Changes in caveolin-1, caveolin-3 and vascular endothelial growth factor expression and protein content after botulinum toxin A injection in the right masseter muscle of dystrophin deficient (mdx-) mice.

Progressive muscle wasting, frequently associated with inflammation, muscle fibre degeneration and fibrosis, is a characteristic of DMD (Duchenne muscular dystrophy). Its most common used animal model, the mdx mouse, however can overcome muscle degeneration by regeneration processes and is for this reason not suitable to answer all scientific questions. The aim of this study was to evaluate the ability of botulinum toxin A (BTX-A) in breaking down muscle regeneration in mdx mice. For this purpose, the right masseter muscle of 100 days old mdx and healthy mice was paralyzed by a single specific intramuscular injection of BTX-A. After 21 days, right and left masseter and temporal muscles as well as tongue muscle were carefully dissected, and gene and protein expression of caveolin-1, caveolin-3 and vascular endothelial growth factor (VEGF) were determined using quantitative RT-PCR and Western blot technique. Statistics were performed using Student's t-test and Mann Whitney U-test (significance level: P ≤ 0.05). After BTX-A injection, in both mice strains and for all three studied genes, no significant differences in mRNA amount could be detected between treated and untreated masseter muscles. A significant increase in caveolin-1, caveolin-3 and VEGF mRNA expression could only be found in the right temporal muscle of control mice compared to the left side. All three investigated proteins were more frequent to be found in dystrophic masseter muscle samples compared to the corresponding control samples, whereas significant decreased caveolin-3 protein levels could only be detected in the treated masseter versus untreated masseter muscle of controls. In contrast to previous conclusions, with this study it was not possible to prove a BTX-A-induced dystrophic phenotype in control animals, in which only the known decreases of caveolin-3 protein expression could be verified due to denervation. At the same time, however, gene and protein expression in dystrophic mice was not changed after BTX-A injection.

Differential expression of genes involved in the calcium homeostasis in masticatory muscles of MDX mice.

Duchenne Muscular Dystrophy (DMD) and its murine model, mdx, are characterized by Ca(2+) induced muscle damage and muscle weakness followed by distorted dentofacial morphology. In both, DMD patients and in mdx mice, could be proven so far that only the extraocular muscles (EOM) are not affected by muscular dystrophy. The EOMs are protected against calcium overload by enhanced expression of genes involved in the Ca(2+) homeostasis. We could recently demonstrate that masticatory muscles of mdx mice are differentially affected by muscle dystrophy. The dystrophic masseter and temporalis shows muscle histology comparable to all other skeletal muscles in this animal model, whereas dystrophic tongue muscles seem to develop a milder phenotype. Due to this fact it is to hypothesize that an altered Ca(2+) homeostasis seems to underlie the mdx masticatory muscle pathology. Aim of this study was to examine the mRNA and protein levels of the sarcoplasmic reticulum Ca(2+) ATPases SERCA1 and SERCA2, the plasma membrane Ca(2+) ATPases Atp2b1 and Atp2b4, the sodium/calcium exchanger NCX1, the ryanodine receptor 1, parvalbumin, sarcolipin, phospholamban and the L-type Ca(2+) channel alpha-1 subunit (Cacna1s) in Musculus masseter, temporalis, and tongue of 100 day old control and mdx mice. In mdx masseter muscle significant increased mRNA levels of NCX1 and Cacna1s were found compared to control mice. In contrast, the mRNA amount of RYR1 was significant reduced in mdx temporalis muscle, whereas ATP2b4 was significant increased. In mdx tongue a down-regulation of the ATP2b1, sarcolipin and parvalbumin mRNA expression was found, whereas the phospholamban mRNA level was significantly increased compared to controls. These data were verified by western blot analyses. Our findings revealed that mdx masticatory muscles showed an unequally altered expression of genes involved in the Ca(2+) homeostasis that can support the differences in masticatory muscles response to dystrophin deficiency.