Mechanism Of Perfluorooctane Sulfonate Damage Mitochondria Of Mouse Embryonic Stem Cell-Derived Cardiomyocytes

Perfluorooctane sulfonate ?PFOS? is a representative compound of polyfluorinated alkyl substances, which has been identified in various environment sectors, even in the body of animals and human. PFOS is a persistent organic contaminant and resistant to degradation. Previous studies showed that PFOS induced several adverse organic effects. Currently, concerns had accumulated regarding its development toxicity, liver toxicity, neurotoxicity, cardiovascular toxicity and so on. The most bio-accumulation of PFOS in liver led more concerns on the liver toxicity, and recent studies revealed the involvement of PPARa activation in PFOS-induced hepatic toxicity. As previously reported, the bio-accumulation of PFOS was greatest in the liver, followed by the heart. Heart is one of the earliest formed organs during embryogenesis. Any abnormality during this period could result in the malformation of the organ, and worse, embryo lethal. In zebrafish and medaka models, PFOS-induced abnormalities had been observed in heart development, including an increase in the sinus venosus bulbus arteriosus distance, an alteration of heart rate and a decrease in hatch rate. Although numerous studies had suggested the cardiovascular toxicity of PFOS, little was known about the underlying molecular mechanisms. Herein, it was urgent to start the research.Since murine embryonic stem ?ES? cells were first found in the inner cell mass of blastocysts, they had been widely used in various researches because of their infinite multiplication capacity in suitable circumstances and the ability of differentiating into any kinds of somatic cells in vivo or in vitro. ES cells could differentiate into functional cardiomyocytes with myocardial specific markers. Cardiomyocyte differentiation could mimic the embryonic development, thus providing an accurate and accessible method that facilitated embryotoxic evaluation and research in vitro. In our previous work, the proteomics analysis identified 176 differentially expressed proteins, of which,67 were up-regulated and 109 were down-regulated. The differentially expressed proteins were mainly related to metabolic pathways, including glucose metabolism ?4.5%?, nucleic acid metabolism ?18.7%? and lipid metabolism ?18.7%?. According to the cell location, the differentially expressed proteins were localized in mitochondrion ?11.4%?. Herein, we considered the toxicity of PFOS in cardiomyocytes might be closely associated with mitochondria damage.Mitochondria provide ATP by oxidative phosphorylation, thereby satisfy the energy demand of the electrical activity and contractile action of cardiac. At the same time, mitochondria participate in calcium homeostasis, metabolism, signal transduction and other cellular activity. Mitochondria-associated endopasmic reticulum ?ER? membrane ?MAM? is a subcompartment of the ER that forms a coupling structure with mitochondria. In mamalian MAM, the ER and mitochondria are tethered to each other by dimers of mitofusin proteins Mfns and by IP3 receptor ?IP3R?-Grp75-VDAC trimeric complex. The main function of MAM is to facilitate the transfer of lipids and calcium between the two organells. Ca²⁺ in mitochondria mainly affects several respiratory dehydrogenase, which can promote the synthesis of ATP. Mammalian target of rapamycin complex 2 ?mTORC2? is a multiprotein complex composed of subunits including mTOR, rictor, mLST8, PRR5 and mSinl, which is located in ER. Studies had shown that in Rictor knockout MEFs, phosphorylation of IP3R was significantly reduced. IP3R has Akt binding sites, and mTORC2-Akt inhibits Ca²⁺ release from sarcoplasmic reticulum to the mitochondria combinated with IP3R. If the coupling structure of the mitochondria and ER is destroyed or more binging of Akt and IP3R receptor, both will cause the reduction of the ER Ca²⁺ release to the mitochondria and the ATP synthesis. In addition, the reduced expression of mitochondrial fusion protein Mfn2 results in the decrease of mitochondrial membrane potential. Mfn2 is activated by PGC-1?, then affects the MAM structure. Compared with other types of cells, cardiomyocyte demand for higher energy and more abundant mitochondria to maintain its normal metabolism and contracting action, therefore, cardiomyocytes are extremely sensitive to mitochondrial damage of exogenous compounds. To ensure the normal cardiomyocyte differentiation and cardiomyocyte contraction activity during cardiogenesis, cells switch from glycolytic to P-oxidation. Disordered energy metabolism, such as low level of mitochondrial (3-oxidation activity, results in a decrease of ATP production, furthermore disorders pump function, reduces systolic function and the transport of Ca²⁺, finally leads to heart disease. Studies had reported PFOS could lead to mitochondrial injury in rats, mainly related to its lowered expression of mitochondrial ATP synthetase, but there were still no reports about clarifying the underlying molecular mechanisms of PFOS-induced mitochondria damage of mouse ES cell-derived cardiomyocytes in vitro.Rictor is related to cell growth, apoptosis and metabolism, which is involved in metabolism reprogramming of tumor cell, and promotes chemotherapy resistance through the activation of Akt ?Ser473? phosphorylation in response to growth factor signaling. EGFR is one of the epidermal growth factor receptor families, which is involved in the regulation of cell proliferation and differentiation. EGFR signaling pathway plays an important role in the development of embryonic stage. It had been reported that prenatal SD rats exposed to PFOS led to the increase of EGFR expression and the damage of weaning SD rats heart function. The binging of EGFR and ligand promotes EGFR tyrosine auto-phosphorylation and p-EGFR ?Tyr1086?, then activates Rictor/mTORC2 signal. In addition, Akt phosphorylation promotes HK2 binding to the mitochondrial outer-membrane, which stimulates glycolysis and inbibits the mitochondrial membrane potential. On the other hand, Akt phosphorylation can also increases the expression of Acaca and FASN by increasing the SREBP1 cracking, which results in the accumulation of fatty acid and further mitochondrial damage. Whether PFOS affects the mitochondria Ca²⁺, the energy metabolism and mitochondria by increasing the p-EGFR and activating Rictor/mTORC2 signaling had not been reported.Therefore, based on the results of the differentially expressed proteins during cardiac differentiation, our research was to further explore the mitochondria damage and the related mechanism induced by PFOS, to clarify whether PFOS influence the molecular events of mitochondria-ER coupling structure, Ca²⁺ shuttle and the mitochondrial energy metabolism process. The results could provide experimental basis for further revealing the sensitive targets of PFOS, and was in favor of EST application to assess environmental toxicants of cardiac toxicity as a kind of effective toxicity target appraisal system.Objective:To investigate the effects of mitochondria damage induced by PFOS on mouse ES cell-derived cardiomyocytes, and provide the experimental basis for further revealing the sensitive targets of PFOS.Methods:This study were based on the classical ES cell cardiomyogenesis model:firstly, hanging drops with the embryoid bodies ?EBs? for 3 days, then culture EBs in bacteriological dishes for 2 days, finally plating EBs for further differention. The western-blot, flow cytometry and immunostaining experiments were used to detect developmental cardiotoxicity of PFOS. The intracellular Ca²⁺concentration, Ca²⁺ transients and mitochondrial Ca²⁺ transient were determined by the living cell workstation. The ATP production of mouse ES cell-derived cardiomyocytes was detected after exposed to PFOS, and JC-1 was used to assess ??m of cardiomyocytes. Furthermore the electron microscopy was used to observe mitochondria structure. Then, the ratio of Bax/Bcl-2 was detected by western-blot. According to the results of electron microscopy, the expression level of mitochondrial function related proteins were tested by western blot. At the same time, the co-immunoprecipitation was used to detect the effect of PFOS on IP3R-Grp75-VDAC complex of MAM structure and IP3R-Akt binding. To explore the relationship between mTORC2 signaling pathway and mitochondrial damage in cardiomyocytes, the expression of Rictor, p-EGFR ?Tyr1086?, p-Akt ?ser473?, HK2, SREBP-1, FASN and Acaca were assessed by western-blot analysis.Results:1.40 ?M PFOS inhibited the formation of myocardial with 18±7%,29±6% and 42±5% beating EBs at day 5+3, day 5+4 and day 5+5, much more lower than the beating percent 44±10%,60±3% and 79±3% in the control group. The specific protein marker of mesoderm brachyury, myocardial structure protein a-Actinin and myosin heavy chain MYH10 significantly decreased. And PFOS hindered the silence of Oct4 expression. The intracellular Ca²⁺ concentration and amplitude of the intracellular Ca²⁺ transients fell in the mouse ES cell-derived cardiomyocytes. Western blot showed that the protein expression of L-type Ca²⁺ channel was decreased after PFOS exposure, while the protein level of RyR2 was no difference between two groups. These suggested that PFOS reduced the intracellular calcium transients in mouse ES cell derived cardiomyocytes.2. PFOS reduced the ATP production, intracellular mitochondrial membrane potential of mouse ES cell-derived cardiomyocytes. The electron microscopy results showed that the mitochondria appeared to be swollen with vacuolar structure and with loss of cristae in PFOS treated group. And there was no complete MAM structure in PFOS treated group, while there were more complete MAM structures in control group. At the same time, the immunostaining results showed that PFOS reduced the co-localization of ER and mitochondria. The ration of apoptosis regulatory proteins Bax and Bcl-2 was no changed in PFOS treated mouse ES cell-derived cardiomyocytes, meanwhile, the expression of cytochrome C in mitochondria was no difference between two groups. PFOS increased lactic acid production, lactic acid productions of PFOS group were 0.46±0.12,0.33±0.02 and 0.17±0.04 mmol/gprot respectively on on day 5, day 5+5 and ESC-CMs, which were more than 0.15±0.03,0.14±0.03 and 0.10±0.02 mmol/gprot in the control group. The content of fatty acid in the cardiomyocytes was increased significantly after PFOS treatment, which was detected by TLC. These results suggested that PFOS damaged the mitochondria of ESC-CMs, increased the intracellular lactate and fatty acids, but did not induce apoptosis.3. PFOS significantly reduced the expression of Mfn2 of MAM structure, while the expression of Mfnl had no significant change. At the same time, PFOS significantly reduced IP3R-Grp75-VDAC complex, while significantly increased the binding of IP3R and Akt. PFOS significantly decreased mitochondrial Ca²⁺ transient of ESC-CMs after ATP stimulation. It indicated that PFOS reduced Ca²⁺ released from the ER to the mitochondrial which was associated with MAM structural damage. The western blot results showed that PFOS increased p-EGFR ?Tyr1086?, Rictor and p-Akt ?ser473? expression, and the expression of HK2 in PFOS group mitochondria, which was significantly higher than that of the control group. At the same time, the co-expression of HK2 and mitotracker was significantly increased in PFOS group compared with the control group by immunofluorescence analysis. The expressions of LDHA, cracking SREBP-1, FASN and Acaca in PFOS group were also significantly higher than that of the control group. It suggested that PFOS could activate Rictor through interaction with EGFR, and mediate HK2 binding to the mitochondrial membrane, so as to promote glycolysis. At the same time, Rictor promoted pyrolysis of SREBP-1, thereby resulting in intracellular fatty acid accumulation. On the other hand, PFOS destructed MAM structure, which resulting in blocking of mitochondrial Ca²⁺transient amplitude triggered by ATP.Conclusion1. During the differentiation of mouse ES cells into cardiomyocytes, PFOS exposure led to swollen mitochondria with vacuolar and lower ATP production, but did not cause apoptosis. At the same time, PFOS decreased the expression of L-type Ca²⁺channel to disturb [Ca²⁺]c transient amplitude triggered by caffeine in ESC-CMs.2. PFOS increased EGFR phosphorylation, activated Rictor signaling, mediated HK2 binding to mitochondrial membrane, thereby stimulated glycolysis and inhibited ATm of ESC-CMs. At the same time, PFOS accumulated intracellular fatty acid by activating Rictor, thereby attenuated PGC-la and Mfn2 expression, then destroyed MAM, resulting in the blocking of mitochondrial Ca²⁺ transient amplitude triggered by ATP.