| Experiment Id | GSE164453 | Name | Gene expression profiling of Hif1alpha defective hearts in E12.5 mice |
| Experiment Type | RNA-Seq | Study Type | WT vs. Mutant |
| Source | GEO | Curation Date | 2023-02-20 |
| description | Hypoxia is an important environmental cue in heart development. Despite of extensive characterization of gain and loss of function models, there is disagreement about the impact of HIF1 signaling elimination during cardiac development. Here, we used new conditional knock out models of Hif1alpha in Nkx2.5 cardiac progenitors and cardiomyocytes to assess the morphological and functional consequences of HIF1alpha loss in the developing heart. By combining histology, electron microscopy and high-throughput genomics and proteomics, we found that deletion of Hif1alpha leads to impaired embryonic glycolysis without influencing cardiomyocyte proliferation and results in an increased mitochondrial number, activation of a transient amino acid response and temporal upregulation of HIF2alpha and ATF4 by E12.5. Hif1alpha mutants display normal expression of genes and proteins involved in fatty acid oxidation and do not show any sign of cardiac dysfunction in the adulthood. Our results demonstrate that cardiac HIF1 signaling is dispensable for heart development and reveal the metabolic flexibility of the embryonic myocardium, opening the potential application of alternative energy sources as therapeutic interventions during ischemic events. 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 2 KOs and 2 controls from the same litter. For RNA extraction QIAzol Lysis Reagent (Qiagen; CA; USA) and the miRNeasy Mini Kit (Qiagen; CA; USA). 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. |
