BACKGROUND: Understanding the mechanisms regulating human cardiomyocyte proliferation holds significant promise for developing effective therapies to enhance cardiac regeneration and repair. This study investigates the role of circALPK2, a circular RNA derived from the back-splicing of the 4th exon of alpha protein kinase 2 (ALPK2), in regulating cardiomyocyte proliferation and its therapeutic efficacy in myocardial infarction (MI) treatment. METHODS: Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) were used to assess the expression and function ofcircALPK2. Lentiviral shRNA-mediated knockdown of circALPK2 was performed in hESC-CMs, followed by RNA sequencing to identify targeted genes and biological processes. The proliferative capacity of wild-type and circALPK2 knockdown hESC-CMs was evaluated using CCK-8 assay, EdU staining and RT-qPCR analysis of cell cycle-related genes. Dual luciferase assays were conducted to validate the predicted miRNA targets and their downstream effects. For in vivo evaluation, MI mice were injected with either wild-type or circALPK2 knockdown hESC-CMs, and the therapeutic potential was assessed by echocardiographic and histological analyses. RESULTS: We identified circALPK2 as a negative regulator of cell proliferation in hESC-CMs. CircALPK2 was abundantly expressed in hESC-CMs. Knockdown of circALPK2 significantly enhanced cell proliferation in hESC-CMs, as demonstrated by CCK-8 assays (p <
0.002) and EdU staining (p <
0.002), and accelerated the expression of cell cycle-related genes, including CCNA2(p <
0.05) and CDK1 (p <
0.01). Furthermore, circALPK2 was found to function as a sponge to inhibit miR-9 activity, while miR-9 mimics significantly boosted the proliferative capacity of hESC-CMs. Glycogen synthase kinase 3β (GSK3B), a key inhibitor of WNT signaling, was identified as a direct target of miR-9, mediating the regulation of cardiomyocyte proliferation. Importantly, circALPK2 knockdown improved the myocardial repair potential of hESC-CMs when injected into infarcted mouse hearts, as indicated by improved left ventricular ejection fraction (p <
0.01) and fractional shortening (p <
0.05). CONCLUSIONS: Our study identifies the circALPK2/miR-9/GSK3B axis as a novel target for promoting cardiomyocyte proliferation and enhancing cardiac regeneration.