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Age-Dependent Transcriptional Alterations in Cardiac Endothelial Cells [bulk RNA-seq]

ABSTRACT: Purpose: The goal of this study is to determine alterations in heart endothelial cell transcripts associated with aging. This is accomplished by comparing endothelial transcriptomes from young and aged organs (heart, brain and kidney). Methods: Endothelial mRNA profiles of 3 month and 24 month old wild-type mice were generated by next generation sequencing using libraries generated via SmartSeq V4 chemistry (Takara) and sequenced on an HiSeq 2500 (Illuina). Tophat2 with default parameters was used to align the sequenced reads against the mouse genome using the GRCm38. HTSeq python software with default parameters was used to quantify the transcripts of the aligned reads using the corresponding GRCm38 gene annotation model from Ensembl. Results: Highly enriched populations of endothelial cells (ECs) isolated from the heart, brain and kidney of young (3 months) and old (24 months) C57/BL6 mice were profiled for RNA expression via bulk RNA sequencing. Approximately 700 cardiac endothelial transcripts significantly differ by age. Gene set enrichment analysis indicated similar patterns for cellular pathway perturbations. Receptor-ligand comparisons indicated parallel alterations in age-affected circulating factors and cardiac endothelial-expressed receptors. Single-cell RNA-seq analysis identified 9 distinct endothelial cell subtypes in the heart with an age-associated population shift observed for an Aplnr-enriched endothelial cell clusters. Conclusions: Gene and pathway enrichment analyses show that age-related transcriptional response of cardiac endothelial cells is distinct from that of endothelial cells derived from the brain or kidney vascular bed. Furthermore, single-cell analysis identified 9 distinct EC subtypes, and shows that Aplnr-enriched subtype is reduced with age in mouse heart. Finally, we identify age-dysregulated genes in specific aged cardiac endothelial subtypes.

ORGANISM(S): Mus musculus

PROVIDER: GSE163821 | GEO | 2021/05/28

REPOSITORIES: GEO

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Project description:Transcriptional profiling of 50% epiboly zebrafish embryos comparing wildtype (control) to Aplnra/b morpholino injected embryos. Goal was to determine any gene expression changes in embryos deficient for the Apelin receptor. Publication summary: The Apelin receptor (Aplnr) has been shown to be essential for proper cardiac progenitor migration during gastrulation. Loss of the Aplnr results in a delay in the ingression of lateral marginal cells and the complete absence of the heart and early cardiac gene expression. How the Aplnr is required at a molecular level for cardiac progenitor migration remains completely unknown. We present evidence to suggest that the Aplnr enhances Nodal signaling to initiate the migration program at the correct time in development. Apelin receptor loss of function embryos exhibit a reduction in a wide range of Nodal target genes while activation of the receptor is capable of boosting their expression. Furthermore, Aplnr deficient embryos are exquisitely sensitive to Nodal receptor inhibitors. In order to directly interrogate the functional consequence of reduced Nodal signaling in the Aplnr cardiac phenotype, we raised the level of Nodal activity in aplnr mutants and found that this was able to provide a near complete rescue. We next sought to determine a mechanism by which lower Nodal signaling levels could translate into a delay in migration. Remarkably, we observe a delay in the expression of mesp family members in Aplnr loss of function embryos at the beginning of gastrulation. The mesp family has previously been implicated in mesodermal migration and we show that they are downstream targets of Nodal. We propose that in the absence of the Aplnr the appropriate Nodal threshold required to initiate the downstream cardiac progenitor “specification/migration” program requires a longer length of time to be attained. This translates into a delay in migration and consequently cardiac progenitors are not capable of reaching their final destination. In this study we demonstrate how the fine tuning of a key signaling pathway is critical for the proper development of the early embryo.

Project description:Abstract: Dysregulation of the imprinted H19/IGF2 locus can lead to Silver-Russell Syndrome (SRS) in humans. However, the mechanism of how abnormal H19/IGF2 expression contributes to various SRS phenotypes remains unclear, largely due to incomplete understanding of the developmental functions of these two genes. We previously generated a mouse model with humanized H19/IGF2 ICR (hIC1) on the paternal allele that exhibited H19/Igf2 dysregulation together with SRS-like growth restriction and perinatal lethality. Here we dissect the role of H19 and Igf2 in cardiac and placental development utilizing multiple mouse models with varying levels of H19 and Igf2. We report severe cardiac defects such as ventricular septal defects (VSDs) and thinned myocardium, placental anomalies including thrombosis and vascular malformations, together with growth restriction in mouse embryos that correlated with the extent of H19/Igf2 dysregulation. Transcriptomic analysis using cardiac endothelial cells of these mouse models shows that H19/Igf2 dysregulation disrupts pathways related to extracellular matrix (ECM) and proliferation of endothelial cells. Our work links the heart and placenta through regulation by H19 and Igf2, demonstrating that accurate dosage of both H19 and Igf2 is critical for normal embryonic development, especially related to the cardiac-placental axis. Methods: E12.5 hearts were lysed with Collagenase, Dispase II, and DNase I. Cardiac endothelial cells were collected using MACS CD31 microbeads and RNA was isolated using RNeasy Micro kit. After confirming RNA integrity using Bioanalyzer, mRNA library was generated from 25ng RNA using NEBNext Poly(A) mRNA Magnetic Isolation Module and Ultra II RNA Library Prep Kit. Library quality was assessed by Bioanalyzer and TapeStation. Sequencing was performed on NovaSeq 6000. Quality of raw fastq reads was assessed using FastQC version 0.11.5. Reads were aligned to the GRCm38/mm10 reference using STAR version 2.4.0i with default parameters and maximum fragment size of 2000 bp. Properly paired primary alignments were retained for downstream analysis using Samtools version 1.9. Count matrices were generated using FeatureCounts version 1.6.2 against RefSeq gene annotation and read into DESeq2 to perform normalization and statistical analysis.

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Age-Dependent Transcriptional Alterations in Cardiac Endothelial Cells [bulk RNA-seq]

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