Transcriptomic and epigenetic assessment of ageing male skin identifies disruption of Ca2+ homeostasis; exacerbated by smoking and UV exposure

Skin ageing has been widely associated with the formation and presence of increasing quantities of senescent cells, the presence of which are thought to reduce cell renewal. This study aimed to identify key factors influencing fibroblast and skin aging in European males using RNA-seq data. Key differences in study designs included known sources of biological differences (sex, age, ethnicity), experimental differences, and environmental factors known to accelerate skin ageing (smoking, UV exposure) as well as study specific batch effects which complicated the analysis. To overcome these complications samples were stratified by these factors and differential expression assessed using Salmon and CuffDiff. Functional enrichment and consistency across studies, stratification’s and tools identified age related alterations in the transcriptomes of fibroblasts and skin. Functional enrichment of results identified alterations in protein targeting to membranes and the ER, and altered calcium homeostasis in aged fibroblasts. Extension to skin controlled for differences in fibroblast culturing methods confirming transient age related alterations in intracellular calcium homeostasis. In middle aged males (40-65) increased keratinisation, skin, epithelial and epidermal development was seen in conjunction with alterations to ER Ca2+ uptake, leading to the identification of related processes including; an unfolded protein response, altered metabolism, increased MMP expression, and altered Ca2+ handling, which were further exacerbated by UV-exposure. Interestingly the genes and processes subsequently decreased in old males (> 65), which exhibited signs of increased senescence. Extension to Illumina 450k array data from ageing skin uncovered evidence of epigenetic regulation; genes and isoforms with overlapping differentially methylated CpGs were differentially expressed. Smoking led to additional enrichment of genes relating to tissue development, cell adhesion, vasculature development, peptide cross-linking, calcium homeostasis, cancer and senescence. The results consistently identified alterations in ER and golgi Ca2+ uptake, which disrupt intracellular and extracellular calcium gradients that regulate metabolic and differentiation signalling in skin and fibroblasts, leading to age related declines skin structure and function. Interestingly many diseases and infections with overlapping molecular consequences, (ER Ca2+ stress, reduced protein targeting to membranes) including COVID-19 are identified by the analysis, suggesting that COVID-19 infection compounds pre-existing cellular stresses in aged males, which could help explain higher COVID-19 mortality rates in aged males, as well as highlighting potential ways to reduce them. ### Competing Interest Statement The authors have declared no competing interest.