ACE2 Knock‐out Mice Show Decreased Blood Pressure Response and Increased Anxiety‐like Behavior to Acute Stress

Abstract

Stress increases Ang-II production and elevates sympathetic outflow which results in anxiety-related behavior and hypertension. Here, we investigated a potential contribution of Angiotensin Converting Enzyme-2 (ACE2) in the regulation of stress and anxiety-related behavior, in conscious freely moving mice. Previous studies reported that overexpression of ACE2 attenuates HPA axis and decreases anxiety-related behavior (Wang et al., 2018); however, deletion of ACE2 and its implication for such responses are largely unknow. Male mice (30-33 g), 6 ACE2 knock-outs (KO) and 6 wild type (WT) controls were chronically implanted with blood pressure (BP) transducers for continuous recording of BP, heart rate (HR) and locomotor activity. Following a 1-week recovery, mice were subjected to resident-intruder test for 10 min, for the measurement of stress-induced cardiovascular changes. During the dark/active period, a naïve intruder-mouse was suddenly introduced in the experimental mice's cages and their cardiovascular responses were analyzed using the Ponemah software (DSI). In response to intruder-intrusion for 10 min, ACE2 KO mice showed significant (p<0.05) attenuation in BP increases (+15 ±7 mmHg) compared to their WT controls (+39 ±7 mmHg). However, no significant difference was observed in HR increase in ACE2 KO versus WT mice (+166 ±40 and +173±38 bpm, respectively, p=0.45). In contrast, ACE2 KO mice exhibited a significant increase in locomotor activity compared to controls (179 ±19 and 111 ±14 arbitrary units, respectively, p<0.05). These mice were later subjected to the elevated plus maze (EPM) and light-dark box (LDB) tests for 5 min to evaluate anxiety-related behavior. Interestingly ACE2 KO mice were observed to spend more time in closed arms (244 ±19 s) in EPM compared to WT controls (218 ±22 s, p<0.05). However, in the LDB test, ACE2 KO mice showed no difference (286 ±18 s) compared to WT (279 ±19 s, ns). All mice were then chronically implanted subcutaneously with osmotic minipumps filled with an AT1 receptor antagonist (losartan, 10 mg/kg). A week later, mice were again subjected to the intruder test. ACE2 KO mice showed substantial attenuation in BP increase to intruder-intrusion compared to WT mice (+6±3 and +19±6 mmHg respectively, p<0.05); with no significant difference in HR increase in both the groups. Interestingly, pre-treatment with losartan attenuated the intruder-induced locomotor activity in ACE2 KO mice (149±29, p=0.11). In addition, losartan treatment did not attenuate the time ACE2 KO mice spent in closed arms in EPM; but exhibited a decreased time spent in the dark compartment in the LDB test. Surprisingly, our results did not show exacerbated BP responses to acute stress in the absence of ACE2 but instead produced decreased BP responses suggesting other compensatory mechanisms involved. Losartan-mediated attenuation of the increased locomotor activity and anxiety-related behaviors in ACE2 KO mice strongly suggests an Ang-II mediated anxiety-related behaviors. Our experimental findings further extend the current notion that ACE2 potentially contributes to the regulation of stress and anxiety.