AMPK Promotes The Maturation Of HiPSC-CMs By Regulating Mitochondrial Function

Objective: To observe the changes of mitochondrial structure and function after hiPSCs differentiated into hiPSC-CMs and evaluate the relationship between AMPK and maturation of hiPSC-CMs.To investigate the effects for the activation of AMPK on mitochondria of hiPSC-CMs and its possible mechanism.Methods:(1)hiPSCs were resuscitated and cultured in PGM1 medium.Microscope was used to observe the growth status of hiPSCs.The pluripotency of hiPSCs were detected by immunofluorescence.(2)hiPSCs were induced into hiPSC-CMs by regulating Wnt signaling pathway.The cardiac specific markers of hiPSC-CMs were detected by immunofluorescence staining and flow cytometry.(3)The hiPSC-CMs were processed at a specific time,and the following experimental groups were carried out: control group;AMPK inhibition group(Compound C);AMPK activation group(AICAR);AMPK activation and PDK4 inhibition group(AICAR+DCA).(4)Western blot and q RT-PCR were used to detect the expression of AMPK,energy metabolism related factors,CM-specific markers,and electrophysiological related genes;The morphology and sarcomere structure of hiPSC-CMs were observed by immunofluorescence;Hexokinase kit and lactate assay kit were used to validate hexokinase activities and lactate levels.Calcium transient was detected by loading Fluo-4AM.(5)The ultrastructural and morphology of mitochondria were observed by transmission electron microscope and Mitotracker staining;The changes of mitochondrial membrane potential were evaluated by JC-1staining;ATP assay kit was used to detect intracellular ATP content;Evaluation of OCR and fatty acid oxidation(FAO)by mitochondrial stress test and substrate oxidative stress;The expression of mitochondrial fusion and fission were detected;q RT-PCR was used to detect mitochondrial DNA copy number.Results:(1)hiPSCs formed colonies with tight arrangement and clear boundaries.hiPSCs consistently stained positive for the pluripotent stem cell-specific markers Nanog and Sox2.(2)hiPSC-CMs began spontaneous contraction after 8 days of differentiation and strongly beat around the 12 th day of differentiation.The flow analysis indicated that hiPSC-CMs containing more than 92% c Tn T-positive cells.Immunostaining demonstrated that hiPSC-CMs expressed CM-specific markers,?-actinin,Cx43,and c Tn T.Immunofluorescence detection of MLC2 v,and MLC2 a showed that hiPSC-CMs mainly consist of ventricular CMs and a few other atrial CMs.(3)Transmission electron microscopy and Mitotracker staining showed that the mitochondria in hiPSCs were granular and sparsely cristae,the mitochondria in hiPSC-CMs were more mature,slender and connected in network,with more dense cristae and dense matrix.hiPSC-CMs had significantly higher OCRs associated with basal respiration,ATP production,maximal respiration,and mitochondrial membrane potential to those in hiPSCs.(4)Inhibition of AMPK decreased the OCR values of basal respiration,maximal respiration and ATP production of hiPSC-CMs,inhibited the expression of CM-specific,and decreased the mitochondrial DNA copy number.The results showed that AMPK was related to the maturity of hiPSC-CMs.(5)Activation of AMPK promoted the expression of metabolism-related targets,and decreased hexokinase activities and the production of lactate in hiPSC-CMs;Activation of AMPK had longer sarcomeres and lower circularity index in hiPSC-CMs;Activation of AMPK promoted the expression of CM-specific markers,and electrophysiological related genes in hiPSC-CMs;Activation of AMPK enhanced the function of calcium transient in hiPSC-CMs.(6)Transmission electron microscopy showed that the number of mitochondria were increased in AICAR-treated hiPSC-CMs and AICAR-treated hiPSC-CMs contained more mitochondrial crista than those in the control.In addition,the myofibrils were plentiful and well organized,and the Z-lines of the muscle fibers were visible in AICAR-treated hiPSC-CMs.Mito Tracker staining revealed that hiPSC-CMs contained isolated mitochondria with fragments,and AICAR-treated hiPSC-CMs consisting of an extensively interconnected filamentous network.(7)Activation of AMPK promoted the expression of mitochondrial fusion protein,inhibited the expression of mitochondrial fission protein,and increased the mitochondrial DNA copy number.;Activation of AMPK increased the mitochondrial membrane potential,increased the OCR value of mitochondrial basal respiration,maximum respiration,and ATP production,and promoted fatty acid oxidation.(8)Activation of AMPK cooperated with inhibition of PDK4 promoted the expression of metabolism-related factors,and increased the OCR value of mitochondrial basal respiration,maximum respiration,and ATP production.Conclusion: The enhancement of mitochondrial oxidative metabolism of hiPSC-CMs is related to the activation of AMPK.Activation of AMPK can promote the maturation of hiPSC-CMs by regulating mitochondrial function,and may be achieved by regulating the expression of PDK4.