Elsevier

Toxicology

Volume 382, 1 May 2017, Pages 108-116
Toxicology

Mitochondrial toxicity of perfluorooctane sulfonate in mouse embryonic stem cell-derived cardiomyocytes

https://doi.org/10.1016/j.tox.2017.03.011Get rights and content

Abstract

Perfluorooctane sulfonate (PFOS) is a persistent organic contaminant that may cause cardiotoxicity in animals and humans. However, little is known about the underlying mechanism by which it affects the organelle toxicity in cardiomyocytes during the cardiogenesis. Our previous proteomic study showed that differences of protein expression mainly existed in mitochondria of cardiomyocytes differentiated from embryonic stem (ES) cells after exposure to PFOS. Here, we focused on mitochondrial toxicity of PFOS in ES cell-derived cardiomyocytes. The cardiomyogenesis from ES cells in vitro was inhibited, and the expression of L-type Ca2+ channel (LTCC) was decreased to interrupt [Ca2+]c transient amplitude in cardiomyocytes after PFOS treatment. Transmission electron microscope revealed that swollen mitochondrion with vacuole in PFOS-treated cells. Meanwhile, mitochondrial transmembrane potential (ΔΨm) was declined and ATP production was lowered. These changes were related to the increased EGFR phosphorylation, activated Rictor signaling, then mediated HK2 binding to mitochondrial membrane. Furthermore, PFOS reduced the interaction of IP3R-Grp75-VDAC and accumulated intracellular fatty acids by activating Rictor, thereby attenuating PGC-1α and Mfn2 expressions, then destroying mitochondria-associated endoplasmic reticulum membrane (MAM), which resulted in the decrease of [Ca2+]mito transient amplitude triggered by ATP. In conclusion, mitochondrial structure damages and abnormal Ca2+ shuttle were the important aspects in PFOS-induced cardiomyocytes toxicity from ES cells by activating Rictor signaling pathway.

Introduction

Perfluorooctane sulfonate (PFOS) is a fluorine-saturated eight-carbon compound. PFOS is used worldwide in food packaging bags, cosmetics and as wetting agents (Butenhoff et al., 2006, Kantiani et al., 2010, Parsons et al., 2008, Prevedouros et al., 2006). In recent years, PFOS has been detected in many organisms (Nordén et al., 2016, Parolini et al., 2016, Riebe et al., 2016, Schuetze et al., 2010), including humans (Olsen et al., 2007). PFOS is resistant to degradation with an approximate 5-year half-life in human serum (Olsen et al., 2007). Numerous toxicological and epidemiological reports suggested that PFOS caused several adverse effects (Elcombe et al., 2012, Li et al., 2016b, Wang et al., 2010, Wang et al., 2015a), including cardiovascular toxicity (Huang et al., 2011). Cardiac development is one of the earliest processes during embryogenesis. PFOS exposure could lead to the decrease of cardiac-development related genes and proteins in this period (Cheng et al., 2013, Zhang et al., 2016). The cardiomyocyte differentiation of embryonic stem (ES) cell is widely used as a model to assess embryotoxicity in vitro (Radaszkiewicz et al., 2016, Theunissen et al., 2013). Our previous study of proteomics identified 176 differentially expressed proteins, of which 11.4% were mainly localized in the mitochondrion of cardiomyocytes derived from ES cells after PFOS treatment (Zhang et al., 2016). It is urgent to identify whether PFOS-mediated cardiomyocyte toxicity is closely associated with the mitochondrial dysfunction. In cardiomyocytes, there is a higher density of mitochondria compared with other organelles, thereby satisfying the energy demand of the electrical activity and contractile action of the heart. Normal mitochondrial morphology is closely associated with ATP production and cellular functions (Daum et al., 2013). The mitochondrial transmembrane potential (ΔΨm) depends on the cellular oxidative phosphorylation, and mitochondrial swelling may cause depolarization of ΔΨm, which result in lower ATP production (Buckman and Reynolds, 2001, Wang et al., 2015b). In Sprague Dawley rat model, prenatal PFOS exposure induced mitochondrial injury and even the apoptosis of the weaned offspring heart through the mitochondria-mediated apoptotic pathway (Xia et al., 2011, Zeng et al., 2015). Though PFOS-induced mitochondrial damage had been acknowledged as a key organelle target for its cardiac-development toxicity, the underlying mechanisms were not fully elucidated yet.

Mitochondria-associated endoplasmic reticulum membrane (MAM) is a structure of necessity between the endoplasmic reticulum (ER) and the mitochondria, participating in lipid metabolism, mitochondrial calcium fluxes, and other cellular activities (Rizzuto et al., 1998). In mammalian MAM, the ER and mitochondria are tethered to each other by dimers of mitofusin proteins (Mfns) and the IP3 receptor (IP3R)-Grp75-VDAC trimeric complex (de Brito and Scorrano., 2008; Szabadkai et al., 2006). Mammalian target of rapamycin complex 2 (mTORC2) is a multi-protein complex composed of subunits including mTOR, Rictor, mLST8, PRR5, and mSinl, which might be activated by epidermal growth factor receptor (EGFR). Rictor is essential for the activity of mTORC2 (Oh and Jacinto, 2011, Tanaka et al., 2011). Rictor/mTORC2 might be the core of MAM signaling hub that controlled mitochondrial function via Akt meditated phosphorylation of IP3R and hexokinase 2 (HK2) (Betza et al., 2013). Meanwhile, Rictor/mTORC2 activated lipogenesis through SREBP1 (Hagiwara et al., 2012). Fatty acid excess was associated with the decreased expression of PGC-1α, which was believed to directly modulate Mfn2 expression in many tissues (Elezaby et al., 2015). Once MAM structure was disrupted, ATP production was decreased, accompanying with Ca2+ shuttling decrease through IP3R into the mitochondrial matrix (Li et al., 2016a). In the present study, whether Rictor/mTORC2 signaling participated in PFOS-induced cardiotoxicity through affecting the mitochondrial function was valuable to be explored.

In this study, the model of cardiomyocyte differentiation from mouse ES cells was employed to investigate the mechanism for the mitochondria damage in ES cell-derived cardiomyocytes after PFOS treatment during the cardiogenesis. The structure and the function of mitochondria were assessed during the cardiogenesis. PFOS-induced destruction of MAM structure was examined by the transmission electron microscope, immunofluorescence and coimmunoprecipitate analysis. In addition, the calcium fluxes of the differentiated cardiomyocytes were evaluated after PFOS exposure. Finally, whether Rictor/mTORC2 signaling pathway took part in damaging mitochondria and inhibiting calcium fluxes by PFOS were confirmed by immunofluorescence and western blotting analysis. The results demonstrated that abnormal mitochondrial structure and calcium fluxes were involved in the PFOS-induced cardiomyocytes toxicity from ES cells by activating Rictor signaling pathway, which might provide the experimental basis for further revealing the sensitive targets and mechanisms of PFOS-induced cardiotoxicity.

Section snippets

ES cell culture and PFOS treatment

Mouse ES cell line D3 (ERL-1934, American Type Culture collection of the Chinese Academy of Sciences) were conducted as previously described (Wo et al., 2008, Zhang et al., 2016). Briefly, ES cells were treated with 750 cells in a droplet of 20 μL, placed on the inner cover of petri dishes and cultured as inverted hanging drops for 3 days in the presence of 40 μM PFOS (Sigma Aldrich, USA) (the ID50 concentration of PFOS) or 1‰ DMSO (control group) (Zhang et al., 2016). On day 3, the embryoid

PFOS impaired mitochondrial structure and function of ESC-CMs

PFOS inhibited cardiomyocyte differentiation from mouse ES cells, as shown in Fig. 1A, the differentiation ratio of the beating EBs was significantly decreased by 18%, 29%, and 42% after exposure to PFOS (40 μM) when compared with DMSO treatment (44%, 60%, and 79%) on day 5 + 3, 5 + 4, and 5 + 5, respectively. The analysis of α-Actinin expression by flow cytometry demonstrated that only about 14% of α-Actinin positive expression in PFOS-treated cells, while control group (DMSO) contained 35% of

Discussion

PFOS is a persistent, bio-accumulative and toxic organic pollutant, which has been found ubiquitously in the environment (Xiao et al., 2012). Dietary intake is the predominant route for human exposure to PFOS (Van Asselt et al., 2013). PFOS has been found in maternal, umbilical cord and newborn infant blood samples (Zhang and Qin, 2014). Moreover, PFOS can penetrate the placental barrier and enter the fetus in rats (Hinderliter et al., 2005). The general population data from National Health and

Competing interests

The authors have declared that no competing interest exists.

Acknowledgements

This work was supported by grants from the National Natural Sciences Foundation of China (No 81573426); Public Welfare Project of Zhejiang Provincial Science and Technology Department (No 2016C33157); Zhejiang Provincial Natural Science Foundation of China (No LY13H310001).

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