Skin capillary endothelial cells form a network of spatiotemporally conserved Ca(2+) activity.

Ca(2+) signaling and its regulation are important for endothelial cell (EC) functions, including local blood flow control, mechanotransduction, and barrier function. Yet the spatiotemporal organization of Ca(2+) activity and its regulation across a vascular plexus is poorly understood in an in vivo mammalian context, largely due to technical barriers. To overcome this gap in knowledge, we developed an approach to resolve Ca(2+) activity with single cell resolution in the skin vasculature of live adult mice by multi-photon imaging. Here, we tracked thousands of Ca(2+) events in the skin capillary plexus during homeostasis and observed signaling heterogeneity between ECs, with just over half displaying Ca(2+) activity over minutes. Longitudinal tracking of the same mice revealed that the same ECs maintain Ca(2+) activity over days to weeks. Interestingly, activity dynamics, such as frequency and event duration, are not conserved at a single cell level but at an EC population level. To identify the molecular underpinning of this spatiotemporal Ca(2+) activity, we conditionally deleted in ECs the most expressed gap junction protein - Connexin 43 (Cx43). We found that loss of Cx43 initially causes a subset of ECs to display sustained Ca(2+) activity and biases the dynamics of the whole network towards chronically persistent activity over time. Lastly, through pharmacological targeting of a small panel of known Ca(2+) mediators, we showed that inhibition of L-type Voltage Gated Ca(2+) channels largely restores physiological Ca(2+) activity after loss of Cx43, but has no effect on signaling dynamics in homeostatic settings.