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Eur J Cardiothorac Surg 2004;26:981-987
© 2004 Elsevier Science NL

Enhancement of Na⁺,K⁺-ATPase and Ca²⁺-ATPase activities in multi-cycle ischemic preconditioning in rabbit hearts

Department of Anesthesiology and Resuscitology, Ehime University School of Medicine, Shitsukawa, Shigenobu-cho, Onsen-gun, Ehime 791-0295, Japan

Received 10 January 2004; received in revised form 31 May 2004; accepted 16 June 2004.

^* Corresponding author. Tel.: +81-89-960-5383; fax: +81-89-960-5386
e-mail: yoro{at}hypnos.m.ehime-u.ac.jp

	Abstract

Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion References

 
Objective: Ischemic preconditioning (IP) has been shown to attenuate intracellular Na⁺ accumulation and Ca²⁺ overload during ischemia and reperfusion, both of which are closely related to the outcome of myocardial damage. We compared the effects of single- and four-cycle IP in Na⁺,K⁺-activated adenosine 5'-triphosphatase (Na⁺,K⁺-ATPase) and Ca²⁺-activated adenosine 5'-triphosphatase (Ca²⁺-ATPase) activities in in vivo rabbit hearts, correlating these differences to the quality of protection against subsequent ischemia. Methods: The morphological outcome was evaluated in in vivo rabbit hearts subjected to 30 min of coronary occlusion and reperfusion for 180 min by assessing the ratio of infarct volume to risk zone volume. The effects of single- and four-cycle preconditioning ischemia were then examined. Another set of in vivo rabbit hearts was subjected to the measurement of ATPase activities at the conclusion of final preconditioning ischemia and at 60 min after reperfusion following 30 min of ischemia. Results: The infarct volume was reduced by single-cycle IP to 38% of that in the control group. The four-cycle IP further reduced the infarct volume, which was 11% of that in the control group. Na⁺,K⁺-ATPase activity at 60 min after reperfusion in the four-cycle group was increased to 172% of that in the control group (10.8 µmol ADP/h/mg protein), whereas no difference was found in the single-cycle group. On the other hand, Ca²⁺-ATPase activity at the conclusion of IP was increased by single-cycle IP, the value being 255% of that in the control group (4.9 µmol ADP/h/mg protein). The four-cycle IP further increased the activity, and the value was 158% of that in the single-cycle group. Conclusions: Since increases in Na⁺,K⁺-ATPase and Ca²⁺-ATPase activities contribute to the decrease in intracellular Ca²⁺ concentration, the enhancement of these activities by four-cycle IP may be involved in the additional protection.

Key Words: Ca²⁺-pump • Infarction • Na⁺/K⁺-pump • Preconditioning • Sarcolemma

Abbreviations: IP, ischemic preconditioning • ATP, adenosine 5'-triphosphate • ADP, adenosine 5'-diphosphate • Na⁺, K⁺-ATPase, Na⁺, K⁺-activated adenosine 5'-triphosphatase • Ca²⁺-ATPase, Ca²⁺-activated adenosine 5'-triphosphatase • TTC, triphenyl tetrazolium chloride • HPLC, high-performance liquid chromatography

	1. Introduction

Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion References

 
Transient ischemia for a short duration has been shown to produce cardioprotection against subsequent prolonged ischemic insult, termed ischemic preconditioning (IP) [1–9]. In myocardial ischemia, Ca²⁺ and Na⁺ influx into myocardium, and Ca²⁺ overload is regarded as a crucial factor in the development of ischemic myocardial damage. IP has been shown to attenuate intracellular Na⁺ accumulation and Ca²⁺ overload during ischemia and reperfusion [10]. Na⁺,K⁺-activated adenosine 5'-triphosphatase (Na⁺,K⁺-ATPase) facilitates transportation of Na⁺ from the intracellular space. The decrease in the intracellular Na⁺ concentration enhances removal of Ca²⁺ from the intracellular space by facilitating Na⁺/Ca²⁺ exchange mechanism. Therefore, prevention of Ca²⁺ overload by IP may involve changes in Na⁺,K⁺-ATPase activity. In contrast, the inhibition of Na⁺,K⁺-ATPase activity has been shown to attenuate the beneficial effect of IP [11]. Sandhu et al. [12] reported that three-cycle IP was more effective against myocardial infarction than single-cycle IP in an experimental model. It is likely that multi-cycle IP also changes ion movements to exert a stronger influence than single-cycle IP. In the present study, we compared the quality of cardioprotection between single- and multi-cycle IP using an in vivo model of rabbit hearts. We also determined the activities of Na⁺,K⁺-ATPase and Ca²⁺-activated adenosine 5'-triphosphatase (Ca²⁺-ATPase), measures of membrane functional integrity.

	2. Materials and methods

Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion References

 
2.1. Experiment 1
2.1.1. Surgical preparation
This study was approved by the Committee on Animal Experimentation of Ehime University School of Medicine (Ehime, Japan) and conformed with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1996). Male, Japanese white rabbits, weighing 2.4–3.4 kg, were anesthetized by an intravenous injection of sodium pentobarbital (30 mg/kg) with additional intermittent dosing by observing the palpebral reflex and limb movement. No muscle relaxants were given to any of the animals so that the depth of anesthesia could be observed. After tracheostomy, all rabbits were ventilated with 100% oxygen. The femoral artery and carotid artery cannulae were established for aortic pressure monitoring and arterial blood sampling. Arterial blood gas tensions were kept within physiologic limits. The esophageal temperature was monitored and maintained at 38–39 °C. Electro-cardiogam, blood pressure and heart rate were monitored using a model AB-621G bioelectric amplifier and a model AP-641G blood pressure amplifier (Nihon Kohden, Tokyo, Japan), respectively. Arterial blood gas tensions were analyzed using the ABL^TM 505 blood gas and electrolyte system (Radiometer, Copenhagen, Denmark).

After a left thoracotomy, the heart was suspended in a pericardial cradle. A 4-0 silk ligature was passed underneath a prominent branch of the left coronary artery, at a level approximately one third of the distance from the base to the apex. Both ends of the ligature were passed through soft tubing to form a snare. Myocardial ischemia was confirmed by regional cyanosis, and successful reperfusion was confirmed by blushing of the previously cyanotic myocardium.

2.1.2. Experimental protocol
Three groups of rabbits were subjected to 30 min of coronary occlusion and reperfusion for 180 min (n=7 in each group). Control animals underwent 30 min of coronary occlusion without pretreatment. The IP-1 group was preconditioned with 5 min of coronary occlusion and 10 min of reperfusion preceding 30 min of occlusion. The IP-4 group was preconditioned with four cycles of 5-min occlusion (Fig. 1). Heart rate and mean arterial blood pressure were recorded at baseline, at 20 min of occlusion, at 60 min of reperfusion, and at 180 min of reperfusion.

View larger version (16K): [in this window] [in a new window]   Fig. 1. Experimental protocols for in vivo rabbit hearts (Section 2.1). Interventions are shown on the time course. Black boxes indicate ischemia and white boxes indicate reperfusion. Durations of ischemia and reperfusion are shown. IP-1, one cycle of preconditioning ischemia; IP-4, four cycles of preconditioning ischemia.

2.2. Experiment 2
2.2.1. Experimental protocol
Another set of 30 rabbits, weighing 2.5–3.7 kg, was subjected to the measurement of Na⁺,K⁺-ATPase and Ca²⁺-ATPase activities. Surgical preparation was performed as described in Section 2.1. After making a snare for regional ischemia, rabbits were randomly allocated to one of six groups (n=5 in each). The hearts of three groups were excised and frozen without receiving ischemia-reperfusion injury. Within the three groups, hearts of the control group were excised and frozen in liquid nitrogen without any pretreatments. In the IP-1 group, the hearts were frozen at the conclusion of preconditioning with a single cycle of 5-min ischemia and 10-min reperfusion. In the IP-4 group, the hearts were preconditioned with four cycles of 5-min ischemia (Fig. 2A). The hearts were then frozen immediately after conclusion of final transient ischemia following 10-min reperfusion.

View larger version (13K): [in this window] [in a new window]   Fig. 2. Experimental protocols for ATPase assays in in vivo rabbit hearts (Section 2.2). Black boxes indicate ischemia and white boxes indicate reperfusion. Durations of ischemia and reperfusion are shown. The arrows indicate the point of excision and freezing of the hearts. IP-1, one cycle of preconditioning ischemia; IP-4, four cycles of preconditioning ischemia.

2.2.2. Membrane preparation
Using part of the ischemic area of each frozen heart, we evaluated the activities of Na⁺,K⁺-ATPase and Ca²⁺-ATPase. Cardiac membrane vesicles were prepared according to the methods of Jones et al. [13] and Nawada et al. [11]. Protein concentrations of membrane vesicles were measured according to the method described in Peterson et al. [14], using bovine serum albumin as the standard.

2.2.3. Measurement of Na⁺,K⁺-ATPase activity
Fifty microliters of purified membrane vesicles were added to each of two test tubes, one containing 900 µl of 120 mmol/l NaCl, 5 mmol/l KCl, 5 mmol/l MgCl₂, 1 mmol/l ouabain, and 100 mmol/l Tris–HCl (pH 7.0), the other containing the same medium without ouabain. Since ouabain inhibits Na⁺,K⁺-ATPase activity, the difference in enzyme activity represents Na⁺,K⁺-ATPase activity. The medium was preincubated at 37 °C for 10 min, and the reaction catalyzed by adding 50 µl of 50 mmol/l ATP in buffer to each tube for a final ATP concentration of 2.5 mmol/l. The reaction was terminated after 10 min by the addition of 50 µl 60% perchloric acid. After 20 min, the pH was increased to 9.0–9.5 with 70 µl of 8 mol/l NaOH to prevent the spontaneous degeneration of ATP in acid. The suspensions were centrifuged at 20,000xg for 30 min. The supernatant was injected into a high-performance liquid chromatography (HPLC) system to determine the amount of ADP [15]. Na⁺,K⁺-ATPase activity was determined to be the difference between ATPase activity measured in the absence and in the presence of ouabain, expressed as the amount of ADP produced per hour per milligram of protein. To evaluate Na⁺,K⁺-ATPase activity in an acidic buffer, an identical procedure was performed using an incubation medium adjusted to a pH of 6.5. Two values were obtained from each membrane preparation.

2.2.4. Measurement of Ca²⁺-ATPase activity
Activity of Ca²⁺-ATPase was measured using the basal medium containing 120 mmol/l NaCl, 5 mmol/l KCl, 5 mmol/l MgCl₂, and 100 mmol/l Tris–HCl (pH 6.8). Ca²⁺-ATPase activity was determined to be the difference between ATP hydrolysis measured in the basal medium containing 50 µmol/l Ca²⁺ and that containing 1 mmol/l ethylene glycol-bis(ß-aminoethyl ether) -N,N,N',N'-tetraacetic acid (EGTA), expressed as the amount of ADP produced per hour per milligram of protein. To evaluate Ca²⁺-ATPase activity in an acidic buffer, an identical procedure was performed using an incubation medium adjusted to a pH of 6.5. Two data were obtained from each membrane preparation.

2.3. Statistical analysis
Differences in hemodynamic variables, risk zone, infarct size, and enzyme activities among groups were assessed with one-way analysis of variance (ANOVA), followed by a Scheffé's post hoc test. The threshold for statistical significance was set at P<0.05.

	3. Results

Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion References

 
3.1. Hemodynamic data
There were no significant differences in baseline hemodynamic variables (heart rate, mean arterial blood pressure, and rate pressure product) among experimental groups in Section 2.1 (Table 1). There were no significant differences in hemodynamic variables at 20 min of occlusion, 60 min of reperfusion, and at 180 min of reperfusion compared to baseline variables within each group. The basal body temperatures in the control, IP-1, and IP-4 groups were 38.8±0.6, 38.5±0.6, and 38.6±0.7 °C (mean±SD, n=7 each), respectively. There were no significant differences among the groups.

View larger version (15K): [in this window] [in a new window]   Fig. 3. The activities of Na+,K+-ATPase at the conclusion of final preconditioning ischemia (Conclusion of IP, A) and 60 min after reperfusion (60 min Rep., B) in in vivo rabbit hearts. Na+,K+-ATPase activity was determined as the difference between enzyme activity measured in the absence of and the presence of ouabain. Enzyme activities were determined at pH 7.0 (black bars) and pH 6.5 (gray bars), and expressed as the amount of ADP produced per hour per milligram of protein. Each value represents the mean±SD from ten measurements. C, control; IP-1, one cycle of preconditioning ischemia; IP-4, four cycles of preconditioning ischemia. *P<0.05 compared with the corresponding control group. P<0.01 compared with the corresponding IP-1 group.

The activity of Ca²⁺-ATPase at the conclusion of preconditioning ischemia was 4.9±3.2 µmol ADP/h/mg protein (n=10) at pH 6.8 in the control groups. Ca²⁺-ATPase activity was increased by single-cycle IP, the value being 255% of that in the control group. The activity was further increased by four-cycle IP, and the value was 158% of that in the IP-1 group (Fig. 4A). When the pH of the medium was 6.5, the activities of Ca²⁺-ATPase were suppressed in all groups. Similar to the activity at pH 6.8, Ca²⁺-ATPase activity was increased by single-cycle IP, the value being 248% of that in the control group. A further increase was observed in the IP-4 group, the value being 186% of that in the IP-1 group (Fig. 4A).

View larger version (16K): [in this window] [in a new window]   Fig. 4. The activities of Ca2+-ATPase at the conclusion of final preconditioning ischemia (Conclusion of IP, A) and 60 min after reperfusion (60 min Rep., B) in in vivo rabbit hearts. Ca2+-ATPase activity was determined as the difference between enzyme activity measured in the absence of and the presence of Ca2+. Enzyme activities were determined at pH 6.8 (black bars) and pH 6.5 (gray bars), and expressed as the amount of ADP produced per hour per milligram of protein. Each value represents the mean±SD from ten measurements. C, control; IP-1, one cycle of preconditioning ischemia; IP-4, four cycles of preconditioning ischemia. *P<0.05 compared with the corresponding control group. **P<0.01 compared with the corresponding control group. P<0.05 compared with the corresponding IP-1 group. P<0.01 compared with the corresponding IP-1 group.

	4. Discussion

Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion References

 
This study has shown that four-cycle IP provided stronger protection against myocardial necrosis than single-cycle IP. This finding is consistent with the results of Sandhu et al. [12] which showed a frequency-dependency of IP in reducing myocardial necrosis by subsequent ischemia. Likewise, a cumulative increase in the protective effect against arrhythmias by 1–3 cycles of IP has been reported in the rat heart [16], suggesting a frequency-dependency of IP in functional aspects as well as in morphology (infarct size). Nevertheless, considering that a single 5-min coronary occlusion attenuated ventricular arrhythmias during a subsequent coronary occlusion without improving the morphologic outcome [17], the effects of IP against arrhythmias may be caused in a manner dissimilar to that of infarction.

There are some contradictory reports which showed an all-or-nothing effect of IP against myocardial necrosis [2–4,18]. In these reports, preconditioning ischemia more frequent than a threshold is regarded to provide no further benefit. The threshold is thought to be dependent on animal species, models, collateralization, heart size, and body temperature [18]. In one study on rabbit hearts, no significant difference in infarct size was shown between 1, 2, and 4 cycles of IP [19]. Although statistical significance was not shown in this study, there was still a tendency of frequency-dependency. Because the magnitude of improvement with single-cycle IP was rather strong, the protective effect may have been elevated by a single-cycle IP to an extent good enough to provide almost the maximum protective effect. Their results, therefore, do not disprove the cumulative effect of IP.

Two ion pumps play an important role in myocardial action by transporting Na⁺, K⁺, and Ca²⁺ through the membrane: one is Na⁺,K⁺-ATPase in the sarcolemma, and the other is Ca²⁺-ATPase in the sarcoplasmic reticulum [20]. In the present study, there were no significant differences in Na⁺,K⁺-ATPase activity at the conclusion of IP among three groups. On the other hand, Na⁺,K⁺-ATPase activity after ischemia-reperfusion challenge was significantly enhanced by four-cycle IP, whereas single-cycle IP did not exhibit a remarkable increase. When activation of sarcolemmal Na⁺,K⁺-ATPase facilitates Na⁺ efflux, the intracellular concentration of Na⁺ decreases. This facilitates the Na⁺/Ca²⁺ exchange system and results in a decrease in the intracellular concentration of Ca²⁺. Because Na⁺ accumulation and Ca²⁺ influx during ischemia are detrimental in reperfusion injury [21], the enhanced activity of Na⁺,K⁺-ATPase may be a factor in cardioprotection. Moreover, since morphological damage develops during the reperfusion period, the facilitation of enzyme activity in the late phase of reperfusion with four cycles of IP may be involved in the additional cardioprotection. The membrane fraction that we obtained using this method has been shown to contain both sarcolemmal and sarcoplasmic membrane [13,20], Na⁺,K⁺-ATPase activity being primarily predominant in the sarcolemmal membrane [20]. Therefore, the facilitation of Na⁺,K⁺-ATPase activity in the present study reflects an increase in sarcolemmal Na⁺,K⁺-ATPase activity by four-cycle IP. The additional effect of four-cycle IP may be partly caused by the facilitation of sarcolemmal Na⁺,K⁺-ATPase activity during reperfusion.

Nawada et al. [11] demonstrated that the inhibition of sarcolemmal Na⁺,K⁺-ATPase by digoxin reversed the beneficial effect of IP in rabbit hearts, further suggesting the relationship between Na⁺,K⁺-ATPase activity and the improvement by IP. The inhibition of enzyme activity may abolish the protective effect of single-cycle IP. Hence, it seems that the action of Na⁺,K⁺-ATPase is essential for the effect of IP, and the increased element of Na⁺,K⁺-ATPase activity contributes to the effect of multi-cycle IP. Imahashi et al. [22] reported that Na⁺ efflux by activation of mitochondrial K_ATP channels, which is mediated by Na⁺,K⁺-ATPase, mainly contributes to functional protection in preconditioned hearts. Four-cycle IP may be able to induce this signal cascade, whereas single-cycle IP is insufficient to induce it. Taken together with these findings, the enhancement of Na⁺,K⁺-ATPase activity may be an important factor in the strong limitation of infarct size with multiple cycles of IP.

There was no difference in Ca²⁺-ATPase activity after ischemia-reperfusion injury. However, Ca²⁺-ATPase activity at the conclusion of IP was markedly increased by both single- and four-cycle IP, and Ca²⁺-ATPase activity in the four-cycle IP group was much higher than that in the single-cycle IP group. Similar results were observed even in an acidic buffer. ATPase activity determined in an acidic condition may well reflect ATPase activity in in vivo hearts subjected to ischemia, since intracellular lactic acidosis associated with anaerobic metabolism develops during ischemia,. Besides the action of the Na⁺/Ca²⁺ exchange system, Ca²⁺-ATPase is involved in the removal of cytosolic Ca²⁺. Ca²⁺-ATPase exists on both the sarcolemmal and sarcoplasmic membrane, and it contributes to the removal of Ca²⁺ from the cytosol to the extracellular space and sarcoplasmic reticulum, respectively. Because our membrane fraction included both elements, individual changes in Ca²⁺-ATPase activity could not be evaluated. However, both single- and four-cycle of IP seem to affect the total amount of Ca²⁺ transported by the Ca²⁺ pump during subsequent ischemia, and the strong protection may be provided by further activation of Ca²⁺-ATPase by multiple-cycle IP.

The enhancement of Na⁺,K⁺-ATPase activity in the reperfusion phase and the enhancement of Ca²⁺-ATPase activity immediately following preconditioning ischemia may be involved in the additional protection induced by multi-cycle IP. Further, since Na⁺,K⁺-ATPase activity in the reperfusion phase is prominent at the physiologic intracellular pH value, restoration of pH after reperfusion may also be an important factor in cardioprotection in clinical situations.

	Acknowledgments

 
The authors would like to acknowledge the technical assistance of Dr Michihiro Sumida, Ms Michiko Dote, and Dr Takeshi Takaku. This work was supported, in part, by the department of Anesthesiology and Resuscitology, Ehime University School of Medicine.

	References

Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion References

 

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Personal Folders Download to citation manager Permission Requests Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Yorozuya, T. Articles by Arai, T. Search for Related Content PubMed PubMed Citation Articles by Yorozuya, T. Articles by Arai, T. Related Collections Cardiac - physiology Molecular biology Myocardial infarction Myocardial protection