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Myocardial gene expression in mice with cardiac-restricted knockout of COP9 signalosome subunit 8 (CSN8)

ABSTRACT: Background: The COP9 signalosome (CSN) consisting of 8 protein subunits (CSN1 through CSN8) is a deneddylase for Cullin-RING ligases (CRLs), essential to the functioning of the ubiquitin-proteasome system. CSN8 is encoded by the Cops8 gene. We have shown that CSN8 plays a key role in the regulation of cardiac protein quality control (PQC), and cardiomyocyte-restricted Cops8 knockout (CSN8-CKO) in perinatal and adult mice causes massive cardiomyocyte necrosis and acute heart failure in mice. Objective: To identify the underlying mechanisms at the transcriptional level, we examined the impact of perinatal CSN8-CKO on myocardial gene expression. Design: Whole mouse genome DNA microarrays (CodeLink) were used to assess myocardial gene expression profiles in Control (CTL), heterozygous (Het-CKO), and homozygous (Hom-CKO) cardiac Cops8 knockout littermate mice at 2- and 3-weeks-of-age. GeneSpring GS software (v.12.5) was used to analyze differentially expressed genes (DEGs). DEGs were analyzed using the GeneGo MetaCore Analytical Suite (http://genego.com; GeneGo, St. Joseph, MI) to identify significantly enriched pathways. Network analysis was performed using R package WGCNA on the expression profiles of DEGs. IPA analysis was also performed. Results: The microarray data revealed 521 DEGs at 3-weeks-of-age with cut-off log2 |fold change|≧1. Among these genes, 271 genes were upregulated and 250 were downregulated. Similarly, at 2-weeks-of-age, out of 78 DEG, 35 genes were upregulated and 43 were downregulated. The transcript levels of many substrate receptors of CRLs (Fbxo14, Fbxo31, Klhdc1, Klhdc7a, Klh31, Klh41) were significantly down-regulated by CSN8-CKO. Consistent with cardiac pathology and prior RNA dot blot analysis results, the microarray analysis revealed significant upregulation of fetal genes (e.g., Nppa, Nppb, Acta1) and down-regulation of many postnatal genes (Actc1, Atp2a2, Pln, Tpm1). Our results suggest that CSN8-CKO-induced decrease in cardiac contractility could be due to decreased transcription of cacna1s, ptgfr and mylk2 genes. Pathways related to cellular stress particularly oxidative stress and vesicular trafficking were also observed to be enriched following CSN8 deficiency in cardiomyocytes. Enrichment of DEGs in chromatin remodeling pathway corroborates the involvement of CSN8 in gene expression regulation. Conclusions: Myocardial gene expression profiles are dynamically affected by CSN8-CKO in mice. Our data strongly suggest that CSN8 is involved in the regulation of myocardial gene expression programs that regulate ubiquitination, chromatin remodeling, and vesicle trafficking in cardiomyocytes.

ORGANISM(S): Mus musculus

PROVIDER: GSE100104 | GEO | 2017/08/23

SECONDARY ACCESSION(S): PRJNA390725

REPOSITORIES: GEO

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Project description:Rationale: The Id1 and Id3 genes play major roles during cardiac development, despite their expression being confined to non-myocardial layers (endocardium â?? endothelium - epicardium). We previously described that Id1â??/â??Id3â??/â?? double knockout (dKO) mouse embryos die at mid-gestation from multiple cardiac defects, but early demise precluded the studies of the roles of Id in the adult mice. Objective: To elucidate postnatal roles of Id genes in the heart. Methods and Results: We ablated Id1 gene in the vasculature and Id3 gene globally to generate Tie2Cre+Id1F/â??Id3â??/â?? and Tie2Cre+Id1F/FId3â??/â?? conditional KO (Id cKO) embryos. Half of the Id cKO mice die at birth. Postnatal demise was associated with cardiac underdevelopment, enlargement, muscular ventricular septal and endothelial defects. Surviving Id cKO mice exhibited dilated, fibrotic cardiomyopathy associated with defects in the vasculature. The adult cardiac phenotype progressed into heart failure and resembled endomyocardial fibroelastosis. An abnormal vascular response was also observed in the healing process of excisional skin wounds of Id cKO mice. Expression patterns of vascular, fibrotic and hypertrophic markers were altered in the Id cKO hearts, but addition of Insulin-Like Growth Factor binding protein-3 (IGFbp3) reversed gene expression profiles of vascular and fibrotic, but not hypertrophic markers. Conclusions: Conditional ablation of Id genes in the vasculature leads to dilated fibrotic cardiomyopathy. The findings could reveal important insights into the role(s) of the endocardial network of the endothelial lineage to the development of dilated fibrotic cardiomyopathy and identify a potential therapeutic target, IGFbp3, in its treatment. Total RNA from heart tissue was isolated (RNeasy, QIAGEN) from P180 WT, Id control, Id cKO, IGFbp3 incubated Id cKO, and control incubated Id cKO. RNA was converted to cDNA, cRNA, and hybridized to DNA sequences contained in the GeneChip Mouse Gene 1.0 ST Array (Affymetrix). Information from at least duplicate samples was compared and filtered by fold change >2 (Id cKO vs WT, Id control vs WT, IGFbp3 incubated Id cKO vs. control incubated Id cKO) and statistical p-value<0.001.

Project description:DEHP study Phthalate plasticizers are ubiquitous chemicals linked to several cardiovascular diseases in animal models and in humans. In spite of this, the mechanisms by which phthalate exposures cause adverse cardiac health outcomes are unclear. In particular, whether phthalate exposures during pregnancy interfere with normal developmental programming of the cardiovascular system, and the resulting implications this may have for long-term disease risk, are unknown. Recent studies suggest that the effects of phthalates on metabolic and neurobehavioral outcomes are sex-specific. However, the influence of sex on cardiac susceptibility to phthalate exposures has not been investigated. One mechanism by which developmental exposures may influence long-term health is through altered programming of DNA methylation. In this work, we utilized an established mouse model of human-relevant perinatal exposure and enhanced reduced representation bisulfite sequencing to investigate the long-term effects of diethylhexyl phthalate (DEHP) exposure on DNA methylation in the hearts of adult male and female offspring at 5 months of age (n=5-7 mice per sex and exposure). Perinatal DEHP exposure led to hundreds of sex-specific, differentially methylated cytosines (DMCs) and differentially methylated regions (DMRs) in the heart. Pathway analysis of DMCs revealed enrichment for several pathways in females, including insulin signaling, regulation of histone methylation, and tyrosine phosphatase activity. In males, DMCs were enriched for glucose transport, energy generation, and developmental programs. Notably many sex-specific genes differentially methylated with DEHP exposure in our mouse model were also differentially methylated in published data of heart tissues collected from human heart failure patients. Together these data highlight the potential role for DNA methylation in DEHP-induced cardiac effects, and emphasize the importance of sex as a biological variable in environmental health studies. Lead study Environmental factors play an important role in the etiology of cardiovascular diseases. Cardiovascular diseases exhibit marked sexual dimorphism; however, the sex-specific effects of environmental exposures on cardiac health are incompletely understood. Perinatal and adult exposures to the metal lead (Pb) are linked to several adverse cardiovascular outcomes, but the sex-specific effects of this toxicant on the heart have received little attention. Perinatal environmental exposures can lead to disease through disruption of the normal epigenetic programming that occurs during early development. Using a mouse model of human-relevant perinatal environmental exposure, we investigated the effects of exposure to Pb during gestation and lactation on DNA methylation in the hearts of adult offspring mice (n=6 per sex). Two weeks prior to mating, dams were assigned to control or Pb acetate (32 ppm) water, and exposure continued until offspring were weaned at 3 weeks of age. Enhanced reduced-representation bisulfite sequencing was used to measure DNA methylation in the hearts of offspring at 5 months of age. Although Pb exposure stopped at 3 weeks of age, we discovered hundreds of differentially methylated cytosines (DMCs) and regions (DMRs) in males and females at 5 months of age. DMCs/DMRs and their associated genes were sex-specific, with a small, but statistically significant subset overlapping between sexes. Pathway analysis revealed altered methylation of genes important for cardiac and other tissue development in males, and histone demethylation in females. Together, these data demonstrate that perinatal exposure to Pb induces sex-specific changes in cardiac DNA methylation that are present long after cessation of exposure, and highlight the importance of considering sex in environmental epigenetics and mechanistic toxicology studies.

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Myocardial gene expression in mice with cardiac-restricted knockout of COP9 signalosome subunit 8 (CSN8)

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