ACE2 Ubiquitination in the Paraventricular Nucleus of the Hypothalamus Contributes to Angiotensin‐II‐Induced Hypertension in Mice

Abstract

Ubiquitination is a post‐translational modification of proteins where the ubiquitin moiety is covalently attached at the C‐terminus to specific lysine residues on the target protein, leading to degradation in proteasome or lysosomes. Angiotensin converting enzyme 2 (ACE2) is a transmembrane protein with a primary role in converting Angiotensin (Ang)‐II into Ang‐(1‐7) and thereby reducing blood pressure (BP). We previously reported that ACE2 undergoes internalization and degradation via ubiquitination in response to Ang‐II. Here we investigated whether ACE2 ubiquitination in the paraventricular nucleus of the hypothalamus (PVN), contributes to Ang‐II mediated hypertension in mice. Wildtype C57BL/6J (WT, n=6) and ACE2 KO (n=6) male mice were bilaterally injected into the PVN with an adeno‐associated virus vector expressing a ubiquitin resistant ACE2 mutant in which 5 lysine residues from the C‐terminus of the human ACE2 protein were replaced with arginine (hACE2‐5R). Six‐weeks later, anesthetized mice were connected to a stereotaxic setup and BP was recorded from the common carotid artery. Focal microinjections of Ang‐II (100 ng in 100 nL) were administered into PVN, and BP responses were analyzed. In another group, mice were chronically implanted subcutaneously with BP telemetry and administered Ang‐II via osmotic minipump (600 ng/kg/day) for 2‐weeks. In comparison to WT mice (n=6), acute microinjections of Ang‐II into PVN resulted in a significantly attenuated BP response in hACE2‐5R transfected WT mice (9±1 vs. 14±2 mmHg; n=6, p<0.05). A similarly attenuated response was observed in ACE2 KO mice (n=8) expressing hACE2‐5R when compared to ACE2 KO mice (7±2 vs. 16±3, n=5, p<0.05). Furthermore, chronic Ang‐II‐infused WT and ACE2 KO mice expressing hACE2‐5R exhibited a similar reduction of hypertension (125±2 and 121±1 mmHg, respectively) in comparison to their naïve WT and ACE2 KO controls (137±1 and 148±2 mmHg, respectively, p<0.05). Protein analysis revealed a significant attenuation of AT1receptor expression and increased ACE2 activity in the hypothalamus of mice transfected with hACE2‐5R in the PVN. Our results demonstrate that ACE2 is ubiquitinated by circulating Ang‐II and overexpression of ubiquitination resistant ACE2 enhances the enzyme’s activity and decreases BP in hypertensive mice.