Selective blockade of AT1R and B1R modulates the expression of ACE2 ubiquitination partners

Abstract

Angiotensin Converting Enzyme 2 (ACE2) is an important compensatory enzyme in the renin‐angiotensin system (RAS), converting Angiotensin (Ang)‐II to Ang‐(1‐7). We previously reported that Ang‐II mediates ACE2 internalization through its type 1 receptor (AT1R), followed by ubiquitination and degradation in lysosomes. We recently identified UBR1 and BRCC36, as potential ubiquitinase and deubiquitinase, respectively, modulating ACE2 expression in hypertensive conditions. Here, we aimed to evaluate the role of bradykinin B1R receptor in Ang‐II‐mediated ACE2 internalization and the enzyme’s ubiquitination partners in hypertension.C57BI/6J mice (3‐month‐old, n=3/group) were divided into 3 groups: control (Ctrl), B1R antagonist SSR240612 (SSR), AT1R antagonist Losartan (Los). Mice were implanted with blood pressure (BP) probes (telemetry) and upon recovery, baseline BP was recorded for 24 hours. Mice were then implanted subcutaneously with an osmotic pump containing Ang‐II (490 ng/Kg/min/4 weeks) or saline. BP was recorded weekly, for 4 weeks. After 2weeks of Ang‐II infusion, the mice were implanted with an additional osmotic pump containing Losartan (30 mg/Kg/min) or SSR240612 (10 mg/Kg/min) for 2 weeks. Animals were sacrificed and the heart was isolated from all groups and protein expression for UBR1, BRCC36 and ACE2 was assessed using capillary western. Clinical relevance was further assessed in human heart left ventricle tissues of normal and hypertensive donors.Ang‐II infusion induced a significant increase in mean BP in Ctrl group (159.2±1.5 vs. 100.6±0.5 mmHg; one‐way ANOVA, **P<0.001) after 4 weeks of infusion. This was significantly attenuated with blockade of B1R or AT1R (SSR: 98.8±0.6; Los: 99.6±0.8 mmHg; n=3: one‐way ANOVA, **P<0.001). Capillary western with mouse heart tissue confirmed that Ang‐II‐induced a significant 2‐fold increase (*P<0.05) in UBR1 expression and the SSR240612 treatment contributed to a significant 4‐fold increase (**P<0.001). However, losartan had no effect. BRCC36 expression in Ang‐II, losartan and SSR240612 treated group was significantly decreased by 2‐3‐fold (*P<0.05). However, Ang‐II induced decrease in ACE2 levels was significantly attenuated by losartan and SSR240612 (**P<0.001), indicating both B1R and AT1R receptors contribute to ACE2 internalization and degradation. In human’s, the UBR1 expression was 2 folds (*P<0.05) higher in hypertensive CA male and AA female and BRCC36 expression showed 2 folds decrease (*P<0.05) in CA hypertensive females. However, ACE2 expression showed 3 folds decrease in hypertensive CA males, indicating elevated levels of UBR1 mediated ACE2 ubiquitination and degradation in hypertensive conditions.Our data show that Ang‐II‐mediated hypertension was attenuated with inhibition of both AT1R and B1R receptor, while B1R contributed to upregulation of UBR1 and both AT1R and B1R contributes to downregulation of BRCC36. Therefore, inhibition of both AT1R and B1R modulates the expression of ACE2 ubiquitination partners and also inhibits ACE2 internalization and degradation. Therefore, developing a treatments strategy and simultaneously targeting AT1R, B1R, UBR1 and BRCC36, involved in the regulation of ACE2 expression may be effective for the treatment of cardiovascular disorders.