Abstract: Adult mammalian hearts have limited regenerative capacity, unlike zebrafish and neonatal mouse hearts, which can fully regenerate after cardiac injury. In adults, heart regeneration primarily relies on cardiomyocyte proliferation, but the low proliferative capacity of adult cardiomyocytes hinders heart regeneration after myocardial infarction (MI) and limits our understanding of the regulatory mechanisms involved. Our previous work demonstrated that the small-molecule cocktail 5SM promotes cardiomyocyte proliferation and cardiac regeneration in adult rats post-MI. Based on these findings, we hypothesize that 5SM can help identify proliferating cardiomyocytes and uncover key signaling pathways involved in cardiac regeneration. To investigate the molecular mechanisms of 5SM, we employed single-cell RNA sequencing and spatial transcriptomics. Our results revealed that 5SM treatment significantly increased cardiomyocyte proliferation in the infarct border zone and Wt1+ non-myocytes in the epicardium, indicating a dual effect on both cell populations. Further analysis identified two primary mechanisms through which 5SM promotes cardiomyocyte proliferation. First, 5SM indirectly promoted cardiomyocyte proliferation by activating FGF signaling from Wt1+ non-myocytes to cardiomyocytes, as evidenced by elevated Fgf1 expression in Wt1+ cells and Fgfr1 in cardiomyocytes. Overexpression of Fgf1 in Wt1+ epicardial cells amplified the proliferative effects of 5SM, while Fgfr1 knockdown in cardiomyocytes impaired cardiac recovery. Second, 5SM directly increased Caveolin-1 (Cav1) expression in cardiomyocytes, and Cav1 knockdown inhibited cardiomyocyte proliferation. These two mechanisms are interconnected, as 5SM enhanced the interaction between Cav1 and FGFR1 in cardiomyocytes, activating the ERK and PI3K-AKT signaling pathways to synergistically promote cardiomyocyte proliferation. In conclusion, the 5SM-induced FGF1-FGFR1-CAV1 signaling pathway between Wt1+ cells and cardiomyocytes plays a crucial role in heart regeneration. This study highlights the importance of intercellular signaling in cardiac repair, offering a promising therapeutic strategy for MI and new insights into heart regeneration mechanisms.
