Btg2 is a Negative Regulator of Cardiomyocyte Hypertrophy through a Decrease in Cytosolic RNA.
Btg2 is a Negative Regulator of Cardiomyocyte Hypertrophy through a Decrease in Cytosolic RNA.
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DOI：
10.1038/srep28592
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发表时间：
2016-06-27
影响因子：
4.6
通讯作者：
Sakata Y
中科院分区：
文献类型：
--
作者：
Masumura Y;Higo S;Asano Y;Kato H;Yan Y;Ishino S;Tsukamoto O;Kioka H;Hayashi T;Shintani Y;Yamazaki S;Minamino T;Kitakaze M;Komuro I;Takashima S;Sakata Y
在肥厚刺激下，心肌细胞进入高代谢状态，加速生物量积累。尽管调控蛋白水平的分子途径已经得到了很好的研究，但心肌细胞中RNA积累的功能意义及其调控机制仍然难以捉摸。在这里，我们通过单细胞成像和使用培养心肌细胞的c-Myc （Myc）介导的高代谢分析模型阐明了心肌细胞中RNA的定量动力学。新生RNA标记结合单细胞成像表明，Myc蛋白显著增加了每个心肌细胞的总RNA产量。高通量测序的染色质免疫沉淀澄清了过表达的Myc与一组特定基因结合并招募RNA聚合酶II。在这些基因中，我们发现Btg2是Myc的新靶点。Btg2过表达显著降低心肌细胞表面积。相反，shrna介导的Btg2的下调加速了肾上腺素能刺激诱导的肥大。通过质谱分析，我们确定Btg2结合了一系列组成mRNA死基化复合物的蛋白质。有趣的是，Btg2特异性地抑制细胞质内的RNA水平，而不是核内的RNA水平。Btg2敲低进一步增强了肾上腺素能刺激下心肌细胞胞质内RNA的积累，提示Btg2通过负向调节RNA积累负向调节反应性肥厚。我们的发现为心肌细胞中RNA水平调节机制的功能意义提供了见解。
Under hypertrophic stimulation, cardiomyocytes enter a hypermetabolic state and accelerate biomass accumulation. Although the molecular pathways that regulate protein levels are well-studied, the functional implications of RNA accumulation and its regulatory mechanisms in cardiomyocytes remain elusive. Here, we have elucidated the quantitative kinetics of RNA in cardiomyocytes through single cell imaging and c-Myc (Myc)-mediated hypermetabolic analytical model using cultured cardiomyocytes. Nascent RNA labeling combined with single cell imaging demonstrated that Myc protein significantly increased the amount of global RNA production per cardiomyocyte. Chromatin immunoprecipitation with high-throughput sequencing clarified that overexpressed Myc bound to a specific set of genes and recruits RNA polymerase II. Among these genes, we identified Btg2 as a novel target of Myc. Btg2 overexpression significantly reduced cardiomyocyte surface area. Conversely, shRNA-mediated knockdown of Btg2 accelerated adrenergic stimulus-induced hypertrophy. Using mass spectrometry analysis, we determined that Btg2 binds a series of proteins that comprise mRNA deadenylation complexes. Intriguingly, Btg2 specifically suppresses cytosolic, but not nuclear, RNA levels. Btg2 knockdown further enhances cytosolic RNA accumulation in cardiomyocytes under adrenergic stimulation, suggesting that Btg2 negatively regulates reactive hypertrophy by negatively regulating RNA accumulation. Our findings provide insight into the functional significance of the mechanisms regulating RNA levels in cardiomyocytes.
DOI：
10.3791/51420
发表时间：
2014-05-13
期刊：
Journal of visualized experiments : JoVE
影响因子：
--
作者：
Biddlestone J;Druker J;Shmakova A;Ferguson G;Swedlow JR;Rocha S
通讯作者：
Rocha S