Regulation of circular RNAs in patients with cardiogenic shock

Abstract Background The mortality rate of cardiogenic shock (CS) is around 50%. There is a need for biomarkers to predict the prognosis of CS. Due to their shape, circular RNAs (circRNAs) exhibit higher stability than other classes of RNA. Regulation of circRNAs has been observed in cardiovascular diseases such as myocardial infarction, but their regulation in CS is largely unknown. Monocytes can be classified into different populations based on their surface markers. Non-classical and intermediate monocytes are characterized by CD16 expression and have been associated with non-inflammatory function such as vascular healing. Methods and Results Blood samples were taken from patients with CS after myocardial infarction (MI) (n=4) on admission and 24 hours after revascularization and from healthy controls. Bioinformatic analysis by RNA-Sequencing revealed the expression of a total of 1819 circRNAs in the blood. 457 circRNAs showed altered expression in CS at the time of admission compared to controls, with 35 circRNAs upregulated and 422 circRNAs downregulated. Of these 457 circRNAs, 80 circRNAs were no longer regulated 24 hours after revascularization. This indicates dynamic regulation of circRNA during CS. Using divergently oriented primers spanning the backsplice junction of selected circRNA candidates, the presence of 17 of the most highly regulated or expressed circRNAs was confirmed by RT-PCR in peripheral blood mononuclear cells. We confirmed the circularity of the 17 circRNA candidates by RNAse-R digestion and oligo-dT selection. For one of the top candidates, circRNA-4, we confirmed upregulation in a larger group of patients with CS after MI (n=52) compared to healthy controls (n=38, 10.9-fold, p < 0.0001). Interestingly, patients with MI without CS (n=37) showed no increase in circRNA-4 compared to healthy controls (n=31, p=0.2), suggesting a potential CS-specific regulation of circRNA-4 independent of myocardial infarction. Single cell sequencing of CS patients (N=4) revealed an increase in CD16+ monocytes and enriched expression of the circRNA-4 host gene. Interestingly, knockdown of circRNA-4 in human monocytes using siRNA targeting the circRNA-4-specific backsplice site resulted in a reduction of the non-classical monocyte marker CD16, suggesting a role for circRNA-4 in monocyte subpopulation switching. Conclusion In conclusion, the data reveal a dynamic and significant regulation of circRNAs in the blood of patients with CS after MI. circRNA-4 showed a CS-specific upregulation that was not primarily dependent on MI. After knockdown of circRNA-4, a reduced expression of the surface marker CD16 was also shown in monocytes indicating the involvement in monocyte subpopulation switch toward non-classical monocytes. Thus, these data set the stage to further study circRNAs as potential new specific markers in acute diseases such as cardiogenic shock.