Matrix and cytoskeletal tension gate stretch-induced calcium signaling

The extracellular matrix (ECM) and mechanical loading shape cellular behavior, yet their interaction remains obscure. We developed a dynamic proto-tissue model using human tendon fibroblasts and live-cell calcium imaging to study how ECM and cell mechanics regulate mechanotransduction. Stretch-induced calcium signaling served as a functional readout. We discovered that ascorbic acid-dependent ECM deposition is essential for proto-tissue maturation and the recovery of stretch-induced calcium signaling at physiological strains. ECM synthesis and mechanical integration enhanced stretch sensitivity, reducing the strain needed to trigger a calcium response from ∼40 % in isolated cells to ∼5 % in matured proto-tissues. A strong correlation between tissue rupture and onset of calcium signaling indicates a mechanistic link to ECM damage. Disrupting ECM crosslinking, ECM integrity, cell alignment, or cytoskeletal tension reduced mechanosensitivity, demonstrating that stretch-induced calcium signaling depends critically on ECM-cytoskeleton integration and mechanics. Fundamentally, our work closely replicates stretch-induced calcium signaling observed in rodent tendon explants in an in vitro model and bridges the gap between cell-scale and tissue-scale mechanotransduction. STATEMENT OF SIGNIFICANCE: The dysregulation of the tendon extracellular matrix is central to tendon disease, with controlled mechanical loading via physical therapy as the only established treatment. Tendon cells repair and maintain the matrix based on mechanical demands, yet how they sense loading and how matrix or cytoskeletal mechanics influence this process remains unclear. Animal models are often impractical, and existing in vitro models lack physiological relevance. We developed a dynamic in vitro model that replicates load-induced calcium signaling, a physiological tendon cell response observed in rodent tendons, and show that matrix and cytoskeletal mechanics are key to load sensation. Anchored to a validated sensory response, our model enhances physiological relevance and offers a platform to study tendon degeneration and recovery mechanisms.