Mitochdrial Metabolism And Regulation During Differention Of HiPSCs Into Cardiomyocytes

Part I Changes of mitochondrial metabolism and protein expression after hiPSCs differentiated into cardiomyocytesObjective: To observe the correlation between the changes of mitochondrial metabolism and the expression of PGC-1? and its downstream oxidative metabolism-related proteins after hiPSCs differentiated into hiPSC-CMs.Methods:(1)hiPSCs were resuscitated and cultured in PGM1 human pluripotent stem cell culture medium,and the medium was changed every day.The pluripotency markers of hiPSCs were detected by immunofluorescence staining and flow cytometry.(2)hiPSCs were induced to differentiate into cardiomyocytes by sequential transient activation/ inhibition of Wnt signaling pathway.Observed cell growth status under an inverted phase contrast microscope.The cardiac specific markers of hiPSCCMs were detected by qRT-PCR,immunofluorescence staining and flow cytometry.(3)The experiment was divided into two groups: hiPSCs group and hiPSC-CMs group.Mitochondrial stress test kit was applied to detect the oxygen consumption rate(OCR);ATP assay kit and JC-1 probe were used to measure intracellular ATP content and mitochondrial membrane potential,respectively;transmission electron microscope(TEM)and Mitotracker Red staining were employed to observe mitochondrial morphology;the protein expression levels of PGC-1? and its downstream oxidative metabolismrelated proteins were detected by Western blot.Results:(1)hiPSCs grow in colonies with smooth colony edges,tightly arranged cells,and rapid proliferation.Immunofluorescence staining results showed that hiPSCs express pluripotency marker OCT4,SOX2,SSEA-4,and TRA-1-60,and the positive expression of OCT4 was 95% detected by flow cytometry in hiPSCs.(2)The first beating of hiPSC-CMs was observed on day8-10 of induction culture,and robust spontaneous contraction were observed on day12.qRT-PCR results showed that hiPSC-CMs expressed cardiac specific markers TNNT2 and MYH6;immunofluorescence staining results showed that cardiac structural markers cTnT,?-actinin and Cx43 were positive in hiPSC-CMs;flow analysis result showed that cTnT positive expression rate of hiPSC-CMs reached 98.6%.(3)Compared with hiPSCs,the OCR of hiPSC-CMs increased,including basal respiration,maximum respiration,ATP-related respiration and non-mitochondrial respiration,and ATP content and mitochondrial membrane potential of hiPSC-CMs also increased;mitochondrial morphology changed,mitochondria were mainly fragmented in hiPSCs,while rod-shaped in hiPSC-CMs;the expressions of PGC-1? and its downstream transcription factors NRF1,ERR? and mitochondrial proteins involved in metabolic pathways such as tricarboxylic acid cycle,oxidative phosphorylation,fatty acid oxidation,and mitochondrial pyruvate transport in hiPSC-CMs were significantly increased.Conclusion:(1)We successfully induced undifferentiated hiPSCs to specifically differentiate into cardiomyocytes expressing cardiac-specific markers.(2)The enhancement of mitochondrial respiratory function after hiPSCs differentiated into hiPSC-CMs was positively correlated with the expression of PGC-1? and its downstream oxidative metabolism-related proteins.Part ? Effect of PGC-1? on mitochondrial metabolism of hi PSC-CMs and its possible mechanismObjective: To investigate the role of PGC-1? in regulating mitochondrial metabolism of hi PSC-CMs and its possible mechanism.Methods:(1)PGC-1?-si RNA was used to transfect hi PSC-CMs,q RTPCR and Western blot detected the expression of PGC-1?,and mitochondrial stress test kit assessed oxygen consumption rate(OCR).(2)Treated hi PSCCMs with different concentrations of ZLN005,q RT-PCR and Western blot were used to detect PGC-1? expression,CCK8 assay was employed to measure cell viability,and screened out the optimal working concentration and used this concentration for subsequent experiments.Divided the experiment into two groups: control group and ZLN005 group,and treated hi PSC-CMs with ZLN005 at optimal working concentration.Tested oxygen consumption rate(OCR)using mitochondrial stress test kit.JC-1 probe was applied to detect mitochondrial membrane potential.Observed and recorded hi PSC-CMs' beating frequency under an inverted microscope.Observed mitochondrial morphology using Mitotracker Red staining,and analyzed the fluorescence intensity to reflect mitochondria content.The expression of metabolism-related genes were detected by q RT-PCR and Western blot.(3)q RT-PCR and Western blot were used to detect the effect of PGC-1?-si RNA on the expression of ERR?.Co-IP was employed to detect protein interaction between PGC-1? and ERR?.Divided the experiment into three groups: control group,ZLN005 group and ZLN005 + XCT790 group,inhibited ERR? protein expression by XCT790 when activating PGC-1? though ZLN005,and then detected mitochondrial respiratory function and protein expression by mitochondrial stress test and Western blot,respectively.Results:(1)The use of PGC-1?-si RNA effectively knocked down the expression of PGC-1? and suppressed the OCR of basal respiration,ATPrelated respiration and non-mitochondrial respiration in hi PSC-CMs.(2)8 ?mol/L is the optimal working concentration of ZLN005,could effectively activate the expression of PGC-1? without cytotoxicity.Compared with the control group,the OCR of basal respiration,maximum respiration,ATPrelated respiration and non-mitochondrial respiration,mitochondrial membrane potential,beating frequency and mitochondrial content increased;mitochondrial morphology changed into a network,and mitochondria scattered throughout the cytoplasm in the ZLN005 group.q RT-PCR and Western blot results showed that ZLN005 promoted the express of mitochondrial fusion genes,inhibited the express of mitochondrial fission genes,and promoted the expression of transcription factors NRF1,ERR? and key mitochondrial proteins such as CPT-1?/?,CS,COXIV,COX5 B,Cyt C and MPC1.(3)In hi PSC-CMs,PGC-1?-si RNA induced decreased expression of ERR?,additionally PGC-1? could combine with ERR? to form a complex.Compared with the ZLN005 group,the basal respiration,maximum respiration,and ATP-associated respiration of the ZLN005 + XCT790 group were significantly reduced,and mitochondrial protein expressions of MFN1/2,CS,COX4,COX5 B,and Cyt C were also significantly decreased,while there was almost no significant difference compared with the control group.Conclusion:(1)PGC-1? plays an important role in regulating mitochondrial metabolism.Activating PGC-1? expression through ZLN005 can promote the maturation of hi PSC-CMs mitochondrial metabolism: enhanced mitochondrial respiration,improved mitochondrial membrane potential,increased mitochondria content,advanced mitochondrial fusion,and increased expression of oxidative metabolism-related proteins.(2)The possible mechanism of PGC-1? regulating mitochondrial metabolism is to promote the expression of downstream oxidative metabolism-related proteins,of which ERR? plays an important role in the mitochondrial oxidative metabolism induced by PGC-1?.PGC1-? can directly regulate ERR? expression and combines with it to form a coactivation complex to regulate the expression of downstream proteins to enhance mitochondrial function.