Cardiovascular Research

Cardiovascular Research 2002 55(4):739-748; doi:10.1016/S0008-6363(02)00461-3
© 2002 by European Society of Cardiology

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Copyright © 2002, European Society of Cardiology

Effect of inhibition of Na⁺/Ca²⁺ exchanger at the time of myocardial reperfusion on hypercontracture and cell death

Javier Inserte^a, David Garcia-Dorado^a,*, Marisol Ruiz-Meana^a, Ferran Padilla^a, José A Barrabés^a, Pilar Pina^a, Luis Agulló^a, Hans Michael Piper^b and Jordi Soler-Soler^a

^aServicio de Cardiologia, Hospital Universitari Vall d’Hebron, Passeig Vall d’Hebron 119-129, 08035 Barcelona, Spain
^bPhysiologisches Institut Justus-Liebig-Universität, Giessen, Germany

^* Corresponding author. Tel.: +34-93-489-4038; fax: +34-93-489-4032 dgdorado{at}hg.vhebron.es

Received 8 October 2001; accepted 29 April 2002

	Abstract

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
Objective: There is recent evidence that Ca²⁺ influx via reverse mode Na⁺/Ca²⁺ exchange (NCX) at the time of reperfusion can contribute to cardiomyocyte hypercontracture. However, forward NCX is essential for normalization of [Ca²⁺]_i during reperfusion, and its inhibition may be detrimental. This study investigates the effect of NCX inhibition with KB-R7943 at the time of reperfusion on cell viability. Methods: The effect of several concentrations of KB-R7943 added at reperfusion was studied in Fura-2 loaded quiescent cardiomyocytes submitted to 40 min of simulated ischemia (NaCN 2 mM, pH 6.4), and in rat hearts submitted to 60 min of ischemia. [Ca²⁺]_i and cell length were monitored in myocytes, and functional recovery and LDH release in isolated hearts. From these experiments an optimal concentration of KB-R7943 was identified and tested in pigs submitted to 48 min of coronary occlusion and 2 h of reperfusion. Results: In myocytes, KB-R7943 at concentrations up to 15 µM reduced [Ca²⁺]_i rise and the probability of hypercontracture during re-energization (P<0.01). Nevertheless, in rat hearts, the effects of KB-R7943 applied during reperfusion after 60 min of ischemia depended on concentration and timing of administration. During the first 5 min of reperfusion, KB-R7943 (0.3–30 µM) induced a dose-dependent reduction in LDH release (half-response concentration 0.29 µM). Beyond 6 min of re-flow, KB-R7943 had no effect on LDH release, except at concentrations 15 µM, which increased LDH. KB-R7943 at 5 µM given during the first 10 min of reflow reduced contractile dysfunction (P = 0.011), LDH release (P = 0.019) and contraction band necrosis (P = 0.014) during reperfusion. Intracoronary administration of this concentration during the first 10 min of reperfusion reduced infarct size by 34% (P = 0.033) in pigs submitted to 48 min of coronary occlusion. Conclusions: These results are consistent with the hypothesis that during initial reperfusion NCX activity results in net reverse mode operation contributing to Ca²⁺ overload, hypercontracture and cell death, and that NCX inhibition during this phase is beneficial. Beyond this phase, NCX inhibition may impair forward mode-dependent Ca²⁺ extrusion and be detrimental. These findings may help in the design of therapeutic strategies against lethal reperfusion injury, with NCX as the target.

KEYWORDS Calcium (cellular); Ischemia; Myocytes; Na/Ca-exchanger; Reperfusion

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This article is referred to in the Editorial by S. Anderson (pages 706–707) in this issue.

	1. Introduction

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
Cell death occurring during the first minutes of reperfusion is explained as a consequence of the combined action of mechanical and chemical stress on cardiomyocytes in which previous ischemia has induced sarcolemmal and cytoskeletal fragility [1]. Excessive contractile activation, secondary to increased energy availability together with increased intracellular Ca²⁺ concentration ([Ca²⁺]_i), plays a central role among the causes of stress [2,3]. Excessive contractile activation causes hypercontracture, which in myocardium in situ results in sarcolemmal rupture, massive enzyme release, and a pathologic pattern known as contraction band necrosis [4,5].

The mechanisms responsible for altered Ca²⁺ handling in reperfused cardiomyocytes have not been completely elucidated. [Ca²⁺]_i rises during prolonged anoxia or ischemia [6,7]. During reoxygenation or reperfusion, Ca²⁺ sequestration by the sarcoplasmic reticulum (SR) tends to rapidly normalize [Ca²⁺]_i [8]. Ca²⁺ uptake by the SR may be followed by Ca²⁺ release and re-uptake resulting in Ca²⁺ oscillations that contribute to the development of hypercontracture [8]. It has been proposed that Ca²⁺ influx at reperfusion can contribute to reperfusion injury [9–11] at least in part by increasing Ca²⁺ oscillations.

In mammalian cardiomyocytes, sarcolemmal Na⁺/Ca²⁺ exchange (NCX) takes place through the NCX1 isoform, with a Ca²⁺:Na⁺ coupling ratio of 1:3 and a net movement of a positive charge. The direction of NCX operation depends on the difference between trans-membrane potential (V_M) and reversal potential (RP), determined by the intra and extracellular concentrations of Na⁺ and Ca²⁺. During diastole RP is close to –60 mV, and positive with respect to resting V_M (close to –80 mV), which results in forward (Ca²⁺ out) NCX. During systolic depolarization, RP is negative with respect to V_M, which results in reverse NCX. The contribution of reverse NCX to Ca²⁺ transients is negligible in normal conditions in most animal species [12]. However, reverse NCX may have an important pathophysiological role in situations in which [Na⁺]_i is elevated. The role of reverse NCX in the genesis of cytosolic Ca²⁺ overload during ischemia has been recently established in cardiomyocytes [13].

The highly increased [Na⁺]_i observed after re-energization is likely to be the result of three major factors: first, Na⁺ gain during prior metabolic inhibition [14]; second, additional Na⁺ influx associated with normalization of pH_i through Na⁺/H⁺ exchange and Na⁺/HCO₃^– cotransport; and third, Na⁺ entry from adjacent myocytes via gap-junctions [15]. Moreover, Na⁺/K⁺-ATPase activity may be impaired in myocardium that is reperfused after prolonged ischemia [16]. The concept that Ca²⁺ influx at the time of re-energization may contribute to cell injury secondary to transient anoxia or ischemia is supported by the protective effect observed when extracellular Ca²⁺ concentration is reduced during reperfusion [9,11], and by the fact that high extracellular Na⁺ during reperfusion attenuates post-ischemic contractile dysfunction [17]. However, evidence of the long suspected contribution of reverse NCX to Ca²⁺ overload during reperfusion [10,17] has remained elusive until recently [18], due in part to the absence of sufficiently selective inhibitors of this exchanger.

An isothiourea derivative, 2-[2-4[(nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulphonate (KB-R7943; Kanebo, Osaka, Japan), was recently characterized as a fairly selective inhibitor of NCX in cardiomyocytes [19–22]. Under ionic conditions allowing alternate net forward and net reverse NCX transport during the cardiac cycle in response to changes in membrane potential, KB-R7943 inhibits forward and reverse exchange at a virtually identical half-maximal inhibition concentration (IC₅₀) (1 µM) [21]. However, in cells under ionic conditions allowing a preponderance of NCX reverse operation ([Na⁺]_out=0), the inhibitory ability of KB-R7943 is significantly higher (IC₅₀=0.32 µM) than in cells exposed to ionic conditions allowing a preponderance of forward transport via NCX ([Ca²⁺]_out0, IC₅₀=17 µM) or in cells exposed to ionic conditions allowing alternate net forward or net reverse NCX transport (IC₅₀=1 µM) [19–21]. The mechanism responsible for these differences is not known, but it could be related to a different prevalence of distinct exchange transport states under the different ionic conditions. Inhibition becomes higher as the prevalence of the intracellular 3Na⁺-loaded conformation increases [23].

In a recent study we showed that the presence of 10 µM KB-R7943 in the reoxygenation buffer reduced Ca²⁺ oscillations and hypercontracture in cardiomyocytes previously exposed to hypoxia, and attenuated LDH release in isolated hearts exposed to 60 min of ischemia [18]. The potentially harmful effects of NCX inhibition using forward NCX to extrude Ca²⁺ in reperfused cells, the dependence of these effects on the time of administration, and the effects of NCX inhibition during in vivo reperfusion were not analyzed in that study. These important points are addressed in the present study. The effects of different concentrations of KB-R7943 added at the time of reperfusion were analyzed in cardiomyocytes exposed to simulated ischemia, and in isolated rat hearts submitted to non-flow ischemia. To evaluate the potential relevance of the effects of NCX inhibition to in vivo conditions, selected concentrations of KB-R7943 were administered to pigs during coronary reperfusion.

	2. Methods

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
The experimental procedures conformed with the Guide for the Care and Use of Laboratory Animals published by the United States National Institute of Health (NIH Publication No. 85-23, revised 1996), and were approved by the Research Commission on Ethics of the Hospital Vall d’Hebron.

2.1 Studies in isolated cardiomyocytes
Ventricular myocytes were isolated from adult male Sprague–Dawley rats and plated in glass-bottom dishes coated with 199/HEPES medium containing 4% fetal calf serum (Gibco), as previously described [24]. Then 2 h after plating, non-attached cells were discarded by changing the medium (in mM: NaCl 140, KCl 3.6, MgSO₄ 1.2, CaCl₂ 1, HEPES 20, pH 7.4).

2.1.1 Experimental protocols
The inhibitory effect of KB-R7943 on NCX during conditions allowing a preponderance of reverse transport was studied by monitoring [Ca²⁺]_i in normoxic myocytes during Na⁺ withdrawal ([Na⁺]_out=0; osmolarity corrected by addition of Li⁺) for 30 s after functional blockade of the SR with 150 nM thapsigargin and 10 µM ryanodine [18,25].

To analyze the effect of KB-R7943 during simulated reperfusion, cardiomyocytes were first subjected to 40 min of metabolic inhibition (in mM: NaCl 140, KCl 3.6, MgSO₄ 1.2, CaCl₂ 1, HEPES 20, NaCN 2, 2-deoxyglucose 20, pH 6.4). The inhibitor and 2-deoxyglucose were then removed, 5 mM glucose was added, and pH was normalized at 7.4. KB-R7943 at 0 (control), 5, 10 or 15 µM was added to the simulated reperfusion buffer. The contribution of inhibition of Ca²⁺ oscillations to the effects of inhibition of NCX with KB-R7943 was analyzed in additional experiments in which simulated reperfusion was performed in the presence of either 10 µM KB-R7943 or SR functional blockade, both these conditions, or neither of them. The changes in osmolarity of the medium associated with the addition or removal of metabolic inhibition were not corrected.

2.1.2 Measurement of isolated cell morphology and [Ca²⁺]_i
All experiments were performed at 37 °C on the stage of an inverted microscope [15]. Individual myocytes were imaged throughout the experiment, and changes in cell length and [Ca²⁺]_i were simultaneously monitored using color-coded 340/380 ratio fluorescence images (QuantiCell900, Visitech, UK) in myocytes loaded with Fura-2 [15]. Cardiomyocyte hypercontracture was defined as a reduction in cell length >10% of the length at the end of the metabolic inhibition period.

2.2 Studies in the isolated perfused rat heart
Hearts from male Sprague–Dawley rats (n = 40) were perfused with a modified Krebs-Henseleit bicarbonate buffer (in mM: NaCl 140, NaHCO₃ 24, KCl 2.7, KH₂PO₄ 0.4, MgSO₄ 1, CaCl₂ 1.8, and glucose 11) at a constant pressure of 60 mmHg [24]. Left ventricle (LV) pressure was monitored during equilibration through the use of a water-filled latex balloon inserted into the LV and inflated to obtain an end-diastolic pressure (LVEDP) between 6 and 8 mmHg [24]. Coronary flow was measured at regular intervals. Lactate dehydrogenase (LDH) activity was measured in samples of coronary effluent by spectrophotometry, as previously described [24].

2.2.1 Experimental protocol
In the ischemia–reperfusion experiments, hearts were subjected to 37 °C non-flow ischemia for 60 min, and reperfused for 60 min. KB-R7943 at concentrations ranging from 0 (control) to 30 µM was added to the perfusion buffer during the first 10 min of reperfusion. In additional experiments (n = 8), 15 µM KB-R7943 or its vehicle was added only during the first 4 min of reperfusion. The balloon was deflated during ischemia [26] and at the end of the reperfusion period it was filled again with the same amount of water as had been used during the preischemic perfusion.

In a separate series of experiments, the effect of 5 µM KB-R7943 (vs. vehicle) was investigated in 12 hearts in which LV pressure was monitored throughout the experiment by maintaining the LV balloon inflated during ischemia and reperfusion.

2.2.2 Histological analysis
Cross-sectional 4-µm midventricular sections were stained with Masson's trichrome. Contraction band necrosis was morphometrically quantified as previously described [24]. Two perpendicular lines crossing at the center of the LV cavity were drawn for each section. Serial microphotographs of adjacent optical fields (x400) were obtained along these lines, and digitized for subsequent analysis (Olympus DP10 camera and Micro Image^TM, Olympus Optical, Japan). Each microphotograph was graded using a semiquantitative scoring system from 0 to 3 (0, 1, 2 and 3 correspond to contraction band necrosis involving <1/4, 1/4–1/2, 1/2–3/4 and >3/4 of the photographed frame, respectively) [24], and the average score was calculated for each heart.

2.3 Transient coronary occlusion in the in situ pig heart
A total of 12 Large White pigs weighing 30–40 kg were premedicated with 10 mg/kg azaperone i.m., anesthetized with thiopental 30 mg/kg i.v., followed by continuous infusion, intubated, mechanically ventilated with room air, and monitored as previously described [23]. A midline sternotomy was performed and the left anterior descending coronary artery (LAD) was dissected free at its midpoint and surrounded by an elastic snare. Two pairs of 1-mm diameter ultrasonic crystals were inserted into the inner third of the LV wall in the LAD and circumflex territory, respectively, and LV pressure and LAD coronary blood flow were measured [4,24]. A 2.5F catheter for intracoronary infusion was advanced through a Judkins 7F guiding catheter into the LAD until its tip was placed immediately proximal to the occlusion site [24].

2.3.1 Study protocol
A total of 10 animals were submitted to 48 min of LAD occlusion and randomly allocated to receive an intracoronary infusion of saline alone or saline containing 35 µM KB-R7943 during the first 10 min of reperfusion. In both groups, the infusion rate was continuously adjusted to obtain a final concentration of 5 µM KB-R7943. In additional experiments, animals were allocated to receive KB-R7943 at 15 µM (n = 4) or saline (n = 2). Finally, coronary occlusion was not performed in two pigs and KB-R7943 at a final concentration of 5 µM was infused into the mid LAD for 10 min.

2.3.2 Infarct size measurement
After 2 h of reperfusion the LAD was re-occluded and 5 ml of 10% fluorescein was injected into the left atrium. The heart was excised, cooled at 4 °C, and cut into 5–7-mm slices that were imaged (Olympus Digital Camera C-1400L) under UV light to outline the area at risk. The slices were then incubated at 37 °C for 10 min in 1% triphenyltetrazolium chloride (pH 7.4) and imaged again under white light to outline the area of necrosis. The area at risk and the area of necrosis were measured on the digitized images, and the mass of myocardium at risk and the mass of necrotic myocardium were calculated as described [2,24].

2.4 Statistical analysis
Differences between groups were assessed by means of one-way ANOVA. Individual comparisons between groups were performed by the Student's t-test for independent samples. Changes along time were assessed by multiple ANOVA. Significance was set at a P-value of 0.05. Results are expressed as mean±S.E.M.

	3. Results

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
3.1 Effect of KB-R7943 on hypercontracture and [Ca²⁺]_i kinetics in isolated cardiomyocytes
In normoxic myocytes, KB-R7943 inhibited [Ca²⁺]_i rise induced by Na⁺ withdrawal with a calculated IC₅₀ of 0.15±0.02 µM (Fig. 1).

View larger version (21K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Inhibition of reverse NCX by KB-R7943 in isolated cardiomyocytes. [Ca2+]i rise induced by 30 s of Na+ withdrawal was measured in the presence of different concentrations of KB-R7943. n = 6–9 cells per group. Data are mean±S.E.M.

 
After 40 min of metabolic inhibition, re-energization induced hypercontracture in 77.2% of cells within 5 min. The presence of KB-R7943 at 5, 10 or 15 µM in the re-energization media significantly reduced the proportion of cells undergoing hypercontracture (60.9, 41.2 and 35.3%, respectively, P<0.05).

During the first minute of reoxygenation, a decrease in [Ca²⁺]_i was observed in all the treatment groups, with no between-group differences. After this time, [Ca²⁺]_i increased in the control group but continued to decrease in cells re-energized in the presence of KB-R7943 during the first 5 min of reoxygenation, remaining stable thereafter (Fig. 2). There were no differences in the change in Fura-2 ratio during the first 10 min of re-energization between cells treated with 5, 10 or 15 µM KB-R7943 (1.02±0.10, 0.86±0.08 and 0.92±0.07, respectively).

View larger version (32K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Effect of 10 µM KB-R7943 and 150 nM thapsigargin plus 10 µM ryanodine (T&R) on diastolic Fura-2 ratio (a.u., arbitrary units) during re-energization of isolated cardiomyocytes after 40 min of simulated ischemia. Pre-SI (, n = 19) indicates Fura-2 ratio before simulated ischemia. Data are mean±S.E.M. *P<0.001 for KB-R7943 groups in 2x2 factorial ANOVA.

 
SR blockade with thapsigargin and ryanodine was not associated with a significant reduction in the number of cells undergoing hypercontracture. Hypercontracture was significantly less frequent in KB-R7943 treated cells, and was virtually absent (one out of 22) in cells receiving both KB-R7943 and SR functional blockade (Fig. 3). The frequency of Ca²⁺ oscillations was lower in cells re-energized in the presence of 10 µM KB-R7943 than in the controls (27.2±2.8 vs. 35.6±2.8 oscillations/min, P = 0.060). There were no Ca²⁺ oscillations in cells in which SR function had been blocked by treatment with thapsigargin plus ryanodine.

View larger version (25K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Effect of inhibition of NCX with 10 µM KB-R7943 (KB) and functional blockade of the sarcoplasmic reticulum with 150 nM thapsigargin plus 10 µM ryanodine (T&R) on cardiomyocyte hypercontracture during re-energization after 40 min of simulated ischemia. Error bars indicate 95% CI. *P<0.05 versus control.

 
3.2 Studies in perfused rat heart
The concentration- and time-dependent effects of KB-R7943 exposure during initial myocardial reperfusion were investigated in the perfused rat heart model.

In control hearts subjected to transient ischemia, LV developed pressure (LVdevP), defined as LV peak systolic pressure–LVEDP measured at 60 min of reperfusion, recovered to 9.5±2.6% of its initial value. LVdevP recovery increased by the addition of KB-R7943 at concentrations of 0.3–10 µM during the first 10 min of reperfusion (to values between 19.3±3.2 and 25.7±6.35.1%, P<0.01). This effect was lost at higher concentrations, but was sustained when perfusion with 15 µM KB-R7943 was restricted to the first 4 min of reperfusion (21.5±4.3%, P = 0.0.012) (Fig. 4).

View larger version (24K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Effect of addition of KB-R7943 during the initial 4 () or 10 min of reperfusion on LVdevP recovery in isolated rat hearts (B). n = 4 per group. Data are mean±S.E.M.

 
LDH release during the 60 min of reperfusion decreased by more than 25% with exposure to KB-R7943 at 0.3–10 µM (P<0.05). Concentrations of 15 or 30 µM KB-R7943 exacerbated LDH release (212 and 233% of control value, respectively, P<0.01). However, 15 µM KB-R7943 was beneficial (P = 0.038) when added only during the first 4 min of reperfusion (Fig. 5). Separate analysis of the effects of the different concentrations of KB-R7943 during the first 5 min of reperfusion and thereafter (minutes 6–12) disclosed a protective effect on the initial period with a calculated half-response concentration of 0.29±0.09 µM. After this initial period, KB-R7943 had detrimental effect at concentrations 15 µM (Fig. 6).

View larger version (28K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 Effect of addition of KB-R7943 during the initial 4 or 10 min of reperfusion on LDH release in isolated rat hearts. n = 4 per group. Data are mean±S.E.M.

 

View larger version (20K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 6 Effect of KB-R7943 on LDH release during the initial 5 min of reperfusion (panel A) and during subsequent reperfusion (minutes 6–12, panel B) in isolated rat hearts. Control and 0.1 µM KB-R7943 groups were not included in panel B because large LDH release during the initial 5 min made them not comparable with the remaining groups. EC50, concentration of KB-R7943 eliciting half-maximal increase of LDH release; IC50, concentration of KB-R7943 eliciting half-maximal inhibition of LDH release. Data are mean±S.E.M.

 
In the series of hearts in which LV pressure was monitored throughout the experiment, perfusion with 5 µM KB-R7943 significantly reduced the LVEDP peak during early reperfusion (126.1±7.6 mmHg in the control group vs. 101.1±3.9 mmHg in 5 µM KB-R7943 group, P = 0.014, Fig. 7A) and increased the LVdevP measured at 60 min of reperfusion (14.5±3.3% with respect to pre-ischemic values in controls vs. 30.5±4.4% in KB-R7943 group, P = 0.016, Fig. 7B). Ventricular fibrillation or tachycardia occurred frequently during reperfusion, but resolved spontaneously within the first 5 min of reflow. These events occurred in 73% of control hearts and 41% of hearts receiving 5 µM KB-R7943 during the first 10 min of reperfusion (P = 0.151).

View larger version (23K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 7 Changes in left ventricular end-diastolic pressure (LVEDP, panel A) and left ventricular developed pressure (LVdevP, panel B) during ischemia and reperfusion in control hearts (Control, n = 6) and in hearts receiving 5 µM KB-R7943 during the first 10 min of reperfusion (KB-R7943, n = 6). The period of drug infusion is denoted by KB. Data are presented as mean±S.E.M. *P<0.05 versus control group.

 
In this series of experiments, LDH release during reperfusion was 79.3±9.8 U/gdw per 60 min in the control group and 50.0±5.6 U/gdw per 60 min in the KB-R7943 group (P = 0.016). Histological analysis revealed extensive areas of contraction band necrosis (mean score (0–3) of 2.17±0.24) in control hearts, and significantly smaller areas of contraction band necrosis in hearts that had received 5 µM KB-R7943 during initial reperfusion (mean score: 1.42±0.15, P = 0.014).

3.3 Intracoronary KB-R7943 in the pig heart in situ
The potential therapeutic relevance of NCX inhibition during coronary reperfusion was investigated in the pig heart in situ model.

In animals not submitted to coronary occlusion, intracoronary infusion of 5 µM KB-R7943 for 10 min did not induce arrhythmia, changes in heart rate, mean aortic pressure or coronary blood flow, or systolic shortening in the LAD territory.

3.3.1 Transient coronary occlusion
There were no differences between groups in hemodynamic variables throughout the experiment (Table 1). Three controls and one treated animal presented ventricular fibrillation during ischemia, and one animal from each group presented ventricular fibrillation during reperfusion.

View this table: [in this window] [in a new window]   Table 1 Haemodynamic and regional contractility data during coronary occlusion and reperfusion in the in situ pig heart

 
3.3.2 Infarct size
There were no significant differences between groups in the mass of myocardium at risk (10.3±0.9% of ventricular mass in the control group vs. 9.1±0.7% in the KB-R7943 group, P = 0.340). Necrosis involved 7.4±0.9% of the ventricular mass in controls and 4.4±1.0% in animals receiving 5 µM KB-R7943; infarct size (expressed as percentage of area at risk developing necrosis) was 37.8% smaller in treated animals (P = 0.033, Fig. 8). At 15 µM, KB-R7943 had no beneficial effect on the extent of the necrosis (6.8±2.0% of ventricular mass, P = 0.233).

View larger version (17K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 8 Infarct size after 48 min of coronary occlusion and 2 h of reperfusion in pigs. Data are mean±S.E.M. *P<0.05 versus control group.

 

	4. Discussion

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
The present study confirms our previous results indicating that inhibition of NCX with KB-R7943 during re-energization reduces [Ca²⁺]_i and the probability of hypercontracture in isolated quiescent myocytes submitted to simulated ischemia, and demonstrates that KB-R7943 addition during initial reperfusion limits hypercontracture in rat and pig myocardium, and in vivo. In intact, contracting myocardium, these beneficial effects were observed at low drug concentrations (0.5–10 µM) lacking significant functional effects on normoxic myocardium, but were lost at higher concentrations. These results are consistent with the hypothesis that reverse NCX contributes to Ca²⁺ mediated cell injury during initial reperfusion.

4.1 Effect of KB-R7943 on [Ca²⁺]_i oscillations and hypercontracture in quiescent myocytes
The results in isolated cardiomyocytes confirm previous observations suggesting that NCX activity may result in additional Ca²⁺ influx and injury during initial reperfusion [18]. The initial decay in cytosolic Ca²⁺ in the control group has been explained by the higher rate of Ca²⁺ sequestration by the SR as compared to the rate of extracellular Ca²⁺ influx. Accumulation of Ca²⁺ in the SR has been shown to result in repetitive cycles of Ca²⁺ release and re-uptake leading to Ca²⁺ oscillations [8,18], and the late increase in [Ca²⁺]_i observed in myocytes reperfused under control conditions could reflect a net Ca²⁺ flux from SR to cytoplasm during this period. The sustained reduction in [Ca²⁺]_i observed in the presence of KB-R7943 can be thus explained as the consequence of reduced extracellular Ca²⁺ influx during initial reperfusion resulting in attenuated SR Ca²⁺ overload, less Ca²⁺ oscillation, and reduced [Ca²⁺]_i. Previous studies have shown that SR functional blockade has a beneficial effect on reoxygenation-induced hypercontracture as a consequence of a reduction in Ca²⁺ oscillations with no significant effect on [Ca²⁺]_i [8]. In an earlier work we demonstrated that KB-R7943 reduced the frequency of Ca²⁺ oscillations in isolated myocytes submitted to hypoxia/reoxygenation [18].

Together with results from previous studies [8], the present findings suggest that reduced diastolic [Ca²⁺]_i protects against reperfusion-induced hypercontracture, independently of the potentially protective effect of reduced Ca²⁺ oscillations. The mechanism by which [Ca²⁺]_i declines in myocytes during the first minute of re-energization even in the presence of thapsigargin and ryanodine is not clear [8]. The role of Ca²⁺ stores other than the SR (e.g. the mitochondria), cannot be ruled out. We speculate that mitochondria contribute to Ca²⁺ buffering during initial reperfusion and that mitochondrial Ca²⁺ overload, resulting in mitochondrial injury and subsequent Ca²⁺ release, contributes to the late increase in [Ca²⁺]_i (Fig. 2). In this context, reduced influx during initial reoxygenation in the presence of KB-R7943 could attenuate mitochondrial Ca²⁺ overload and prevent the opening of mitochondria transition pore and mitochondrial driven cell death [27]. KB-R7943 might also modify mitochondrial Ca²⁺ overload through a direct effect on the mitochondrial Na⁺/Ca²⁺ exchanger, a molecule not identical to the sarcolemmal exchanger NCX1, for which specific inhibitors have been described [28]. However, the relevance of this potential effect would be limited by the poor diffusion of KB-R7943 across cell membranes [23]. So far, there are no available data documenting any effect of the drug on mitochondrial Na⁺/Ca²⁺ exchanger in intact cells.

4.2 Concentration-dependent effect of KB-R7943 on reperfused myocardium
In reperfused rat hearts, NCX inhibition with KB-R7943 at concentrations of 0.3–10 µM during the first 10 min of reperfusion improved functional recovery and markedly reduced enzyme release and contraction band necrosis. However, in contrast to what was observed in quiescent cells, KB-R7943 was clearly detrimental at 15 or 30 µM. The increase in late LDH release with high KB-R7943 concentrations cannot be explained as a mere consequence of reduced initial release, as it was prevented by limiting drug infusion to the first 4 min of reperfusion. The contrasting effect of high versus low KB-R7943 concentrations in intact myocardium can be explained by the different effect of the drug in cells presenting continuous reverse NCX operation during initial reperfusion and in cells that survive to this period. In cardiomyocytes that have not yet recovered the trans-sarcolemmal Na⁺ gradient and in which there is a net Ca²⁺ influx through reverse mode NCX activity, the potentially detrimental effect of high concentrations of KB-R7943 on forward mode need not be considered, since there is no forward NCX activity. In these cells, even low concentrations of KB-R7943 should have a beneficial effect on Ca²⁺ homeostasis, since KB-R7943 has an IC₅₀ close to 0.3 µM under these conditions. This interpretation is consistent with the concentration–response curve of LDH release in isolated hearts in the present study, with a calculated half response concentration of 0.29 µM.

In myocytes surviving initial reperfusion and recovering normal trans-membrane Na⁺ gradient the RP returns to normal values, above diastolic V_M, resulting in alternating net forward (diastole) and reverse (systole) NCX operation during the cardiac cycle. Under these conditions KB-R7943 is expected to inhibit both modes of operation similarly, although with a higher IC₅₀ than in cells under ionic conditions resulting in continuous net reverse NCX [20]. Since forward NCX is important for recovery of Ca²⁺ homeostasis [6], KB-R7943 could have detrimental effects in this situation. The concentration–response curve for LDH release between minutes 6 and 12 of reflow is consistent with this prediction. The fact that enhanced LDH release only occurs at 15 µM, a concentration substantially larger than the IC₅₀ for forward or reverse NCX under normal (‘bi-directional’), ionic conditions, suggests that near-complete inhibition of forward NCX is necessary to elicit the adverse effect.

In the present study, the dose and time dependence of the effects of KB-R7943 on reperfused myocardium were analyzed in unloaded hearts. Hearts were maintained unloaded (with LV balloons deflated) to avoid the potentially confounding effects of the extremely high, non-physiological LV pressure reached in the isovolumic heart during reperfusion. Nevertheless, the protective effect of 5 µM KB-R7943 was similar in hearts submitted to ischemia–reperfusion under isovolumic conditions.

Finally, in isolated cardiomyocytes [22] or isolated rat hearts under normal conditions, 15 µM of KB-R7943 had no effect on Ca²⁺ transients or contraction (data not shown). The dissimilar effects of 15 µM on reperfused and normoxic cells can be explained by a higher dependence of reperfused cells on Ca²⁺ extrusion via forward NCX [6]. It is also possible that NCX1 activity is reduced during early reperfusion as a consequence of changes in phosphorylation states, pH, and other derangements [16,22], as has been proven true during prolonged ischemia [13].

4.3 Effects of KB-R7943 during in vivo myocardial reperfusion
The relative importance of NCX in Ca²⁺ homeostasis may vary between species and be larger in humans than in rats [22]. The therapeutic implications of the present study are further increased by the reduction in infarct size induced by inhibition of NCX during initial reperfusion in the pig heart. Importantly, this beneficial effect was observed in the absence of any detectable influence in hemodynamic status or in reperfusion arrhythmias. In previous studies, intracoronary infusion of KB-R7943 at final concentrations up to 15 µM had no effect on coronary blood flow or regional wall function of normally perfused pig myocardium (unpublished data). Furthermore, although this high concentration did not limit infarct size, it was not detrimental.

Overall, the present results are consistent with the hypothesis that reverse NCX may result in additional Ca²⁺ influx and injury during myocardial reperfusion, and they demonstrate that inhibition of NCX during initial reperfusion attenuates reperfusion injury and reduces myocardial infarction without interfering with normal cell function. The development of new inhibitors able to target NCX in cells exhibiting continuous net reverse mode exchange during initial reperfusion at a wide range of concentrations, without affecting forward mode exchange in other cells, could have important therapeutic applications.

Time for primary review 22 days.

	Acknowledgements

 
This work was supported by a CICYT grant (SAF99/0102). We thank Yolanda Puigfel and Lourdes Trobo for their excellent technical work, and Dr Alberto Fernandez for his contribution to the discussion of pharmacologic issues.

	References

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 

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