Upregulation of Epithelial Na + Channel Beta Subunit in Isolated Mouse Middle Cerebral Artery Enhances Pressure‐Induced Constriction

Abstract

Pressure-induced constriction (PIC), also referred to as myogenic constriction, is an intrinsic response to the vascular smooth muscle cell (VSMC) of small arteries and arterioles. The response is mechano-dependent and initiated by intraluminal pressure-induced VSMC stretch. While numerous molecules are required for the overall signaling response, our laboratories have focused on the role of Degenerin (Deg) proteins, such as Epithelial Na+Channel (ENaC), because of their close evolutionary link to known mechanosensors in the nematode. Loss of function phenotypes, using broad spectrum pharmacological inhibition and specific gene silencing, have been demonstrated in cerebral arteries. However, a gain of function phenotype has not been shown. Demonstration of a gain of function phenotype is critical to establishing a role for loss of vascular degenerin function in certain diseases, such as pre-eclampsia. Thus, the aim of this study was to determine if upregulation of full-length βENaC in the isolated middle cerebral artery (MCA) enhances the PIC response. To address this aim, MCA segments were dissected from mature, female mice (24 weeks old, mixed genetic background) and transiently transfected with βENaC enhanced green fluorescent protein (EGFP) fusion vector (βENaC-EGFP, n=5) or with EGFP alone (EGFP, n=5) using Lipofectamine 3000 (Thermo Fisher Scientific) using a 1:1 ratio. Vessel segments were incubated for 18 hours at 37 °C and 5% CO2, then pressurized in a pressure-flow myograph system with physiological salt solution. Responses to KCl (20-80 mM), phenylephrine (PE, 10-7-10-4 M), and active (+Ca2+) and passive (-Ca2+) diameters measured under graded pressure steps (15-90 mm Hg, 15 mm Hg increments, 5 min) were determined to assess vasoconstriction to depolarizing and α-adrenergic agonists and intraluminal pressure, respectively. Data were analyzed using repeated measures ANOVA. MCA vasoconstrictor responses to KCl (80 mM, 30.3±2.2% vs 34.3±4.2%, main effect of treatment p=0.55) and PE (10-4 M, 40.3±4.0% vs 35.6.0±3.1%, main effect of treatment p=0.73) were identical in βENaC-EGFP and EGFP transfected segments, respectively. Circumferential stress/strain, wall thickness and wall-to-lumen ratio were also similar. In contrast, PIC responses were greater in βENaC-EGFP group at >30 mm Hg (main effect of treatment p=0.0015). Peak myogenic tone was nearly 2 fold greater in βENaC-EGFP treated segments (11.6±0.4% vs 6.3±0.4% at 75 mmHg, p=0.0006). These data confirm previous findings that βENaC is an important mediator of the PIC response. Moreover, they provide proof-of-concept that upregulating βENaC can potentially improve PIC responses in cerebral vessels and will allow us to test the hypothesis that downregulation of vascular βENaC in inflammatory conditions, such as preeclampsia, contributes to cerebrovascular dysfunction.