Acta Physiologica Sinica, June 25, 2003, 55(3): 245－250
Received 2003-01-18 Accepted 2003-04-29
This study was supported by grants from National Natural Science Foundation, and the Science and Technology Committee of Shanghai Municipality (02JC14038).
Corresponding author. Tel: +86-21-54920305; Fax: +86-21-54920306; E-mail: znzhou@sever.shcnc.ac.cn
Research Paper
Inhibitors of
Na+/H+ and Na+/Ca2+ exchange depress intracellular calcium elevation induced by
ischemia/reperfusion in rat cardiac myocytes
DONG Jian-Wen, ZHU Hai-Feng, ZHOU Zhao-Nian*
Laboratory
of Hypoxic Cardiovascular Physiology,
Abstract: An increase in cytosolic free calcium has been shown to occur during ischemia in perfused hearts and plays a pivotal role in ischemia/reperfusion injury. The objective of this study was to investigate the contributions of Na+/H+exchange and Na+/Ca2+ exchange to changes in intracellular calcium ([Ca2+]i) during simulated ischemia and reperfusion in quiescent isolated rat cardiac myocytes. [Ca2+]i was measured by laser confocal microscope using the fluorescent indicator Fluo 3 and expressed as the corrected intensity of Fluo 3 fluorescence. [Ca2+]i increased to 140.3±13.0% (P<0.05 vs preischemic control 100%) after 5 min simulated ischemia, and remained at high level of 142.8±15.5% (P<0.05) after the following 15 min reperfusion. The increase in [Ca2+]i during simulated ischemia and reperfusion was suppressed by 100 μmol/L amiloride (inhibitor of Na+/H+exchanger), 5 mmol/L NiCl2 (inhibitor of Na+/Ca2+ exchanger) and calcium-free solution; [Ca2+]i was 101.4±16.3%, 110.4±11.1% and 107.1±10.8%, respectively, after 5 min simulated ischemia, and 97.8±14.3%, 106.2±14.5% and 106.6±15.7%, respectively, after 15 min reperfusion. Compared with control cells, the amplitude of spontaneous calcium oscillation was lessened in cells treated with Ca-free perfusion and NiCl2 during reperfusion. In addition, no calcium oscillation was observed in cells pretreated with amiloride. These results suggest that Na+/H+exchange and Na+/Ca2+ exchange are activated during simulated ischemia in isolated quiescent cardiac myocytes, leading to the elevation of [Ca2+]i induced by simulated ischemia and reperfusion.
Key words: cardiac myocytes; ischemia/reperfusion; calcium; Na+/H+exchanger; sodium-calcium exchanger
Amiloride和Ni2+抑制缺血/复灌引起的大鼠心肌细胞内钙的增加
董建文, 朱海峰, 周兆年*
中国科学院上海生命科学研究院低氧心血管生理实验室, 上海 200031
摘要: 本文旨在研究Na+/H+交换以及Na+/Ca2+交换对模拟缺血/复灌引起的大鼠心肌细胞内游离钙水平变化的调节作用。分别利用模拟缺血液和正常台氏液对大鼠心肌细胞进行缺血/复灌处理, 在缺血期间分别应用Na+/H+交换抑制剂阿米洛利（amiloride）、 Na+/Ca2+交换抑制剂NiCl2以及无钙液, 观察它们对细胞内游离Ca2+浓度变化的影响。利用Zeiss-LSM-510激光共聚焦显微镜检测、采集细胞内游离Ca2+的指示剂Fluo 3-AM的荧光信号, 计算出相对于正常(缺血前)的相对荧光强度, 以表示胞内游离Ca2+浓度的变化。结果显示, 模拟缺血引起大鼠心肌细胞内游离Ca2+持续上升, 缺血前的相对荧光强度值为100% , 模拟缺血5 min后为 140.3±13.0% (P<0.05), 复灌15 min后为 142.8±15.5% (P<0.05)。经100 μmol/L amiloride、 5 mmol/L NiCl2和无钙液分别预处理, 模拟缺血5 min后的相对荧光强度分别为101.4±16.3% (P<0.05)、 110.4±11.1% (P<0.05)和107.1±10.8 (P<0.05); 复灌15 min后则分别为 97.8±14.3% (P<0.05)、 106.2±14.5% (P<0.0 5)和106.6±15.7 (P<0.05)。另外, 与对照组细胞相比, 再灌注期间NiCl2和无钙液处理的细胞钙振荡的产生幅度明显减弱, amiloride处理组细胞无明显钙振荡出现。上述结果表明, 在静息心肌细胞模拟缺血/再灌注损伤模型中, Na+/H+交换和Na+/Ca2+交换在缺血期被激活; Na+/H+交换和Na+/Ca2+交换的激活对于缺血复灌引起的细胞内钙的升高是非常重要的。
关键词: 心肌细胞; 缺血再灌注; 钙离子; Na+/H+交换; Na+/Ca2+交换
中图分类号: Q463
The mechanisms of reperfusion injury in ischemic myocardium have been studied extensively. Among several mechanisms that have been proposed for the reperfusion injury, an excessive accumulation of calcium in reperfused ischemic cardiac myocytes appears to be a final pathway to cellular injury[1,2]. However, the exact mechanisms of calcium overload and its relation to cell injury remain undetermined. There are considerable data to support the general hypothesis that accumulation of [Na+]i during ischemia and early reperfusion leads, via Na+/Ca2+ exchange, to elevated [Ca2+]i, resulting in myocardial damage[3－5]. In addition, excessive calcium influx through L-type calcium channels has been suggested as one pathway by which calcium overload occurs. However, the exact time at which Na+/H+exchange and Na2+/Ca2+ exchange are activated remains unclear. Furthermore, most studies of function and structure changes in ischemia and reperfusion myocardium have used whole hearts in vivo or in vitro as injury models. Studies at the level of single cells focus on effects of metabolic inhibition, hypoxia/anoxia and oxygen free radials; and little efforts have been taken to determine the effects of simulated ischemia and reperfusion on single cardiac myocytes. The objective of the present study was to investigate the changes of intracellular calcium in a cellular model of simulated ischemia/reperfusion in isolated rat ventricular myocytes. Furthermore, roles of myocardial Na+/H+exchange and Na+/Ca2+ exchange in the regulation of [Ca2+]i during simulated ischemia/reperfusion were determined by using corresponding inhibitor, amiloride and NiCl2.
1 MATERIALS AND METHODS
1.1 Isolation of single ventricular cells of rats. Ventricular myocytes were isolated from adult male Sprague-Dawley rats, weighing 250－300 g, as previously described[6]. In brief, heart was quickly excised and perfused in a retrograde fashion with oxygenated Ca2+-free Tyrode's solution (in mmol/L): NaCl 135.0, KCl 5.4, MgCl2 1.0, NaH2PO4 0.33, HEPES 5.0, glucose 10.0 gassed with pure O2 (pH 7.4, and maintained at 37 ℃) for 5 min. Then the perfusion solution was switched to Ca2+-free Tyrode's solution containing 0.1% collagenase I, 0.01% protease and 0.1% bovine serum albumin for 20 min. The ventricles were minced and filtered through a nylon mesh. Cells were washed with Ca2+-free Tyrode's solution, after which CaCl2 was added to the medium at 5 min intervals to achieve respective [Ca2+]i values of 0.25, 0.5, 1.0, and ultimately 1.25 mmol/L. With this method, we obtained over 80% yield of calcium-tolerant rod shaped myocytes.
1.2 Loading of fluorescent calcium indicator[7]. Myocytes were allowed to settle down and adhere to a poly-L-lysine treated glass cover slip, which was attached to a flow-through chamber (0.2 ml volume), for 10 min. Then the cardiomyocytes were loaded with acetoxymethylester (AM) of Fluo3 (10 μmol/L) (Molecular Probe Inc.,USA) for 30 min at 37℃. The stock solution of Fluo 3-AM was prepared by mixing 50 μg of fluorescent dye in 50 μl dry dimethyl sulfoxide (DMSO) and kept frozen in aliquots until use. Fluo 3-AM loaded myocytes were then washed with dye-free Tyrode's solution twice, and incubated for further 30 min at 37℃ for complete hydrolysis of dye. The perfusion chamber was mounted on the stage of an invert microscope (Zeiss Axiovert100M, Carl Zeiss Co., Germany) and perfused with normal Tyrode's solution at 2 ml/min.
1.3 Fluorescent recording by laser confocal microscopy. Single myocytes with loaded fluorescent probes were imaged with a Zeiss LSM-510 laser scanning confocal microscope equipped with an Argon/Keron laser (15 mW, Coherent Co. USA). The Fluo 3-loaded cells were excited at a wavelength of 488 nm and the fluorescence was detected at 525 nm. Two-dimensional confocal images were acquired by scanning an image of 512×512 pixels. Images of fluorescence intensity were obtained sequentially at 525 nm after background subtraction in a defined cytosolic cell area. Image-acquisition rate was 6 frames per minute. An electrically controlled shutter limited exposure to excitation light to the time of actual data collection (1 sec for each collection). The fluorescence excitation and images acquisitions were monitored by software (LSM 510, Version 2.01 Carl Zeiss Co., Germany). Time course of fluorescence changes in the cell regions could be obtained automatically with analysis software (TimeSeries, Carl Zeiss Co., Germany).
In the present study, the corrected fluorescence intensity was used to present the change in [Ca2+]i. It was expressed as the percentage changes of fluorescence intensity of Fluo 3 against control level: corrected fluorescence intensity=[F525/Fbase,525]×100, where F525 is the measured fluorescence intensity emitted at 525 nm and Fbase,525 is the fluorescence intensity emitted at 525 nm at the beginning of the experiment.
1.4 Simulated ischemia /reperfusion in single cell models． Single cell model of myocardial ischemia was simulated by changing from perfusion with normal Tyrode's solution to perfusion with glucose-free simulated ischemic buffer containing 20 mmol/L sodium lactate (mmol/L: NaCl 137, KCl 5.4, MgCl2 1.0, CaCl2 1.8, NaHCO3 3.8, NaH2PO4 0.9, sodium lactate 20, pH 6.8, equilibrated with 95% N2 + 5% CO2)[8]. Reperfusion was achieved by switching the simulated ischemic buffer back to the normal Tyrode's solution. The cells were exposed to simulated ischemia for 5 min and then reperfused for 15 min. All experiments were carried out at room temperature (22－25 ℃).
1.5 Experiment protocol for measuring [Ca2+]i． Myocytes were randomly assigned to one of the three protocols. Myocytes were exposed to normal Tyrode's solution for 3 min, then to simulated ischemia at pH 6.8 for 5 min followed by 15 min reperfusion with normal Tyrode's solution (Protocol 1). In protocol 2, cells were exposed to calcium free ischemic solution (Ca2+ was substituted by Mg2+ 2.5 mmol/L and containing EGTA 0.5 mmol/L) instead of ischemic solution followed by reperfusion (Ca2+-free group). In protocol 3, inhibitor of Na+/H+exchange (amiloride 100 μmol/L, purchased from sigma) or Na+/Ca2+ exchange (NiCl2 5 mmol/L) was included in perfusion from 3 min before and during simulated ischemia, then myocytes were reperfused with normal Tyrode's solution.
1.6 Statistical analysis． All values were expressed as mean±SD. Comparisons between groups were assessed by one-way ANOVA with post hoc analysis using the Student-Newman-Keuls test. Student's-t test was used in comparison between only two groups. Statistical significance was defined as P<0.05.
2 RESULTS
2.1 Manifestation of [Ca2+]i changes during ischemia/reperfusion
Figure 1A shows typical recording of the time course of Fluo-3 fluorescence in an individual cell subjected to 5 min simulated ischemia followed by 15 min reperfusion. A progressive increase of fluorescence intensity was observed when the cells were exposed to simulated ischemia. It increased to 140.5±10.31% of control at the end of ischemia (Fig.1B).
Fig.1. A. Original trace of changes in fluorescence intensity recorded from a single ventricular cell exposed to simulated ischemia/reperfusion. B. Different responses of ventricular cells to normal Ca2+ Tyrode's solution (n=29) and modified zero Ca2+ Tyrode's solution (n=27).
Almost all cells showed similar responding pattern when subjected to ischemia. It was shown that there were two major manifestations of changes in [Ca2+]i upon reperfusion. Firstly, a burst of spontaneous calcium oscillation occurred upon reperfusion. Secondly, during the initial period of reperfusion there was a rapid decline in fluorescence in most cells (32 out of 49), which lasted no more than 2 min, but the elevated level of [Ca2+]i did not return to preischemia level after the fast phase of decline and remained at a higher level compared to that of preischemia. It was elevated to 142.8±15.5% of pre-ischemic value after 15 min of reperfusion (Fig.1B).
2.2 Effects of different pretreatment on elevated cytosolic calcium during simulated ischemia/reperfusion
In addition, Fig.1B shows no marked elevation of [Ca2+]i occurred when the cells were perfused with Ca2+-free solution; and the intensity of Fluo 3 fluorescence presented slight fluctuation. And pretreatment with Na+/H+exchange inhibitor (amiloride, 100 μmol/L) and Na+/Ca2+ exchange inhibitor (NiCl2, 5 mmol/L), no significant change in [Ca2+]i was noted when the cells were exposed to ischemia (Fig.2). Amiloride and Ni2+ almost eliminated the ischemia-induced calcium accumulation during this period. In contrast to the sustained elevation of [Ca2+]i during reperfusion in control group, no further calcium elevation overload was observed when the cells were pretreated with amiloride and Ni2+ (Fig.2).
2.3 Effects of pretreatment on calcium oscillation
As shown in Fig.2, no calcium oscillation was observed in the cells pretreated with amiloride during reperfusion. Spontaneous calcium oscillation was observed in 10 cells out of 37 cells perfused with Ca2+-free solution and 14 cells out of 47 cells pretreated with NiCl2. However, the amplitude of spontaneous calcium oscillation was much lower in both amiloride and Ca2+-free groups compared to that of control (Fig.3). This effect was more apparent in Ca2+-free group than that of Ni2+ group.
Fig.2.Effects pf pretreatment with Na+/H+exchange inhibitor (amiloride, 100 mmol/L, n=40) and Na+/Ca2+ exchange inhibitor (NiCl2, 5 mmol/L, n=33) on calcium accumulation induced by simulated ischemia/reperfusion.
Fig.3.Original trace of calcium oscillation upon reperfusion with or without pretreatment.
3 DISCUSSION
We observed the alteration in intracellular calcium during simulated ischemia/reperfusion in a single cells model. In our study, the simulated ischemia was achieved by perfusing freshly isolated quiescent cardiomyocytes with modified glucose-free Tyrode's solution, which mimics hypoxia, acidosis, lactate accumulation, and substrate deprivation, and the metabolic changes occurred during myocardial ischemia/reperfusion in vivo.
Changes in the activity of Na+/H+exchange during ischemia/reperfusion were yet unclear. Lazdunski et al.[4] originally hypothesized that protons will accumulate in the cell and in the extracellular space during ischemia, then the low pH would inhibit Na+/H+exchange; upon reperfusion, restoration of normal pH would stimulate Na+/H+exchange, leading to a rapid increase in [Na+]i, which in turn stimulate Na+/ Ca2+ exchange, leading to [Ca2+]i overload. Some studies have shown that inhibitors of Na+/H+exchange during the reperfusion alone offer significant protection to myocardium after ischemia and delay the rise in cytosolic-free calcium in perfused rat hearts exposed to global ischemia, but have no effects on the [Na+]i during ischemia[9－11]. These results were consistent with the assumption of Lazdunski et al. that Na+/H+exchange was not activated until reperfusion. However, several studies have reported that inhibitors of Na+/H+exchange must be present before and during ischemia to abolish the rise of [Na+]i during ischemia[12－14], suggesting that ischemia activated the Na+/H+exchange. In the present study, we found that amiloride depressed the elevation of [Ca2+]i induced by simulated ischemia and reperfusion in isolated cardiac myocytes. It suggests that Na+/H+exchange is activated from the initial of the simulated ischemia, contributing to calcium overload in ischemia and reperfusion.
Despite calcium overload in the myocardium as a key element in ischemia and reperfusion induced injury, the precise mechanism leading to the increase in [Ca2+]i remains uncertain. Our study suggested that source of calcium for the overload is likely from outside the cell because calcium overload during ischemia and reperfusion was abolished in a zero calcium medium. In addition, excessive calcium influx through L-type calcium channels has been suggested as one pathway by which calcium overload occurs[1]. However, it seems that the calcium entry through voltage-sensitive L-type calcium channel was negligible because many investigators have failed to demonstrate a reduction of calcium overload when calcium channel blockade was employed either during ischemia or reperfusion[8,15]. The prevalent pathway for calcium overload during reperfusion is thought to be via Na+/Ca2+ exchange secondary to an increase in [Na+]i. When the Na+/Ca2+ exchange is activated is still controversial. Imahashi K et al.[5] found that KB-R7943, a selective inhibitor of Na+/Ca2+ exchange, only decelerated the recovery of [Na+]i during three minutes of reperfusion, but had no effects on the increase of [Na+]i during ischemia. Tani M et al.[3] found that Ca2+ uptake increased only during reperfusion while intracellular Na+ concentration increased during ischemia and recovered during reperfusion. Shigematsu S et al.[16] provided more convinced evidence that exposure to anoxia significantly decreased both the inward and outward directed Na+/Ca2+ exchange currents, while subsequent reoxygenation rapidly restored the amplitude. These results suggest that the cardiac Na+/Ca2+ exchange was suppressed during anoxia and that the inhibition was reversed by reoxygenation. In contrast, other studies[17,18] reported that the Na+/Ca2+ exchanges were activated during ischemia when the influx of Na+ was introduced through the Na+/H+exchange. In the present study, the observation that Ca2+-free perfusion and inhibitor of Na+/Ca2+ exchange before and during ischemia could abolish the calcium overload supports the conclusion that Na+/Ca2+ exchanges were activated during ischemia.
In conclusion, our study suggests that calcium overload may be due to the activation of Na+/H+exchange and Na+/Ca2+ exchange in the model of simulated ischemia and reperfusion in quiescent isolated cardiac myocytes. Thus, inhibition of Na+/H+exchange and/or Na+/Ca2+ exchange during ischemia and/or reperfusion may be a promising therapeutic target.
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