Regulation of microcirculatory homeostasis in the endothelium by Connexin 43 hemichannel activity

Endothelial Ca 2+ increase plays a key role in the regulation of vascular activity in the microcirculation. This includes processes such as hyperpermeability in veins /capillaries, as well as the regulation of vasomotor tone in resistance arteries. Therefore, the study of Ca 2+ -permeable channels that regulate endothelial activity is a critical concept in vascular research. We recently demonstrated that TRPV4 and connexin 43 hemichannels (Cx43h) contribute to endothelial Ca 2+ influx upon TRPV4 activation. However, it remains unclear whether Cx43h mediate other endothelial vascular responses, whether this represents a general canonical pathway independent of agonist stimulation (e.g., ACh, VEGF, others), or whether genetic tools targeting Cx43h could further refine our findings. We hypothesize that Cx43h are critical Ca 2+ -permeable channels in the endothelium, regulating hyperpermeability and vasomotor tone in the mesenteric bed of mice. Blocking Cx43 hemichannel reduced VEGF-induced hyperpermeability in mouse mesenteric vascular beds in vivo and endothelial cultures. VEGF stimulation increased hemichannel activity in mesenteric endothelial cells (ECs), triggering rapid Ca 2+ influx. This effect was nullified by Gap19 (Cx43h blocker) or NG-monomethyl-L-arginine (LNMMA), blocking NO production. Indicating that NO-SCx43h are critical for mediating hyperpermeability. To explore the role of NO-mediated S-nitrosylation in VEGF-induced hyperpermeability, we used knock-in mouse models incapable of Cx43 S-nitrosylation. These mice showed reduced VEGF-stimulated hyperpermeability in vivo and in vitro. To confirm Cx43h, we used mice with suppressed hemichannel activity, while preserving gap junction function under a VEGF and I-R protocol. Our results show that hyperpermeability was abolished in these KI mice. These results show: 1) Cx43 hemichannels activate crucially in VEGF-induced hyperpermeability, and 2) S-nitrosylated Cx43 regulates I-R inflammatory damage significantly. Finally, using intact mesenteric arterioles and primary ECs, inhibition of Cx43 hemichannels through pharmacological and genetic approaches attenuated endothelial hyperpolarization, a key component of arteriolar relaxation. To test the effect of blunted Cx43h activity on vasomotion in intact in vivo vessels, we used intravital microscopy of mesenteric arterioles. Inhibition of Cx43 hemichannels with Gap19 or in knock-in mice with suppressed hemichannel activity reduced relaxation in response to ACh, emphasizing the physiological role of Cx43 in arteriolar diameter regulation. In summary, we propose that endothelial Cx43h is a critical new Ca 2+ -permeable channel regulating microcirculation activity. Supported by the American Heart Association (AHA) Career Development Award (#932684) and Research Supplement to Promote Diversity in Science (#23DIVSUP1054931) This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.