Developmental ablation of Id1 and Id3 genes in the vasculature leads to postnatal cardiac phenotypes

Rationale: The Id1 and Id3 genes play major roles during cardiac development, despite their expression being confined to non-myocardial layers (endocardium - endothelium - epicardium). We previously described that Id1-/-;Id3-/- double knockout (dKO) mouse embryos die at mid-gestation from multiple cardiac defects, but early demise precluded the studies of the roles of Id in the adult mice. Objective: To elucidate postnatal roles of Id genes in the heart. Methods and Results: We ablated Id1 gene in the vasculature and Id3 gene globally to generate Tie2Cre+;Id1F-;Id3-/- and Tie2Cre+;Id1F/F;Id3-/- conditional KO (Id cKO) embryos. Half of the Id cKO mice die at birth. Postnatal demise was associated with cardiac underdevelopment, enlargement, muscular ventricular septal and endothelial defects. Surviving Id cKO mice exhibited dilated, fibrotic cardiomyopathy associated with defects in the vasculature. The adult cardiac phenotype progressed into heart failure and resembled endomyocardial fibroelastosis. An abnormal vascular response was also observed in the healing process of excisional skin wounds of Id cKO mice. Expression patterns of vascular, fibrotic and hypertrophic markers were altered in the Id cKO hearts, but addition of Insulin-Like Growth Factor binding protein-3 (IGFbp3) reversed gene expression profiles of vascular and fibrotic, but not hypertrophic markers. Conclusions: Conditional ablation of Id genes in the vasculature leads to dilated fibrotic cardiomyopathy. The findings could reveal important insights into the role(s) of the endocardial network of the endothelial lineage to the development of dilated fibrotic cardiomyopathy and identify a potential therapeutic target, IGFbp3, in its treatment. Total RNA from heart tissue was isolated (RNeasy, QIAGEN) from P180 WT, Id control, Id cKO, IGFbp3 incubated Id cKO, and control incubated Id cKO. RNA was converted to cDNA, cRNA, and hybridized to DNA sequences contained in the GeneChip Mouse Gene 1.0 ST Array (Affymetrix). Information from at least duplicate samples was compared and filtered by fold change >2 (Id cKO vs WT, Id control vs WT, IGFbp3 incubated Id cKO vs. control incubated Id cKO) and statistical p-value<0.001.研究背景：Id1与Id3基因虽仅表达于非心肌层（心内膜-内皮-心外膜），却在心脏发育过程中发挥核心作用。本团队此前已报道，Id1-/-;Id3-/-双基因敲除（double knockout, dKO）小鼠胚胎会于妊娠中期因多种心脏缺陷死亡，但该过早死亡模型阻碍了对Id基因在成年小鼠体内功能的研究。 研究目的：阐明Id基因在心脏中的产后调控功能。 方法与结果：我们通过在血管系统中特异性敲除Id1基因、全局敲除Id3基因，成功构建了Tie2Cre+;Id1F-;Id3-/-与Tie2Cre+;Id1F/F;Id3-/-条件性敲除（conditional KO, cKO）小鼠胚胎。半数Id cKO小鼠在出生时即死亡，其产后死亡与心脏发育不全、心脏扩大、肌性室间隔缺损及内皮功能缺陷密切相关。存活的Id cKO小鼠表现出扩张性纤维化心肌病，并伴随血管系统结构与功能异常。成年Id cKO小鼠的心脏表型会逐步进展为心力衰竭，其病理特征与心内膜弹力纤维增生症高度相似。此外，在Id cKO小鼠的切除性皮肤伤口愈合过程中，也观察到异常的血管应答反应。对Id cKO小鼠心脏组织的血管、纤维化及肥大标志物的表达模式分析显示，上述标志物的表达均发生显著改变；而添加胰岛素样生长因子结合蛋白3（Insulin-Like Growth Factor binding protein-3, IGFbp3）可逆转血管与纤维化相关标志物的基因表达谱，但无法改善肥大相关标志物的异常表达。 结论：在血管系统中条件性敲除Id基因可诱发扩张性纤维化心肌病。本研究结果可为内皮谱系心内膜网络在扩张性纤维化心肌病发生发展中的作用提供关键理论依据，并为该疾病的治疗确定潜在治疗靶点IGFbp3。 实验方法：我们从P180龄野生型（WT）、Id对照组、Id cKO组、IGFbp3处理的Id cKO组及溶剂对照处理的Id cKO组小鼠的心脏组织中提取总RNA（采用"RNeasy"试剂盒，QIAGEN公司）。将提取的RNA逆转录为cDNA并合成cRNA，随后与GeneChip Mouse Gene 1.0 ST Array（Affymetrix公司）搭载的DNA序列进行杂交。对至少两份重复样本的检测数据进行比较分析，并通过差异倍数>2（Id cKO vs WT、Id对照组vs WT、IGFbp3处理的Id cKO组vs溶剂对照处理的Id cKO组）及统计学p值<0.001的标准进行筛选。