Abstract 10947: Highly Enhanced Cardiac Reprogramming from Fibroblasts by MicroRNA
Background Cardiomyocytes are terminally differentiated cells and their regenerative capacity is limited. We recently found that functional cardiomyocytes can be directly generated (reprogrammed) from fibroblasts by transduction of three cardiac transcription factors, Gata4, Mef2c and Tbx5 (GMT). MicroRNAs (miRs) are highly conserved small RNAs that control cell fate including cardiogenesis by suppressing specific mRNAs post-transcriptionally. This study was designed to investigate whether miRs promote direct cardiac reprogramming.
Methods and Results MicroRNA microarray analyses were performed to identify cardiac-enriched miRs (cardiac miRs). Six miRs were highly expressed in cardiomyocytes than fibroblasts by more than 10-fold. To determine if miRs induce cardiac reprogramming, we used mouse embryonic and neonatal tail-tip fibroblasts (MEF and TTF) from α-myosin heavy chain (αMHC)-GFP transgenic mouse in which only cardiomyocytes express GFP. We first transfected cardiac miRs alone into αMHC-GFP MEF and TTF, but GFP or cardiac troponinT (cTnT) was not induced, suggesting cardiac miRs alone are not sufficient for cardiac reprogramming in MEF and TTF. Next, we transduced cardiac miRs plus GMT into MEF and TTF. Compared with control miR, a pool of cardiac miRs greatly enhanced generation of αMHC-GFP+ cells. By screening of cardiac miRs, we identified one cardiac miR (mirX) is sufficient for increasing cardiac induction. Addition of mirX increased generation of cTnT+ cells by 5 folds than control miR. Whole transcriptome analyses demonstrated that mirX plus GMT changed global gene expression pattern of αMHC-GFP+ cells toward a cardiomyocyte-like state after 1 week of transduction. Moreover, spontaneous cellular contractions began after 10 days of mirX and GMT introduction, which generally took 4 weeks in GMT transduction. MirX addition increased the number of spontaneously contracting cardiomyocyte-like cells nearly 10-fold. Molecularly, we also found that overexpression of gene Y, a target of mirX, suppressed cardiac induction. Conclusions These results indicate that microRNAs and transcription factors can cooperate in a powerful way to reprogram non-cardiac fibroblasts into functional cardiomyocytes.
- © 2012 by American Heart Association, Inc.