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EMF protects cardiomyocytes against hypoxia-induced injury via heat shock protein 70 activation
Chemico-Biological Interactions 248 (2016) 8e17
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EMF protects cardiomyocytes against hypoxia-induced injury via heat shock protein 70 activation Jinhong Wei, Jie Tong, Liying Yu, Jianbao Zhang* The Key Laboratory of Biomedical Information Engineering of Ministry of Education, School of Life Science and Technology, Xi'an Jiaotong University, Xi'an, 710049, People's Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 September 2015 Received in revised form 4 January 2016 Accepted 7 February 2016 Available online 11 February 2016
Intracellular calcium (Ca2þi) overload induced by chronic hypoxia alters Ca2þi homeostasis, whereas ameliorating calcium homeostasis is believed to be responsible for cardioprotection. We hypothesize that cardioprotection by electromagnetic fields (EMF) exposure may restore Ca2þi homeostasis altered by hypoxia insults. Cardiomyocytes isolated from neonatal Sprague-Dawley rats were exposed to chronic hypoxia (1% O2, 5% CO2, 37 C). We observed that cardiomyocytes injury and hypertrophy were alleviated in hypoxic cardiomyocytes exposed with EMF preconditioning. Compared with hypoxic cardiomyocytes, the diastolic [Ca2þ]i was decreased, the amplitude of Ca2þi oscillations was recovered when cardiomyocytes exposed with EMF. In addition, we also found that EMF exposure significantly increased heat shock protein 70 (HSP70) mRNA expression in hypoxic cardiomyocytes. However, treatment with HSP70 blocker KNK437, almost completely inhibited the EMF induced-cardioprotection and the beneficial effects of Ca2þ oscillation in hypoxic cardiomyocytes. These results suggest that EMF preconditioning ameliorates Ca2þi homeostasis through activating HSP70, thereby producing the cardioprotective effect and reduction in hypoxic cardiomyocytes damage. © 2016 Elsevier Ireland Ltd. All rights reserved.
Keywords: EMF Hypoxia Cardioprotection Calcium homeostasis HSP70
1. Introduction Hypoxia is the natural consequence of some environments (e.g., high altitude, diving), it is also a common feature of many clinical diseases (e.g., sleep apnea syndrome, chronic obstructive pulmonary disease, heart failure, vascular diseases, stroke, sepsis, metabolic myopathies). Hypoxia is generally associated with cardiovascular diseases, and it elicits a variety of functional responses in cardiomyocytes, including cell proliferation [1], hypertrophy [2] and death [3]. Many responses of cells on hypoxia are mediated by Ca2þ signals [4,5]. Intracellular calcium (Ca2þi) plays a central role on regulating contractility, gene transcription, energy balance, hypertrophic growth, and apoptosis in the heart [6e8]. In many instances, one of the constant responses to hypoxia is an increase in intracellular calcium concentration ([Ca2þ]i) via the activation of various plasma membrane Ca2þ conductances, such as voltage-gated Ca2þ channels, ligand-operated Ca2þ channels, and non-specific cation channels [9]. Ca2þi homeostasis is very critical to cell survival and hypoxia can induce cell death by increasing
* Corresponding author. E-mail address: [email protected] (J. Zhang). http://dx.doi.org/10.1016/j.cbi.2016.02.003 0009-2797/© 2016 Elsevier Ireland Ltd. All rights reserved.
[Ca2þ]i [10]. Cardiomyocytes Ca2þ loading is recognized as a major factor in acute hypoxia pathology, and then promotes cell death, contractile dysfunction and arrhythmogenic activity [5]. Dysregulation of Ca2þi homeostasis can lead not only to loss of normal physiological control mechanisms but also to pathological changes in cell growth. If a tool to manipulate Ca2þi was available, it might be a promising approach for protection of cardiomyocytes against hypoxia-mediated injury. Since extremely low frequency electromagnetic fields (ELF-EMF) can penetrate into tissues, ELF-EMF affects cellular functions, such as RNA transcription, DNA synthesis, protein expression, protein phosphorylation, microvesicle motility, proliferation, differentiation and apoptosis. As early as 1977, pulsed electromagnetic fields (PEMF) have been successfully used to treat chronic non-union bone fracture [11]. The Food and Drug Administration of USA has approved EMF as a safe and effective mean for treatment of osteoporosis and bone non-unions [12]. In the past two decades, effects of EMF exposure on the cardiovascular system have also been investigated. Dicarlo et al. showed that ELF-EMF induced stress responses that protect chick embryo myocardium from anoxia damage [13]. Barzelai et al. revealed that an EMF of 80 nT at 15.95e16.00 Hz protected against coronary artery occlusion [14].
J. Wei et al. / Chemico-Biological Interactions 248 (2016) 8e17
Albertini et al. 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 [15]. These results suggested that EMF exposure may be an effective and noninvasive way to treat cardiovascular-related diseases, although the mechanisms are not clear. Calcium signaling is a possible target of ELF-EMF on biological systems according to a hypothesized ion-protein interaction [16]. Numerous studies showed that EMF changed [Ca2þ]i levels in rat pituitary cells, osteoblasts and cardiac cells, even though the mechanisms underlying these effects have not been fully understood [16e19]. Furthermore, our previous studies have found that ELF-EMF can regulate Ca2þi oscillations via affecting Ca2þ associated protein activities in cardiomyocytes [20]. It suggests that EMF exposure may be one of the tools to manipulate the Ca2þi handling under pathological conditions. However, the effects of EMF exposure on Ca2þi handling and mechanisms involved in its cytoprotective effects during myocardial injury induced by hypoxia remain largely unknown. The present study examined the protective effect of EMF exposure and the mechanism underlying this protection against hypoxia-induced injury in neonatal rat cardiomyocytes. 2. Materials and methods 2.1. 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 (Xi'an, Shaanxi, China). Animals were supplied by the Animal Center of the Fourth Military Medical University (Xi'an, Shaanxi, China). Neonatal ventricular myocytes were isolated from 1-day-old Sprague-Dawley rats as described previously [10,20]. In brief, neonatal rats were anesthetized with isoflurane (1.5e2.0% 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, USA) for 30 min at 37 C. After the cells were precipitated by centrifugation (at room temperature, 50xg for 5 min) and the collagenase solution was removed, the pellet of cells was resuspended in Kraft-Brühe (KB) solution (composed of (in mM): KCl 85, K2HPO4 30, MgSO4 5, 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, USA) and incubated in Dulbecco's modified Eagle medium (DMEM; Invitrogen Corporation, Grand Island, NY, USA) supplemented with 15% fetal calf serum (FCS; Hyclone, Logan, UT, USA) in 5% CO2 at 37 C for 24 h. The medium was replaced with a serum-free medium before the cells were exposed to hypoxia. 2.2. EMF exposure An EMF exposure system that provided a relatively uniform electromagnetic field for cells exposure as described previously was used [20]. Briefly, the exposure consists of a waveform generator, amplifier and solenoid. The waveform generator was an extremelylow frequency function generator, which provided rectangular
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waveforms. After being amplified, the signals were output to the solenoid. A cylindrical solenoid (25 cm long and 5 cm diameter) made of 1000 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, USA). Frequencies (pulse-width and interpulse-interval in msec) of the ELF-EMF used in the experiments were 15 Hz. The flux density was 2 mT. Culture plates were placed on the plexiglass shelves in the center of solenoid within an incubator. The cells were perpendicular to the long axis of the solenoid. At the same time, control plates were placed in an identical incubator on the plexiglass shelves with unpowered solenoid. The CO2 concentration, humidity, and temperature of the control, treatment incubators were totally the same and were not affected by EMF. The EMF exposure system was checked daily with an oscilloscope. During hypoxia culture, cells were placed in a hypoxic (1% O2, 5% CO2, 37 C) incubator (Galaxy oxygen control incubator, RS Biotech, Irvine, UK) for 12 h. Control (Normoxia group) were incubated for equivalent periods under normoxic conditions (21% O2, 5%CO2, 37 C). EMF exposure cells (EMF þ Hypoxia group) were exposed with ELF-EMF (15 Hz, 2mT) for 30 min before hypoxic conditions. KNK437 (N-formyl-3,4-methylenedioxy-benzylidene-g-butyrolactam; Sigma, St. Louis, MO, USA), a heat shock protein 70 (HSP70) inhibitor, was added to the culture medium 1 h before EMF exposure (EMF þ Hypoxia þ KNK437). The final concentration of KNK437 was 100 mM in EMF þ Hypoxia þ KNK437 group, the same concentration of DMSO was used as control in Hypoxia group and EMF þ Hypoxia group. The final concentration of DMSO in each culture medium was 0.25% (v/v), and this concentration DMSO had no effect on results. 2.3. Cardiomyocytes morphological analysis Cardiomyocytes images were captured with a CCD (600ES-CU; Pixera, Los Gatos, CA, USA) camera fixed to an inverted microscope (Leica DMIRB, Leica Microsystems, Wetzlar, Germany). From each coverslip, 30 regular myocytes were selected at random and viewed at 200 magnification. Cardiomyocytes were outlined and the cell surface area (CSA) was measured with a Simple PCI software (High Performance Imaging Software, Compix, Cranberry, PA, USA). 2.4. Protein content Cultured cardiomyocytes were treated with the three conditions for 12 h. The cells were washed with PBS and then treated with 10% trichloroacetic acid (Sigma, St. Louis, MO, USA) at 4 C for 1 h to precipitate the protein. The precipitates were dissolved in 0.15 M NaOH. Thereafter, the protein content was determined using a BCA protein assay kit (Beyotime, Haimen, Jiangsu, China). 2.5. Cytotoxicity assay Cell death was assessed by the trypan blue exclusion assay performed in each treated group. Trypan blue stain (Sigma, St. Louis, MO, USA) 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, St. Louis, MO, USA) for 5 min and centrifuged at 50xg 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 cell death rate was expressed as a percentage of the trypan blue-positive cells. Cell viability was assessed by MTS (3-(4,5-dimethylthiazol-2yl)-5-(3-carboxymethylphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay (CellTiter 96® AQueous One Solution Cell Proliferation
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Assay, Promega, Madison, WI, USA) performed in each treated group. Briefly, purified neonatal rat cardiomyocytes were seeded in a 96-well cell culture plates at 104 cells per well and were exposed to EMF and/or hypoxia. After treatment, 20 mL of the CellTiter 96® AQueous One Solution was added to each well containing 100 mL medium, and the cells were incubated at 37 C for 3 h. The plates were then agitated on a plate shaker. The absorbance was measured at 490 nm using a microplate reader (Multiskan MK3, Thermo, Waltham, MA, USA). The relative number of viable cells was determined by comparison to untreated cells, in which viability was assumed to be 100%. Cell proliferation was quantified using a DNA bromodeoxyuridine (BrdU) incorporation assay (Roche Applied Science, Mannheim, Germany). The amount of incorporated BrdU is a measure of the rate of DNA synthesis of the cells and thus indirectly of cell proliferation. Briefly, purified neonatal rat cardiomyocytes were seeded in a 96-well cell culture plates at 104 cells per well and were exposed to EMF and/or hypoxia. After treatment, BrdU labelling solution was added to each well at a final concentration of 10 mM. At the end of the stimulation period, the cells were fixed (60 min) and then incubated for 90 min. At room temperature, with 1/100 dilution of peroxidase-labelled anti-BrdU antibody. The wells were then washed three times and incubated for 5 min, and the luminescence was measured using a microplate reader (Multiskan MK3, Thermo, Waltham, MA, USA). Proliferating cells were detected by the incorporation of BrdU into their DNA according to the manufacturer's protocol. 2.6. Calcium imaging The cardiomyocytes were placed on a 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, 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 RC26 chamber (Warner Instruments, Hamden, CT, USA) and PH-1 heated platform (Warner Instruments, Hamden, CT, USA) that was mounted on an inverted microscope (Olympus America, Melville, NY, USA). Fura-2 fluorescence was alternately excited at the wavelengths of 340 nm and 380 nm with a monochrometer (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 Simple PCI software. Intracellular free calcium concentration was calculated with the formula: [Ca2þ]i ¼ Kd [(RRmin)/(RmaxR)] b [21], 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 Ca2þ-saturating and Ca2þ-free conditions measured after cells were treated with 0.1% Triton X-100 and 10 mM ethylene glycol tetraacetic acid (EGTA), and b is the fluorescence ratio at 380 nm under Ca2þ-free condition to that under Ca2þ-saturating condition. 2.7. RNA extraction, reverse transcription, and real-time PCR Total RNA was isolated from cultured myocytes using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Single-stranded cDNA was synthesized using a Mastercycler gradient thermocycler (Eppendorf, Hamburg, Germany). Real-time PCR was performed on cDNA using 20 ml reaction volumes with SYBR Premix Ex Taq Takara (TaKaRa, Dalian, China) using the 7900HT Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). All primer pairs for
DNA sequence of proteins are shown in Table 1. The mixtures were heated to and held at 95 C for 30 s followed by 40 cycles at 95 C for 5 s, and 60 C for 30 s. All reactions were performed in triplicate, and the mRNA values were normalized to the 18s ribosome RNA, which is a housekeeping gene. We carried out a study to analyze the relative quantity of the expression data based on 2DDCt method, where DDCt ¼ (CT, targetCT, ER)Time x(CT, targetCT, ER)Time 0. Time x is any time point and time 0 represents the 1 expression of the target gene normalized to the endogenous reference (ER). 2.8. Statistical analysis All data are represented as the mean ± SEM. The significance of differences between individual groups was determined by using a one-way analysis of variance (ANOVA). Data analysis was carried out by SPSS software 11.5 (SPSS Inc, Chicago, IL, USA), and a difference was considered statistically significant when p < 0.05. 3. Results 3.1. Effect of EMF exposure on hypoxia-induced injury in cardiomyocytes Cardiomyocytes were randomly divided into 3 groups and incubated in a serum-free medium for 12 h with normoxic conditions (21% O2, 5%CO2, 37 C, Normoxia group), hypoxic conditions (1% O2, 5%CO2, 37 C, Hypoxia group), EMF þ Hypoxic conditions (EMF þ Hypoxia group), respectively. Fig. 1 showed that EMF mediated cardioprotective action against cytotoxicity in cardiomyocytes. The inhibition of cell death by EMF exposure was confirmed with the trypan blue exclusion assay (Fig. 1A). The cell viability and DNA synthesis significantly decreased in Hypoxia group, compared to Normoxia group, and increased in EMF þ Hypoxia group (Fig. 1B and 1C), compared with Hypoxia group (p < 0.05). As shown in Fig. 1D, Hypoxia group increased mRNA expression of pro-apoptotic proteins such as Bcl-2associated X protein (Bax) and caspase-3 and decreased mRNA expression of the anti-apoptotic protein, B cell leukemia/ lymphoma-2 (Bcl-2). Furthermore, the level of mRNA expressions of Bax and caspase-3 were down-regulated, and the expression of Bcl-2 was significantly up-regulated when the cells were exposed with EMF. These results suggested that EMF exposure had a protective effect on hypoxia-induced injury in cardiomyocytes. 3.2. Effect of EMF exposure on hypoxia-induced hypertrophy in cardiomyocytes Fig. 2 clearly showed that EMF exposure inhibited the hypoxiainduced cardiomyocytes hypertrophy (Fig. 2A and Fig. 2B).
Table 1 Sequences of primers used for quantitative real-time PCR. Target gene
Primer sequence
Bcl-2
Forward: 50 -AGGAAGTGAACATTTCGGTGAC-30 Reverse: 50 -GCTCAGTTCCAGGACCAGGC-30 Forward: 50 -TGCTTCAGGGTTTCATCCAG-30 Reverse: 50 -GGCGGCAATCATCCTCTG-30 Forward: 50 -ACATGGCGTGTCATAAAATACC-30 Reverse: 50 -CACAAAGCGACTGGATGAAC-30 Forward: 50 -CGCTCAGTCAGGCGGAT -30 Reverse: 50 - GCCCCAAATGCAGCCAT-30 Forward: 50 -AGGACCCACCATTGAGGAAGTG-30 Reverse: 50 -TCAAGCAAATCTCCACGTACATAC-30 Forward: 50 -GCCGCTAGAGGTGAAATTCTTG-30 Reverse: 50 -CTTTCGCTCTGGTCCGTCTT-30
Bax Caspase-3
b-MHC HSP70 18S
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Fig. 1. EMF exposure reduces hypoxia-induced injury in cardiomyocytes as demonstrated by cell death rate (A), cell viability (B) and BrdU incorporation (C). Data are represented as the mean ± SEM (n ¼ 5, for each group). EMF also improved mRNA expression level of genes related to apoptosis (D). Data are represented as the mean ± SEM of 3 replicate measurements in 3 different cell cultures. *p < 0.05 vs. Normoxia group, #p < 0.05 vs. Hypoxia group.
Moreover, the results of cell total protein content (Fig. 2C) were consistent with them. b-myosin heavy chain (b-MHC) were used as markers of cardiomyocytes hypertrophy [22]. We measured the mRNA expression
after cardiomyocytes were cultured for 12 h with Normoxia, Hypoxia and EMF þ Hypoxia, respectively (Fig. 2D). Our findings indicated that the mRNA expression of b-MHC were up-regulated in Hypoxia group compared to the Normoxia group, whereas the
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Fig. 2. EMF inhibited the hypoxia-induced cardiomyocytes hypertrophy. Cells were observed with phase-contrast microscopy (A). The photographs were captured using a 20 objective, and the magnification bar corresponds to 20 mm. Effects of EMF on cell surface areas (B), protein content (C) and expression of marker genes for cardiac hypertrophy (D) were determined. Data are represented as the mean ± SEM (n ¼ 30 cells or 3 replicate measurements in 3 different cell cultures). *p < 0.05 vs. Normoxia group, #p < 0.05 vs. Hypoxia group.
expression of these markers were down-regulated in EMF þ Hypoxia group compared to Hypoxia group. These suggested that EMF exposure prevents cardiomyocytes hypertrophy caused by hypoxia. 3.3. Effect of EMF on intracellular calcium oscillations in hypoxic cardiomyocytes The Ca2þi oscillations in spontaneously beating cells, dyed with Fura-2/AM, were measured. Fig. 3A showed the representative tracings of F340/F380 fluorescence ratio. Baseline of [Ca2þ]i oscillations, which reflects intracellular resting Ca2þ level and recovery level of Ca2þ after depolarization (diastolic [Ca2þ]i). Fig. 3B showed that intracellular resting Ca2þ level of Normoxia group, Hypoxia group and EMF þ Hypoxia group were 72.32 ± 7.91 nM, 95.06 ± 9.75 nM and 74.25 ± 8.11 nM, respectively (n ¼ 6 for each group). The intracellular resting Ca2þ level was significantly increased in Hypoxia group compared to Normoxia group, while it was markedly decreased in EMF þ Hypoxia group (Fig. 3B) compared with Hypoxia group (p < 0.05). Those results indicated that EMF prevented the rise of diastolic [Ca2þ]i in the heart cells caused by hypoxia administration. Fig. 3C showed changes of the Ca2þi oscillations amplitude. Compared with the Normoxia group,
the amplitude of Ca2þi oscillations significantly decreased by 23.41 ± 2.11% in Hypoxia group, while it markedly increased by 20.02 ± 2.16% (n ¼ 6 for each group) in EMF þ Hypoxia group (Fig. 3C) compared with Hypoxia group (p < 0.05). It also indicated that EMF prevents the decline of Ca2þi oscillations amplitude in Hypoxia group. Fig. 3D showed that the frequencies of Ca2þi oscillations in Normoxia group, Hypoxia group and EMF þ Hypoxia group were 0.50 ± 0.022 Hz, 0.33 ± 0.018 Hz and 0.40 ± 0.019 Hz, respectively (n ¼ 6 for each group). With the same trend, EMF prevented the drop of Ca2þi oscillations frequency in Hypoxia group. These data indicated that treatment with EMF exposure improves the depression of hypoxia-induced Ca2þ oscillations and prevents the dysregulation of Ca2þi homeostasis in hypoxiainduced myocardial injury. 3.4. Assessment of Ca2þ content in sarcoplasmic reticulum (SR) In 0 Naþ- 0 Ca2þ solution, the caffeine-induced Ca2þi transients is an index of the SR Ca2þ content because caffeine depletes the Ca2þ in SR [23]. Cardiomyocytes were perfused with normal Tyrode solution for 30 s and rapidly switched to 0 Naþ- 0 Ca2þ (140 mM LiCl and 10 mM EGTA substituted for NaCl and CaCl2, pH ¼ 7.4 with the addition of LiOH) solution for another 30 s, then 10 mM caffeine
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Fig. 3. Effects of EMF exposure on Ca2þi oscillations in hypoxic cardiomyocytes. (A) Representative tracings of F340/F380 fluorescence ratio. (B) [Ca2þ]i oscillations baseline, (C) amplitude of Ca2þi oscillations and (D) frequency of Ca2þi oscillations. Data are presented as the mean ± SEM (n ¼ 6, for each group). *p < 0.05 vs. Normoxia group, #p < 0.05 vs. Hypoxia group.
was added to release Ca2þ in SR. Ca2þ content of each group were measured using the aforementioned method [10,20]. Fig. 4A showed representative traces of C[Ca2þ]i estimated in 0 Naþ- 0 Ca2þ solution, Fig. 4B showed that Hypoxia group and EMF þ Hypoxia group reduced the Ca2þ content in SR by 27.12 ± 3.64% and 9.73 ± 2.26% (n ¼ 6 for each group), respectively. Compared with the Normoxia group, Ca2þ content in SR was dramatically reduced during exposure to hypoxia and was recovered when treatment with EMF in cardiomyocytes. These data indicated that treatment with EMF prevents the decrease of SR Ca2þ contents in hypoxiainduced myocardial injury of rats.
3.5. Effects of EMF exposure on the expression of heat shock protein 70 (HSP70) HSP70 mediates delayed cardioprotection of preconditioning [24]. Our findings indicated that the mRNA expression of HSP70 were up-regulated in Hypoxia group. However, there were significantly increased in mRNA expression levels of HSP70 when treatment with EMF in hypoxic cardiomyocytes (Fig. 5).
3.6. HSP70 inhibitors rescue cardioprotection of EMF to hypoxic cardiomyocytes KNK437, a novel inhibitor of HSP70 [25], was added to the culture medium before 1 h of EMF exposure. As presented in Fig. 6, when pretreatment with KNK437 by the EMF þ Hypoxia group, the
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Fig. 4. Representative tracings of caffeine-induced Ca2þi transients (C[Ca2þ]i) estimated in 0 Naþ- 0 Ca2þ solution (A); and sarcoplasmic reticulum (SR) Ca2þ content (B). Horizontal bars above the tracing indicate the presence of 10 mM caffeine. Data are represented as the mean ± SEM (n ¼ 6, for each group). *p < 0.05 vs. Normoxia group, #p < 0.05 vs. Hypoxia group.
recovered to the levels of Hypoxia group. The final concentration of KNK437 was 100 mM in EMF þ Hypoxia þ KNK437 group, the same concentration of DMSO was used as control in Hypoxia group and EMF þ Hypoxia group. The final concentration of DMSO in each culture medium was 0.25% (v/v), and this concentration DMSO had no effect on results [26]. These results clearly showed that the EMF exposure induced cardioprotection almost completely inhibited by the presence of KNK437 in hypoxic cardiomyocytes.
3.7. Effects of KNK437 on intracellular calcium oscillations in cardiomyocytes
Fig. 5. Analysis of the expression of heat shock protein 70 (HSP70). Data are represented as the mean ± SEM of 3 replicate measurements in 3 different cell cultures. *p < 0.05 vs. Normoxia group, ##p < 0.01 vs. Hypoxia group.
cell death rate, cell viability and DNA synthesis were dramatically
The performance of Ca2þ oscillations can be found in Fig. 7A. It showed that [Ca2þ]i baseline of Hypoxia group, EMF þ Hypoxia group and EMF þ Hypoxia þ KNK437 group were 95.06 ± 9.75 nM,74.25 ± 8.11 nM and 99.23 ± 8.96 nM, respectively (Fig. 7B). The results indicated that [Ca2þ]i baseline of EMF þ Hypoxia þ KNK437, compared with EMF þ Hypoxia group, markedly increased (P < 0.05), even a little higher than Hypoxia group. Fig. 7C showed changes of the Ca2þi oscillations amplitude. Compared with Hypoxia group, the amplitude of Ca2þi oscillations significantly increased by 20.02 ± 2.16% in EMF þ Hypoxia group, while it was markedly decreased by 27.031 ± 2.36% (n ¼ 5 for each group) in EMF þ Hypoxia þ KNK437 group compared with EMF þ Hypoxia group (p < 0.05). With the same trend, KNK437 effectively suppressed the frequency of Ca2þ oscillations in EMF þ Hypoxia þ KNK437 group (Fig. 7D). These data indicated that pretreatment with KNK437 can almost completely inhibits the Ca2þ handling effects of EMF exposure in EMF þ Hypoxia þ KNK437 group.
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Fig. 6. KNK437 rescue cardioprotection of EMF in hypoxic cardiomyocytes as demonstrated by cell death rate (A), cell viability (B) and BrdU incorporation (C). Data are represented as the mean ± SEM (n ¼ 5, for each group). *p < 0.05 vs. Hypoxia group, #p < 0.05 vs. EMF þ Hypoxia group, N.S. ¼ not statistically different.
4. Discussion The main findings of the study are that: (1) EMF preconditioning significantly protected against myocardial damage, and prevented cardiomyocytes hypertrophy caused by hypoxia exposure; (2) EMF exposure ameliorated Ca2þi homeostasis in hypoxic cardiomyocytes; (3) EMF exposure exerted cardioprotection via activated HSP70 in hypoxic cardiomyocytes. Hypoxia has been shown to cause myocardial hypertrophy [2], myocyte damage [3] and cardiomyopathy [27]. Albertini et al. reported that EMF exposure has been shown to limit necrotic area in rats subjected to acute myocardial infarction [15]. We have demonstrated that hypoxia-induced cardiomyocytes hypertrophy is related to the intracellular calcium overload and EMF can regulate intracellular oscillation [10,20]. To test whether EMF exposure protects cardiomyocytes from hypoxia-induced injury and prevents cell death, we measured cell death rate, cell viability, DNA synthesis and mRNA expression level of genes related to apoptosis in the cardiomyocytes of rats. Our results indicated that pretreatment with EMF improved the damage and hypertrophy of the cardiomyocytes induced by hypoxia, indicating that EMF exposure exerts a pronounced preventative effect on myocardial injury. Although many studies related to cardioprotection of EMF exposure against a potentially lethal stress have been reported [13e15], but a few is involved in Ca2þ handling and especially, the underlying mechanism is unexplored. It is well known that Ca2þ plays an important role in regulating contractility, gene transcription, energy balance, hypertrophic growth, and damage in heart [6e8]. The SR and sarcolemma of the cardiomyocytes are major components of normal Ca2þ homeostasis in the heart [28]. The depolarization of the action potential activates L-type Ca2þ channels and induce the increase of [Ca2þ]i levels. A small rise in [Ca2þ]i triggers Ca2þ release from SR via RyR2 by the process known as Ca2þ-induced Ca2þ release [29]. During relaxation, most of the Ca2þ released is sequestered by the SR via SERCA2a and partly extruded out of the cell via the sarcolemmal NCX [6]. Therefore, we investigated whether intracellular Ca2þ handling was altered by EMF exposure of rat cardiomyocytes. The results showed that EMF exposure prevented the rise of intracellular resting Ca2þ
level caused by hypoxia administration (Fig. 3B). The rise in resting [Ca2þ]i tends to block many vital enzymatic functions and leads to cell death [30]. The prevention of the increase in diastolic [Ca2þ]i by EMF exposure may, at least in part, account for the beneficial effects of EMF on myocardial injury. Secondly, we also found that it markedly increased the amplitude and the frequency of Ca2þi oscillation in EMF þ Hypoxia group (Fig. 3C and 3D), compared with Hypoxia group. A similar result was obtained in a previous study, which demonstrated that EMF exposure significantly reduced the amplitude of Ca2þi oscillation during hypoxia in rat cardiomyocytes [19]. The amplitude of S[Ca2þ]i reflects the Ca2þ release during excitation-contraction coupling and directly correlates with contraction in rat cardiomyocytes [23]. The frequency of Ca2þ oscillations represents the cardiomyocytes spontaneously beating frequency, while also is an indicator of cell contractility. These results suggested attenuated myocardial Ca2þ oscillations and contractility in Hypoxia group, while it significantly recovered after treatment with EMF. Thus, our results demonstrated that EMF exposure plays a cardioprotective role by improving calcium homeostasis in hypoxic cardiomyocytes. EMF changed intracellular Ca2þ transients, but its mechanism is unclear. Our results showed that EMF exposure elevated gene transcript levels including Ca2þ handling proteins and HSP70. The interaction mechanism of EMF with gene regulation remains unknown. Several theoretical approaches to EMF mechanism have been proposed including cyclotron resonance [31] and forced vibration of ions [32]. There is evidence from biochemical reactions that EMFs can accelerate electron transfer and move within DNA [33]. The initial interaction could involve the displacement of electrons in the H-bonds that hold DNA together, thereby causing chain separation and initiating transcription and translation. An important clue to EMF stimulation of biosynthesis comes from identification of a specific EMF-sensitive DNA sequence on both the c-myc and the HSP70 gene promoters [34]. Numerous studies also verified that a twofold induction of HSP70 improves heart muscle cell resistance to ischemia and hypoxia [35]. EMF exposure has been shown to induce heat shock proteins, which help to maintain the conformation of cellular proteins during periods of stress [36]. Fan et al. reported that anti-apoptotic mechanisms of stress
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Fig. 7. Effects of KNK437 on Ca2þi oscillations in hypoxic cardiomyocytes. (A) Representative tracings of F340/F380 fluorescence ratio. (B) [Ca2þ]i oscillations baseline, (C) amplitude of Ca2þi oscillations and (D) frequency of Ca2þi oscillations. Data are presented as the mean ± SEM (n ¼ 5, for each group). *p < 0.05 vs. Hypoxia group, #p < 0.05 vs. EMF þ Hypoxia group, N.S. ¼ not statistically different.
response proteins via attenuation of the ASK-JNK/p38 signaling cascades [37]. There is evidence that HSP70 has a direct effect on apoptosis by preventing caspase-3 activation, poly (ADP-ribose) polymerase (PARP) cleavage, DNA laddering, and cell death in vitro, but its actions appear to be downstream of cytochrome C release [38]. Our results clearly showed that the EMF exposure induced cardioprotection, but it is almost completely abolished by HSP70 inhibitor (KNK437) in hypoxic cardiomyocytes. There is also evidence showing a link between HSP70 and Ca2þ handling. For example, deleting the inducible 70 kDa heat shock genes impairs cardiac contractile function and Ca2þ handling associated with hypertrophy [39]. The NCX known to play an important determinant of myocardial contractility in heart failure, is acted on by
HSP70, leading to desensitization by a reduction in Vmax [40]. Marx et al. showed that HSP70 also modulates other components of Ca2þ-handling, such as SERCA2a and phosphorylation of RyR [41]. Ca2þ handling-related proteins such as RyR2 and SERCA2a in the membrane of SR and NCX in the sarcolemmal membrane are involved in maintaining Ca2þ homeostasis. It is very likely that HSP70 may stabilize or facilitate the activities of these proteins as a molecule chaperone, thus restoring the Ca2þ homeostasis impaired by hypoxia insults. These literatures and our findings suggest interaction of HSP70 with elements of the Ca2þ handling mechanism that result in ameliorated calcium homeostasis and enhanced myocardial contractility. Overall, our results indicated that activation of HSP70 by EMF preconditioning, it not only diminishes the
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extent of cardiac injury but also enhances the contractile function via remaining Ca2þ homeostasis in hypoxic cardiomyocytes. In summary, the study shows that EMF exposure had beneficial effects on preventing histological damage and cardiomyocytes hypertrophy in hypoxic myocardial. EMF preconditioning inhibited the increase of diastolic [Ca2þ]i, prevented the decrease of SR Ca2þ contents in hypoxic cardiomyocytes. We have also found that EMF significantly activates HSP70, which not only weakens the extent of cardiac injury, but also enhances the contractile function by remaining Ca2þ homeostasis in hypoxic cardiomyocytes. Thus, we believe that EMF protects cardiomyocytes against hypoxia induced alterations in Ca2þ homeostasis via HSP70 activation. Acknowledgements This work was supported by a grant from the National Natural Science Foundation of China, (No.31170893 and No. 11372244). Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.cbi.2016.02.003 Declaration of interest The authors report no declarations of interest. References [1] W. Tong, F. Xiong, Y. Li, L. Zhang, Hypoxia inhibits cardiomyocyte proliferation in fetal rat hearts via upregulating TIMP-4, Am. J. Physiol. Regul. Integr. Comp. Physiol. 304 (2013) R613eR620. [2] K. Shyu, W. Cheng, B. Wang, H. Chang, Hypoxia activates muscle-restricted coiled-coil protein (MURC) expression via transforming growth factor-beta in cardiac myocytes, Clin. Sci. 126 (2014) 367e375. [3] M. Tanaka, H. Ito, S. Adachi, H. Akimoto, T. Nishikawa, T. Kasajima, F. Marumo, M. Hiroe, Hypoxia induces apoptosis with enhanced expression of Fas antigen messenger RNA in cultured neonatal rat cardiomyocytes, Circ. Res. 75 (1994) 426e433. [4] D. Mottet, G. Michel, P. Renard, N. Ninane, M. Raes, C. Michiels, Role of ERK and calcium in the hypoxia-induced activation of HIF-1, J. Cell. Physiol. 194 (2003) 30e44. [5] K.A. Seta, Y. Yuan, Z. Spicer, G. Lu, J. Bedard, T.K. Ferguson, P. Pathrose, A. ColeStrauss, A. Kaufhold, D.E. Millhorn, The role of calcium in hypoxia-induced signal transduction and gene expression, Cell calcium 36 (2004) 331e340. [6] D.M. Bers, Cardiac excitation-contraction coupling, Nature 415 (2002) 198e205. [7] D.M. Bers, Calcium cycling and signaling in cardiac myocytes, Annu. Rev. Physiol. 70 (2008) 23e49. [8] H. Xu, Y. Zhang, J. Sun, J. Wei, L. Sun, J. Zhang, Effect of distinct sources of Ca2þ on cardiac hypertrophy in cardiomyocytes, Exp. Biol. Med. 237 (2012) 271e278. [9] E.C. Toescu, Hypoxia sensing and pathways of cytosolic Ca2þ increases, Cell calcium 36 (2004) 187e199. [10] J. Wei, H. Xu, L. Shi, J. Tong, J. Zhang, Trimetazidine protects cardiomyocytes against hypoxia-induced injury through ameliorates calcium homeostasis, Chemico Biol. Interact. 236 (2015) 47e56. [11] C. Bassett, A. Pilla, R. Pawluk, A non-operative salvage of surgically-resistant pseudarthroses and non-unions by pulsing electromagnetic fields: a preliminary report, Clin. Orthop. Relat. Res. 124 (1977) 128e143. [12] R.H.W. Funk, T. Monsees, N. Oezkucur, Electromagnetic effects:From cell biology to medicine, Prog. Histochem. Cytochem. 43 (2009) 177e264. [13] A. DiCarlo, J. Farrell, T. Litovitz, Myocardial protection conferred by electromagnetic fields, Circulation 99 (1999) 813e816. [14] S. Barzelai, A. Dayan, M.S. Feinberg, R. Holbova, S. Laniado, M. Scheinowitz, Electromagnetic field at 15.95-16 Hz is cardio protective following acute myocardial infarction, Ann. Biomed. Eng. 37 (2009) 2093e2104. [15] A. Albertini, P. Zucchini, G. Noera, R. Cadossi, C.P. Napoleone, A. Pierangeli, Protective effect of low frequency low energy pulsing electromagnetic fields on acute experimental myocardial infarcts in rats, Bioelectromagnetics 20
17
(1999) 372e377. [16] E. Barbier, B. Veyret, B. Dufy, Stimulation of Ca2þ influx in rat pituitary cells under exposure to a 50 Hz magnetic field, Bioelectromagnetics 17 (1996) 303e311. [17] X. Zhang, X. Liu, L. Pan, I. Lee, Magnetic fields at extremely low-frequency (50Hz, 0.8 mT) can induce the uptake of intracellular calcium levels in osteoblasts, Biochem. Biophys. Res. Commun. 396 (2010) 662e666. € ker, M. Deniz, Y. Nergiz, Intracellular Ca2þ levels in rat ventricle [18] C. Sert, S. So cells exposed to extremely low frequency magnetic field, Electromagn. Biol. Med. 30 (2011) 14e20. [19] S. Yitzhaki, A. Shainberg, M. Shaked, Z. Schuss, D. Fixler, Weak magnetic field at 16 Hz affects cardiac myocyte Ca2þ transients and reduces cells damage caused by hypoxia, Open Opt. J. 5 (2011) 33e39. [20] J. Wei, J. Sun, H. Xu, L. Shi, L. Sun, J. Zhang, Effects of extremely low frequency electromagnetic fields on intracellular calcium transients in cardiomyocytes, Electromagn. Biol. Med. 34 (2015) 77e84. [21] G. Grynkiewicz, M. Poenie, R.Y. Tsien, A new generation of Ca2þ indicators with greatly improved fluorescence properties, J. Biol. Chem. 260 (1985) 3440e3450. [22] N. Yamaguchi, N. Takahashi, L. Xu, O. Smithies, G. Meissner, Early cardiac hypertrophy in mice with impaired calmodulin regulation of cardiac muscle Ca2þ release channel, J. Clin. Investig. 117 (2007) 1344e1353. [23] T.R. Shannon, S.M. Pogwizd, D.M. Bers, Elevated sarcoplasmic reticulum Ca2þ leak in intact ventricular myocytes from rabbits in heart failure, Circ. Res. 93 (2003) 592e594. [24] J. Zhou, J. Pei, G. Wang, S. Wu, W. Wang, C. Cho, T. Wong, Inducible HSP70 mediates delayed cardioprotection via U-50488H pretreatment in rat ventricular myocytes, Am. J. Physiol. Heart Circ. Physiol. 281 (2001) H40eH47. [25] Y. Teng, L. Ngoka, Y. Mei, L. Lesoon, J.K. Cowell, HSP90 and HSP70 proteins are essential for stabilization and activation of WASF3 metastasis-promoting protein, J. Biol. Chem. 287 (2012) 10051e10059. [26] S.J. Haggarty, T.U. Mayer, D.T. Miyamoto, R. Fathi, R.W. King, T.J. Mitchison, S.L. Schreiber, Dissecting cellular processes using small molecules: identification of colchicine-like, taxol-like and other small molecules that perturb mitosis, Chem. Biol. 7 (2000) 275e286. [27] S. Murarka, M.R. Movahed, Diabetic cardiomyopathy, J. cardiac Fail. 16 (2010) 971e979. [28] D.H. MacLennan, M. Abed, C. Kang, Structure-Function Relationships in Ca2þ Cycling Proteins, J. Mol. Cell. Cardiol. 34 (2002) 897e918. [29] B. Chamberlain, P. Volpe, S. Fleischer, Calcium-induced calcium release from purified cardiac sarcoplasmic reticulum vesicles. General characteristics, J. Biol. Chem. 259 (1984) 7540e7546. [30] J. Gilloteaux, J. Bissler, P. Kondolios, D. Jarjoura, Cardiomyocyte aging and hypertrophy: atrial and ventricular changes in normal and myopathic Syrian hamsters, J. Submicrosc. Cytol. Pathol. 22 (1990) 249e264. [31] A.R. Liboff, Geomagnetic cyclotron resonance in living cells, J. Biol. Phys. 13 (1985) 99e102. [32] D.J. Panagopoulos, A. Karabarbounis, L.H. Margaritis, Mechanism for action of electromagnetic fields on cells, Biochem. Biophys. Res. Commun. 298 (2002) 95e102. [33] M. Blank, R. Goodman, A mechanism for stimulation of biosynthesis by electromagnetic fields: charge transfer in DNA and base pair separation, J. Cell. Physiol. 214 (2008) 20e26. [34] H. Lin, M. Blank, K. Haseroth, R. Goodman, Regulating genes with electromagnetic response elements, J. Cell. Biochem. 81 (2001) 143e148. [35] R.J. Heads, D.M. Yellon, D.S. Latchman, Differential cytoprotection against heat stress or hypoxia following expression of specific stress protein genes in myogenic cells, J. Mol. Cell. Cardiol. 27 (1995) 1669e1678. [36] M. Blank, R. Goodman, Electromagnetic fields stress living cells, Pathophysiology 16 (2009) 71e78. [37] G. Fan, X. Ren, J. Qian, Q. Yuan, P. Nicolaou, Y. Wang, W.K. Jones, G. Chu, E.G. Kranias, Novel cardioprotective role of a small heat-shock protein, Hsp20, against ischemia/reperfusion injury, Circulation 111 (2005) 1792e1799. [38] S.D. Westerheide, R.I. Morimoto, Heat shock response modulators as therapeutic tools for diseases of protein conformation, J. Biol. Chem. 280 (2005) 33097e33100. [39] Y.K. Kim, J. Suarez, Y. Hu, P.M. McDonough, C. Boer, D.J. Dix, W.H. Dillmann, Deletion of the inducible 70-kDa heat shock protein genes in mice impairs cardiac contractile function and calcium handling associated with hypertrophy, Circulation 113 (2006) 2589e2597. [40] J.G. Kiang, X.Z. Ding, D.E. McClain, Overexpression of HSP-70 Attenuates Increases in [Ca2þ]i and Protects Human Epidermoid A-431 Cells after Chemical Hypoxia, Toxicol. Appl. Pharmacol. 149 (1998) 185e194. [41] S.O. Marx, S. Reiken, Y. Hisamatsu, T. Jayaraman, D. Burkhoff, N. Rosemblit, A.R. Marks, PKA phosphorylation dissociates FKBP12. 6 from the calcium release channel (ryanodine receptor): defective regulation in failing hearts, Cell 101 (2000) 365e376.
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EMF protects cardiomyocytes against hypoxia-induced injury via heat shock protein 70 activation Jinhong Wei, Jie Tong, Liying Yu, Jianbao Zhang* The Key Laboratory of Biomedical Information Engineering of Ministry of Education, School of Life Science and Technology, Xi'an Jiaotong University, Xi'an, 710049, People's Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 September 2015 Received in revised form 4 January 2016 Accepted 7 February 2016 Available online 11 February 2016
Intracellular calcium (Ca2þi) overload induced by chronic hypoxia alters Ca2þi homeostasis, whereas ameliorating calcium homeostasis is believed to be responsible for cardioprotection. We hypothesize that cardioprotection by electromagnetic fields (EMF) exposure may restore Ca2þi homeostasis altered by hypoxia insults. Cardiomyocytes isolated from neonatal Sprague-Dawley rats were exposed to chronic hypoxia (1% O2, 5% CO2, 37 C). We observed that cardiomyocytes injury and hypertrophy were alleviated in hypoxic cardiomyocytes exposed with EMF preconditioning. Compared with hypoxic cardiomyocytes, the diastolic [Ca2þ]i was decreased, the amplitude of Ca2þi oscillations was recovered when cardiomyocytes exposed with EMF. In addition, we also found that EMF exposure significantly increased heat shock protein 70 (HSP70) mRNA expression in hypoxic cardiomyocytes. However, treatment with HSP70 blocker KNK437, almost completely inhibited the EMF induced-cardioprotection and the beneficial effects of Ca2þ oscillation in hypoxic cardiomyocytes. These results suggest that EMF preconditioning ameliorates Ca2þi homeostasis through activating HSP70, thereby producing the cardioprotective effect and reduction in hypoxic cardiomyocytes damage. © 2016 Elsevier Ireland Ltd. All rights reserved.
Keywords: EMF Hypoxia Cardioprotection Calcium homeostasis HSP70
1. Introduction Hypoxia is the natural consequence of some environments (e.g., high altitude, diving), it is also a common feature of many clinical diseases (e.g., sleep apnea syndrome, chronic obstructive pulmonary disease, heart failure, vascular diseases, stroke, sepsis, metabolic myopathies). Hypoxia is generally associated with cardiovascular diseases, and it elicits a variety of functional responses in cardiomyocytes, including cell proliferation [1], hypertrophy [2] and death [3]. Many responses of cells on hypoxia are mediated by Ca2þ signals [4,5]. Intracellular calcium (Ca2þi) plays a central role on regulating contractility, gene transcription, energy balance, hypertrophic growth, and apoptosis in the heart [6e8]. In many instances, one of the constant responses to hypoxia is an increase in intracellular calcium concentration ([Ca2þ]i) via the activation of various plasma membrane Ca2þ conductances, such as voltage-gated Ca2þ channels, ligand-operated Ca2þ channels, and non-specific cation channels [9]. Ca2þi homeostasis is very critical to cell survival and hypoxia can induce cell death by increasing
* Corresponding author. E-mail address: [email protected] (J. Zhang). http://dx.doi.org/10.1016/j.cbi.2016.02.003 0009-2797/© 2016 Elsevier Ireland Ltd. All rights reserved.
[Ca2þ]i [10]. Cardiomyocytes Ca2þ loading is recognized as a major factor in acute hypoxia pathology, and then promotes cell death, contractile dysfunction and arrhythmogenic activity [5]. Dysregulation of Ca2þi homeostasis can lead not only to loss of normal physiological control mechanisms but also to pathological changes in cell growth. If a tool to manipulate Ca2þi was available, it might be a promising approach for protection of cardiomyocytes against hypoxia-mediated injury. Since extremely low frequency electromagnetic fields (ELF-EMF) can penetrate into tissues, ELF-EMF affects cellular functions, such as RNA transcription, DNA synthesis, protein expression, protein phosphorylation, microvesicle motility, proliferation, differentiation and apoptosis. As early as 1977, pulsed electromagnetic fields (PEMF) have been successfully used to treat chronic non-union bone fracture [11]. The Food and Drug Administration of USA has approved EMF as a safe and effective mean for treatment of osteoporosis and bone non-unions [12]. In the past two decades, effects of EMF exposure on the cardiovascular system have also been investigated. Dicarlo et al. showed that ELF-EMF induced stress responses that protect chick embryo myocardium from anoxia damage [13]. Barzelai et al. revealed that an EMF of 80 nT at 15.95e16.00 Hz protected against coronary artery occlusion [14].
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Albertini et al. 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 [15]. These results suggested that EMF exposure may be an effective and noninvasive way to treat cardiovascular-related diseases, although the mechanisms are not clear. Calcium signaling is a possible target of ELF-EMF on biological systems according to a hypothesized ion-protein interaction [16]. Numerous studies showed that EMF changed [Ca2þ]i levels in rat pituitary cells, osteoblasts and cardiac cells, even though the mechanisms underlying these effects have not been fully understood [16e19]. Furthermore, our previous studies have found that ELF-EMF can regulate Ca2þi oscillations via affecting Ca2þ associated protein activities in cardiomyocytes [20]. It suggests that EMF exposure may be one of the tools to manipulate the Ca2þi handling under pathological conditions. However, the effects of EMF exposure on Ca2þi handling and mechanisms involved in its cytoprotective effects during myocardial injury induced by hypoxia remain largely unknown. The present study examined the protective effect of EMF exposure and the mechanism underlying this protection against hypoxia-induced injury in neonatal rat cardiomyocytes. 2. Materials and methods 2.1. 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 (Xi'an, Shaanxi, China). Animals were supplied by the Animal Center of the Fourth Military Medical University (Xi'an, Shaanxi, China). Neonatal ventricular myocytes were isolated from 1-day-old Sprague-Dawley rats as described previously [10,20]. In brief, neonatal rats were anesthetized with isoflurane (1.5e2.0% 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, USA) for 30 min at 37 C. After the cells were precipitated by centrifugation (at room temperature, 50xg for 5 min) and the collagenase solution was removed, the pellet of cells was resuspended in Kraft-Brühe (KB) solution (composed of (in mM): KCl 85, K2HPO4 30, MgSO4 5, 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, USA) and incubated in Dulbecco's modified Eagle medium (DMEM; Invitrogen Corporation, Grand Island, NY, USA) supplemented with 15% fetal calf serum (FCS; Hyclone, Logan, UT, USA) in 5% CO2 at 37 C for 24 h. The medium was replaced with a serum-free medium before the cells were exposed to hypoxia. 2.2. EMF exposure An EMF exposure system that provided a relatively uniform electromagnetic field for cells exposure as described previously was used [20]. Briefly, the exposure consists of a waveform generator, amplifier and solenoid. The waveform generator was an extremelylow frequency function generator, which provided rectangular
9
waveforms. After being amplified, the signals were output to the solenoid. A cylindrical solenoid (25 cm long and 5 cm diameter) made of 1000 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, USA). Frequencies (pulse-width and interpulse-interval in msec) of the ELF-EMF used in the experiments were 15 Hz. The flux density was 2 mT. Culture plates were placed on the plexiglass shelves in the center of solenoid within an incubator. The cells were perpendicular to the long axis of the solenoid. At the same time, control plates were placed in an identical incubator on the plexiglass shelves with unpowered solenoid. The CO2 concentration, humidity, and temperature of the control, treatment incubators were totally the same and were not affected by EMF. The EMF exposure system was checked daily with an oscilloscope. During hypoxia culture, cells were placed in a hypoxic (1% O2, 5% CO2, 37 C) incubator (Galaxy oxygen control incubator, RS Biotech, Irvine, UK) for 12 h. Control (Normoxia group) were incubated for equivalent periods under normoxic conditions (21% O2, 5%CO2, 37 C). EMF exposure cells (EMF þ Hypoxia group) were exposed with ELF-EMF (15 Hz, 2mT) for 30 min before hypoxic conditions. KNK437 (N-formyl-3,4-methylenedioxy-benzylidene-g-butyrolactam; Sigma, St. Louis, MO, USA), a heat shock protein 70 (HSP70) inhibitor, was added to the culture medium 1 h before EMF exposure (EMF þ Hypoxia þ KNK437). The final concentration of KNK437 was 100 mM in EMF þ Hypoxia þ KNK437 group, the same concentration of DMSO was used as control in Hypoxia group and EMF þ Hypoxia group. The final concentration of DMSO in each culture medium was 0.25% (v/v), and this concentration DMSO had no effect on results. 2.3. Cardiomyocytes morphological analysis Cardiomyocytes images were captured with a CCD (600ES-CU; Pixera, Los Gatos, CA, USA) camera fixed to an inverted microscope (Leica DMIRB, Leica Microsystems, Wetzlar, Germany). From each coverslip, 30 regular myocytes were selected at random and viewed at 200 magnification. Cardiomyocytes were outlined and the cell surface area (CSA) was measured with a Simple PCI software (High Performance Imaging Software, Compix, Cranberry, PA, USA). 2.4. Protein content Cultured cardiomyocytes were treated with the three conditions for 12 h. The cells were washed with PBS and then treated with 10% trichloroacetic acid (Sigma, St. Louis, MO, USA) at 4 C for 1 h to precipitate the protein. The precipitates were dissolved in 0.15 M NaOH. Thereafter, the protein content was determined using a BCA protein assay kit (Beyotime, Haimen, Jiangsu, China). 2.5. Cytotoxicity assay Cell death was assessed by the trypan blue exclusion assay performed in each treated group. Trypan blue stain (Sigma, St. Louis, MO, USA) 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, St. Louis, MO, USA) for 5 min and centrifuged at 50xg 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 cell death rate was expressed as a percentage of the trypan blue-positive cells. Cell viability was assessed by MTS (3-(4,5-dimethylthiazol-2yl)-5-(3-carboxymethylphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay (CellTiter 96® AQueous One Solution Cell Proliferation
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Assay, Promega, Madison, WI, USA) performed in each treated group. Briefly, purified neonatal rat cardiomyocytes were seeded in a 96-well cell culture plates at 104 cells per well and were exposed to EMF and/or hypoxia. After treatment, 20 mL of the CellTiter 96® AQueous One Solution was added to each well containing 100 mL medium, and the cells were incubated at 37 C for 3 h. The plates were then agitated on a plate shaker. The absorbance was measured at 490 nm using a microplate reader (Multiskan MK3, Thermo, Waltham, MA, USA). The relative number of viable cells was determined by comparison to untreated cells, in which viability was assumed to be 100%. Cell proliferation was quantified using a DNA bromodeoxyuridine (BrdU) incorporation assay (Roche Applied Science, Mannheim, Germany). The amount of incorporated BrdU is a measure of the rate of DNA synthesis of the cells and thus indirectly of cell proliferation. Briefly, purified neonatal rat cardiomyocytes were seeded in a 96-well cell culture plates at 104 cells per well and were exposed to EMF and/or hypoxia. After treatment, BrdU labelling solution was added to each well at a final concentration of 10 mM. At the end of the stimulation period, the cells were fixed (60 min) and then incubated for 90 min. At room temperature, with 1/100 dilution of peroxidase-labelled anti-BrdU antibody. The wells were then washed three times and incubated for 5 min, and the luminescence was measured using a microplate reader (Multiskan MK3, Thermo, Waltham, MA, USA). Proliferating cells were detected by the incorporation of BrdU into their DNA according to the manufacturer's protocol. 2.6. Calcium imaging The cardiomyocytes were placed on a 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, 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 RC26 chamber (Warner Instruments, Hamden, CT, USA) and PH-1 heated platform (Warner Instruments, Hamden, CT, USA) that was mounted on an inverted microscope (Olympus America, Melville, NY, USA). Fura-2 fluorescence was alternately excited at the wavelengths of 340 nm and 380 nm with a monochrometer (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 Simple PCI software. Intracellular free calcium concentration was calculated with the formula: [Ca2þ]i ¼ Kd [(RRmin)/(RmaxR)] b [21], 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 Ca2þ-saturating and Ca2þ-free conditions measured after cells were treated with 0.1% Triton X-100 and 10 mM ethylene glycol tetraacetic acid (EGTA), and b is the fluorescence ratio at 380 nm under Ca2þ-free condition to that under Ca2þ-saturating condition. 2.7. RNA extraction, reverse transcription, and real-time PCR Total RNA was isolated from cultured myocytes using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Single-stranded cDNA was synthesized using a Mastercycler gradient thermocycler (Eppendorf, Hamburg, Germany). Real-time PCR was performed on cDNA using 20 ml reaction volumes with SYBR Premix Ex Taq Takara (TaKaRa, Dalian, China) using the 7900HT Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). All primer pairs for
DNA sequence of proteins are shown in Table 1. The mixtures were heated to and held at 95 C for 30 s followed by 40 cycles at 95 C for 5 s, and 60 C for 30 s. All reactions were performed in triplicate, and the mRNA values were normalized to the 18s ribosome RNA, which is a housekeeping gene. We carried out a study to analyze the relative quantity of the expression data based on 2DDCt method, where DDCt ¼ (CT, targetCT, ER)Time x(CT, targetCT, ER)Time 0. Time x is any time point and time 0 represents the 1 expression of the target gene normalized to the endogenous reference (ER). 2.8. Statistical analysis All data are represented as the mean ± SEM. The significance of differences between individual groups was determined by using a one-way analysis of variance (ANOVA). Data analysis was carried out by SPSS software 11.5 (SPSS Inc, Chicago, IL, USA), and a difference was considered statistically significant when p < 0.05. 3. Results 3.1. Effect of EMF exposure on hypoxia-induced injury in cardiomyocytes Cardiomyocytes were randomly divided into 3 groups and incubated in a serum-free medium for 12 h with normoxic conditions (21% O2, 5%CO2, 37 C, Normoxia group), hypoxic conditions (1% O2, 5%CO2, 37 C, Hypoxia group), EMF þ Hypoxic conditions (EMF þ Hypoxia group), respectively. Fig. 1 showed that EMF mediated cardioprotective action against cytotoxicity in cardiomyocytes. The inhibition of cell death by EMF exposure was confirmed with the trypan blue exclusion assay (Fig. 1A). The cell viability and DNA synthesis significantly decreased in Hypoxia group, compared to Normoxia group, and increased in EMF þ Hypoxia group (Fig. 1B and 1C), compared with Hypoxia group (p < 0.05). As shown in Fig. 1D, Hypoxia group increased mRNA expression of pro-apoptotic proteins such as Bcl-2associated X protein (Bax) and caspase-3 and decreased mRNA expression of the anti-apoptotic protein, B cell leukemia/ lymphoma-2 (Bcl-2). Furthermore, the level of mRNA expressions of Bax and caspase-3 were down-regulated, and the expression of Bcl-2 was significantly up-regulated when the cells were exposed with EMF. These results suggested that EMF exposure had a protective effect on hypoxia-induced injury in cardiomyocytes. 3.2. Effect of EMF exposure on hypoxia-induced hypertrophy in cardiomyocytes Fig. 2 clearly showed that EMF exposure inhibited the hypoxiainduced cardiomyocytes hypertrophy (Fig. 2A and Fig. 2B).
Table 1 Sequences of primers used for quantitative real-time PCR. Target gene
Primer sequence
Bcl-2
Forward: 50 -AGGAAGTGAACATTTCGGTGAC-30 Reverse: 50 -GCTCAGTTCCAGGACCAGGC-30 Forward: 50 -TGCTTCAGGGTTTCATCCAG-30 Reverse: 50 -GGCGGCAATCATCCTCTG-30 Forward: 50 -ACATGGCGTGTCATAAAATACC-30 Reverse: 50 -CACAAAGCGACTGGATGAAC-30 Forward: 50 -CGCTCAGTCAGGCGGAT -30 Reverse: 50 - GCCCCAAATGCAGCCAT-30 Forward: 50 -AGGACCCACCATTGAGGAAGTG-30 Reverse: 50 -TCAAGCAAATCTCCACGTACATAC-30 Forward: 50 -GCCGCTAGAGGTGAAATTCTTG-30 Reverse: 50 -CTTTCGCTCTGGTCCGTCTT-30
Bax Caspase-3
b-MHC HSP70 18S
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Fig. 1. EMF exposure reduces hypoxia-induced injury in cardiomyocytes as demonstrated by cell death rate (A), cell viability (B) and BrdU incorporation (C). Data are represented as the mean ± SEM (n ¼ 5, for each group). EMF also improved mRNA expression level of genes related to apoptosis (D). Data are represented as the mean ± SEM of 3 replicate measurements in 3 different cell cultures. *p < 0.05 vs. Normoxia group, #p < 0.05 vs. Hypoxia group.
Moreover, the results of cell total protein content (Fig. 2C) were consistent with them. b-myosin heavy chain (b-MHC) were used as markers of cardiomyocytes hypertrophy [22]. We measured the mRNA expression
after cardiomyocytes were cultured for 12 h with Normoxia, Hypoxia and EMF þ Hypoxia, respectively (Fig. 2D). Our findings indicated that the mRNA expression of b-MHC were up-regulated in Hypoxia group compared to the Normoxia group, whereas the
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Fig. 2. EMF inhibited the hypoxia-induced cardiomyocytes hypertrophy. Cells were observed with phase-contrast microscopy (A). The photographs were captured using a 20 objective, and the magnification bar corresponds to 20 mm. Effects of EMF on cell surface areas (B), protein content (C) and expression of marker genes for cardiac hypertrophy (D) were determined. Data are represented as the mean ± SEM (n ¼ 30 cells or 3 replicate measurements in 3 different cell cultures). *p < 0.05 vs. Normoxia group, #p < 0.05 vs. Hypoxia group.
expression of these markers were down-regulated in EMF þ Hypoxia group compared to Hypoxia group. These suggested that EMF exposure prevents cardiomyocytes hypertrophy caused by hypoxia. 3.3. Effect of EMF on intracellular calcium oscillations in hypoxic cardiomyocytes The Ca2þi oscillations in spontaneously beating cells, dyed with Fura-2/AM, were measured. Fig. 3A showed the representative tracings of F340/F380 fluorescence ratio. Baseline of [Ca2þ]i oscillations, which reflects intracellular resting Ca2þ level and recovery level of Ca2þ after depolarization (diastolic [Ca2þ]i). Fig. 3B showed that intracellular resting Ca2þ level of Normoxia group, Hypoxia group and EMF þ Hypoxia group were 72.32 ± 7.91 nM, 95.06 ± 9.75 nM and 74.25 ± 8.11 nM, respectively (n ¼ 6 for each group). The intracellular resting Ca2þ level was significantly increased in Hypoxia group compared to Normoxia group, while it was markedly decreased in EMF þ Hypoxia group (Fig. 3B) compared with Hypoxia group (p < 0.05). Those results indicated that EMF prevented the rise of diastolic [Ca2þ]i in the heart cells caused by hypoxia administration. Fig. 3C showed changes of the Ca2þi oscillations amplitude. Compared with the Normoxia group,
the amplitude of Ca2þi oscillations significantly decreased by 23.41 ± 2.11% in Hypoxia group, while it markedly increased by 20.02 ± 2.16% (n ¼ 6 for each group) in EMF þ Hypoxia group (Fig. 3C) compared with Hypoxia group (p < 0.05). It also indicated that EMF prevents the decline of Ca2þi oscillations amplitude in Hypoxia group. Fig. 3D showed that the frequencies of Ca2þi oscillations in Normoxia group, Hypoxia group and EMF þ Hypoxia group were 0.50 ± 0.022 Hz, 0.33 ± 0.018 Hz and 0.40 ± 0.019 Hz, respectively (n ¼ 6 for each group). With the same trend, EMF prevented the drop of Ca2þi oscillations frequency in Hypoxia group. These data indicated that treatment with EMF exposure improves the depression of hypoxia-induced Ca2þ oscillations and prevents the dysregulation of Ca2þi homeostasis in hypoxiainduced myocardial injury. 3.4. Assessment of Ca2þ content in sarcoplasmic reticulum (SR) In 0 Naþ- 0 Ca2þ solution, the caffeine-induced Ca2þi transients is an index of the SR Ca2þ content because caffeine depletes the Ca2þ in SR [23]. Cardiomyocytes were perfused with normal Tyrode solution for 30 s and rapidly switched to 0 Naþ- 0 Ca2þ (140 mM LiCl and 10 mM EGTA substituted for NaCl and CaCl2, pH ¼ 7.4 with the addition of LiOH) solution for another 30 s, then 10 mM caffeine
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Fig. 3. Effects of EMF exposure on Ca2þi oscillations in hypoxic cardiomyocytes. (A) Representative tracings of F340/F380 fluorescence ratio. (B) [Ca2þ]i oscillations baseline, (C) amplitude of Ca2þi oscillations and (D) frequency of Ca2þi oscillations. Data are presented as the mean ± SEM (n ¼ 6, for each group). *p < 0.05 vs. Normoxia group, #p < 0.05 vs. Hypoxia group.
was added to release Ca2þ in SR. Ca2þ content of each group were measured using the aforementioned method [10,20]. Fig. 4A showed representative traces of C[Ca2þ]i estimated in 0 Naþ- 0 Ca2þ solution, Fig. 4B showed that Hypoxia group and EMF þ Hypoxia group reduced the Ca2þ content in SR by 27.12 ± 3.64% and 9.73 ± 2.26% (n ¼ 6 for each group), respectively. Compared with the Normoxia group, Ca2þ content in SR was dramatically reduced during exposure to hypoxia and was recovered when treatment with EMF in cardiomyocytes. These data indicated that treatment with EMF prevents the decrease of SR Ca2þ contents in hypoxiainduced myocardial injury of rats.
3.5. Effects of EMF exposure on the expression of heat shock protein 70 (HSP70) HSP70 mediates delayed cardioprotection of preconditioning [24]. Our findings indicated that the mRNA expression of HSP70 were up-regulated in Hypoxia group. However, there were significantly increased in mRNA expression levels of HSP70 when treatment with EMF in hypoxic cardiomyocytes (Fig. 5).
3.6. HSP70 inhibitors rescue cardioprotection of EMF to hypoxic cardiomyocytes KNK437, a novel inhibitor of HSP70 [25], was added to the culture medium before 1 h of EMF exposure. As presented in Fig. 6, when pretreatment with KNK437 by the EMF þ Hypoxia group, the
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Fig. 4. Representative tracings of caffeine-induced Ca2þi transients (C[Ca2þ]i) estimated in 0 Naþ- 0 Ca2þ solution (A); and sarcoplasmic reticulum (SR) Ca2þ content (B). Horizontal bars above the tracing indicate the presence of 10 mM caffeine. Data are represented as the mean ± SEM (n ¼ 6, for each group). *p < 0.05 vs. Normoxia group, #p < 0.05 vs. Hypoxia group.
recovered to the levels of Hypoxia group. The final concentration of KNK437 was 100 mM in EMF þ Hypoxia þ KNK437 group, the same concentration of DMSO was used as control in Hypoxia group and EMF þ Hypoxia group. The final concentration of DMSO in each culture medium was 0.25% (v/v), and this concentration DMSO had no effect on results [26]. These results clearly showed that the EMF exposure induced cardioprotection almost completely inhibited by the presence of KNK437 in hypoxic cardiomyocytes.
3.7. Effects of KNK437 on intracellular calcium oscillations in cardiomyocytes
Fig. 5. Analysis of the expression of heat shock protein 70 (HSP70). Data are represented as the mean ± SEM of 3 replicate measurements in 3 different cell cultures. *p < 0.05 vs. Normoxia group, ##p < 0.01 vs. Hypoxia group.
cell death rate, cell viability and DNA synthesis were dramatically
The performance of Ca2þ oscillations can be found in Fig. 7A. It showed that [Ca2þ]i baseline of Hypoxia group, EMF þ Hypoxia group and EMF þ Hypoxia þ KNK437 group were 95.06 ± 9.75 nM,74.25 ± 8.11 nM and 99.23 ± 8.96 nM, respectively (Fig. 7B). The results indicated that [Ca2þ]i baseline of EMF þ Hypoxia þ KNK437, compared with EMF þ Hypoxia group, markedly increased (P < 0.05), even a little higher than Hypoxia group. Fig. 7C showed changes of the Ca2þi oscillations amplitude. Compared with Hypoxia group, the amplitude of Ca2þi oscillations significantly increased by 20.02 ± 2.16% in EMF þ Hypoxia group, while it was markedly decreased by 27.031 ± 2.36% (n ¼ 5 for each group) in EMF þ Hypoxia þ KNK437 group compared with EMF þ Hypoxia group (p < 0.05). With the same trend, KNK437 effectively suppressed the frequency of Ca2þ oscillations in EMF þ Hypoxia þ KNK437 group (Fig. 7D). These data indicated that pretreatment with KNK437 can almost completely inhibits the Ca2þ handling effects of EMF exposure in EMF þ Hypoxia þ KNK437 group.
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Fig. 6. KNK437 rescue cardioprotection of EMF in hypoxic cardiomyocytes as demonstrated by cell death rate (A), cell viability (B) and BrdU incorporation (C). Data are represented as the mean ± SEM (n ¼ 5, for each group). *p < 0.05 vs. Hypoxia group, #p < 0.05 vs. EMF þ Hypoxia group, N.S. ¼ not statistically different.
4. Discussion The main findings of the study are that: (1) EMF preconditioning significantly protected against myocardial damage, and prevented cardiomyocytes hypertrophy caused by hypoxia exposure; (2) EMF exposure ameliorated Ca2þi homeostasis in hypoxic cardiomyocytes; (3) EMF exposure exerted cardioprotection via activated HSP70 in hypoxic cardiomyocytes. Hypoxia has been shown to cause myocardial hypertrophy [2], myocyte damage [3] and cardiomyopathy [27]. Albertini et al. reported that EMF exposure has been shown to limit necrotic area in rats subjected to acute myocardial infarction [15]. We have demonstrated that hypoxia-induced cardiomyocytes hypertrophy is related to the intracellular calcium overload and EMF can regulate intracellular oscillation [10,20]. To test whether EMF exposure protects cardiomyocytes from hypoxia-induced injury and prevents cell death, we measured cell death rate, cell viability, DNA synthesis and mRNA expression level of genes related to apoptosis in the cardiomyocytes of rats. Our results indicated that pretreatment with EMF improved the damage and hypertrophy of the cardiomyocytes induced by hypoxia, indicating that EMF exposure exerts a pronounced preventative effect on myocardial injury. Although many studies related to cardioprotection of EMF exposure against a potentially lethal stress have been reported [13e15], but a few is involved in Ca2þ handling and especially, the underlying mechanism is unexplored. It is well known that Ca2þ plays an important role in regulating contractility, gene transcription, energy balance, hypertrophic growth, and damage in heart [6e8]. The SR and sarcolemma of the cardiomyocytes are major components of normal Ca2þ homeostasis in the heart [28]. The depolarization of the action potential activates L-type Ca2þ channels and induce the increase of [Ca2þ]i levels. A small rise in [Ca2þ]i triggers Ca2þ release from SR via RyR2 by the process known as Ca2þ-induced Ca2þ release [29]. During relaxation, most of the Ca2þ released is sequestered by the SR via SERCA2a and partly extruded out of the cell via the sarcolemmal NCX [6]. Therefore, we investigated whether intracellular Ca2þ handling was altered by EMF exposure of rat cardiomyocytes. The results showed that EMF exposure prevented the rise of intracellular resting Ca2þ
level caused by hypoxia administration (Fig. 3B). The rise in resting [Ca2þ]i tends to block many vital enzymatic functions and leads to cell death [30]. The prevention of the increase in diastolic [Ca2þ]i by EMF exposure may, at least in part, account for the beneficial effects of EMF on myocardial injury. Secondly, we also found that it markedly increased the amplitude and the frequency of Ca2þi oscillation in EMF þ Hypoxia group (Fig. 3C and 3D), compared with Hypoxia group. A similar result was obtained in a previous study, which demonstrated that EMF exposure significantly reduced the amplitude of Ca2þi oscillation during hypoxia in rat cardiomyocytes [19]. The amplitude of S[Ca2þ]i reflects the Ca2þ release during excitation-contraction coupling and directly correlates with contraction in rat cardiomyocytes [23]. The frequency of Ca2þ oscillations represents the cardiomyocytes spontaneously beating frequency, while also is an indicator of cell contractility. These results suggested attenuated myocardial Ca2þ oscillations and contractility in Hypoxia group, while it significantly recovered after treatment with EMF. Thus, our results demonstrated that EMF exposure plays a cardioprotective role by improving calcium homeostasis in hypoxic cardiomyocytes. EMF changed intracellular Ca2þ transients, but its mechanism is unclear. Our results showed that EMF exposure elevated gene transcript levels including Ca2þ handling proteins and HSP70. The interaction mechanism of EMF with gene regulation remains unknown. Several theoretical approaches to EMF mechanism have been proposed including cyclotron resonance [31] and forced vibration of ions [32]. There is evidence from biochemical reactions that EMFs can accelerate electron transfer and move within DNA [33]. The initial interaction could involve the displacement of electrons in the H-bonds that hold DNA together, thereby causing chain separation and initiating transcription and translation. An important clue to EMF stimulation of biosynthesis comes from identification of a specific EMF-sensitive DNA sequence on both the c-myc and the HSP70 gene promoters [34]. Numerous studies also verified that a twofold induction of HSP70 improves heart muscle cell resistance to ischemia and hypoxia [35]. EMF exposure has been shown to induce heat shock proteins, which help to maintain the conformation of cellular proteins during periods of stress [36]. Fan et al. reported that anti-apoptotic mechanisms of stress
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Fig. 7. Effects of KNK437 on Ca2þi oscillations in hypoxic cardiomyocytes. (A) Representative tracings of F340/F380 fluorescence ratio. (B) [Ca2þ]i oscillations baseline, (C) amplitude of Ca2þi oscillations and (D) frequency of Ca2þi oscillations. Data are presented as the mean ± SEM (n ¼ 5, for each group). *p < 0.05 vs. Hypoxia group, #p < 0.05 vs. EMF þ Hypoxia group, N.S. ¼ not statistically different.
response proteins via attenuation of the ASK-JNK/p38 signaling cascades [37]. There is evidence that HSP70 has a direct effect on apoptosis by preventing caspase-3 activation, poly (ADP-ribose) polymerase (PARP) cleavage, DNA laddering, and cell death in vitro, but its actions appear to be downstream of cytochrome C release [38]. Our results clearly showed that the EMF exposure induced cardioprotection, but it is almost completely abolished by HSP70 inhibitor (KNK437) in hypoxic cardiomyocytes. There is also evidence showing a link between HSP70 and Ca2þ handling. For example, deleting the inducible 70 kDa heat shock genes impairs cardiac contractile function and Ca2þ handling associated with hypertrophy [39]. The NCX known to play an important determinant of myocardial contractility in heart failure, is acted on by
HSP70, leading to desensitization by a reduction in Vmax [40]. Marx et al. showed that HSP70 also modulates other components of Ca2þ-handling, such as SERCA2a and phosphorylation of RyR [41]. Ca2þ handling-related proteins such as RyR2 and SERCA2a in the membrane of SR and NCX in the sarcolemmal membrane are involved in maintaining Ca2þ homeostasis. It is very likely that HSP70 may stabilize or facilitate the activities of these proteins as a molecule chaperone, thus restoring the Ca2þ homeostasis impaired by hypoxia insults. These literatures and our findings suggest interaction of HSP70 with elements of the Ca2þ handling mechanism that result in ameliorated calcium homeostasis and enhanced myocardial contractility. Overall, our results indicated that activation of HSP70 by EMF preconditioning, it not only diminishes the
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extent of cardiac injury but also enhances the contractile function via remaining Ca2þ homeostasis in hypoxic cardiomyocytes. In summary, the study shows that EMF exposure had beneficial effects on preventing histological damage and cardiomyocytes hypertrophy in hypoxic myocardial. EMF preconditioning inhibited the increase of diastolic [Ca2þ]i, prevented the decrease of SR Ca2þ contents in hypoxic cardiomyocytes. We have also found that EMF significantly activates HSP70, which not only weakens the extent of cardiac injury, but also enhances the contractile function by remaining Ca2þ homeostasis in hypoxic cardiomyocytes. Thus, we believe that EMF protects cardiomyocytes against hypoxia induced alterations in Ca2þ homeostasis via HSP70 activation. Acknowledgements This work was supported by a grant from the National Natural Science Foundation of China, (No.31170893 and No. 11372244). Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.cbi.2016.02.003 Declaration of interest The authors report no declarations of interest. References [1] W. Tong, F. Xiong, Y. Li, L. Zhang, Hypoxia inhibits cardiomyocyte proliferation in fetal rat hearts via upregulating TIMP-4, Am. J. Physiol. Regul. Integr. Comp. Physiol. 304 (2013) R613eR620. [2] K. Shyu, W. Cheng, B. Wang, H. Chang, Hypoxia activates muscle-restricted coiled-coil protein (MURC) expression via transforming growth factor-beta in cardiac myocytes, Clin. Sci. 126 (2014) 367e375. [3] M. Tanaka, H. Ito, S. Adachi, H. Akimoto, T. Nishikawa, T. Kasajima, F. Marumo, M. Hiroe, Hypoxia induces apoptosis with enhanced expression of Fas antigen messenger RNA in cultured neonatal rat cardiomyocytes, Circ. Res. 75 (1994) 426e433. [4] D. Mottet, G. Michel, P. Renard, N. Ninane, M. Raes, C. Michiels, Role of ERK and calcium in the hypoxia-induced activation of HIF-1, J. Cell. Physiol. 194 (2003) 30e44. [5] K.A. Seta, Y. Yuan, Z. Spicer, G. Lu, J. Bedard, T.K. Ferguson, P. Pathrose, A. ColeStrauss, A. Kaufhold, D.E. Millhorn, The role of calcium in hypoxia-induced signal transduction and gene expression, Cell calcium 36 (2004) 331e340. [6] D.M. Bers, Cardiac excitation-contraction coupling, Nature 415 (2002) 198e205. [7] D.M. Bers, Calcium cycling and signaling in cardiac myocytes, Annu. Rev. Physiol. 70 (2008) 23e49. [8] H. Xu, Y. Zhang, J. Sun, J. Wei, L. Sun, J. Zhang, Effect of distinct sources of Ca2þ on cardiac hypertrophy in cardiomyocytes, Exp. Biol. Med. 237 (2012) 271e278. [9] E.C. Toescu, Hypoxia sensing and pathways of cytosolic Ca2þ increases, Cell calcium 36 (2004) 187e199. [10] J. Wei, H. Xu, L. Shi, J. Tong, J. Zhang, Trimetazidine protects cardiomyocytes against hypoxia-induced injury through ameliorates calcium homeostasis, Chemico Biol. Interact. 236 (2015) 47e56. [11] C. Bassett, A. Pilla, R. Pawluk, A non-operative salvage of surgically-resistant pseudarthroses and non-unions by pulsing electromagnetic fields: a preliminary report, Clin. Orthop. Relat. Res. 124 (1977) 128e143. [12] R.H.W. Funk, T. Monsees, N. Oezkucur, Electromagnetic effects:From cell biology to medicine, Prog. Histochem. Cytochem. 43 (2009) 177e264. [13] A. DiCarlo, J. Farrell, T. Litovitz, Myocardial protection conferred by electromagnetic fields, Circulation 99 (1999) 813e816. [14] S. Barzelai, A. Dayan, M.S. Feinberg, R. Holbova, S. Laniado, M. Scheinowitz, Electromagnetic field at 15.95-16 Hz is cardio protective following acute myocardial infarction, Ann. Biomed. Eng. 37 (2009) 2093e2104. [15] A. Albertini, P. Zucchini, G. Noera, R. Cadossi, C.P. Napoleone, A. Pierangeli, Protective effect of low frequency low energy pulsing electromagnetic fields on acute experimental myocardial infarcts in rats, Bioelectromagnetics 20
17
(1999) 372e377. [16] E. Barbier, B. Veyret, B. Dufy, Stimulation of Ca2þ influx in rat pituitary cells under exposure to a 50 Hz magnetic field, Bioelectromagnetics 17 (1996) 303e311. [17] X. Zhang, X. Liu, L. Pan, I. Lee, Magnetic fields at extremely low-frequency (50Hz, 0.8 mT) can induce the uptake of intracellular calcium levels in osteoblasts, Biochem. Biophys. Res. Commun. 396 (2010) 662e666. € ker, M. Deniz, Y. Nergiz, Intracellular Ca2þ levels in rat ventricle [18] C. Sert, S. So cells exposed to extremely low frequency magnetic field, Electromagn. Biol. Med. 30 (2011) 14e20. [19] S. Yitzhaki, A. Shainberg, M. Shaked, Z. Schuss, D. Fixler, Weak magnetic field at 16 Hz affects cardiac myocyte Ca2þ transients and reduces cells damage caused by hypoxia, Open Opt. J. 5 (2011) 33e39. [20] J. Wei, J. Sun, H. Xu, L. Shi, L. Sun, J. Zhang, Effects of extremely low frequency electromagnetic fields on intracellular calcium transients in cardiomyocytes, Electromagn. Biol. Med. 34 (2015) 77e84. [21] G. Grynkiewicz, M. Poenie, R.Y. Tsien, A new generation of Ca2þ indicators with greatly improved fluorescence properties, J. Biol. Chem. 260 (1985) 3440e3450. [22] N. Yamaguchi, N. Takahashi, L. Xu, O. Smithies, G. Meissner, Early cardiac hypertrophy in mice with impaired calmodulin regulation of cardiac muscle Ca2þ release channel, J. Clin. Investig. 117 (2007) 1344e1353. [23] T.R. Shannon, S.M. Pogwizd, D.M. Bers, Elevated sarcoplasmic reticulum Ca2þ leak in intact ventricular myocytes from rabbits in heart failure, Circ. Res. 93 (2003) 592e594. [24] J. Zhou, J. Pei, G. Wang, S. Wu, W. Wang, C. Cho, T. Wong, Inducible HSP70 mediates delayed cardioprotection via U-50488H pretreatment in rat ventricular myocytes, Am. J. Physiol. Heart Circ. Physiol. 281 (2001) H40eH47. [25] Y. Teng, L. Ngoka, Y. Mei, L. Lesoon, J.K. Cowell, HSP90 and HSP70 proteins are essential for stabilization and activation of WASF3 metastasis-promoting protein, J. Biol. Chem. 287 (2012) 10051e10059. [26] S.J. Haggarty, T.U. Mayer, D.T. Miyamoto, R. Fathi, R.W. King, T.J. Mitchison, S.L. Schreiber, Dissecting cellular processes using small molecules: identification of colchicine-like, taxol-like and other small molecules that perturb mitosis, Chem. Biol. 7 (2000) 275e286. [27] S. Murarka, M.R. Movahed, Diabetic cardiomyopathy, J. cardiac Fail. 16 (2010) 971e979. [28] D.H. MacLennan, M. Abed, C. Kang, Structure-Function Relationships in Ca2þ Cycling Proteins, J. Mol. Cell. Cardiol. 34 (2002) 897e918. [29] B. Chamberlain, P. Volpe, S. Fleischer, Calcium-induced calcium release from purified cardiac sarcoplasmic reticulum vesicles. General characteristics, J. Biol. Chem. 259 (1984) 7540e7546. [30] J. Gilloteaux, J. Bissler, P. Kondolios, D. Jarjoura, Cardiomyocyte aging and hypertrophy: atrial and ventricular changes in normal and myopathic Syrian hamsters, J. Submicrosc. Cytol. Pathol. 22 (1990) 249e264. [31] A.R. Liboff, Geomagnetic cyclotron resonance in living cells, J. Biol. Phys. 13 (1985) 99e102. [32] D.J. Panagopoulos, A. Karabarbounis, L.H. Margaritis, Mechanism for action of electromagnetic fields on cells, Biochem. Biophys. Res. Commun. 298 (2002) 95e102. [33] M. Blank, R. Goodman, A mechanism for stimulation of biosynthesis by electromagnetic fields: charge transfer in DNA and base pair separation, J. Cell. Physiol. 214 (2008) 20e26. [34] H. Lin, M. Blank, K. Haseroth, R. Goodman, Regulating genes with electromagnetic response elements, J. Cell. Biochem. 81 (2001) 143e148. [35] R.J. Heads, D.M. Yellon, D.S. Latchman, Differential cytoprotection against heat stress or hypoxia following expression of specific stress protein genes in myogenic cells, J. Mol. Cell. Cardiol. 27 (1995) 1669e1678. [36] M. Blank, R. Goodman, Electromagnetic fields stress living cells, Pathophysiology 16 (2009) 71e78. [37] G. Fan, X. Ren, J. Qian, Q. Yuan, P. Nicolaou, Y. Wang, W.K. Jones, G. Chu, E.G. Kranias, Novel cardioprotective role of a small heat-shock protein, Hsp20, against ischemia/reperfusion injury, Circulation 111 (2005) 1792e1799. [38] S.D. Westerheide, R.I. Morimoto, Heat shock response modulators as therapeutic tools for diseases of protein conformation, J. Biol. Chem. 280 (2005) 33097e33100. [39] Y.K. Kim, J. Suarez, Y. Hu, P.M. McDonough, C. Boer, D.J. Dix, W.H. Dillmann, Deletion of the inducible 70-kDa heat shock protein genes in mice impairs cardiac contractile function and calcium handling associated with hypertrophy, Circulation 113 (2006) 2589e2597. [40] J.G. Kiang, X.Z. Ding, D.E. McClain, Overexpression of HSP-70 Attenuates Increases in [Ca2þ]i and Protects Human Epidermoid A-431 Cells after Chemical Hypoxia, Toxicol. Appl. Pharmacol. 149 (1998) 185e194. [41] S.O. Marx, S. Reiken, Y. Hisamatsu, T. Jayaraman, D. Burkhoff, N. Rosemblit, A.R. Marks, PKA phosphorylation dissociates FKBP12. 6 from the calcium release channel (ryanodine receptor): defective regulation in failing hearts, Cell 101 (2000) 365e376.