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

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 Ca levels ([Ca ]i) caused by hypoxia and abolished [Ca 2 ]i elevation caused by high extracellular Ca levels ([Ca ]o) or by caffeine. Addition of TPEN returned synchronized beating of cardiomyocytes desynchronized by [Ca ]o elevation. To discover the mechanism by which TPEN reduces [Ca ]i in cardiomyocytes, the cells were treated with thapsigargin, which inhibits Ca uptake into the sarcoplasmic reticulum (SR). TPEN successfully reduced [Ca ]i elevated by thapsigargin, indicating that TPEN did not sequester Ca in the SR. However, TPEN did not reduce [Ca ]i in the Na -free medium in which the Na /Ca exchanger was inhibited. Taken together, the results show that activation of sarcolemmal Na /Ca exchanger by TPEN increases Ca extrusion from the cytoplasm of cardiomyocytes, preventing cytosolic Ca overload, which explains the beneficial effects of TPEN on postischemic cardiac status. N,N,N ,N -Tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN) is a chelator of divalent cations, lipid-soluble, and hence, cell-permeable. TPEN is a chelator of transition metals with a high affinity and also binds Ca with a low affinity. Its structure is similar to 1,2-bis(2-aminophenoxy)ethane-N,N,N ,N -tetraacetic acid (Arslan et al., 1985), which binds Ca with high affinity and was found to protect heart tissue from ischemic damage (Billman, 1993). TPEN was proposed as a heart-protective active ingredient for hypothermic cardiac preservative solution, a protective drug against myocardial ischemia-reperfusion injury, and as a reducer of postischemic cardiac damage, but the mechanism of protection is not known (Appelbaum et al., 1990). It was demonstrated in an ex vivo model of ischemia-reperfusion that TPEN is capable of decreasing the incidence of reperfusion-induced damage and improving cardiac recovery. In addition, TPEN reduced metabolic damage after prolonged global ischemia (Appelbaum et al., 1990; Karck et al., 1992; Ferdinandy et al., 1998). Reperfusion of an occluded coronary artery usually results in ventricular arrhythmias such as extra-systoles (ES), ventricular tachycardia (VT), and ventricular fibrillation (VF). It was found that TPEN provides almost total (greater than 90%) protection against ischemiareperfusion-induced arrhythmias (Chevion, 1991). In isolated rat hearts, incubation with TPEN before ischemia caused a decrease of up to 75% in VF and VT (Ferdinandy et al., 1998). In this study, prevention of an overload in intracellular calcium ([Ca ]i), which generally occurs during ischemia, This work was partially supported by the Ministry of Health in Israel and by the Horowitz Foundation of Bar-Ilan University. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. 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 Ca -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 / Ca exchanger. 0022-3565/05/3133-1046–1057$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 313, No. 3 Copyright © 2005 by The American Society for Pharmacology and Experimental Therapeutics 81281/1199527 JPET 313:1046–1057, 2005 Printed in U.S.A. 1046 at A PE T Jornals on O cber 2, 2017 jpet.asjournals.org D ow nladed from has been hypothesized as a mechanism of cardioprotection by TPEN. Increase of [Ca ]i is one of the most important factors of tissue injury caused by oxygen deprivation (Piper et al., 1993). [Ca ]i elevation in cardiac cells occurs through three ways: via the Ca channels (Schroder et al., 1998), via the reverse mode of the Na /Ca exchanger (Dipla et al., 1999), and on Ca 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 Ca 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 [Ca ]i has already been demonstrated in noncardiac cells. TPEN significantly reduced the level of free Ca 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 M (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 Ca overload following hypoxia by TPEN was verified by in vitro study. It is proposed that TPEN activates SR Ca -ATPase (SERCA2a) or Na /Ca exchanger to remove surplus Ca and thereby protect the cardiac cells from injury caused by Ca overload during hypoxia. Materials and Methods