Introduction:
Cardiovascular diseases are the leading cause of death worldwide however, there is increasing evidence that a subtype of non-coding RNA, circular RNAs (circRNAs), formed through backsplicing, are emerging as important regulators of cardiac development and cardiovascular diseases. CircRNAs are known to regulate gene expression through a multitude of functions such as; acting as miRNA sponges, transcription enhancers, interacting with RNA binding proteins, or interfering with the expression of the linear mRNA host. Prevalent circRNAs involved in cardiac development and potential regeneration can be determined by focusing on their differential expression in regeneration competent versus non-competent neonatal mouse hearts.

Methods and Results:
RNA-seq was performed to determine the expression levels of circRNA candidates in day 1 and day 7 mouse hearts. Several candidates underwent further tests for circRNA validation until a potential candidate for regeneration and proliferation, circREGEN, was found to be downregulated on day 7 both in RNA-seq analysis as well as in neonatal mouse tissue samples using RT-PCR. Tissue distribution was measured for circREGEN, showing high expression levels in the heart, specifically, neonatal cardiomyocytes. Importantly, the function of circRNAs can be partially determined by their localization. Through subcellular fractionation as well as CRISPR/dCas13 live cell tracking studies, we showed that circREGEN is localized in the cytoplasm.

Primarily in neonatal mammal hearts, cardiomyocytes (CMs) are highly proliferative but quickly withdraw from the cell-cycle leaving the heart without significant regenerative capacity. This withdrawal correlates with the transition from the hypoxic uterine to normoxic postnatal environment. Therefore, we subjected HL-1 cells to hypoxia and observed an increase in circREGEN expression. This data was confirmed in neonatal mouse and rat CMs, neonatal living myocardial tissue, and human induced pluripotent stem cell (iPSC)-derived CMs, showing a potential for not only sequence conservation, but for circREGEN to be functionally conserved across species as well.

Further experiments were performed to functionally validate circREGEN using siRNAs and overexpression plasmids in neonatal mouse and rat CMs.

It is important to note, that hypoxia studies are an applicable in vitro assay by which to simulate an infarct. Therefore, we decided to further examine the expression of circREGEN in living myocardial tissue, an ex-vivo model, after cryoinjury and saw an up-regulation.

Conclusion:
In conclusion, we have promising in vitro data, which warrants further functional investigation both in vitro and in vivo, with the ultimate goal to determine if circREGEN has therapeutic potential to regenerate damaged heart tissue after a myocardial infarct by stimulating the proliferation of CMs in the adult heart.