Inhibition of circALPK2 enhances proliferation and therapeutic potential of human pluripotent stem cell-derived cardiomyocytes in myocardial infarction

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 < 0.001) and EdU staining (p < 0.001), 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 3 beta (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.