Introduction

Structural remodeling in heart failure involves with complex interactions of cardiac myocytes and non-myocytes. Cardiac endothelial cells are key players in this process, mediating inflammation and fibrosis, controlling angiogenesis and nutrition supply, and modulating the function of other cell types by paracrine signaling. Aldosterone is a known driver of adverse cardiac remodeling acting on the on the mineralocorticoid receptor (MR), a ligand-activated transcription factor. Thus, we explored the epigenetic landscape of cardiac endothelial cells to uncover modes of transcriptional regulation in experimental heart failure with focus on the MR.

Methods and results

We isolated cardiac endothelial cells from wildtype mice after transverse aortic constriction (TAC) by fluorescence-activated cell sorting (FACS) to assess changes in gene expression after chronic pressure overload. We found 944 genes differentially expressed (q<0.05). To uncover modes of transcriptional regulation in cardiac endothelial cells we conducted an integrated analysis of RNA-seq, ChIP-seq, ATAC-seq and DNA-methylation data. We identified 8145 regions as active cis-regulatory regions. Using Hi-C, we assessed chromatin interactions and linked active enhancer regions to differentially regulated genes. Comparison of the endothelial cell transcriptome after pressure overload or aldosterone stimulation (ALDO) revealed a high proportion of genes showing the same direction of regulation, both after TAC and ALDO. Analysis of enriched biological processes among those genes revealed an association to angiogenesis and regulation of immune responses. Using Hi-C data, we identified cis-regulatory regions harboring MR binding sites and thereby defined direct MR target genes.

To assess the functional relevance of these findings, mice with endothelial cell-specific deletion of the mineralocorticoid receptor (MRCdh5Cre) were generated using the Cre/loxP system. MRCdh5Cre and Cre-negative littermates (MRwildtype) underwent transverse aortic constriction (TAC, n=5-7 per group). After two weeks of pressure overload cardiac hypertrophy (ventricle weight 143.2 ± 5.2 vs. MRwildtype 167.3 ± 6.7 mg, P<0.001) and interstitial fibrosis (sirius red stained area 8.2 ± 4.7 vs. MRwildtype 13.5 ± 4.5 %, P<0.05) were attenuated in MRCdh5Cre mice. Gene expression analysis revealed that out of 442 genes differentially regulated after TAC and aldosterone treatment, 93% were restored by endothelial-specific MR deletion.

Conclusion

Endothelial cell-specific MR deletion has protective effects on left ventricular inflammation and remodeling after pressure overload. Integrated analysis of the epigenetic landscape of cardiac endothelial cells as well as the 3D genome architecture provide insight into regulation of gene expression by MR and epigenetic modifications.