Yamak Abir, Hu Dongjian, Mittal Nikhil, Buikema Jan W, Ditta Sheraz, Lutz Pierre G, Moog-Lutz Christel, Ellinor Patrick T, Domian Ibrahim J
Harvard Medical School, Boston, MA 02115, USA; Cardiovascular Research Center, Massachusetts General Hospital, 185 Cambridge Street, CPZN3200, Boston, MA 02114, USA; Cardiovascular Disease Initiative, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.
Cardiovascular Research Center, Massachusetts General Hospital, 185 Cambridge Street, CPZN3200, Boston, MA 02114, USA; Department of Biomedical Engineering, Boston University, Boston, MA 02215, USA.
iScience. 2020 Mar 27;23(3):100959. doi: 10.1016/j.isci.2020.100959. Epub 2020 Mar 4.
Defining the pathways that control cardiac development facilitates understanding the pathogenesis of congenital heart disease. Herein, we identify enrichment of a Cullin5 Ub ligase key subunit, Asb2, in myocardial progenitors and differentiated cardiomyocytes. Using two conditional murine knockouts, Nkx.Asb2 and AHF-Cre.Asb2, and tissue clarifying technique, we reveal Asb2 requirement for embryonic survival and complete heart looping. Deletion of Asb2 results in upregulation of its target Filamin A (Flna), and concurrent Flna deletion partially rescues embryonic lethality. Conditional AHF-Cre.Asb2 knockouts harboring one Flna allele have double outlet right ventricle (DORV), which is rescued by biallelic Flna excision. Transcriptomic and immunofluorescence analyses identify Tgfβ/Smad as downstream targets of Asb2/Flna. Finally, using CRISPR/Cas9 genome editing, we demonstrate Asb2 requirement for human cardiomyocyte differentiation suggesting a conserved mechanism between mice and humans. Collectively, our study provides deeper mechanistic understanding of the role of the ubiquitin proteasome system in cardiac development and suggests a previously unidentified murine model for DORV.
明确控制心脏发育的信号通路有助于理解先天性心脏病的发病机制。在此，我们发现在心肌祖细胞和分化的心肌细胞中，Cullin5泛素连接酶关键亚基Asb2高度富集。利用两种条件性小鼠基因敲除模型（Nkx.Asb2和AHF-Cre.Asb2）以及组织透明技术，我们揭示了Asb2对胚胎存活和心脏完全成环的必要性。Asb2的缺失导致其靶标细丝蛋白A（Flna）上调，同时Flna的缺失部分挽救了胚胎致死性。携带一个Flna等位基因的条件性AHF-Cre.Asb2基因敲除小鼠出现右心室双出口（DORV），而双等位基因Flna切除可挽救这一情况。转录组学和免疫荧光分析确定Tgfβ/Smad是Asb2/Flna的下游靶点。最后，利用CRISPR/Cas9基因组编辑技术，我们证明了Asb2对人类心肌细胞分化的必要性，这表明小鼠和人类之间存在保守机制。总的来说，我们的研究为泛素蛋白酶体系统在心脏发育中的作用提供了更深入的机制理解，并提出了一种此前未被识别的DORV小鼠模型。
