Abstract 14233: Checkpoint Kinase 1 Combines PKM2 C-domain to Stimulate Endogenous Cardiomyocyte Renewal and Cardiac Repair by Activating Cardiac Metabolic Reprogramming in a Porcine Model of Myocardial Ischemia/Reperfusion Injury

Abstract

Background: The regenerative capacity of adult mammalian hearts is limited. We previously reported CHK1 in adult mice could promote cardiac regeneration after MI. However, the specific mechanism of CHK1 needs more comprehensive and in-depth interpretation. Methods: I/R injury was induced in pigs, recombinant human checkpoint kinase 1 (rhCHK1) protein encapsulated in hydrogel was injected into the infarct border zone. The therapeutic efficacy of rhCHK1 was evaluated by echocardiography, cMRI, hematological indices and immunofluorescence. Multi-omics analysis were performed to explore the potential mechanisms of rhCHK1. Further, protein interaction prediction and CO-IP experiments were conducted to verify the specific interaction pattern and domains between CHK1 and PKM2. Hipsc-CMs and porcine were used for confirming whether rhCHK1 promotes cardiac regeneration through combining with the PKM2 C-domain. Results: RhCHK1 protein stimulated CMs proliferation and reduced cardiac inflammation at 3 dpi, improved cardiac function and attenuated ventricular remodeling, and reduced the infarct area at 28 dpi. Mechanistically, multiomics analysis showed that glycolysis and mTOR pathways were significantly enriched in this process. Further CO-IP experiments and protein docking prediction showed that CHK1 directly bound to and activated the S37 and Y105 sites of PKM2 to promote metabolic reprogramming. We further constructed plasmids that knock out different CHK1 and PKM2 domains, and transfected them into H293T cells for CO-IP experiments. It was found that the 391-476 domain of CHK1 directly binds to the 157-400 amino acids of PKM2. Further hipsc-CMs and pig experiments both demonstrated that CHK1 stimulates CMs renewal and cardiac repair by activating PKM2 C-domain-mediated cardiac metabolic reprogramming and mTORC1 pathway. Conclusion: This study demonstrates that rhCHK1 could combine with PKM2 391-476 amino acid domain to mediate metabolic reprogramming and mTORC1 pathways to promote CM proliferation and myocardial repair. This study is the first to clarify the specific functional mechanism of CHK1 in pigs. Our results underscore the potential of the local applied rhCHK1 as a safe and simple feasible treatment option for repairing heart.