Targeted Intrathecal Nos1 Inhibition Rescues Pulmonary Hypertension and Right Ventricular Failure in Rats by Reducing Neuroinflammation, Apoptosis and Sympathoexcitation

Introduction: Pulmonary hypertension (PH) is associated with aberrant sympathoexcitation leading to RV failure (RVF), arrhythmias and death. NOS1 is known to induce neuroinflammation, nitrosative stress and apoptosis in neural tissue. In spinal cord, NOS1 is activated by afferent signaling via Ca 2+ influx through activated NMDAR in dorsal horn by increased glutamate release via K + channels. Hypothesis: Cardiopulmonary afferent signaling in PH-RVF leads to NOS1-mediated neuroinflammation, nitrosative stress and apoptosis in thoracic spinal cord (TSC) resulting in aberrant sympathoexcitation. Targeted intrathecal NOS1-inhibition may rescue PH and RVF. Methods: Male rats received s.c. Monocrotaline (MCT, 60mg/kg, n=8, 4wk) or Sugen (SuHx, 20mg/kg, n=8, 3wk hypoxia+2wk normoxia). Controls received PBS (n=8). A group of MCT-rats received either daily intrathecal specific NOS1-inhibitor S-Methyl-L-thiocitrulline (SMTC; 0.5mg/kg, n=7) or PBS (n=7) from day 14-28. Echo and RV cath were performed. TSC and RV RNASeq, RT-qPCR, NOS1 activity, NOS1 immunolocalization, microglia+astrocyte quantification and TUNEL were performed. Plasma catecholamines were measured by ELISA. Values are mean±SD. Results: Rats developed severe PH-RVF (Increased RVSP, Fulton-index, decreased RVFAC; p<0.01). TSC transcriptome showed common genes and pathways linked to neuroinflammation, nitrosative stress and apoptosis and identified NOS1 as one of the top-most upregulated genes (MCT / SuHx:8 fold; p<0.05) in TSC but not lumbar. We found significantly increased microglia+astrocyte activation, NMDAR-1, Kv3.4, CX3CL1, nitrosative stress markers AP1B1, AP1M1 and pro-apoptotic p53, TGFβ1 expression in TSC. We observed increased NOS1 activity, its colocalization with microglia+astrocyte, apoptosis and norepi/epi in both models. SMTC decreased RVSP, Fulton-index, RVID d , pulmonary vascular wall thickness, increased RVFAC by reducing TSC NOS1 activity, CX3CL1/TGF β 1, microglia/astrocyte activation, apoptosis and plasma norepi/epi (p<0.05). RV transcriptome demonstrated that SMTC decreased pro-fibrotic, pro-apoptotic, SNS & RAAS related and pro-inflammatory RV gene expression profile. Conclusions: Intrathecal NOS1 inhibition rescues PH and RVF.