Abstract 1839: MicroRNA-499 Promotes the Differentiation of Cardiac Progenitor Cells into Myocytes
Myocardial regeneration mediated by cardiac progenitor cells (CPCs) results in the partial recovery of the infarcted heart but the newly formed myocytes within the necrotic tissue have fetal-neonatal characteristics. In contrast, CPC activation in the remote viable myocardium results in the formation of mature myocytes, suggesting that CPC differentiation is conditioned by the surrounding cells. Thus, the hypothesis is raised that microRNAs (miRs) that are highly expressed in myocytes and are absent in CPCs, may translocate through gap junctions to adjacent CPCs promoting their differentiation. By employing miR array and Q-RT-PCR, miR-499 was found to be ~500-fold more expressed in myocytes than CPCs. Additionally, we demonstrated that miR-499 translocates from neighboring cells to CPCs through the formation of gap junctions. The translocated miR-499 was functional and repressed the expression of target genes. Among 200 putative targets of miR-499, we have elected to study Sox6 and Rod1. The validation of these putative miR-499-targets was obtained by reporter assays; cells transfected with miR-499 together with plasmids carrying luciferase and the 3′-UTR region of Sox6 or Rod1 show the expected decrease in luciferase activity. Transcripts of Sox6 and Rod1 were measured by Q-RT-PCR in myocytes and CPCs; Sox6 mRNA was 2-fold higher and Rod1 mRNA was 98% lower in myocytes than CPCs. However, the protein levels of Sox6 and Rod1 were significantly lower in myocytes than CPCs suggesting that miR-499 promotes degradation and/or inhibition of translation of these target genes. To document miR-499 function, CPCs were transfected with a miR-499-expression vector and cell proliferation and differentiation were evaluated 3 days later. BrdU incorporation decreased 60% and the cells displayed a marked upregulation of the myocyte-specific transcription factors Nkx2.5 and MEF2C. Similar results were obtained when Sox6 and Rod1 were selectively blocked with siRNA. In both cases, the number of Nkx2.5- and MEF2C-positive cells increased 2–3-fold. Thus, our data indicate that miR-499 translocates via gap junction from myocytes to CPCs where miR-499 is a crucial modulator of the differentiation of CPCs into cardiomyocytes through the repression of Sox6 and Rod1.