Transcriptional and Electrical Identity in Endothelial Cells Is Orchestrated by Intercellular Coupling

Pia Burboa¹Veronica Kuzdowicz¹Stefany Ordenes²Helmuth A Sánchez¹Mauricio A Lillo^1,3*

• ¹Rutgers University Newark, Newark, United States
• ²Centro Interdisciplinario De Neurociencia De Valparaiso, Valparaíso, Chile
• ³Rutgers Biomedical and Health Sciences, Rutgers, The State University of New Jersey, Newark, United States

The membrane potential (Vm) of vascular cells is a fundamental determinant of vasomotor tone, particularly in resistance arteries and arterioles, where precise electrical signaling ensures tissue perfusion and blood pressure regulation. While the electrophysiological role of vascular smooth muscle cells (VSMCs) is well defined, the bioelectrical contribution of endothelial cells (ECs)—especially their Vm dynamics, calcium responsiveness, and transcriptional adaptability—remains incompletely understood. Here, we combined intracellular microelectrode recordings, calcium imaging, RNAscope, and immunofluorescence to dissect the transcriptional and electrical landscape of ECs in murine mesenteric arterioles and primary cultures. ECs grown in isolation exhibited a depolarized Vm (~–25 mV), disorganized ion channel expression, and diminished levels of key regulators such as FOXO3, MEF2C, KCa2.3, KCa3.1, and Kir2.1. Functionally, these cells failed to hyperpolarize or elevate intracellular calcium in response to acetylcholine (ACh) or the KCa activator SKA-31. In contrast, electrically coupled ECs displayed more negative and heterogeneous Vm values (–65 mV to –40 mV), robust hyperpolarization, and markedly enhanced calcium responses. This phenotype correlated with spatially coordinated upregulation of ion channels and transcription factors, supporting a coupling-dependent transcriptional program that sustains endothelial bioelectrical competence. Time-resolved heatmaps and 3D activity maps revealed that coupled ECs generate synchronized calcium waves upon ACh stimulation, with larger ΔF/F₀ amplitudes and greater spatiotemporal coordination compared with non-coupled counterparts. These organized calcium dynamics mirrored peripheral ion-channel clustering and supported a hyperpolarization-competent phenotype. Notably, about 30% of ECs in coupled conditions exhibited Vm below –55 mV. Consistently, endothelium removal in intact arterioles abolished the hyperpolarized component and depolarized the vessel wall toward –30 mV, indicating that the endothelium plays a dominant role in establishing arteriolar Vm. Altogether, our findings uncover a transcriptionally regulated, calcium-sensitive, and electrically competent endothelial phenotype critically dependent on cell-cell coupling. We propose that connexin-mediated communication not only enables ionic and metabolic exchange but also synchronizes transcriptional programs that define endothelial identity and maintain vascular integration. Disruption of this electro-transcriptional coupling may represent an early hallmark of endothelial dysfunction and impaired vasodilatory conduction.