Transcriptional reprogramming of cardiac cells holds great promise for developing novel therapeutic strategies through deep rewiring of cellular behavior. We hypothesized that endogenous activation of Cyclin genes could promote cardiomyocyte proliferation, as previously suggested by classical overexpression studies. To test this, we employed a CRISPR-based activation system using catalytically inactive Cas9 (dCas9) fused to the VPR transactivation domain, targeted via guide RNAs (gRNAs) to the promoter regions of CCNA2, CCNB1, CCND1, and CCND2. This approach was implemented in a mouse model with cardiomyocyte-restricted dCas9-VPR expression and in human iPSC-derived cardiomyocytes (hiPSC-CMs) constitutively expressing dCas9VPR. Up to four gRNAs per gene were screened in HEK293T (human) and Neuro2a (mouse) cells to identify effective target-specific activators. Furthermore, we generated and characterized a novel hiPSC line co-expressing dCas9VPR, endogenously GFP-tagged alpha-actinin-2 and a FUCCI-based cell cycle reporter, enabling simultaneous visualization of cardiomyocyte identity and proliferative activity. Using this system, we found that activation of CCNA2, but not CCNB1, CCND1 or CCND2, significantly enhanced cell cycle re-entry in hiPSC-CMs, as shown by confocal FUCCI imaging. To extend these findings in vivo, we systemically injected postnatal day 1 (P1) mice with adeno-associated virus (AAV) vectors encoding FUCCI and CCNA2-specific gRNAs. At P5, within the cardiac regenerative window, CCNA2 expression was upregulated, accompanied by an increased proportion of G1-phase cardiomyocytes compared to non-targeting controls, which was not the case for P30 hearts (outside of the regenerative window). However, few cells progressed into S/G2 phases in P5 hearts, suggesting intrinsic mechanisms limiting full cell cycle progression in vivo. Proteomic analysis of P5 hearts revealed that CCNA2 activation led to sustained metabolic remodeling toward glycolysis, indicating a link between CCNA2-driven G1 re-entry and cardiomyocyte metabolic state. In summary, our study integrates CRISPR/dCas9-based endogenous gene activation with advanced cell cycle imaging to reveal a previously unrecognized role of CCNA2 in promoting cardiomyocyte G1 re-entry and metabolic rewiring. These findings suggest that additional regulatory factors are required to achieve a full cardiomyocyte proliferation capacity postnatally.
