Abstract 17836: High Throughput Functional Screening Identifies microRNAs Inducing Cardiac Regeneration
Whereas enlargement of the heart during mammalian development relies on cardiomyocyte proliferation, at birth cardiomyocytes stop dividing and further growth occurs through hypertrophy. As a consequence, repair of cardiac damage through myocardial regeneration is limited.
By high-content, fluorescence microscopy-based high-throughput screening in rat neonatal cardiomyocytes using a library of microRNA mimics corresponding to all the annotated microRNAs (~1000 microRNAs), we have recently identified 40 microRNAs able to increase cardiomyocyte proliferation, as evaluated by analyzing EdU incorporation (DNA synthesis), G2/M phase of the cell cycle (phospho-H3 positivity) and karyokinesis (Aurora B staining in midbodies). Deep sequencing of endogenous microRNAs revealed that several of the identified microRNAs were expressed in neonatal, replicating cardiomyocytes but not in adult cardiomyocytes. Two of these microRNAs were tested in vivo by injecting either the synthetic microRNA or the corresponding AAV9 vector intracardially (n=6 per group) into neonatal hearts. Both microRNAs induced marked proliferation of cardiomyocytes (EdU+ cardiomyocytes 0.1±0.009% in control, 2±0.5 and 3±0.8% in miRNA-reated hearts). The same microRNAs, administered after myocardial infarction in adult mice (n=8 per group), were also effective in reducing infarct areas and improving cardiac function (EF at 60 days: 38±8% in controls, 54±6 and 59±9% in miRNA-treated hearts).
In vivo experiments using transgenic animals, genetically labelled to overexpress the GFP gene selectively in adult, MHC+ cardiomyocytes, indicate that the selected miRNAs directly act on differentiated cardiac cells. Indeed, their administration to adult rat cardiomyocytes triggers their proliferation up to 2 weeks (EdU+ cells: from 0% in control cells up to 20±1% in the miRNA-treated cells). The selected microRNAs target the mRNAs for several cytoskeletal proteins; accordingly, siRNA-mediated silencing of these proteins, individually or in combination, mimicksthe proliferative effect of miRNAs.
In conclusion, it is possible to achieve adult cardiomyocyte proliferation in vivo through the delivery of selected miRNAs. These results have obvious therapeutic implications.
- © 2013 by American Heart Association, Inc.