Spatial transcriptomics for CD36/FABP5/PPARd signaling axis defines spontaneously proliferating cardiomyocytes in neonatal stages

Spontaneously cycling cardiomyocytes (CMs) represent a rare and unique population in the neonatal heart with the potential to drive cardiac regeneration in adulthood. While previous studies have observed their existence, the mechanisms regulating their cell cycle activity remain poorly understood. Here, we aim to identify the signaling axis that defines the spontaneously proliferating CMs in the neonatal stage. Overall design: Using single-cell RNA sequencing of neonatal mice CMs at postnatal day 1 (NMCMs-P1) we identified CD36 as a marker of the spontaneously proliferating CMs and those who respond to the cell cycle induction. Both Cd36 global and CMs-specific knockout mice (CD36KO, CD36CKO) exhibited smaller hearts, fewer CMs, impaired CM proliferation, and limited regenerative capacity at P1 following apical resection. Bulk-RNAseq in CD36KO and CD36CKO showed downregulation of cell cycle genes, as well as Fabp5, Ppard, and Rxrs. Spatial transcriptomics for P1 wild-type (WT) mice hearts identified and localized a subpopulation of CMs with high cell cycle activity and co-expression of Cd36, Fabp5, and Ppard. To define the upstream modulator of this signaling pathway, we conducted spatial metabolomics which revealed a decrease in CD36KO and CD36CKO cardiac intracellular contents of retinyl ester which was further confirmed with quantitative internalization assays. At Nano molar concentrations, retinoic acid (RA) enhanced the cell cycle activity of WT-P1 NMCMs but not the CD36KO. Knockdown of Fabp5 abolished the RA effect on CM proliferation, while its overexpression enhanced the effect of RA. Crossbreeding CD36KO mice with Ppard-cardiac specific overexpressing mice rescued the proliferative phenotype, restoring CM proliferation and regenerative capacity after apical resection in P1 to the WT levels. Furthermore, Ppard overexpression in neonatal mice resulted in enhanced CM proliferation and improved regenerative capacity following apical resection.