Saxon Jacob G, Baer Daniel R, Barton Julie A, Hawkins Travis, Wu Bingruo, Trusk Thomas C, Harris Stephen E, Zhou Bin, Mishina Yuji, Sugi Yukiko
Department of Regenerative Medicine and Cell Biology and Cardiovascular Developmental Biology Center, Medical University of South Carolina, Charleston, SC 29425, USA; College of Charleston, Honors College, Undergraduate Student. Charleston, SC 29425, USA.
Department of Regenerative Medicine and Cell Biology and Cardiovascular Developmental Biology Center, Medical University of South Carolina, Charleston, SC 29425, USA.
Dev Biol. 2017 Oct 1;430(1):113-128. doi: 10.1016/j.ydbio.2017.08.008. Epub 2017 Aug 6.
Distal outgrowth, maturation and remodeling of the endocardial cushion mesenchyme in the atrioventricular (AV) canal are the essential morphogenetic events during four-chambered heart formation. Mesenchymalized AV endocardial cushions give rise to the AV valves and the membranous ventricular septum (VS). Failure of these processes results in several human congenital heart defects. Despite this clinical relevance, the mechanisms governing how mesenchymalized AV endocardial cushions mature and remodel into the membranous VS and AV valves have only begun to be elucidated. The role of BMP signaling in the myocardial and secondary heart forming lineage has been well studied; however, little is known about the role of BMP2 expression in the endocardial lineage. To fill this knowledge gap, we generated Bmp2 endocardial lineage-specific conditional knockouts (referred to as Bmp2 cKO) by crossing conditionally-targeted Bmp2 mice with a Cre-driver line, Nfatc1, wherein Cre-mediated recombination was restricted to the endocardial cells and their mesenchymal progeny. Bmp2 cKO mouse embryos did not exhibit failure or delay in the initial AV endocardial cushion formation at embryonic day (ED) 9.5-11.5; however, significant reductions in AV cushion size were detected in Bmp2 cKO mouse embryos when compared to control embryos at ED13.5 and ED16.5. Moreover, deletion of Bmp2 from the endocardial lineage consistently resulted in membranous ventricular septal defects (VSDs), and mitral valve deficiencies, as evidenced by the absence of stratification of mitral valves at birth. Muscular VSDs were not found in Bmp2 cKO mouse hearts. To understand the underlying morphogenetic mechanisms leading to a decrease in cushion size, cell proliferation and cell death were examined for AV endocardial cushions. Phospho-histone H3 analyses for cell proliferation and TUNEL assays for apoptotic cell death did not reveal significant differences between control and Bmp2 cKO in AV endocardial cushions. However, mRNA expression of the extracellular matrix components, versican, Has2, collagen 9a1, and periostin was significantly reduced in Bmp2 cKO AV cushions. Expression of transcription factors implicated in the cardiac valvulogenesis, Snail2, Twist1 and Sox9, was also significantly reduced in Bmp2 cKO AV cushions. These data provide evidence that BMP2 expression in the endocardial lineage is essential for the distal outgrowth, maturation and remodeling of AV endocardial cushions into the normal membranous VS and the stratified AV valves.
房室（AV）管内心内膜垫间充质的远端生长、成熟和重塑是四腔心形成过程中必不可少的形态发生事件。间充质化的房室心内膜垫产生房室瓣和膜性室间隔（VS）。这些过程的失败会导致多种人类先天性心脏缺陷。尽管具有这种临床相关性，但关于间充质化的房室心内膜垫如何成熟并重塑为膜性室间隔和房室瓣的机制才刚刚开始被阐明。骨形态发生蛋白（BMP）信号在心肌和第二心脏形成谱系中的作用已得到充分研究；然而，关于BMP2在心脏内膜谱系中的表达作用却知之甚少。为了填补这一知识空白，我们通过将条件性靶向Bmp2小鼠与Cre驱动系Nfatc1杂交，生成了Bmp2心脏内膜谱系特异性条件性敲除小鼠（称为Bmp2 cKO），其中Cre介导的重组仅限于心内膜细胞及其间充质后代。Bmp2 cKO小鼠胚胎在胚胎第（ED）9.5 - 11.5天初始房室心内膜垫形成过程中未出现失败或延迟；然而，与ED13.5和ED16.5的对照胚胎相比，在Bmp2 cKO小鼠胚胎中检测到房室垫大小显著减小。此外，从心脏内膜谱系中删除Bmp2持续导致膜性室间隔缺损（VSDs）和二尖瓣缺陷，出生时二尖瓣分层缺失证明了这一点。在Bmp2 cKO小鼠心脏中未发现肌性VSDs。为了了解导致垫大小减小的潜在形态发生机制，对房室心内膜垫的细胞增殖和细胞死亡进行了检测。用于细胞增殖的磷酸化组蛋白H₃分析和用于凋亡细胞死亡的TUNEL检测未揭示对照和Bmp2 cKO在房室心内膜垫方面的显著差异。然而，细胞外基质成分versican、Has2、胶原蛋白9a1和骨膜蛋白的mRNA表达在Bmp2 cKO房室垫中显著降低。参与心脏瓣膜发生的转录因子Snail₂、Twist1和Sox9的表达在Bmp2 cKO房室垫中也显著降低。这些数据提供了证据，表明心脏内膜谱系中BMP2的表达对于房室心内膜垫向正常膜性室间隔和分层房室瓣的远端生长、成熟和重塑至关重要。
