Regulation Of Energy Metabolism By Nrf2 During The Maturation Of Cardiomyocytes Derived From Human Induced Pluripotent Stem Cells

Background: For myocardial injury diseases,due to the non-renewable myocardium,the source of organ donation is scarce.Even if actively treated,the mortality rate is still high.At that time,the emergence of stem cell replacement therapy provides new possibilities for the treatment of myocardial injury diseases.Among a variety of stem cells that can be induced into cardiomyocytes,human induced pluripotent stem cells(hiPSCs)have received more attention due to their many advantages.However,the study shows that although the isolated myocardial cells(hiPSCs-CMS)have spontaneous beating of cardiomyocytes,express myocardial specific ion channel related genes and the appearance of myocardial sarcoid structure.However,its metabolism is still high in glycolysis and extremely low in fatty acid oxidation,which is more like immature cardiomyocytes during fetal period.This has severely hindered the application of hiPSC-CMs in research systems such as myocardial replacement therapy,drug screening,and disease modeling.Studies have shown that glucose can inhibit the maturation of cardiomyocytes,and fatty acids can promote the adaptive transformation of cardiomyocytes’ metabolism to be dominated by fatty acid beta oxidation metabolism.This corresponds to the process in which the fetus’ s energy source changes from blood sugar to fatty acid-rich milk after birth,and stimulates the development and maturation of cardiomyocytes.Therefore,by adding fatty acids to the medium,the mitochondrial metabolism can be more mature,and finally the maturity of the structure and function of hiPSC-CMs can be improved.Furthermore,through literature review and preliminary experimental studies,we found that the expression level of nuclear factorrelated factor 2(NRF2)was significantly increased in hiPSC-CMs where fatty acids promote maturation.As a transcription factor,NRF2 participates in the regulation of a huge functional network of antioxidants,mitochondrial respiration,and drug metabolism.In recent years,more and more evidences have shown that NRF2 plays a very important role in regulating mitochondrial respiration and can promote mitochondrial fatty acid oxidation.Therefore,we inhibited NRF2 in the process of culturing hiPSCCMs with fatty acids,and explored its role in the process of fatty acid promoting the maturation of cardiomyocytes.Further literature review found that some researchers intervened in the expression of related cytokines(such as HIF-1α,Let-7,PGC-1α,etc.)that can regulate the conversion of energy metabolism to promote the conversion of hiPSC-CMs from glycolysis to oxidative phosphorylation,and ultimately improve the maturity of hiPSC-CMs.Studies have shown that NRF2 helps regulate mitochondrial morphology and function through intermolecular association.NRF2 can promote mitochondrial biogenesis by directly upregulating mitochondrial transcription factors,and participates in the regulation of mitochondrial respiration through mitochondrial activation.In addition,some mitochondrial proteins are involved in regulating NRF2,thus forming a mutual regulation loop between mitochondria and NRF2.Under steady-state conditions,NRF2 affects mitochondrial membrane potential,fatty acid oxidation,substrate(NADH and FADH2/succinic acid)respiratory availability,and ATP synthesis.This role is even more critical when stimulated by stress or growth factors.Our research has also shown that the inhibition of NRF2 in the process of using fatty acids to promote the maturation of hiPSC-CMs will cause severe hinder the maturation of cell.Therefore,we further inhibited and activated NRF2 in hiPSC-CMs to explore whether NRF2 could promote the maturation of hiPSC-CMs and its effect on growth and development.Objective: To clarify the energy metabolism of the hiPSC-CMs we induced.By adding fatty acids to the medium,the metabolism of hiPSCCMs will be further matured,and finally the structure and function of hiPSCCMs will be improved,and the role of NRF2 in the maturation of hiPSCCMs promoted by fatty acids will be explored.Then we will further study whether regulating NRF2 alone can promote the transformation of hiPSCCMs mitochondrial metabolism,and ultimately improve the maturity of hiPSC-CMs structure and function.Methods:(1)Regulate Wnt signal at different time points of differentiation to induce myocardial differentiation of hiPSCs.Detection of myocardial specific markers,cell morphology,gene expression related to energy metabolism,mitochondrial staining,and hippocampal real-time energy detection,to identify myocardial characteristics and mitochondrial morphology and respiratory function of differentiated cells.(2)By adding fatty acids to the sugar-free hiPSC-CMs maintenance medium and continuously culturing for 7 days,the cell structure and function of the obtained hiPSC-CMs,mitochondrial fusion,membrane potential,respiration,fatty acid oxidation levels were detected to assess the maturity of the cells.And inhibit the expression of NRF2 in the process of fatty acid culture of hiPSC-CMs,analyze the structure and function of cells and the morphological structure and function of mitochondria,and evaluate the maturity of hiPSC-CMs.(3)Inhibit and activate NRF2 respectively,and detect the cellular structure of hiPSC-CMs,calcium transients,mitochondrial biosynthesis,mitochondrial fusion,mitochondrial membrane potential,mitochondrial respiration,and fatty acid oxidation levels.Results and Conclusion:(1)After induction,hiPSCs successfully differentiated into cardiomyocytes capable of spontaneously beating,with myocardial-specific structure and expressing myocardial-specific ion channel-related genes.Detection of hiPSC-CMs mitochondria showed that although hiPSC-CMs mitochondrial respiratory level increased,several enzymes that catalyzed fatty acid β oxidation did not increase significantly after differentiation.It shows that the fatty acid metabolism level of cells is not enough,and external intervention is needed to increase the fatty acid oxidation level of cells,and thereby improve the maturity of hiPSC-CMs.(2)By adding fatty acids to the medium,we obtained hiPSC-CMs with significantly improved cell structure maturity,and the mitochondrial respiration capacity of the cells was also significantly increased.After inhibiting the expression of NRF2 in the process of using fatty acids to promote the maturation of hiPSC-CMs,the cell structure became naiver,the sarcomere became shorter,and the cells became smaller and rounder.When studying the ability of mitochondrial respiration and fatty acid oxidation,we found that after NRF2 is inhibited,the mitochondrial metabolic maturation of hiPSC-CMs is also inhibited.These results indicate that FA can promote the maturation of hiPSC-CMs,and NRF2 plays a very important role in the process of FA promoting the maturation of hiPSC-CMs.(3)After NRF2 is inhibited,the structural maturation of hiPSC-CMs is blocked,and the morphological structure and function of mitochondria become more immature.After activating NRF2,the mitochondrial respiration and fatty acid oxidation capabilities of hiPSC-CMs were significantly enhanced,the degree of mitochondrial fusion was higher,the number of mitochondria significantly increased,the myocardial structure and calcium transient ability of the cells were improved,and the overall maturity of the cells was improved.These results indicate that NRF2 plays a very important role in promoting the mitochondrial maturation of hiPSCCMs and ultimately affecting the overall maturation state of cells.It provides in-deeper insights into the molecular mechanisms regulating the metabolism of hiPSC-CMs,and lays a scientific theoretical and experimental basis for the clinical application of stem cell replacement therapy,drug screening and disease modeling.And provide a research model that can be used for reference for this type of research,and ultimately hope to solve the clinical cardiac dysfunction and improve the prognosis of patients.