High-throughput single cardiomyocyte transcriptomics identify novel gene programs underlying the normal developmental process of terminal cardiomyocyte differentiation

It is generally accepted that G2M cell cycle arrest occurs around birth (P3 in mice) as a natural process of cardiomyocyte (CM) terminal differentiation resulting in binucleation/polyploidization that renders the mammalian heart non-regenerative after damage. Mechanisms underlying this postnatal switch in CM proliferation remain unclear. Herein, we developed a novel approach combining fluorescence-activated cell sorting (FACS) with single cell RNA sequencing (scRNA-seq) of fixed CMs and specifically identified transcription factors (TFs) involved in G2M cell cycle completion of mouse CMs. Samples for scRNA-seq were collected at E16.5, P1, and P5 by dissociation of the left ventricle of the heart whereafter fixed cardiomyocytes were selectively enriched and divided into diploid and tetraploid fractions using FACS of fixed cardiac cells.