Three ways to try and heal a diseased muscle: macrophages, exon skipping and cell transplantation

Muscular dystrophies have been the main interest of our group for many years. Here we will present three lines of research that deal both with basic features of muscle pathologies and with the development of possible therapeutic approaches. The first one has to do with the study of the role of macrophages in the context of muscle regeneration. To this aim, we use the murine macrophage cell line J774 to obtain a serum-free, conditioned medium (mMCM) that we previously found to enhance the proliferation rate and the differentiation of rat and human myoblast (these latter both normal and dystrophic) (1-2). We are now characterizing its mechanism(s) of action in the murine model. We confirmed the pro-proliferative effect of mMCM on murine satellite cells, plus a pro-differentiation activity, and at the same time had a distinct anti-proliferative effect on primary fibroblasts from dystrophic muscle (i.e., from mdx mice). We also investigated the effects of mMCM on macrophages polarization, using human monocytes from blood that were differentiated and then stimulated to acquire either pro- or anti-inflammatory phenotype. Results so far indicate that mMCM seemed to have mixed effects, indicating that its composition might affect both populations. Similar findings were obtained in vivo, when the administration of mMCM to wt regenerating muscle seemed to decrease the number of both pro- and anti-inflammatory macrophages. Importantly, when applied in experiment of cell transplantation in dystrophic muscle, macrophagic factors led to much better grafting of donor wt satellite cells. At the same time, we are also using the mdx dystrophic model to study the endogenous mechanisms that lead to the formation of dystrophin-positive fibers in the context of a dystrophic background, the so-called “revertant” fibers. Such phenomenon has been reported in man as well in animal models and is thought to occur thanks to alteration of the splicing mechanisms. We have analyzed a wide cohort of mdx mice of different ages and looked at different muscle types, showing that, as opposed to what had been suggested previously, the formation of new revertant fibers is a phenomenon that occurs throughout the animals’ life. Besides, we found evidences that the number of revertant fibers increases with age not only in skeletal muscle but also in the heart, something that had never been reported before. We are now trying to clone satellite cells in which the ‘reversion’ phenomenon has occurred, in order to be able to study the biological mechanisms behind it. 1) Malerba et al., (2009) Selection of multipotent cells and enhanced muscle reconstruction by myogenic macrophage-secreted factors. Exp Cell Res 315: 915-27. 2) Malerba et al., (2008). Macrophage-secreted factors enhance the in vitro expansion of DMD muscle precursor cells while preserving their myogenic potential. Neurol Res, 32(1):55-62