INHIBITORY EFFECT OF OCTYL-PHENOL AND BISPHENOL A ON CALCIUM SIGNALING IN CARDIOMYOCYTE DIFFERENTIATION OF MOUSE
EMBRYONIC STEM CELLS

Embryonic stem cells (ESCs) are pluripotent cells derived from the inner cell mass of a blastocyst, an early-stage preimplantation embryo, and are useful for in vitro model systems in the study of embryogenesis (1). ESCs can differentiate into all derivatives of the three primary germ layers (ectoderm, endoderm, and mesoderm). For example, embryonic stem cells can differentiate into liver, pancreas, neurons, and cardiomyocytes. In particular, mouse stem cells can naturally differentiate into cardiomyocytes (2, 3).

Progesterone (P4) is an endogenous steroid hormone that has a variety of important functions in the body. Progesterone is involved in the regulation of the menstrual cycle, in maintaining pregnancy, and in embryogenesis. In the brain, progesterone has a neuroprotective effect, and in the heart, progesterone reportedly contributes to the cardiac repolarization processes (4). Some studies have indicated a risk of fetal anomalies and toxicity with progesterone therapy during pregnancy (5, 6).

Calcium plays an important role in signal transduction pathways and acts as a second messenger in fertilization and in the contraction of all muscle cell types (7). Calcium is controlled by calcium transport proteins, as well as by calcium channels, carriers, and pumps. Steroid hormones control the expression of many cell regulatory factors and likely regulate calcium channel expression as they are involved in the regulation of cardiac excitability (8). Transient receptor potential cation channel subfamily V member2 (TRPV2) is expressed in a variety of tissues including the heart (cardiomyocyte), lung, prostate, and small intestine. Cardiomyocytes express not only TRPV2 calcium channel but also ryanodine receptor (RyR) and sarcoplasmic reticulum Ca2+-ATPase (SERCA) (9). Maintaining intracellular calcium homeostasis is important in many aspects of cardiac function. In cardiomyocytes, calcium homeostasis is regulated by intracellular calcium uptake and release, primarily via membrane potential and sarcoplasmic reticulum (SR) function through the integrated function of calcium entry and exit pathways. This process controls the contraction and relaxation of the heart (10). Intracardiac calcium enters the calcium channel and causes a substantial release of calcium through the RyR channel in the SR, resulting in cardiac contraction. After cardiac contraction, calcium is released from the filaments and returns to the SR through the SERCA pump, initiating cardiac relaxation (11).

Endocrine-disrupting chemicals (EDCs) have structures similar to endogenous steroid hormones such as progesterone and estrogen and disrupt the action of various endogenous hormones. EDCs can also combine with hormone receptors to produce different responses. There are various types of EDCs, such as octylphenol (OP), nonylphenol (NP), bisphenol A (BPA), and some plastics, pesticides, and paint, which are commonly found in the environment.

In previous studies, progesterone was shown to affect embryonic development, including cardiac differentiation (12). However, the effect of OP and BPA on mESCs is unknown, and the specific effects of OP and BPA on the differentiation of mESCs into functional cardiomyocytes during early differentiation has not been reported. In this study, to examine the effect of OP and BPA on early differentiation of mESCs into cardiomyocytes, we assessed the beating ratio, the expression of calcium channels and contraction-related genes, and the cytosolic calcium level during differentiation in the presence of OP and BPA.

MATERIALS AND METHODS

mESCs cell culture and maintenance

mESCs (ES-E14TG2a) were purchased from the American Type Culture Collection. Pluripotent mESCs were cultured in basal medium [DMEM/F-12/phenol red-free (Gibco, Logan, UT, USA) with leukemia inhibitory factor (mLIF, 10 ng/mL; Millipore] with 10% heat-inactivated and charcoal-dextran treated certified FBS (CD-FBS), non-essential amino acids (NEAA, 1X, Gibco), 2-mercaptoethanol (10–4 M), penicillin (100 U/ml) and streptomycin (100 µg/ml) on mitomycin C-treated mouse embryonic fibroblasts in a 60-mm plate (Falcon, Glendale, AZ, USA) at 37ºC in a 5% CO2 humidified tissue culture incubator (Sanyo, San Diego, CA, USA).

Differentiation into cardiomyocytes

To induce differentiation, mESCs were suspended in differentiation medium containing 15% CD-FBS without leukemia inhibitory factor (LIF). For the formation of embryo bodies (EBs), a cell suspension containing 800 cells/drop was prepared and 25 µl X 84 drops were plated on the lid of a 90-mm Petri dish (SPL Inc, South Korea). PBS, 6 ml was added to the bottom plate and cells were cultured as hanging drops after turning over the lids. After 3 days, the mEBs formed on the lid of 2 plates were transferred to an uncoated Petri dish with 6 ml of differentiation medium without LIF. After suspension culture for one day, the EBs were transferred and attached to a 6-well plate with 2 mL of differentiation medium (6 ~ 7 EBs per well). EBs differentiated into cardiomyocytes for 12 days. The exchange with a differentiation medium (DMEM/F-12/phenol red-free + 15% CD-FBS + NEAA + 2-mercaptoethanol (10–4 M), penicillin (100 U/ml) and streptomycin (100 µg/ml)) every two days. The mouse stem cells are easily differentiated into cardiomyocytes, and the known methods are used (12).

Chemicals and treatment

P4, OP, BPA and mifepristone (RU486) were purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). Stock solutions were made by dissolving in DMSO (dimethyl sulfoxide; Santa Cruz Biotechnology, CA, USA). On day 2 after attachment, mESCs were treated with P4 (10–8 M), OP (10–6 M), and BPA (10–7 M) for 10 days. The exchange with a differentiation medium containing the chemical (P4, OP, and BPA) every two days. On day 11 after attachment of mEBs, cells were exposed to progesterone antagonist (RU486, 10–6 M) for one day. The control used DMSO included medium. All experiments were performed in triplicate.

Assessment of cardiomyocyte differentiation by beating ratio

Spontaneous contraction of differentiated mESCs was observed manually using a phase-contrast microscope (Olympus, IX71). Beating ratio was determined by the number of contracting cell populations to the number of attached mEBs. One cell population means differentiated cells from one mEB. Beating ratio was the percentage (%) of beating EBs/total EBs. It is graphed by repeated test.

Total RNA extraction and real-time PCR

Total RNA was extracted using TRI reagent (Ambion, Austin, TX, USA) according to the manufacturer’s protocol. The total RNA concentration was measured from the absorbance at 260 nm using an Epoch Microplate Spectrophotometer (BioTek Instruments, Inc.). RNA (1 µg) was transcribed using MMLV (Moloney murine leukemia virus) reverse transcriptase (Invitrogen, Carlsbad, CA, USA) with random primers (9-mers; TaKaRa Bio Inc., Shiga, Japan) to produce first-strand complementary DNA (cDNA). The cDNA template (1 µl) was assayed using SYBR PCR (TaKaRa Bio Inc.) real-time PCR according to the manufacturer’s protocol. Real-time PCR was performed under the following conditions: 40 cycles of denaturation at 95ºC for 30 s, annealing at 58ºC for 30 s, and extension at 72ºC for 30 s. Fluorescence intensity was measured at the end of the extension phase of each cycle. The threshold value for fluorescence intensity for all samples was set manually. The PCR cycle at which the fluorescence intensity threshold was in the exponential phase of PCR amplification was designated as the CT (threshold cycle). The CT value was determined automatically from the exponential phase of the delta CT fluorescence detection graph. The PCR product of Rn18S (18S ribosomal RNA) was used as an internal control for normalization. The gene primer sequences for Tbx20, Ctn1 (cardiac troponin I), Rbp4 (retinol binding protein 4), Ly6e (lymphocyte antigen 6 complex), Gata4 (GATA binding protein 4), Pgr (progesterone receptor), Trpv2 (transient receptor potential cation channel subfamily V member 2), Ryr2 (ryanodine receptor 2), Calm2 (calmodulin 2), Mylk3 (myosin light chain kinase 3) and Rn18S are shown in Table 1. The amount of transcript present was inversely related to the observed CT, and CT was expected to increase by 1 for every 2-fold dilution in the amount of transcript. Relative expression was calculated using the equation R = 2–(ΔCTsample – ΔCTcontrol). To determine a normalized arbitrary value for each gene, every data point was normalized to the control gene, as well as to their respective controls.

Table 1. Oligonucleotide sequences for quantitative real-time PCR

F, forward; R, reverse.

Western blotting analysis

Proteins were extracted from differentiated cardiomyocytes using RIPA buffer (50 mM Tris pH 7.4, 1 mM EDTA, 1 mM PMSF, 1% NP-40, 150 mM NaCl and 0.25% sodium deoxycholate) supplemented with protease inhibitor cocktail. BCA assay (Sigma-Aldrich) was performed at 562 nm to determine protein concentration. Fifty µg of protein were treated by mixing with sodium dodecyl sulfate (SDS) sample buffer and heating at 65ºC for 20 min. Protein samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The separated proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Merck Millipore, Taunton, MA, USA). Membranes were blocked with 5% skim milk (dissolved in TBS-T) for 1 hour. The membranes were then incubated with primary antibodies for 3 hours at RT or overnight at 4ºC: anti-PGR (SC-538, 1:800 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-TRPV2 (SC-22520, 1:500 dilution, Santa Cruz) and anti-α-tubulin (#2144, 1:1000 dilution, Cell Signaling Technology, Beverly, MA, USA) diluted in 1% BSA (bovine serum albumin). Next, the membrane was washed four times for 10 min each with TBS-T. The blot was subsequently incubated for 1 hour with anti-goat secondary antibody (1:3000) and anti-rabbit secondary antibody (1:3000) diluted in 2.5% non-fat milk in TBS-T. The membrane was washed four times for 10 min each with TBS-T. Protein bands were detected using ECL solution with Chemi Doc GenGnome 5 (Syngene, Cambridge, UK). The optical density of the target band was analyzed by Image J (NIH, Bethesda, MD, USA).

Calcium imaging with confocal microscopy

Calcium imaging was performed using a confocal laser scanning microscope (Zeiss, LSM 710). Following treatment with trypsin, mEBs that had differentiated into cardiomyocytes were separated into single cells, and individually isolated cardiomyocyte cells (3 × 103 cells) were attached to a coverglass bottom dish (SPL 100350) in differentiation medium for 3 hour. Cardiomyocytes were loaded with fluo-4-AM (5 µM; Invitrogen) for 1 h at 37ºC, then transferred to Ca2+-free BSS buffer solution (5.4 mM KCl, 5.5 mM d-glucose, 1 mM MgSO4, 130 mM NaCl, and 20 mM HEPES at pH 7.4). Detection of calcium imaging was performed at 3-s intervals for 20 min using a confocal microscope. Cardiomyocytes were treated with 2 mM CaCl2 on the holder at 1 min to measure the inflow rate of calcium in the cytosol, and cardiomyocytes were treated with the L-type Ca2+ channel blocker, nifedipine (10 µM), for 5 min.

Statistical analysis

Significant differences were detected by using ANOVA. The analysis was performed using the Prism Graph Pad v5.0 (Graph Pad Software, San Diego, CA, USA). Values are expressed as means ± SD of at least three separate experiments, in which case a representative result is depicted in the figures. P values < 0.05 were considered statistically significant.

RESULTS

Inhibitory effects of progesterone, octyl-phenol, and bisphenol A on the contracting cardiomyocytes of mouse embryonic stem cells

To investigate the inhibitory effects of P4, OP, and BPA, the contracting cardiomyocytes were treated with P4, OP, and BPA. The beating ratio of the differentiated cardiomyocytes was decreased in the P4-, OP-, and BPA-treated groups (Fig. 1A), indicating that P4, OP, and BPA suppress the contracting cardiomyocytes of mESCs. Tbx20, a marker for cardiac progenitor cells in secondary cardiomyocytes, and Ctn1, a cardiac-specific marker, were decreased by P4, OP, and BPA treatments and recovered with RU486 plus P4, OP, or BPA treatments (Fig. 1B and 1C). Also, we confirmed the expression of cardiac development and morphogenesis genes such as Rbp4, Ly6e, and Gata4. Cardiac development and morphogenesis genes were decreased by P4, OP, and BPA treatments and recovered with RU486 plus P4, OP, or BPA treatments (Fig. 1D and 1F). These results show that P4, OP, and BPA inhibited differentiation into cardiomyocytes.

Fig. 1. Effects of progesterone (P4), octylphenol (OP), and bisphenol A (BPA) in cardiac differentiation. (A) Inhibitory effect of P4, OP, and BPA on beating ratio. Beating ratio was the percentage (%) of beating EBs/total EBs. It is graphed by repeated test. Expression levels of (B) cardiac progenitor marker (Tbx20), (C) cardiac specific marker (Ctn1), (D) retinol binding protein 4 (Rbp4), (E) lymphocyte antigen 6 complex (Ly6e), and (F) GATA binding protein 4 (Gata4) on 12 days after attachment. mRNA level was measured by real-time PCR and normalized by Rn18s *P < 0.05 versus vehicle group (VE); #P < 0.05 versus agonist. P4 (10–8 M), OP (10–6 M), and BPA (10–7 M) were treated from the 2 to 12 days after attachment, and they were exchanged with medium containing chemical every two days. The RU486 (10–6 M) was treated on the 11th day after attachment. The control used DMSO included medium.

Expression of progesterone receptor and calcium channel with progesterone, octyl-phenol, and bisphenol A treatment

The mRNA expression levels of progesterone receptor (Pgr) and calcium channel (Trpv2) following treatments with P4, OP, and BPA were analyzed by qRT-PCR (Fig. 2A and 2B). The Pgr mRNA levels were significantly increased by P4, OP, and BPA compared to vehicle control and these increases were reversed by RU486, a progesterone blocker. Trpv2 mRNA levels were significantly decreased in the P4-, OP-, and BPA-treated groups and restored by RU 486. Protein expression levels of PGR and TRPV2 were confirmed by western blotting (Fig. 2C). Protein expression of PGR was increased by P4, OP, and BPA, and reversed by RU486 (Fig. 2D). Protein expression of TRPV2 was decreased in the P4-, OP-, and BPA-treated groups and recovered in the RU486-treated group (Fig. 2E). The pattern of mRNA and protein expressions of Trpv2 was opposite that of the Pgr gene following P4, OP, and BPA treatments. These data indicate that PGR expression is regulated by P4, OP, and BPA, whereas the expression of Trpv2 gene is independent of PGR regulation by treatments with P4, OP, and BPA.

Fig. 2. Effects of P4, OP, and BPA on progesterone receptor and Ca2+ channel on 12 days after attachment. The effects of P4 and its antagonist (RU486) on the transcriptional level of (A) progesterone receptor (Pgr) and (B) transient receptor potential cation channel subfamily V member 2 (Trpv2). mRNA level was measured by real-time PCR and normalized to Rn18s. (C) Effect of P4 and RU486 on expressions of PGR and TRPV2 proteins by Western blotting. Quantification of (D) PGR, (E) TRPV2 protein levels using NIH ImageJ software. Protein levels were normalized to α-tubulin. *P < 0.05 versus vehicle group (VE); #P < 0.05 versus agonist. P4 (10–8 M), OP (10–6 M), and BPA (10–7 M) were treated from the 2 to 12 days after attachment, and they were exchanged with medium containing chemical every two days. The RU486 (10–6 M) was treated on the 11th day after attachment. The control used DMSO included medium.

Expression of contraction-related genes with progesterone, octyl-phenol, and bisphenol A treatment

To confirm the cytosolic calcium changes in contraction signaling of the contracting cardiomyocytes of mESCs, mRNA expression levels of contraction-related genes such as Ryr2, Calm2 and Mylk3 were analyzed. Ryr2, Calm2 and Mylk3 mRNA expressions were significantly decreased in the P4-, OP-, and BPA-treated groups and recovered with RU486 treatment (Fig. 3A-3C). This result showed that P4, OP, and BPA reduce the cardiomyocyte contraction of mESCs via down-regulation of Trpv2, Ryr2, Calm2, and Mylk3 mRNA expressions.

Fig. 3. Effects of P4, OP, and BPA on contraction-related genes on 12 days after attachment. Effects of P4 and its antagonist (RU486) on transcriptional levels of (A) ryanodine receptor 2 (Ryr2), (B) calmodulin 2 (Calm2), and (C) myosin light-chain kinase 3 (Mylk3). mRNA level was measured by real-time PCR and normalized to Rn18s. *P < 0.05 versus vehicle group (VE); #P < 0.05 versus agonist. P4 (10–8 M), OP (10–6 M), and BPA (10–7 M) were treated from the 2 to 12 days after attachment, and they were exchanged with medium containing chemical every two days. The RU486 (10–6 M) was treated on the 11th day after attachment. The control used DMSO included medium.

Effect of progesterone, octyl-phenol, and bisphenol A on the cytosolic Ca2+ level

Cytosolic calcium levels were determined by confocal microscopy to assess the effects of P4, OP, and BPA during cardiomyocyte differentiation of mESCs (Fig. 4A and 4B). Cytosolic calcium levels were decreased by P4, OP, and BPA and reversed with RU486. To confirm whether the decrease in cytosolic calcium level was related to L-type calcium channel, cardiomyocytes were treated with nifedipine, a calcium channel blocker. Decreases in cytosolic calcium level were observed with nifedipine in the vehicle group and RU486-treated group. However, almost no response was observed in the P4-, OP-, and BPA-treated groups. These results suggest that P4, OP, and BPA reduce the cytosolic Ca2+ level.

Fig. 4. Decreased cytosolic Ca2+ level with P4, OP, and BPA treatment on 12 days after attachment. (A) Effect of nifedipine, a Ca2+ channel blocker, on the cytosolic Ca2+ level. (B) Comparison of cytosolic Ca2+ level measured by confocal microscopy. *P < 0.05 versus vehicle group (VE); #P < 0.05 versus agonist. P4 (10–8 M), OP (10–6 M), and BPA (10–7 M) were treated from the 2 to 12 days after attachment, and they were exchanged with medium containing chemical every two days. The RU486 (10–6 M) was treated on the 11th day after attachment. The control used DMSO included medium.

DISCUSSION

Stem cells have been differentiated into cardiomyocytes, and they are widely used in toxicity assessment (13), regenerative cardiology (14), and disease studies such as QT syndrome (15). Previous studies have examined the effects of endocrine-disrupting chemicals such as OP and BPA using mature cardiomyocytes cells or neonatal cardiomyocyte (16). Since there is a difference between mature cardiomyocytes and differentiated cardiomyocytes (16), we have identified the effects of OP and BPA exposure in differentiating stem cells into cardiomyocytes. The relationship between heart disease and steroid hormone action is poorly understood. In previous studies, steroid hormones were shown to regulate heart growth and function. Estrogen and estrogenic endocrine-disrupting chemicals (bisphenol A) promote arrhythmia in the female heart via alteration of calcium handling (17). Progesterone has been investigated independently of estrogen and affects blood pressure and the cardiovascular system. The progesterone effect on vessel tissue can be mediated by calcium channel current activation and adjustment of the free, cytosolic calcium content (18). Progesterone treatment during cardiomyocyte differentiation of mESCs resulted in decreases in the beating ratio, the mRNA levels of calcium channel and contraction-related genes, and the cytosolic calcium level (12). Endocrine-disrupting chemicals and steroid hormones have similar effects (19, 20).

This study examined the effects of P4, OP, and BPA on cardiomyocyte differentiation of mESCs. Important methods of confirming the differentiation into cardiomyocytes demonstrate the gene expressions of cardiomyocytes markers and beating, etc. (21, 22). BPA changes gene expression of intestinal and metabolic factors and glucose transporters (23). Acute BPA exposure modifies cardiomyocyte function, resulting in a slowed in spontaneous beating rate in the neonatal rat ventricular myocytes and an increase in the beat rate variability (16). BPA exposure also impairs intracellular calcium handling, resulting in reduced calcium transient amplitude, increased calcium transient uptake, and prolonged duration. In this study, we measured the beating to confirm that it was differentiated. The beating ratio of the differentiated cardiomyocytes was decreased by the P4, OP, and BPA treatments, indicating that OP and BPA affect cardiac differentiation of mESCs and the contraction of differentiated cardiomyocytes. The previous study has shown that acute BPA exposure modifies cardiomyocyte function, resulting in slowed spontaneous beating rate in the neonatal rat ventricular myocytes and increased beat rate variability (16). Treatment of canine ventricular cardiac cells with calcium chelators determines the action potential durations in these cells (24). Zimna et al. demonstrate the relationship between expression of Hif-1a and proangiogenic genes such as Vegf-a and Plgf and their receptors during myocardial hypoxia (25). BPA exposure also impairs intracellular calcium handling, resulting in reduced calcium transient amplitude, increased calcium transient uptake, and prolonged duration. In previous studies, P4 decreases the mRNA levels of Ca2+ binding-related genes (Ryr2, Trpv2, Calm2, and Mylk3) and the cardiac development and morphogenesis genes (Rbp4, Ly6e, and Gata4). The co-administration of the antagonist RU486 and P4 restores the decreased mRNA levels of these genes. Those results demonstrate the cardiac development is controlled through progesterone receptor signaling (12, 18). In the present result, progesterone receptor expression was increased by OP and BPA treatments and reversed with RU486, indicating that the effects of OP, BPA, and P4 are dependent on the progesterone receptors. TRPV2 expression was down-regulated by P4, OP, and BPA treatments and this expression was restored by RU 486. TRPV2 channel is critical for the maintenance of cardiac structure and function (21). TRPV2 ablation from the heart of adult mice decreases cardiac function and then results in the destruction (21) of intercalated discs of cardiac muscle and the impairment of calcium handling in single cardiac myocytes (26).

Calcium contraction-related genes including RyR2, Calm2, and Mylk3 were down-regulated by P4, OP, and BPA, and the reductions in these three genes were restored by RU486. These results indicate that, as endocrine-disrupting substances, OP and BPA are functionally similar to P4; these chemicals can control cardiac development through the progesterone receptor. A previous study showed that exposure to progesterone during pregnancy increased the incidence of malformations (6). These results suggest that progesterone is modestly required for the early development of the fetus and that high levels of progesterone may interfere with normal differentiation into myocardial cells.

The measurement of the intracellular calcium levels using confocal microscopy showed that treatment with P4, OP, and BPA delayed the time required to reach maximal levels of intracellular calcium. Thus, the expression of TRPV2 and RyR2 are reduced by P4, OP, and BPA, and calcium in the cells is not smoothly regulated. The cytosolic calcium level in the RU486-treated group was similar to that of the vehicle-treated group. With nifedipine, an L-type calcium channel blocker (27), the cytosolic calcium level was decreased in the VE and RU486-treated groups, but there was no significant change in the groups treated with P4, OP, and BPA. In P4, OP, and BPA treatments, the expression of TRPV2 is low and its function is defective, so no significant changes are expected with nifedipine treatment. In previous studies, cardiomyocytes from TRPV2-knockout mice did not form intercalated discs, and low cytosolic calcium levels were detected. These results indicate that calcium influx through TRPV2 affects cardiac differentiation and differentiated cardiomyocytes.

Beating is the endpoint of cardiomyocyte differentiation and is related to calcium influx (28). Intracellular calcium levels are regulated by SR calcium channels including RYR and SERCAs. Intracellular calcium then forms a complex with calmodulin (CaM); this Ca-CaM complex can activate MLCK, which causes contraction of the sarcomere (29). Treatment with P4, OP, and BPA decreased the expression of the calcium contraction-related gene RyR2, Calm2, and Mylk3 and reduced the beating ratio of differentiated cardiomyocytes. When cells were co-treated with RU486, RyR2, Calm2, and Mylk3 mRNA expressions were restored.

In summary, the actions of OP and BPA are quite similar to P4 function. P4, OP, and BPA have an inhibitory effect on cardiomyocyte differentiation of mESCs via the progesterone receptor. P4, OP, and BPA decreased the expression levels of contraction-related genes such as Ryr2, Calm2, Mylk3, and the intracellular calcium inlet channel TRPV2. This effectively decreases the intracellular calcium level, resulting in inhibition of cardiomyocyte differentiation and decreased beating ratio.

Acknowledgments: This research was funded by National Research Foundation of Korea (NRF) (2017R1A2B2005031).

Conflict of interests: None declated.

REFERENCES

1. Doetschman TC, Eistetter H, Katz M, Schmidt W, Kemler R. The in vitro development of blastocyst-derived embryonic stem cell lines: formation of visceral yolk sac, blood islands and myocardium. J Embryol Exp Morphol 1985; 87: 27-45.
2. Robbins J, Gulick J, Sanchez A, Howles P, Doetschman T. Mouse embryonic stem cells express the cardiac myosin heavy chain genes during development in vitro. J Biol Chem 1990; 265: 11905-11909.
3. Liu H, Jiang Q, Ju Z, Guan S, He B. Pentraxin 3 promotes cardiac differentiation of mouse embryonic stem cells through JNK signaling pathway. Cell Biol Int 2018; 42: 1556-1563.
4. Kurokawa J, Furukawa T. Non-genomic action of sex steroid hormones and cardiac repolarization. Biol Pharm Bull 2013; 36: 8-12.
5. Check JH, Rankin A, Teichman M. The risk of fetal anomalies as a result of progesterone therapy during pregnancy. Fertil Steril 1986; 45: 575-577.
6. Harlap S, Prywes R, Davies AM. Letter: birth defects and oestrogens and progesterones in pregnancy. Lancet 1975; 1: 682-683.
7. Viola HM, Hool LC. How does calcium regulate mitochondrial energetics in the heart? - new insights. Heart Lung Circ 2014; 23: 602-609.
8. Johnson BD, Zheng W, Korach KS, Scheuer T, Catterall WA, Rubanyi GM. Increased expression of the cardiac L-type calcium channel in estrogen receptor-deficient mice. J Gen Physiol 1997; 110: 135-140.
9. Fijnvandraat AC, van Ginneken AC, de Boer PA, et al. Cardiomyocytes derived from embryonic stem cells resemble cardiomyocytes of the embryonic heart tube. Cardiovasc Res 2003; 58: 399-409.
10. Sejersted OM. Calcium controls cardiac function - by all means! J Physiol 2011; 589 (Pt 12): 2919-2920.
11. Liu W, Yasui K, Opthof T, et al. Developmental changes of Ca(2+) handling in mouse ventricular cells from early embryo to adulthood. Life Sci 2002; 71: 1279-1292.
12. Kang HY, Choi YK, Jeung EB. Inhibitory effect of progesterone during early embryonic development: suppression of myocardial differentiation and calcium-related transcriptome by progesterone in mESCs: Progesterone disturb cardiac differentiation of mESCs through lower cytosolic Ca(2). Reprod Toxicol 2016; 64: 169-179.
13. Higa A, Hoshi H, Yanagisawa Y, et al. Evaluation system for arrhythmogenic potential of drugs using human-induced pluripotent stem cell-derived cardiomyocytes and gene expression analysis. J Toxicol Sci 2017; 42: 755-761.
14. Park M, Yoon YS. Cardiac regeneration with human pluripotent stem cell-derived cardiomyocytes. Korean Circ J 2018; 48: 974-988.
15. Sala L, Gnecchi M, Schwartz PJ. Long QT. Syndrome modelling with cardiomyocytes derived from human-induced pluripotent stem cells. Arrhythm Electrophysiol Rev 2019; 8:105-110.
16. Ramadan M, Sherman M, Jaimes R, Chaluvadi A, Swift L, Posnack NG. Disruption of neonatal cardiomyocyte physiology following exposure to bisphenol-A. Sci Rep 2018; 8: 7356. doi: 10.1038/s41598-018-25719-8
17. Yan S, Chen Y, Dong M, Song W, Belcher SM, Wang HS. Bisphenol A and 17beta-estradiol promote arrhythmia in the female heart via alteration of calcium handling. PLoS One 2011; 6: e25455. doi: 10.1371/journal.pone.0025455
18. Barbagallo M, Dominguez LJ, Licata G, et al. Vascular effects of progesterone : role of cellular calcium regulation. Hypertension 2001; 37: 142-147.
19. Rehan M, Ahmad E, Sheikh IA, et al. Androgen and progesterone receptors are targets for bisphenol A (BPA), 4-methyl-2,4-bis-(p-hydroxyphenyl)pent-1-ene - a potent metabolite of BPA, and 4-tert-octylphenol: a computational insight. PLoS One 2015; 10: e0138438. doi: 10.1371/journal.pone.0138438
20. Baker ME, Chandsawangbhuwana C. 3D models of MBP, a biologically active metabolite of bisphenol A, in human estrogen receptor alpha and estrogen receptor beta. PLoS One 2012; 7: e46078. doi: 10.1371/journal.pone.0046078
21. Seki T, Yuasa S, Kusumoto D, et al. Generation and characterization of functional cardiomyocytes derived from human T cell-derived induced pluripotent stem cells. PLoS One 2014; 9: e85645. doi: 10.1371/journal.pone.0085645
22. Mummery C, Ward D, van den Brink CE, et al. Cardiomyocyte differentiation of mouse and human embryonic stem cells. J Anat 2002; 200: 233-242.
23. Ahangarpour A, Afshari G, Mard SA, Khodadadi A, Hashemitabar M. Preventive effects of procyanidin A2 on glucose homeostasis, pancreatic and duodenal homebox 1, and glucose transporter 2 gene expression disturbance induced by bisphenol A in male mice. J Physiol Pharmacol 2016; 67: 243-252.
24. Horvath B, Szentandrassy N, Veress R, et al. Effect of the intracellular calcium concentration chelator BAPTA acetoxy-methylester on action potential duration in canine ventricular myocytes. J Physiol Pharmacol 2018; 69: 99-107.
25. Zimna A, Wiernicki B, Kolanowski T, et al. Influence of hypoxia prevailing in post-infarction heart on proangiogenic gene expression and biological features of human myoblast cells applied as a pro-regenerative therapeutic tool. J Physiol Pharmacol 2018; 69: 859-874.
26. Katanosaka Y, Iwasaki K, Ujihara Y, et al. TRPV2 is critical for the maintenance of cardiac structure and function in mice. Nat Commun 2014; 5: 3932. doi: 10.1038/ncomms4932
27. Rast G, Weber J, Disch C, Schuck E, Ittrich C, Guth BD. An integrated platform for simultaneous multi-well field potential recording and Fura-2-based calcium transient ratiometry in human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes. J Pharmacol Toxicol Methods 2015; 75: 91-100.
28. Puceat M, Jaconi M. Ca2+ signalling in cardiogenesis. Cell Calcium 2005; 38:
29. Martinsen A, Dessy C, Morel N. Regulation of calcium channels in smooth muscle: new insights into the role of myosin light chain kinase. Channels (Austin) 2014; 8: 402-413.