Abstract Background/Introduction Cardiovascular diseases remain the number one cause of death worldwide (1). As effective curative treatments are lacking, there is an urgent need for novel therapies. One promising new approach is the utilization of circular RNAs (circRNAs), which represent a subtype of non-coding RNAs, and are emerging as regulators of cardiovascular diseases (2). These circRNAs may be utilized as a novel therapeutic approach after heart injury by means of inducing proliferation of existing cardiomyocytes to ultimately achieve cardiac regeneration. Purpose Neonatal mouse hearts can fully regenerate after injury up to seven days after birth (3). Our aim was therefore to identify circRNA candidates that was regulated in neonatal mouse hearts compared to non-regenerating hearts. The main rationale was, that modulation of such circRNAs could impact proliferation and regeneration of cardiomyocytes in the postnatal heart after injury. This would allow us to find potential novel treatment options to treat cardiac injury in the future as efficient treatments are currently missing. Using our approach allowed us to identify a novel and highly conserved circRNA, which we termed circREGEN. Methods We performed knockdown studies in vitro and subsequent whole transcriptome analysis. Further, we utilized in vitro transcribed circREGEN for therapeutic overexpression in vitro to stain for cell cycle markers. To understand the role of circREGEN in the context of heart regeneration, myocardial infarction (MI) surgery will be performed by permanent left anterior descending artery ligation in neonatal mice, and lipid nanoparticles encapsulating circREGEN will be delivered to the heart. Finally, cardiac function improvement (ie. Regeneration) and gene regulation of cell cycle genes will be investigated. Thereby we aim to prolong the "regenerative window" to ultimately be able to use our circRNA candidate for therapeutic application also in the adult heart. Results Whole transcriptome analysis of circREGEN knockdown in human iPSC-derived cardiomyocytes (hiPSC-CMs) revealed a downregulation of proliferation and regeneration markers, while apoptosis genes and cell cycle inhibitors were upregulated, indicating an anti-proliferative phenotype when circREGEN is abscent. Further, we confirmed pro-proliferative effects of circREGEN in vitro in neonatal mouse cardiomyocytes and in hiPSC-CMs. Next, we will study the role of circREGEN in vivo with an MI model in neonatal mice. Conclusion CircREGEN is a circular RNA present in neonatal and therefore proliferating mouse hearts. The fact that knockdown of this candidate leads to cell cycle impairment, and overexpression using in vitro transcribed circREGEN shows increased proliferation in hiPSC-CMs, makes this a promising candidate for therapeutic application after heart injury in the future.
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