The epi-drug RVX-208 restores autophagy in cardiometabolic heart failure with preserved ejection fraction

Abstract Background Cardiometabolic heart failure with preserved ejection fraction (cHFpEF) is a deadly condition whose mechanisms remain poorly understood. Key biological processes such as autophagy appear to be heavily involved in the pathophysiology of the disease, with epigenetic modulators acting as a bridge between environmental stressors and transcriptional changes. Epigenetic readers, namely BET proteins, bind acetylated histone motifs and play a critical role in regulating gene transcription in disease states. Whether BET proteins are involved in autophagy modulation in the heart is largely unknown. Purpose we posit that BET proteins modulate autophagy in cHFpEF and that their pharmacological inhibition by BET inhibitors (BETi) restores autophagic flux in this setting. Methods An established experimental mouse model of cHFpEF combining metabolic and hemodynamic stress for 15 weeks was employed. To investigate the in vivo effects of BETi on cardiac autophagy, cHFpEF mice were treated with vehicle or with a selective BETi (RVX-208) for 14 days. Single nuclei RNA sequencing (snRNAseq) was employed to detect cell-specific transcriptional changes. Autophagic genes were detected by a custom real-time PCR array followed by validation with immunoblotting. ChiP-seq and CUT&RUN assays were performed to study the enrichment of active chromatin marks (H3K27ac, H3K4me3) and reader proteins (BRD2, BRD4) at gene loci in cardiac specimens from the different experimental groups. Results Autophagy was impaired in cHFpEF mice, as indicated by the upregulation of mTOR signaling and reduced expression of autophagosome genes Atg7 and Atg13. Single-nucleus RNA sequencing (snRNA-seq) identified four main clusters of cardiomyocytes, each exhibiting suppressed autophagic pathways. Gene expression profiling revealed significant dysregulation of autophagic flux-related genes, including Atg5, TMEM74, and PS6K (involved in mTOR signaling). ChiP-seq showed a genome-wide increase of histone acetylation (H3K27ac) in HFpEF as compared to control hearts, suggesting the potential of BET-modulating strategies in this setting. Specifically, in cHFpEF hearts ChIP-seq revealed a marked enrichment of active chromatin marks and BET proteins on mTOR promoter, thus confirming a BET-driven transcriptional regulation of autophagy-related genes. Of note, in vivo treatment of cHFpEF mice with RVX-208, an FDA-approved BETi, was able to reset autophagy-related transcriptional alterations in the cHFpEF myocardium thus rescuing autophagic flux. Autophagy restoration was associated with a significant improvement of diastolic dysfunction and exercise tolerance in cHFpEF mice treated with RVX-208. Conclusions We show a BET-driven mechanism regulating autophagy in cHFpEF. Pharmacological inhibition of BET proteins restores autophagic flux while improving cardiac function in cHFpEF mice. Our results set the stage for preclinical studies testing FDA-approved BET inhibitors in cHFpEF.