Exploring Replicative Senescence and Oxidative Stress-Induced Remodelling of Mitochondrial-Associated Membranes in Human Skin Fibroblasts

(1) Background: Calcium transfer between the endoplasmic reticulum (ER) and mitochondria through the IP3R-VDAC1 complex at mitochondria-associated ER membranes (MAMs) is essential for cellular homeostasis. Alterations in this signalling axis have been implicated in ageing and cellular senescence. (2) Methods: We developed an in vitro human dermal fibroblast (HDF) model combining replicative senescence and acute oxidative stress to investigate the role of ER-mitochondria coupling in skin ageing and to enable biomolecule screening. (3) Results: In situ proximity ligation assays revealed that replicative senescence significantly increased the number of VDAC1/IP3R complexes per cell (+85% and +72%, p < 0.01), together with elevated cellular reactive oxygen species (+47% and +74%, p < 0.05). Consistently, acute oxidative stress (50 µM t-BHP, 30 min) rapidly increased VDAC1/IP3R complexes (+48%, p < 0.001) and intra-mitochondrial calcium levels (+19%, p < 0.001). These effects persisted for 24 h post-treatment and were associated with impaired mitochondrial function (-27% in the Bioenergetic Health Index, p < 0.05). We also established a flexibility index capturing both acute and long-term adaptations and detecting the protective effects of an orchid extract. (4) Conclusions: ER-mitochondria coupling disruption via the IP3R-VDAC1 complex may contribute to oxidative stress-induced senescence and represent a key mechanism in extrinsic skin ageing.