SKELETAL MUSCLE REGENERATION IN THE ADULT MOUSE AND RAT: STUDY ON CONNEXIN EXPRESSION AND ROLE IN NORMAL AND REGENERATING SKELETAL MUSCLE AND ON LOW

Abstract

The first aim of present work was to perform a comparative spatial and temporal analysis of connexin (Cx) Cx37, Cx39, Cx40, Cx43, and Cx45 expression in developing skeletal muscle and in the adult regenerating skeletal muscle in response to crush injury. Among the Cxs examined, only the Cx39, Cx43 and Cx45 were found expressed during embryonic life and progressively reduced during early postnatal life to become dramatically expressed at very low levels like Cx43 and Cx45, or to be undetectable like Cx39 in the adult muscle. Cx37 and Cx40 were found expressed at low levels and were localized in the endothelial cells. In the adult skeletal muscle, various kinds of trauma promote proliferation of satellite cells that differentiate into myoblasts forming new myofibers, or to repair the damaged one. Within 24h from injury, Cx37 expression was upregulated in the endothelial cells of blood vessels, and, 5 days after injury, Cx37-expressing cells were found inside the area of lesion and formed clusters generating new blood vessels with endothelial cells expressing Cx37. Three days after injury, Cx39 mRNA was selectively expressed in myogenin-positive cells, forming rows of closely apposed cell nuclei fusing in myotubes. Cx40 mRNA-labeled cells were observed within 24h from injury in the endothelium of blood vessels, and, 5 days after lesion, Cx40-labeled cells were found inside the area of lesionforming rows of myogenin-positive, closely apposed cells coexpressing Cx39. Within 24h from lesion, both Cx43 and Cx45 mRNAs were upregulated in individual cells, and some of them were positive for M-cadherin. Three days after injury, a large number of both Cx43 and Cx45 mRNA-labeled and myogenin-positive cells were found inside the area of lesion. Taken together, these results show that at least four Cxs, out of five expressed in regenerating skeletal muscle, can be differentially involved in communication of myogenic cells during the process of cell proliferation, aggregation, and fusion to form new myotubes or to repair damaged myofibers. The second aim of this study was to examine how low-intensity endurance exercise affects the regeneration process in dystrophin-deficient skeletal muscle. The lack of dystrophin in mdx mice, an animal model for Duchenne muscular dystrophy, leads to cycles of muscle degeneration and regeneration processes. Male adult mdx and wild type mice were subjected to low-intensity endurance exercise by running on a motorized Rota-Rod for 5 v Dott.ssa M. Frinchi days/week, for 4 weeks at progressively increasing loads. Exercised mdx mice showed a trend to lower body weight gain and positive effect on the degree of fatigue. Histomorphological analysis showed a significant reduction of both muscle necrosis foci and regeneration processes in the gastrocnemius and quadriceps muscles of exercised mdx mice. The reduction of regeneration process was also evaluated by examining the protein expression of Cx39, as a specific gene expressed during regeneration process of injured muscles. While Cx39 was not expressed both in wild type exercised nor in sedentary mice, it was markedly increased in sedentary mdx mice, because of active degeneration/regenerating process, and dropped to very low levels in exercised mdx mice, suggesting a reduction of muscle regeneration process. This study has shown that specific low intensity endurance exercise induces a strong beneficial effect on the regeneration of dystrophic muscle and may have therapeutic value at least to decrease progression of muscular dystrophy and in less extend for strengthening dystrophic muscle.