Abstract Body (Do not enter title and authors here): Rationale: Pulmonary arterial hypertension (PAH) is a rare cardiopulmonary disease that can cause right heart failure and even death. In PAH, excessive mitochondrial fission is responsible for the hyperproliferation of pulmonary artery smooth muscle cells (PASMC), which is incompletely mediated by the large GTPase dynamin-related protein 1 (Drp1). We hypothesize that another GTPase, Dynamin 2 (DNM2), elevated in PASMC of PAH patients, is responsible for the final stage of mitochondrial fission.
Objectives: 1. Identify the differentially expressed gene (DEG) signature in PAH PASMC after silencing DNM2 using small interfering RNA (siRNA) by means of RNA sequencing (RNA-seq); 2. Identify the biological processes or pathways associated with these DEGs.
Methods: Human PAH PASMCs (n=5 cell line) were transfected with control siRNA or siRNA targeting DNM2. 3’RNA-seq was used to create libraries which we sequenced on the Illumina Nextseq550. Bioinformatic analysis revealed DEGs which we placed into context using the STRING: protein query database and visualized using Cytoscape 3.10.2. Network functional enrichment analysis was performed using STRING.
Results: Inhibiting DNM2 significantly reduced mitochondrial fission and slowed PAH PASMC proliferation. RNA-seq identified 156 DEGs (80 down-regulated, 56 up-regulated). The top 5 down-regulated DEGs were DNM2, RGCC, TICAM1, RASSF3, MRPL39 and the top 5 up-regulated DEGs were ITIF1, ARL6IP1, POLR3G, CBX1, CMPK2. We tested the biologic relevance of RGCC which encodes the Regulator of Cell Cycle using siRNA and showed inhibition of cell proliferation in human PAH PASMC. Network analysis identified 152 edges amongst all DEGs. The largest subnetwork contains 77 nodes and 151 edges. PXDN, which encodes Peroxidasin, a heme-containing peroxidase that is secreted into the extracellular matrix, has the highest degree of connectivity (15 connections), the highest betweenness centrality (0.266), and the highest closeness centrality (0.441). Enrichment analysis found that the p53 signaling pathway and the cytokine signaling in the immune system are the top KEGG and Reactome pathways affected by siDNM2, respectively.
Conclusions: DNM2 appears to be an important regulator of the increased mitochondrial fission in PAH. Silencing DNM2 not only inhibits fission but also slows cell proliferation, apparently by reducing the expression of RGCC. Silencing DNM2 in human PAH PASMC results in the disruption of 136 genes.