The aging of skeletal muscle: morphological experimental approaches

The research project of my doctorate was to further investigate on myonuclear involvement in skeletal muscle aging as well as age-related changes in muscle ECM. In this purpose, two different methodological approaches were used. First, ultrastructural immunocytochemistry revealed age-related differences in the distribution and density of some molecular factors involved in RNA pathways between old (28 months) mice and their late adult counterpart (19 months). Second, morphological and morphometrical evaluation (performed by fluorescent and transmission electron microscopy) combined with proteomic approach showed higher amount of several muscle ECM components in old (24 months) compared to adult mice (12 months), suggesting that muscle ECM remodelling may hinder muscle cell-matrix interplay as well as its structural organization. Once age-related muscular dysfunctions had been identified, we investigated two strategies potentially able to counteract sarcopenia: physical exercise (as a non-pharmacological approach) and nanotechnology (considered as a suitable tool for pharmacological intervention). Preliminary data based on morphometrical and morphological evaluations performed at transmission and fluorescence microscopy showed that in old (24 months) trained mice physical activity limit/prevent age-related modifications of myofiber (at cellular and sub-cellular level) as well as muscle ECM organization. Further, a pilot study tested the suitability of phospholipid-based nanoparticles (NPs: ethosomes, transethosomes) as nanocarriers to deliver vitamin D3, based on evidence suggesting that vitamin D supplementation may improve muscle functionality in elderly. We have evaluated NPs biocompatibility in three different cell line representative of epithelial, connective and muscle tissue. Then, observation at bright-field, fluorescence and transmission electron microscopy were performed to assess NPs internalization, intracellular fate and the potentially associated subcellular alteration. Our preliminary results set the stage for in vivo investigations aimed at testing vitamin D transdermal administration as a novel strategy to address age-related muscular disease. Finally, during my research period abroad I collaborated in setting up an in vitro co-culture system containing primary macrophages and murine myoblast cells which were previously exposed to etoposide (a drug able to induce DNA damage). Muscle cells were investigated by real-time PCR, to determine whether myoblast gain or lose their intrinsic function in presence of DNA damage and in presence or absence of macrophages. Lastly, immunofluorescence investigations were carried out in muscle cells to investigate the ability of macrophages to counteract DNA damage. The purpose of this preliminary investigation was to describe in vitro the crosstalk between macrophages and muscle cells under DNA damage condition.