http://informahealthcare.com/ebm ISSN: 1536-8378 (print), 1536-8386 (electronic) Electromagn Biol Med, Early Online: 1–8 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/15368378.2014.881744
ORIGINAL ARTICLE
Effects of extremely low frequency electromagnetic fields on intracellular calcium transients in cardiomyocytes Jinhong Wei, Junqing Sun, Hao Xu, Liang Shi, Lijun Sun, and Jianbao Zhang
Electromagn Biol Med Downloaded from informahealthcare.com by Dalhousie University on 01/02/15 For personal use only.
The Key Laboratory of Biomedical Information Engineering of Ministry of Education, School of Life Science and Technology, Xi’an Jiaotong University, Xi’an, People’s Republic of China Abstract
Keywords
Calcium transients play an essential role in cardiomyocytes and electromagnetic fields (EMF) and affect intracellular calcium levels in many types of cells. Effects of EMF on intracellular calcium transients in cardiomyocytes are not well studied. The aim of this study was to assess whether extremely low frequency electromagnetic fields (ELF-EMF) could affect intracellular calcium transients in cardiomyocytes. Cardiomyocytes isolated from neonatal Sprague-Dawley rats were exposed to rectangular-wave pulsed ELF-EMF at four different frequencies (15 Hz, 50 Hz, 75 Hz and 100 Hz) and at a flux density of 2 mT. Intracellular calcium concentration ([Ca2+]i) was measured using Fura-2/AM and spectrofluorometry. Perfusion of cardiomyocytes with a high concentration of caffeine (10 mM) was carried out to verify the function of the cardiac Na+/Ca2+ exchanger (NCX) and the activity of sarco(endo)-plasmic reticulum Ca2+-ATPase (SERCA2a). The results showed that ELF-EMF enhanced the activities of NCX and SERCA2a, increased [Ca2+]i baseline level and frequency of calcium transients in cardiomyocytes and decreased the amplitude of calcium transients and calcium level in sarcoplasmic reticulum. These results indicated that ELF-EMF can regulate calcium-associated activities in cardiomyocytes.
Cardiomyocyte, ELF-EMF, Ca2+ transients, Na+/Ca2+ exchanger, sarcoplasmic reticulum
Introduction It has been reported that extremely low frequency electromagnetic fields (ELF-EMF) can affect cell functions, such as cellular proliferation and differentiation (Esmail et al., 2012; Foletti et al., 2009; Ross, 1990; Ventura et al., 2005), apoptosis (Santini et al., 2005), DNA synthesis (Focke et al., 2010), RNA transcription (Goodman et al., 1983), protein expression (Goodman & Henderson, 1988), protein phosphorylation (Sun et al., 2001), microvesicle motility (Go¨lfert et al., 2001) and inhibition of cellular adherence (Jandova´ et al., 2001). Since ELF-EMF can penetrate into tissues, its effect on an organism can be extensive. As early as 1977, pulsed electromagnetic fields (EMF) have been successfully used to treat chronic non-union bone fracture (Bassett et al., 1977). The Food and Drug Administration of the United States has approved EMF as a safe and effective mean for treatment of osteoporosis and bone non-unions (Funk et al., 2009). In the past two decades, effects of EMF on the cardiovascular system have also been investigated. Dicarlo et al. (1999) showed that ELF-EMFs induced stress responses that protect chick embryo myocardium from anoxia damage. Barzelai et al. (2009) revealed that an EMF of 80 mT at 15.95–16.00 Hz protected against coronary artery occlusion. Address correspondence to Jianbao Zhang, School of Life Science and Technology, Xi’an Jiaotong University, Xi’an 710049, People’s Republic of China. Tel: 0086-29-82665942. Fax: 0086-29-82668664. E-mail: [email protected]
History Received 22 August 2013 Revised 6 January 2014 Accepted 7 January 2014 Published online 5 February 2014
Albertini et al. (1999) found that exposure to a 3-mT 75-Hz magnetic field for 18 h significantly reduced necrotic area in rats subjected to acute myocardial infarction. These results suggested that EMF exposure may be an effective and noninvasive way to treat cardiovascular-related diseases, even though the mechanisms are not clear. Intracellular calcium (Ca2þ i ) plays an important role in the regulation of excitation–contraction coupling, gene transcription, energy balance and hypertrophic growth in the heart (Bers, 2002, 2008; Xu et al., 2012). Recently, Cui et al. (2013) showed that ELF-EMF (50 Hz, 0.2 mT) exposure inhibited T-type calcium channel in HEK293 cells. Calcium signaling is a possible target of ELF-EMF on biological systems according to a hypothesized ion–protein interactions (Kaiser, 1995; Lednev, 1991). In fact, some in vitro studies showed that EMF changed intracellular calcium concentration ([Ca2+]i) levels in T-lymphocytes, Jurkat cells, neuronal cells, rat pituitary cells, cardiac cells and osteoblasts, although the mechanisms underlying these effects have not been fully understood (Barbier et al., 1996; Craviso et al., 2002; Grassi et al., 2004; Lindstrom et al., 1995; Sert et al., 2011; Yitzhaki, 2011; Zhang et al., 2010). Recently, the effects of ELF-EMF on [Ca2+]i levels were studied in cardiac cells. Exposure to a 50-Hz 0.25 mT EMF in vivo has been shown to increase [Ca2+]i in cardiac ventricle cells of rats (Sert et al., 2011). Exposure to a 16-Hz 40 nT EMF decreased the amplitude of cytosolic Ca2+ transients by affecting the activity of ATP-sensitive potassium channel
2
J. Wei et al.
in cardiac cells (Yitzhaki, 2011). Intracellular calcium is regulated in cardiomyocytes by many ways (Xu et al., 2012). Kaiser (1995) proposed models that focus on calcium as the probable candidate for interaction of EMF in biosystems. In addition to membrane-bound enzymes and pumps, other membrane macromolecules also have been considered as the potential units of cellular EMF interaction. In this study, different frequencies of ELF-EMF were used to stimulated neonatal cardiomyocytes, and intracellular calcium transient characteristics were examined to further investigate the mechanism of interaction of EMF with cardiomyocytes.
Materials and methods
Electromagn Biol Med Downloaded from informahealthcare.com by Dalhousie University on 01/02/15 For personal use only.
Animals and primary cultures of neonatal rat ventricular cardiomyocytes This investigation conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication No. 85-23, revised 1996) and was approved by the Animal Administrative Committee of Xi’an Jiaotong University. Animals were supplied by the Laboratory Animal Center of the Fourth Military Medical University. Neonatal ventricular myocytes were isolated from 1-day-old Sprague-Dawley rats as described previously (Xu et al., 2012). In brief, neonatal rats were anesthetized with isoflurane (1.5–2.0% vol/vol in air) and disinfected with 75% ethanol and then decapitated under aseptic conditions. The heart was quickly removed and placed in ice-cold phosphate buffered saline (PBS) containing 100 U/ml penicillin and 100 mg/ml streptomycin. The ventricles were minced into approximately 1 mm3 fragments and dissociated with 0.1% collagenase II (GIBCO, Grand Island, NY) for 30 min at 37 C. After the cells were precipitated by centrifugation (at room temperature, 50 g for 5 min) and the collagenase solution was removed, the pellet of cells was resuspended in Kraft-Bru¨he solution (composed of (in mM) KCl: 85, K2HPO4: 30, MgSO4: 5, ethylene glycol tetraacetic acid (EGTA): 1, Na2ATP: 5, pyruvate: 5, creatin: 5, taurin: 20 and glucose: 20; titrated to pH ¼ 7.3 with KOH) at room temperature for 15 min. Cardiomyocytes were then plated on a 22 22 mm2 glass coverslip (Fisher Scientific, Pittsburgh, PA) and incubated in Dulbecco’s modified Eagle medium (DMEM; Invitrogen Corporation, Grand Island, NY) supplemented with 15% fetal calf serum (FCS; Hyclone, Logan, UT) in 5% CO2 at 37 C for 24 h. The medium was replaced with a serum-free medium before the cells were exposured to ELF-EMF. Cell viability Cell viability was assessed by the trypan blue exclusion assay performed in each treated group. Trypan blue stain (Sigma, St. Louis, MO) was prepared fresh as a 0.4% solution in 0.9% sodium chloride. The cells were washed in PBS twice and then suspended in 0.25% trypsin (Sigma) for 5 min and centrifuged at 50 g for 5 min. Supernatants were removed, and pellets were resuspended in 100 mL 0.4% trypan blue solution and incubated for 5 min at room temperature. Cells were microscopically counted in a hemocytometer, and the
Electromagn Biol Med, Early Online: 1–8
cell viability was expressed as a percentage of the trypan blue-negative cells. EMF exposure An EMF exposure system that provided a relatively uniform EMF for cells exposure as described previously (Zhang et al., 2007) was used. Briefly, the exposure consists of a waveform generator, amplifier and solenoid. The waveform generator was an extremely-low frequency function generator, which provided rectangular waveforms. After being amplified, the signals were output to the solenoid. A cylindrical solenoid (6 cm long and 8 cm diameter) made of 600 turns of 1 mm diameter copper wire on a plastic tube. The intensity of EMF at the position of the coverslip was measured with a Model 455 DSP Gaussmeter (Lakeshore, Westerville, OH). Frequencies (pulse-width and interpulse-interval in msec) of the ELF-EMF used in the experiments were 15 (10, 57), 50 (3, 17), 75 (2, 11) and 100 (1, 9) Hz. The average flux density was 2 mT. The coverslip, on which cardiomyocytes were grown, was placed in a Warner model RC-26 chamber (Warner Instruments, Hamden, CT), which was fixed in a Warner PH-1 heated platform (Warner Instruments). The platform was held by the stage of an Olympus iX-71 (Olympus Corporation, Tokyo, Japan) inverted microscope in order to measure intracellular calcium. The solenoid was placed on top of the chamber, and cells were located in the bottom center of the solenoid during experiments. The coverslip was perpendicular to the long axis of the solenoid. During the experiment, cardiomyocytes were perfused with normal Tyrode solution (containing in mM NaCl: 140, KCl: 4, CaCl2: 2, MgCl2: 1, glucose: 10 and HEPES: 5; pH 7.4) at a flow rate of 0.8 ml/min and kept at 37 ± 0.1 C by the Warner PH-1 heated platform. The EMF performance of the exposure system was checked daily with an oscilloscope. Figure 1(a) depicts the protocol of EMF exposure. In this study, cardiomyocytes were exposed at different frequencies (0, 15, 50, 75 and 100 Hz). Each condition was repeated 5–7 times, and intracellular calcium transients of 3–5 cardiomyocytes were analyzed for every time. First, normal calcium transients were measured for 60 s as the baseline. And then, the cardiomyocytes were exposure for 180 s with ELF-EMF. Calcium transients were monitored for another 60 s after the field was turned off. Controls were sham-exposed. In the presence of 10 mM caffeine, sarco(endo)-plasmic reticulum Ca2+-ATPase (SERCA2a) is inhibited (Bers, 2000) and other removal pathways (e.g. sarcolemmal Ca2+-ATPase, mitochondrial Ca2+ uniporter) are negligible (Bers, 2002). Then, Ca2+ clearance from the cytoplasm is mainly through Na+/Ca2+ exchanger (NCX) in the plasma membrane. The half-decay time (T1/2) of caffeine-induced Ca2+ transients (C[Ca2+]i) could be used as an index of NCX function (Kohlhaas et al., 2006). The T1/2 of spontaneous Ca2þ i transients (S[Ca2+]i) reflects Ca2+ removal rate from cytoplasm and can be used to evaluate SERCA2a activity (Schaeffer et al., 2009; Shannon et al., 2003). Thus, the function of cardiac SERCA2a and NCX activities could be assessed by the calcium response to the subsequent addition of high concentration caffeine (final concentration of 10 mM). During the experiment of each group, ELF-EMF was applied,
ELF-EMFs and Ca2+ transients
DOI: 10.3109/15368378.2014.881744
3
Statistical analysis All data are represented as the mean ± SEM. Data obtained during exposure (designated as ‘‘exposure’’) were compared with corresponding data obtained before exposure (designated as ‘‘spontaneous’’). Statistical analyses were performed using the paired Student’s t-test or one-way analysis of variance (ANOVA). The Student’s t-test was applied for single comparisons with the spontaneously beating cells group. Comparisons among more than two groups were performed using one-way ANOVA, followed by the Tukey’s post hoc test. Data analysis was carried out by SPSS software (SPSS Inc., Chicago, IL), and a difference was considered statistically significant when p50.05.
Electromagn Biol Med Downloaded from informahealthcare.com by Dalhousie University on 01/02/15 For personal use only.
Results
Figure 1. Experimental protocol to measure [Ca2+]i in cardiomyocytes in the presence and absence of ELF-EMFs. (a) Cardiomyocytes subjected to sham exposure or EMF exposure and (b) caffeine-induced Ca2þ transients (C[Ca2+]i) estimated in normal Tyrode solution or 0 i Na+–0 Ca2+ solution.
and calcium imaging was monitored for the entire process. Cardiomyocytes were perfused with normal Tyrode solution or 0 Na+–0 Ca2+ solution (with LiCl 140 mM and EGTA 10 mM substituted for NaCl and CaCl2; titrated to pH ¼ 7.4 with LiOH) for 30 s and then rapidly with added high concentration caffeine for another 30 s (Figure 1b). Calcium imaging The cardiomyocytes were placed on a glass coverslip and were incubated with 1 mM fura-2/AM in HEPES-buffered physiological saline solution (HPSS, containing in mM, NaCl: 120, KCl: 5.4, Mg2SO4: 0.8, HEPES: 20, CaCl2: 1.8 and Glucose: 10; pH 7.4) for 30 min at room temperature, and then washed three times with HPSS to remove extracellular dye. The coverslip was fixed in the Warner model RC-26 chamber and PH-1 heated platform, which was mounted on an inverted microscope. Fura-2 fluorescence was alternately excited at the wavelengths of 340 nm and 380 nm with a monochromator (TILL Photonics, Polychrome V, Munich, Bavaria, Germany) and focused on the cells via a 40 oil objective (NA ¼ 1.35, U/340, Olympus). Emitted fluorescence at 510 nm was collected by a high-speed cooled CCD camera (Hamamastsu C9100, Shizuoka, Japan) and was recorded with a Simple PCI software (High Performance Imaging Software, Compix, Cranberry, PA). Intracellular free calcium concentration was calculated with the formula: [Ca2+]i ¼ Kd [(R – Rmin)/(Rmax – R)] b (Grynkiewicz et al., 1985), where R is the ratio of 510 nm emitted fluorescence excited at 340 nm and 380 nm, Kd represents the dissociation constant, Rmax and Rmin are the fluorescence ratios under Ca2+-saturating and Ca2+-free conditions measured after cells were treated with 0.1% Triton X-100 and 10 mM EGTA, and b is the fluorescence ratio at 380 nm under Ca2+-free condition to that under Ca2+-saturating condition. All experiments were performed at 37 ± 0.1 C.
No significant changes in calcium parameters were observed during sham exposure. Therefore, sham-exposure data are not presented. Effects of exposure to ELF-EMF at different frequencies on Ca2+ transients The Ca2þ i transients in spontaneously beating cells, dyed with Fura-2/AM, were measured before (named as spontaneous group; left side in Figure 2a) and during (named as exposure group; right side in Figure 2b) ELF-EMF exposure. Figure 2(a) shows representative tracings of F340/F380 fluorescence ratio. Figure 2(b) shows that intracellular resting calcium level increased by 9.23 ± 3.23%, 5.22 ± 1.75%, 12.63 ± 3.09% and 4.77 ± 1.19%, respectively (n ¼ 7 for each group) when cardiomyocytes were exposed to 15, 50, 75 and 100 Hz ELF-EMF. Figure 2(c) shows changes of the Ca2þ transients amplitudes. Compared with the spontaneous i group, the amplitudes of Ca2þ transients significantly i decreased by 39.99 ± 3.91%, 31.51 ± 3.27%, 49.45 ± 3.73% and 31.04 ±2.41%, respectively (n ¼ 7 for each group) when cardiomyocytes were respectively exposured to 15, 50, 75 and 100 Hz ELF-EMF. Figure 2(d) shows that the frequencies of Ca2þ transients in the spontaneous group were significantly i increased by 56.71 ± 2.08%, 26.12 ± 1.17%, 130.28 ± 1.95% and 29.38 ± 4.93%, respectively (n ¼ 7 for each group) when cardiomyocytes were exposed to 15, 50, 75 and 100 Hz ELF-EMF. Altered function of NCX and SERCA2a by ELF-EMF Application of a high concentration of caffeine (10 mM) caused a rapid Ca2+ increase due to release of calcium from the sarcoplasmic reticulum (SR) (Figure 3a). With caffeine treatment, the clearance of calcium from the cytoplasm is mainly through NCX in the plasma membrane. Thus, the halfdecay time (T1/2) of C[Ca2+]i could be used as an index of NCX function (Kohlhaas et al., 2006). Figure 3(b) shows that the different ELF-EMF frequencies reduced T1/2 of C[Ca2+]i by 38.26 ± 1.54%, 52.18 ± 4.00%, 60.79 ± 2.32% and 43.22 ± 3.25% (n ¼ 5 for each group), respectively. In the rat cardiomyocyte, immediately after contraction, 92% of the released Ca2+ is sequestered back to the SR via SERCA2a, close to 7% is pumped out of the cell via NCX and 1% by the slow systems (Bers, 2002). The T1/2 of S[Ca2+]i
Electromagn Biol Med Downloaded from informahealthcare.com by Dalhousie University on 01/02/15 For personal use only.
4
J. Wei et al.
Electromagn Biol Med, Early Online: 1–8
Figure 2. Effects of ELF-EMF at different frequencies (15, 50, 75 and 100 Hz) on intracellular calcium transients in cardiomyocytes. (a) Representative tracings of F340/F380 fluorescence ratio, left side is the spontaneous group and right side is the exposure group. (b) [Ca2+]i transients baseline, (c) amplitudes of Ca2þ transients and (d) frequency of Ca2þ transients. Data are presented as the mean ± SEM (n ¼ 7, for each group). *p50.05 vs. i i spontaneous group.
reflects Ca2+ removal rate from cytoplasm can be used to evaluate SERCA2a activity (Schaeffer et al., 2009; Shannon et al., 2003). Figure 2(a) shows the Ca2þ transients under i spontaneous conditions. Accordingly, Figure 3(c) shows that the T1/2 of S[Ca2+]i during exposure of ELF-EMF at different frequencies decreased by 59.03 ± 3.13%, 47.85 ± 2.74%, 71.9 ± 1.49% and 41.4 ± 2.41% (n ¼ 5 for each group), respectively.
depletes the calcium in SR (Shannon et al., 2003). Figure 4(a) shows representative traces of C[Ca2+]i estimated in 0 Na+–0 Ca2+ solution, and Ca2+ content in SR was dramatically reduced in cardiomyocytes during exposure to ELF-EMF at different frequencies. Figure 4(b) shows that 15, 50, 75 and 100 Hz ELF-EMF reduced the Ca2+ content in SR by 35.25 ± 2.16%, 25.94 ± 3.62%, 53.08 ± 3.09% and 18.00 ± 4.29% (n ¼ 6 for each group), respectively.
Assessment of Ca2+ content in SR
Discussion
+
2+
solution, the caffeine-induced Ca2þ In 0 Na –0 Ca i transients is an index of the SR Ca2+ content because caffeine
In this study, effects of ELF-EMF exposure on intracellular calcium transients in neonatal cardiomyocytes were
ELF-EMFs and Ca2+ transients
Electromagn Biol Med Downloaded from informahealthcare.com by Dalhousie University on 01/02/15 For personal use only.
DOI: 10.3109/15368378.2014.881744
5
Figure 2. Continued.
investigated. Our results showed that ELF-EMF at 2 mT and 15, 50, 75 and 100 Hz increased the resting [Ca2+]i and the frequency of calcium transients and decreased the amplitude of calcium transients and calcium content in SR. Although many studies related to the effects of ELF-EMF on intracellular calcium have been reported, the literature remains controversial and the underlying mechanism has been only explored to a limited extent. For example, ELF-EMF exposure has been found to cause an positive change of intracellular calcium in neurons, cardiomyocytes and osteoblasts (Barbier et al., 1996; Pessina et al., 2001; Sert et al., 2011; Zhang et al., 2010), while others reported a negative effect on intracellular calcium signal after ELF-EMF exposure in human Jurkat cells and bovine chromaffin cells (Galvanovskis et al., 1999; Ikehara et al., 2002). Moreover, there are studies that reported no significant effect of ELF-EMF on bovine chromaffin cells, HL-60 cells and human Jurkat cells (Craviso et al., 2002; Lyle et al., 1997; Sontag, 1998). These results indicated that there exists a complicated mechanism for biological effects of EMF and the effects of EMFs on intracellular calcium. It is necessary to perform well-designed experiments for different kind of cells and EMF. Many of the biological effects of ELF-EMF are thought to be mediated by changes in intracellular Ca2+ (Fanelli
et al., 1999; Karabakhtsiana et al., 1994; Zhou et al., 2002). In our study, changes in Ca2+ transients within individual cardiomyocytes induced by ELF-EMF were demonstrated in real time. Our results showed that ELF-EMFs markedly increased the resting [Ca2+]i, which is consistent with the result of Sert et al. (2011). Other than an elevated resting [Ca2+]i, we also found that EMF changed the activities of NCX and SERCA2a, and increased the frequency of calcium transients and decreased the amplitude of calcium transients. Intracellular Ca2+ is regulated by the Ca2+ handling proteins, namely, voltage-gated Ca2+ channels, ryanodine receptors, SERCA2a and NCX. ELF-EMF exposure can modify the biophysical properties of cell membranes, including their permeability to Ca2+ ions (Grassi et al., 2004; Panagopoulos et al., 2002). It was known that ELF-EMF upregulated L-type calcium channel expression (Piacentini et al., 2008) and inhibited T-type calcium channel expression (Cui et al., 2013), but there has been no study on the effects of ELF-EMF on SERCA2a and NCX. Both SERCA2a and NCX are involved in the removal of the released calcium in rat cardiomyocytes after contraction. Our results showed that NCX function and SERCA2a activity are significantly enhanced during exposure of ELF-EMF (Figure 3). The changed activities of NCX and SERCA2a
J. Wei et al.
Electromagn Biol Med, Early Online: 1–8
Electromagn Biol Med Downloaded from informahealthcare.com by Dalhousie University on 01/02/15 For personal use only.
6
Figure 3. (a) Representative tracings of caffeine-induced Ca2þ transients (C[Ca2+]i) estimated in Tyrode solution. The horizontal bars above the i tracings indicate the presence of 10 mM caffeine. (b) and (c) show results of changes in half-decay time (T1/2) of caffeine-induced Ca2þ transients i (C[Ca2+]i) and spontaneous Ca2þ transients (S[Ca2+]i), respectively. ‘‘Spontaneous’’ is pooled data from the different treatment groups. Data are i represented as the mean ± SEM (n ¼ 5, for each group). *p50.05 vs. spontaneous group.
will make calcium leak easily and then increased the frequency of calcium transients. Diastolic Ca2+ leak and intracellular Ca2+ overload in cardiomyocytes are usually recognized as an increased frequency of spontaneous Ca2+ sparks/transients (Gangopadhyay & Ikemoto, 2010; Neef et al., 2010; Ogrodnik & Niggli, 2010). Moreover, decreases
in amplitude of calcium transients and calcium content in SR could be due to a shorter time for refilling of SR and intracellular calcium. The mechanism underlying the effects of EMF on activities of NCX and SERCA2a is not clear. Tsong et al. (1992) proposed an electro-conformational coupling for
Electromagn Biol Med Downloaded from informahealthcare.com by Dalhousie University on 01/02/15 For personal use only.
DOI: 10.3109/15368378.2014.881744
ELF-EMFs and Ca2+ transients
7
2+ + 2+ Figure 4. Representative tracings of caffeine-induced Ca2þ solution (a); and sarcoplasmic reticulum i transients (C[Ca ]i) estimated in 0 Na –0 Ca (SR) Ca2+ content (b). Horizontal bars above the tracing indicate the presence of 10 mM caffeine. ‘‘Spontaneous’’ is pooled data from the different treatment groups. Data are represented as the mean ± SEM (n ¼ 6, for each group). *p50.05 vs. spontaneous group.
cellular enzymatic systems. Karimov et al. (1999) proposed a model for the mechanism of interactions of weak EMF with biomolecules. The focus of this model was based on the dependence of natural resonances of biomolecules on the shape of the EMF signal and features of biomolecular structures and active centers containing metal atoms. Grundler & Kaiser (1992) and Kaiser (1995) considered coupling of EMF to intracellular Ca2+ oscillations as a possible mechanism and they explained how non-linear oscillators could manifest specific phenomena including synchronization, sub- and superharmonic resonances and frequency and intensity sensitivity. An explanation of enhanced ion mobility in ion aqueous solutions in the presence of EMF was given by Del Giudice et al. (1998) and Lacy-Hulbert et al. (1998). These processes may act when a cardiomyocyte is exposed to ELF-EMF, but what specific mechanism is really involved will need to be verified by future experiments.
Conclusion Data from the present experiment indicate that ELF-EMFs exposure can alter Ca2þ signaling in cardiomyocytes by i affecting the activities of NCX and SERCA2a.
Declaration of interest The authors report no declarations of interest. This work was supported by a grant from the National Natural Science Foundation of China (No. 31170893).
References Albertini, A., Zucchini, P., Noera, G., et al. (1999). Protective effect of low frequency low energy pulsing electromagnetic fields on acute experimental myocardial infarcts in rats. Bioelectromagnetics 20: 372–377.
Barbier, E., Dufy, B., Veyret, B. (1996). Stimulation of Ca2+ influx in rat pituitary cells under exposure to a 50 Hz magnetic field. Bioelectromagnetics 17:303–311. Barzelai, S., Dayan, A., Feinberg, M. S., et al. (2009). Electromagnetic field at 15.95–16 Hz is cardio protective following acute myocardial infarction. Ann. Biomed. Eng. 37:2093–2104. Bassett, C. A., Pilla, A. A., Pawluk, R. J. (1977). A non-operative salvage of surgically-resistant pseudarthroses and non-unions by pulsing electromagnetic fields: A preliminary report. Clin. Orthop. Relat. Res. 124:128–143. Bers, D. M. (2000). Calcium fluxes involved in control of cardiac myocyte contraction. Circ. Res. 87:275–281. Bers, D. M. (2002). Cardiac excitation-contraction coupling. Nature 415: 198–205. Bers, D. M. (2008). Calcium cycling and signaling in cardiac myocytes. Annu. Rev. Physiol. 70:23–49. Craviso, G. L., Poss, J., Lanctot, C., et al. (2002). Intracellular calcium activity in isolated bovine adrenal chromaffin cells in the presence and absence of 60 Hz magnetic fields. Bioelectromagnetics 23:557–567. Cui, Y., Liu, X., Yang, T., et al. (2013). Exposure to extremely lowfrequency electromagnetic fields inhibits T-type calcium channels via AA/LTE4 signaling pathway. Cell Calcium. [Epub ahead of print]. doi: 10.1016/j.ceca.2013.11.002. Del, G. E., Preparata, G., Vitiello, G. (1988). Water as a free electric dipole laser. Phys. Rev. Lett. 61:1085–1088. DiCarlo, A. L., Farrell, J. M., Litovitz, T. A. (1999). Myocardial protection conferred by electromagnetic fields. Circulation 99:813–816. Esmail, M. Y., Sun, L., Yu, L., et al. (2012). Effects of PEMF and glucocorticoids on proliferation and differentiation of osteoblasts. Electromagn. Biol. Med. 31:375–381. Fanelli, C., Coppola, S., Barone, R., et al. (1999). Magnetic fields increase cell survival by inhibiting apoptosis via modulation of Ca2+ influx. FASEB J. 13:95–102. Focke, F., Schuermann, D., Kuster, N., et al. (2010). DNA fragmentation in human fibroblasts under extremely low frequency electromagnetic field exposure. Mutat. Res. 683:74–83. Foletti, A., Lisi, A., Ledda, M., et al. (2009). Cellular ELF signals as a possible tool in informative medicine. Electromagn. Biol. Med. 28: 71–74. Funk, R. H., Monsees, T., Ozkucur, N. (2009). Electromagnetic effects: From cell biology to medicine. Prog. Histochem. Cytochem. 43: 177–264. Go¨lfert, F., Hofer, A., Thu¨mmler, M., et al. (2001). Extremely low frequency electromagnetic fields and heat shock can increase microvesicle motility in astrocytes. Bioelectromagnetics 22:71–78.
Electromagn Biol Med Downloaded from informahealthcare.com by Dalhousie University on 01/02/15 For personal use only.
8
J. Wei et al.
Galvanovskis, J., Sandblom, J., Bergqvist, B., et al. (1999). Cytoplasmic Ca2+ oscillations in human leukemia T-cells are reduced by 50 Hz magnetic fields. Bioelectromagnetics 20:269–276. Gangopadhyay, J. P., Ikemoto, N. (2010). Intracellular translocation of calmodulin and Ca2+/calmodulin-dependent protein kinase II during the development of hypertrophy in neonatal cardiomyocytes. Biochem. Biophys. Res. Commun. 396:515–521. Goodman, R., Bassett, C., Henderson, A. (1983). Pulsing electromagnetic fields induce cellular transcription. Science 220:1283–1285. Goodman, R., Henderson, A. (1988). Exposure of salivary gland cells to low-frequency electromagnetic fields alters polypeptide synthesis. Proc. Natl. Acad. Sci. USA 85:3928–3932. Grassi, C., D’Ascenzo, M., Torsello, A., et al. (2004). Effects of 50 Hz electromagnetic fields on voltage-gated Ca2+ channels and their role in modulation of neuroendocrine cell proliferation and death. Cell Calcium 35:307–315. Grundler, W., Kaiser, F. (1992). Experimental evidence for coherent excitations correlated with cell growth. Nanobiology 1:163–176. Grynkiewicz, G., Poenie, M., Tsien, R. Y. (1985). A new generation of Ca2+ indicators with greatly improved fluorescence properties. J. Biol. Chem. 260:3440–3450. Ikehara, T., Park, K. H., Yamaguchi, H., et al. (2002). Effects of a time varying strong magnetic field on release of cytosolic free Ca2+ from intracellular stores in cultured bovine adrenal chromaffin cells. Bioelectromagnetics 23:505–515. Jandova´, A., Hurych, J., Pokorny´, J., et al. (2001). Effects of sinusoidal magnetic field on adherence inhibitionv of leukocytes. Electromagn. Biol. Med. 20:397–413. Kaiser, F. (1995). Coherent oscillations: their role in the interaction of weak ELM-fields with cellular systems. Neural Network World 5: 751–762. Karabakhtsiana, R., Broudea, N., Shaltsa, N., et al. (1994). Calcium is necessary in the cell response to EM fields. FEBS Lett. 349:1–6. Karimov, A., Reshetnyak, S., Shcheglov, V. (1999). Two mechanisms of electromagnetic-radiation interaction with information-carrying biomacromolecules. J. Russian Laser Res. 20:1–6. Kohlhaas, M., Zhang, T., Seidler, T., et al. (2006). Increased sarcoplasmic reticulum calcium leak but unaltered contractility by acute CaMKII overexpression in isolated rabbit cardiac myocytes. Circ. Res. 98:235–244. Lacy-Hulbert, A., Metcalfe, J. C., Hesketh, R. (1998). Biological responses to electromagnetic fields. FASEB J. 12:395–420. Lednev, V. V. (1991). Possible mechanism for the influence of weak magnetic fields on biological systems. Bioelectromagnetics 12:71–75. Lindstrom, E., Lindstrom, P., Berglund, A., et al. (1995). Intracellular calcium oscillations in a T-cell line after exposure to extremelylow-frequency magnetic fields with variable frequencies and flux densities. Bioelectromagnetics 16:41–47. Lyle, D. B., Fuchs, T. A., Casamento, J. P., et al. (1997). Intracellular calcium signaling by Jurkat T-lymphocytes exposed to a 60 Hz magnetic field. Bioelectromagnetics 18:439–445. Neef, S., Dybkova, N., Sossalla, S., et al. (2010). CaMKII-dependent diastolic SR Ca2+ leak and elevated diastolic Ca2+ levels in right atrial myocardium of patients with atrial fibrillation. Circ. Res. 106: 1134–1144. Ogrodnik, J., Niggli, E. (2010). Increased Ca2+ leak and spatiotemporal coherence of Ca2+ release in cardiomyocytes during beta-adrenergic stimulation. J. Physiol. 588:225–242.
Electromagn Biol Med, Early Online: 1–8
Panagopoulos, D. J., Karabarbounis, A., Margaritis, L. H. (2002). Mechanism for action of electromagnetic fields on cells. Biochem. Biophys. Res. Commun. 298:95–102. Pessina, G. P., Aldinucci, C., Palmi, M., et al. (2001). Pulsed electromagnetic fields affect the intracellular calcium concentrations in human astrocytoma cells. Bioelectromagnetics 22: 503–510. Piacentini, R., Ripoli, C., Mezzogori, D., et al. (2008). Extremely low-frequency electromagnetic fields promote in vitro neurogenesis via upregulation of Ca(v)1-channel activity. J. Cell. Physiol. 215: 129–139. Ross, S. M. (1990). Combined DC and ELF magnetic fields can alter cell proliferation. Bioelectromagnetics 11:27–36. Santini, M. T., Ferrante, A., Rainaldi, G., et al. (2005). Extremely low frequency (ELF) magnetic fields and apoptosis: A review. Int. J. Radiat. Biol. 81:1–11. Schaeffer, P. J., Desantiago, J., Yang, J., et al. (2009). Impaired contractile function and calcium handling in hearts of cardiac-specific calcineurin b1-deficient mice. Am. J. Physiol. Heart. Circ. Physiol. 297:H1263–H1273. Sert, C., Soker, S., Deniz, M., et al. (2011). Intracellular Ca2+ levels in rat ventricle cells exposed to extremely low frequency magnetic field. Electromagn. Biol. Med. 30:14–20. Shannon, T. R., Pogwizd, S. M., Bers, D. M. (2003). Elevated sarcoplasmic reticulum Ca2+ leak in intact ventricular myocytes from rabbits in heart failure. Circ. Res. 93:592–594. Sontag, W. (1998). Action of extremely low frequency electric fields on the cytosolic calcium concentration of differentiated HL-60 cells: Nonactivated cells. Bioelectromagnetics 19:32–40. Sun, W., Chiang, H., Fu, Y., et al. (2001). Exposure to 50 Hz electromagnetic fields induces the phosphorylation and activity of stress-activated protein kinase in cultured cells. Electromagn. Biol. Med. 20:415–423. Tsong, T. Y. (1992). Molecular recognition and processing of periodic signals in cells: Study of activation of membrane ATPases by alternating electric fields. Biochim. Biophys. Acta 1113: 53–71. Ventura, C., Maioli, M., Asara, Y., et al. (2005). Turning on stem cell cardiogenesis with extremely low frequency magnetic fields. FASEB J. 19:155–157. Xu, H., Zhang, Y., Sun, J., et al. (2012). Effect of distinct sources of Ca2+ on cardiac hypertrophy in cardiomyocytes. Electromagn. Biol. Med. 237:271–278. Yitzhaki, S. (2011). Weak magnetic field at 16 Hz affects cardiac myocyte Ca2+ transients and reduces cells damage caused by hypoxia. Open Optics J. 5:33–39. Zhang, X., Liu, X., Pan, L., et al. (2010). Magnetic fields at extremely low-frequency (50 Hz, 0.8 mT) can induce the uptake of intracellular calcium levels in osteoblasts. Biochem. Biophys. Res. Commun. 396: 662–666. Zhang, X., Zhang, J., Qu, X., et al. (2007). Effects of different extremely low-frequency electromagnetic fields on osteoblasts. Electromagn. Biol. Med. 26:167–177. Zhou, J., Yao, G., Zhang, J., et al. (2002). CREB DNA binding activation by a 50Hz magnetic field in HL60 cells is dependent on extra- and intracellular Ca2+ but not PKA, PKC, ERK, or p38 MAPK. Biochem. Biophys. Res. Commun. 296:1013–1018.
ORIGINAL ARTICLE
Effects of extremely low frequency electromagnetic fields on intracellular calcium transients in cardiomyocytes Jinhong Wei, Junqing Sun, Hao Xu, Liang Shi, Lijun Sun, and Jianbao Zhang
Electromagn Biol Med Downloaded from informahealthcare.com by Dalhousie University on 01/02/15 For personal use only.
The Key Laboratory of Biomedical Information Engineering of Ministry of Education, School of Life Science and Technology, Xi’an Jiaotong University, Xi’an, People’s Republic of China Abstract
Keywords
Calcium transients play an essential role in cardiomyocytes and electromagnetic fields (EMF) and affect intracellular calcium levels in many types of cells. Effects of EMF on intracellular calcium transients in cardiomyocytes are not well studied. The aim of this study was to assess whether extremely low frequency electromagnetic fields (ELF-EMF) could affect intracellular calcium transients in cardiomyocytes. Cardiomyocytes isolated from neonatal Sprague-Dawley rats were exposed to rectangular-wave pulsed ELF-EMF at four different frequencies (15 Hz, 50 Hz, 75 Hz and 100 Hz) and at a flux density of 2 mT. Intracellular calcium concentration ([Ca2+]i) was measured using Fura-2/AM and spectrofluorometry. Perfusion of cardiomyocytes with a high concentration of caffeine (10 mM) was carried out to verify the function of the cardiac Na+/Ca2+ exchanger (NCX) and the activity of sarco(endo)-plasmic reticulum Ca2+-ATPase (SERCA2a). The results showed that ELF-EMF enhanced the activities of NCX and SERCA2a, increased [Ca2+]i baseline level and frequency of calcium transients in cardiomyocytes and decreased the amplitude of calcium transients and calcium level in sarcoplasmic reticulum. These results indicated that ELF-EMF can regulate calcium-associated activities in cardiomyocytes.
Cardiomyocyte, ELF-EMF, Ca2+ transients, Na+/Ca2+ exchanger, sarcoplasmic reticulum
Introduction It has been reported that extremely low frequency electromagnetic fields (ELF-EMF) can affect cell functions, such as cellular proliferation and differentiation (Esmail et al., 2012; Foletti et al., 2009; Ross, 1990; Ventura et al., 2005), apoptosis (Santini et al., 2005), DNA synthesis (Focke et al., 2010), RNA transcription (Goodman et al., 1983), protein expression (Goodman & Henderson, 1988), protein phosphorylation (Sun et al., 2001), microvesicle motility (Go¨lfert et al., 2001) and inhibition of cellular adherence (Jandova´ et al., 2001). Since ELF-EMF can penetrate into tissues, its effect on an organism can be extensive. As early as 1977, pulsed electromagnetic fields (EMF) have been successfully used to treat chronic non-union bone fracture (Bassett et al., 1977). The Food and Drug Administration of the United States has approved EMF as a safe and effective mean for treatment of osteoporosis and bone non-unions (Funk et al., 2009). In the past two decades, effects of EMF on the cardiovascular system have also been investigated. Dicarlo et al. (1999) showed that ELF-EMFs induced stress responses that protect chick embryo myocardium from anoxia damage. Barzelai et al. (2009) revealed that an EMF of 80 mT at 15.95–16.00 Hz protected against coronary artery occlusion. Address correspondence to Jianbao Zhang, School of Life Science and Technology, Xi’an Jiaotong University, Xi’an 710049, People’s Republic of China. Tel: 0086-29-82665942. Fax: 0086-29-82668664. E-mail: [email protected]
History Received 22 August 2013 Revised 6 January 2014 Accepted 7 January 2014 Published online 5 February 2014
Albertini et al. (1999) found that exposure to a 3-mT 75-Hz magnetic field for 18 h significantly reduced necrotic area in rats subjected to acute myocardial infarction. These results suggested that EMF exposure may be an effective and noninvasive way to treat cardiovascular-related diseases, even though the mechanisms are not clear. Intracellular calcium (Ca2þ i ) plays an important role in the regulation of excitation–contraction coupling, gene transcription, energy balance and hypertrophic growth in the heart (Bers, 2002, 2008; Xu et al., 2012). Recently, Cui et al. (2013) showed that ELF-EMF (50 Hz, 0.2 mT) exposure inhibited T-type calcium channel in HEK293 cells. Calcium signaling is a possible target of ELF-EMF on biological systems according to a hypothesized ion–protein interactions (Kaiser, 1995; Lednev, 1991). In fact, some in vitro studies showed that EMF changed intracellular calcium concentration ([Ca2+]i) levels in T-lymphocytes, Jurkat cells, neuronal cells, rat pituitary cells, cardiac cells and osteoblasts, although the mechanisms underlying these effects have not been fully understood (Barbier et al., 1996; Craviso et al., 2002; Grassi et al., 2004; Lindstrom et al., 1995; Sert et al., 2011; Yitzhaki, 2011; Zhang et al., 2010). Recently, the effects of ELF-EMF on [Ca2+]i levels were studied in cardiac cells. Exposure to a 50-Hz 0.25 mT EMF in vivo has been shown to increase [Ca2+]i in cardiac ventricle cells of rats (Sert et al., 2011). Exposure to a 16-Hz 40 nT EMF decreased the amplitude of cytosolic Ca2+ transients by affecting the activity of ATP-sensitive potassium channel
2
J. Wei et al.
in cardiac cells (Yitzhaki, 2011). Intracellular calcium is regulated in cardiomyocytes by many ways (Xu et al., 2012). Kaiser (1995) proposed models that focus on calcium as the probable candidate for interaction of EMF in biosystems. In addition to membrane-bound enzymes and pumps, other membrane macromolecules also have been considered as the potential units of cellular EMF interaction. In this study, different frequencies of ELF-EMF were used to stimulated neonatal cardiomyocytes, and intracellular calcium transient characteristics were examined to further investigate the mechanism of interaction of EMF with cardiomyocytes.
Materials and methods
Electromagn Biol Med Downloaded from informahealthcare.com by Dalhousie University on 01/02/15 For personal use only.
Animals and primary cultures of neonatal rat ventricular cardiomyocytes This investigation conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication No. 85-23, revised 1996) and was approved by the Animal Administrative Committee of Xi’an Jiaotong University. Animals were supplied by the Laboratory Animal Center of the Fourth Military Medical University. Neonatal ventricular myocytes were isolated from 1-day-old Sprague-Dawley rats as described previously (Xu et al., 2012). In brief, neonatal rats were anesthetized with isoflurane (1.5–2.0% vol/vol in air) and disinfected with 75% ethanol and then decapitated under aseptic conditions. The heart was quickly removed and placed in ice-cold phosphate buffered saline (PBS) containing 100 U/ml penicillin and 100 mg/ml streptomycin. The ventricles were minced into approximately 1 mm3 fragments and dissociated with 0.1% collagenase II (GIBCO, Grand Island, NY) for 30 min at 37 C. After the cells were precipitated by centrifugation (at room temperature, 50 g for 5 min) and the collagenase solution was removed, the pellet of cells was resuspended in Kraft-Bru¨he solution (composed of (in mM) KCl: 85, K2HPO4: 30, MgSO4: 5, ethylene glycol tetraacetic acid (EGTA): 1, Na2ATP: 5, pyruvate: 5, creatin: 5, taurin: 20 and glucose: 20; titrated to pH ¼ 7.3 with KOH) at room temperature for 15 min. Cardiomyocytes were then plated on a 22 22 mm2 glass coverslip (Fisher Scientific, Pittsburgh, PA) and incubated in Dulbecco’s modified Eagle medium (DMEM; Invitrogen Corporation, Grand Island, NY) supplemented with 15% fetal calf serum (FCS; Hyclone, Logan, UT) in 5% CO2 at 37 C for 24 h. The medium was replaced with a serum-free medium before the cells were exposured to ELF-EMF. Cell viability Cell viability was assessed by the trypan blue exclusion assay performed in each treated group. Trypan blue stain (Sigma, St. Louis, MO) was prepared fresh as a 0.4% solution in 0.9% sodium chloride. The cells were washed in PBS twice and then suspended in 0.25% trypsin (Sigma) for 5 min and centrifuged at 50 g for 5 min. Supernatants were removed, and pellets were resuspended in 100 mL 0.4% trypan blue solution and incubated for 5 min at room temperature. Cells were microscopically counted in a hemocytometer, and the
Electromagn Biol Med, Early Online: 1–8
cell viability was expressed as a percentage of the trypan blue-negative cells. EMF exposure An EMF exposure system that provided a relatively uniform EMF for cells exposure as described previously (Zhang et al., 2007) was used. Briefly, the exposure consists of a waveform generator, amplifier and solenoid. The waveform generator was an extremely-low frequency function generator, which provided rectangular waveforms. After being amplified, the signals were output to the solenoid. A cylindrical solenoid (6 cm long and 8 cm diameter) made of 600 turns of 1 mm diameter copper wire on a plastic tube. The intensity of EMF at the position of the coverslip was measured with a Model 455 DSP Gaussmeter (Lakeshore, Westerville, OH). Frequencies (pulse-width and interpulse-interval in msec) of the ELF-EMF used in the experiments were 15 (10, 57), 50 (3, 17), 75 (2, 11) and 100 (1, 9) Hz. The average flux density was 2 mT. The coverslip, on which cardiomyocytes were grown, was placed in a Warner model RC-26 chamber (Warner Instruments, Hamden, CT), which was fixed in a Warner PH-1 heated platform (Warner Instruments). The platform was held by the stage of an Olympus iX-71 (Olympus Corporation, Tokyo, Japan) inverted microscope in order to measure intracellular calcium. The solenoid was placed on top of the chamber, and cells were located in the bottom center of the solenoid during experiments. The coverslip was perpendicular to the long axis of the solenoid. During the experiment, cardiomyocytes were perfused with normal Tyrode solution (containing in mM NaCl: 140, KCl: 4, CaCl2: 2, MgCl2: 1, glucose: 10 and HEPES: 5; pH 7.4) at a flow rate of 0.8 ml/min and kept at 37 ± 0.1 C by the Warner PH-1 heated platform. The EMF performance of the exposure system was checked daily with an oscilloscope. Figure 1(a) depicts the protocol of EMF exposure. In this study, cardiomyocytes were exposed at different frequencies (0, 15, 50, 75 and 100 Hz). Each condition was repeated 5–7 times, and intracellular calcium transients of 3–5 cardiomyocytes were analyzed for every time. First, normal calcium transients were measured for 60 s as the baseline. And then, the cardiomyocytes were exposure for 180 s with ELF-EMF. Calcium transients were monitored for another 60 s after the field was turned off. Controls were sham-exposed. In the presence of 10 mM caffeine, sarco(endo)-plasmic reticulum Ca2+-ATPase (SERCA2a) is inhibited (Bers, 2000) and other removal pathways (e.g. sarcolemmal Ca2+-ATPase, mitochondrial Ca2+ uniporter) are negligible (Bers, 2002). Then, Ca2+ clearance from the cytoplasm is mainly through Na+/Ca2+ exchanger (NCX) in the plasma membrane. The half-decay time (T1/2) of caffeine-induced Ca2+ transients (C[Ca2+]i) could be used as an index of NCX function (Kohlhaas et al., 2006). The T1/2 of spontaneous Ca2þ i transients (S[Ca2+]i) reflects Ca2+ removal rate from cytoplasm and can be used to evaluate SERCA2a activity (Schaeffer et al., 2009; Shannon et al., 2003). Thus, the function of cardiac SERCA2a and NCX activities could be assessed by the calcium response to the subsequent addition of high concentration caffeine (final concentration of 10 mM). During the experiment of each group, ELF-EMF was applied,
ELF-EMFs and Ca2+ transients
DOI: 10.3109/15368378.2014.881744
3
Statistical analysis All data are represented as the mean ± SEM. Data obtained during exposure (designated as ‘‘exposure’’) were compared with corresponding data obtained before exposure (designated as ‘‘spontaneous’’). Statistical analyses were performed using the paired Student’s t-test or one-way analysis of variance (ANOVA). The Student’s t-test was applied for single comparisons with the spontaneously beating cells group. Comparisons among more than two groups were performed using one-way ANOVA, followed by the Tukey’s post hoc test. Data analysis was carried out by SPSS software (SPSS Inc., Chicago, IL), and a difference was considered statistically significant when p50.05.
Electromagn Biol Med Downloaded from informahealthcare.com by Dalhousie University on 01/02/15 For personal use only.
Results
Figure 1. Experimental protocol to measure [Ca2+]i in cardiomyocytes in the presence and absence of ELF-EMFs. (a) Cardiomyocytes subjected to sham exposure or EMF exposure and (b) caffeine-induced Ca2þ transients (C[Ca2+]i) estimated in normal Tyrode solution or 0 i Na+–0 Ca2+ solution.
and calcium imaging was monitored for the entire process. Cardiomyocytes were perfused with normal Tyrode solution or 0 Na+–0 Ca2+ solution (with LiCl 140 mM and EGTA 10 mM substituted for NaCl and CaCl2; titrated to pH ¼ 7.4 with LiOH) for 30 s and then rapidly with added high concentration caffeine for another 30 s (Figure 1b). Calcium imaging The cardiomyocytes were placed on a glass coverslip and were incubated with 1 mM fura-2/AM in HEPES-buffered physiological saline solution (HPSS, containing in mM, NaCl: 120, KCl: 5.4, Mg2SO4: 0.8, HEPES: 20, CaCl2: 1.8 and Glucose: 10; pH 7.4) for 30 min at room temperature, and then washed three times with HPSS to remove extracellular dye. The coverslip was fixed in the Warner model RC-26 chamber and PH-1 heated platform, which was mounted on an inverted microscope. Fura-2 fluorescence was alternately excited at the wavelengths of 340 nm and 380 nm with a monochromator (TILL Photonics, Polychrome V, Munich, Bavaria, Germany) and focused on the cells via a 40 oil objective (NA ¼ 1.35, U/340, Olympus). Emitted fluorescence at 510 nm was collected by a high-speed cooled CCD camera (Hamamastsu C9100, Shizuoka, Japan) and was recorded with a Simple PCI software (High Performance Imaging Software, Compix, Cranberry, PA). Intracellular free calcium concentration was calculated with the formula: [Ca2+]i ¼ Kd [(R – Rmin)/(Rmax – R)] b (Grynkiewicz et al., 1985), where R is the ratio of 510 nm emitted fluorescence excited at 340 nm and 380 nm, Kd represents the dissociation constant, Rmax and Rmin are the fluorescence ratios under Ca2+-saturating and Ca2+-free conditions measured after cells were treated with 0.1% Triton X-100 and 10 mM EGTA, and b is the fluorescence ratio at 380 nm under Ca2+-free condition to that under Ca2+-saturating condition. All experiments were performed at 37 ± 0.1 C.
No significant changes in calcium parameters were observed during sham exposure. Therefore, sham-exposure data are not presented. Effects of exposure to ELF-EMF at different frequencies on Ca2+ transients The Ca2þ i transients in spontaneously beating cells, dyed with Fura-2/AM, were measured before (named as spontaneous group; left side in Figure 2a) and during (named as exposure group; right side in Figure 2b) ELF-EMF exposure. Figure 2(a) shows representative tracings of F340/F380 fluorescence ratio. Figure 2(b) shows that intracellular resting calcium level increased by 9.23 ± 3.23%, 5.22 ± 1.75%, 12.63 ± 3.09% and 4.77 ± 1.19%, respectively (n ¼ 7 for each group) when cardiomyocytes were exposed to 15, 50, 75 and 100 Hz ELF-EMF. Figure 2(c) shows changes of the Ca2þ transients amplitudes. Compared with the spontaneous i group, the amplitudes of Ca2þ transients significantly i decreased by 39.99 ± 3.91%, 31.51 ± 3.27%, 49.45 ± 3.73% and 31.04 ±2.41%, respectively (n ¼ 7 for each group) when cardiomyocytes were respectively exposured to 15, 50, 75 and 100 Hz ELF-EMF. Figure 2(d) shows that the frequencies of Ca2þ transients in the spontaneous group were significantly i increased by 56.71 ± 2.08%, 26.12 ± 1.17%, 130.28 ± 1.95% and 29.38 ± 4.93%, respectively (n ¼ 7 for each group) when cardiomyocytes were exposed to 15, 50, 75 and 100 Hz ELF-EMF. Altered function of NCX and SERCA2a by ELF-EMF Application of a high concentration of caffeine (10 mM) caused a rapid Ca2+ increase due to release of calcium from the sarcoplasmic reticulum (SR) (Figure 3a). With caffeine treatment, the clearance of calcium from the cytoplasm is mainly through NCX in the plasma membrane. Thus, the halfdecay time (T1/2) of C[Ca2+]i could be used as an index of NCX function (Kohlhaas et al., 2006). Figure 3(b) shows that the different ELF-EMF frequencies reduced T1/2 of C[Ca2+]i by 38.26 ± 1.54%, 52.18 ± 4.00%, 60.79 ± 2.32% and 43.22 ± 3.25% (n ¼ 5 for each group), respectively. In the rat cardiomyocyte, immediately after contraction, 92% of the released Ca2+ is sequestered back to the SR via SERCA2a, close to 7% is pumped out of the cell via NCX and 1% by the slow systems (Bers, 2002). The T1/2 of S[Ca2+]i
Electromagn Biol Med Downloaded from informahealthcare.com by Dalhousie University on 01/02/15 For personal use only.
4
J. Wei et al.
Electromagn Biol Med, Early Online: 1–8
Figure 2. Effects of ELF-EMF at different frequencies (15, 50, 75 and 100 Hz) on intracellular calcium transients in cardiomyocytes. (a) Representative tracings of F340/F380 fluorescence ratio, left side is the spontaneous group and right side is the exposure group. (b) [Ca2+]i transients baseline, (c) amplitudes of Ca2þ transients and (d) frequency of Ca2þ transients. Data are presented as the mean ± SEM (n ¼ 7, for each group). *p50.05 vs. i i spontaneous group.
reflects Ca2+ removal rate from cytoplasm can be used to evaluate SERCA2a activity (Schaeffer et al., 2009; Shannon et al., 2003). Figure 2(a) shows the Ca2þ transients under i spontaneous conditions. Accordingly, Figure 3(c) shows that the T1/2 of S[Ca2+]i during exposure of ELF-EMF at different frequencies decreased by 59.03 ± 3.13%, 47.85 ± 2.74%, 71.9 ± 1.49% and 41.4 ± 2.41% (n ¼ 5 for each group), respectively.
depletes the calcium in SR (Shannon et al., 2003). Figure 4(a) shows representative traces of C[Ca2+]i estimated in 0 Na+–0 Ca2+ solution, and Ca2+ content in SR was dramatically reduced in cardiomyocytes during exposure to ELF-EMF at different frequencies. Figure 4(b) shows that 15, 50, 75 and 100 Hz ELF-EMF reduced the Ca2+ content in SR by 35.25 ± 2.16%, 25.94 ± 3.62%, 53.08 ± 3.09% and 18.00 ± 4.29% (n ¼ 6 for each group), respectively.
Assessment of Ca2+ content in SR
Discussion
+
2+
solution, the caffeine-induced Ca2þ In 0 Na –0 Ca i transients is an index of the SR Ca2+ content because caffeine
In this study, effects of ELF-EMF exposure on intracellular calcium transients in neonatal cardiomyocytes were
ELF-EMFs and Ca2+ transients
Electromagn Biol Med Downloaded from informahealthcare.com by Dalhousie University on 01/02/15 For personal use only.
DOI: 10.3109/15368378.2014.881744
5
Figure 2. Continued.
investigated. Our results showed that ELF-EMF at 2 mT and 15, 50, 75 and 100 Hz increased the resting [Ca2+]i and the frequency of calcium transients and decreased the amplitude of calcium transients and calcium content in SR. Although many studies related to the effects of ELF-EMF on intracellular calcium have been reported, the literature remains controversial and the underlying mechanism has been only explored to a limited extent. For example, ELF-EMF exposure has been found to cause an positive change of intracellular calcium in neurons, cardiomyocytes and osteoblasts (Barbier et al., 1996; Pessina et al., 2001; Sert et al., 2011; Zhang et al., 2010), while others reported a negative effect on intracellular calcium signal after ELF-EMF exposure in human Jurkat cells and bovine chromaffin cells (Galvanovskis et al., 1999; Ikehara et al., 2002). Moreover, there are studies that reported no significant effect of ELF-EMF on bovine chromaffin cells, HL-60 cells and human Jurkat cells (Craviso et al., 2002; Lyle et al., 1997; Sontag, 1998). These results indicated that there exists a complicated mechanism for biological effects of EMF and the effects of EMFs on intracellular calcium. It is necessary to perform well-designed experiments for different kind of cells and EMF. Many of the biological effects of ELF-EMF are thought to be mediated by changes in intracellular Ca2+ (Fanelli
et al., 1999; Karabakhtsiana et al., 1994; Zhou et al., 2002). In our study, changes in Ca2+ transients within individual cardiomyocytes induced by ELF-EMF were demonstrated in real time. Our results showed that ELF-EMFs markedly increased the resting [Ca2+]i, which is consistent with the result of Sert et al. (2011). Other than an elevated resting [Ca2+]i, we also found that EMF changed the activities of NCX and SERCA2a, and increased the frequency of calcium transients and decreased the amplitude of calcium transients. Intracellular Ca2+ is regulated by the Ca2+ handling proteins, namely, voltage-gated Ca2+ channels, ryanodine receptors, SERCA2a and NCX. ELF-EMF exposure can modify the biophysical properties of cell membranes, including their permeability to Ca2+ ions (Grassi et al., 2004; Panagopoulos et al., 2002). It was known that ELF-EMF upregulated L-type calcium channel expression (Piacentini et al., 2008) and inhibited T-type calcium channel expression (Cui et al., 2013), but there has been no study on the effects of ELF-EMF on SERCA2a and NCX. Both SERCA2a and NCX are involved in the removal of the released calcium in rat cardiomyocytes after contraction. Our results showed that NCX function and SERCA2a activity are significantly enhanced during exposure of ELF-EMF (Figure 3). The changed activities of NCX and SERCA2a
J. Wei et al.
Electromagn Biol Med, Early Online: 1–8
Electromagn Biol Med Downloaded from informahealthcare.com by Dalhousie University on 01/02/15 For personal use only.
6
Figure 3. (a) Representative tracings of caffeine-induced Ca2þ transients (C[Ca2+]i) estimated in Tyrode solution. The horizontal bars above the i tracings indicate the presence of 10 mM caffeine. (b) and (c) show results of changes in half-decay time (T1/2) of caffeine-induced Ca2þ transients i (C[Ca2+]i) and spontaneous Ca2þ transients (S[Ca2+]i), respectively. ‘‘Spontaneous’’ is pooled data from the different treatment groups. Data are i represented as the mean ± SEM (n ¼ 5, for each group). *p50.05 vs. spontaneous group.
will make calcium leak easily and then increased the frequency of calcium transients. Diastolic Ca2+ leak and intracellular Ca2+ overload in cardiomyocytes are usually recognized as an increased frequency of spontaneous Ca2+ sparks/transients (Gangopadhyay & Ikemoto, 2010; Neef et al., 2010; Ogrodnik & Niggli, 2010). Moreover, decreases
in amplitude of calcium transients and calcium content in SR could be due to a shorter time for refilling of SR and intracellular calcium. The mechanism underlying the effects of EMF on activities of NCX and SERCA2a is not clear. Tsong et al. (1992) proposed an electro-conformational coupling for
Electromagn Biol Med Downloaded from informahealthcare.com by Dalhousie University on 01/02/15 For personal use only.
DOI: 10.3109/15368378.2014.881744
ELF-EMFs and Ca2+ transients
7
2+ + 2+ Figure 4. Representative tracings of caffeine-induced Ca2þ solution (a); and sarcoplasmic reticulum i transients (C[Ca ]i) estimated in 0 Na –0 Ca (SR) Ca2+ content (b). Horizontal bars above the tracing indicate the presence of 10 mM caffeine. ‘‘Spontaneous’’ is pooled data from the different treatment groups. Data are represented as the mean ± SEM (n ¼ 6, for each group). *p50.05 vs. spontaneous group.
cellular enzymatic systems. Karimov et al. (1999) proposed a model for the mechanism of interactions of weak EMF with biomolecules. The focus of this model was based on the dependence of natural resonances of biomolecules on the shape of the EMF signal and features of biomolecular structures and active centers containing metal atoms. Grundler & Kaiser (1992) and Kaiser (1995) considered coupling of EMF to intracellular Ca2+ oscillations as a possible mechanism and they explained how non-linear oscillators could manifest specific phenomena including synchronization, sub- and superharmonic resonances and frequency and intensity sensitivity. An explanation of enhanced ion mobility in ion aqueous solutions in the presence of EMF was given by Del Giudice et al. (1998) and Lacy-Hulbert et al. (1998). These processes may act when a cardiomyocyte is exposed to ELF-EMF, but what specific mechanism is really involved will need to be verified by future experiments.
Conclusion Data from the present experiment indicate that ELF-EMFs exposure can alter Ca2þ signaling in cardiomyocytes by i affecting the activities of NCX and SERCA2a.
Declaration of interest The authors report no declarations of interest. This work was supported by a grant from the National Natural Science Foundation of China (No. 31170893).
References Albertini, A., Zucchini, P., Noera, G., et al. (1999). Protective effect of low frequency low energy pulsing electromagnetic fields on acute experimental myocardial infarcts in rats. Bioelectromagnetics 20: 372–377.
Barbier, E., Dufy, B., Veyret, B. (1996). Stimulation of Ca2+ influx in rat pituitary cells under exposure to a 50 Hz magnetic field. Bioelectromagnetics 17:303–311. Barzelai, S., Dayan, A., Feinberg, M. S., et al. (2009). Electromagnetic field at 15.95–16 Hz is cardio protective following acute myocardial infarction. Ann. Biomed. Eng. 37:2093–2104. Bassett, C. A., Pilla, A. A., Pawluk, R. J. (1977). A non-operative salvage of surgically-resistant pseudarthroses and non-unions by pulsing electromagnetic fields: A preliminary report. Clin. Orthop. Relat. Res. 124:128–143. Bers, D. M. (2000). Calcium fluxes involved in control of cardiac myocyte contraction. Circ. Res. 87:275–281. Bers, D. M. (2002). Cardiac excitation-contraction coupling. Nature 415: 198–205. Bers, D. M. (2008). Calcium cycling and signaling in cardiac myocytes. Annu. Rev. Physiol. 70:23–49. Craviso, G. L., Poss, J., Lanctot, C., et al. (2002). Intracellular calcium activity in isolated bovine adrenal chromaffin cells in the presence and absence of 60 Hz magnetic fields. Bioelectromagnetics 23:557–567. Cui, Y., Liu, X., Yang, T., et al. (2013). Exposure to extremely lowfrequency electromagnetic fields inhibits T-type calcium channels via AA/LTE4 signaling pathway. Cell Calcium. [Epub ahead of print]. doi: 10.1016/j.ceca.2013.11.002. Del, G. E., Preparata, G., Vitiello, G. (1988). Water as a free electric dipole laser. Phys. Rev. Lett. 61:1085–1088. DiCarlo, A. L., Farrell, J. M., Litovitz, T. A. (1999). Myocardial protection conferred by electromagnetic fields. Circulation 99:813–816. Esmail, M. Y., Sun, L., Yu, L., et al. (2012). Effects of PEMF and glucocorticoids on proliferation and differentiation of osteoblasts. Electromagn. Biol. Med. 31:375–381. Fanelli, C., Coppola, S., Barone, R., et al. (1999). Magnetic fields increase cell survival by inhibiting apoptosis via modulation of Ca2+ influx. FASEB J. 13:95–102. Focke, F., Schuermann, D., Kuster, N., et al. (2010). DNA fragmentation in human fibroblasts under extremely low frequency electromagnetic field exposure. Mutat. Res. 683:74–83. Foletti, A., Lisi, A., Ledda, M., et al. (2009). Cellular ELF signals as a possible tool in informative medicine. Electromagn. Biol. Med. 28: 71–74. Funk, R. H., Monsees, T., Ozkucur, N. (2009). Electromagnetic effects: From cell biology to medicine. Prog. Histochem. Cytochem. 43: 177–264. Go¨lfert, F., Hofer, A., Thu¨mmler, M., et al. (2001). Extremely low frequency electromagnetic fields and heat shock can increase microvesicle motility in astrocytes. Bioelectromagnetics 22:71–78.
Electromagn Biol Med Downloaded from informahealthcare.com by Dalhousie University on 01/02/15 For personal use only.
8
J. Wei et al.
Galvanovskis, J., Sandblom, J., Bergqvist, B., et al. (1999). Cytoplasmic Ca2+ oscillations in human leukemia T-cells are reduced by 50 Hz magnetic fields. Bioelectromagnetics 20:269–276. Gangopadhyay, J. P., Ikemoto, N. (2010). Intracellular translocation of calmodulin and Ca2+/calmodulin-dependent protein kinase II during the development of hypertrophy in neonatal cardiomyocytes. Biochem. Biophys. Res. Commun. 396:515–521. Goodman, R., Bassett, C., Henderson, A. (1983). Pulsing electromagnetic fields induce cellular transcription. Science 220:1283–1285. Goodman, R., Henderson, A. (1988). Exposure of salivary gland cells to low-frequency electromagnetic fields alters polypeptide synthesis. Proc. Natl. Acad. Sci. USA 85:3928–3932. Grassi, C., D’Ascenzo, M., Torsello, A., et al. (2004). Effects of 50 Hz electromagnetic fields on voltage-gated Ca2+ channels and their role in modulation of neuroendocrine cell proliferation and death. Cell Calcium 35:307–315. Grundler, W., Kaiser, F. (1992). Experimental evidence for coherent excitations correlated with cell growth. Nanobiology 1:163–176. Grynkiewicz, G., Poenie, M., Tsien, R. Y. (1985). A new generation of Ca2+ indicators with greatly improved fluorescence properties. J. Biol. Chem. 260:3440–3450. Ikehara, T., Park, K. H., Yamaguchi, H., et al. (2002). Effects of a time varying strong magnetic field on release of cytosolic free Ca2+ from intracellular stores in cultured bovine adrenal chromaffin cells. Bioelectromagnetics 23:505–515. Jandova´, A., Hurych, J., Pokorny´, J., et al. (2001). Effects of sinusoidal magnetic field on adherence inhibitionv of leukocytes. Electromagn. Biol. Med. 20:397–413. Kaiser, F. (1995). Coherent oscillations: their role in the interaction of weak ELM-fields with cellular systems. Neural Network World 5: 751–762. Karabakhtsiana, R., Broudea, N., Shaltsa, N., et al. (1994). Calcium is necessary in the cell response to EM fields. FEBS Lett. 349:1–6. Karimov, A., Reshetnyak, S., Shcheglov, V. (1999). Two mechanisms of electromagnetic-radiation interaction with information-carrying biomacromolecules. J. Russian Laser Res. 20:1–6. Kohlhaas, M., Zhang, T., Seidler, T., et al. (2006). Increased sarcoplasmic reticulum calcium leak but unaltered contractility by acute CaMKII overexpression in isolated rabbit cardiac myocytes. Circ. Res. 98:235–244. Lacy-Hulbert, A., Metcalfe, J. C., Hesketh, R. (1998). Biological responses to electromagnetic fields. FASEB J. 12:395–420. Lednev, V. V. (1991). Possible mechanism for the influence of weak magnetic fields on biological systems. Bioelectromagnetics 12:71–75. Lindstrom, E., Lindstrom, P., Berglund, A., et al. (1995). Intracellular calcium oscillations in a T-cell line after exposure to extremelylow-frequency magnetic fields with variable frequencies and flux densities. Bioelectromagnetics 16:41–47. Lyle, D. B., Fuchs, T. A., Casamento, J. P., et al. (1997). Intracellular calcium signaling by Jurkat T-lymphocytes exposed to a 60 Hz magnetic field. Bioelectromagnetics 18:439–445. Neef, S., Dybkova, N., Sossalla, S., et al. (2010). CaMKII-dependent diastolic SR Ca2+ leak and elevated diastolic Ca2+ levels in right atrial myocardium of patients with atrial fibrillation. Circ. Res. 106: 1134–1144. Ogrodnik, J., Niggli, E. (2010). Increased Ca2+ leak and spatiotemporal coherence of Ca2+ release in cardiomyocytes during beta-adrenergic stimulation. J. Physiol. 588:225–242.
Electromagn Biol Med, Early Online: 1–8
Panagopoulos, D. J., Karabarbounis, A., Margaritis, L. H. (2002). Mechanism for action of electromagnetic fields on cells. Biochem. Biophys. Res. Commun. 298:95–102. Pessina, G. P., Aldinucci, C., Palmi, M., et al. (2001). Pulsed electromagnetic fields affect the intracellular calcium concentrations in human astrocytoma cells. Bioelectromagnetics 22: 503–510. Piacentini, R., Ripoli, C., Mezzogori, D., et al. (2008). Extremely low-frequency electromagnetic fields promote in vitro neurogenesis via upregulation of Ca(v)1-channel activity. J. Cell. Physiol. 215: 129–139. Ross, S. M. (1990). Combined DC and ELF magnetic fields can alter cell proliferation. Bioelectromagnetics 11:27–36. Santini, M. T., Ferrante, A., Rainaldi, G., et al. (2005). Extremely low frequency (ELF) magnetic fields and apoptosis: A review. Int. J. Radiat. Biol. 81:1–11. Schaeffer, P. J., Desantiago, J., Yang, J., et al. (2009). Impaired contractile function and calcium handling in hearts of cardiac-specific calcineurin b1-deficient mice. Am. J. Physiol. Heart. Circ. Physiol. 297:H1263–H1273. Sert, C., Soker, S., Deniz, M., et al. (2011). Intracellular Ca2+ levels in rat ventricle cells exposed to extremely low frequency magnetic field. Electromagn. Biol. Med. 30:14–20. Shannon, T. R., Pogwizd, S. M., Bers, D. M. (2003). Elevated sarcoplasmic reticulum Ca2+ leak in intact ventricular myocytes from rabbits in heart failure. Circ. Res. 93:592–594. Sontag, W. (1998). Action of extremely low frequency electric fields on the cytosolic calcium concentration of differentiated HL-60 cells: Nonactivated cells. Bioelectromagnetics 19:32–40. Sun, W., Chiang, H., Fu, Y., et al. (2001). Exposure to 50 Hz electromagnetic fields induces the phosphorylation and activity of stress-activated protein kinase in cultured cells. Electromagn. Biol. Med. 20:415–423. Tsong, T. Y. (1992). Molecular recognition and processing of periodic signals in cells: Study of activation of membrane ATPases by alternating electric fields. Biochim. Biophys. Acta 1113: 53–71. Ventura, C., Maioli, M., Asara, Y., et al. (2005). Turning on stem cell cardiogenesis with extremely low frequency magnetic fields. FASEB J. 19:155–157. Xu, H., Zhang, Y., Sun, J., et al. (2012). Effect of distinct sources of Ca2+ on cardiac hypertrophy in cardiomyocytes. Electromagn. Biol. Med. 237:271–278. Yitzhaki, S. (2011). Weak magnetic field at 16 Hz affects cardiac myocyte Ca2+ transients and reduces cells damage caused by hypoxia. Open Optics J. 5:33–39. Zhang, X., Liu, X., Pan, L., et al. (2010). Magnetic fields at extremely low-frequency (50 Hz, 0.8 mT) can induce the uptake of intracellular calcium levels in osteoblasts. Biochem. Biophys. Res. Commun. 396: 662–666. Zhang, X., Zhang, J., Qu, X., et al. (2007). Effects of different extremely low-frequency electromagnetic fields on osteoblasts. Electromagn. Biol. Med. 26:167–177. Zhou, J., Yao, G., Zhang, J., et al. (2002). CREB DNA binding activation by a 50Hz magnetic field in HL60 cells is dependent on extra- and intracellular Ca2+ but not PKA, PKC, ERK, or p38 MAPK. Biochem. Biophys. Res. Commun. 296:1013–1018.
