A species conserved circular RNA influences cardiac regeneration through cardiomyocyte proliferation, regulation of oxidative stress, and TGFβ induced wound healing

Sarah Cushman (Hannover)1, D. Lu (Hannover)1, A. Schmidt (Hannover)1, K. Schmidt (Hannover)1, A.-K. Pralas (Hannover)1, S. K. Schröder (Hannover)1, C.-K. Huang (Hannover)1, N. Abbas (Hannover)1, I. Riedel (Hannover)1, K. Xiao (Hannover)1, T. Thum (Hannover)1, C. Bär (Hannover)1

1Medizinische Hochschule Hannover Institut für Molekulare und Translationale Therapiestrategien, OE-8886 Hannover, Deutschland


A class of non-coding RNAs, circular RNAs (circRNAs), which form stable, covalently closed loop structures through backsplicing, are emerging as important regulators of cardiac development and cardiovascular diseases. CircRNAs are known to regulate gene expression and influence key factors involved in regeneration; such as cardiomyocyte (CM) differentiation and proliferation. Predominant circRNAs involved in cardiac development and potential regeneration can be identified 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.
RNA-seq was performed in mouse hearts at day 1 and 7, and the circRNA, circREGEN, showed an increased expression at day 1 both in RNA-seq analysis and in neonatal mouse tissue samples. CircREGEN showed high expression levels in the heart, particularly in CMs, and was found 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, cardiac organoids, and human induced pluripotent stem cell (hiPSC)-derived CMs, were subjected to hypoxia (simulating the low-oxygen, uterine environment) and a consistent increase in circREGEN expression was observed. As the oxygenated state is known to influence cardiac regenerative potential, we further investigated whether CMs could also be induced to renter the cell cycle with increased levels of circREGEN.
CircREGEN was functionally validated using both knockdown and overexpression techniques in several cardiac cell lines. These data showed a decrease in proliferation after circREGEN knockdown. Overexpression showed an increase in CM proliferation, as well as reduced ROS levels under basal conditions and after H2O2 challenge. TGFβ stimulation allowed for minimal activation of fibrosis markers for potential wound healing effects in cardiac fibroblasts upon circREGEN overexpression. Together, these loss- and gain-of-function experiments demonstrate potential protective and regenerative effects of circREGEN.
To determine a potential mode of action for circREGEN after the localization was found to be restricted to the cytoplasm, RNA immunoprecipitation was performed. CircREGEN interacted with an RNA binding protein (RBP) circREGEN-IP1 and protein expression was additionally increased upon overexpression of circREGEN.
In addition, circREGEN expression was examined in adult mouse myocardial tissue after a myocardial infarction (MI), and a downregulation was observed. Decreased expression in heart failure tissue highlights the need for circREGEN overexpression as a therapeutic strategy.
Further studies into the exact interaction between this RBP and circREGEN are still warranted, however, this RBP is already known to regulate the cell cycle through non-coding RNA sponging which will be further explored for this candidate. CircREGEN overexpression will also be induced in an in vivo MI model to determine if the pro-proliferative effects and TGFβ induced wound healing that were observed in the in vitro setting could be replicated after cardiac injury.
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