Background – The SARS-CoV-2 pandemic has emerged as a dangerous infectious disease that most commonly acts as a respiratory illness. Additional effects on other organ-systems such as the myocardium are often neglected. Clinical studies have shown that an infection with SARS-CoV-2 can lead to cardiac inflammation and detrimental cardiac remodelling. ACE2 and TMPRSS2 are important endogenous entry routes for the virus. Expression profiles of human heart failure confirmed an upregulation of ACE2 and TMPRSS2. In silico analysis identified a potential non-coding RNA-based (miRNA) regulatory mechanism. We identified miRNA-362-5p to have a potential dual regulatory role and subsequently tested miRNA modulation in various in vitro and ex vivo model systems based on myocardial slices in a high-throughput configuration.

Methods and Results – Data mining for ACE2 and TMPRSS2 expression in human heart samples (healthy vs. diseased) was conducted on the basis of RNA-Seq datasets. A multi-modal in silico analysis of human ACE2 and TMPRSS2 3´-UTR identified miRNA-362-5p as a potential inhibitor of ACE2/TMPRSS2 expression. We validated this finding in vitro via miRNA reporter gene assay. Since miRNAs are known to regulate many different mRNAs, further in silico analysis additionally predicted the following miRNA-dependent pathways VEFGA-VEFGR2, VEGF and mTOR. We then transferred the miRNA experiments ex vivo into cultured living myocardial slices (LMS). In brief, fresh rat and human hearts were cut and trimmed into 300 μm thick LMS and then cultured for 24h with and without miR-362-5p overexpression (different doses of 3 nM and 30 nM). qPCR-based miRNA detection revealed successful overexpression of miRNA-362-5p in rat and human LMS. To confirm the intracellular effect of miR-362-5p mimics in the LMS, we chose to investigate the expression of the miRNA processing enzyme DICER to see a possible reaction due to higher miRNA concentration in the cells. In qPCR, we could see an upregulation of DICER in human LMS indicating the effectiveness of miRNA-362-5p as a translational modulator.

Conclusion – In summary, we report the in silico prediction and in vitro validation of miR-362-5p as a potent post-transcriptional regulator of ACE2 and TMPRSS2. We herein highlight this combination of in silico analytics and ex vivo cardiac modelling as a possible high-throughput testing platform for miR-dependent therapeutic approaches. Given the potential of cardio-inflammatory side effects caused by SARS-CoV-2 infection, we established a miRNA-based model-system in ex vivo LMS. These findings may lead to the development of novel RNA-based therapeutics counteracting SARS-CoV-2 viral entry.