News in Muscular Dystrophy

A.C. Söderpalma, A.K. Kroksmarka, P. Magnussonb, A.C. Åhlandera, J. Karlssona, M. Tuliniusa, D. Swolin-Eidea - Sweden

Duchenne muscular dystrophy (DMD) is an inherited X-linked recessive disorder that leads to reduced bone turnover and an increased risk of osteoporosis due to progressive muscular impairment. Becker muscular dystrophy is caused by a deletion in the same gene, but shows a relatively milder clinical course. Muscle mass and bone mass are closely related and it is known that inactivity and immobilisation lead to loss of mineral from the skeleton. Six boys with Becker (10.8–18.9 years at baseline) and 18 boys with DMD (2.3–19.7 years at baseline) were followed 4 years with respect to areal bone mineral density (BMD) in the hip and axial skeleton measured by DXA and calcaneal BMD measured by DXL Calscan BMD at all sites; total body (TB) (P=0.0002), TB head excluded (TBHE) (P<0.0001), spine (P=0.0001), hip (P=0.002) and calcaneal (P<0.0001), were significantly lower in the DMD group compared with Becker at baseline and follow-up for all sites, P<0.0001. Bone mineral accretion was significantly less in the DMD group at all sites after the study period in comparison with Becker; TB (P=0.002), TBHE (P<0.0001), spine (P=0.0001), hip (P=0.002) and calcaneal (P<0.05). BMD increased at all sites in Becker during the follow-up period, but only significantly for TB and spine, P<0.05. A smaller increase was found in the DMD group for TB, TBHE and spine, P<0.05; but decreased BMD in the hip (P<0.05) after 4 years. Summary: A greater bone mineral accretion was observed in the Becker group in comparison with DMD and we even observed a decreased hip BMD in the DMD group after the study period. We suggest that these findings could, in part, be explained by the better preserved muscle strength in Becker and the earlier appearing weakness in DMD of the lower extremities compared with the upper body.

A. Rufoa, A.Del Fattorea,M.L.Bianchib, L.Morandif, E. Bertinic, A.Musaròd, S. Ferrarie, D. Pierroze, M. Capullia, N. Ruccia, F. De Benedettic, A. Tetia - Italy

Muscular dystrophies are characterized by inflammation, osteoporosis and increased risk of fractures other than myofiber necrosis and reduced muscular strength. We observed muscular atrophy and bone loss in mice overexpressing the pro-inflammatory cytokine IL-6 and propose that IL-6 may link the muscular and the bone phenotype in muscular dystrophies. Duchenne Muscular Dystrophy (DMD) is an X-linked disease due to mutations of the dystrophin gene. In DMD patients, we observed increased IL-6 in muscle biopsies and in sera. Similar to osteoblasts from IL-6 overexpressing mice and to osteoblasts treated with IL-6, human osteoblasts exposed to DMD sera failed to mineralize the extracellular matrix and showed reduced Osterix and Osteocalcin mRNA expression, despite normal alkalinephosphatase activity and Runx2 mRNA. The circulating RANKL/OPG protein ratio was low in DMD patients and inversely correlated with bone density. Transcriptional analysis revealed a similar reduction in RANKL/OPG ratio and increased IL-6 in osteoblasts exposed to DMD sera, along with up-regulation of further 26 genes and downregulation of further 90 genes associated with osteoblast function and osteoblast–osteoclast cross-talk. Despite low RANKL/OPG ratio, peripheral blood monocytes from patients and those from healthy donors exposed to DMD sera exhibited increased osteoclastogenesis similar to that observed in IL-6 overexpressing mice. In addition, mature osteoclasts expressed dystrophin, co-localized with F-actin in podosomes and actin rings, suggesting a role in cytoskeletal remodelling and bone resorption. Dystrophin-deficient mice (MDX) showed reduced tibial trabecular and cortical bone compared to WT, due to decreased osteoblast and increased osteoclast activity. Similar increase of osteoclast activity was observed in MDX calvariae. These latter bones are not subjected to muscular traction, suggesting systemic induction of osteoclastogenesis. We therefore propose that, besides mechanical failure, additional factors induce low bone density in DMD, among which we hypothesize relevant roles for systemic IL-6 and osteoclast dystrophin.

A. Peacock, B. Oldroyd, A. Shaw, A.M. Childs, T Mushtaq - UK

Background: Duchenne Muscular Dystrophy (DMD) is characterised by progressive muscle wasting. Glucocorticoids (GCs) are used to slow the progression of the disease and prolong ambulation, but they also suppress growth and bone metabolism. Aim: A retrospective study that assessed serial changes in body composition and bone mineral density (BMD) as measured by dualenergy absorptiometry (DXA) in boys with DMD before and after GC treatment. Method: 11 boys were treated with prednisolone (0.75 mg/kg/ day: 10 days on/10 days off). Standard deviation scores (SDS) were calculated from local reference data for total body BMD (TBBMD), Total Body Less Head BMD (TBLH), Lumbar Spine (LS) BMD (LSBMD) and LS Bone Mineral Apparent Density (LSBMAD). Serial scans were repeated if symptomatic or initial low BMD. Results:

By scan 3 there was a trend for decreasing BMD at all sites. The TBLH is lower than the TBBMD indicating the influence of the skull in the growing child. The LSBMAD is lower than the TBBMD which could reflect the detrimental GC effects on trabecular bone. Due to the limited number of scans there were no significant differences in the DXA BMD results. As there is no control group it is not possible to compare the effects of GC to those with reducing mobilisation. Conclusion: Serial BMD measurements in boyswith DMD may show a progressive reduction in bone density with time. (⁎Pre-steroids.)

S. Vaia, L. Morandib, M.L. Bianchia - Italy

A frequent complication of Duchenne Muscular Dystrophy (DMD) is an increased susceptibility to fractures, causing immobilization and worsening of muscle hypotrophy and weakness. There are very few data on bone density (BMD) and bone metabolism in DMD patients and it is unknown if DMD per se – independently of glucocorticosteroid (GC) treatment – causes bone metabolism derangements and osteoporosis. In a group of 15 DMD children (aged 3–6 years), we evaluated BMD (with DXA) and bone metabolism before starting GCs, at baseline and after 6 and 12 months. In most children, BMD (spine and total body) was lower than normal for age, and in 8 (53.3%) it was significantly reduced (spine BMAD Z-score −2.4±0.6). Low BMD was present in 2 (28.5%) of the 7 children aged 3– 4 years, but in 6 of 8 (75%) of those aged 5–6 years. We also measured the pituitary-adrenal response with a 1 mg dexamethasone overnight suppression test, to evaluate whether the acute response to GCs could predict the long-term GCs impact on bone. The suppressed morning serum cortisol and the increased insulin levels were correlated with changes in BMD and in bone turnover markers during the 12-month follow-up. Upon multiple regression analysis, cortisol suppression and insulin increase were correlated with spine BMD Z-score (p=0.03), BMD loss rate (p=0.02), and bone marker changes (NTx: p=0.02; BSAP: p=0.03). In children with suppressed cortisol levels below 50th percentile, spine BMD Z-score decreased (−0.7±0.5) after 12 months. These findings indicate that: BMD is often decreased in DMD even before GCs; Reduced BMD might be more prevalent in older than in younger boys; Cortisol suppression test may be used to evaluate the sensitivity to exogenous GCs and their ability to induce bone side  effects.

Research Grant UILDM - TELETHON (GUP 0300537).

H.H. Kecskemethy, H.T. Harckea,, S.J. Bachracha, - USA

This study examines the history of fracture in relation to BMD and ambulation as measured by group mean Z-scores for lateral distal femur (LDF) and lumbar spine (LS) in three cohorts of children: non-ambulatory DMD and CP and ambulatory DMD. Fracture history of 49 children with DMD (23 ambulatory, 26 nonambulatory) and 47 non-ambulatory children with CP was obtained at time of first BMD assessment. Mean LS and LDF Zscores (3 regions) were calculated for positive or negative fracture history.

Positive fracture history in ambulatory DMD was 4/23 with half occurring in the lower extremity. Non-ambulatory DMD fracture history was positive in 13/26 (with 69% in the lower extremity). Fracture history was positive in 19/47 of the CP group (lower extremity 89%). Both non-ambulatory groups had mean LDF Z-scores well below normal regardless of fracture history, whereas ambulatory DMD subjects had LDF values in normal/low-normal range. Ambulatory status did not influence LS Z-scores of DMD patients compared to LDF Z-scores. Differences in LDF Z-scores were noted in fracture and nonfracture groups except for the non-ambulatory DMD group. LDF BMD is easily obtained on disabled children and provides another DXA parameter for use in evaluating fracture risk.

N.J. Crabtreea,b, K.A. Wardc, H. Roperd, J.E. Adamsc, M.Z. Mughale, N.J. Shawb  - UK

DMD is the most common childhood neuromuscular disorder causing loss of ambulation in early life. Steroids are currently used to improve muscle strength and prolong ambulation although the effect on bone health in this group of children is still unclear. The aim of this study was to compare the longitudinal changes in bone strength in healthy children with those observed in children with DMD, who either remained ambulant or who lost independent ambulation during the period of follow-up. Forty children were studied, 17 healthy boys (9.1±1.5 years) and 23 boys with DMD (8.6±2.1 years), taking intermittent steroids. PQCT was used to measure bone geometry, density and strength of the non-dominant tibia. Measurements were made at the distal metaphysis and mid-diaphysis sites. Data were adjusted for age, height and duration of steroids. After 15.0±3.1 months, 7 DMD boys lost independent ambulation. Longitudinal growth between the groups remained constant. In DMD boys, who remained ambulant, there was a slowing down in periosteal bone growth at the mid diaphysis (0.8 vs. 2.6 mm2/month; p<0.05). Whereas for DMD boys who lost ambulation, there was a significant reduction in the rate of bone growth at the mid-diaphysis (0.4 mm2/month; p<0.05) and at the distal metaphysis (2.8 vs. 6.2 mm2/month; p<0.05). In contrast, the rate of change in bone density at the distal metaphysis (−2.8 vs. 0.3 mg/cm3/month; p<0.001) and cortical bone mass (−0.2 vs. 1.3 mg/mm/month; p<0.001) and stress-strain index (2.0 vs. 9.9 mm3/month; p<0.05) at the mid diaphysis was only significantly different from the healthy boys in the 7 boys who lost independent ambulation. These data suggest that ambulation and hence muscle function and gravitational load have the greatest effect on bone strength and density in boys with DMD. Whilst they remain ambulant the effect of the relatively high dose steroids appears to be negligible. However, when they eventually lose independent ambulation significant losses in bone strength occur as a direct result of diminished periosteal bone growth and bone mineral accrual.

Y. Urade, M. Hayashi, T. Maruyama, S. Kamauchi, I. Mohri and K. Aritake - Japan

Duchenne muscular dystrophy (DMD) is an X-linked muscular abnormality caused by the loss of dystrophin and is one of the most gravely genetic disorders. We have recently found that hematopoietic prostaglandin (PG) D synthase (H-PGDS) was induced in grouped necrotic muscle fibers in DMD patients (Okinaga T. et al., Acta Neuropathol. 2002; 104: 377–384). We developed novel H-PGDS inhibitors based on the X-ray crystallographic analysis of human H-PGDS complexed with its prototype inhibitor, HQL-79 (Aritake K. et al., J. Biol. Chem. 2006: 281: 15277–15286). In this study, we developed a novel therapy for DMD by inhibition of H-PGDS. H-PGDS was localized in the necrotic muscle fibers and accumulated macrophages in mdx mice. Oral administration of H-PGDS inhibitors for 5 days prevented the expansion of muscular necrosis in an mdx mouse model, as measured by X-ray computed tomography (CT) imaging enhanced by non-ionic contrast media. The treatment with H-PGDS inhibitors also decreased the expression of mRNAs of pro-inflammatory cytokines. These results indicate that PGD2 produced by H-PGDS plays important pathological roles on the expansion of muscle necrosis. H-PGDS inhibitor also accelerated the accumulation and activation of macrophages in the necrotic area. These results indicate that PGD2 produced by H-PGDS is involved in the expansion of muscle necrosis in DMD and that inhibition of H-PGDS is a novel therapy for DMD.

Myofiber degeneration, inflammation, and fibrosis are remarkable features of Duchenne muscular dystrophy. We hypothesized that the administration of imatinib mesylate, an inhibitor of tyrosine kinase and TGF-β profibrogenic activity, could improve the muscular conditions in mdx mice. Four-week old mdx mice were treated and exercised for 6 weeks. Gastrocnemius and diaphragm histopathology, strength, creatine kinase, and cytokine levels were evaluated. The treated group presented increased muscular strength and decreased CK levels, injured myofibers, and inflammatory infiltrates. Pro-inflammatory cytokines and TGF-β were also reduced, while IL-10 was increased, suggesting an immunomodulatory effect of imatinib, which can ameliorate the dystrophic phenotype in mdx mice.

A. Rufoa,  A. Del Fattorea, M.L. Bianchib, L. Morandic, E. Bertinid, A. Musaròe, S. Ferrarif, D. Pierrozf, M. Capullia, N. Ruccia, F. De Benedettid, A. Tetia - Italy

Duchenne Muscular Dystrophy (DMD) is induced by mutations of the dystrophin gene that cause disruption of sarcolemmal integrity, myofiber necrosis and inflammation. Besides muscular damage, patients show osteoporosis and increased risk of fractures, that we hypothesize are due to determinants other than mechanical unloading. Consistently, in DMD children we observed increased levels of IL-6, an inflammatory cytokine known to reduce osteoblast and increase osteoclast activity in vitro and in pre-pubertal mice. When exposed to DMD sera, osteoblasts from healthy donors failed to mineralize matrix nodules and showed reduced osterix and osteocalcin mRNA expression, despite normal alkaline phosphatase activity and Runx2 mRNA. Surprisingly, the circulating RANKL/OPG protein ratio was low in DMD patients and inversely correlated with bone density. Similar reduced transcriptional RANKL/OPG ratio and increased IL-6 mRNA expression were observed in osteoblasts exposed to DMD sera, along with up-regulation of further 26 genes and down-regulation of further 90 genes associated with osteoblast function and osteoblast–osteoclast cross-talk. Despite low RANKL/OPG ratio, peripheral blood monocytes from patients and those from healthy donors exposed to DMD sera exhibited increased ability to differentiate into osteoclasts. Mature osteoclasts expressed dystrophin, co-localized with F-actin, first in filopodia, then in podosomes and finally in actin rings, suggesting a role of the dystrophin complex in the organization of the sealing zone cytoskeleton. Dystrophin-deficient mice (MDX) showed reduced cortical and trabecular bone compared to WT, as assessed in both genders by microCT and histomorphometry, which was due to decreased osteoblast and increased osteoclast activity. Interestingly, similar alterations, especially for the osteoclast lineage, were observed in calvariae from MDX mice, in which mechanical forces play negligible roles as they are not subjected to muscular traction. In conclusion, we suggest that osteoporosis in DMD patients could be explained by factors other than mechanical unloading due to muscular failure, among which we underscored a possible role for IL-6 and osteoclast dystrophin.

Evans NP, Misyak SA, Robertson JL, Bassaganya-Riera J, Grange RW - USA

Duchenne muscular dystrophy is a debilitating genetic disorder characterized by severe muscle wasting and early death in affected boys. The primary cause of this disease is mutations in the dystrophin gene that result in the absence of the protein dystrophin and the associated dystrophin-glycoprotein complex in the plasma membrane of muscle fibers. In normal muscle, this complex forms a link between the extracellular matrix and the cytoskeleton that is thought to protect muscle fibers from contraction-induced membrane lesions and to regulate cell signaling cascades. Although the primary defect is known, the mechanisms that initiate disease onset have not been characterized. Data collected during early maturation suggest that inflammatory and immune responses are key contributors to disease pathogenesis and may be initiated by aberrant signaling in dystrophic muscle. However, detailed time course studies of the inflammatory and immune processes are incomplete and need to be characterized further to understand the disease progression. The purposes of this review are to examine the possibility that initial disease onset in dystrophin-deficient muscle results from aberrant inflammatory signaling pathways and to highlight the potential clinical relevance of targeting these pathways to treat Duchenne muscular dystrophy.

19 - (Experimental Cell Research, 2009) Engraftment of Mesenchymal Stem Cells into Dystrophin-Deficient Mice is not Accompanied by Functional Recovery

Joshua T. Selsby1,2, Kevin Morine2, Klara Pendrak2, Z. Tian3, Erica Blanco3, Elisabeth R Barton3, H Lee Sweeney2. 1Animal Science, Iowa State University, Ames, IA, 2Physiology, 3Anatomy and Cell Biology, University of Pennsylvania, Philadelphia, PA

Increased PGC-1α, either through a transgenic animal or gene transfer, provides therapeutic benefit to dystrophic skeletal muscle  likely by increasing slow and oxidative protein expression. Resveratrol has been shown to activate SIRT-1, a known deacetylase capable of acting on and increasing the activity of PGC-1α. The purpose of this investigation was to determine if resveratrol supplementation could rescue dystrophic skeletal muscle. We hypothesized that daily feeding of resveratrol would improve muscle function in a similar fashion as PGC-1α overexpression. To test this hypothesis, 1-mo old mdx mice were either fed a control diet (Con) or a diet containing resveratrol at 100 mg/kg/day (Res) for 8 wks. Following intervention, absolute muscle mass was reduced 18-30% in the Res group compared to Con, however, body mass was also reduced in the Res group by 20% compared to Con. Relative muscle mass was similar between groups, except for the EDL, which was significantly smaller in the Res group. Solei in the Res group were more fatigue resistant than in the Con group, however, resistance to contraction induced injury was similar between groups. These data indicate that partial rescue of dystrophic pathology is possible through resveratrol. Perhaps, alternative sources or doses of resveratrol will provide a greater response.

Joshua T. Selsby1,2, Kevin Morine2, Klara Pendrak2, Z. Tian3, Erica Blanco3, Elisabeth R Barton3, H Lee Sweeney2. 1Animal Science, Iowa State University, Ames, IA, 2Physiology, 3Anatomy and Cell Biology, University of Pennsylvania, Philadelphia, PA

PGC-1α has received a great deal of attention due to its potential to induce oxidative and slow proteins. The purpose of this investigation was to: 1) extend observations made in an initial study demonstrating transgenic over-expression of PGC-1α reduced dystrophic pathology 2) eliminate effects that could have taken place during development. Neonatal mdx mice were injected in the right hind limb with 1x1011 gc of AAV causing overexpression of PGC-1α and sacrificed at 4 (n=6) or 6 wks of age (n=5). Muscle mass was reduced in limbs over-expressing PGC- 1α at 4 and 6 wks, however, tetantic force and specific force in the soleus and EDL were either maintained or improved when compared to control limbs. PGC-1α over-expression caused EDLs to be more resistant to damage at 6 wks and the soleus to be more fatigue resistant at 4 and 6 wks compared to control limbs. PGC- 1α increased expression of slow proteins as utrophin was increased nearly 2-fold and type I myosin heavy chain nearly 3-fold as well as expression of oxidative proteins as cytochrome C and uncoupling protein-1 were increased approximately 2-fold, complex IV subunit IV (cytochrome C oxidase) was increased 1.5- fold and myoglobin was increased 3-fold in limbs over-expressing PGC-1α compared to control limbs. These data demonstrate the potential therapeutic role of the PGC-1α pathway for dystrophic skeletal muscle.

Jong-Hee Kim1, BR Macias2, C Canon2, S Courtney2, John M Lawler2. 1Health and Kinesiology, Texas A&M University, College Station, TX, 2Texas A&M University, College Station, TX Duchenne muscular dystrophy (DMD) is characterized by devastating muscle degeneration associated with oxidative stress, loss of contractile tissue, muscle atrophy, muscle weakness and increased fibrosis in respiratory and locomotor muscles. We tested the hypothesis that protection against oxidative stress via the catalase/superoxide dismutase mimetic EUK-134 will prevent reduction of diaphragm and skeletal muscle mass/body mass during the early inflammatory phase (20-28 days) in mdx mice. C57BL(wild type) and mdx mice were given EUK-134 (30mg/kg body weight/day, i.p., injection) for 8 days, beginning at 20 days of age. Body mass was significantly lower in mdx mice (-37.7%) than wild type but, EUK-134 increased body mass by 15.5% in mdx mice. Absolute muscle mass was lower in diaphragm (-44.3%), gastrocnemius (-53.6%), tibialis anterior (-58.1%) in mdx mice. Muscle mass/body mass was lower in diaphragm (-11.4%), gastrocnemius (-26.3%), tibialis anterior (-34.4%), but not heart, plantaris, soleus, extensor digitorum longus in mdx mice. EUK- 134 had a significant positive effect in protecting against reduced muscle mass/body mass in diaphragm (+38.7%) and gastrocnemius (+27.1%) in mdx mice. These data indicate that EUK-134 provide protection against reduced muscle mass/body mass in diaphragm and gastrocnemius in mdx mice during this early phase of muscular dystrophy.

Marni Della Boppart1, Stephen J Kaufman2. 1Kinesiology and Community Health, 2Cell and Developmental Biology, University of Illinois, Urbana, IL

MCK-driven transgenic expression of the alpha7 integrin can ameliorate pathology in a mouse model of Duchenne muscular dystrophy (mdx/utrn-/-) and thus compensate for the loss of dystrophin in diseased mice. In spite of the beneficial effects of the alpha7 integrin in protecting mice from dystrophy, identification of molecular signaling events responsible for these changes remain to be established. PURPOSE: To determine a role for signaling in the amelioration of muscular dystrophy by alpha7 integrin. METHODS: Five wk wild type, mdx/utrn-/-, and alpha7BX2-mdx/utrn-/- gastrocnemius muscles (n=3-8/group) were dissected and extracted. Activation of PI3K, ILK, AKT, mTOR, p70S6K, GSK-3, and p38 was measured using in vitro activity assays or phosphospecific antibodies and western blotting. RESULTS: Significant increases in ILK activity (2.0-fold), AKT- (P) (2.3-fold), mTOR-(P) (57%), p70S6K-(P) (11.7-fold), and ERK-(P) (66%) were observed in dystrophic mdx/utrn-/- muscle compared to wild type. Significant decreases in GSK-3-(P) (57%) and p38-(P)(2.9-fold) were also observed. Most of these signaling events were similar in dystrophic mice over-expressing the alpha7 integrin. However, a further increase in p70S6K-(P) (18-fold) and decrease in GSK-3-(P)(3.7-fold) were detected in alpha7BX2- mdx/utrn-/- compared to wild type mice and these changes were significant compared to mdx/utrn-/- mice. CONCLUSION: The alpha7beta1 integrin confers a protective effect in dystrophic mice and increased p70S6K activity appears important to this.

Candace M. Parchen1, Justin M. Percival2, Dao-Fu Dai3, Heidi N Gray1, Stanley C. Froehner2, Joseph A. Beavo1. 1Pharmacology, 2Physiology and Biophysics, 3Pathology, University of Washington, Seattle, WA

Duchenne muscular dystrophy (DMD) is the most prevalent type of muscular dystrophy and is the result of an X-linked mutation in the dystrophin gene. The progression of skeletal muscle damage is rapid in DMD patients and cardiomyopathy soon follows. We have investigated whether or not sildenafil citrate, a phosphodiesterase 5 (PDE5) inhibitor, can be used to ameliorate the age-related cardiac dysfunction in dystrophin-null (mdx) mice, a mouse model of DMD. Using echocardiography, we show that chronic sildenafil treatment improves several functional deficits in the cardiac performance of aged mdx mice. Collagen VI levels are also lower in the hearts of sildenafil-treated mdx mice, suggesting a remodeling of the extracellular matrix. This is the first study to report a cardioprotective effect of PDE5 inhibition in aged mdx mice. Overall, the data suggest that PDE5 inhibitors could be a useful treatment for the cardiomyopathy suffered by DMD patients.

Joseph M Vitale, Elizabeth Stillwell, Farah Khadim, Genie Elson, Aneela Altaf, Joel Schneider, Ghassan Yehia, Diego Fraidenraich. Cell Biology & Molecular Medicine, University of Medicine & Dentistry of New Jersey, Newark, NJ

Duchenne muscular dystrophy (DMD) is an incurable neuromuscular degenerative disease, caused by a mutation in the dystrophin gene. Mdx mice recapitulate DMD features. Here we show that injection of wild-type (WT) embryonic stem cells (ESCs) into mdx blastocysts produces mice with improved pathology. A small fraction of WT ESCs incorporates into the mdx mouse nonuniformly to upregulate protein levels of dystrophin in the skeletal muscle. The chimeric muscle shows reduced regeneration and restores dystrobrevin, a dystrophin-related protein, in areas with high and with low dystrophin content. WT ESC injection also normalizes the amount of fat, a tissue that does not express dystrophin. ESC injection without dystrophin does not prevent the appearance of phenotypes in the skeletal muscle or in the fat. Thus, dystrophin supplied by the ESCs reverses disease in mdx mice globally.

7) Murine mammary tumor growth is blunted in dystrophin deficient mdx mice

Mary Pat Meaney, Robert W. Grange, Young H. Ju. Human Nutrition, Foods and Exercise, Virginia Tech, Blacksburg, VA

Breast cancer (BC) is the second leading cause of cancer death in women in the US. Changes to the dystrophin glycoprotein complex (DGC), a multi-protein structure that likely plays mechanical and signaling roles, have been reported in BC cells. For example, expression of α- and β-dystroglycan appears to be inversely related to tumor stage. However, systemic absence of the DGC, as seen in the mdx mouse model of Duchenne muscular dystrophy, does not appear to induce spontaneous mammary tumor formation, indicating that presence of the DGC may be necessary for breast cancer initiation. We therefore hypothesized that breast tumor growth would be altered in mdx mice compared to wild type (C57BL/6) mice. We injected E0771 murine mammary adenocarcinoma cells into mdx and C57BL/6 mice. After 3 weeks of tumor growth, blood, skeletal muscles and tumors were collected and analyzed. Growth of E0771 tumors and serum content of migration and invasion chemokine markers, RANTES and MCP-1, was dramatically blunted in mdx mice. The dystrophin protein was not detectable in E0771 cells or tumors, suggesting that its expression, like that of other DGC components, may be suppressed or altered in this cancer cell line. The exact mechanism(s) of tumor inhibition in mdx mice is not presently known; however, our results suggest a mechanism for suppressing BC growth and progression may depend on the absence of one or more proteins in the DGC.

Duchenne-Meryon muscular dystrophy (DMD) is the most common and lethal genetic muscle disease. Ameliorating muscle necrosis, inflammation, and fibrosis represents an important therapeutic approach for DMD. Imatinib, an antineoplastic agent, demonstrated antiinflammatory and antifibrotic effects in liver, kidney, lung, and skin of various animal models. This study tested antiinflammatory and antifibrotic effects of imatinib in mdx mice, a DMD mouse model. We treated mdx mice with intraperitoneal injections of imatinib at the peak of limb muscle inflammation and the onset of diaphragm fibrosis. Controls received PBS vehicle or were left untreated. Muscle necrosis, inflammation, fibrosis, and function were evaluated by measuring serum CK activity, endomysial CD45 immunoreactive inflammation area, endomysial collagen III deposition, and hind limb grip strength. Phosphorylation of the tyrosine kinase targets of imatinib was assessed by Western blot in diaphragm tissue and in primary cultures of peritoneal macrophages and skeletal muscle fibroblasts. Imatinib markedly reduced muscle necrosis, inflammation, and fibrosis, and significantly improved hind limb grip strength in mdx mice. Reduced clinical disease was accompanied by inhibition of c-abl and PDGFR phosphorylation and suppression of TNF-alpha and IL-1 expression. Imatinib therapy for DMD may hold promise for ameliorating muscle necrosis, inflammation, and fibrosis by inhibiting c-abl and PDGFR signaling pathways and downstream inflammatory cytokine and fibrotic gene expression.

Duchenne muscular dystrophy is characterized by progressive muscle weakness and early death resulting from dystrophin deficiency. Loss of dystrophin results in disruption of a large dystrophin glycoprotein complex, leading to pathological calcium (Ca2+)-dependent signals that damage muscle cells1–5. We have identified a structural and functional defect in the ryanodine receptor (RyR1), a sarcoplasmic reticulum Ca2+ release channel, in the mdx mouse model of muscular dystrophy that contributes to altered Ca2+ homeostasis in dystrophic muscles. RyR1 isolated from mdx skeletal muscle showed an age-dependent increase in S-nitrosylation coincident with dystrophic changes in the muscle. RyR1 S-nitrosylation depleted the channel complex of FKBP12 (also known as calstabin-1, for calcium channel stabilizing binding protein), resulting in ‘leaky’ channels. Preventing calstabin-1 depletion from RyR1 with S107, a compound that binds the RyR1 channel and enhances the binding affinity of calstabin-1 to the nitrosylated channel, inhibited sarcoplasmic reticulum Ca2+ leak, reduced biochemical and histological evidence of muscle damage, improved muscle function and increased exercise performance in mdx mice. On the basis of these findings, we propose that sarcoplasmic reticulum Ca2+ leak via RyR1 due to S-nitrosylation of the channel and calstabin-1 depletion contributes to muscle weakness in muscular dystrophy, and that preventing the RyR1-mediated sarcoplasmic reticulum Ca2+ leak may provide a new therapeutic approach.

While Focusing On Heart Disease, Researchers Discover New Tactic Against Fatal Muscular Dystrophy