Methods of senescence induction in human AC16 Cardiomyocytes and associated pathway and phenotypic changes

Cellular senescence, induced by stress factors within a cell, is a state of cell growth arrest that has been found to contribute to age-related diseases. Senescent cells exhibit a particular phenotype that is generically characterized by increased expression of cell-cycle regulator proteins, increased expression of senescence associated beta-galactosidase, and increased oxidative mitochondrial stress. Particularly in the cardiomyocyte, this state of growth arrest contributes to many prevalent cardiomyopathies. However, while it is broadly known that oxidative stress and DNA damage inducing conditions can contribute to a cell becoming senescent, methods to induce senescence in cardiomyocytes effectively have yet to be systematically investigated. In particular, the particular lesions and concentrations that can adequately induce senescence as well as the particular pathway through which senescence is induced in each induction method are unknown. Accordingly, the aim of this study is to optimizethe dosage and regimen of three pro-senescence insults (hydrogen peroxide, doxorubicin, and UV-C light) that will best induce senescence as measured by the associated senescent phenotype while gaining deeper understanding of differences in the pathways that are employed within a cell in the process of inducing senescence from different forms of stress. To do so, we used western blots and staining techniques in order to analyze relative expression of classic senescence markers in cells that were exposed to stress conditions as compared to healthy cells. The most relevant proteins that were compared were p16 and p21, cell cycle regulator proteins, and H2A.X, a double-stranded DNA damage protein. We found that in conditions of 500 μM H2O2, 0.1 μM Dox, and 5 mJ/cm^2 UV-C light, p21 and H2A.X were significantly upregulated and p16 was interestingly found to be significantly downregulated in all conditions. Treated samples were then filtered and tested via mass spectrometry in order to determine relative protein expressions. Preliminary mass spectrometry data used to compare the different insults showed some evidence of activation of different senescent pathways. Samples insulted with Dox showed an enrichment in the apoptotic pathway while those treated with UV-C or H2O2 showed enrichment in translation elongation and nonsense mediated decay related pathways. Furthermore, we found that combinatorial treatment of H2O2, UV-C light and doxorubicin increased expressed mitochondrial oxidative stress as seen by MitoSox staining techniques. In summary, our results showed the different concentrations that can induce senescent phenotypes in cardiomyocytes and the differences in the phenotypes through different senescence induction methods.