Defining the major signaling mechanism which controls spontaneous cardiomyocyte proliferation in the Neonatal Stage - Project Summary Spontaneous cardiomyocyte (CM) regeneration has been demonstrated in embryonic and neonatal mammals; however, adult CMs go into cell cycle arrest as their turnover has been reported as minimal in human hearts and rodents. Several recent discoveries to induce CM proliferation in adult CMs, such as Cyclin A2, mir199, YAP- 5SA, or the combination of CDK1, CDK4, cyclin B1, and cyclin D1 (collectively known as 4F), have provided new mechanistic insights of our understanding of the cell cycle block in adult CMs. However, a pertinent question is what is the major signaling pathway that dictates CMs to spontaneously proliferate in fetal/neonatal stages and be abolished in adulthood. Our recently published temporal single-cell RNA-seq data from 60-day mature hiPS-CMs indicates the presence of a unique subpopulation of primed starting CMs that respond to the 4F. One of the major characteristics of this primed population is the expression of the transmembrane scavenger receptor; CD36. CD36 is a fatty acid internalization receptor with recent indications of regulating several signaling pathways. Our single-cell RNAseq from primary CMs isolated from P1 hearts demonstrated that CD36 is expressed only in the spontaneously proliferating CMs population and the responders to the 4F cell cycle stimulation. Our preliminary data show that loss of CD36 global and CM-specific knockouts are born with smaller hearts, which contain fewer CMs compared to their WT littermates. Furthermore, compared to their WT littermates that completely regenerate the heart apex following apical resection at P1, CD36KO showed minimal regeneration of the apex. Mechanistically, bulk RNAseq data from the P1 CD36KO hearts showed significant downregulation in the expression of the cell cycle genes and the retinoic acid (RA)-cell cycle induction signaling genes, such as FABP5, PPARd, and RXR that coordinately play a critical role in cell decision between proliferation and growth. Spatial metabolomics analysis demonstrated a decline in retinoic acid esters within the CD36CKO hearts, which indicates a deficiency in retinoic acid internalization. To confirm the causality of this proposed mechanism, in vivo, crossing CD36KO with CM-specific PPARd overexpression (PPARdCTG) only in CMs rescued the proliferation deficiency phenotype in CD36KO in the P1 hearts. We hypothesize that CD36 modulates RA signaling through esterification and internalization of the RA to bind to FABP5 to activate the PPARd/RXR transcriptional program, thereby regulating the decision of CMs to progress through the cell cycle when needed. To test this hypothesis, we will address these aims: Aim 1: To address the hypothesis that CD36 internalizes RA to bind to FABP5 to initiate the signaling mechanism. Aim 2: To test the hypothesis that RA/FABP5 binding is the rate-limiting step in the proposed pathway to activate the PPARd/RXR transcriptional activity. Aim 3: To test the hypothesis that FABP5/PPARd overexpression is a therapeutic target for ischemic heart failure.
