Functional and Structural Improvements in mdx Skeletal Muscle Following Chronic Daily Administration of Pyrrolidine Dithiocarbomate (PDTC)

34th Annual Meeting of Society for Neuroscience; October/2004, San Diego

C. Carlson, A. Siegel, A. Samadi, S. Vulcanar. - USA

The presence of severe dystrophy in the mdx triangularis sterni (TS) indicates that passive stretch activates pathogenic mechanisms that do not involve generalized increases in resting Ca2+ influx (Carlson et al., Neurobiology of Disease, 14, 229,2003).To test whether nuclear activation of NFkB (Kumar and Boriek, FASEB J., 17,386, 2003) is directly involved in dystrophic pathogenesis, mdx mice were treated daily (29-82 days)with 50 mg/kg PDTC (ip),an antioxidant which inhibits the nuclear activation of NFB by stabilizing cytosolic IkB-a. Western blots indicated that a single dose increased cytosolic IkB-a in mdx diaphragm for at least 5 hrs.Chronically treated mdx mice exhibited modest increases in whole body tension (Carlson and Makiejus, Muscle and Nerve, 13,480,1990),a significant (p<0.05) decrease in fatigue,and small increases in gastrocnemius twitch tension.Untreated mdx TS fibers (5-12 m)had an average RP of -43 mV (+1.4 SE, N=77 fibers)which was less (p<0.001) than that seen in nondystrophic TS fibers (5-17 m,-51 mV + 1.4,N=61).PDTC treated mdx muscle fibers isolated at 8 to 9 months had more obvious striations and an average RP (-52 +1.7 mV,N= 48) that was identical to that seen in nondystrophic TS fibers.Older PDTC treated mdx mice (11-22 m) had significantly improved RPs in both caudal (-41 mV+ 2.8, N=52;p<0.001) and middle (-42 + 2.8 mV, N=44; p<0.05) regions of the TS in comparison to age matched saline-injected mice (caudal: -23 + 2.0 mV, N=93; middle: -34.2 +2.5 mV, N=50).These TS muscles also exhibited an increase in the appearance of healthy intact fibers, particularly in the middle region of the muscle.These results indicate that a stretch-induced inflammatory reaction involving NFkB nuclear activation contributes significantly to dystrophic pathology, and that agents which inhibit the nuclear activation of NFkB should have pronounced value in the treatment of muscular dystrophy

C. Carlson, A. Siegel, A. Samadi, S. Vulcanar. FUNCTIONAL AND STRUCTURAL IMPROVEMENTS IN MDX SKELETAL MUSCLE FOLLOWING CHRONIC DAILY ADMINISTRATION OF PYRROLIDINE DITHIOCARBAMATE (PDTC) Program No. 684.18. 2004 Abstract Viewer/Itinerary Planner. Washington, DC: Society for Neuroscience, 2004. Online

Effects of exercise and steroid on skeletal muscle apoptosis in the mdx mouse

Muscle and Nerve, 2004

Jeong-Hoon Lim, MD, Dai-Youl Kim, MD, Moon Suk Bang, MD, PhD - Republic of Korea

Abstract:

Reports concerning the influence of exercise loading and steroid administration on dystrophinopathy are inconsistent. To investigate the effect of muscle exercise in Duchenne muscular dystrophy (DMD), 15 control and 15 mdx mice, an animal model of DMD, were divided into free-living (n = 6), exercise (n = 6), and immobilization (n = 3) groups. Free- living and exercise groups were further divided into steroid- treated and sham-treated groups to evaluate the effect of steroid administration. We measured apoptotic changes by in

situ DNA nick-end labeling (TUNEL), DNA fragmentation assay, and Western blotting for Bcl-2 and BAX. Apoptosis was most prominent in the sham-treated exercise group, and it was significantly reduced in the steroid-treated exercise group. The steroid-treated free-living group showed a higher rate of apoptotic change than the sham-treated free-living group. Apoptosis was minimized in the free-living condition, whereas exercise loading and immobilization caused apoptotic change in this muscular dystrophy animal model. Steroid administration induced apoptosis in muscle of free-living mice, but alleviated the apoptotic damage caused by exercise loading in mdx mice. These findings suggest that steroid administration may be effective in preventing a postexercise deterioration of skeletal muscle in animal models of DMD.

Administration of Insulin-Like Growth Factor-I Improves Fatique Resistance of Skeletal Muscles from Dystrophic mdx Mice

Muscle and Nerve

PAUL GREGOREVIC, , DAVID R. PLANT, , and GORDON S. LYNCH, - Australia

Abstract:

Muscle fatigue occurs in many neuromuscular diseases, including the muscular dystrophies, and it contributes to a loss of functional capacity and reduced quality of life for affected patients. An improvement in fatigue resistance has been observed in diaphragm muscles of mdx mice following insulin-like growth factor-I (IGF-I) administration. Whether similar treatment can improve locomotor muscle function in mdx mice is not known. We examined the ef.cacy of IGF-I administration (1 mg/kg daily s.c. for 8 weeks) on structural, metabolic, and functional properties of extensor digitorum longus (EDL) and soleus muscles of mdx mice, and tested the hypothesis that IGF-I treatment would improve function in these muscles. After treatment, muscles were more resistant to fatigue during repeated maximal contractions than muscles from untreated mice, an improvement associated with increased muscle .ber succinate dehydrogenase activity in the absence of changes in cellular (single-.ber) contractile activation characteristics. The .ndings have important clinical implications, not just for the dystrophinopathies, but for all neuromuscular pathologies where fatigue of locomotor muscles limits functional capacity and decreases quality of life.

Immune Parameters in Three Duchenne Muscular Dystrophy Patients Following Intramuscular Transplantation of Normal Myogenic Cells Under FK506 Immunosuppression

Transplantation, 2004

D. Skuk, R. Roy, B. Roy, M. Goulet, F.J. Dugre´, J.P.Tremblay – Canada

Summary:

Aims: To test the efficacy of FK506 immunosuppression to control acute rejection in the context of myoblast allotransplantation in Duchenne muscular dystrophy (DMD) patients.

Methods: Three DMD patients received injections of myoblasts obtained from skeletal muscle biopsies of normal donors. The cells (30 x 10⁶) were injected in 1 cm³ of the Tibialis anterior by 25 parallel injections. Similar patterns of saline injections were performed in the contralateral muscles. The patients received FK506 for immunosuppression, the doses being adjusted to maintain a blood concentration between 8 to 20 ug/L. Periodical monitoring of the patients included weight, hemogram, glycemia, lipid profile and serum levels of creatinine, urea, sodium, potassium and chlorides. Patients 1 and 2 were previously taking deflazacort to delay the progression of the disease and we maintained this treatment during the study. Muscle biopsies were performed at the injected sites 4 weeks later. Histological studies were done to analyze the success of the graft, and to characterize the immune cells present in the tissue. RT-PCR analysis of the biopsies were done to observe the expression of INFgamma, TNF-alpha, granzyme B, IL-6 and FasL. The presence of antibodies against the donor cells in blood samples taken before and at 2, 4 and 8 weeks after cell transplantation was analyzed by cytofluorometry.

Results: We observed dystrophin-positive myofibers in the cellgrafted sites of the three patients: 9 % (patient 1), 6.8 % (patient 2), and 11 % (patient 3). Since patients 1 and 2 had identified dystrophin-gene deletions, these results were obtained using monoclonal antibodies specifically to the deleted exons. The grafted site exhibited some pockets of leukocyte infiltration composed of macrophages and CD4 and CD8 lymphocytes in patient 1. In patient 2, similar leukocyte infiltrations were smaller and scarce. No leukocyte accumulations were observed in patient 3. No antibodies against the donor-cells and the donor MHC class-I and class II were detected in the 3 cases. Only the IL-6 mRNA was significantly increased in one grafted site (patient 1). The only potential side effect of the immunosuppression was some weight increase in patient 3.

Conclusions: Significant dystrophin expression can be obtained in the skeletal muscles of DMD patients following specific conditions of cell delivery and immunosuppression. Only the presence of some macrophage/T-lymphocyte accumulations in the vicinity of dystrophin-positive myofibers, and an increase in IL-6 expression could suggest to a moderate, non-completely controlled cellular acute rejection in patient 1. For the rest, the immunosuppression seems quite appropriate. Since patients 1 and 2 received chronic corticosteroid therapy, an additive effect of this treatment on the FK506-based immunosuppression is perhaps possible. The long-term benefit of a FK506-based immunosuppression for myoblast transplantation in DMD patients remains to be tested.