Exon skipping using antisense oligonucleotides (AONs) has successfully been used to reframe the mRNA in various Duchenne muscular dystrophy patients carrying deletions in the DMD gene. In this study we tested the feasibility of the exon skipping approach for patients with small mutations in in-frame exons. We first identified 54 disease-causing point mutations. We selected five patients with nonsense or frameshifting mutations in exons 10, 16, 26, 33, and 34. Wild-type and mutation specific 2′OMePS AONs were tested in cell-free splicing assays and in cultured cells derived from the selected patients. The obtained results confirm cell-free splicing assay as an alternative system to test exon skipping propensity when patients’ cells are unavailable. In myogenic cells, similar levels of exon skipping were observed for wild-type and mutation specific AONs for exons 16, 26, and 33, whereas for exon 10 and exon 34 the efficacy of the AONs was significantly different. Interestingly, in some cases skipping efficiencies for mutated exons were quite dissimilar when compared with previous reports on the respective wild-type exons. This behavior may be related to the effect of the mutations on exon skipping propensity, and highlights the complexity of identifying optimal AONs for skipping exons with small mutations. Hum Mutat 30:1-8, 2009. (c) 2009 Wiley-Liss, Inc.

We have earlier shown that antisense morpholino oligomers are able to restore dystrophin expression by systemic delivery in body-wide skeletal muscles of dystrophic mdx mice. However, the levels of dystrophin expression vary considerably and, more importantly, no dystrophin expression has been achieved in cardiac muscle. In this study, we investigate the efficiency of morpholino-induced exon skipping in cardiomyoblasts and myocytes in vitro, and in cardiac muscle in vivo by dose escalation. We showed that morpholino induces targeted exon skipping equally effectively in both skeletal muscle myoblasts and cardiomyoblasts. Effective exon skipping was achieved in cardiomyocytes in culture. In the mdx mice, morpholino rescues dystrophin expression dose dependently in both skeletal and cardiac muscles. Therapeutic levels of dystrophin were achieved in cardiac muscle albeit at higher doses than in skeletal muscles. Up to 50 and 30% normal levels of dystrophin were induced by single systemic delivery of 3 g kg(-1) of morpholino in skeletal and cardiac muscles, respectively. High doses of morpholino treatment reduced the serum levels of creatine kinase without clear toxicity. These findings suggest that effective rescue of dystrophin in cardiac muscles can be achieved by morpholino for the treatment of Duchenne muscular dystrophy.Gene Therapy advance online publication, 17 September 2009; doi:10.1038/gt.2009.120.

Skeletal muscle produces a myriad of mitogenic factors possessing cardiovascular regulatory effects that can be explored for cardiac repair. Given the reported findings that VEGF may modulate muscle regeneration, we investigated the therapeutic effects of chronic injections of low doses of human recombinant VEGF-A165 (0.1-1 microg/kg) into the dystrophic hamstring muscle in a hereditary hamster model of heart failure and muscular dystrophy. In vitro, VEGF stimulated proliferation, migration, and growth factor production of cultured C2C12 skeletal myocytes. VEGF also induced production of HGF, IGF2, and VEGF by skeletal muscle. Analysis of skeletal muscle revealed an increase in myocyte nuclear (531+/-12 VEGF 1 microg/kg vs. 364+/-19 number/mm(2) saline) and capillary (591+/-80 VEGF 1 microg/kg vs. 342+/-21 number/mm(2) saline) densities. Skeletal muscle analysis revealed an increase in Ki67(+) nuclei in the VEGF 1 microg/kg group compared to saline. In addition, VEGF mobilized c-kit(+), CD31(+), and CXCR4(+) progenitor cells. Mobilization of progenitor cells was consistent with higher SDF-1 concentrations found in hamstring, plasma, and heart in the VEGF group. Echocardiogram analysis demonstrated improvement in left ventricular ejection fraction (0.60+/-0.02 VEGF 1 microg/kg vs. 0.45+/-0.01 saline) and an attenuation in ventricular dilation (5.59+/-0.12 VEGF 1 microg/kg vs. 6.03+/-0.09 mm saline) five weeks after initiating therapy. Hearts exhibited higher cardiomyocyte nuclear (845+/-22 VEGF 1 microg/kg vs. 519+/-40 number/mm(2) saline) and capillary (2159+/-119 VEGF 1 microg/kg vs. 1590+/-66 number/mm(2) saline) densities. Myocardial analysis revealed ~2.5 fold increase in Ki67+ cells and ~2.8 fold increase in c-kit+ cells in the VEGF group which provides evidence for cardiomyocyte regeneration and progenitor cell expansion. This study provides novel evidence of a salutary effect of VEGF in the cardiomyopathic hamster via induction of myogenic growth factor production by skeletal muscle and mobilization of progenitor cells which resulted in attenuation of cardiomyopathy and repair of the heart. Key words: vascular endothelial growth factor, heart failure, delta-sarcoglycan, skeletal muscle.

Laminin alpha2-deficient Congenital Muscular Dystrophy, called MDC1A, is a rare, devastating genetic disease characterized by severe neonatal hypotonia (”floppy infant syndrome”), peripheral neuropathy, inability to stand or walk, respiratory distress and premature death in early life. Transgenic overexpression of the apoptosis inhibitor protein BCL-2, or deletion of the pro-apoptotic Bax gene in a mouse model for MDC1A prolong survival and mitigate pathology, indicating that apoptotic events are involved in the pathology. Here we demonstrate that the pro-apoptotic glyceraldehyde 3-phosphate dehydrogenase (GAPDH)-Siah1-CBP/p300-p53 pathway is activated in a mouse model for MDC1A. Moreover, we show that omigapil, which inhibits GAPDH-Siah1 mediated apoptosis, ameliorates several pathological hallmarks in the MDC1A mouse model. Specifically, we demonstrate that treatment with omigapil inhibits apoptosis in muscle, reduces body weight loss and skeletal deformation, increases locomotive activity and protects from early mortality. These data qualify omigapil, which is in late phase of clinical development for human use, as a drug candidate for the treatment of MDC1A.

Facioscapulohumeral muscular dystrophy (FSHD) is caused by contractions of D4Z4 repeats at 4q35.2 thought to induce misregulation of nearby genes, one of which, DUX4, is actually localized within each repeat. A conserved ORF (mDUX), embedded within D4Z4-like repeats, encoding a double-homeodomain protein, was recently identified on mouse chromosome 10. We show here that high level mDUX expression induces myoblast death, while low non-toxic levels block myogenic differentiation by down-regulating MyoD and Myf5. Toxicity and MyoD/Myf5 expression changes were competitively reversed by overexpression of Pax3 or Pax7, implying mechanistic similarities with the anti-myogenic activity of human DUX4. We tested the effect of mDUX expression on Xenopus development, and found that global overexpression led to abnormalities in gastrulation. When targeted unilaterally into blastomeres fated to become tail muscle in 16-cell embryos, mDUX caused markedly reduced tail myogenesis on the injected side. These novel cell and animal models highlight the myopathic nature of sequences within the FSHD-related repeat array.

BACKGROUND: Patients with muscular dystrophy have been reported to experience a variety of life-threatening complications during and after general anesthesia. We performed a systematic analysis to define the spectrum of anesthetic-related complications in patients with muscular dystrophy, with an emphasis on malignant hyperthermia susceptibility. METHODS: A literature search was undertaken using multiple search engines and the appropriate articles were reviewed by the authors to determine anesthetic-associated complications in patients with muscular dystrophy. Of all the types of muscular dystrophy, Duchenne muscular dystrophy (DMD) and Becker dystrophy (BD) represent nearly all the anesthesia-related reports. RESULTS: Anesthetic complications in patients with DMD and BD include intraoperative heart failure, inhaled anesthetic-related rhabdomyolysis (absence of succinylcholine), and succinylcholine-induced rhabdomyolysis and hyperkalemia. CONCLUSION: We did not find an increased risk of malignant hyperthermia susceptibility in patients with DMD or BD compared with the general population. However, dystrophic patients who are exposed to inhaled anesthetics may develop disease-related cardiac complications, or rarely, a malignant hyperthermia-like syndrome characterized by rhabdomyolysis. This latter complication may also occur postoperatively. Succinylcholine administration is associated with life-threatening hyperkalemia and should be avoided in patients with DMD and BD.

The purpose of this report is to outline the process, obstacles, and outcomes for the development and successful implementation of a service-learning experience between the Department of Physical Therapy at the University of Texas Southwestern Medical Center and the Muscular Dystrophy Association of Dallas. The rationale for initiation of the partnership is outlined, and curriculum planning concerns and integration are presented. These include development of community partnerships, identification of curricular placement, and development of academic and community roles, including didactic and clinical objectives for the participants. The importance of understanding the service-learning philosophy and evaluation of the student and community impact is emphasized. Outcomes report students’ assessments encompassing issues of both prior and future didactic education, development of clinical skills and problem solving, and evaluation of professional and personal issues. It is suggested that this model of service learning project has the ability to be implemented across a wide spectrum of allied health care curricula.

A non-randomised retrospective study to compare the results of surgical correction of scoliosis in Duchenne’s muscular dystrophy (DMD) patients using three different instrumentation systems-Sublaminar instrumentation system (Group A), a hybrid of sublaminar and pedicle screw systems (Group B) and pedicle screw system alone (Group C). Between 1993 and 2003, 43 patients with DMD underwent posterior spinal fusion and instrumentation. Group A (n = 19) had sublaminar instrumentation system, Group B (n = 13) had a hybrid construct and Group C (n = 11) was treated with pedicle system. The mean blood loss in Group A was 4.1 l, 3.2 l in Group B and 2.5 l in Group C. Average operating times in Group A, B and C were 300, 274 and 234 min, respectively. Mean pre-operative, post-operative and final Cobb angle in Group A was 50.05 +/- 15.46 degrees , 15.68 +/- 11.23 degrees and 21.57 +/- 11.63 degrees , Group B was 17.76 +/- 8.50 degrees , 3.61 +/- 2.53 degrees and 6.69 +/- 4.19 degrees and Group C was 25.81 +/- 9.94 degrees , 5.45 +/- 3.88 degrees , 8.90 +/- 5.82 degrees , respectively. Flexibility index or the potential correction calculated from bending radiographs were 60 +/- 6.33, 70 +/- 4.65 and 67 +/- 6.79% for Group A, Group B and Group C respectively. The percentage correction achieved was 72.5 +/- 14.5% in Group A, 82 +/- 6% in Group B and 82 +/- 8% in Group C. The difference between percentage correction achieved and the flexibility index was 12.45 +/- 8.22, 12.05 +/- 1.3 and 15.00 +/- 1.21% in Group A, B and C, respectively The percentage loss of correction in Cobb angles at final follow-up in Group A, B and C was 12.5 +/- 3.5, 16.5 +/- 1. and 12.5 +/- 2.5%, respectively. Complications seen in Group A were three cases of wound infection and two cases of implant failure; Group B had a single case of implant failure and Group C had one patient with wound infection and one case with a partial screw pull out. Early surgery and smaller curve corrections appears to be the current trend in the management of scoliosis in DMD. This has been possible due to early curve detection and surgery thus having the advantage of less post-operative respiratory complications and stay in paediatric intensive care. Also, early surgery avoids development of pelvic deformity and extension of instrumentation to the pelvis thereby reducing blood loss. This trend reflects the advent of newer and safer instrumentation systems, advanced techniques in anaesthesia and cord monitoring. Sublaminar instrumentation system group had increased operating times and blood loss compared to both the hybrid and pedicle screw instrumentation systems due to increased bleeding from epidural vessels and pelvic instrumentation. Overall, the three instrumentation constructs appear to provide and maintain an optimal degree of correction at medium to long term follow up but the advantages of lesser blood loss and surgical time without the need for pelvic fixation seem to swing the verdict in favour of the pedicle screw system.

Chaperones assist the metamorphosis of polypeptides to fully functional proteins. The family of heat shock proteins 70 (HSP70) bind at an early stage, while the HSP90 bind to proteins near their active conformation. We studied HSP90 and HSP70 in muscle biopsies from controls and patients diagnosed with the three main idiopathic inflammatory myopathies (IIM), namely dermatomyositis (DM), sporadic inclusion body myositis (IBM), and polymyositis (PM). Human skeletal muscle displayed constitutive levels of protein, but in IIM both HSP90 and HSP70 were upregulated. Regenerating muscle fibers of IIM and muscular dystrophy patients showed increased expression of HSP90 and HSP70, as did atrophic perifascicular muscle fibers of DM and vacuolated fibers of IBM. The infiltrates of IIM displayed weak homogeneous HSP70 staining. HSP90 were specifically upregulated in the CD68(+) cells, actively invading non-necrotic muscle fibers of IBM/PM and co-localized with inducible NO synthase. HSP90:HSP70 ratios were increased in IBM and PM tissues displaying high inflammation grade. Our results point to a multifaceted role for chaperones in muscle tissue: a general protective role for both HSP90 and HSP70 families in damaged muscle fibers, and a specific cytotoxic role for HSP90 in muscle fiber invasion typically associated with IBM and PM.

BACKGROUND: Nitrous oxide (N(2)O) acts on supraspinal noradrenergic neurons to produce analgesia, but it is unclear if analgesia contributes to N(2)O’s immobilizing effects. We tested the hypothesis that N(2)O minimum alveolar anesthetic concentration (MAC) is unchanged after selective ablation of supraspinal noradrenergic neurons, or in naïve animals at N(2)O exposure timepoints when analgesia is absent. METHODS: We determined tailflick latency (TFL) and hindpaw withdrawal latency (HPL) under 70% N(2)O, N(2)O MAC, and isoflurane MAC before and after intracerebroventricular injections of anti-dopamine-beta hydroxylase conjugated to saporin (SAP-DBH; n = 7), or a control antibody conjugated to saporin (n = 5). In a separate group of naive rats (n = 8), N(2)O MAC was determined at 25-45 min after initiation of N(2)O exposure (during peak analgesia) and again at 120-140 min (after TFL and HPL returned to baseline). RESULTS: After 30 min of N(2)O exposure, TFL and HPL increased significantly but declined back to baseline within 120 min. N(2)O did not produce analgesia in rats that received SAP-DBH. However, N(2)O and isoflurane MAC were not significantly different between SAP-DBH and control-injected animals (Mean +/- sd for N(2)O: 1.7 +/- 0.1 atm vs 1.7 +/- 0.2 atm; isofurane: 1.6 +/- 0.2% vs 1.7 +/- 0.2%). In naïve animals, N(2)O MAC was not different at the 30 min period compared with the 120 min period (1.8 +/- 0.1 atm vs 1.8 +/- 0.2 atm). CONCLUSIONS: Destroying brainstem noradrenergic neurons or prolonged exposure to N(2)O removes its analgesic effects, but does not change MAC. The immobilizing mechanism of N(2)O is independent from its analgesic effects.