Conditional Ablation of Renin in the Nucleus Ambiguus Impairs Vagal Cardioinhibitory Function

Background: The brain renin-angiotensin system (RAS) plays a critical role in autonomic function. For decades, lack of solid evidence supporting renin expression in the brain hampered the complete understanding of how this system operates. Recently, we identified renin-expressing neurons within the nucleus ambiguus (NuAm), a region with known functions of parasympathetic cardiac modulation. We hypothesized that conditional ablation of renin from the NuAm impacts autonomic function in mice. Methods: To ablate renin from the NuAm (Ren NuAm-KO ), 10-13-week-old Ren flox males were subjected to bilateral microinjections of adeno-associated viruses (AAV) targeting the NuAm (50 nL; coordinates from lambda: -2.2 mm AP, ±1.25 mm ML, -6.1 mm DV). These AAV carry either Cre-recombinase under the control of NuAm-specific promoter (AAV-PRSx8-Cre, n=7) or a control AAV (AAV-Empty, n=6). Radiotelemetry transducers were implanted 3 weeks after stereotaxic surgery. 24-hour heart rate (HR) and blood pressure (BP) were acquired. On separate days, BP and HR were measured before and after injection of methylatropine (2 mg/kg, IP), propranolol (5 mg/kg, IP), or saline (control, IP). The difference in HR and BP before and after injection was also calculated. Results: During 24-hour recordings, Ren NuAm-KO had significantly higher HR during the light cycle (550±6 bpm vs. 518±10 bpm; p<0.05), presumably due to either diurnal cardiac parasympathetic withdrawal or augmented sympathetic tone. Then, mice were subjected to autonomic blockade studies to assess autonomic function. At baseline, Ren NuAm-KO mice consistently exhibited elevated HR compared to WT (577±12 bpm vs. 468±22 bpm; p<0.01). However, parasympathetic blockade with methylatropine abolished this difference in HR (630±8 bpm vs. 610±23 bpm; p=0.45). There was a trend toward a decrease in Ren NuAm-KO ’s chronotropic response to methylatropine when compared to WT (ΔHR: 53±10 bpm vs. 142 ± 36 bpm; p=0.056), suggesting that these mice exhibit an impaired vagal cardioinhibitory tone. No differences in responses to propranolol or saline (vehicle) between groups were observed, suggesting sympathetic tone is preserved. Conclusions: These data indicate that the novel renin-expressing neurons might contribute to maintaining autonomic balance under healthy conditions. Future studies will further address the contribution of sex as a biological variable and the role of renin in the NuAm under pathological conditions such as hypertension.