Introduction:

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. Prevalent circRNAs involved in cardiac development and potential regeneration can be determined by focusing on their differential expression in regenerative neonatal versus postnatal mouse hearts. We identified a circRNA that was both highly expressed in neonatal hearts and under hypoxic conditions (simulating the uterine environment), and hypothesized a high potential for regeneration.

Methods and Results:

RNA-seq was performed in day 1 and 7 mouse hearts and the circRNA, circREGEN, showed an increased expression on day 1 both in RNA-seq analysis and in neonatal mouse tissue samples. CircREGEN showed high expression levels in the heart, specifically, in cardiomyocytes (CMs), and was seen to be highly conserved between species. Due to the fact that postnatal CMs lose their ability to regenerate by day 5, a circRNA candidate that was highly expressed in 1-3 day old neonatal cardiomyocytes, was determined to be a potential target for proliferation and regeneration.

HL-1 cells, H9c2 cells, primary mouse and rat CMs, neonatal rat living myocardial tissue, and human induced pluripotent stem cell (iPSC)-derived CMs, were subjected to hypoxia (simulating this low oxygen, uterine environment) and a consistent increase in circREGEN expression was observed. This data suggested that in addition to having high sequence conservation, circREGEN is functionally conserved across species as well.

Further experiments were performed in multiple cell types to functionally validate circREGEN using knockdown and overexpression systems. This data showed a decrease in proliferation and increase in caspase activity after circREGEN knockdown, in addition to a reduction in ROS and apoptosis after overexpression. These loss- and gain-of-function experiments together show potential protective and regenerative effects of circREGEN.

Additionally, the expression of circREGEN was examined in adult mouse myocardial tissue after a myocardial infarct (MI), and a downregulation was observed. Decreased expression in heart failure tissue highlights the need for circREGEN overexpression as a therapeutic strategy.

Conclusion: