Adult ovine cardiomyocytes express the cell cycle-inhibiting gene Meis1. A potential target for cardiac regeneration based on cardiomyocyte division.

LOCATELLI P; GIMENEZ S; OLEA FD; HNATIUK A; CROTTOGINI A; GHIRINGHELLI D; BELAICH M

Buenos Aires

Congreso; ISHR XXII Word Congress; 2016

International Society for Heart research

Introduction: Meis1 is a transcription factor known to regulate adult cardiomyocyte cell cycle. In mice it keeps cell cycle arrest through interactions with cell cycle inhibiting proteins (p15, p16, p19, p21), and its decreased expression is a mitogenic stimulus for postnatal cardiomyocytes. Meis1 inhibition may therefore be a potential means to promote adult cardiomyocytes division and this, in turn, could represent a potential cardiac regenerative therapy. We thus aimed at searching for Meis1 and other cell cycle regulatory genes in adult sheep cardiomyocytes Methods: Primers were designed targeting meis 1, cdkn1a, cdkn2aip, cdk2, cdk4, cdk6, ciclin E1, ciclin D2 and gapdh genes using the information of exon junctions to detect only mRNAs. End point RT-PCRs were performed from adult healthy sheep myocardium RNA. PCR products were recovered and molecularly cloned into a generic plasmid. The plasmids, each containing the target sequences of one gene, were sequenced to confirm that the insert corresponded to the desired fragment. These plasmids were used as calibrators of real-time-PCR with SyBrGreen to obtain efficiency and dynamic range parameters. Results: The genes meis1, cdkn1a, cdkn2aip, cdk2, cdk4, cdk6, ciclin E1 and ciclin D2 were expressed in ovine myocardium. Real time PCR was optimized employing the calibrator plasmids, obtaining a dynamic range of 102?108 for all genes, and adequeate efficiencies for quantitative estimations. The ratio target gene/GAPDH for the analyzed genes was: Meis1: 2.7, cdk4: 2.5, cdk6: 2.8, Ciclin D2: 3.6. Conclusion: Meis 1 is expressed in adult sheep myocardium, thus being a potential target for silencing strategies aimed at fostering adult cardiomyocyte cell cycle reentry and divison.