8Identification of cardiomyocyte-specific lncRNAs as potential regulators of cardiac regeneration

Funding Acknowledgements: Sigrid Juselius Foundation, Tekes (project no 40395/13), The Finnish Foundation for Cardiovascular Research, Academy of Finland Introduction: During a myocardial infarction nearly one billion cardiomyocytes (CMs) die. In the neonatal mammalian hearts the lost CMs are renewed after injury, but this regenerative capacity is lost within seven days after birth, and the adult mammalian heart is unable to regenerate. Several signaling pathways and factors have been identified as regulators of CM cell cycle activity and proliferation. Nevertheless, the understanding of the factors and genetic programs, including long non-coding RNAs (lncRNAs), regulating CM proliferation is limited. Purpose: The purpose of this work is to identify lncRNAs that regulate CM proliferation and could therefore be targeted for induction of CM proliferation and cardiac regeneration. Methods: RNA sequencing (RNAseq) was performed in cardiac tissue samples obtained from 1-, 4-, 9- and 23-day old mice (P1, P4, P9 and P23, respectively) and differential expression of lncRNAs was calculated with DESeq2 software. Cardiac enrichment was evaluated using Gene database by NCBI and Expression Atlas by EMBL-EBI. Ten cardiac-enriched lncRNAs with significant expression changes within the early postnatal period were chosen for validation with quantitative reverse transcription PCR (qRT-PCR). The relative lncRNA expression was calculated with the ΔΔCt method using Rn18s and Actb as the reference genes. Statistical significance was determined using one-way ANOVA and a Tukey HSD. To identify cardiomyocyte-specific lncRNAs, neonatal mouse CMs and non-CMs were isolated and purified using magnetic-activated cell sorting and lncRNA expression was analyzed using qRT-PCR.