Identification and characterization of distinct cell cycle stages in cardiomyocytes using the FUCCI transgenic system

Abstract

Understanding the regulatory mechanism by which cardiomyocyte proliferation transitions to endoreplication and cell cycle arrest during the neonatal period is crucial for identifying proproliferative factors and developing regenerative therapies. We used a transgenic mouse model based on the fluorescent ubiquitination-based cell cycle indicator (FUCCI) system to isolate and characterize cycling cardiomyocytes at different cell cycle stages at a single-cell resolution. Single-cell transcriptome analysis of cycling and noncycling cardiomyocytes was performed at postnatal days 0 (P0) and 7 (P7). The FUCCI system proved to be efficient for the identification of cycling cardiomyocytes with the highest mitotic activity at birth, followed by a gradual decline in the number of cycling and mitotic cardiomyocytes during the neonatal period. Cardiomyocytes showed premature cell cycle exit at G1/S shortly after birth and delayed G1/S progression during endoreplication at P7. Single-cell RNA-seq confirmed previously described signaling pathways involved in cardiomyocyte proliferation (Erbb2 and Hippo/YAP), cardiomyocyte motility, and maturation-related transcriptional changes during postnatal development, including the metabolic switch from glycolysis to fatty acid oxidation in cardiomyocytes. Additionally, we generated transcriptional profiles specific to cell division and endoreplication in cardiomyocytes. Deciphering transcriptional changes at different developmental stages and in a cell cycle-specific manner may facilitate the identification of genes important for adult cardiomyocyte proliferation and heart regeneration. Main findingsFUCCI reliably identifies cycling cardiomyocytes at distinct cell cycle stages in neonatal, juvenile, and adult hearts. Cell cycle activity decreases as the metabolic switch transitions from glycolysis to fatty acid metabolism in postnatal cardiomyocytes. Cell cycle arrest at G1/S is linked to the DNA damage response in postnatal cardiomyocytes. Distinct gene expression patterns are linked to different cell cycle phases in dividing and endoreplicating postnatal cardiomyocytes.