Dynamic regulation of single-cell contractile heterogeneity in the human trabecular meshwork

Altered trabecular meshwork (TM) mechanics contribute to increased aqueous humor outflow resistance and elevated intraocular pressure in glaucoma, yet how contractility is regulated across individual TM cells remains poorly understood. Using a high-throughput single-cell contractility assay, we quantified time-resolved contractile behavior in primary human TM cells under defined extracellular matrix (ECM) compositions, substrate stiffnesses, and pharmacologic perturbations, and related functional mechanical behavior to intracellular protein abundance. Single-cell analyses revealed that TM contractility is not a uniform population property but a context-dependent contractile state shaped by both extracellular and intracellular regulation. ECM composition established distinct contractile programs, with collagen IV promoting rapid convergence toward a highly contractile state, collagen I supporting a slower but similar response, and fibronectin maintaining a persistently heterogeneous low-contractility state. In contrast, the intracellular perturbation dexamethasone (DEX) did not uniformly increase contractility but selectively prolonged contractile activity within the most mechanically active cells, thereby sustaining highly contractile states. This response was progressively constrained as ECM stiffness increased and was absent on pathologically stiff substrates, demonstrating that extracellular mechanical context conditions glucocorticoid responsiveness. Conversely, ROCK inhibition overrode both basal and DEX-induced contractile programs, collapsing the heterogeneous population into a uniformly low-contractility state. Finally, intracellular protein abundance poorly predicted functional mechanical behavior, with weak pooled associations reflecting divergent donor-specific relationships rather than consistent molecular-mechanical coupling. Together, these findings show that TM contractility emerges from integrating intrinsic cellular state and the extracellular mechanical environment rather than from either alone. More broadly, they identify single-cell contractile state as a functional dimension of TM cell identity that cannot be inferred from static molecular marker abundance or population-averaged mechanical measurements.