Regulation of Postnatal Cardiomyocyte Maturation by an RNA Splicing Regulator RBFox1.-猫眼课题宝

DOI：

10.1161/circulationaha.122.061602

发表时间：

2023-10

期刊：

Circulation

影响因子：

37.8

通讯作者：

中科院分区：

医学1区

文献类型：

作者：

Jijun Huang;Josh Z Lee;Christoph D. Rau;A. Pezhouman;Tomohiro Yokota;H. Miwa;Matthew Feldman;Tsz Kin Kong;Ziyue Yang;Woan Ting Tay;Ivan Pushkarsky;Kyungsoo Kim;Shan S Parikh;Shreya Udani;B. Soh;Chen Gao;L. Stiles;O. Shirihai;Bjorn C. Knollmann;R. Ardehali;Dino Di Carlo;Yibin Wang

心肌细胞从新生到成年的成熟过程涉及到心肌细胞形态、增殖能力、代谢和生理的复杂变化。然而，人们对潜在的监管机制仍然知之甚少。1，2尽管有几个外在因素，如机械负荷，电刺激，激素和营养物质，已被牵连在这个过程中，2内在的调节电路，心肌细胞出生后成熟在很大程度上是未知的。为了寻找答案，我们分析了新生大鼠和成年大鼠心脏之间的整体转录组变化。差异表达基因的基因本体分析揭示了细胞周期、代谢和收缩性的预期变化。此外，mRNA剪接是最丰富的差异表达基因途径之一，其在心肌细胞成熟中的功能意义尚未报道（图[A]）。在心脏富集的RNA剪接调节因子中，我们发现Rbfox 1（RNA结合fox-1同源物1）在新生大鼠心脏中的表达水平可忽略不计，但在成年大鼠心脏中显著诱导3（图[B]）。对先前发表的出生后单细胞RNA测序（RNA-seq）数据集的检查（P1至P14）小鼠心脏4（图[C]）还显示Rbfox 1在围产期心脏中表达较低，但在主要存在于P14心脏中的心肌细胞亚群中高度诱导（图[D]，左上），其也具有细胞周期相关基因的最低表达和与成熟心肌细胞相关的基因的最高表达（图[D]）。Rbfox 1在新生大鼠心室心肌细胞中的异位表达导致细胞尺寸增大（图[E]），参与钙处理（Ryr 2、Atp 2a 2和Pln）、代谢（Ckm）和细胞-细胞偶联（Cnx 43;图[F]）的基因表达升高，肌节组织化增强（图[G]和[H]），以及双核化百分比升高（图[I]）。然而，未观察到对T-小管形成的显著影响。与这些分子和形态学特征一致，表达Rbfox 1的心肌细胞显示出更稳健的收缩（图[J]和[K]），并且在2 Hz起搏条件下通过达到峰值的时间和衰减时间测量的细胞内钙瞬变得到更好的维持（图[L]和[N]）。此外，全场光学标测显示Rbfox 1表达的新生大鼠心室心肌细胞的动作电位时程显著缩短（图[O]和[P]），这是啮齿动物心肌细胞电生理成熟的特征。Rbfox 1表达还增强了心肌细胞中的耗氧量（图[Q]），而不影响基于线粒体DNA与基因组DNA比率的线粒体含量（数据未显示）。在人多能干细胞衍生的心肌细胞中，Rbfox 1的表达低于检测。Rbfox 1的异位表达显著增加了与成熟相关的心脏基因的表达，包括MYH 6、MYH 7、SERCA 2A和CKM（图11）。

Cardiomyocyte maturation from neonatal to adult stage involves complex changes in myocyte morphology, proliferative capacity, metabolism, and physiology. However, the underlying regulatory mechanism remains poorly understood. 1, 2 Whereas several extrinsic factors, such as mechanical load, electrical stimulation, hormones, and nutrients, have been implicated in this process, 2 the intrinsic regulatory circuit governing cardiomyocyte postnatal maturation is largely unknown. In search of answers, we analyzed the global transcriptome changes between neonatal and adult rat hearts. Gene Ontology analysis from differentially expressed genes revealed expected changes in cell cycle, metabolism, and contractility. In addition, mRNA splicing was among the top enriched differentially expressed gene pathways, the functional significance of which in cardiomyocyte maturation is unreported (Figure [A]). Among the top cardiac enriched RNA splicing regulators, we found Rbfox1 (RNA binding fox-1 homolog 1) showed a negligible level of expression in neonatal rat heart but was dramatically induced in adult rat heart3 (Figure [B]). Examination of a previously published single-cell RNA sequencing (RNA-seq) dataset from postnatal (P1 to P14) mouse hearts4 (Figure [C]) also revealed that Rbfox1 expression was low in perinatal hearts, but highly induced in a subpopulation of cardiomyocytes present predominantly in P14 hearts (Figure [D], top left), which also have the lowest expression of cell-cycle–related genes and the highest expression of genes associated with mature cardiomyocytes (Figure [D]). Ectopic expression of Rbfox1 in neonatal rat ventricular cardiomyocytes resulted in enlarged cell size (Figure [E]), elevated expression genes involved in calcium handling (Ryr2, Atp2a2, and Pln), metabolism (Ckm), and cell–cell coupling (Cnx43; Figure [F]), enhanced sarcomere organization (Figure [G] and [H]), and higher percentage of binucleation (Figure [I]). However, no significant effect on T-tubule formation was observed. In line with these molecular and morphologic features, Rbfox1-expressing cardiomyocytes showed more robust contraction (Figure [J] and [K]) and better maintained intracellular calcium transients measured by both time to peak and time to decay under 2 Hz pacing condition (Figure [L] and [N]). In addition, whole-field optical mapping showed significantly reduced action potential duration in Rbfox1-expressing neonatal rat ventricular cardiomyocytes (Figure [O] and [P]), a characteristic feature of electrophysiologic maturation in rodent cardiomyocytes. Rbfox1 expression also enhanced oxygen consumption in cardiomyocytes (Figure [Q]) without affecting mitochondrial content on the basis of mito-DNA to genomic DNA ratio (data not shown). In human pluripotent stem cell–derived cardiomyocytes, Rbfox1 expression was below detection. Ectopic expression of Rbfox1 significantly increased the expression of cardiac genes associated with maturation, including MYH6, MYH7, SERCA2A, and CKM (Figure

Regulation of Postnatal Cardiomyocyte Maturation by an RNA Splicing Regulator RBFox1.

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