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CARDIOVASCULAR

N,N,N',N'-Tetrakis(2-pyridylmethyl)-ethylenediamine Improves Myocardial Protection against Ischemia by Modulation of Intracellular Ca²+ Homeostasis

Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel (Y.A.S., V.S., A.S.); and D-Pharm Ltd., Rehovot, Israel (R.K., G.O., A.K., Y.J.A.)

Received November 24, 2004; accepted January 20, 2005.

	Abstract

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N,N,N',N'-Tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN), a transition-metal chelator, was recently found to protect against myocardial ischemia-reperfusion injury. The goals of this study were to investigate the in vivo antiarrhythmic and antifibrillatory potential of TPEN in rats and guinea pigs and to study the in vitro effects of TPEN on calcium homeostasis in cultured newborn rat cardiac cells in normoxia and hypoxia. We demonstrated on an in vivo rat model of ischemia-reperfusion that TPEN abolishes ventricular fibrillation incidence and mortality and decreases the incidence and duration of ventricular tachycardia. To elucidate the mechanism of cardioprotection by TPEN, contraction, synchronization, and intracellular calcium level were examined in vitro. We have shown for the first time that TPEN prevented the increase in intracellular Ca²⁺ levels ([Ca²⁺]_i) caused by hypoxia and abolished [Ca²⁺]_i elevation caused by high extracellular Ca²⁺ levels ([Ca²⁺]_o) or by caffeine. Addition of TPEN returned synchronized beating of cardiomyocytes desynchronized by [Ca²⁺]_o elevation. To discover the mechanism by which TPEN reduces [Ca²⁺]_i in cardiomyocytes, the cells were treated with thapsigargin, which inhibits Ca²⁺ uptake into the sarcoplasmic reticulum (SR). TPEN successfully reduced [Ca²⁺]_i elevated by thapsigargin, indicating that TPEN did not sequester Ca²⁺ in the SR. However, TPEN did not reduce [Ca²⁺]_i in the Na⁺-free medium in which the Na⁺/Ca²⁺ exchanger was inhibited. Taken together, the results show that activation of sarcolemmal Na⁺/Ca²⁺ exchanger by TPEN increases Ca²⁺ extrusion from the cytoplasm of cardiomyocytes, preventing cytosolic Ca²⁺ overload, which explains the beneficial effects of TPEN on postischemic cardiac status.

In this study, prevention of an overload in intracellular calcium ([Ca²⁺]_i), which generally occurs during ischemia, has been hypothesized as a mechanism of cardioprotection by TPEN. Increase of [Ca²⁺]_i is one of the most important factors of tissue injury caused by oxygen deprivation (Piper et al., 1993). [Ca²⁺]_i elevation in cardiac cells occurs through three ways: via the Ca²⁺ channels (Schroder et al., 1998), via the reverse mode of the Na⁺/Ca²⁺ exchanger (Dipla et al., 1999), and on Ca²⁺ release from sarcoplasmic reticulum (SR) stores. Because the affinity of TPEN for calcium is low, it may be not sufficient to prevent an overload in calcium even by direct binding of calcium in calcium stores of the SR. Therefore, it may be assumed that TPEN activates Ca²⁺ channels and pumps to remove calcium from the cytosol. TPEN may activate them by heavy metal ion chelation as an indirect effect (McNulty and Taylor, 1999) or by activation of signal transduction via G protein-coupled receptors (Webster et al., 2003). The effect of TPEN on [Ca²⁺]_i has already been demonstrated in noncardiac cells. TPEN significantly reduced the level of free Ca²⁺ in the endoplasmic reticulum and increased the activity of calcium-dependent calcium channels in glandular cells (Liu and Ambudkar, 2001) and in bovine adrenal chromaffin cells (Powis and Zerbes, 2002). Beneficial effects of TPEN in cells are found in a narrow concentration range, between 100 nM and 2 µM (Kim et al., 1999).

The aim of this study was to demonstrate the antiarrhythmic and antifibrillatory effects of TPEN in vivo and to investigate the mechanism by which TPEN protects cardiomyocytes from hypoxic damage. Prevention of Ca²⁺ overload following hypoxia by TPEN was verified by in vitro study. It is proposed that TPEN activates SR Ca²⁺-ATPase (SERCA2a) or Na⁺/Ca²⁺ exchanger to remove surplus Ca²⁺ and thereby protect the cardiac cells from injury caused by Ca²⁺ overload during hypoxia.

	Materials and Methods

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Experiments in Vivo
Animal Preparation. This study complied with Israeli legislation regarding experimental procedures on laboratory animals. Adult male Sprague-Dawley rats (n = 38) weighing 270 to 330 g (supplied by Harlan, Indianapolis, IN) were housed in pairs in an environmentally controlled room, with food and water available ad libitum. The rats were anesthetized by i.p. administration of 40 mg/kg pentobarbitone sodium in a volume of 1 ml/kg. Tracheostomy and cannulation were performed, and the animals were artificially ventilated (Harvard rodent ventilator, model 683) using 1 ml of room air per 100 g b.wt. at 54 to 56 cycles/min. A catheter filled with heparinized saline (50 IU/ml) was inserted into the left main carotid artery for arterial pressure measurement using a pressure transducer (UFI, model 1050 bp). The right jugular vein was cannulated for drug administration. ECG (lead II) was monitored with three subcutaneous limb electrodes and recorded along with the arterial pressure using an MP100 data acquisition system (Biopac Systems Inc., Santa Barbara, CA).

Adult male Dunkin Hartley guinea pigs (n = 10) weighing 500 to 700 g were used (Harlan). The preparation procedure included anesthesia, tracheotomy, catheterization of both jugular veins, and ECG recording and was similar to that described above. The guinea pigs were ventilated with 0.8 to 1 ml of room air per 100 g b.wt. at 45 cycles/min.

Ischemia-Reperfusion Induced Arrhythmias in Rats
Preventive Model. The rat's chest was opened by a horizontal cut through the ribs by a rib spreader. The rat was allowed to stabilize for 2 min before the administration of the drug. TPEN dissolved in EtOH was infused at 100 µl/min in the following concentrations: 0.075 mg/ml (n = 11), 0.3 mg/ml (n = 12), and vehicle solution –5% EtOH in saline (n = 11). The final TPEN doses were 0.5 and 2 mg/kg.

An additional 10 mg/kg (1.5 mg/ml) TPEN dose was tested (n = 5), but these experiments were discontinued due to the toxicity of TPEN at this dose. Arterial pressure and heart rate data were collected from four rats that received 10 mg/kg TPEN, without opening their chests. TPEN was administered for 9 to 11 min before the occlusion period, depending on body weight, and administration continued during the 10-min occlusion period. During infusion, a suture was made around the left anterior descending (LAD) coronary artery using a 5-0 silk thread, which was fed through a plastic tube so that the tightening of the tube with the use of tight clips occluded the artery. After 10 min of myocardial ischemia, the ligature was released, the drug infusion was stopped, and reperfusion was initiated. The incidence of arrhythmia was recorded during ischemia and reperfusion in accordance with the Lambeth convention, as ES, salvos, bigeminy, VT, and VF (Walker et al., 1988). The onset and termination times of arrhythmia were also recorded.

Curative Model. In this model, the same procedure as in the preventive paradigm was used with the following alterations. The LAD was first occluded, and then the drug (the same formulation) was administered by manual injection during the 4th and 5th min of the ischemic period. Two animal groups were tested: 2 mg/kg TPEN (n = 7) and control rats (n = 7).

Experiments in Vitro
Solutions. Standard Tyrode's solution, pH 7.4, contained 136.9 mM NaCl, 2.68 mM KCl, 0.42 mM NaH₂PO₄, 11.9 mM NaHCO₃, 1.05 mM MgCl₂, 1.8 mM CaCl₂, and 5.55 mM glucose. Tyrode-choline solution, pH 7.4, contained: 10 mM NaCl, 126.9 mM choline chloride, 2.68 mM KCl, 0.42 mM NaH₂PO₄, 11.9 mM NaHCO₃, 1.05 mM MgCl₂, 1.8 mM CaCl₂, and 5.55 mM glucose. Buffer Na⁺-free solution, pH 7.4, contained: 136.9 mM choline chloride, 2.68 mM KCl, 0.42 mM NaH₂PO₄, 1.05 mM MgCl₂, 1.8 mM CaCl₂, 11 mM HEPES, and 5.55 mM glucose. Standard phosphate-buffered saline (PBS) solution, pH 7.4, contained: 135 mM NaCl, 8.09 mM Na₂HPO₄, 1.47 mM KH₂PO₄, 2.68 mM KCl, 0.9 mM CaCl₂, 0.48 mM MgCl₂, and 5.55 mM glucose. In Ca²⁺-free medium, PBS solution was without CaCl₂ and MgCl₂.

Preparation of Rat Heart Cell Cultures. The rat hearts (1–2 days old) were sterilely removed and washed three times in Ca²⁺- and Mg²⁺-free PBS to remove excess blood cells. The hearts were minced to small fragments and then agitated in a proteolytic enzyme-RDB solution prepared from a fig tree extract as described previously (Brik and Shainberg, 1990; Shneyvays et al., 2001). The RDB was diluted 1:200 in PBS at 25°C for a few cycles of 10 min each. The supernatant suspension containing dissociated cells, to which medium containing 10% horse serum was added, was centrifuged at 500g for 5 min. After centrifugation, the supernatant phase was discarded, and cells were resuspended in Dulbecco's modified Eagle medium supplemented with 10% heat-inactivated horse serum and 0.5% chick embryo extract. The suspension of cells was diluted to 1 x 10⁶ cells/ml, and 1.5 ml was placed in 35-mm collagen/gelatin-coated plastic culture dishes or on collagen/gelatin-coated coverslips. Cultures were incubated in a humidified atmosphere of 5% CO₂ and 95% air at 37°C. A confluent monolayer, which exhibits spontaneous contractions, developed in culture within 2 days. The growth medium was replaced every 3 days. The experiments were performed on these cardiomyocyte cultures after 3 to 5 days in vitro.

Hypoxia. Cardiomyocytes from neonatal rat hearts were cultured and exposed to hypoxia for 90 min with glucose-free PBS (1 ml/dish) in a closed chamber by replacement of air with N₂ (100%) as previously described (Safran et al., 2001). After 90 min of hypoxia, the pO₂ in the PBS was <0.4 torr. Oxygen levels in the N₂ atmosphere were below 1%. Continuous monitoring of intracellular [Ca²⁺]_i during hypoxia was performed in a special barrier well, where cells were protected from oxygen by a laminar counterflow layer of the inert gas argon (Ar 100%). The coverslips with cultured cells were placed at the bottom of the chamber. This chamber was mounted on a specially modified Zeiss inverted epifluorescence microscope (Carl Zeiss GmbH, Jena, Germany) as previously described (Stern et al., 1988). Hypoxic stress injury was characterized by lactate dehydrogenase (LDH) released from cells and by the propidium iodide (PI) staining method.

Experiments on rat cardiomyocyte cultures were performed in three groups. The first group was treated with TPEN (dissolved in DMSO) for 15 min before and during hypoxia. The second group was untreated and underwent hypoxia for various time periods. The third group was the control group, which was untreated and remained in normoxia.

LDH Activity. After hypoxia, LDH activity was determined using LDH-L kit (Thermo Trace, Melbourne, Australia) in the cardiac cell medium (PBS). The product of the enzyme activity was measured in a spectrophotometer at a wavelength of 340 nm at 30°C.

PI and Hoechst measurement. Cultured cardiomyocytes were incubated with PI (5 µg/ml) (Molecular Probes, Eugene, OR) in PBS for 30 min at 37°C to stain nucleus/DNA red of dead cells, washed with PBS twice, and the fluorescence was measured with excitation at 485 nm and emission at 635 nm. Afterward cells were labeled with Hoechst 33342 (Molecular Probes) to stain the nucleus/DNA blue of all cells.

Mitochondrial activity. Living cardiomyocytes grown on coverslips were exposed to 4-(4-(dimethylaminostyryl)-N-methylpyridinium iodide (DASPMI) (Sigma-Aldrich, St. Louis, MO), dissolved in PBS at a final concentration of 10 µg/ml, for 10 min at a temperature of 37°C. The coverslips were then washed and mounted on the hypoxic chamber containing dye-free medium. DASPMI fluorescence was excited at 460 nm, and emission was measured at a wavelength of 540 nm as previously described (Shneyvays et al., 2002). The signal intensity was quantified by using densitometry analysis software ImageJ (National Institutes of Health, Bethesda, MD).

Measurement of contractility. Culture dishes were placed in a specially designed Plexiglas chamber at 37°C. The chamber was placed on the stage of an inverted phase contrast microscope (Olympus, Tokyo, Japan). Measurement of the contractions of the cardiomyocytes at baseline and in response to interventions was conducted using a video motion detector system as described in detail by Zangen and Shainberg (1997). Analog tracing was recorded using an oscilloscope joined through a specially designed interface to an IBM computer, and kinetic data were analyzed by the Microsoft Excel program.

Intracellular Ca²⁺ measurements. Intracellular free calcium ([Ca²⁺]_i) from an individual cell was measured during hypoxia by using the indicator Indo-1 under an epifluorescent microscope. The ratiometric method for the experiments has been described previously (Shneyvays et al., 2001). Cardiomyocytes were incubated with 3 µM Indo-1/AM and 1.5 µM pluronic acid for 30 min in glucose-enriched PBS at 25°C. Indo-1 was excited at 355 nm, and the emitted light was then split by a dichroic mirror to two photomultipliers (Hamamatsu Corporation, Bridgewater, NJ) with input filters at 405:495 nm. The fluorescence ratios of 405:495 nm were fed to a SAMPLE program written by Dr. D. Kaplan from the Biological Institute (Ness-Ziona, Israel). The accepted ratios were considered in an arbitrary scale as previously described (Shneyvays et al., 2001). The time integral of Ca²⁺ influx was determined as area under curve via the same program, SAMPLE, which gives the integral during any specified time window. From this value, the baseline offset, measured at the diastolic level, was subtracted. The time window was the same for each experiment. Calcium transient amplitude R was measured as R = R_max – R_baseline, where R = fluorescence ratio R_405:490 as described previously (Shneyvays et al., 2004).

Statistical analysis. In each experimental group, in vivo measurements of onset and termination times, heart rate, and arterial pressure and in vitro results are expressed as mean ± S.D. n represents the number of experiments. The incidence of VF and death are shown as a percentage of total animals tested and compared by means of the ² method. In vitro data were analyzed by analysis of variance with application of a post hoc Tukey-Kramer test. p < 0.05 was accepted as indicating statistical significance. Each experiment was repeated at least three times and performed each time on three separate dishes per treatment.

Chemicals. Pentobarbitone sodium (Sanofi Veterinaria, Lisbon, Portugal), Dulbecco's modified Eagle's medium, penicillin/streptomycin, and fetal calf serum were purchased from Beit Haemek (Israel). RDB solution was purchased from Biological Institute (Ness-Ziona, Kibbutz Bcit Haemek, Israel). Indo-1/AM was obtained from Teflabs (Austin, TX). All other chemicals were purchased from Sigma-Aldrich.

	Results

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Antiarrhythmic and Antifibrillatory Effects of TPEN
Ischemia-reperfusion was performed on two rat models: the preventive paradigm model, where animals were infused with TPEN before the LAD occlusions and the curative model, where TPEN was given during LAD occlusion.

TPEN in concentrations of 0.5 mg/kg had no effect on hemodynamics (heart rate and arterial pressure). Treatment with 2 mg/kg TPEN caused a small but significant decline in the heart rate throughout the whole experiment compared with nontreated animals, and a decrease in mean arterial pressure was seen only during the early occlusion period (Fig. 1, A and B). TPEN (10 mg/kg) had a severe hemodynamic effect, which was exemplified by bradycardia and a decrease in arterial pressure but was not lethal. This hemodynamic effect was reduced after about 20 min following termination of drug infusion (data not shown).

View larger version (34K): [in this window] [in a new window]   Fig. 1. The hemodynamic effect of TPEN throughout the experimental procedure of ischemia and reperfusion in a preventive paradigm. Animals were infused with TPEN for 20 min (10 min before and 10 min during LAD occlusion). Both heart rate and arterial pressure data were calculated during periods of sinus rhythm only. Results are presented as mean ± S.D. Significance: *, p < 0.05; **, p < 0.005 versus vehicle. BPM, beats per minute.

Results from the preventive treatment model are shown in Table 1. Within the experimental control group, the ischemia-reperfusion procedure resulted in cardiac arrest in the middle of the ischemic period in 2 of 11 rats. The remaining animals displayed arrhythmia following LAD occlusion and reperfusion, whereas most of them experienced reversible or irreversible VF periods. All nontreated rats demonstrated long VT intervals during both the occlusion and the reperfusion periods.

With 0.5 mg/kg TPEN, VF occurred in only 1 of the 11 group members (p < 0.05 versus nontreated rats), and two rats died during the ischemia-reperfusion period (Table 1). TPEN (0.5 mg/kg) significantly decreased the incidence and duration of VT during the ischemic period but not during the reperfusion period (Table 1; Fig. 2). The incidence and duration of other kinds of arrhythmia (ES, salvos, and bigeminy) were not significantly affected in either TPEN-treated or nontreated animals.

View larger version (20K): [in this window] [in a new window]   Fig. 2. Duration of ventricular tachycardia in TPEN-treated (0.5 and 2 mg/kg) and nontreated rats during the occlusion and reperfusion periods. Animals were infused with TPEN for 20 min (10 min prior and 10 min during LAD occlusion). Results are presented as mean log ± S.D. Significance: *, p < 0.05; **, p < 0.01; ***, p < 0.0005 versus vehicle.

Within the nontreated group, the first arrhythmic incidents were single ES or short salvos periods that occurred 5 ± 0.2 min from the moment of LAD occlusion. Arrhythmic incidents occurred throughout the ischemic period. Reperfusion-induced arrhythmias began seconds from blood reflow in all the experimental groups and lasted for 7.5 ± 1.3 min in the control animals. As seen in Fig. 3, both 0.5 and 2 mg/kg TPEN treatments significantly decreased the ischemia-induced arrhythmia, and 2 mg/kg TPEN reduced reperfusion-induced arrhythmias (p < 0.001).

View larger version (24K): [in this window] [in a new window]   Fig. 3. Onset of arrhythmia in the occlusion period, termination of arrhythmia in the reperfusion period, and total time of arrhythmia in TPEN-treated and nontreated rats. Animals were infused with TPEN for 20 min (10 min prior to LAD occlusion and 10 min during occlusion). Results are presented as mean ± S.D. Significance: *, p < 0.05; **, p < 0.001; ***, p < 0.0001 versus vehicle.

Acute Treatment in Rats. As shown in Table 2, 2 mg/kg TPEN decreased the incidence of VF and death when compared with nontreated animals, but the results did not reach statistical significance. TPEN-treated rats showed a significant decrease in VT duration during the ischemic period, but no significant difference appeared during the reperfusion period, in contrast to the preventive treatment.

Effects of TPEN on Cardiomyocyte Cultures Subjected to Hypoxia
TPEN Prevents LDH Leakage. To verify the cardioprotective ability of TPEN, cardiac cells grown in vitro were subjected to hypoxia. The cells were pretreated with TPEN for 15 min before and during various periods of hypoxia. LDH release from cardiomyocytes increased in a time-dependent manner with hypoxia duration ranging from 1 h to 135 min (Fig. 4A). However, in cells treated with 1 µM TPEN before and during hypoxia, the amount of LDH released was reduced 2-fold (from 161 ± 4% to 86 ± 10% after 1 h of hypoxia and in the same proportion after 135 min of hypoxia, Fig. 4A). Protective effect of TPEN was obtained also in a dose-dependent manner beginning at 100 nM TPEN (LDH leakage decreased from 527 ± 7% to 203 ± 14% during 105 min of hypoxia, Fig. 4, A and B). In the range of 0.1 to 3 µM TPEN, 1 µM was the most efficient in reducing LDH leakage especially during prolonged (120 min) hypoxia (Fig. 4B).

View larger version (37K): [in this window] [in a new window]   Fig. 4. Effect of TPEN on LDH release during hypoxia. Four-day-old cardiomyocytes were incubated with 100 nM and 1 µM TPEN for 15 min before and during different times of hypoxia (A) and with 0.1 to 3 µM TPEN during two times of hypoxia (B). Untreated cells served as a control. The LDH release was determined immediately after hypoxia. The release in the control cultures was defined as 100%. Results are expressed as percentage of total cell LDH level and are the mean ± S.D. Significance: *, p < 0.01; **, p < 0.005 versus control; #, p < 0.01; ##, p < 0.005 versus hypoxia.

View larger version (68K): [in this window] [in a new window]   Fig. 5. Effect of TPEN on cardiac cell loss measured by PI entry and on DASPMI fluorescence in mitochondria after hypoxia. Four-day-old cardiomyocytes were incubated with 1 µM TPEN for 15 min before and during 1.5 h of hypoxia (D and H) or subjected to hypoxia in the absence of TPEN treatment (C and G). Untreated cells (A and E) and treated with TPEN (B and F) served as control. After hypoxia, cells were stained with PI and Hoechst (A–D), and PI levels were ascertained (bar graph). Results are expressed as OD and are the mean ± S.D. Significantly different: *, p < 0.005 versus control; #, p < 0.005 versus hypoxia. DASPMI was added immediately after hypoxia and incubated for 15 min (E–H). The densitometry data were expressed as fluorescence intensity (line graph), and arrows indicate location of quantification. n = 6. Bars = 10 µm. Results of one representative experiment from six separate tests.

TPEN Protects the Cardiomyocytes from Ca²⁺ Overload during Hypoxia. Because the increase of intracellular Ca²⁺ ([Ca²⁺]_i) levels in cardiomyocytes is considered a major factor in causing hypoxic damage (Borgers et al., 1988), the ability of TPEN to reduce [Ca²⁺]_i during hypoxia was elucidated with the fluorometric calcium probe Indo-1. Continuous monitoring of [Ca²⁺]_i during hypoxia was realized in a special barrier well, where cells were protected from oxygen by a laminar counterflowing layer of the inert gas argon. Intracellular Ca²⁺ levels were measured every 5 min during hypoxia in nontreated cells and cells treated with TPEN (each measurement lasted 10 s). In untreated cells, a gradual increase in baseline (diastolic) Ca²⁺ levels (R_bas, baseline ratio 405:495) and a decrease of amplitude in [Ca²⁺]_i transients were observed during hypoxia, up to cessation of Ca²⁺ oscillations (Fig. 6A). In cells pretreated with 1 µM TPEN, Ca²⁺ levels during hypoxia remained significantly lower than in nontreated cells (mean R_bas± S.D., 1.35 ± 0.08 after 30 min of hypoxia with 1 µM TPEN, Fig. 6B, compared with 2.24 ± 0.17 after 30 min of hypoxia without TPEN, Fig. 6A), and Ca²⁺ transients were less interrupted.

View larger version (15K): [in this window] [in a new window]   Fig. 6. Effect of TPEN on [Ca]i overload during hypoxia. Four-day-old untreated cardiomyocytes and pretreated with 1 µM TPEN for 15 min cardiomyocytes were exposed to hypoxia. Hypoxia induced a gradual rise in [Ca2+]i, elevation of diastolic [Ca2+]i, and termination of beating activity after 30 min of exposure (A). Pretreatment of cultures with TPEN prevented the basal calcium elevation and maintained myocyte contractility (B). Ca2+ level in cytoplasm was measured by Indo-1 fluorescence. Each measurement during hypoxia was 10 s with intervals of 5 to 10 min between measurements.

TPEN Decreased Caffeine-Induced Calcium Level Elevation in the Cytosol. To verify whether TPEN regulates the amount of calcium in cytosol, myocytes were paced by electric stimulation for 20 s to ensure SR calcium release. After pacing was stopped, caffeine (10 mM) was rapidly applied. Addition of caffeine in this system caused Ca²⁺ release from SR. However, the high level of [Ca²⁺]_i was decreased during the next few minutes. Treatment with TPEN after [Ca²⁺]_i elevation by caffeine caused a slow decrease in [Ca²⁺]_i and Ca²⁺ oscillations were restored (Fig. 7A). We have quantified the effect of TPEN by calculating the time integral of free calcium level in determined time window. TPEN significantly decreased the amount of free Ca²⁺ released by caffeine from SR (3.63 ± 1.24 µM/s, n = 6 versus 6.55 ± 2.04 µM/s, n = 9) in caffeine-treated cells. Obtained data suggest that 1 µM TPEN can successfully remove surplus Ca²⁺ released from the SR.

View larger version (33K): [in this window] [in a new window]   Fig. 7. Effect of TPEN on [Ca]i transients. Four-day-old cardiomyocytes were used. TPEN (1 µM) decreased [Ca2+]i elevated by 10 mM caffeine (arrows) and reestablished contractility (A). Increase of extracellular calcium from 1.8 to 3.8 mM (arrow) increased diastolic [Ca2+]i and ceased calcium oscillations, and application of 1 µM TPEN (arrow) reduced baseline of [Ca2+]i (B). Administration of thapsigargin (TG; 5 µM, arrow) increased diastolic [Ca2+]i, and application of 1 µM TPEN (arrow) reduced baseline of free calcium in regular PBS and did not reduce in Na+-free buffer (C). The quantification of data with thapsigargin is shown in D. Representative results from nine experiments for each test are shown.

These results suggest that 1 µM TPEN increased Ca²⁺ extrusion activity. Because of the low affinity of TPEN to Ca²⁺, its chelating effect may be neglected, and Ca²⁺ extrusion may be postulated either by SR calcium uptake or by activation of the Na⁺/Ca²⁺ exchanger on the sarcolemma.

TPEN Activates Na⁺/Ca²⁺ Exchanger. To test whether TPEN stimulated SR calcium uptake, we examined its effect in the presence of the SERCA2a inhibitor, thapsigargin. Blockade of SR Ca²⁺ uptake by administration of 5 µM thapsigargin in regular Tyrode medium significantly elevated intracellular [Ca²⁺]_i level (R_bas = 2.17 ± 0.19, n = 9) compared with control (R_bas = 1.03 ± 0.09, n = 9). The increased baseline level of [Ca²⁺]_i was significantly reduced after application of 1 µM TPEN (R_bas = 1.08 ± 0.12, n = 9), indicating that blockade of SR calcium uptake did not prevent the effect of TPEN. To examine the role of the Na⁺/Ca²⁺ exchanger in TPEN activity, we studied the Ca²⁺ response in Na⁺-free medium. Obtained results show that 1 µM TPEN reduced the basal level of elevated Ca²⁺ with thapsigargin, R_bas only to 1.56 ± 0.1 in low-Na⁺ medium (20 mM), and did not change the Ca²⁺ level in the Na⁺-free medium in which the Na⁺/Ca²⁺ exchanger was inhibited (n = 9, Fig. 7, C and D). These results show that TPEN reduces Ca²⁺ overload by activation of Na⁺/Ca²⁺ exchanger to extrude Ca²⁺ from the cytosol (Fig. 7, C and D).

Effects of TPEN on Synchronization of Cardiomyocyte Contraction. The cellular contraction activity of cultured cardiomyocytes was analyzed by a video technique previously described (Zangen and Shainberg, 1997; Shneyvays et al., 1998). The majority of the cultured cells exhibited synchronized spontaneous contractions (Fig. 8). Increase of [Ca²⁺]_o, from 1.8 to 3.8 mM violated synchronized beating activity of cardiomyocytes up to total desynchronization with fibrillating contractions and termination of cell movement. Addition of 1 µM TPEN after [Ca²⁺]_o elevation rapidly restored the synchronized cell movement. Figure 8 demonstrated cardiomyocyte contraction of four different cells under normal conditions, following an increase of [Ca²⁺]_o to 3.8 mM and administration of 1 µM TPEN.

View larger version (58K): [in this window] [in a new window]   Fig. 8. Effect of TPEN on cardiomyocytes contractility after extracellular Ca2+ elevation. Simultaneous recording of cellular contraction of four different cells (A–C) from culture in normal conditions (control), following increase of [Ca2+]o to 3.8 mM (Ca 4 mM) and following administration of 1 µM TPEN (TPEN). One experiment is representative of six.

	Discussion

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TPEN is known as a cell-permeable metal ion chelator. In addition to binding Cu²⁺, Zn²⁺, and Fe²⁺ with a high affinity, TPEN binds Ca²⁺ and Mg²⁺ with a low affinity. Much research has focused on the use of specific and nonspecific transition metal ion chelators as cardioprotective agents in reperfusion-induced arrhythmias. Iron chelators such as deferoxamine and copper chelators such as bathocuproine and neocuproine (which is also an iron chelator) are the best studied groups so far. The cardioprotective properties of these compounds were shown in several studies both in vitro and in vivo (Ambrosio et al., 1987; Reddy et al., 1989; Appelbaum et al., 1990; Williams et al., 1991; Spencer et al., 1998; Shadid et al., 1999), but the mechanism of the protective effect is still not known.

Possible Mechanisms of TPEN Cardioprotection. TPEN has been found as a cardioprotective drug only in ex vivo models, using the Langendorff model (Appelbaum et al., 1990; Chevion, 1991; Karck et al., 1992; Ferdinandy et al., 1998). Due to the different properties of TPEN, it can most probably prevent cardiac damages by more than one mechanism. TPEN can act as an antioxidant during reoxygenation because metals such as iron may convert weakly reactive molecules to strongly reactive molecules, and the chelator of metals could reduce damage caused by free radical production (Galey et al., 1998). On the other hand, the antiarrhythmic effects of TPEN and reduction of VT duration in the ischemic period, when oxygen deprivation leads to energetic exhaustion and Ca²⁺ overloading in the myocardium, could be the result of vasodilatation or modulation of heart contractility and oxygen consumption. The increase in intracellular level of Zn²⁺ under pathological conditions may be an important factor in hypoxic damage (Powell et al., 1994), and chelation of Zn²⁺ may be included in the cardioprotective mechanism of TPEN (Turan, 2003). Additionally, TPEN can prevent Ca²⁺ overload in cardiac cells by direct Ca²⁺ binding or by activating mechanisms of Ca²⁺ removal. The last option was examined for the first time in this work.

Research on TPEN in Vivo. This study was performed to verify the cardioprotective effects of TPEN in vivo and to investigate the mechanism of protection in vitro. Our results in vivo have shown that TPEN successfully improves cardiac recovery of perfused hearts after regional and prolonged ischemia. TPEN applied i.v. to anesthetized rats at the lowest dose used (0.5 mg/kg) displayed protection against fibrillation and ischemia-induced arrhythmias and partially protected against reperfusion-induced arrhythmia and mortality. At 2 mg/kg, TPEN provided overall cardiac protection for all the parameters measured. However, 10 mg/kg TPEN proved to have a hemodynamic toxic effect that accompanied the cardiac protection. The lethal dose of TPEN determined in this study, 30 to 40 mg/kg, is in accord with published data (Evangelou and Kalfakakou, 1993).

Two in vivo modes of treatment were employed here, a preventive paradigm in which the drug was infused prior and during the ischemic period, and a curative paradigm in which a bolus injection was given during the expected onset of arrhythmia in the ischemic period. In the preventive model, TPEN effectively reduced ischemia-reperfusion-induced arrhythmia and successfully decreased the mortality rate. The antiarrhythmic properties of TPEN were not fully sustained in the curative treatment model. It is possible that the ability of TPEN to prevent arrhythmia in the acute treatment paradigm decreased because of a requirement for additional time to penetrate into the cells.

Research on TPEN in Vitro. To elucidate the cellular mechanism of cardioprotection of TPEN, we designed and carried out an in vitro study on primary cardiac cell cultures. The results of in vitro LDH leakage and propidium iodide staining after hypoxia demonstrate that the treatment with 0.1 to 3 µM TPEN reduces damage of cardiomyocyte membranes in a dose-dependent manner. TPEN also successfully preserved the structure and energetic capability of mitochondria, which were significantly damaged during 90 min of hypoxia. In cells pretreated with 1 µM TPEN for 15 min before and during hypoxia, mitochondria appeared much more active than in nontreated cells subjected to hypoxia.

TPEN Preserves Ca²⁺ Homeostasis. In oxygen-deprived cardiomyocytes, Ca²⁺ inserted from the medium and from SR release accumulated in the cytosol and caused cell damage (Piper et al., 1993). Therefore, we tested the ability of TPEN to prevent cytosolic Ca²⁺ overload during hypoxia. As was shown in this study, 1 µM TPEN delayed the increase of intracellular free calcium in cardiomyocytes caused by hypoxia (Fig. 6, A and B).

In previous studies, it was suggested that Ca²⁺ regulation by TPEN occurs by direct binding of Ca²⁺ in the calcium stores (endoplasmic reticulum). Very high concentrations of TPEN were used because of its low affinity to Ca²⁺ (K_d = 4.0 x 10^–5 M). Direct measurements of luminal free Ca²⁺ in BNK-21 fibroblasts have demonstrated intraluminal binding of Ca²⁺ by 200 µM TPEN (Hofer et al., 1998; Caroppo et al., 2003). In the SR of rat cardiomyocytes (Zucchi et al., 2001), the Ca²⁺ level is also high, but these concentrations of TPEN are toxic for cardiac cells. TPEN (1 µM), which was found to protect cardiomyocytes from hypoxic damage, is unlikely to be sufficient to significantly affect the millimolar Ca²⁺ concentration in the SR by binding Ca²⁺. We suggest an alternative and more useful mechanism of Ca²⁺ regulation by TPEN. We suppose that low concentration of TPEN may protect cardiomyocytes by regulation of calcium homeostasis in a nonbinding way. Prevention of Ca²⁺ overload may be due to inhibition of Ca²⁺ influx from the medium or efflux from SR or by activation of the extrusion systems. Other previous studies have shown that TPEN can strongly inhibit the L-type Ca²⁺ channel current (Turan, 2003) and thereby inhibit Ca²⁺ influx to the cells. As is known, in cardiac cells, Ca²⁺ influx through dihydropyridine receptors is necessary for contractions, and Ca²⁺-induced Ca²⁺ release from SR is thought to be the dominant mechanism (Evangelou and Kalfakakou, 1993). However, inhibition of Ca²⁺ influx may explain the prevention of intracellular calcium overload but cannot explain the decrease of Ca²⁺ after Ca²⁺ overload.

TPEN Decreases [Ca²⁺]_i Overload by Activation of Na⁺/Ca²⁺ Exchanger. We examined the hypothesis that a decrease of cytosol calcium by TPEN may occur due to removal of Ca²⁺ by activation of SERCA2a or Na⁺/Ca²⁺ exchanger (NCX). We applied TPEN the moment after [Ca²⁺]_i increase, so the role of L-type Ca²⁺ channel current inhibition became irrelevant. We have shown that TPEN decreased [Ca²⁺] in the cytosol after caffeine application, which not only induces Ca²⁺ efflux from SR but also closed Ca²⁺ influx to SR (Bassani et al., 1994). These data were confirmed by the direct inhibition of the SERCA2a by thapsigargin. The lowering of cytosolic [Ca²⁺] by TPEN in the presence of the SERCA2a inhibitor thapsigargin suggests that SERCA2a is not mediated in decrease of [Ca²⁺] in cytosol. However, TPEN did not decrease [Ca²⁺]_i after elevation in the absence of Na⁺ in the buffer medium, conditions which inhibit the NCX. These results suggest that TPEN prevents Ca²⁺ overload via activation of the NCX.

Earlier in our laboratory, it was shown that elevation of extracellular [Ca²⁺]_o from 1.9 to 3.9 mM increased [Ca²⁺]_i and transformed the synchronized cell movement to an asynchronous one (Manoach et al., 1997). We used this model in our study and found that addition of 1 µM TPEN after elevation of [Ca²⁺]_o returned the beating of cells to the normal rate and promoted synchronization of cell contractions.

In conclusion, in vivo antiarrhythmic and antifibrillatory effects of TPEN are demonstrated here. Given the cardioprotective properties of TPEN against ischemia-reperfusion-induced arrhythmias, therapeutically useful doses for i.v. administration are defined. In vitro results suggest that TPEN activates the NCX, but not SERCA2a, to work more effectively and in this way preserve homeostasis of Ca²⁺ during hypoxia. TPEN protects cardiac cells against Ca²⁺ overload damage during hypoxia by activating Ca²⁺ surplus extrusion to the medium and not by regulating Ca²⁺ influx to the SR. Thus, TPEN has considerable therapeutic potential for use in some surgical cardiovascular procedures. TPEN may be useful in the future as an antiarrhythmic and antifibrillatory drug. TPEN has the potential to protect against Ca²⁺ overload damage caused by ischemia and cardiotoxic drugs such as anticancer treatments (Alloatti et al., 1998; Shneyvays et al., 2001; Wang et al., 2001; Suter et al., 2004; Yeh et al., 2004). Additionally, this study opens a new direction for a therapeutic strategy by direct activation of the NCX forward mode to circumvent the problems of Ca²⁺ overload in other excitable cells such as the central nervous system (Petty and Wettstein, 1999).

	Acknowledgements

 
We thank S. Moshel, T. Zinman, and A. Isaac for technical support. We also thank M. Eichler and S. Victor for help in preparing this article.

	Footnotes

 
This work was partially supported by the Ministry of Health in Israel and by the Horowitz Foundation of Bar-Ilan University.

doi:10.1124/jpet.104.081281.

ABBREVIATIONS: TPEN, N,N,N',N'-tetrakis(2-pyridylmethyl)-ethylenediamine; ES, extra-systoles; VT, ventricular tachycardia; VF, ventricular fibrillation; SR, sarcoplasmic reticulum; SERCA2a, SR Ca²⁺-ATPase; LAD, left anterior descending; PBS, phosphate-buffered saline; LDH, lactate dehydrogenase; PI, propidium iodide; DASPMI, 4-(4-(dimethylaminostyryl)-N-methylpyridinium iodide; RPP, rate-pressure product; NCX, Na⁺/Ca²⁺ exchanger.

Address correspondence to: Prof. Asher Shainberg, Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel. E-mail: shaina{at}mail.biu.ac.il

	References

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