Embryonic VHL-HIF signaling defines dynamic heart metabolic compartments essential for cardiac maturation

While gene regulatory networks involved in cardiogenesis have been characterized, the role of bioenergetics remains less studied. Here we show that until midgestation, myocardial metabolism is compartmentalized, with a glycolytic signature restricted to compact myocardium contrasting with increased mitochondrial oxidative activity in the trabeculae. HIF1a regulation mirrors this pattern, with expression predominating in compact myocardium and scarce in trabeculae. By midgestation, the compact myocardium downregulates HIF1a and switches toward oxidative metabolism. Deletion of the E3 ubiquitin ligase Vhl results in HIF1a hyperactivation, disrupting metabolic compartmentalization and blocking the midgestational shift toward oxidative phosphorylation. Moreover, the altered glycolytic signature induced by HIF1 trabecular activation precludes regulation of genes essential for cardiac conduction system establishment. Our findings reveal VHL-HIF-mediated metabolic compartmentalization in the developing heart and the connection between metabolism and myocardial differentiation. These results highlight the importance of bioenergetics in ventricular myocardium specialization and its potential relevance to congenital heart disease. Overall design: RNA was isolated from individual E12.5 embryonic hearts after removal of the atria and valvular region. KOs and control littermates were matched by somite count, and a total number of 3 KOs and 3 controls from 3 independent litters were used. For RNA extraction, QIAzol Lysis Reagent (Qiagen; CA; USA) and the miRNeasy Mini Kit (Qiagen; CA; USA) were used. RNA was quantified and its purity checked with a NanoDrop ND-1000 spectophotometer (Thermo Scientific; MA; USA). RNA integrity was verified with an Agilent 2100 Bioanalyzer (Agilent Technologies; CA; USA). Index-tagged cDNA libraries were constructed from 500 ng of total RNA using the TruSeq RNA Sample Preparation v2 Kit (Illumina; CA; USA). Libraries were quantified by Quant-iT™ dsDNA HS assay in a Q-bit fluorometer (Life Technologies; CA; USA). Average library size and size distribution were determined by DNA 1000 assay in an Agilent 2100 Bioanalyzer. Libraries were normalized to 10nM using 10mM Tris-HCl, pH8.5 containing 0.1% Tween 20 and then applied to an Illumina flow cell for cluster generation (True Seq SR Cluster Kit V2 cBot) and sequencing-by-synthesis. Single reads of length 75bp were generated with the TruSeq SBS Kit v5 (Illumina; CA; USA) on the Genome Analyzer IIx platform, following the standard RNA sequencing protocol. Reads were further processed using the CASAVA package (Illumina; CA; USA) to split reads according to adapter indexes and produce fastq files.

尽管参与心脏发生的基因调控网络已得到充分表征，但生物能学的相关作用仍较少被研究。本研究发现，在妊娠中期之前，心肌代谢呈现分区化特征：糖酵解特征仅局限于致密层心肌，而肌小梁区域的线粒体氧化活性则显著升高。缺氧诱导因子1α（HIF1a）的调控模式与此一致，其在致密层心肌中高表达，而在肌小梁中表达稀少。至妊娠中期时，致密层心肌会下调HIF1a的表达，并转向氧化代谢。删除E3泛素连接酶Vhl（Vhl）会导致HIF1a过度激活，破坏代谢分区化，并阻断妊娠中期向氧化磷酸化的代谢转换。此外，由HIF1a介导的肌小梁糖酵解特征改变，会阻碍心脏传导系统建立所必需的基因调控。我们的研究结果揭示了发育中心脏中VHL-HIF通路介导的代谢分区化现象，以及代谢与心肌分化之间的关联。这些结果强调了生物能学在心室心肌特化中的重要性，以及其与先天性心脏病的潜在相关性。
实验设计：从去除心房和瓣膜区域的E12.5胚胎心脏中分离总RNA。基因敲除（KO）样本与同窝野生型对照样本通过体节数进行匹配，共使用来自3个独立胎次的3例KO样本与3例对照样本。RNA提取采用QIAzol裂解试剂（Qiagen；加利福尼亚州；美国）与miRNeasy迷你试剂盒（Qiagen；加利福尼亚州；美国）。通过NanoDrop ND-1000分光光度计（Thermo Scientific；马萨诸塞州；美国）对RNA进行定量并检测纯度。采用安捷伦2100生物分析仪（Agilent Technologies；加利福尼亚州；美国）验证RNA完整性。取500 ng总RNA，使用TruSeq RNA样本制备v2试剂盒（Illumina；加利福尼亚州；美国）构建带有索引标签的cDNA文库。通过Qbit荧光计（Life Technologies；加利福尼亚州；美国）搭载Quant-iT™ dsDNA高灵敏度检测试剂盒对文库进行定量。采用安捷伦2100生物分析仪的DNA 1000检测试剂盒测定文库的平均片段长度与片段分布。使用含0.1% Tween 20的10mM Tris-HCl（pH 8.5）将文库标准化至10nM，随后将文库加载至Illumina流动槽进行簇生成（采用TrueSeq SR簇生成试剂盒V2 cBot），并进行边合成边测序。使用TruSeq SBS试剂盒v5（Illumina；加利福尼亚州；美国）在Genome Analyzer IIx平台上生成75bp的单端reads，遵循标准RNA测序流程。后续使用CASAVA软件包（Illumina；加利福尼亚州；美国）对reads进行处理，根据接头索引拆分reads并生成fastq文件。