Investigating circular RNA as therapeutic targets in ischemia-reperfusion injury

Ischemic heart disease, including myocardial infarction (MI), is the leading cause of death worldwide. Current treatment options for completely obstructed arteries leading to a MI include reperfusion therapy. However, while reperfusion is occasionally required dependent on the severity of the blockage, there are disadvantageous side-effects resulting from sudden re-oxygenation, known as ischemia-reperfusion injury (IRI) resulting in exacerbated cell and tissue damage in the infarcted area of the heart. 

Circular RNAs (circRNAs) are demonstrating increasing potential as therapeutic targets in cardiovascular diseases (CVDs). This class of non-coding RNAs (ncRNAs) mainly derive from pre-mRNA by back-splicing, forming a RNA loop that lacks free 3’ and 5’ ends and that is resistant against exonucleolytic RNase degradation. CircRNAs display a strong cell- and tissue specificity with a wide conservation across species. Several studies indicate that circRNAs can regulate gene expression through their interactions with microRNAs (miRNAs) or regulatory proteins. Importantly, it was found that circRNA expression is (dys)regulated in pathologies, including CVDs, and consequently exhibit a promising therapeutic potential.

An in vitro-model of IRI was established in human induced pluripotent stem cell derived-cardiomyocytes (hiPSC-CMs) and in neonatal mouse cardiomyocytes (NMCMs) to identify and further test circRNA as putative disease target candidates. Multiple cellular-based assays were employed in this model to illustrate the phenotypic alterations characteristic of reperfused cardiac tissue injury including; increased levels of ROS generation and cell death.  

Previously identified therapeutic circRNAs in other CVDs were then tested in this in vitro IRI model. Here, we first used circINSR, derived from the insulin receptor locus, which we found to therapeutically  inhibit apoptosis when overexpressed, in a doxorubicin-induced cardiotoxicity model. Hence, we tested, if circINSR overexpression has similar cardioprotective effects after reperfusion injury.  Initial data, first in the in vitro model, demonstrated that circINSR overexpression results in decreased number of apoptotic cells and retained sarcomeric structure in hiPSC-CMs. 

Furthermore, an in vivo-model of IRI in mice was established and circINSR was overexpressed via an adeno-associated virus (AAV9). 24h after IRI surgery, overexpression of the circINSR and a decreased infarct size was detected via Triphenyltetrazolium (TTC) staining.  

 

Further in vitro and in vivo studies will be performed to confirm the beneficial effects of circINSR in IRI as a promising therapeutic target for multiple CVDs. In addition, hiPSC-CMs harvested from the established in vitro disease model will be subjected to total RNA sequencing to identify additionally regulated circular RNAs after reperfusion. Promising circRNA candidates will then be subsequently tested and validated in the in vitro model before the most promising targets are functionally verified in the in vivo IRI mouse model.