Background: Right ventricular (RV) dysfunction is a significant prognostic determinant of morbidity and mortality in pulmonary arterial hypertension (PAH). Despite the importance of RV function in PAH, the underlying molecular mechanisms of RV dysfunction secondary to PAH remain unclear. We aim to identify and compare molecular determinants of RV failure using RNA sequencing of RV tissue from 2 clinically relevant animal models of PAH. Methods: We performed RNA sequencing on RV from rats treated with monocrotaline or Sugen with hypoxia/normoxia. PAH and RV failure were confirmed by catheterization and echocardiography. We validated the RV transcriptome results using quantitative real-time polymerase chain reaction, immunofluorescence, and Western blot. Immunohistochemistry and immunofluorescence were performed on human RV tissue from control (n=3) and PAH-induced RV failure patients (n=5). Results: We identified similar transcriptomic profiles of RV from monocrotaline- and Sugen with hypoxia-induced RV failure. Pathway analysis showed genes enriched in epithelial-to-mesenchymal transition, inflammation, and metabolism. Histological staining of human RV tissue from patients with RV failure secondary to PAH revealed significant RV fibrosis and endothelial-to-mesenchymal transition, as well as elevated Cellular Communication Network Factor 2 (top gene implicated in epithelial-to-mesenchymal transition/endothelial-to-mesenchymal transition) expression in perivascular areas compared with normal RV. Conclusions: Transcriptomic signature of RV failure in monocrotaline and Sugen with hypoxia models showed similar gene expressions and biological pathways. We provide translational relevance of this transcriptomic signature using RV from patients with PAH to demonstrate evidence of epithelial-to-mesenchymal transition/endothelial-to-mesenchymal transition and protein expression of Cellular Communication Network Factor 2 (CTGF [connective tissue growth factor]). Targeting specific molecular mechanisms responsible for RV failure in monocrotaline and Sugen with hypoxia models may identify novel therapeutic strategies for PAH-associated RV failure.

Larval zebrafish possess a number of molecular and genetic advantages for rigorous biological analyses of learning and memory. These advantages have motivated the search for novel forms of memory in these animals that can be exploited for understanding the cellular and molecular bases of vertebrate memory formation and consolidation. Here, we report a new form of behavioral sensitization in zebrafish larvae that is elicited by an aversive chemical stimulus [allyl isothiocyanate (AITC)] and that persists for ≥30 min. This form of sensitization is expressed as enhanced locomotion and thigmotaxis, as well as elevated heart rate. To characterize the neural basis of this nonassociative memory, we used transgenic zebrafish expressing the fluorescent calcium indicator GCaMP6 ( Chen et al., 2013 ); because of the transparency of larval zebrafish, we could optically monitor neural activity in the brain of intact transgenic zebrafish before and after the induction of sensitization. We found a distinct brain area, previously linked to locomotion, that exhibited persistently enhanced neural activity following washout of AITC; this enhanced neural activity correlated with the behavioral sensitization. These results establish a novel form of memory in larval zebrafish and begin to unravel the neural basis of this memory.

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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.

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