Circular RNAs dysregulation in ischemic heart failure

Abstract Introduction Circular RNAs (circRNAs) are an emerging class of non-coding RNAs originating from the splicing and circularization of pre-mRNAs and long non-coding RNAs. CircRNAs can regulate transcription and splicing, sequester microRNAs acting as “sponge” and inducing the respective targets, and bind to RNA binding proteins. CircRNAs have been found deregulated in several cardiovascular diseases, including heart failure (HF). However, incomplete and sometimes contradictory results have been reported on their regulation and function in HF, possibly for the heterogeneity of patients analyzed, indicating that our understanding of the regulation and role of cardiac circRNAs is still very limited. Purpose We aim to identify novel circRNA candidates deregulated in ischemic HF and to functionally characterize them in HF. Methods To define the circRNA expression pattern in ischemic HF, a high-depth RNA-seq was performed on the remote zone of left ventricle (LV) samples of 20 non end-stage ischemic HF patients with reduced LV ejection fraction and 20 matched controls. Differentially expressed circRNAs identified using CIRIquant tool were validated by qRT-PCR. Samples from the border zone of non end-stage HF patients and LV biopsies from end-stage HF patients were also used to measure circRNAs expression. Results By RNA-seq, we identified 17 circRNAs as differentially expressed with FDR<0.1. qRT-PCR results confirmed the differential expression of 4 circRNAs, cSLC6A6, cNAA16, cMLIP and cHDCA9, which were upregulated in HF samples (t-test, p-value <0.001). To evaluate circRNAs deregulated independently of their linear counterparts, we also estimated the ratio between circular and linear isoforms. cSLC6A6, cMLIP and cHDCA9 showed also a statistically significant (t-test, p-value <0.002) increase of the circular-to-linear ratio between HF and control patients. To complement the unbiased strategy of RNA-seq, we evaluated the modulation of 18 candidate circRNAs identified by a literature analysis as dysregulated in ischemic or not-ischemic cardiomyopathies or in biological mechanisms relevant for ischemic HF. cPVT1, cANKRD17, cBPTF, displaying a concordant deregulation in different stages of the disease (non end-stage and end-stage HF) and in different regions of LV myocardium (remote and border zone), were chosen for further analysis. Using siRNAs targeting the backsplice junction, cPVT1 was specifically knockdown in a human cardiomyocyte cell line. RNA-seq analysis of cPVT1 knockdown samples identified 3577 significantly differentially expressed genes (FDR<0.01). Gene ontology and pathway analysis showed that these genes were mainly involved in the formation of extracellular matrix, fibrosis and in cellular senescence, suggesting the involvement of cPVT1 in these HF-related disease mechanisms. Conclusion We identified new deregulated circRNAs in ischemic HF patients that might play a pathogenic role in HF. Funding Acknowledgement Type of funding sources: Private hospital(s). Main funding source(s): Italian Ministry of Health, Ricerca Corrente 2021-2023 “CircularRNAs in heart failure: regulation, function and interaction with the transcriptome”