Two N‐glycosylated Asparagines within Endothelial αENaC are Crucial for Shear Force Sensing and Blood Pressure Regulation in vivo
Authors: Knoepp Fenja , Ashley Zoe , Weissmann Norbert , Fronius Martin 
Published: June 2021

Flow‐mediated vascular reactivity contributes to blood pressure regulation, but the underlying mechanisms are not yet fully elucidated. An arising candidate for vascular flow‐sensing is the shear force (SF)‐sensitive epithelial Na+‐channel (ENaC) that is expressed in endothelial cells. However, experimental evidence for the functional significance of ENaC in flow‐mediated blood pressure regulation is scarce. Here we explored the molecular mechanism of how ENaC senses SF and if this mechanism contributes to systemic blood pressure regulation.

Using site directed mutagenesis in combination with heterologous expression in Xenopus oocytes, two asparagines (N) within the αENaC subunit were identified whose glycosylation was crucial for SF‐responsiveness of ENaC, without changing the basic channel properties (open propability, surface expression). Based on these findings, an alpha subunit lacking these two asparagines (ΔN339/538) was overexpressed in endothelial cells of mice. Therefore, adenoviral vectors (AdVs) were used that drive transgene and eGFP expression by the endothelial cell‐specific VE‐cadherin promoter. Three groups of mice were transduced with AdVs, carrying the transgene for either 1) wildtype αENaC (αWT), 2) αΔN339/538 or 3) an empty vector that served as control. Systolic blood pressure and heart rate was determined before and 2 weeks after transduction via tail cuff measurements. Cardiac function was assessed by echocardiography and flow‐mediated responses of isolated carotid arteries analyzed by pressure myography. Fluorescence microscopy was utilized for quantifying protein expression and to prove endothelial cell specific transgene‐localization.

Mice developed hypertension after αWT expression (before: 115 ± 3 mmHg, after: 157 ± 5 mmHg, n= 14, p ≤ 0.01), with no change in heart rate. By contrast, the increase in blood pressure was blunted in mice expressing αΔN339/538 (before: 114 ± 2 mmHg, after: 125 ± 2 mmHg, n = 14, p ≤ 0.01) and absent in controls (before: 112 ± 2 mmHg, after: 114 ± 2 mmHg, n = 15, p = 0.9), although protein‐expression was similar in all three groups. To increase SF within the vasculature, mice underwent treadmill‐exercise. Before AdV‐transduction, exercise elevates blood pressure by 17 ± 2 % in all groups. After transgene expression, exercise‐induced increase in blood pressure was unchanged in αWT and control (15 ± 3% and 17 ± 2 %, resp.). Interestingly, no exercise‐induced increase of blood pressure was observed in αΔN339/538 mice (before exercise: 125 ± 2 mmHg, after exercise: 129 ± 1 mmHg, n = 14, p = 0.5). These findings suggest a critical role for αENaC in SF‐sensing in arteries. Accordingly, the flow‐induced vasoreactivity of isolated carotid arteries from αΔN339/538 mice was augmented compared to αWT and control. Moreover, amiloride (an ENaC inhibitor) had no effect on flow‐induced response of αΔN339/538 but αWT and control arteries.

In conclusion, we show that endothelial αENaC senses SF via two asparagines within its extracellular domain. This enables ENaC to act as endothelial flow sensor that contributes to systemic blood pressure regulation in vivo.

Support or Funding Information

This study was supported by Government funding administered by the Royal Society of New Zealand (Marsden Funding) and a scholarship from the Excellence Cluster Cardio‐Pulmonary System (ECCPS)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.