Introduction. Cellular senescence, characterised by irreversible cell cycle arrest and a secretory phenotype, is linked to age-related metabolic disorders such as Type 2 diabetes, obesity, non-alcoholic fatty liver disease and many more (1). Emerging evidence highlights calcium signal ling's role in senescence and its therapeutic potential. Repurposing calcium channel modulators offers promise for addressing senescence and associated metabolic disorders.
Aims. To study the re-modelling of calcium signalling pathways in different models of cellular senescence. To test calcium channel modulators for their potential senotherapeutic effect.
Methods. Human derived fibroblasts (HDF, < passage 10) were induced to senescence via Mitomycin C (MMC) (DNA damage) or D-galactose (oxidative stress). Cytosolic calcium was measured using Cal Red™ R525/650 AM (AAT Bioquest) by fluorescence microscopy. Endoplasmic reticulum (ER) Ca2+ release and store-operated Ca2+ entry (SOCE) were measured using Fluo-4 AM (Thermo Fisher Scientific) by plate reader and fluorescence imaging. Gene and protein expression were assessed by qPCR. For drug screening, 140 small-molecule compounds targeting different calcium channels, were tested in our MMC-induced senescence. Senescence markers including SA-β-gal, nuclei area, cell area, P21, and EDU incorporation, were measured by fluorescence microscopy.
Results. Cytosolic Ca2+ increased 2-fold in MMC-induced senescence and 1.5-fold in D-galactose-induced senescence. SOCE response showed a 1.5-fold decrease in MMC-treated cells. This is while, ER Ca2+ release remained unchanged. SOCE components' expression increased over 2-fold in MMC-induced senescence. Markers of senescence-associated secretory phenotype (SASP) were upregulated in MMC-induced senescence and pharmacological suppression of SOCE reduced the expression of some of these markers, including IL-1α, MMP-1, and IL-8, by around two folds. Drug screening revealed specific Ca2+ channel modulators with senomorphic (reducing senescence marker levels) or senolytic (selectively killing senescent cells) abilities.
Discussion. This study identified a remodeling of Ca2+ signalling in two senescence models. In addition, calcium channel drug screening identified potential senotherapeutic compounds. Given that senescence is a cellular phenomenon observed across varied pathological conditions, current hits from our library screen can be repurposed for targeting age related metabolic disorders.