Common regulatory pathways mediate activity of microRNAs inducing cardiomyocyte proliferation

Loss of functional cardiomyocytes is a major determinant of heart failure after myocardial infarction. Previous high throughput screening studies have identified a series of microRNAs that induce cardiomyocyte proliferation and stimulate cardiac regeneration after myocardial infarction. Here we investigate the mechanism of action of the top ten most effective of these miRNAs. Analysis of the transcriptional profile of miRNA-treated cardiomyocytes revealed an involvement of the Hippo-YAP pathway in their action. All the investigated miRNAs activated YAP-mediated transcription, nuclear localization of active YAP and increased expression of YAP responsive genes. In particular, miR-199a-3p, one of the most effective miRNAs, directly downregulated two mRNA targets impinging on the Hippo pathway, the upstream inhibitory kinase TAOK1and the E3 ubiquitin ligase destroying YAP, b-TrCP. Most of the miRNAs that promoted proliferation also modulated the dynamics of the cardiomyocyte actin cytoskeleton. In particular, four miRNAs targeted Cofilin2 and increased the ratio between polymerized F-actin and monomeric G-actin. Cells treated with the most effective miRNAs (including miR-199a-3p) were round-shaped, with formation of gross bundles of actin fibers at the cytoplasm periphery. During mitosis, these miRNA-treated cardiomyocytes displayed disruption of the sarcomeric architecture. Downregulation of Cofilin2 itself was sufficient to promote cardiomyocyte proliferation, activate nuclear translocation of YAP and stimulate transcription of TEAD-responsive genes. Inhibition of F-actin polymerization decreased YAP activation. Collectively, these results indicate that activation of YAP and modulation of the acting cytoskeleton are major components of the pro-proliferative effect of miR-199a-3p and other miRNAs inducing cardiomyocyte proliferation Cardiomyocyte transcriptomic profiles of 0-day old wild type (WT) rats were generated by deep sequencing using Illumina Hiseq 2000

功能性心肌细胞丢失是心肌梗死（myocardial infarction）后心力衰竭的主要决定性因素。既往高通量筛选研究已鉴定出一系列可诱导心肌细胞增殖、促进心肌梗死术后心脏再生的微小RNA（microRNA, miRNA）。本研究针对其中效果最优的10种miRNA的作用机制展开深入探究。对经miRNA处理的心肌细胞进行转录组分析后发现，Hippo-YAP通路（Hippo-YAP pathway）参与了其调控过程。所有受试miRNA均可激活YAP介导的转录、促进活性YAP的核定位，并上调YAP靶基因的表达。其中，效果最为突出的miR-199a-3p可直接下调两个作用于Hippo通路的mRNA靶点：上游抑制性激酶TAOK1以及降解YAP的E3泛素连接酶b-TrCP。
多数可促进心肌细胞增殖的miRNA还可调控其肌动蛋白细胞骨架（actin cytoskeleton）动力学。具体而言，4种miRNA靶向Cofilin2，并提高了聚合态F-肌动蛋白（F-actin）与单体态G-肌动蛋白（G-actin）的比值。经效果最优的miRNA（包括miR-199a-3p）处理的细胞呈圆形，胞质周边可见粗大的肌动蛋白纤维束。在有丝分裂过程中，经miRNA处理的心肌细胞可见肌小节结构破坏。单独下调Cofilin2即可促进心肌细胞增殖、激活YAP的核转位，并刺激TEAD响应基因的转录。抑制F-肌动蛋白聚合可降低YAP的激活水平。综上，上述结果表明，YAP的激活与肌动蛋白细胞骨架的调控是miR-199a-3p及其他诱导心肌细胞增殖的miRNA发挥促增殖作用的核心环节。
本研究采用Illumina Hiseq 2000（Illumina Hiseq 2000）进行深度测序，获取了0日龄野生型（wild type, WT）大鼠的心肌细胞转录组谱。