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Mis-expression of Igf2 and H19 work independently on distinct cell types to cause cardiomyopathy in a Beckwtih Wiedemann mouse model

ABSTRACT: Igf2 and H19 are linked, reciprocally imprinted genes that play critical roles in mammalian development. Igf2 encodes a peptide hormone, Insulin-like Growth Factor 2, that binds to the InsR and Igf1R receptor kinases to regulate cell growth and metabolism through well-established pathways. H19 encodes a 2.3 kb lncRNA whose biochemical actions are only now being identified. Here we use a mouse model to investigate cardiomyopathies associated with maternal loss of imprinting at the locus. Increased circulating levels of IGF2 during fetal development result in activation of pAKT/mTOR signaling in cardiomyocytes to cause cellular hypertrophy and hyperplasia in neonatal hearts. This neonatal hypertrophy is unaffected by H19 RNA levels. However, loss of H19 does cause fibrosis and progressive cardiac pathology in adult mice. In hearts, H19 expression is concentrated in endothelial cells associated with blood vessels and capillaries and loss of H19 results in high incidence of trans differentiation of these cells to smooth muscle. H19 encoded miRNAs 675-3p and -5p are not sufficient to promote normal development. Thus these experiments define a novel role for the H19 lncRNA in regulating cell fate.

ORGANISM(S): Mus musculus

PROVIDER: GSE111418 | GEO | 2020/03/01

REPOSITORIES: GEO

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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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Mis-expression of Igf2 and H19 work independently on distinct cell types to cause cardiomyopathy in a Beckwtih Wiedemann mouse model

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