Tamoxifen increases muscular strength of the mdx dystrophic mice

Abstract from 44th Annual Meeting - American Society for Cell Biology (December, 4-8, 2004, Washington)

Cavalsan,¹ R. F. Vasquez,¹ R. C. Lin,¹ S. B. Zyngier,¹ F. J. Velloso,² B. H. Santos,² L. L. Fogaca,² M. Vainzof,² D. Feder¹ ; ¹ Pharmacology, ABC Faculty of Medicine, Santo Andre, Brazil, ² Human Genome Research Center - IB USP, Sao Paulo, Brazil

The mdx mice is a well-known model of Xp21 dystrophin-deficient muscular dystrophy. Although a good genetic and biochemical model, the mdx shows no muscle weakness, but under physical exercise, a loss of muscular strength can be detected. Here we have tested the possible therapeutic beneficial effect of Tamoxifen in the degenerative process of the dystrophic muscle, analyzing muscle strength of tamoxifen treated mdx mice, under intensive physical exercise. A total of 22 mices aged 4 weeks were divided into 3 groups: control (n= 8) treated with 0.5 mL saline ip, Tamoxifen 5 mg/Kg body weight ip (n= 8) daily and Tamoxifen 10 mg/Kg ip (n=6) daily. The exercise protocol was done in a wheel revolving at 18 cm/s, for 10 minutes, twice a day, 5 days/week, up to 12 weeks. Whole-body strength was measured weekly using a force transducer coupled to a computer. Mice tails were attached via a non-flexibile nylon cord to the transducer, the animals were electricly stimulated to run, and the force to pull the cord was registered continuously. Results were analyzed by the Kruskal-Wallis test. The mice treated either with 5 mg/Kg (6.26+ 1.44 dynes/g body weight) or 10 mg/Kg (6.46+ 2.52) showed a significant increase of muscular strength (p<0.05) compared to the control group (3.66+ 0.77) starting in the 5^th week, and maintaining significance up to the end of the experiment. Histological and histochemical analysis of the complex of the gastrocnemius muscle are under analysis, but preliminary results suggest a less degree of degeneration in the Tamoxifen treated groups.

Tamoxifen is an anti tumoral drug and act on TGF-beta. Its possible therapeutic effect in the degenerative process of the dystrophic muscle could ameliorates the clinical course of dystrophic patients. FAPESP-CEPID, CNPq, PRONEX, NEPAS-FMABC.

Human Retinal Dystrophin Transgene Converts Lethal Muscular Dystrophy into Viable Mild Myopathy in Dystrophin-Utrophin Null Mice

Abstracts from 44th Annual Meeting - American Society for Cell Biology (December, 4-8, 2004, Washington)

R. Gaedigk,^1,2 D. J. Law,³ K. M. Fitzgerald-Gustafson,^4,2 S. G. McNulty,¹ N. N. Nsumu,¹ A. C. Modrcin,⁵ R. J. Rinaldi,⁵ D. Pinson,⁶ S. C. Fowler,⁷ M. Bilgen,^4,8 J. Burns,⁹ S. D. Hauschka,¹⁰ R. A. White^1,2 ; ¹ Medical Research, Children's Mercy Hospitals & Clinics, Kansas City, MO, ² Pediatrics, UMKC School of Medicine, Kansas City, MO, ³ School of Biological Sciences, Univ. of Missouri-Kansas City, Kansas City, MO, ⁴ Hoglund Brain Imaging Center, Univ. of Kansas Medical Center, Kansas City, KS, ⁵ Rehabilitation Medicine, Children's Mercy Hospitals & Clinics, Kansas City, MO, ⁶ Pathology & Laboratory Medicine, Univ. of Kansas Medical Center, Kansas City, KS, ⁷ Pharmacology & Toxicology, Univ. of Kansas, Lawrence, KS, ⁸ Molecular & Integrative Physiology, Univ. of Kansas Medical Center, Kansas City, KS, ⁹ Veterinary Imaging Services, Topeka, KS, ¹⁰ Biochemistry, Univ. of Washington, Seattle, WA

Duchenne muscular dystrophy (DMD) is a progressive muscle disease caused by severe dystrophin gene mutations that often result in death by the third decade. The mdx mouse is the most commonly used DMD model. Although they lack dystrophin and have underlying muscle disease, mdx mice appear physically normal. This may be partially due to compensatory expression of the dystrophin-related protein, utrophin. In contrast, double mutant mice (DM), deficient for both dystrophin and utrophin (mdx/+, utrn -/-), die by 3 months of age and suffer from severe muscle weakness, pronounced growth retardation, kyphosis, weight loss, slack posture, and immobility (Deconinck et al. (1997) Cell 90: 717-727; Grady et al. (1997) Cell 90: 729-738). These features make them an excellent physiological model for DMD research. The capacity of a naturally occurring isoform, human retinal dystrophin (Dp260), to compensate for the missing muscle isoform Dp427 was tested in DM mice. The expression of this transgene prevents premature death and reduces the severe muscular dystrophy phenotype to a mild myopathy. Electromyography (EMG), histology, radiography, magnetic resonance imaging, and behavior studies show that DM transgenic mice grow normally, have normal spinal curvature and locomotion, and have reduced muscle pathology. EMG and histologic data from transgenic DM mice are typical of mild myopathy, while the DM mice exhibit severe abnormalities commonly seen in human dystrophinopathies. The expression of human Dp260 in DM mice converts a severe and lethal muscular dystrophy into a non-lethal mild myopathy. Muscle-specific expression of Dp260 could have several advantages over other treatments of DMD

Visualization of Ectopic Calcification in mdx Mouse Skeletal Muscle
N. Kikkawa, T. Ohno, M. Shiozuka, R. Matsuda; The Graduate School of Arts and Sciences, The University of Tokyo, Tokyo, Japan

It has been demonstrated that osteogenic differentiation of skeletal muscle cells are induced by osteogenic factors, such as bone morphogenetic proteins, both in vitro and in vivo. Spontaneous ectopic bone formation in vivo has also been reported in, for instance, fibrodysplasia ossificans progressiva, which is a rare bone disorder. Another example of self-generating ectopic calcification has been found in skeletal muscle of mdx mouse, a model of Duchenne muscular dystrophy (DMD).

We observed the ectopic calcification in mdx mouse thigh muscle by using x-ray micro CT-scanner SkyScan-1074, which gives resolution of 22um. The x-ray images were reconstructed into three dimentional visions. Calcifications were found as spicular structures running parallel to the muscle fiber. Paraffin sections of the regions found by X-ray microtomography were Von Kossa stained to confirm calcium deposits. We also detected ectopic calcifications of living mice with mouse-whole-body x-ray CT-scanner of Aloka. No ectopic calcification was observed in skeletal muscle of B10 mouse, which possesses normal dystrophin gene and was used as a negative control.
Calcified regions of mdx mouse thigh did not overlap with the Evans blue-positive areas but corresponded to some of the regenerating areas. Thus, ectopic calcification can be a diagnostic marker of muscle regeneration in mdx mice, and be available to determine the effects of drug or gene therapies.

Muscle Proteoglycan Levels in Duchenne Muscular Dystrophy Differ from Other Muscular Dystrophies
S. Zanotti, E. Canioni, F. Cornelio, L. Morandi, M. Mora; Neuromuscular Diseases and Neuroimmunology, National Neurological Institute C. Besta, Milano, Italy

Biglycan and decorin are small leucine-rich extracellular proteoglycans that interact with several matrix proteins particularly collagens, and also with cytokines whose activity they may modulate. To better understand the role of these proteoglycans in muscle fibrosis in muscular dystrophies, we investigated the expression of their transcripts and proteins in several forms of muscular dystrophy compared to age-matched controls. mRNAs of both proteoglycans were significantly downregulated in Duchenne muscular dystrophy and significantly upregulated in Becker muscular dystrophy, sarcoglycanopathies and dysferlinopathy. By immunohistochemistry, decorin and biglycan were mainly localized in connective tissue and apparently increased with age. Their presence increased in relation to increased fibrosis in all types of dystrophic muscle.
The increase in biglycan and decorin in most muscle diseases indicates a role of these proteoglycans in the extracellular matrix organization. The significant decrease of decorin and biglycan mRNA in DMD distinguishes this disease from the other studied and may be related to the high levels of TGF-beta1 and TNF-alpha found in this disease.

Sex Differences in Muscle Stem Cell Transplantation Efficiency
B. Deasy,¹ A. Lu,¹ K. Urish,¹ J. Tebbets,¹ B. Gharaibeh,¹ R. Rubin,² J. Huard¹ ; ¹ University of Pittsburgh, Pittsburgh, PA, ² Allegheny General Hospital, Pittsburgh, PA

We previously demonstrated that muscle-derived stem cells (MDSCs) efficiently regenerate skeletal muscle tissue after transplantation into dystrophic mdx mice, which model muscular dystrophy. Here we show that transplantation of female MDSC (F-MDSC) rather than male MDSCs (M-MDSC) significantly improves skeletal muscle regeneration.

We transplanted F-MDSCs into the gastrocnemius muscles of female mdx mice and the same number of M-MDSCs into the gastrocnemius muscles of male mdx mice. We used a previously described protocol to measure the cells’ regeneration index (RI)_the number of dystrophin-positive fibers in the host muscle per 10⁵ donor cells_two weeks after transplantation. The F-MDSCs elicited significantly superior muscle regeneration equivalent to a 6-fold increase in efficiency (F-MDSC RI=686±120 versus M-MDSCs RI=105±25, P< 0.01). Sex-crossed transplantation also demonstrated the same trend for the donor cells. We hypothesize that this difference may be related to reduced ability of the M-MDSC to self-renew or to tolerate hypoxic stress. F-MDSCs maintained CD34 expression (>80% of total cells) for up to 200 population doublings, whereas M-MDSCs exhibited a rapid drop in CD34 expression (from >80% to <30% of total cells) after only 45 population doublings. To support the notion that the F-MDSC are more tolerant to stress, we also found that proliferation of F-MDSC was less affected by low O₂ conditions as compared with M-MDSC.

Our discovery of sex-related differences in muscle stem cell biology reveals clear limitations to using male stem and progenitor cells for cell-tracking purposes. This finding also warrants careful consideration by researchers working to identify optimal populations of stem and progenitor cells for use in cell therapy and tissue engineering, clinicians who perform bone marrow stem cell transplantation, and basic scientists investigating cell and developmental biology.