Because pretreatment with ischemic or pharmacological preconditioning is seldom possible in patients with ongoing acute myocardial infarction, strategies that directly target reperfusion could be more reliable. In this regard, ischemic postconditioning (I-Post) and pharmacological postconditioning (P-Post) have generated considerable interest recently. I-Post, which entails brief repetitive mechanical interruptions during early reperfusion, has reduced myocardial infarction in dogs [1], rodents [2], and even humans [3]. The infarct limitation effect by I-Post is as effective as that of ischemic preconditioning (I-Pre) [1]. In addition, P-Post with various adjuvants, such as opioid, bradykinin, insulin, and urocontin, has also been shown to provide cardioprotection [4-7].

N,N,N'N'-Tetrakis-[2-pyridylmethyl]-ethylenediamine (TPEN) is a high-affinity chelator of transition metals, such as Zn²⁺. TPEN also binds Ca²⁺ with a low affinity, and its fast diffusion across membranes renders it an intracellular Ca²⁺ buffer [8]. It has been proposed that ischemic pretreatment with TPEN confers cardioprotection in rat cardiomyocytes [9] and hearts [10], and TPEN treatment before and during hypoxia induces activation of a sarcolemmal Na⁺/Ca²⁺ exchanger and increases Ca²⁺ extrusion from the cytoplasm of cardiomyocytes, preventing cytosolic Ca²⁺ overload in cultured cardiomyocytes [9]. Furthermore, TPEN administered prior to ischemia has exerted beneficial effects on post-ischemic cardiac function; it has also reduced ventricular fibrillation (VF) incidence as well as the incidence and duration of ventricular tachycardia in in vivo rat hearts [10]. However, the TPEN induced myocardial infarct limitation effect, which is considered a more reliable endpoint than functional recovery [11], has not been characterized.

Interestingly, some drugs protect the myocardium against ischemic injury but not against reperfusion injury and vice versa [12-14]. In this regard, it should be determined whether TPEN also provides cardioprotection when administered during the reperfusion period, which is a more suitable management time in clinical settings. Thus far, however, there is scant information about targeting TPEN during the reperfusion phase and the subsequent effects it would have on infarct limitation and cardiac function recovery.

Therefore, in this study, we examined whether P-Post with TPEN at the concentrations that are cardioprotective against ischemia could reduce myocardial infarct size and induce cardiodynamic functional recovery in an isolated rat heart model.

All hearts for this experiment were perfused within 30 s after excision. A total of 19 rat hearts were used for the infarct size measurement study. Two hearts were excluded for the following reasons: HR < 250 beats/min (1) and CF > 18 ml/min (1) after the stabilization period. Therefore, we report the data for 17 hearts that successfully completed the infarct experiment study. The incidence of VF during the first 30 min after reperfusion was 67% (6/9) in the control group and 62% (5/8) in the TPEN group, respectively. Statistical analysis was not performed for the occurrence of VF, because of the small sample size. Hearts experiencing VF after reperfusion usually reverted to sinus rhythm spontaneously or by finger flick cardioversion.

TPEN is a drug that was proposed to protective against myocardial ischemic injury and reduce post-ischemic cardiac damage [17]. Until now, most of the studies on TPEN's cardioprotective effect were performed on ischemic pretreatment, and the effect of P-Post with TPEN remained. The present study has shown for the first time that targeting the use of TPEN, a metal chelator, during the reperfusion phase limits myocardial infarction. In addition, the study showed that there were no differences in cardiodynamic parameters, such as LVDP, RPP, +dP/dt_max, and -dP/dt_min, between TPEN-treated and non-treated hearts. Our data showed that P-Post with TPEN reduces myocardial infarct size but does not improve the functional recovery after reperfusion in isolated rat hearts.

Although tissue salvage by an anti-infarct intervention leads to improvement of cardiac function, infarct size limitation does not always produce mechanical functional recovery of hearts after myocardial ischemic/reperfusion (I/R) injury. For example, a potent opioid such as fentanyl, or angiotensin II reduces infarct size but does not protect against myocardial dysfunction in isolated rat hearts [18,19]. In addition, there are also several lines of evidence that I-Pre and I-Post also limit infarct size but do not reduce myocardial dysfunction [20,21]. The adenosine receptor agonist AMP579 or nitric oxide donors, on the other hand, alleviate both myocardial infarct size and dysfunction [22,23]. The discrepancy between infarct limitation and cardiac function recovery may be explained by continued stunning, or the possibility that the salvaged tissue is not yet normal [11]. Myocardial stunning is prolonged myocardial dysfunction with the histological absence of necrosis after I/R injury. It is proposed that stunning occurs in reversibly injured cells or via nonlethal injury to the epicardial border zones surrounding infarcted myocardium [24]. Although cellular ATP levels or free radicals have been proposed to be important mediators for stunning [25], the exact mechanism responsible for stunning remains unclear. Another possible mechanism for myocardial stunning is an increase in the concentration of intracellular Ca²⁺. The increase in the Ca²⁺ concentrations in myocardial cells is likely explains the depressed ability of I/R hearts to generate contractile force [26]. In our present study, targeting TPEN during the reperfusion period reduced myocardial infarction but did not attenuate post-ischemic systolic dysfunction, even though TPEN has a Ca²⁺ buffer action. We do not know the exact reason for this, but the Ca²⁺ buffer action of the TPEN concentration (10 µM) used during reperfusion might not be enough to attenuate myocardial systolic dysfunction.

It has been previously demonstrated that TPEN reduces basal cardiac nitric oxide (NO) content and prevents the accumulation of NO during myocardial I/R [9]. In addition it has been proposed that lowering sarcolemmal Ca²⁺ content may be a mechanism underlying the recently reported cardioprotective and anti-arrhythmic features of TPEN [9,10]. A chelator is a substance consisting of molecules that bind tightly to metal atoms, thus forcing the metal atoms to go wherever the chelator goes. Though TPEN has been used mainly as a heavy metal (e.g. Zn²⁺) chelator, it has a low affinity for Ca²⁺ as well and its fast diffusion across membranes make TPEN a membrane permeable buffer for Ca²⁺ within intracellular stores [8]. Recently, Shmist et al. [9] demonstrated that TPEN improves myocardial protection against ischemia by modulation of intracellular Ca²⁺ homeostasis. Jung et al. [27] also demonstrated that myocardial protection by TPEN has been related to its modulation of Ca²⁺ homeostasis through the activation of the Na⁺/Ca²⁺ exchanger leading to increased extrusion of Ca²⁺ from the cytoplasm in ventricular myocytes. However, the exact mechanism of the cardioprotective effect achieved by targeting the administration of TPEN during the reperfusion period has not yet been well established and should be determined in the future.

Interestingly, some drugs protect the myocardium when used during the ischemic phase but not in the reperfusion phase and vice versa [12-14]. In this study, administrating TPEN during the reperfusion phase effectively reduced myocardial infarction. Taken together with other previous reports on the effect of PEN treatments targeting ischemia [8,9], one can propose that TPEN has anti-ischemic and reperfusion injury protective effects.

It is also interesting that TPEN acts as a membrane permeable Zn²⁺ chelator, i.e., TPEN reduces intracellular Zn²⁺ concentrations. Zn²⁺ is essential in maintaining the structure and function of cell membranes, and it plays an important role in the maintenance of the activity of various enzymes [28]. Recently, several lines of evidence documented that exogenous Zn²⁺ may lead to cardioprotection against reperfusion injury [29,30]. In our study, TPEN administration during the reperfusion phase significantly reduced myocardial infarction, even though TPEN reduces intracellular Zn²⁺ concentrations. The reason for this is unclear, but we can propose that the Ca²⁺ buffering effect of TPEN may be a more important mechanism for the limitation of infarction than the ability to lower the Zn²⁺ concentration.

We conclude that P-Post with a 10 µM concentration of TPEN, which is proposed to have a cardioprotective effect during ischemia, limits myocardial infarction but does not attenuate myocardial dysfunction in isolated rat hearts. Further studies are needed to determine the exact mechanism for this discrepancy.