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Gene expression data from CITED4 knockout and control mouse hearts after transverse aortic constriction

ABSTRACT: Rationale: Cardiac CITED4 is induced by exercise and is sufficient to cause physiological hypertrophy and mitigate adverse ventricular remodeling after ischemic injury. However, the role of endogenous CITED4 in response to physiological or pathological stress is unknown. Objective: To investigate the role of CITED4 in murine models of exercise and pressure overload. Methods and Results: We generated cardiomyocyte-specific CITED4 knockout mice (C4KO) and subjected them to an intensive swim exercise protocol as well as transverse aortic constriction (TAC). Echocardiography, western blotting, qPCR, immunohistochemistry, immunofluorescence, and transcriptional profiling for mRNA and miRNA expression were performed. Cellular crosstalk was investigated in vitro. CITED4 deletion in cardiomyocytes did not affect baseline cardiac size or function in young adult mice. C4KO mice developed modest cardiac dysfunction and dilation in response to exercise. After TAC, C4KOs developed severe heart failure with left ventricular dilation, impaired cardiomyocyte growth accompanied by reduced mammalian target of rapamycin (mTOR) activity and maladaptive cardiac remodeling with increased apoptosis, autophagy, and impaired mitochondrial signaling. Interstitial fibrosis was markedly increased in C4KO hearts after TAC. RNAseq revealed induction of a pro-fibrotic miRNA network. miR30d was decreased in C4KO hearts after TAC and mediated crosstalk between cardiomyocytes and fibroblasts to modulate fibrosis. miR30d inhibition was sufficient to increase cardiac dysfunction and fibrosis after TAC. Conclusions: CITED4 protects against pathological cardiac remodeling by regulating mTOR activity and a network of miRNAs mediating cardiomyocyte to fibroblast crosstalk. Our findings highlight the importance of CITED4 in response to both physiological and pathological stimuli.

ORGANISM(S): Mus musculus

PROVIDER: GSE149584 | GEO | 2020/04/30

REPOSITORIES: GEO

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Project description:BACKGROUND: Heart failure (HF) is one of the leading causes of mortality worldwide. Extracellular vesicles (EVs) including small EVs, or exosomes and their molecular cargo are known to modulate cell to cell communication during multiple cardiac diseases. However, the role of systemic EV biogenesis inhibition in the models of HF is not well documented and remains unclear. METHODS: We investigated the role of circulating exosomes during cardiac dysfunction and remodeling in a mouse transverse aortic constriction (TAC) model of HF. Importantly, we investigate the efficacy of Tipifarnib (Tip), a recently identified exosome biogenesis inhibitor that targets the critical proteins (Rab27a, nSmase2 and Alix) involved in exosome biogenesis for this mouse model of HF. In this study, 10-week-old male mice underwent TAC surgery, were randomly assigned to groups with and without Tip treatment (10 mg/kg three times/week) and monitored for 8 weeks and a comprehensive assessment was conducted through performed echocardiographic, histological, and biochemical studies. RESULTS: TAC significantly elevated circulating plasma exosomes and markedly increased cardiac left ventricular (LV) dysfunction, cardiac hypertrophy, and fibrosis. Furthermore, injection of plasma exosomes from TAC mice induced LV dysfunction and cardiomyocyte hypertrophy in uninjured mice without TAC. On the contrary, treatment of Tip in TAC mice reduced circulating exosomes to baseline and remarkably improved LV functions, hypertrophy, and fibrosis. Tip treatment also drastically altered the miRNA profile of circulating post-TAC exosomes, including miR331-5p which was highly downregulated both in TAC circulating exosomes and in TAC cardiac tissue. Mechanistically, miR331-5p is crucial for inhibiting fibroblast-to-myofibroblast transition by targeting HOXC8, a critical regulator of fibrosis. Tipifarnib treatment in TAC mice upregulated the expression of miR331-5p that acts as potent repressor for one of the fibrotic mechanisms mediated by HOXC8. CONCLUSIONS: Our study underscores the pathological role of exosomes in HF and fibrosis in response to pressure overload. Tipifarnib mediated inhibition of exosome biogenesis and cargo sorting may serve as a viable strategy to prevent progressive cardiac remodeling to HF.

Project description:Aims: We investigate sex differences and the role of oestrogen receptor beta (ERbeta) in a mouse model of pressure overload-induced myocardial hypertrophy. Methods and results: We performed transverse aortic constriction (TAC) or sham surgery in male and female wild-type (WT) and ER knockout (ERbeta-/-) C57Bl6 mice. All mice were characterised by echocardiography and haemodynamic measurements and were sacrificed nine weeks after surgery. Left ventricular (LV) samples were analysed by microarray profiling, real-time RT-PCR and histology. After nine weeks, WT males showed more hypertrophy and heart failure signs than WT females. Notably, WT females developed a concentric form of hypertrophy, while males developed eccentric hypertrophy. These sex differences were abolished in ERbeta-/- mice. ERbeta deletion augmented the TAC-induced increase in cardiomyocyte diameter in both sexes. Gene expression profiling revealed that male WT hearts had a stronger induction of matrix-related genes and a stronger repression of mitochondrial genes than female hearts. ERbeta-/- mice exhibited a different transcriptome. Induction of pro-apoptotic genes after TAC occurred in ERbeta-/- mice of both sexes with a stronger expression in ERbeta-/- males. Histological analysis revealed, that cardiac fibrosis was more pronounced in male WT TAC than in female mice. This was abolished in ERbeta-/- mice. Apoptosis was significantly induced in both sexes of ERbeta-/- TAC mice, but it was most prominent in males. Conclusion: Female sex offers protection against ventricular chamber dilation in the TAC model. Both the female sex and ER attenuate the development of fibrosis and apoptosis; thus slowing the progression to heart failure. The influence of sex (male/female) and estrogen receptor beta expression (ERbeta knockout/wildtype) on cardiac hypertrophy (transverse aortic constriction/sham operated) was investigated. The left ventricular transcriptome of four individual mice for each combination of the three factors (sex, genotype, surgery) was detected with Affymetrix RAE 430 2.0 GeneChip arrays.

Project description:Purpose: Cardiac fibroblasts (CFs) contribute to pathological remodeling by proliferating and secreting extracellular matrix. In contrast, physiological remodeling is an adaptive response to excessive demands of exercise that results in increased heart size without accompany fibrosis or deterioration of cardiac function. We hypothesize that these divergent outcomes are partially a function of cell-autonomous and paracrine cardioprotection afforded by CFs specifically during physiological remodeling. Our objective is to define the gene expression program (GEP) of CFs isolated from mouse models of physiological (swim) and pathological (thoracic aortic constriction (TAC) or myocardial infarction (MI)) remodeling to identify novel mechanisms underlying the development of pathological cardiac fibrosis and the cardioprotective benefits of exercise. Methods: Cardiomyocytes (CMs) were isolated by gravity sedimentation and CFs were isolated by differential plating for 2 hours from single-cell suspensions of 10-14 week old male C57BL/6 hearts subjected to the following 7 conditions: sedentary CFs (n=3), sham surgery CFs (n=2), 3 day TAC CFs (n=3), 10 day TAC CFs (n=3), 4 day swim CFs (n=3), 10 day swim CFs (n=3), and 5 day MI CFs (n=2). CMs were isolated from sedentary 10-14 week old male C57BL/6 hearts (n=3). Total RNA was isolated using the Qiagen RNeasy Plus Kit and genomic DNA removed using Qiagen gDNA columns. RNA quality was assessed with the Agilent Bioanalyzer and only samples with RIN >8.5 were used for sequencing. Library construction was performed using the TruSeq Stranded mRNA Sample Preparation Kit from 100ng poly-A mRNA. An Illumina HiSeq 2500 was used to generate 20-25 million single end reads of 100nt for each sample. The sequence reads that passed quality filters (Trimmomatic-0.32) were analyzed at the transcript isoform level using STAR_2.4.2a for SHRiMP_2_2_3 mapping to mouse genome assembly GRCm38.p4 version mm10 and processed with Cufflinks-2.0.2. Results: We determined that CFs display distinct GEPs that are inversely regulated in pathological and physiological remodeling. Spearman correlation and Principal Component Analysis (PCA) of genes with FPKM >1 and q >0.05 revealed consistency between biological replicates and highlighted active changes in GEPs during physiological and pathological remodeling both of which diverged from control samples in principal component (PC) 1 and PC2. CFs isolated from mice 5 days after initiation of MI also diverged from controls, TAC, and swim samples, demonstrating a different fibroblast GEP in ischemic and pressure overload induced remodeling. This analysis also revealed that our control models (sham surgery and sedentary) were indistinguishable from each other, therefore we typically treated sham and sedentary mice as a single control group in further studies. These initial results reveal that CFs differentially respond to pathological and physiological cues in as few as 3 days by enacting dynamic transcriptional changes in vivo. Consistent with previous reports of myofibroblast activation, Ingenuity Pathway Analysis (IPA) revealed that the most significantly enriched GO terms in pathological fibroblasts included stimulation of Rho-dependent contractile pathways and integrin-linked kinase (ILK) signaling, the latter of which was downregulated in a comparison between 10-day swim vs. control CFs. IPA transcription factor analysis comparing 10-day swim and 10-day TAC CFs further identified upregulation of detoxification and inflammatory transcription factors and downregulation of the serum response factor (SRF) after exercise. Furthermore, independent motif analysis using HOMER 2.0 identified the CArG box (SRF binding site) as the most enriched transcription factor binding site within 5kb of genes that are upregulated in 10-day TAC vs sham CFs. Conclusions: We have defined the GEPs of CFs that underlie physiological and pathological cardiac remodeling. Not only is the GEP of pathological CFs enriched in Rho-dependent pathways and SRF target genes, but the GEP of physiological CFs is enriched in detoxification and inflammatory target genes. We also demonstrate that the CFs change their transcriptional signature consistently and rapidly. Within 3-4 days of initiating extracellular stimuli, distinct profiles are observed in CFs isolated from swim-trained, pressure overload, or ischemic models of cardiac remodeling.

Project description:Background: Pathological cardiac overload triggers maladaptive myocardial remodeling that predisposes to the development of heart failure. The contribution of long non-coding RNAs (lncRNAs) to intercellular signaling during cardiac remodeling is largely unknown. Methods: We analyzed the expression of Gadlor 1 and Gadlor2 lncRNAs in mouse hearts, mouse cardiac cells, extracellular vesicles (EVs), human failing hearts as well as patient serum. Gadlor knock-out (KO) mice were generated and analyzed. The effect of Gadlor knock-out and Gadlor overexpression during cardiac pressure overload induced by transverse aortic constriction (TAC) was analyzed by echocardiography, histological analyses and RNA sequencing in isolated cardiac cells. Gadlor1/2 interaction partners were identified by RNA antisense purification coupled with mass-spectrometry (RAP-MS). Results: In the heart, the related lncRNAs Gadlor1 and 2 are mainly expressed in endothelial cells and to a lesser extent in fibroblasts. Gadlor1/2 are upregulated in failing mouse hearts as well as in the myocardium and in serum of heart failure patients. Interestingly, Gadlor1 and 2 are secreted from endothelial cells within EVs, which are taken up by cardiomyocytes. Gadlor-KO mice exerted reduced cardiomyocyte hypertrophy, diminished myocardial fibrosis and improved cardiac function, but paradoxically suffered from sudden death during prolonged overload. Gadlor overexpression, in turn, triggered hypertrophy, fibrosis and cardiac dysfunction. Mechanistically, Gadlor1 and Gadlor2 inhibit angiogenic gene expression in endothelial cells, while promoting the expression of pro-fibrotic genes in cardiac fibroblasts. In cardiomyocytes, Gadlor1/2 upregulate mitochondrial genes, but downregulate angiogenesis genes, while interacting with the transcriptional regulator Glyr1 and the calcium/calmodulin-dependent protein kinase type II (CaMKII), entailing cardiomyocyte hypertrophy and perturbed cardiomyocyte calcium dynamics. Conclusions: We describe that Gadlor1 and 2, two related, novel lncRNAs, are upregulated in cardiac pathological overload and are secreted from endothelial cells within EVs. Gadlor1/2 induce cardiac dysfunction, cardiomyocyte hypertrophy and myocardial fibrosis by exerting heterocellular effects on cardiac cellular gene-expression and by affecting calcium dynamics in cardiomyocytes, which take up the Gadlor1/2 by EV mediated transfer from endothelial cells. Targeted inhibition of Gadlor lncRNAs in endothelial cells or fibroblasts might serve as therapeutic strategy in the future.

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Gene expression data from CITED4 knockout and control mouse hearts after transverse aortic constriction

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