Center for Pharmacogenomics, Department of Internal Medicine, Washington University School of Medicine, St. Louis, MO, United States.
Center for Pharmacogenomics, Department of Internal Medicine, Washington University School of Medicine, St. Louis, MO, United States.
J Mol Cell Cardiol. 2018 Aug;121:60-68. doi: 10.1016/j.yjmcc.2018.06.009. Epub 2018 Jun 30.
G-protein receptor kinases (GRKs) regulate adult hearts by modulating inotropic, chronotropic and hypertrophic signaling of 7-transmembrane spanning neurohormone receptors. GRK-mediated desensitization and downregulation of β-adrenergic receptors has been implicated in adult heart failure; GRKs are therefore a promising therapeutic target. However, germ-line (but not cardiomyocyte-specific) GRK2 deletion provoked lethal fetal heart defects, suggesting an unexplained role for GRKs in heart development. Here we undertook to better understand the consequences of GRK deficiency on fetal heart development by creating mice and cultured murine embryonic fibroblasts (MEFs) having floxed GRK2 and GRK5 alleles on the GRK6 null background; simultaneous conditional deletion of these 3 GRK genes was achieved using Nkx2-5 Cre or adenoviral Cre, respectively. Phenotypes were related to GRK-modulated gene expression using whole-transcriptome RNA sequencing, RT-qPCR, and luciferase reporter assays. In cultured MEFs the atypical 7-transmembrane spanning protein and GRK2 substrate Smoothened (Smo) stimulated Gli-mediated transcriptional activity, which was interrupted by deleting GRK2/5/6. Mice with Nkx2-5 Cre mediated GRK2/5/6 ablation died between E15.5 and E16.5, whereas mice expressing any one of these 3 GRKs (i.e. GRK2/5, GRK2/6 or GRK5/6 deleted) were developmentally normal. GRK2/5/6 triple null mice at E14.5 exhibited left and right heart blood intermixing through single atrioventricular valves or large membranous ventricular septal defects. Hedgehog and GATA pathway gene expression promoted by Smo/Gli was suppressed in GRK2/5/6 deficient fetal hearts and MEFs. These data indicate that GRK2, GRK5 and GRK6 redundantly modulate Smo-GATA crosstalk in fetal mouse hearts, orchestrating transcriptional pathways previously linked to clinical and experimental atrioventricular canal defects. GRK modulation of Smo reflects convergence of conventional neurohormonal signaling and transcriptional regulation pathways, comprising an unanticipated mechanism for spatiotemporal orchestration of developmental gene expression in the heart.
G 蛋白偶联受体激酶（GRKs）通过调节 7 跨膜神经激素受体的变力、变时和肥大信号来调节成人心脏。GRK 介导的β肾上腺素能受体脱敏和下调已被牵连到成人心力衰竭中；因此，GRKs 是一个很有前途的治疗靶点。然而，种系（但不是心肌细胞特异性）GRK2 缺失引发了致命的胎儿心脏缺陷，这表明 GRKs 在心脏发育中存在尚未解释的作用。在这里，我们通过在 GRK6 缺失背景下创建具有 floxed GRK2 和 GRK5 等位基因的小鼠和培养的鼠胚胎成纤维细胞（MEFs），以及分别使用 Nkx2-5 Cre 或腺病毒 Cre 实现这些 3 个 GRK 基因的条件性缺失，来更好地了解 GRK 缺失对胎儿心脏发育的影响。使用全转录组 RNA 测序、RT-qPCR 和荧光素酶报告基因分析将表型与 GRK 调节的基因表达相关联。在培养的 MEFs 中，非典型 7 跨膜蛋白和 GRK2 底物 Smoothened（Smo）刺激 Gli 介导的转录活性，而删除 GRK2/5/6 则中断了这种活性。用 Nkx2-5 Cre 介导的 GRK2/5/6 缺失的小鼠在 E15.5 和 E16.5 之间死亡，而表达这 3 种 GRK 中的任何一种（即 GRK2/5、GRK2/6 或 GRK5/6 缺失）的小鼠发育正常。在 E14.5 时，GRK2/5/6 三重缺失小鼠的左、右心血液通过单一房室瓣或大的膜性室间隔缺损混合。在 GRK2/5/6 缺失的胎儿心脏和 MEFs 中，Smo/Gli 促进的 Hedgehog 和 GATA 通路基因表达受到抑制。这些数据表明，GRK2、GRK5 和 GRK6 可冗余地调节胎儿小鼠心脏中的 Smo-GATA 串扰，协调先前与临床和实验性房室管缺陷相关的转录途径。GRK 对 Smo 的调节反映了传统神经激素信号和转录调节途径的融合，构成了心脏发育时空基因表达协调的一种意外机制。
