Perturbation of muscle regeneration by small molecules

Skeletal muscle plays fundamental roles for locomotion, posture maintenance and breathing and to preserve its function, skeletal muscle has developed a remarkable capacity to regenerate also after severe damage. Several studies aimed at understanding the cellular and molecular mechanisms involved in muscle repair that are deregulated in muscular dystrophy-associated fibrosis and in aging-related muscle dysfunction. However, the cellular and molecular effectors of muscle repair remain largely unknown. This doctoral thesis aims at understanding molecular mechanisms and the interplay between different muscle populations, by perturbing muscle regeneration and differentiation with small molecules. Recent studies have suggested that muscle regenerative process is improved when AMPK is activated. In the muscle of young and old mice a low calorie diet, which activates AMPK, markedly enhances muscle regeneration. Remarkably, intraperitoneal injection of AICAR, an AMPK agonist, improves the structural integrity of muscles of dystrophin-deficient mdx mice. Building on these observations we asked whether metformin, a powerful anti-hyperglycemic drug, which indirectly activates AMPK, affects the response of skeletal muscle to damage. In our conditions, metformin treatment did not significantly influence muscle regeneration. On the other hand we observed that the muscles of metformin treated mice are more resilient to cardiotoxin injury displaying lesser muscle damage. Accordingly myotubes, originated in vitro from differentiated C2C12 myoblast cell line, become more resistant to cardiotoxin damage after pre-incubation with metformin. Our results indicate that metformin limits cardiotoxin damage by protecting myotubes from necrosis. Although the details of the molecular mechanisms underlying the protective effect remain to be elucidated, we report a correlation between the ability of metformin to promote resistance to damage and its capacity to counteract the increment of intracellular calcium levels induced by cardiotoxin treatment. Since increased cytoplasmic calcium concentrations characterize additional muscle pathological conditions, including dystrophies, metformin treatment could prove a valuable strategy to ameliorate the conditions of patients affected by dystrophies. Moreover, in order to understand and control the differentiation decisions of muscle cell populations, we used automated fluorescence microscopy to screen the Prestwick library of small molecules 100% approved by the U.S. Food and Drug Administration (FDA). We have developed fluorescence microscopy readouts to monitor cell proliferation and differentiation into skeletal muscle, adipocyte or osteoblasts both in mesoangioblasts (MABS), a muscle multipotent cell line, and in a heterogeneous mixture of diverse muscle cell populations. We performed the high-throughput and highcontent screening, in order to identify compounds that either promote or inhibit the differentiation process. To date we have screened 240 molecules and we produced a list of drugs that affect the differentiation of muscle cells in skeletal muscle, adipocytes or osteoblasts. We have performed experiments to validate the list of putative interfering small molecules and in parallel we have built a similarity tree from the collection of transcriptional expression profile data from cultured human cells treated with bioactive small molecules, developed by the Connectivity Map team in The Broad Institute of MIT. By highlighting the obtained putative drugs in the similarity tree, we can discriminate if drugs affecting a specific differentiation phenotype may do so via similar or different molecular pathway and we can identify molecular mechanisms involved in differentiation decision.