Regulation of Protein Inhibitor of Neuronal Nitric Oxide Synthase in the Paraventricular Nucleus during Chronic Heart Failure: Role for Angiotensin II‐ AT1R
Authors: Sharma Neeru M , Liu Xuefei , Zheng Hong , Patel Kaushik P 
Published: November 2023

Previously we have shown that decreased expression and activity of neuronal nitric oxide synthase (nNOS) within the paraventricular nucleus (PVN) of rats with chronic heart failure (CHF) leads to enhance sympathetic activation. nNOS is a highly regulated enzyme requiring homodimerization to become active and is degraded by the ubiquitin‐proteasome system. We have demonstrated that nNOS dimer/monomer ratio is decreased whereas expression of PIN (a protein inhibitor of nNOS, known to dissociate nNOS dimers into monomers) is increased in the PVN of rats with CHF. However, PIN transcripts levels remain unchanged. Further, the significant increase in PIN expression in the PVN of CHF rats was ameliorated by Losartan (Los) treatment, suggesting the involvement of angiotensin (Ang) II/AT1R signaling in the post‐transcriptional stabilization of PIN. To elucidate the molecular mechanism/s responsible for the increased PIN expression post‐transcriptionally, we used NG108‐15 hybrid neuronal cell line as an in vitro model. PIN translation was inhibited using cycloheximide (CHX) for 0–4h after 20h of pretreatment with Ang II. CHX mediated decrease in PIN expression was ameliorated with Ang II (0.19±0.04 CHX vs 0.41±0.06* CHX AngII 4h). Proteasome inhibitor lactacystin (LC) treatment dramatically elevated PIN level suggesting the involvement of proteasome system in PIN regulation. In vitro ubiquitination assay in cells transfected with pCMV‐(HA‐Ub)8 vector revealed a reduction of HA‐Ub‐PIN conjugates after Ang II treatment (9.2 ± 2.2 LC vs. 4.5 ± 0.6* LC Ang II). TUBE (Tandem Ubiquitin‐Binding Entities) assay showed decrease PIN‐Ub conjugates in Ang II‐treated cells (1.23 ± 0.05 LC vs. 0.82 ± 0.12* LC AngII) while Los treatment diminishes the Ang II‐mediated stabilization of PIN (1.21 ± 0.07 LC Los vs. 1.14 ± 0.04* LC AngII Los). Using coronary artery ligation‐induced CHF model, we found that there was decreased accumulation of PIN‐Ub conjugates in the PVN of CHF rats compared to Sham (1 ± 0.10 Sham vs. 0.63 ± 0.05* CHF). These results suggest that PIN is targeted for rapid degradation by the ubiquitin–proteasome pathway and Ang II via AT1R delays the rate of degradation resulting in accumulation of PIN leading to a decrease in the dimeric active form of nNOS. Additionally, we found decreased GTP cyclohydrolase 1 expression: a rate‐limiting enzyme for BH4 biosynthesis (0.74±0.03 Sham vs 0.48±0.02* CHF) and reduced BH4 levels in the PVN of CHF rats, suggesting that perhaps reduced BH4 availability may also contribute to decreased nNOS dimers. Taken together, our studies reveal post‐translational regulation of nNOS by protein‐protein interactions involving PIN and BH4, and provide significant insight into the possible molecular mechanism(s) within the PVN that may contribute to the over‐activation of the sympathetic drive during CHF.Support or Funding InformationSupported by AHA ‐ 14SDG19980007 and NIH grants HL124104 & HL62222.