Skin capillary endothelial cells form a network of spatiotemporally conserved Ca²⁺ activity

Ca²⁺ signaling and its regulation are important for endothelial cell (EC) function and signaling. Yet, the spatiotemporal organization of Ca²⁺ activity and its regulation across a vascular plexus is poorly understood in an in vivo mammalian context. To overcome this gap in knowledge, we developed an intravital imaging approach to resolve Ca²⁺ activity with single-cell resolution in skin vasculature of adult mice via multiphoton microscopy. Here, we tracked thousands of Ca²⁺ events in the skin capillary plexus during homeostasis and observed signaling heterogeneity between ECs, with just over half displaying Ca²⁺ activity at any given time. Longitudinal tracking of the same mice revealed that the same capillary ECs maintain Ca²⁺ activity over days to weeks. Interestingly, activity dynamics, such as frequency and event duration, are not conserved at a single-cell level but are maintained at an EC population level. Molecularly, conditional deletion of the gap junction protein Connexin 43 (Cx43cKO) in ECs leads to a subset of ECs displaying sustained Ca²⁺ activity, biasing signaling dynamics of the whole network toward chronically persistent activity over time. Sustained capillary Ca²⁺ activity results in vascular permeability and flow dysregulation. Last, through pharmacological targeting of known agonists/antagonists, we showed that inhibition of L-type Voltage Gated Ca²⁺ channels non-cell-autonomously restores Ca²⁺ activity, blood flow, and barrier function in Cx43cKO mice. Collectively, our work provides insight into the spatial and temporal characteristics, extent, and regulation of Ca²⁺ activity in skin capillaries of live mice.