New Gap Junction Activity Assay Using Mouse Coronary Endothelial Cells for High-content Screening

Gap junctions (GJs) are crucial for coordinating vascular endothelial cell (EC) function, regulating vascular tone, and ensuring cell survival. GJs are composed of connexins (Cx), primarily Cx37, Cx40, and Cx43 in vascular ECs. Downregulation of connexins is associated with cardiovascular diseases, such as coronary microvascular disease, pulmonary hypertension, and diabetic vascular complications. Enhancing connexin function or expression could serve as a therapeutic strategy. The Scrape Loading/Dye Transfer Assay is commonly used to assess GJ activity, but its limitations in quantification and reproducibility highlight the need for more efficient high-content screening (HTS) methods. This study aims to develop and optimize HTS approaches for assessing GJ function in endothelial cells. Mouse coronary endothelial cells (MCECs) were cultured in donor and recipient flasks. Donor cells were stained with 0.5 µM calcein AM, a green gap junction-permeable dye indicating cell-to-cell communication, and 1 µM Vybrant DiI, a red dye that stains cell membranes, for 30 minutes at 37°C. After staining, donor cells were co-cultured with unstained recipient cells in a 4-well plate at varying donor-to-recipient ratios of 1:5, 1:10, 1:20, or 1:100. Based on initial dye transfer, the 1:20 ratio was selected for further testing. The calcein AM dosage was tested at 0.5 µM and 1 µM, with cell densities of 10 5 , 7.5 x 10 4 , 5 x 10 4 , and 2.5 x 10 4 cells/well assessed. For HTS, donor cells were stained with 1 µM calcein AM and Hoechst 33342 (a blue nuclear stain), while recipient cells were stained with SOYT Deep Red (a red nuclear stain). After 30 minutes, cells were washed and plated in 96-well plates at a donor-to-recipient ratio of 1:20. Following a 6-hour incubation, cells were treated with 50 µM 18β-Glycyrrhetinic acid (18β-GA), a connexin inhibitor, or DMSO as a control. After 15 hours, cells were washed, fixed, and imaged using a fluorescence microscope to assess dye transfer as a measure of GJ activity. The 1:20 donor-to-recipient ratio and 1 µM calcein AM yielded optimal dye transfer. The density of 7.5 x 10 4 cells/well resulted in approximately 100% confluency, forming a monolayer of MCECs. Hoechst 33342 provided superior cell visualization and more accurate cell counting compared to Vybrant DiI. In the 96-well plate assay, cells treated with 50 µM 18β-GA showed a significant decrease in calcein AM dye transfer, confirming the inhibitory effect of 18β-GA on GJ activity in MCECs. This high-content assay efficiently measures gap junction activity in MCECs, enabling quantitative assessments. We plan to use the HTS method to screen potential therapeutic compounds, including various FDA-approved drugs, to modulate GJ communication and treat vascular diseases. Supported by NIH R01HL142214 and DOD W81XWH2110472. 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.