Cardiovascular Research

Cardiovascular Research 2001 51(2):241-250; doi:10.1016/S0008-6363(01)00282-6
© 2001 by European Society of Cardiology

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Copyright © 2000, European Society of Cardiology

Role of the reverse mode of the Na⁺/Ca²⁺ exchanger in reoxygenation-induced cardiomyocyte injury

C Schäfer^a, Y Ladilov^a, J Inserte^b, M Schäfer^a, S Haffner^a, D Garcia-Dorado^b and H.M Piper^a,*

^aPhysiologisches Institut, Justus-Liebig-Universität, Aulweg 129 D-35392 Giessen, Germany
^bHospital General Universitari, Vall d'Hebron, Barcelona, Spain

^* Corresponding author. Tel.: +49-641-9947-241; fax: +49-641-9947-239 michael.piper{at}physiology.med.uni-giessen.de

Received 13 December 2000; accepted 15 March 2001

	Abstract

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Objective: We have recently shown that spontaneous Ca²⁺ oscillations elicit irreversible hypercontracture of cardiomyocytes during reoxygenation. The aim of this study was to investigate whether influx of exterior Ca²⁺ through the reverse mode of the Na⁺/Ca²⁺ exchanger (NCE) contributes to the development of these oscillations and, therefore, to reoxygenation-induced hypercontracture. Methods: Isolated cardiomyocytes and hearts from rats were used as models. Cardiomyocytes were exposed to 60 min simulated ischemia (pH_o 6.4) and 10 min reoxygenation (pH_o 7.4). During reoxygenation cardiomyocytes were superfused with medium containing 1 mmol/l Ca²⁺ (control), with nominally Ca²⁺-free medium or with medium containing 10 µmol/l KB-R 7943 (KB), a selective inhibitor of the reverse mode of the NCE. Results: In reoxygenated cardiomyocytes rapid Ca²⁺ oscillations occurred which were reduced under Ca²⁺-free conditions or in presence of KB. Hypercontracture was also significantly reduced under Ca²⁺-free conditions or in presence of KB. After 30 min of normoxic perfusion isolated rat hearts were subjected to 60 min global ischemia and reperfusion. KB (10 µmol/l) was present during the first 10 min of reperfusion. LVEDP, LVdevP and lactate dehydrogenase (LDH) release were measured. Presence of KB reduced post-ischemic LVEDP and improved left ventricular function (LVdevP). In KB treated hearts the reperfusion induced release of LDH was markedly reduced from 81.1±9.9 (control) to 49.3±8.8 U/60 min/g dry weight. Conclusion: Our study shows that inhibition of the reverse mode of the NCE, during reperfusion only, protects cardiomyocytes and whole hearts against reperfusion injury.

KEYWORDS NCE, Na⁺/Ca²⁺ exchanger; KB, KB-R 7943; SR, sarcoplasmic reticulum; LVEDP, left ventricular endiastolic pressure; LVdevP, left ventricular developed pressure; LDH, lactate dehydrogenase

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This article is referred to in the Editorial by S.J. Conway and S.V. Koushik (pages 194–197) in this issue.

	1 Introduction

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Resupply of oxygen to the myocardium after extended periods of ischemia or hypoxia can rapidly aggravate the already existing injury [1,2]. We showed in previous investigations, using the model of isolated ventricular cardiomyocytes of the rat, that acute reoxygenation injury is based on sudden development of hypercontracture upon reoxygenation [3]. Reoxygenation-induced hypercontracture represents a major cause for acute lethal cell injury in reperfused myocardium [4,5]. Earlier studies of our group showed that during the early phase of reoxygenation oscillations of cytosolic Ca²⁺ occur spontaneously. It has been shown that agents, which inhibit Ca²⁺ uptake in or release from the sarcoplasmatic reticulum (SR) and thereby reduce these oscillations, protect reoxygenated cardiomyocytes against hypercontracture and reduce reperfusion-induced injury in whole myocardium. The oscillations are due to the cycling of a large amount of Ca²⁺ between cytosol and SR. The Ca²⁺ overload is a result of a preceding prolonged ischemic period. The oscillations are initiated by the re-supply of energy to the SR Ca²⁺ pump. The frequency of these oscillations with high cytosolic peak Ca²⁺ concentrations correlates with the extent of hypercontracture [6,7]. It is therefore important to understand the factors that modulate Ca²⁺ oscillations in reoxygenated cardiomyocytes.

The aim of the present study was to investigate on the cellular level (i) whether the development of these Ca²⁺ oscillations is influenced by transsarcolemmal influx of extracellular Ca²⁺ during the early phase of reoxygenation, and in case of a positive answer, (ii) whether extracellular Ca²⁺ enters into the cells via the reverse mode of the Na⁺/Ca²⁺ exchanger (NCE) and (iii) whether knowledge of this mechanism can lead to a new approach for protection of cardiomyocytes against reoxygenation-induced injury. Experiments were carried out with isolated cardiomyocytes from the ventricular myocardium of adult rats and isolated perfused rat hearts. A novel compound, KB-R 7943 (KB), was used in our study. It inhibits preferentially the reverse mode of NCE and has lower potency for the forward mode of NCE or other ion transport systems, such as the Na⁺/H⁺ exchanger, L-type Ca²⁺ channels, voltage-gated Na⁺ channels, and inward rectifier K⁺ channels [8–11]. The inhibitor was applied to cardiomyocytes or hearts solely during the reoxygenation/reperfusion period.

	2 Methods

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
2.1 Experimental models
2.1.1 Isolated cardiomyocytes
The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23 1985). Ventricular heart muscle cells were isolated from 200–250 g adult male Wistar rats [12], and plated in medium 199 with 4% fetal calf serum on glass cover-slips that had been preincubated overnight with 4% fetal calf serum. Four hours after plating, the cover-slips were washed with medium 199. As a result of the wash, damaged cells were removed, leaving a homogeneous population of rod-shaped quiescent cardiomyocytes (<95%) attached to the cover-slip. From each isolation 2–3 coverslips were used. On each coverslip 4–6 cells were investigated. Only cells exhibiting a rod-shaped morphology and no signs of sarcolemmal blebbing were used for the experiments. These cells were found to have a low resting [Ca²⁺]_i.

2.1.2 Isolated hearts
Hearts from 300–350 g adult male Sprague–Dawley rats were mounted on a Langendorff system. The hearts were perfused with a Krebs–Henseleit bicarbonate buffer at 37°C using a non-circulating Langendorff apparatus, at a constant pressure of 60 mmHg. Left ventricular pressure was monitored by means of a water-filled latex balloon placed in the left ventricle and connected to a pressure transducer 43600 F (Baxter, The Netherlands) through a Cordis 5F catheder (Cordis, Miami, Fl, USA). The left ventricular end-diastolic pressure (LVEDP) was set at 8 mmHg by adjusting the filling of the balloon which was then left at that filling throughout the experiment. The signal obtained was digitised and recorded continuously. The variables measured included LVEDP and LVdevP (left ventricular developed pressure), calculated as the difference between left ventricular peak systolic pressure and LVEDP. Lactate dehydrogenase (LDH) activity was measured in samples collected from the coronary effluent at different times throughout the reperfusion period. LDH activity was assayed spectrophotometrically in 0.5 mmol/l phosphate buffer, 1 mmol/l sodium pyruvate and 0.3 mmol/l NADH.

2.2 Media
The perfusion chamber (1 ml filling volume) placed on the microscope stage was perfused at a flow-rate of 0.5 ml/min with modified, glucose-free normoxic bicarbonate-buffered solution at 37°C containing (mmol/l): NaCl 118.0, KCl 2.6, KH₂PO₄ 1.2, MgSO₄ 1.2, CaCl₂ 1.0 and NaHCO₃ 22.0; the medium was gassed with 5% CO₂–95% O₂, the resulting pH was 7.4. In the anoxic medium the bicarbonate concentration was reduced to 2.2 mmol/l; resulting in a pH of 6.4 when medium was gassed with 5% CO₂–95% N₂. NaCl concentration of the anoxic medium was elevated to 137.8 mmol/l, in order to equalise Na⁺ concentrations of anoxic and normoxic media. Medium was made anoxic by autoclaving as described previously [13]. The Na⁺-free medium contained (mmol/l): N-methylglucamin 125.0, KCl 2.6, KH₂PO₄ 1.2, MgSO₄ 1.2, CaCl₂ 1.0 and HEPES 25.0. pH was adjusted to 7.4 with HCl. When made anoxic, the medium was autoclaved and gassed with 100% N₂.

2.3 Ca²⁺, pH and cell length measurements
To measure [Ca²⁺]_i or [H⁺]_i, cardiomyocytes were loaded at 35°C with fura-2 or BCECF, respectively. For loading, cells attached to the glass coverslips were incubated for 30 min in medium 199 with the acetoxymethyl ester of fura-2 (2.5 µmol/l) and for 15 min with BCECF (1.25 µmol/l). After the loading, the cells were washed twice with medium 199 for 30 min to allow hydrolysis of the acetoxymethyl esters within the cell. The fluorescence from dye-loaded cells was 20–30 times higher than background fluorescence, i.e. fluorescence from cells not loaded with the dye. The loading protocols used provided the highest yield in fluorescence and minimal dye compartmentation.

The coverslip with the loaded cells was introduced into a gas-tight, temperature-controlled (37°C), transparent perfusion chamber positioned in the light path of an inverted microscope (Diaphot TMD, Nikon). Alternating excitation of the fluorescent dye at wavelengths of 340 and 380 nm for fura-2 and 440 and 490 nm for BCECF was performed with an AR-cation measurement system adapted to the microscope (Spex Industries). Emitted light (500–520 nm for fura-2 and 520–560 nm for BCECF) from an area of 10x10 mm within a single fluorescent cell was collected by the photomultiplier of the system. The light signal was recorded and analysed by an IBM PC/AT-based data analysis system (Model DM3000CM, ISA). Simultaneously to the measurement of fluorescence, the cell's microscopic image was recorded with a video camera and stored on tape. From these recordings, changes of the cell length were determined later. In the case of hypercontracted cells, the cell dimension along its previous longitudinal axis was determined.

2.4 In vivo calibration of BCECF and fura-2
Calibration of the BCECF ratio signal was performed, as previously described by Koop and Piper [14], with 10 µg/ml nigericin, a K⁺/H⁺ ionophore, and incubation media with various pH values. The fura-2 signal was calibrated according to the method described by Li et al. [15]. For this purpose, the cells were exposed to 5 µmol/l ionomycin in modified Tyrode's solution (pH 7.4; composition see below) containing either 3 mmol/l Ca²⁺ or 5 mmol/l bis-(aminoethyl)-glycolether)-N,N,N',N'-tetraacetic acid (EGTA) to obtain the maximum (R_max) and the minimum (R_min) ratio of fluorescence, respectively. To prevent morphological alterations during calibration, cells were ATP-depleted with 1 mmol/l KCN. The free cytosolic Ca²⁺ concentration ([Ca²⁺]_i) was calculated according to Grynkiewicz et al. [16] with use of pH-dependent K_d values for fura-2, determined in intact cardiomyocytes by constructing calibration curves. For the first 10 min of reoxygenation the integral of the fura-2 ratio was determined as area between the actual trace of the fura-2 ratio and the ratio of 0.5, which is the normoxic value of fura-2 ratio.

2.5 Experimental protocols
2.5.1 Protocol 1: Na⁺ withdrawal in isolated cardiomyocytes under normoxic conditions
To estimate the capacity of KB-R 7943 (10 µmol/l) to inhibit the reverse mode of NCE, the following protocol was applied. Under normoxic conditions, cardiomyocytes were pretreated for 30 min with 150 nmol/l thapsigargin, to inhibit the Ca²⁺ ATPase of the sarcoplasmatic reticulum (SR), 5 min with 4 µmol/l ryanodine, to inhibit Ca²⁺ release channels of the SR, 5 min with 4 µmol/l HOE 642, to inhibit the sarcolemmal Na⁺/H⁺ exchanger, and 2 min with 300 µmol/l ouabain, to inhibit the Na⁺/K⁺ ATPase. Then the extracellular Na⁺ was withdrawn in the presence of these substances. The osmolarity of Na⁺-free medium was corrected by the appropriate addition of N-methylglucamin. The rate of cytosolic Ca²⁺ accumulation was monitored with the fluorescence indicator fura-2.

2.5.2 Protocol 2: Simulated ischemia–reperfusion in isolated cardiomyocytes
The protocol of simulated ischemia–reperfusion in single cardiomyocytes was established in our previous studies [6,7,10,17]. This protocol consists of 80 min of anoxia at pH_o 6.4 and 10 min of reoxygenation at pH_o 7.4. This protocol has been shown to produce rigor contracture, cytosolic Ca²⁺ overload and acidosis during anoxia and Ca²⁺ oscillation, pH_i recovery and irreversible hypercontracture during reoxygenation. Because of the sarcolemmal integrity changes in ion homeostasis during reoxygenation can be estimated and compared with the degree of hypercontracture.

Three groups of experiments were performed. In the control group, the standard protocol of anoxia and reoxygenation was performed. The reoxygenation buffer contained the vehicle DMSO (dilution 1/1000). In the second group, reoxygenation was performed in presence of 10 µmol/l KB-R 7943 (solved in DMSO). In the third group, cells were reoxygenated under nominally Ca²⁺-free conditions.

2.5.3 Protocol 3: Inhibition of the Na⁺/Ca²⁺ exchanger in forward and reverse mode by depletion of cardiomyocytes from internal Na⁺ during anoxia and reoxygenation
In these experiments the NCE was inhibited by the depletion of intra- and extracellular Na⁺ as described by Siegmund et al. [18]. Na⁺ was replaced by N-methyl-D-glucamine. The depletion of cardiomyocytes of internal Na⁺ was performed by the following protocol. The cells were first incubated with normoxic Tyrode solution for 5 min, thereafter with a Na⁺ and additionally Ca²⁺ free incubation buffer. Absence of external Ca²⁺ was chosen to avoid the Na⁺ withdrawal contraction. After 20 min of Na⁺- and Ca²⁺-free incubation Ca²⁺ (1 mmol/l) was readmitted. Then the perfusion media was switched to the anoxic Na⁺-free medium. The pH_o of the anoxic media was 7.4. After the fura-2 signal had reached the same end-anoxic level as in anoxic experiments in presence of Na⁺, the cells were reoxygenated with use of Na⁺-free normoxic media. KB-R 7943 (10 µmol/l) was applied with the onset of reoxygenation. Following this protocol, pH_i at the 4th min of reoxygenation was the same as in cells reoxygenated after simulated ischemia without the inhibition of Na⁺ dependent transport processes.

2.5.4 Protocol 4: Ischemia–reperfusion in the whole heart
After 30 min of normoxic perfusion, hearts were subjected to global ischemia for 60 min by clamping the aortic in-flow line, and reperfused for 60 min. Hearts were allocated to one of two groups receiving, respectively, 10 µmol/l KB during the first 10 min of reperfusion (n=6) or only the vehicle DMSO (dilution 1/1000) (control group, n=8). The composition of the Krebs–Henseleit bicarbonate buffer (KH), used for heart perfusion, was as follows (in mmol/l): NaCl 140.0, NaHCO₃ 24.0, KCl 2.7, KH₂PO₄ 0.4, MgSO₄ 1.0, CaCl₂ 1.8, and glucose 11.0. KH was continuously gassed with 95% O₂–5% CO₂ which results in a medium pH 7.4.

2.6 Materials
Medium 199 was purchased from Boehringer Mannheim; fetal calf serum from Gibco; acetoxymethyl esters of fura-2 and BCECF from Paesel and Lorey (Frankfurt, Germany); KB-R 7943 was a gift from Kanebo (Osaka, Japan). All other chemicals were from Merck or Sigma and of highest purity available.

2.7 Statistics
Data are given as mean values±S.E.M. from n individual cells investigated in separate experiments. Statistical comparisons were performed by one-way ANOVA and use of the Student–Newman–Keuls test for posthoc analysis. Differences with P<0.05 were regarded as statistically significant.

	3 Results

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
3.1 Inhibition of the reverse mode of the NCE by KB-R 7943 in acidified cardiomyocytes
When cardiomyocytes were exposed to 80 min simulated ischemia (anoxia at pH_o 6.4) the pH_i at the beginning of reoxygenation was 6.4. During the reoxygenation period (pH_o 7.4) the pH_i recovered to the pre-anoxic value 7.1. After 4 min of reoxygenation, when the frequency of Ca²⁺ oscillation reached its maximum, the pH_i was 6.8. We therefore tested the inhibitory effect of 10 µmol/l KB on the reverse mode of the NCE at pH_i 6.8. We found that this degree of cytosolic acidosis could be established in normoxic cardiomyocytes by incubating them for 10 min with medium adjusted to pH 6.8. In order to activate the reverse mode of the NCE, cardiomyocytes were superfused with Na⁺-free medium in presence of 1 mmol/l Ca²⁺. Under these conditions Na⁺ withdrawal led to a rapid rise of cytosolic Ca²⁺, as shown by the rise of the fura-2 ratio (fura-2 ratio under control conditions: 0.52±0.06 a.u.; n=6; fura-2 ratio after 2 min of Na⁺ withdrawal: 2.33±0.04 a.u.; n=6; P<0.05). A 10-min treatment with 10 µmol/l KB before and during Na⁺-free superfusion was sufficient to completely inhibit the reverse mode of NCE (fura-2 ratio after 2 min of Na⁺ withdrawal in presence of 10 µmol/l KB: 0.49±0.05 a.u.; n=6).

3.2 Influence of KB and Ca²⁺-free media on cytosolic Ca²⁺ recovery during 10 min reoxygenation
The ratio of fura-2 fluorescence was monitored to evaluate changes in cytosolic Ca²⁺ concentration during simulated ischemia and reoxygenation. During simulated ischemia isolated cardiomyocytes developed Ca²⁺ overload. According to the calibration protocol, the initial ratio of 0.4 (a.u.) in normoxic cells corresponds to a [Ca²⁺]_i of 72 nmol/l, the end-anoxic fura-2 ratio of 2.2 (a.u.) corresponds to a [Ca²⁺]_i of 1.9 µmol/l. This level represents severe Ca²⁺ overload. When cells were reoxygenated in medium with pH 7.4, the fura-2 ratio declined to the initial control value within 10 min. Concomitantly with the Ca²⁺ recovery, transient Ca²⁺ oscillations occurred. Fig. 1 shows original recordings of the fura-2 ratio under control conditions and in presence of KB. Under either condition the fura-2 ratio declined within 10 min to the pre-anoxic level. In controls, Ca²⁺ oscillations were much more rapid than in presence of KB. Note, that the envelopes of upper and lower values of the fura-2 ratio recording are similar in both cases. Fig. 2 presents the statistical summary of these upper and lower values for cardiomyocytes reoxygenated under control conditions, in presence of KB or in nominally Ca²⁺-free media. For these parameters significant differences did not occur at any given time. At 10 min reoxygenation upper and lower values coincided since oscillations had ceased at that time. The three experimental conditions nevertheless differed with respect to the frequency of Ca²⁺ oscillations (Fig. 3). Under control conditions the oscillations reached a maximum frequency of about 30 min^–1 between the 2nd and 4th min of reoxygenation and slowed down thereafter. Under Ca²⁺-free conditions or in the presence of KB, the oscillations were less frequent at any time. To estimate net changes in the cytosolic Ca²⁺ balance during reoxygenation, the integral of the fura-2 signal was determined. The integral was calculated from the beginning of reoxygenation to complete recovery of the fura-2 signal (10 min of reoxygenation). The integral in the control cells amounted to 1236±116 a.u., in the KB treated cells to 1346±59 a.u. and in the cells reperfused under nominally Ca²⁺-free conditions to 1332±141 a.u. (n=8, no significant differences). These data indicate that the overall rate of Ca²⁺ removal from reoxygenated cells was comparable under all experimental conditions tested, in spite of the differences in Ca²⁺ oscillations.

View larger version (54K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Time course of fura-2 ratio (original recordings, arbitrary units) under control conditions (top) or in presence of 10 µmol/l KB (below) in cardiomyocytes during the first 10 min of reoxygenation.

 

View larger version (28K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Upper and lower values of the fura-2 ratio signal (a.u.) under control conditions (closed circle), in presence of KB (open square) or in nominally Ca2+-free (open triangle) media in cardiomyocytes during 10 min of reoxygenation. Data are given as mean±S.E.M.; n=8.

 

View larger version (24K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Oscillation frequency of the fura-2 ratio signal under control conditions (closed circle), in presence of KB (open square) or in nominally Ca2+-free media (open triangle) in cardiomyocytes during the first 10 min of reoxygenation. Data are given as mean±S.E.M. *, P<0.05; n=8.

 
3.3 Protection against reoxygenation induced hypercontracture
Fig. 4 shows the cell length after 10 min reoxygenation. The changes in cell length are expressed as relative changes compared to the preceding end-ischemic length. Under control conditions the cell length was reduced to 69±2% of the end-ischemic cell length. The inhibition of the reverse mode of NCE with KB and also the superfusion with nominally Ca²⁺-free media reduced significantly the development of hypercontracture (KB: 80±4%; Ca²⁺-free: 84±3% of the end-ischemic cell-length; both P<0.05 vs. control).

View larger version (24K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Cell length (% of end-ischemic) under control conditions, in presence of KB or in nominally Ca2+-free media in cardiomyocytes after the first 10 min of reoxygenation. Data are given as mean±S.E.M. *, P<0.05; n=8.

 
3.4 Recovery of cytosolic pH during reoxygenation
Under normoxic control conditions pH_i of cardiomyocytes was 7.1. Superfusion of cardiomyocytes with anoxic medium at pH_o 6.4 led to a pronounced acidification of the cytosol. After 80 min, the pH_i was 6.4. During reoxygenation the intracellular pH recovered within 10 min from this end-anoxic value to the pre-anoxic control value. As shown in Fig. 5 neither Ca²⁺-free media nor KB affected the time course of pH_i recovery.

View larger version (19K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 Cytosolic pH under control conditions (closed circle), in presence of KB (open square) or in nominally Ca2+-free media (open triangle) in cardiomyocytes during the first 10 min of reoxygenation. Data are given as mean±S.E.M. *, P<0.05; n=8.

 
3.5 Reoxygenation of Na⁺ depleted cells
Apart from the use of KB and extracellular Ca²⁺ removal, the importance of the Na⁺/Ca²⁺ exchange during the early phase of reoxygenation was tested with yet another protocol. In this protocol cardiomyocytes were first depleted of their Na⁺ contents by incubation in Na⁺-and Ca²⁺-free media, then exposed to anoxic media at pH 7.4 in presence of Ca²⁺ but not Na⁺. Once the fura-2 ratio had reached the plateau level of 2.2 a.u., the cells were reoxygenated in media containing Ca²⁺ but not Na⁺ with pH 7.4. As shown in Table 1 this treatment led to Ca²⁺ oscillations with a frequency much lower than under Na⁺ containing control conditions. Additional presence of KB did not further reduce the oscillation frequency, indicating that KB has no inhibitory effect per se on oscillations. At the 4th minute of reoxygenation, when oscillation frequency was determined, the pH_i was the same in Na⁺-depleted cells and cells not depleted from Na⁺, i.e. 6.8.

View this table: [in this window] [in a new window]   Table 1 Effect of KB-R 7943 (10 µmol/l) on reoxygenation-induced Ca oscillations after 4 min of reoxygenation under Na+-free and Na+ containing conditions. KB was only given during the reoxygenation period

 
3.6 Reduction of reoxygenation-induced injury by KB treatment in the whole heart
Using the Langendorff-perfused heart model we studied whether KB can also protect the whole heart against acute reperfusion injury. KB, 10 µmol/l, was administered for 10 min with onset of the reperfusion. Effects on LVEDP, LVdevP and LDH release were investigated. Presence of KB significantly reduced the LVEDP (Fig. 6) and improved the left ventricular function given as LVdevP. In Fig. 7 the release of LDH during reperfusion following 60 min of ischemia is presented for control hearts and hearts receiving 10 µmol/l KB. In the control group pronounced enzyme release was observed during the early phase of reperfusion. Presence of KB reduced markedly the reperfusion induced release of LDH, from 81.08±9.9 U/60 min/g dry weight under control conditions to 49.25±8.84 U/60 min/g dry weight (P<0.05). In vitro analyses showed that KB does not interfere with the LDH activity assay.

View larger version (33K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 6 Changes in LVEDP (top) and LVdevP (below) during equilibration, 60 min ischemia and 120 min reperfusion in control hearts (closed circle) and in hearts receiving 10 µmol/l KB (open circle) in isolated hearts during the first 10 min of reperfusion. Data are given as mean±S.E.M. *, P<0.05; n=6.

 

View larger version (25K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 7 LDH release during reperfusion following 60 min ischemia in control hearts (closed circle) and in hearts receiving 10 µmol/l KB (open circle) in isolated hearts during the first 10 min of reperfusion. Data are given as mean±S.E.M., n=6. P<0.05 for all data, control vs. KB, between 1 and 15 min.

 

	4 Discussion

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
The aim of this study was to investigate the role of the reverse mode of the NCE in reperfusion injury in isolated cardiomyocytes and in the whole heart. The main findings are the following: Inhibition of reverse mode activation of the NCE during the early phase of reoxygenation (i) reduces Ca²⁺ oscillations in cardiomyocytes (ii) protects cardiomyocytes against reoxygenation-induced hypercontracture and (iii) protects ischemic–reperfused hearts against contracture and enzyme release.

Isolated cardiomyocytes exposed to conditions of simulated ischemia (pH 6.4) and reoxygenation (pH 7.4) have been characterised in several previous studies [7,17,19]. During simulated ischemia the cardiomyocytes lose their energy reserves and, consecutively, undergo rigor shortening. They develop marked cytosolic overload with H⁺, Na⁺ and Ca²⁺. It was shown before that the reverse mode of the NCE can play an important role in the cytosolic accumulation of Ca²⁺ in ischemic cardiomyocytes [10,20–24]. The ischemic disturbance of ion homeostasis can be reversed when the cells are reoxygenated in media with normal extracellular pH [6,7,10]. The transsarcolemmal extrusion of Ca²⁺ from the reoxygenated cells is mediated by a tandem mechanism consisting of the Na⁺ pump, creating a normal transsarcolemmal Na⁺ gradient, and the NCE, using the Na⁺ gradient for the extrusion of Ca²⁺ [20,25]. When acting in this tandem mechanism, the NCE operates in its forward mode, driven by the re-established Na⁺ gradient and membrane potential. At some point during the early phase of reoxygenation, therefore, the operation of the NCE changes its directional mode. The present study was based on the hypothesis that during the first few minutes of reoxygenation the equilibrium conditions at the cell membrane still favour the reverse mode of NCE operation and that this influences significantly the outcome of reoxygenation. The results confirm this hypothesis.

When the reverse mode of NCE was inhibited during the early phase of reoxygenation, the reoxygenation-induced oscillations of cytosolic Ca²⁺ were markedly reduced. Three different protocols were applied in the cell model to reduce oscillations of cytosolic Ca²⁺ in the early reoxygenation phase, namely application of KB, extracellular Ca²⁺ removal and Na⁺ depletion of the cells. All protocols had the same result, i.e. they reduced the oscillatory activity to about one-third of controls. Previous work has demonstrated that these early oscillations of cytosolic Ca²⁺ are due to the uptake and release of Ca²⁺ accumulated during ischemic conditions in the cytosol. They start once resumption of oxidative energy production supplies sufficient amounts of ATP to the SR Ca²⁺ pump [6]. The specific blocker of SR Ca²⁺ release, ryanodine, can suppress these oscillations [6]. The comparison with results of the present study shows that Ca²⁺ influx through the NCE in reverse mode operation is responsible only for a part, but an important part, of these SR-dependent Ca²⁺-oscillations.

The total amount of Ca²⁺ entering the cardiomyocytes through the reverse mode of the NCE during the early phase of reoxygenation seems to be small. This is because, first, the fura-time integral remained unchanged and, second, the time to reach an end of Ca²⁺ oscillations and to re-establish a normal resting level of cytosolic Ca²⁺ concentration was not noticeably changed, when the reverse mode operation was inhibited. The Ca²⁺ influx during this early phase of reoxygenation seems therefore to serve only as trigger for SR Ca²⁺ release, thus accelerating the Ca²⁺ cycling between cytosol and SR. Neither KB nor external Ca²⁺ removal, which both protected reoxygenated cardiomyocytes, had an influence on pH_i recovery during reoxygenation. It was important to analyse the rapidity of pH_i recovery since prolonged intracellular acidosis can also protect reoxygenated cardiomyocytes from too rapid Ca²⁺ oscillations and hypercontracture [7]. In the case of cells subjected to Na⁺ depletion a particular incubation protocol made sure that pH_i was identical to the value observed with the other protocols at the time when Ca²⁺ oscillation were determined, i.e. at the 4th min of reoxygenation. We also used the Na⁺ depletion protocol to show that KB has no direct effect on Ca²⁺ oscillations as it could not affect the oscillatory activity remaining when the NCE had been blocked.

Previous studies have demonstrated that reoxygenation-induced Ca²⁺ oscillations represent the immediate cause for reoxygenation-induced hypercontracture of cardiomyocytes. Reoxygenation-induced hypercontracture has been demonstrated in vitro and in vivo to contribute substantially to acute lethal reperfusion injury in reperfused myocardium [5,26,27]. In tissue, hypercontracture of adjacent cells causes their disruption and leakage of cytosolic enzymes. Agents interfering with SR Ca²⁺ release, such as the specific blocker ryanodine or the anaesthetic halothane, inhibit Ca²⁺ oscillations and hypercontracture in reoxygenated cardiomyocytes [6]. Halothane has also been shown to protect ischemic reperfused hearts against reperfusion-induced contracture and cell death [28]. The present study has discovered another approach to interfere with this pathomechanism of acute reperfusion injury. On the cellular level KB reduces Ca²⁺ oscillations and hypercontracture. On the organ level it reduces diastolic tension and enzyme release and improves contractile function, indicative of a protection against contracture and severe cell injury.

In conclusion, our study shows that pharmacological inhibition of the reverse mode of the NCE with KB-R 7943, applied solely during reperfusion, can be used as a new therapeutical approach to protect myocardial cells against additional Ca²⁺ uptake and cell injury development during the acute phase of reperfusion. The validity of this concept was confirmed by experiments where the NCE was inhibited by extracellular Ca²⁺ removal or cellular Na⁺ depletion.

Time for primary review 21 days.

	Acknowledgements

 
The technical help of D. Schreiber and H. Holzträger is gratefully acknowledged. The study was supported by the European Union through a grant of the Biomed-2 program and grant LA 1159/2-1 of Deutsche Forschungsgemeinschaft.

	References

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 

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