Clin Res Cardiol (2023). https://doi.org/10.1007/s00392-023-02180-w

Identification of Novel Mineralocorticoid Receptor Coregulators

D. Koca1

1Institut für Pharmakologie, Universitätsklinikum Freiburg, Freiburg im Breisgau;

Introduction Mineralocorticoid receptor (MR) antagonists such as eplerenone or spironolactone reduce the morbidity and mortality of patients with congestive heart failure and considered standard therapy.  Recently non-steroidal MR antagonists such as finerenone have been developed that improved cardiovascular outcomes of patients with diabetic kidney disease in large clinical trials. However, the clinical use of MR antagonists is limited by an increased risk for hyperkalemia. MR is a ligand-induced nuclear receptor that is expressed in cardiovascular cells and kidney epithelial cells. The aim of this study was to identify cell type-specific mechanisms of MR induced transcription that might lead to the development of cell selective MR modulators for the treatment of heart failure but sparing renal side effects.  Methods and results We established a cellular model to study MR nuclear shuttling, MR post-translational modifications, MR chromatin interaction, co-regulators and gene expression. For this purpose, doxycycline inducible stable overexpression of GFP-tagged human MR was conducted in T-rex HeLa cell was confirmed by immunoblotting. After aldosterone treatment, translocation of GFP-tagged MR into nucleus was demonstrated by cellular fractionation and confocal microscopy. Furthermore, overexpression of recombinant MR increased the mRNA expression of previously described MR target genes. In total, we identified 2,168 differentially expressed genes upon aldosterone stimulation. To test whether MR antagonists prevented aldosterone-induced upregulation of representative MR target genes (RASD1, FGD4, CDKN1A, TIPARP, ARHGAP29, PDK4), cell were pretreated with eplerenone, spironolactone, esaxerenone, finerenone, AZD9977. Both steroidal and non-steroidal MR antagonists attenuated the upregulation of MR target genes (50 to 90 %) but not nuclear shuttling of MR, indicating that their inhibitory effect involves processes within the nucleus.  To identify direct MR target genes, we performed MR chromatin immunoprecipitation (ChIP-seq). We uncovered 6,855 MR colocalizations that were enriched around the transcription start site (TSS) of target genes. Intersection of the 2,168 differentially expressed genes and the 6,855 MR-bound regions identified 971 differentially expressed, direct MR target genes. These were involved in biological processes of angiogenesis and cell-cell adhesion. To identify coregulators of MR in T-rex HeLa cells, we performed rapid immunoprecipitation mass spectrometry (RIME) of GFP-tagged, chromatin-bound MR and its associated protein complex. Quantitative Proteome analysis revealed that 455 MR-associated proteins in T-rex HeLa cells.  Conclusion The inhibitory effect of MR antagonists involves processes within the nucleus, potentially co-regulator recruitment of MR chromatin interaction. Future studies in cardiac and renal cells will enable us to identify MR-associated proteins that may serve as potential new drug targets.

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