Repression of Ect2 induces cytokinesis failure and decreases heart muscle cell proliferation in congenital heart disease

Proliferation of heart muscle cells (cardiomyocytes) is the basis for heart development and regeneration. In mammals, cardiomyocytes become binucleated, which is thought to limit their proliferative capacity, but mechanisms are unknown. Here, we show the detailed mechanisms of formation of binucleated cardiomyocytes and how these mechanisms are dysregulated in congenital heart disease (CHD). Cardiomyocytes become binucleated by failing the last stage of cytokinesis, abscission. We identified the underlying molecular mechanism with single-cell transcriptional profiling, which showed repression of the cytokinesis gene Ect2, a Rho-guanine-nucleotide exchange factor. Inactivating Ect2 tripled the proportion of binucleated cardiomyocytes and reduced the number of cardiomyocytes by 34% in newborn mice, which was lethal. Cardiomyocyte-specific overexpression of Ect2 in transgenic mice decreased cytokinesis failure. We demonstrate that the Ect2 gene is regulated by the Hippo tumor suppressor pathway, and upstream of that, by ß-adrenergic receptor signaling. In neonatal mice, stimulating ß-adrenergic signaling pathway output with forskolin decreased the number of cardiomyocytes, and administration of ß-blockers increased the number of cardiomyocytes. Finally, we show that patients with tetralogy of Fallot with pulmonary stenosis (ToF/PS), a common type of CHD, develop a 2.2-fold increase in bi- and multi-nucleated cardiomyocytes. Using in vivo labeling of one ToF/PS patient with 15N-thymidine, followed by imaging mass spectrometry, we demonstrate that this increase happens after birth. Organotypic cultures of myocardium from infants with ToF/PS showed that cardiomyocyte binucleation could be reduced with ß-blockers. These results demonstrate how cardiomyocyte proliferation stops and provide the basis for new therapeutic strategies to increase cardiomyocyte proliferation in patients with CHD. Overall design: The cardiomyocytes were isolated from embryonic (E14.5) and postnatal (P5) mouse (FUCCI) hearts, then the single cardiomyocytes were collected through FACS. The cycling cardiomyocytes were identified through Azami Green-Geminin (mAG-hGem) signal. The mRNA was amplified using the antisense RNA (aRNA) amplification method.

心肌细胞（cardiomyocytes）的增殖是心脏发育与再生的核心基础。在哺乳动物体内，心肌细胞会转变为双核状态，该现象被认为会限制其增殖能力，但其潜在分子机制长期以来尚不明确。本研究揭示了双核心肌细胞形成的详细机制，以及此类机制在先天性心脏病（congenital heart disease, CHD）中的失调模式。
心肌细胞通过胞质分裂（cytokinesis）的最终阶段——胞质脱落（abscission）失败而形成双核细胞。我们借助单细胞转录组测序（single-cell transcriptional profiling）鉴定了其潜在分子机制，研究发现Rho鸟苷酸交换因子（Rho-guanine-nucleotide exchange factor）类胞质分裂基因Ect2的表达受到显著抑制。在新生小鼠中，敲低Ect2可使双核心肌细胞的比例增至原本的三倍，并使心肌细胞总数减少34%，该表型具有致死性。而在转基因小鼠中特异性过表达心肌细胞的Ect2，则可降低胞质分裂失败的发生率。
本研究证实，Ect2基因受Hippo肿瘤抑制通路（Hippo tumor suppressor pathway）调控，其上游则受β-肾上腺素能受体（β-adrenergic receptor）信号通路调控。在新生小鼠体内，通过福司柯林（forskolin）激活β-肾上腺素能信号通路会减少心肌细胞数量，而给予β受体阻滞剂则可增加心肌细胞数目。
最终我们发现，患有法洛四联症伴肺动脉狭窄（tetralogy of Fallot with pulmonary stenosis, ToF/PS）这一常见先天性心脏病的患者，其体内双核及多核心肌细胞的数量增加了2.2倍。我们对一名ToF/PS患者使用15N-胸腺嘧啶（15N-thymidine）进行体内标记，随后通过成像质谱（imaging mass spectrometry）分析，证实该细胞数量增加发生于出生之后。
对法洛四联症伴肺动脉狭窄患儿的心肌组织器官型培养实验显示，使用β受体阻滞剂可降低心肌细胞的双核化比例。
上述研究阐明了心肌细胞增殖停止的具体机制，为提升先天性心脏病患者心肌细胞增殖能力的新型治疗策略提供了理论依据。
整体实验设计：从胚胎期（E14.5）和出生后第5天（P5）的FUCCI转基因小鼠心脏中分离心肌细胞，通过荧光激活细胞分选术（fluorescence-activated cell sorting, FACS）收集单个心肌细胞。通过Azami Green-Geminin（mAG-hGem）信号鉴定处于增殖周期的心肌细胞。采用反义RNA（antisense RNA, aRNA）扩增法对mRNA进行扩增。