Endothelial TRPV4/Cx43 Signaling Complex Regulates Vasomotor Tone in Resistance Arteries

Abstract

S-nitrosylation of Cx43 gap junction channels critically regulates communication between smooth muscle cells and endothelial cells. This posttranslational modification also induces the opening of undocked Cx43 hemichannels. However, its specific impact on vasomotor regulation remains unclear. Considering the role of endothelial TRPV4 channel activation in promoting vasodilation through nitric oxide (NO) production, we investigated the direct modulation of endothelial Cx43 hemichannels by TRPV4 channel activation. Using the proximity ligation assay, we identify that Cx43 and TRPV4 are found in close proximity in the endothelium of resistance arteries. In primary endothelial cell cultures from resistance arteries (ECs), GSK-induced TRPV4 activation enhances eNOS activity, increases NO production, and opens Cx43 hemichannels via direct S-nitrosylation. Notably, the elevated intracellular Ca²⁺ levels caused by TRPV4 activation were reduced by blocking Cx43 hemichannels. In ex vivo mesenteric arteries, inhibiting Cx43 hemichannels reduced endothelial hyperpolarization without affecting NO production in ECs, underscoring a critical role of TRPV4/Cx43 signaling in endothelial electrical behavior. We perturbed the proximity of Cx43/TRPV4 by disrupting lipid rafts in ECs using β-cyclodextrin. Under these conditions, hemichannel activity, Ca²⁺ influx, and endothelial hyperpolarization were blunted upon GSK stimulation. Intravital microscopy of mesenteric arterioles in vivo further demonstrated that inhibiting Cx43 hemichannels activity, NO production and disrupting endothelial integrity reduce TRPV4-induced relaxation. These findings underscore a new pivotal role of Cx43 hemichannel associated with TRPV4 signaling pathway in modulating endothelial electrical behavior and vasomotor tone regulation.

Keywords: Caveolae; ECs; hyperpolarization; resting membrane potential.

Figure 1.. TRPV4/Cx43 complex is primarily observed within the endothelial cell layer of resistance arteries.

Proximity Ligation Assay (PLA) was utilized to illustrate the spatial co-localization between TRPV4 and Cx43. Notably, TRPV4 and Cx43 exhibit predominant distribution within the endothelial layer (EC) compared to the smooth muscle cells (SMC). Controls were conducted to validate the specificity of PLA analysis; wherein primary antibodies were omitted as a negative control. Asterisks indicate the luminal region of the vessels, while the green, fluorescent signal corresponds to the internal elastic lamina (IEL). (BF: Brightfield, Scale bar: 80 μm)

Figure 2.. TRPV4 activation promote S-nitrosylated Cx43 hemichannels activity by NO production.

A) Representative immunofluorescence microscopy images illustrating the detection of phosphorylation status at Serine 177 and Threonine 495 residues of endothelial nitric oxide synthase (eNOS), alongside the assessment of protein levels of S-nitrosylated proteins, in vehicle conditions and upon stimulation with 10 nM GSK 1016790A on primary cultures of ECs. B) Representative images from proximity ligation assay demonstrating Cx43 S-nitrosylation levels in both control and 10 nM GSK 1016790A-stimulated conditions. C) Western blot analysis (left) and quantification (right) of S-Nitrosylated Cx43. The S-nitrosylated Cx43 levels were expressed as fold change relative to total Cx43 protein levels per sample. Comparisons between groups were made using Student’s t test. D) Time course analysis to measure the uptake of 5 μM ethidium, assessing hemichannel activity upon 10 nM GSK 1016790A and under pre-treatment of 100 μM L-NAME, 50 μM glycyrrhetinic acid (β-GA), and 50 μM Gap19. Statistical comparisons between the time course curves were performed using one-way ANOVA followed by Tukey post hoc tests. The total number of mice used in the experiments is indicated in parentheses. (scale bar: 80 um)

Figure 3.. TRPV4/Cx43 hemichannels mediate Ca²⁺ increases in endothelial cells in response to TRPV4 activation

A) Left: Representative imaging captures the endothelial cell responses before and after exposure to 10 nM GSK 1016790A stimulation, both under control conditions and in the presence of 50 uM Gap19. Right: Time course analysis illustrates the fluctuations in Fluo-4 signaling subsequent to GSK stimulation. B) Peak Ca²⁺ increases observed in A. Comparisons between groups were made using Student’s t test. C) Analysis conducted to assess the time required to detect the Ca²⁺ peak under control conditions and in the presence of 50 μM Gap19 upon GSK simulation. Comparisons between groups were made using Student’s t test. The total number of mice used in the experiments is indicated in parentheses. (scale bar: 80 um)

Figure 4.. Cx43 hemichannels mediate endothelium hyperpolarization upon TRPV4 activation in intact endothelium.

A) Left: Representative en face imaging of an arteriole showing ECs detected using FITC-Dextran 3KDa in the pipette solution to measure resting membrane potential changes. Right: Hyperpolarization peaks were induced by TRPV4 activation in intact ECs from mesenteric arteries under control conditions, 50 μM Gap19, and KCa channel blockers (300 nM charybdotoxin and 200 nM apamin). Group comparisons were made using nested ANOVA. B) Time to detect hyperpolarization peaks in ECs treated under control and 50 μM Gap19 conditions following stimulation with 10 nM GSK 1016790A. Group comparisons were made using Student’s t-test. C) Resting membrane potential of arteries under control conditions, with 50 μM Gap19, and with 300 nM charybdotoxin and 200 nM apamin. Group comparisons were made using nested ANOVA. The total number of mice used in the experiments is indicated in parentheses. (scale bar: 20 um)

Figure 5.. Critical Role of eNOS/Cx43/TRPV4 proximity in regulating endothelial Ca²⁺ increases and electrical behavior.

A) Representative imaging of proximity ligation assay (PLA) in control conditions and in ECs treated with 5 mM β-Cyclodextrin to assess the spatial proximity between eNOS-TRPV4, Cx43-TRPV4, and Cx43-eNOS. Strong interactions between these proteins are indicated by red dots, while complete disruption of the PLA signal is observed with 5 mM β-Cyclodextrin treatment. B) Ethidium bromide uptake rate of ECs following stimulation with 10 nM GSK 1016790A under control conditions, extracellular zero Ca²⁺, and β-Cyclodextrin treatment. Group comparisons were conducted using nested ANOVA. C) Changes in intracellular calcium peak increases under control and cyclodextrin conditions in cells treated with 10 nM GSK 1016790A. D) Hyperpolarization peak induced by 10 nM GSK 1016790A under control and cyclodextrin conditions. To assess the activity of KCa channels under β-Cyclodextrin conditions, we used 10 μM SKA-3. Group comparisons were made using nested ANOVA. The total number of mice used in the experiments is indicated in parentheses. (scale bar: 80 um)

Figure 6.. Endothelial Cx43 hemichannels mediate vasomotor responses in mesenteric arteries in vivo.

A) Schematic illustrating intravital microscopy approaches. To enhance vessel visualization, 50 μg/kg FITC-dextran (70 kDa) and 10 μg/kg Gap19, to block Cx43 hemichannel activity, were administered via retroorbital injection. B) Representative imaging of the mesenteric bed at baseline, during the phenylephrine (PE) peak, and at the dilation peak in control and 50 μM Gap19 conditions. C) Representative traces of vasomotor responses induced by 100 nM GSK 1016790A, 10 μM PE, 10 μM ACh, and 10 μM NO donor DEANO in control, 50 μM Gap19 and L-NAME conditions. D) Percentage of relaxation in vessels pre-contracted with 10 μM PE upon GSK, 10 μM ACh. Group comparisons were made using Student’s t-test. The total number of mice used in the experiments is indicated in parentheses. E) Percentage of relaxation in vessels-precontracted with 10 μM PE upon 10 μM DEANO in control and Gap19-treated conditions. Group comparisons were made using Student’s t-test. The total number of mice used in the experiments is indicated in parentheses. (scale bar: 500 um).