Transcriptional Suppression of Connexin43 by TBX18 Undermines Cell-Cell Electrical Coupling in Postnatal Cardiomyocytes
Transcriptional Suppression of Connexin43 by TBX18 Undermines Cell-Cell Electrical Coupling in Postnatal Cardiomyocytes
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DOI：
10.1074/jbc.m110.185298
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发表时间：
2011-04-22
影响因子：
4.8
通讯作者：
Cho, Hee Cheol
中科院分区：
文献类型：
--
作者：
Kapoor, Nidhi;Galang, Giselle;Cho, Hee Cheol
T-box转录因子在胚胎心肌细胞谱系中起着重要作用。表达Tbx 18的间充质前体细胞产生心脏起搏器，即窦房结（SAN）。我们试图通过在出生后心肌细胞中强制腺病毒过表达来确定心脏中TBX 18转录调节的靶点。用GFP转导的新生大鼠心肌细胞（NRCMs）显示肌膜的点状Cx43表达。相比之下，TBX 18转导的NRCM表现出稀疏的Cx43表达。在TBX 18转导的2天内，Cx43的转录物和蛋白质水平均显著下调。在豚鼠心脏中直接注射TBX 18在体内抑制Cx43表达。TBX 18对Cx43的抑制活性是高度特异性的; Cx45和Cx40的蛋白水平，其包含SAN和传导系统中的主要间隙连接，未被TBX 18改变。一个基于PCR的启动子分析表明，TBX 18直接抑制Cx43启动子。表型上，TBX 18-NRCM表现出细胞间钙黄绿素染料转移动力学减慢（421 +/- 54 vs对照127 +/- 43 ms）。在对照NRCM单层细胞内钙振荡是高度同步的。相比之下，TBX 18过表达导致异步Ca 2+振荡，表明细胞-细胞偶联减少。耦合降低导致电传播缓慢; TBX 18 NRCM的传导速度相对于对照组减慢50%以上（2.9 +/- 0.5 vs 14.3 +/- 0.9 cm/s）。总之，TBX 18特异性地直接抑制Cx43转录物和蛋白质水平。Cx43抑制导致显著的电解偶联，但其他间隙连接蛋白的保留支持缓慢的动作电位传播，重现了SAN的关键表型标志。
T-box transcription factors figure prominently in embryonic cardiac cell lineage specifications. Mesenchymal precursor cells expressing Tbx18 give rise to the heart's pacemaker, the sinoatrial node (SAN). We sought to identify targets of TBX18 transcriptional regulation in the heart by forced adenoviral overexpression in postnatal cardiomyocytes. Neonatal rat cardiomyocytes (NRCMs) transduced with GFP showed sarcolemmal, punctate Cx43 expression. In contrast, TBX18-transduced NRCMs exhibited sparse Cx43 expression. Both the transcript and protein levels of Cx43 were greatly down-regulated within 2 days of TBX18 transduction. Direct injection of TBX18 in the guinea pig heart in vivo inhibited Cx43 expression. The repressor activity of TBX18 on Cx43 was highly specific; protein levels of Cx45 and Cx40, which comprise the main gap junctions in the SAN and conduction system, were unchanged by TBX18. A reporter-based promoter assay demonstrated that TBX18 directly represses the Cx43 promoter. Phenotypically, TBX18-NRCMs exhibited slowed intercellular calcein dye transfer kinetics (421 +/- 54 versus control 127 +/- 43 ms). Intracellular Ca2+ oscillations in control NRCM monolayers were highly synchronized. In contrast, TBX18 overexpression led to asynchronous Ca2+ oscillations, demonstrating reduced cell-cell coupling. Decreased coupling led to slow electrical propagation; conduction velocity in TBX18 NRCMs slowed by more than 50% relative to control (2.9 +/- 0.5 versus 14.3 +/- 0.9 cm/s). Taken together, TBX18 specifically and directly represses Cx43 transcript and protein levels. Cx43 suppression leads to significant electrical uncoupling, but the preservation of other gap junction proteins supports slow action potential propagation, recapitulating a key phenotypic hallmark of the SAN.