A large-scale functional high-throughput screening identifies miR-515 and miR-519e as potent inducers of human iPSC-cardiomyocyte proliferation

Abstract

Abstract Introduction Ischemic heart failure persists as a global health problem despite optimized medical and adjunctive device therapies. Loss of cardiomyocytes in the absence of a proliferative response comprise a major contributor to pathological remodeling and death in this patient population. Experimental studies have shown that microRNAs (miRNAs) may be used as a therapeutic option to reinduce adult cardiomyocyte proliferation. Purpose This study thought to evaluate proliferative potential in human cardiomyocytes after overexpression and inhibition of 2019 miRNAs. Methods To identify miRNAs that regulate cardiomyocyte proliferation, we performed functional high-throughput screenings in human iPSC-derived cardiomyocytes (hiPSC-CM) after transient hypoxia. Herein, 2019 miRNA-mimics for overexpression and 2019 anti-miRs for inhibition were individually transfected to examine EdU-incorporation in hiPSC-CM. MiR-mimic-515 and miR-mimic-519e that induced the highest EdU-uptake, were further assessed by immunostaining and molecular methods for markers indicative of early and late mitosis. In addition, RNA-Sequencing in hiPSC-CM after overexpression of miR-515 and miR-519e was performed to examine differential gene expression and miRNA-modulated pathways involved in cardiomyocyte proliferation. Results Using a functional high-throughput screening, we assessed differential proliferative potential of 2019 miRNAs after transient hypoxia by transfecting both miR-inhibitor and miR-mimic libraries in human iPSC-derived cardiomyocytes (hiPSC-CM). Overexpression of 28 miRNAs substantially induced proliferative activity in hiPSC-CM, with an overrepresentation of miRNAs belonging to the C19MC-cluster and adjacent miR-371–373 family. Two of these miRNAs, miR-515 and miR-519e increased markers of early and late mitosis, with an additive cardiomyocyte turnover after transient hypoxia and substantially increased Aurora B-kinase activity in midbodies, indicative of cell division. These findings were supported by molecular studies using qRT-PCR, Western blot, and RNA-Sequencing after overexpression of miR-515 and miR-519e showing substantial alterations of signaling pathways relevant for cardiomyocytes proliferation in human iPSC-CM. Conclusion Collectively, these results support a critical role of miR-515 and miR-519e for induction of proliferation in human cardiomyocytes under hypoxic conditions, such as present in patients with ischemia-driven cardiomyopathy. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): This work was supported by the German Centre for Cardiovascular Research (DZHK), Deutsche Stiftung für Herzforschung (DSHF) and OPO Foundation.