Mechanism Of Perfluorooctane Sulfonate Inhibiting Autophagy-Lysosome Pathway To Injury Mouse Embryonic Stem Cell-Derived Cardiomyocytes

Background:Perfluorooctane sulfonate(PFOS)is the most representative and widely distributed member of perfluorinated compounds(PFCs),it is extensively used as industrial materials.PFOS can accumulate in wildlife and human bodies,may cause severe toxic effect to organisms.Since PFOS can pass through the placental barrier,concerns of its developmental toxicity is raising.Studies have shown that exposure to PFOS in zebrafish during embryonic development can cause changes in heart rate and decrease hatching rate.Exposure to PFOS during embryonic development is related to myocardium mitochondrial dysfunction of rats.The cardiac developmental toxicity of PFOS is still in its infancy,and its mechanism still needed to be elucidated in a representative model.Embryonic stem cell test(EST)is a reliable and scientifically animal replacement model for evaluating the embryotoxicity of drugs and environmental toxicants.Based on EST,we have demonstrated that PFOS can inhibit mouse ES cells differentiate into cardiomyocytes in i,vitro and cause mitochondrial dystfunction of embryonic stem cells-derived cardiomyocytes(ESC-CMs).Besides,transmission electron microscop e observation showed that PFOS induced swollen mitochondria and autophagosome accumulation in ESC-CMs.Autophagy is essential for mammalian development and differentiation.Autophagy is also involved in the maturation of mitochondrial functions during heart development.However,it is unclear whether autophagy is involved in the cardiac developmental toxicity of PFOS.Based on our previous research,this study intends to explore the effect of PFOS on autophagosome formation and autophagic degradation pathway of ESC-CMs,and focuses on the role of autophagy-lysosomal pathway in the mitochondrial damage of myocardial cells caused by PFOS,so as to provide experimental basis for further revealing the mechanism of the myocardial developmental toxicity caused by PFOS.At the same time,it may help to promote the application of the EST system as an animal alternative experimental method to evaluate the developmental toxicity of drugs and environmental toxicants.Objective:To explore the mechanism of PFOS inhibiting autophagy-lysosomal pathway to injury ESC-CMs,and provide experimental evidence for revealing the sensitive target of PFOS for cardiac developmental toxicity.Methods and results:1 PFOS inhibitd mouse ES cells differentiate into cardiomyocytes and caused an increase of autophagosomesCardiomyocytes differentiation was induced by using a two-step method,including hanging drop(5 days)culture for embryoid body(EB)formation,then adhering culture for further differentiation.PFOS(40 ?M)was applied to treat ES cells at the beginning of hanging drop.It turned out that PFOS treatment significantly decreased the expression of a-Actinin and beating percentage after adhering culture.Transmission electron microscopy observation reveald that PFOS induced swollen mitochondria and accumulation of autophagosomes in ESC-CMs.With JC-1 staining,we found that PFOS reduced mitochondrial membrane potential of ESC-CMs,at the same time,ATP content was significantly deceased after treated with PFOS.2 PFOS impaired autophagic flux during cardiomyogenesis from ES cellsWestern-blot showed that LC?3-?(autophagy marker protein)and Beclin 1(autophagy initiation complex protein)levels were elevated during cardiomyogenesis from ES cells.PFOS treatment significantly increased the level of LC3-? with unchanged Beclin-1 level and caused an increase in p62(the typical autophagic cargo proteins)level.CQ,a lysosome inhibitor,was used to assess the lysosomal turnover of LC3-? and it turned out that CQ failed to increase the protein level of LC3-? after PFOS treatment.Based on RFP-GFP-LC3 analysis,we found that PFOS treatment blocked the degradation of autophagosomes.These results suggested that autophagy was enhanced during cardiomyogenesis from ES cells,and PFOS treatment impaired autophagic flux of this progress.3 PFOS inhibited mitophagic degradation and impaired mitochondrial biogenesis ofESC-CMs Western-blot showed that PFOS treatment caused an increase in mitochondrial localization of mitophagy-associated protein Parkin.Moreover,immunofluorescence results showed that PFOS treatment caused accumulation of LC3 and p62 on mitochondrial outer membrane.Western-blot and immunofluorescence showed that the expression of PGC-l?(dominant coordination factor for mitochondrial biogenesis)and Tomm 20(mitochondrial outer membrane)were significantly decreased after treated with PFOS.These results suggested that PFOS inhibited mitophagic degradation process and caused a decrease in mitochondrial biogenesis during myocardial differentiation.4 PFOS interfered with the autophagy-lysosomal pathway to block autophagic flux, thereby affected myocardial differentiation and mitochondrial function We labeled autophagosomes with RFP-LC3 and lysosomes with Lyso-Tracker Green dye.Confocal microscope observation showed that the fusion between autophagosomes and lysosomes was blocked after treated with PFOS.Besides,Western-blot showed that PFOS decreased Lamp2a level and inhibited the maturation of Cathepsin D.With LysoSensor staining,we found that the pH of lysosomes was up-regulated in ESC-CMs upon PFOS treatment.PP242+PFOS co-treatment group showed lower p62 level compared with PFOS treated group.Meanwhile,PP242+PFOS co-treatment restored mitochondrial function and increased myocardial differentiation,manifested as improved mitochondrial membrane potential,increased ATP contents,and up-regulated the expression of a-Actinin compared with PFOS treated group.These results suggested that PFOS impaired autophagy-lysosome pathway by causing a decrease in lysosomal function,which may be one of the main mechanisms for inhibiting myocardial differentiation and mitochondrial function of ESC-CMs.Conclusions:1 PFOS reduced the differentiation rate of mouse ES cells towards cardiomyocytes in vitro,blocked autophagic fux and mitophagic degradation during cardiomyogenesis from ES cells.Besides,PFOS caused a decrease of mitochondrial biogenesis and function,manifested as decreased mitochondrial membrane potential and ATP production.2 The mechanism of PFOS inhibiting cardiomyocytes differentiation and causing mitochondrial damage might be related to the increase of lysosomal pH,the inhibition of Cathepsin D maturation,which ultimately resulted in the autophagic flux blockage.