Transcriptomic Signature of Right Ventricular Failure in Experimental Pulmonary Arterial Hypertension: Deep Sequencing Demonstrates Mitochondrial, Fibrotic, Inflammatory and Angiogenic Abnormalities

Abstract: Right ventricular failure (RVF) remains the leading cause of death in pulmonary arterial hypertension (PAH). We investigated the transcriptomic signature of RVF in hemodynamically well-phenotyped monocrotaline (MCT)-treated, male, Sprague-Dawley rats with severe PAH and decompensated RVF (increased right ventricular (RV) end diastolic volume (EDV), decreased cardiac output (CO), tricuspid annular plane systolic excursion (TAPSE) and ventricular-arterial decoupling). RNA sequencing revealed 2547 differentially regulated transcripts in MCT-RVF RVs. Multiple enriched gene ontology (GO) terms converged on mitochondria/metabolism, fibrosis, inflammation, and angiogenesis. The mitochondrial transcriptomic pathway is the most affected in RVF, with 413 dysregulated genes. Downregulated genes included tfam (−0.45-fold), suggesting impaired mitochondrial biogenesis, Cyp2e1 (−3.8-fold), a monooxygenase which when downregulated increases oxidative stress, dehydrogenase/reductase 7C (Dhrs7c) (−2.8-fold), consistent with excessive autonomic activation, and polypeptide N-acetyl-galactose-aminyl-transferase 13 (Galnt13), a known pulmonary hypertension (PH) biomarker (−2.7-fold). The most up-regulated gene encodes Periostin (Postn; 4.5-fold), a matricellular protein relevant to fibrosis. Other dysregulated genes relevant to fibrosis include latent-transforming growth factor beta-binding protein 2 (Ltbp2), thrombospondin4 (Thbs4). We also identified one dysregulated gene relevant to all disordered transcriptomic pathways, Annexin A1. This anti-inflammatory, phospholipid-binding mediator, is a putative target for therapy in RVF-PAH. Comparison of expression profiles in the MCT-RV with published microarray data from the RV of pulmonary artery-banded mice and humans with bone morphogenetic protein receptor type 2 (BMPR2)-mutations PAH reveals substantial conservation of gene dysregulation, which may facilitate clinical translation of preclinical therapeutic and biomarkers studies. Transcriptomics reveals the molecular fingerprint of RVF to be heavily characterized by mitochondrial dysfunction, fibrosis and inflammation.摘要：右心衰竭（Right ventricular failure, RVF）仍是肺动脉高压（pulmonary arterial hypertension, PAH）患者的首要致死原因。本研究针对血流动力学表型明确的、经野百合碱（monocrotaline, MCT）造模的雄性斯普拉格-道利（Sprague-Dawley）大鼠展开实验，该模型患有严重肺动脉高压并伴随失代偿性右心衰竭，具体表现为右心室（right ventricular, RV）舒张末期容积（end diastolic volume, EDV）升高、心输出量（cardiac output, CO）降低、三尖瓣环收缩期位移（tricuspid annular plane systolic excursion, TAPSE）异常以及心室-动脉失耦联，借此探究其右心衰竭的转录组特征。RNA测序结果显示，经MCT造模的右心衰竭大鼠右心室组织中共存在2547个差异表达转录本。富集的基因本体（gene ontology, GO）条目主要集中于线粒体/代谢、纤维化、炎症及血管生成相关通路。其中线粒体转录组通路受影响最为显著，共计413个基因存在表达异常。下调基因包括线粒体转录因子A（tfam，折叠变化为-0.45），提示线粒体生物发生受损；细胞色素P450 2E1（Cyp2e1，折叠变化为-3.8），该单加氧酶表达下调会加剧氧化应激；脱氢酶/还原酶7C（dehydrogenase/reductase 7C, Dhrs7c，折叠变化为-2.8），其表达异常与自主神经过度激活相符；以及多肽N-乙酰半乳糖胺基转移酶13（polypeptide N-acetyl-galactose-aminyl-transferase 13, Galnt13）——这是一种已被证实的肺动脉高压（pulmonary hypertension, PH）生物标志物，折叠变化为-2.7。上调幅度最高的基因为骨膜蛋白（Periostin, Postn，折叠变化为4.5），这是一种与纤维化密切相关的基质细胞蛋白。其他与纤维化相关的差异表达基因还包括潜伏转化生长因子β结合蛋白2（latent-transforming growth factor beta-binding protein 2, Ltbp2）与血小板反应蛋白4（thrombospondin4, Thbs4）。本研究还发现了一个在所有异常转录组通路中均存在表达异常的基因——膜联蛋白A1（Annexin A1）。这种兼具抗炎活性与磷脂结合能力的介质，有望成为右心衰竭合并肺动脉高压的潜在治疗靶点。将本研究中MCT造模大鼠右心室的表达谱，与已发表的肺动脉环扎术小鼠右心室组织、以及携带2型骨形态发生蛋白受体（bone morphogenetic protein receptor type 2, BMPR2）突变的肺动脉高压患者右心室组织的微阵列数据进行对比后发现，二者的基因表达失调模式具有高度保守性，这将有助于推动临床前治疗与生物标志物研究向临床转化。转录组学分析揭示，右心衰竭的分子特征主要表现为线粒体功能障碍、纤维化与炎症反应。