Program in Cardiovascular and Metabolic Disorders, Duke-National University of Singapore Medical School, Singapore (S.C., S.S., E.A., S.V., A.W., S.L., M.W., G.D., S.G.S., B.L.G., S.L., E.Y.C., E.C., J.D., S.A.C., O.J.L.R.).
National Heart Centre Singapore, Singapore (S.S., S.L., J.T., C.J.P., S.A.C.).
Circulation. 2019 Sep 10;140(11):937-951. doi: 10.1161/CIRCULATIONAHA.119.039596. Epub 2019 Jul 9.
Fibrosis is a common pathology in many cardiac disorders and is driven by the activation of resident fibroblasts. The global posttranscriptional mechanisms underlying fibroblast-to-myofibroblast conversion in the heart have not been explored.
Genome-wide changes of RNA transcription and translation during human cardiac fibroblast activation were monitored with RNA sequencing and ribosome profiling. We then used RNA-binding protein-based analyses to identify translational regulators of fibrogenic genes. The integration with cardiac ribosome occupancy levels of 30 dilated cardiomyopathy patients demonstrates that these posttranscriptional mechanisms are also active in the diseased fibrotic human heart.
We generated nucleotide-resolution translatome data during the transforming growth factor β1-driven cellular transition of human cardiac fibroblasts to myofibroblasts. This identified dynamic changes of RNA transcription and translation at several time points during the fibrotic response, revealing transient and early-responder genes. Remarkably, about one-third of all changes in gene expression in activated fibroblasts are subject to translational regulation, and dynamic variation in ribosome occupancy affects protein abundance independent of RNA levels. Targets of RNA-binding proteins were strongly enriched in posttranscriptionally regulated genes, suggesting genes such as MBNL2 can act as translational activators or repressors. Ribosome occupancy in the hearts of patients with dilated cardiomyopathy suggested the same posttranscriptional regulatory network was underlying cardiac fibrosis. Key network hubs include RNA-binding proteins such as Pumilio RNA binding family member 2 (PUM2) and Quaking (QKI) that work in concert to regulate the translation of target transcripts in human diseased hearts. Furthermore, silencing of both PUM2 and QKI inhibits the transition of fibroblasts toward profibrotic myofibroblasts in response to transforming growth factor β1.
We reveal widespread translational effects of transforming growth factor β1 and define novel posttranscriptional regulatory networks that control the fibroblast-to-myofibroblast transition. These networks are active in human heart disease, and silencing of hub genes limits fibroblast activation. Our findings show the central importance of translational control in fibrosis and highlight novel pathogenic mechanisms in heart failure.
纤维化是许多心脏疾病的常见病理，由驻留成纤维细胞的激活驱动。心脏中成纤维细胞向肌成纤维细胞转化的全球转录后机制尚未得到探索。
通过 RNA 测序和核糖体分析监测人类心脏成纤维细胞激活过程中 RNA 转录和翻译的全基因组变化。然后，我们使用 RNA 结合蛋白分析来鉴定纤维化基因的翻译调节剂。与 30 名扩张型心肌病患者的心脏核糖体占有率的整合表明，这些转录后机制在患病的纤维化人类心脏中也是活跃的。
我们在转化生长因子β1驱动的人类心脏成纤维细胞向肌成纤维细胞的细胞转化过程中生成了核苷酸分辨率的翻译组数据。这在纤维化反应的几个时间点确定了 RNA 转录和翻译的动态变化，揭示了瞬时和早期应答基因。值得注意的是，激活的成纤维细胞中所有基因表达变化的约三分之一受到翻译调控，核糖体占有率的动态变化独立于 RNA 水平影响蛋白质丰度。RNA 结合蛋白的靶标在转录后调控基因中强烈富集，表明 MBNL2 等基因可以作为翻译激活剂或抑制剂。扩张型心肌病患者心脏中的核糖体占有率表明，相同的转录后调控网络是心脏纤维化的基础。关键网络枢纽包括 RNA 结合蛋白，如 Pumilio RNA 结合家族成员 2（PUM2）和 Quaking（QKI），它们协同作用调节人类患病心脏中靶转录物的翻译。此外，沉默 PUM2 和 QKI 均可抑制成纤维细胞向转化生长因子β1 反应的促纤维化肌成纤维细胞的转化。
我们揭示了转化生长因子β1 的广泛翻译效应，并定义了控制成纤维细胞向肌成纤维细胞转化的新转录后调控网络。这些网络在人类心脏病中活跃，并且沉默枢纽基因可限制成纤维细胞的激活。我们的研究结果表明翻译控制在纤维化中的重要性，并强调心力衰竭中的新发病机制。
