Human Cardiac Fibroblasts Derived Exosomes Promote Human Induced Pluripotent Stem Cell Derived Cardiomyocytes Maturation Via AMPK Pathway

Objective: To investigate whether human cardiac fibroblast-derived exosomes can promote the maturation of human induced pluripotent stem cell-derived cardiomyocytes(hi PSC-CMs),as well as to investigate the molecular mechanisms of maturation,shorten the maturation time of hi PSC-CMs,and improve cardiac function in mice after hi PSC-CMs treatment for Myocardial Infarction.Methods: Exosomes were extracted from human cardiac fibroblasts(h CFBs)which were cultured with exosome-free FBS medium.The cultured hi PSC-CMs were randomly devided into two groups: co-culture with(EXO)and without(NC)h CFB-EXOs.The morphology of hi PSCCMs was assessed via immunostaining.Mitochondrial functions assay were measured by Seahorse XF.Electrophysiological analysis was performed using a whole cell membrane clamp and Cardio Excyte 96 assay.Potential mechanism was assessed via m RNA-Seq,real-time PCR and western blot.Results: Electron microscopy was used to demonstrate that cardiac fibroblasts are capable of secreting exosomes,Following 48 hours of coculture with hi PSC-CMs of cardiac fibroblast-derived exosomes,the exosome co-culture group(EXO)showed a significant increase in myocardial maturation proteins,including cardiac troponin I(c Tn I)and myosin heavy chain 7(MYH7).Expression was significantly higher and consistent at the transcriptional level.In terms of mitochondrial oxygen consumption,the EXO group consumed significantly more oxygen than the NC group,including basal respiration,proton leakage,ATP production,maximal respiration and spare respiratory capacity.However,there was no significant difference in glycolytic capacity and glycolytic reserve between the two groups.According to electrophysiology,the EXO group exhibited more mature cardiomyocyte characteristics,both in terms of beat rate,maximum upstroke velocity,peak sodium current density,as well as action potential duration.In addition,based on RNA sequencing results and comparison with the KEGG database,we verified that exosomes from CFBs may activate AMPK pathway in hi PSC-CMs.By investigating the differences in expression of carnitine palmitoyltransferase 1(CPT1),acetyl coenzyme A carboxylase(ACACA),and stearoyl coenzyme A desaturase1(SCD1),key rate-limiting enzymes in the AMPK pathway related to fatty acid metabolism.Finally,we confirmed in vivo experiments that cardiac fibroblast-derived exosomes promote the survival of hi PSC-CMs in the infarct region and improve cardiac function in mice.Conclusion: Human cardiac fibroblasts-derived exosomes could significantly facilitate maturation of hi PSC-CMs via activate AMPK pathway.Cardiac fibroblast-derived exosomes promote the maturation of hi PSC-CMs in infarcted mice and improve post-infarct cardiac function.