Abstract Body (Do not enter title and authors here): Introduction: Our lab previously demonstrated the cardiotoxic effect of the transcription factor Krüppel-like factor (KLF) 5 in ischemic cardiomyopathy (ICM) in humans and mice. Investigation of the underlying mechanisms revealed that KLF5 suppresses the abundance of all 5 members of the cardioprotective miR-30 family.

Hypothesis: KLF5 inhibits all miR-30 family members in ICM by inducing non-coding RNA molecules with miRNA sponging activity.

Methods and results: MiRNA array analysis in hearts of cardiomyocyte (CM)-specific KLF5 knockout mice (CM-KLF5-/-) revealed higher expression of all five members of the miR-30-5p family, which are associated with improved survival of heart failure patients. Accordingly, patients with ICM and C57BL/6J mice with Myocardial Infarction (MI) showed elevated cardiac KLF5 and downregulation of all five miR-30s. Similarly, transgenic mice with inducible overexpression of CM-KLF5 (αMHC-rtTA-KLF5) had lower abundance of all 5 members of the miR-30-5p family. In contrast, levels of miR-30-3p and pri-miRs remained unchanged, suggesting that regulation occurs post-transcriptionally. Given the known miRNA sponging role of circular RNAs (circRNAs), we performed circRNA microarrays on hearts from CM-KLF5-/- mice with MI, CM-rtTA-KLF5 and respective controls. Our analysis identified 147 circRNAs that were upregulated with KLF5 induction and downregulated with KLF5 inhibition. In silico analysis of the lead circRNAs for miR-30-5p Response Elements and qRT-PCR validation led to identification of 3 candidate circRNAs conserved between mice and humans that were upregulated in both when KLF5 was induced. Strikingly, all 3 circRNAs are produced from different exons of the Prdm5 gene (circPRDM5^ex4-10, circPRDM5^ex7-12, circPRDM5^ex7-13). The pre-mRNA of PRDM5 was also increased, suggesting that KLF5 induces the transcription of Prdm5. The sponging effect of circPRDM5 on miR-30s was explored with individual and combined transfections of all three circRNAs. We show that all three circular RNAs can suppress miR30s, with circPRDM5^ex7-13 having the most robust effect. Notably, both myocardial KLF5 and circPRDM5^ex4-10 are upregulated in HF patients that do not respond to mechanical unloading, highlighting their cardiotoxic potential.

Conclusions: CM KLF5 activation in humans and mice with ICM stimulates the expression of Prdm5 and three circRNAs from this locus, which suppress the cardioprotective miR-30-5p family through a sponging mechanism.