An Exploratory Study of Kinase Activation Profiles in Hypoxic Human Cardiomyocytes Treated with Protective Extracellular Vesicles

Purpose Myocardial infarction (MI) causes hypoxic cardiomyocyte death, and extracellular vesicles (EVs) offer therapeutic potential. This study aimed to compare kinase activation profiles induced by four human-derived EV types- serum-derived (S-EVs), platelet concentrate-derived (PC-EVs), cardiac stromal cell-derived (CSC-EVs), and bone marrow mesenchymal stromal cell-derived (MSC-EVs)- in hypoxic metabolically matured human iPSC-derived cardiomyocytes (iCMs). Methods Metabolically matured human induced pluripotent stem cell-derived cardiomyocytes (iCMs) were exposed to 12-hour hypoxia ± standardized doses of EVs. Kinase activity was profiled using the PamStation platform, and bioinformatic tools (KRSA, UKA, PTM-SEA, KEA3) identified differentially activated kinases. AKT phosphorylation (Ser473) was measured by ELISA. Results All EVs significantly reduced LDH release versus hypoxia alone (P ≤ 0.0001), with no inter-group differences. Hypoxia globally suppressed kinase activity, while each EV type induced distinct patterns: S-EVs and PC-EVs increased total phosphorylation, whereas CSC-EVs and MSC-EVs further decreased it. Bioinformatics implicated the AKT family in all treatments, but ELISA revealed no change in AKT1/2/3 phosphorylation at Ser473 versus hypoxia controls. Conclusion Despite equivalent cardioprotection, each EV type elicited unique kinase activation profiles, suggesting distinct signaling mechanisms. Kinase activity was not a predictor of protection at the measured time point, highlighting the complexity of EV-mediated pathways.