P65Lack of heme oxygenase-1 affects electrophysiological properties of cardiomyocytes derived from human induced pluripotent stem cells

too low to efficiently compensate for the loss of functional myocardium in heart disease.We hypothesize that cardiomyocyte proliferation is tightly controlled by regulatory mechanisms that can be reactivated to promote endogenous repair after heart injuries.Unequivocal identification of cycling cardiomyocytes is a fundamental requirement for the investigation of these mechanisms.Methods: We used a preexisting transgenic mouse model based on the FUCCI (Fluorescent Ubiquitination-based Cell Cycle Indicator) system to discriminate cycling cardiomyocytes (S/G2/M phases, green fluorescent nuclei) from non-cycling ones (G0/G1 phases, red fluorescent nuclei).In mice ubiquitously expressing FUCCI constructs, we used the myocyte-specific perinuclear marker Pericentriolar Material 1 (PCM-1) to distinguish cardiomyocyte from non-myocyte cells in the heart tissue.Single cardiomyocyte isolations from FUCCI mice neonatal hearts followed by FACS sorting into 96-well plates were performed to obtain single cell transcriptome of cycling and non-cycling cardiomyocytes at different post-natal stages.Results: The presented transgenic strategy proved to be efficient for the identification of cycling cardiomyocytes in neonate, juvenile and adult mouse hearts in vitro and in vivo.The validity of transgenes expression was verified in both systems with immunohistochemistry by co-localization of cardiomyocytes and cell cycle markers with FUCCI fluorescence.The FUCCI system allowed us to confirm previous observations of a gradual decrease in the number of cycling cardiomyocyte during the neonatal period.P0 and P7 cardiomyocyte transcriptome were generated using single cell RNA-sequencing.The cell cycle stage of cardiomyocytes as indicated by their FUCCI fluorescence was highly correlated by their transcriptome profile.Conclusion: Strategies that allow to unambiguously identify cycling cardiomyocytes in vitro and in vivo provide important tools to study the regulation of cardiomyocyte proliferation.With the FUCCI system we seek to identify novel molecular targets that could promote cardiomyocyte proliferation, by using transcriptome profiling (RNA-seq) of isolated cycling cardiomyocytes.