Pax7, Pax3 and Mamstr genes are involved in skeletal muscle impaired regeneration of dy2J/dy2J mouse model of Lama2-CMD.

Congenital muscular dystrophy type-1A (Lama2-CMD) and Duchenne muscular dystrophy (DMD) result from deficiencies of laminin-α2 and dystrophin proteins, respectively. Although both proteins strengthen the sarcolemma, they are implicated in clinically distinct phenotypes. We used RNA-deep sequencing (RNA-Seq) of dy2J/dy2J, Lama2-CMD mouse model, skeletal muscle at 8 weeks of age to elucidate disease pathophysiology. This study is the first report of dy2J/dy2J model whole transcriptome profile. RNA-Seq of the mdx mouse model of DMD and wild-type (WT) mouse was carried as well in order to enable a novel comparison of dy2J/dy2J to mdx. A large group of shared differentially expressed genes (DEGs) was found in dy2J/dy2J and mdx models (1834 common DEGs, false discovery rate [FDR] < 0.05). Enrichment pathway analysis using ingenuity pathway analysis showed enrichment of inflammation, fibrosis, cellular movement, migration and proliferation of cells, apoptosis and necrosis in both mouse models (P-values 3E-10-9E-37). Via canonical pathway analysis, actin cytoskeleton, integrin, integrin-linked kinase, NF-kB, renin-angiotensin, epithelial-mesenchymal transition, and calcium signaling were also enriched and upregulated in both models (FDR < 0.05). Interestingly, significant downregulation of Pax7 was detected in dy2J/dy2J compared to upregulation of this key regeneration gene in mdx mice. Pax3 and Mamstr genes were also downregulated in dy2J/dy2J compared to WT mice. These results may explain the distinct disease course and severity in these models. While the mdx model at that stage shows massive regeneration, the dy2J/dy2J shows progressive dystrophic process. Our data deepen our understanding of the molecular pathophysiology and suggest new targets for additional therapies to upregulate regeneration in Lama2-CMD.

Liposomal steroid nano-drug is superior to steroids as-is in mdx mouse model of Duchenne muscular dystrophy.

Glucocorticosteroids are the most efficacious anti-inflammatory agents and the gold standard treatment in Duchenne muscular dystrophy (DMD). However, their chronic use may lead to severe side effects. We evaluated the use of a novel injectable steroidal nano-drug in mdx mouse model of DMD by comparing the efficacy of nano-liposomes remotely loaded with the steroid prodrug, methylprednisolone hemisuccinate (MPS) with the same steroid as-is, in short (4-weeks) and long-term (58-weeks) treatments. Liposomal-MPS was selectively targeted to the mouse diaphragm, the most dystrophic muscle at early stage of the disease. The bioactivity of the steroidal nano-drug was evidenced by a significant decreased serum TGF-ß and reduced diaphragm macrophage infiltration after short-term treatment. In the long-term, the treatment with liposomal-MPS not only demonstrated improved muscle strength and mobility it also induced lower tibia and lumbar vertebrae osteoporosis indicating much lower bone related adverse effects.

Calmodulin Methyltransferase Is Required for Growth, Muscle Strength, Somatosensory Development and Brain Function.

Calmodulin lysine methyl transferase (CaM KMT) is ubiquitously expressed and highly conserved from plants to vertebrates. CaM is frequently trimethylated at Lys-115, however, the role of CaM methylation in vertebrates has not been studied. CaM KMT was found to be homozygously deleted in the 2P21 deletion syndrome that includes 4 genes. These patients present with cystinuria, severe intellectual disabilities, hypotonia, mitochondrial disease and facial dysmorphism. Two siblings with deletion of three of the genes included in the 2P21 deletion syndrome presented with cystinuria, hypotonia, a mild/moderate mental retardation and a respiratory chain complex IV deficiency. To be able to attribute the functional significance of the methylation of CaM in the mouse and the contribution of CaM KMT to the clinical presentation of the 2p21deletion patients, we produced a mouse model lacking only CaM KMT with deletion borders as in the human 2p21deletion syndrome. No compensatory activity for CaM methylation was found. Impairment of complexes I and IV, and less significantly III, of the mitochondrial respiratory chain was more pronounced in the brain than in muscle. CaM KMT is essential for normal body growth and somatosensory development, as well as for the proper functioning of the adult mouse brain. Developmental delay was demonstrated for somatosensory function and for complex behavior, which involved both basal motor function and motivation. The mutant mice also had deficits in motor learning, complex coordination and learning of aversive stimuli. The mouse model contributes to the evaluation of the role of methylated CaM. CaM methylation appears to have a role in growth, muscle strength, somatosensory development and brain function. The current study has clinical implications for human patients. Patients presenting slow growth and muscle weakness that could result from a mitochondrial impairment and mental retardation should be considered for sequence analysis of the CaM KMT gene.

Losartan, a therapeutic candidate in congenital muscular dystrophy: studies in the dy(2J) /dy(2J) mouse.

OBJECTIVE: Lamininα2-deficient congenital muscular dystrophy type 1A (MDC1A) is a cureless disease associated with severe disability and shortened lifespan. Previous studies have shown reduced fibrosis and restored skeletal muscle remodeling following treatment with losartan, an angiotensin II type I receptor blocker. We therefore evaluated the effect of losartan treatment in the dy(2J) /dy(2J) mouse model of MDC1A. METHODS: Homozygous dy(2J) /dy(2J) and control mice were treated with losartan or placebo for 12 weeks from 6 weeks of age. Outcome measures included hindlimb and forelimb muscle strength by Grip Strength Meter and quantitative muscle fibrosis parameters. Losartan's effects on transforming growth factor ß (TGF-ß) and mitogen-activated protein kinase (MAPK) signaling pathways were evaluated with Western blotting, immunofluorescence, and cytokine measurements. RESULTS: Losartan treatment was associated with significant impressive improvement in muscle strength and amelioration of fibrosis. Administration of losartan inhibited TGF-ß signaling pathway, resulting in decreased serum TGF-ß1 level and reduced downstream phosphorylated (P) Smad2/3 proteins. Moreover, losartan activated Smad7 protein, a key negative regulator of TGF-ß signaling. In addition, losartan treatment inhibited the MAPK cascade as shown by decreased expression of P-p38 MAPK, P-c-jun-N-terminal kinase, and P-extracellular signal-regulated kinases 1 and 2 in the treated mice. INTERPRETATION: Losartan, a commonly prescribed US Food and Drug Administration-approved medication for hypertension, demonstrated clinical improvement and amelioration of fibrosis in the dy(2J) /dy(2J) mouse model of MDC1A via TGF-ß and MAPK signaling pathways. The results of this study support pursuing a clinical trial of losartan treatment in children with MDC1A.

Impaired Regeneration in Dystrophic Muscle-New Target for Therapy.

Muscle stem cells (MuSCs), known as satellite cells (SCs) have an incredible ability to regenerate, which enables the maintenance and growth of muscle tissue. In response to damaging stimuli, SCs are activated, proliferate, differentiate, and fuse to repair or generate a new muscle fiber. However, dystrophic muscles are characterized by poor muscle regeneration along with chronic inflammation and fibrosis. Indications for SC involvement in muscular dystrophy pathologies are accumulating, but their contribution to muscle pathophysiology is not precisely understood. In congenital muscular dystrophy type 1A (LAMA2-CMD), mutations in Lama2 gene cause either complete or partial absence in laminin-211 protein. Laminin-211 functions as a link between muscle extracellular matrix (ECM) and two adhesion systems in the sarcolemma; one is the well-known dystrophin-glycoprotein complex (DGC), and the second is the integrin complex. Because of its protein interactions and location, laminin-211 has a crucial role in muscle function and survival by maintaining sarcolemma integrity. In addition, laminin-211 is expressed in SCs and suggested to have a role in SC proliferation and differentiation. Downstream to the primary defect in laminin-211, several secondary genes and pathways accelerate disease mechanism, while at the same time there are unsuccessful attempts to regenerate as compensation for the dystrophic process. Lately, next-generation sequencing platforms have advanced our knowledge about the secondary events occurring in various diseases, elucidate the pathophysiology, and characterize new essential targets for development of new treatment strategies. This review will mainly focus on SC contribution to impaired regeneration in muscular dystrophies and specifically new findings suggesting SC involvement in LAMA2-CMD pathology.

Improvement of motor conduction velocity in hereditary neuropathy of LAMA2-CMD dy2J/dy2J mouse model by glatiramer acetate.

OBJECTIVE: Glatiramer acetate (GA), an agent modulating the immune system, has been shown to cause significantly improved mobility and hind limb muscle strength in the dy2J/dy2J mouse model for LAMA2-congenital muscular dystrophy (LAMA2-CMD). In view of these findings and the prominent peripheral nervous system involvement in this laminin-α2 disorder we evaluated GA's effect on dy2J/dy2J motor nerve conduction electrophysiologically. METHODS: Left sciatic-tibial motor nerve conduction studies were performed on wild type (WT) mice (n = 10), control dy2J/dy2J mice (n = 11), and GA treated dy2J/dy2J mice (n = 10) at 18 weeks of age. RESULTS: Control dy2J/dy2J mice average velocities (34.49 ±â€¯2.15 m/s) were significantly slower than WT (62.57 ±â€¯2.23 m/s; p < 0.0005), confirming the clinical observation of hindlimb paresis in dy2J/dy2J mice attributed to peripheral neuropathy. GA treated dy2J/dy2J mice showed significantly improved average sciatic-tibial motor nerve conduction velocity versus control dy2J/dy2J (50.35 ±â€¯2.9 m/s; p < 0.0005). CONCLUSION: In this study we show for the first time improvement in motor nerve conduction velocity of LAMA2-CMD dy2J/dy2J mouse model's hereditary peripheral neuropathy following GA treatment. SIGNIFICANCE: This study suggests a possible therapeutic effect of glatiramer acetate on hereditary peripheral neuropathy in this laminin-α2 disorder.

Variable phenotypes of knockin mice carrying the M712T Gne mutation.

GNE myopathy is a recessive adult onset, slowly progressive distal and proximal myopathy, caused by mutations in the GNE gene. The most frequent mutation in GNE myopathy patients is the Middle Eastern founder mutation M712T. We have generated Gne (M712T/M712T) knockin mice. A high mortality rate in the first generation due to renal failure was recorded (as previously described). However, the following Gne (M712T/M712T) offspring generations could be classified into 3 phenotypic categories: severe, mild and without apparent phenotype. By further crossing between mice with no apparent phenotype, we were able to establish a colony of Gne (M712T/M712T) knockin mice with a high- and long-term survival rate, lacking any renal phenotype. These mice did not present any muscle phenotype (clinical or pathological) for up to 18 months. No correlation was found between the expression of any of the two mRNA Gne isoforms in muscle and the mouse genotype or phenotype. However, the expression of isoform 2 mRNA was significantly higher in the kidney of Gne (M712T/M712T) kidney affected mice compared with control. In contrast, the expression of UPR markers Bip, Chop and of the spliced form of XBP1, was upregulated in muscle of Gne (M712T/M712T) mice compared with controls, but was unchanged in the affected kidney. Thus, Gne defects can affect both muscle and kidney in mouse, but probably through different mechanisms.

Nerve growth factor stimulation of ERK1/2 phosphorylation requires both p75NTR and α9ß1 integrin and confers myoprotection towards ischemia in C2C12 skeletal muscle cell model.

The functions of nerve growth factor (NGF) in skeletal muscles physiology and pathology are not clear and call for an updated investigation. To achieve this goal we sought to investigate NGF-induced ERK1/2 phosphorylation and its role in the C2C12 skeletal muscle myoblasts and myotubes. RT-PCR and western blotting experiments demonstrated expression of p75(NTR), α9ß1 integrin, and its regulator ADAM12, but not trkA in the cells, as also found in gastrocnemius and quadriceps mice muscles. Both proNGF and ßNGF induced ERK1/2 phosphorylation, a process blocked by (a) the specific MEK inhibitor, PD98059; (b) VLO5, a MLD-disintegrin with relative selectivity towards α9ß1 integrin; and (c) p75(NTR) antagonists Thx-B and LM-24, but not the inactive control molecule backbone Thx. Upon treatment for 4 days with either anti-NGF antibody or VLO5 or Thx-B, the proliferation of myoblasts was decreased by 60-70%, 85-90% and 60-80% respectively, indicative of trophic effect of NGF which was autocrinically released by the cells. Exposure of myotubes to ischemic insult in the presence of ßNGF, added either 1h before oxygen-glucose-deprivation or concomitant with reoxygenation insults, resulted with about 20% and 33% myoprotection, an effect antagonized by VLO5 and Thx-B, further supporting the trophic role of NGF in C2C12 cells. Cumulatively, the present findings propose that proNGF and ßNGF-induced ERK1/2 phosphorylation in C2C12 cells by functional cooperation between p75(NTR) and α9ß1 integrin, which are involved in myoprotective effects of autocrine released NGF. Furthermore, the present study establishes an important trophic role of α9ß1 in NGF-induced signaling in skeletal muscle model, resembling the role of trkA in neurons. Future molecular characterization of the interactions between NGF receptors in the skeletal muscle will contribute to the understanding of NGF mechanism of action and may provide novel therapeutic targets.

Sustained expression and safety of human GNE in normal mice after gene transfer based on AAV8 systemic delivery.

GNE myopathy is an autosomal recessive adult onset disorder caused by mutations in the GNE gene. GNE encodes the bifunctional enzyme UDP-N-acetylglucosamine 2-epimerase/N-acetyl mannosamine kinase, the key enzyme in the biosynthesis pathway of sialic acid. Additional functions for GNE have been described recently, but the mechanism leading from GNE mutation to this myopathy is unclear. Therefore a gene therapy approach could address all potential defects caused by GNE mutations in muscle. We show that AAV8 viral vectors carrying wild type human GNE cDNA are able to transduce murine muscle cells and human GNE myopathy-derived muscle cells in culture and to express the transgene in these cells. Furthermore, the intravenous administration of this viral vector to healthy mice allows expression of the GNE transgene mRNA and of the coexpressed luciferase protein, for at least 6months in skeletal muscles, with no clinical or pathological signs of focal or general toxicity, neither from the virus particles nor from the wild type human GNE overexpression. Our results support the future use of an AAV8 based vector platform for a safe and efficient therapy of muscle in GNE myopathy.

The Ras antagonist, farnesylthiosalicylic acid (FTS), decreases fibrosis and improves muscle strength in dy/dy mouse model of muscular dystrophy.

The Ras superfamily of guanosine-triphosphate (GTP)-binding proteins regulates a diverse spectrum of intracellular processes involved in inflammation and fibrosis. Farnesythiosalicylic acid (FTS) is a unique and potent Ras inhibitor which decreased inflammation and fibrosis in experimentally induced liver cirrhosis and ameliorated inflammatory processes in systemic lupus erythematosus, neuritis and nephritis animal models. FTS effect on Ras expression and activity, muscle strength and fibrosis was evaluated in the dy(2J)/dy(2J) mouse model of merosin deficient congenital muscular dystrophy. The dy(2J)/dy(2J) mice had significantly increased RAS expression and activity compared with the wild type mice. FTS treatment significantly decreased RAS expression and activity. In addition, phosphorylation of ERK, a Ras downstream protein, was significantly decreased following FTS treatment in the dy(2J)/dy(2J) mice. Clinically, FTS treated mice showed significant improvement in hind limb muscle strength measured by electronic grip strength meter. Significant reduction of fibrosis was demonstrated in the treated group by quantitative Sirius Red staining and lower muscle collagen content. FTS effect was associated with significantly inhibition of both MMP-2 and MMP-9 activities. We conclude that active RAS inhibition by FTS was associated with attenuated fibrosis and improved muscle strength in the dy(2J)/dy(2J) mouse model of congenital muscular dystrophy.

Improved muscle strength and mobility in the dy(2J)/dy(2J) mouse with merosin deficient congenital muscular dystrophy treated with Glatiramer acetate.

The therapeutic effect of Glatiramer acetate, an immune modulating agent, was evaluated in the dy(2J)/dy(2J) mouse with merosin deficient congenital muscular dystrophy, which is a milder variant of the dy/dy mouse. The treated mice showed significant improvement in hind limb muscle strength measured by electronic grip strength meter and in motor performance quantified by video detection software. Glatiramer acetate treatment was associated with significantly increased expression of regeneration transcription factors MyoD and myogenin, and attenuation of the fibrosis markers vimentin and fibronectin. No effective treatment is currently available in congenital muscular dystrophy and Glatiramer acetate may present a new potential treatment for this disorder.