Flk-1(+)Sca-1(-) MSCs) transplantation to repair erectile function in patients suffering from diabetes mellitus (DM)-associated erectile dysfunction (ED).
METHODS: In this study, we isolated Flk-1(+)Sca-1(-) MSCs from bone marrow (bMSCs). Then, newborn male rats were intraperitoneally injected with 5-ethynyl-2-deoxyuridine for the purpose of tracking endogenous Flk-1(+)Sca-1(-) MSCs. Eight weeks later, 8 of these rats were randomly chosen to serve as normal control (N group). The remaining rats were injected intraperitoneally with 60 mg/kg of streptozotocin (STZ) to induce DM. Eight of these rats were randomly chosen to serve as DM control (DM group) while another 8 rats were subject to Flk-1(+)Sca-1(-) MSCs treatment (DM+MSC group). All rats were evaluated for erectile function by intracavernous pressure (ICP) measurement. Afterward, their penile tissues were examined by histology.
RESULTS: Flk-1(+)Sca-1(-) MSCs could differentiate into skeletal muscle cells and endothelial cells in vivo and in vitro. Engrafted Flk-1(+)Sca-1(-) MSCs were shown to home to injured muscle, participate in myofibers repair and could partially reconstitute the sarcolemmal expression of myocardin and ameliorate the level of related specific pathological markers.
CONCLUSIONS: Flk-1(+)Sca-1(-) MSCs could be used in the treatment erectile function in diabetes mellitus associated erectile dysfunction by promoting regeneration of nNOS-positive nerves, endothelium, and smooth muscle in the penis.

Skeletal muscle stem cells, called satellite cells, are a quiescent heterogeneous population. Their heterogeneity is influenced by Pax7, a well-defined transcriptional regulator of satellite cell functions that defines two subpopulations: Pax7(Hi) and Pax7(Lo). However, the mechanisms by which these subpopulations are established and maintained during myogenesis are not completely understood. Here we show that miR-431, which is predominantly expressed in the skeletal muscle, mediates satellite cell heterogeneity by fine-tuning Pax7 levels during muscle development and regeneration. In miR-431 transgenic mice, the Pax7(Lo) subpopulation is enriched, enhances myogenic differentiation and accelerates muscle regeneration. Notably, miR-431 attenuates the muscular dystrophic phenotype in mdx mice and may be a potential therapeutic target in muscular diseases. miR-431 transgenic mice are a unique genetic model for investigating the cellular features and biological functions of Pax7(Lo) satellite cells during muscle development and regeneration.