Cardioprotective effects of normothermic reperfusion with oxygenated potassium cardioplegia: a possible mechanism☆

Abstract

Na⁺/K⁺ pump activation induced by normothermic reperfusion with high potassium cardioplegia may exert a protective effect on reperfusion-induced myocardial damage. We investigated (1) temperature dependency and extracellular potassium dependency of the Na⁺/K⁺ pump current (Ip), (2) effects of high potassium or ouabain during reperfusion on the post-ischemic left ventricular (LV) function. Ip-voltage relation was constructed at 5.0 and 20 mM of KCl (37 °C) using a whole-cell clamp technique in guinea pig myocytes. Ip at –40 mV was measured at 37, 27 and 18 °C (KCl: 5.0 mM). Isolated rat hearts were Langendorff-perfused and subjected to 20 min of global ischemia (37 °C) followed by 35 min of reperfusion (37 °C). The post-ischemic recovery of LV developed pressure (%LVDP) was assessed in the four reperfusate groups (4.8 mM KCl, 10 mM KCl, 20 mM KCl, or 4.8 mM KCl plus 50 μM ouabain during the first 10 min of reperfusion). The 4.8 mM KCl and 10.0 mM KCl groups were compared under metabolic inhibition (glucose-free, NaCN, or hypoxia) during reperfusion. The Ip-voltage relation shifted upward when extracellular KCl was increased from 5.0 to 20 mM. Ip was significantly greater at 37 °C than at 18 °C (114.3±17.2 vs. 22.7±1.2 pA, respectively). %LVDP was significantly greater at the 10.0 mM KCl group than at the 4.8 mM KCl group (54.9±5.5% vs. 34.2±5.9%, respectively). Metabolic inhibition abolished the difference between the two groups. Ouabain significantly decreased %LVDP (15.9±1.6%). Potassium-induced cardiac arrest during normothermic reperfusion may exert a cardioprotective effect by inducing Na⁺/K⁺ pump activation, which may be supported by aerobic metabolism during reoxygenation rather than by energy saving during cardiac arrest.

Myocardial ischemia, Myocardial reperfusion, Potassium-induced cardiac arrest, Na⁺/K⁺ pump, Metabolic inhibition, Ouabain

1. Introduction

Oxygenated warm potassium cardioplegia, infused intermittently or terminally, is widely used for myocardial protection during cardiac surgery. The mechanism of this method has been reported to be related to the preservation of myocardial adenosine triphosphate (ATP) content [1]. ATP breakdown is thought to produce H⁺ (inducing intracellular acidosis) during myocardial ischemia [2], and intracellular acidosis can cause intracellular Na⁺ overload via the Na⁺/H⁺ exchange or Na⁺-HCO₃⁻ co-transport [3].

Several studies have shown that intracellular Na⁺ and Ca²⁺ increase during myocardial ischemia [4], but Ca²⁺ increases even after reperfusion [5]. Excessive Ca²⁺ entry during reperfusion is related to the Na⁺/Ca²⁺ exchange [6]. Therefore, stopping intracellular Na⁺ overload during reperfusion is a key mechanism to inhibit excessive Ca²⁺ entry via the Na⁺/Ca²⁺ exchange during reperfusion. The Na⁺/Ca²⁺ exchange and the Na⁺/K⁺ pump are reported to be strongly correlated through a change in intracellular Na⁺ concentration [7]. Activity of the Na⁺/K⁺ pump has been shown to decrease during myocardial ischemia and reperfusion [8]. Lundmark et al. demonstrated that the increase of myocardial Na⁺/K⁺ pump (i.e. Na⁺/K⁺ ATPase) activity, induced by a protein kinase C inhibitor before ischemia, was associated with a reduced intracellular Na⁺ concentration during ischemia and reperfusion, resulting in increased post-ischemic recovery [9].

Myocardial metabolism is temperature-dependent, which means that a warm (normothermic) myocardium would produce and consume more high-energy phosphate than a cold (hypothermic) myocardium. Active cation transporters could be affected by myocardial temperature as well. The temperature dependency of the cardiac Na⁺/K⁺ pump has been observed electrophysiologically – described as a temperature coefficient (Q₁₀) – sheep cardiac Purkinje cells (Q₁₀=2.9) and guinea pig ventricular myocytes (Q₁₀=2.2) [10]. The intra- and extra-cellular cation concentrations can affect transmembrane cation transport. Extracellular K⁺ and intracellular Na⁺ are known to activate the Na⁺/K⁺ pump, suggesting that a high extracellular K⁺ concentration and intracellular Na⁺ overload during reperfusion may directly activate the Na⁺/K⁺ pump. As a result, the activation of the Na⁺/K⁺ pump should stimulate ATP production by increasing metabolism (ATP-producing process). Otherwise, a high extracellular K⁺ concentration may preserve ATP by inducing cardiac arrest, resulting from membrane depolarization (ATP-saving process), contributing to active transport of Na⁺ by the Na⁺/K⁺ pump during reperfusion.

Few data are available on the relationship between normothermic reperfusion of oxygenated potassium cardioplegia and Na⁺/K⁺ pump activation during reperfusion in terms of myocardial protection during cardiac surgery. We hypothesize that the Na⁺/K⁺ pump activation induced by normothermic reperfusion with high potassium cardioplegia exerts a protective effect on reperfusion-induced myocardial damage. To test this, we investigated three things: (1) temperature dependency and extracellular potassium dependency of the Na⁺/K⁺ pump in a sodium-overloaded myocyte from the aspect of electrophysiology, (2) the effect of high potassium (Na⁺/K⁺ pump activation) or ouabain (Na⁺/K⁺ pump inhibition) during reperfusion on the post-ischemic recovery, and (3) whether the effect of high potassium reperfusion is related to increased oxygen and substrate consumption (ATP-producing process) or preservation of ATP in non-beating hearts (ATP-saving process).

2. Materials and methods

2.1. Temperature dependency and extracellular potassium dependency of the Na⁺/K⁺ pump current

2.1.1. Preparation of ventricular myocytes

Female guinea pigs weighing 300–500 g were used in the study. The animals received humane care in compliance with the ‘Principles of Laboratory Care' formulated by the National Society for Medical Research and the ‘Guide for the Care and Use of Laboratory Animals' published by the National Institutes of Health (NIH publication No. 85-23, revised 1996). Each animal was anesthetized by intraperitoneal injection of sodium pentobarbital (100 mg/kg body weight). Under heparinization (200 U/kg body weight), the heart was excised and then mounted on a Langendorff perfusion system. The heart was given a 3-min infusion of Tyrode solution and then a 5-min infusion of nominally calcium-free Tyrode solution, followed by a 10-min infusion of a nominally calcium-free Tyrode solution containing 0.07 mg/ml of collagenase (Sigma, St Louis, MO, USA) and 0.09 mg/ml of protease (alkaline protease, Nagase Biochemicals, Tokyo, Japan) and finally a 10-min infusion of high-potassium solution for storage of the myocytes (storage solution). The ventricle was cut into small pieces and placed in a beaker containing the storage solution. The myocytes were isolated by gently shaking the beaker to disperse the myocytes and then by filtering with a 210 μm stainless steel mesh.

2.1.2. Solutions for Na⁺/K⁺ pump current measurement

The Na⁺/K⁺ pump currents were measured using the method described by Shattock and Matsuura [11]. The standard Tyrode solution used contained (in mM) 143 NaCl, 5.4 KCl, 1.8 CaCl₂, 0.5 MgCl₂, 0.25 NaH₂PO₄, 5.6 glucose, and 5 HEPES (pH 7.4 adjusted with NaOH). Nominally, calcium-free solution was identical to the standard Tyrode solution except that CaCl₂ was omitted. The storage solution for the isolated myocytes contained (in mM) 70 KOH, 40 KCl, 50 l-glutamic acid, 20 taurine, 20 KH₂PO₄, 3 MgCl₂, 10 glucose, 0.5 EGTA, and 10 HEPES (pH 7.4 adjusted with KOH). The test solution for isolation and measurement of the Na⁺/K⁺ pump current (Ip) contained (in mM) 140 NaCl, 2 CsCl, 2 NiCl₂, 5.5 glucose, 1 MgCl₂, and 5 HEPES (pH 7.4 adjusted with NaOH). The pipette solution contained (in mM) 30 NaCl, 100 CsOH, 100 l-aspartic acid, 20 tetraethyl-ammonium (TEA) (Br), 2 MgCl₂, 5 MgATP, 5 creatine phosphate (Tris), 5 EGTA, 10 glucose, and 10 HEPES (pH 7.4 adjusted with aspartic acid). The calcium current was blocked by using a calcium-free test solution. The possibility of barium passing through the voltage-gated calcium channel was prevented by adding 2 mM NiCl₂ to the calcium-free test solution. The sodium current through the voltage-gated channel was blocked by choosing a ramp pulse voltage protocol. The potassium current was blocked by replacing potassium with cesium and TEA in the pipette solution and also by including barium in the test solution. The Na/Ca exchange current was blocked by adding 2 mM NiCl₂ to the test solution and 10 mM EGTA to the pipette solution.

2.1.3. Recording of Na⁺/K⁺ pump current

The whole-cell clamp technique used was essentially the same as that described by Hamill et al. [12]. The resistance of the pipette filled with the pipette solution was 2.4–4.0 MΩ. A tight seal was established with Tyrode solution. After a whole-cell clamp had been established, the perfusate was changed from the Tyrode solution to the test solution. The Ip was measured either as a constant holding current at –40 mV (test solution: 18, 28, or 37 °C) or during a ramp pulse protocol (test solution: 28 or 37 °C). To determine the relationship between membrane potential and Ip, a voltage-ramp protocol was used. The voltage protocol was sufficiently slow (18 mV/s) to give a quasi-steady-state current-voltage relation. A negative ramp (from +50 to –130 mV) was used to prevent activation of the voltage-gated sodium channel. Current signals were filtered at 5 kHz, stored in a computer (9801 RL, NEC, Tokyo, Japan) using an on-line data acquisition system and a recticorder (OMNIACE RT 3200, Sanei, Japan) for continuous recording of the holding current.

2.2. Effects of high potassium and Na⁺/K⁺ pump inhibition on post-ischemic recovery during reperfusion

2.2.1. In vitro perfusion of isolated hearts

Male Wistar rats weighing 220–280 g were used in the study (animal care: see the section ‘Temperature dependency of the Na⁺/K⁺ pump current’). Each animal was anesthetized with ether and, under heparinization (200 U/kg body weight), the heart was excised and then Langendorff-perfused at a pressure of 100 cm H₂O. The perfusion medium was a modified bicarbonate buffer of the following composition (in mM): 118.5 NaCl, 25.0 NaHCO₃, 4.8 KCl, 1.2 MgSO₄, 1.2 KH₂PO₄, 1.4 CaCl₂, and 11.0 glucose. The buffer was filtered (5 μm in pore size) before use and continuously gassed with 95% oxygen and 5% carbon dioxide (pH 7.4 at 37 °C). During a 5-min stabilizing period, an intraventricular balloon was inserted into the left ventricle (LV) through the mitral valve, and two silver wires were attached to the left atrium and the aortic cannula to enable atrial pacing. The balloon was filled with fluid and attached to a pressure transducer through a fluid-filled tube.

2.2.2. Experimental protocols

During the initial 20 min of the pre-ischemic perfusion, the balloon was inflated with a syringe pump at a rate of 20 μl/min to give an LV end-diastolic pressure (LVEDP) of 10 mmHg during a pacing of 300 beats/min, and then the balloon volume was fixed throughout the experiment. After measurements of LV systolic pressure (LVSP), dp/dt_max, –dp/dt_min, and coronary flow at the end of the pre-ischemic perfusion, the pacing was stopped and then the heart was subjected to 20 min of normothermic (37.0 °C) global ischemia, followed by 35 min of normothermic (37.0 °C) reperfusion. The LV pressure profile was constructed by continuously recording an LV pressure throughout the experiment. The peak pressure (P_peak) of an LV end-diastolic pressure profile during the first 10 min of reperfusion was measured (Fig. 1 ).

Fig. 1.

Representative left ventricular pressure recordings during the first 10 min of reperfusion in a heart subjected to 20 min of normothermic global ischemia and reperfused with buffer solution containing 4.8 mM KCl. P_peak: left ventricular diastolic pressure gradient between the lowest and peak points in the pressure recording.

During the first 10 min of reperfusion, the reperfusate was chosen according to one of four objectives. For example, to elucidate the relationship between the extracellular calcium and the peak formation of an LV end-diastolic pressure profile during reperfusion, a calcium-free (0 mM CaCl₂ and 5.0 mM EGTA) solution or a 2.0 mM NiCl₂ solution (an inhibitor of the calcium channel and the Na⁺/Ca²⁺ exchange) was used in the reperfusate. To determine the effect of higher potassium reperfusion on the post-ischemic recovery, three kinds of potassium concentration (KCl: 4.8, 10.0, and 20.0 mM) were used in the reperfusate. To determine the effect of Na/K pump inhibition during reperfusion on the post-ischemic recovery, ouabain (50 μM) was used in the reperfusate (KCl: 4.8 mM). Finally, to determine the influence of metabolic inhibition on the effect of higher potassium reperfusion, the 4.8 mM KCl group and the 10.0 mM KCl group were compared under the condition of glucose-free, 2 mM NaCN (a mitochondrial electron transporter inhibitor), or hypoxia during the first 10 min of reperfusion.

The pacing was restarted at 300 beats/min 30 min after reperfusion. At the end of reperfusion, LVEDP, LVSP, dp/dt_max, −dp/dt_min, and coronary flow were remeasured during pacing.

2.3. Statistical analysis

Data are reported as mean±S.E. of the mean. Comparisons between two groups were performed using the Student t-test. Multiple comparisons were performed by analysis of variance, and then Dunnett's t-tests were performed when a significant F-value was obtained. A P<0.05 was considered statistically significant. Correlation coefficients between two factors were calculated using covariance analysis. A P<0.05 was considered to represent a statistically significant correlation.

3. Results

3.1. Temperature dependency and extracellular potassium dependency of the Na⁺/K⁺ pump current

The current–voltage (I–V) relationship obtained using the voltage-ramp protocol in the present study is shown in Fig. 2b showing representative tracings of the effects of a change in K⁺ concentration (from 5 mM to 20 mM) and a change in temperature (from 37 °C to 28 °C) on the I–V relationship obtained in an identical myocyte. The I–V relationship shifted upward when the extracellular K⁺ concentration was changed from 5 mM to 20 mM, and then a decrease in temperature of the external solution from 37 °C to 28 °C was associated with a downward shift of the I–V relationship. Fig. 3 shows Ip at –40 mV of membrane potential at 18, 28, or 37 °C in different myocytes, where Ip was significantly greater at 37 °C than at 18 °C (114.3±17.2 vs. 22.7±1.2 pA, respectively; P<0.05).

Fig. 2.

Panel a: current–voltage relationship during the voltage-ramp protocol from +50 to –130 mV (inset) obtained at a temperature of 37 °C in the presence or absence of ouabain (100 μM). Subtraction (a)–(b) represents the ouabain-sensitive current obtained by subtracting current (b) from current (a). Panel b: current–voltage relationship during the voltage-ramp protocol from +50 to –130 mV (inset) obtained at a temperature of 37 °C (K⁺=5 and 20 mM) and 28 °C (K⁺=20 mM).

Fig. 3.

The Na⁺K⁺ pump current at a perfusion temperature of 18, 28, or 37 °C under the conditions of an extracellular potassium concentration of 5 mM and a membrane potential of –40 mV. Values are expressed as means±S.E.M. *P<0.05 vs. the 18 °C group.

3.2. Effects of high potassium and Na⁺/K⁺ pump inhibition during reperfusion on the post-ischemic recovery

LV pressure profile recordings during the first 10 min of reperfusion are shown in Figs. 4 and 5 . Within 10 min after reperfusion, there was a temporal increase in LV diastolic pressure, giving a P_peak in the LV pressure profile (Fig. 4a), which disappeared with a reperfusate of calcium-free (Fig. 4b) or 2.0 mM NiCl₂ (Fig. 4c). Ouabain (50 μM) increased the P_peak in the 4.8 mM KCl group (Fig. 5a, b). An increase in extracellular KCl concentration from 4.8 mM to 10 mM resulted in a decrease in the P_peak (Fig. 5b, c).

Fig. 4.

Representative left ventricular pressure recordings during the first 10 min of reperfusion in a heart subjected to 20 min of normothermic global ischemia and reperfused with buffer (4.8 mM KCl) solution containing 1.4 mM CaCl₂ (panel a), 5.0 mM EGTA with calcium-free (panel b), or 1.4 mM CaCl₂ with 2.0 mM NiCl₂ (panel c).

Fig. 5.

Representative left ventricular pressure recordings during the first 10 min of reperfusion in a heart subjected to 20 min of normothermic global ischemia and reperfused with buffer solution containing 4.8 mM KCl plus 50 μM ouabain (panel a), 4.8 mM KCl (panel b), or 10.0 mM KCl (panel c).

The effects of higher potassium reperfusion and ouabain reperfusion on the post-ischemic recovery are shown in Table 1 . The post-ischemic recovery of LVDP (%LVDP), expressed as a percentage of the pre-ischemic value, was significantly greater in the 10.0 mM KCl and 20.0 mM KCl groups than in the 4.8 mM (control) group (P=0.0137 and P=0.0076, respectively). The post-ischemic increase in LVEDP was significantly less in the 10.0 mM KCl and 20.0 mM groups than in the control group (P<0.0001 and P<0.0001, respectively). Ouabain exhibited a significant decrease in %LVDP (P<0.0001) and a significant increase in LVEDP in the post-ischemic period (P=0.0142). A greater P_peak was associated with a smaller %LVDP and a greater increase in LVEDP in the post-ischemic period with significant correlation coefficients (Fig. 6 ). Table 2 shows the influence of metabolic inhibition on the effect of higher potassium reperfusion. There was no difference in the post-ischemic functional recovery between the 4.8 mM KCl and 10.0 mM KCl groups under the condition of glucose-free, 2 mM NaCN, or hypoxia during the first 10 min of reperfusion.

Table 1

Effects of KCl (4.8, 10.0 and 20.0 mM) and KCl (4.8 mM) plus ouabain (50 μM) on the post-ischemic cardiac function

KCl concentration	4.8 mM	10.0 mM	20.0 mM	4.8 mM+ouabain
Duration of ischemia (min)	20	20	20	20
Developed pressure (mmHg)
Pre-ischemia	92.9±7.7	98.1±3.0	88.0±3.2	105.4±2.2
Post-ischemia	30.5±5.1	53.3±5.0*	49.5±4.8*	16.8±1.7*
Recovery (%)	34.2±5.9	54.9±5.5*	56.8±5.7*	15.9±1.6*
End-diastolic pressure (mmHg)
Pre-ischemia	10	10	10	10
Post-ischemia	42.9±4.2	30.7±3.0*	32.8±4.1*	56.3±1.3*
dp/dt max (mmHg/s)
Pre-ischemia	1375±101	1428±41	1435±68	1383±86
Post-ischemia	435±80	766±84*	782±103*	208±27*
−/dt min (mmHg/s)
Pre-ischemia	–692±55	–725±33	–625±29	–776±47
Post-ischemia	–217±40	–377±37*	–305±51*	–132±20*
Coronary flow (ml/min/g dry weight)
Pre-ischemia	16.5±1.3	15.8±0.7	17.9±0.8	15.3±0.3
Post-ischemia	12.3±1.0	12.3±0.3	14.5±0.5*	11.6±0.5
Recovery (%)	75.0±4.8	78.2±2.7	82.0±4.3	75.5±2.2

Values are expressed as mean±S.E.s of the means. *P<0.05 vs. the 4.8 mM group (20 min of ischemia). n=6/group.

Fig. 6.

Relationship between P_peak and %LVDP (panel a) and relationship between P_peak and the change in LVEDP (panel b) in hearts subjected to 20 min of normothermic global ischemia and reperfused with the buffer solution containing 4.8 mM KCl (circle), 4.8 mM KCl plus 50 μM ouabain (rhombus), 10.0 mM KCl (square), or 20.0 mM KCl (triangle). n=6group. P_peak: left ventricular diastolic pressure gradient between the lowest and peak points in the pressure recording; %LVDP: post-ischemic recovery of left ventricular developed pressure, expressed as a percentage of the pre-ischemic value; LVEDP: left ventricular end-diastolic pressure.

Table 2

Effects of metabolic inhibition on the post-ischemic cardiac function in the 4.8 mM KCl and the 10.0 mM KCl groups

KCl concentration	4.8 mM+	10.0 mM+	4.8 mM+2 mM	10.0 mM+2 mM	4.8 mM+	10.0 mM+
	Glucose-free	Glucose-free	NaCN	NaCN	Hypoxia	Hypoxia
Duration of ischemia (min)	17	17	17	17	20	20
Developed pressure (mmHg)
Pre-ischemia	109.1±2.4	109.2±4.0	103.1±3.1	102.5±1.9	100.6±0.6	102.7±3.1
Post-ischemia	65.1±5.4	58.5±7.7	45.2±4.3	42.3±8.0	32.0±7.0	40.9±5.1
Recovery (%)	59.5±4.2	53.1±6.3	44.3±5.0	41.7±8.2	31.7±6.9	39.8±4.7
End-diastolic pressure (mmHg)
Pre-ischemia	10	10	10	10	10	10
Post-ischemia	27.2±3.1	29.2±4.2	29.4±3.4	24.6±4.4	40.4±3.7	37.8±3.2
dp/dt max (mmHg/s)
Pre-ischemia	1362±48	1455±75	1258±42	1295±53	1678±19	1720±38
Post-ischemia	880±99	833±115	525±60	533±111	500±116	628±94
−dp/dt min (mmHg/s)
Pre-ischemia	–768±32	–725±34	–717±28	–692±20	–907±21	–902±48
Post-ischemia	–458±50	–402±57	–288±22	–300±56	–260±57	–330±46
Coronary flow (ml/min/g dry weight)
Pre-ischemia	15.3±0.3	14.8±0.8	13.5±0.6	13.7±0.3	15.9±0.6	14.9±0.5
Post-ischemia	11.3±0.6	11.2±0.3	10.0±0.5	10.7±0.4	12.0±0.4	11.5±0.3
Recovery (%)	73.7±3.2	75.8±3.0	73.8±1.5	78.6±1.9	75.6±3.5	77.4±2.8

Values are expressed as mean±S.E.s of the means. n=6/group.

4. Discussion

The results from the single-cell preparation in the present study demonstrated two important findings. First, the Na⁺/K⁺ pump current is temperature-dependent, being greater at 37 °C than at 18 °C, which implies that, as already recognized in several cardiac tissues [10], normothermic conditions are more likely to activate the cardiac Na⁺/K⁺ pump than hypothermic conditions are to extrude intracellular Na⁺. Therefore, warmer reperfusion rather than colder reperfusion is likely to enhance the extrusion of the intracellular Na⁺ by activating the Na⁺/K⁺ pump. Ko et al. demonstrated in a study dealing with the effects of warm induction and reperfusion of potassium cardioplegia that 30 min after reperfusion, Na⁺/K⁺ ATPase activity was maintained at the pre-ischemic level by warm (37 °C) cardioplegia but abolished by cold (4 °C) cardioplegia [13].

The second important finding from this study is that the Na⁺/K⁺ pump current increases when extracellular potassium concentration increases, which is supported by previous studies as well [14]. The K_0.5 value (half-maximal pump current activation) has been reported to be higher in Na⁺-containing media ([Na⁺]_o=150 mM) than in Na⁺-free media [14], which means that a higher potassium concentration is required for greater activity of the Na⁺/K⁺ pump in Na⁺-containing media. Based on this study, an extracellular K⁺ concentration 10 mM may be necessary to obtain the maximal Na⁺/K⁺ pump activity seen in the present study ([Na⁺]_o∼140 mM). We chose two kinds of high potassium reperfusion (KCl: 10 and 20 mM) in the whole-heart preparation to enhance Na⁺/K⁺ pump activity during the first 10 min of reperfusion.

Non-linear and time-independent currents remained when Ip was blocked with ouabain (100 μM), which suggests that the I–V relationship contains Ip and residual conductance. According to Shattock and Matsuura, the currents involved in the residual conductance are considered not to be related to Na⁺, K⁺, or Ca²⁺[11]. Although the origin of the residual conductance is unknown, Ip should be unaffected by the residual conductance because there was little influence of changes in temperature on it (data not shown) in the present study.

The results from the whole-heart preparation demonstrated two important factors regarding the time to onset of myocardial damage during reperfusion and the effect of a higher potassium reperfusate on myocardial damage. First, myocardial damage after ischemic insult is likely to occur during early reperfusion, because intracellular Ca²⁺ activity appears to be enhanced within a few minutes after reperfusion, as evidenced by the fact that the transient increase (peak) of diastolic pressure – abolished by a calcium-free reperfusate or a NiCl₂ reperfusate – was found within 10 min after reperfusion in the LV pressure profile. The P_peak was significantly correlated with the post-ischemic recovery, supporting the notion that the peak pressure during early reperfusion may reflect the extent of myocardial damage as a result of intracellular calcium overload.

Second, the Na⁺/K⁺ pump may exert a protective effect if its activity is enhanced but a detrimental effect if its activity is inhibited. For example, the present study demonstrated that ouabain added to the reperfusate during the first 10 min of reperfusion decreased the post-ischemic recovery, suggesting that, during early reperfusion, a process of extruding intracellular Na⁺ to the extracellular space via the Na⁺/K⁺ pump may be important in preventing myocardial damage. Direct Na⁺/K⁺ pump activation induced by high potassium reperfusion may exert a protective effect against ischemia and reperfusion damage. A high concentration of K⁺ is likely to drive the Na⁺/K⁺ pump not only directly (direct activation) but also indirectly (preservation of ATP resulting from reduced ATP consumption in non-beating hearts). Because the amount of ATP consumed by sarcoplasmic reticular (SR) calcium ATPase and myosin ATPase is more dependent on heart rate than is that consumed by Na⁺/K⁺ ATPase [15], it can be assumed that a decrease in heart rate would result in Na⁺/K⁺ ATPase becoming a relatively main consumer of ATP. The SR calcium pump and the myosin ATPase seem to consume much less ATP in an arrested heart than in a beating heart. Therefore, there is a possibility that, in a heart exposed to a higher potassium reperfusate, more ATP can be used for active transport of Na⁺ as a result of saving of myocardial ATP.

The active transport of Na⁺ by the Na⁺/K⁺ pump is associated with ATP utilization, which may be dependent on ATP production as a result of oxygen and substrate consumption or dependent on ATP preservation as a result of saving of myocardial ATP in non-beating hearts. If a cardioprotective effect of high potassium reperfusion is related to preserved ATP, then the cardioprotective effect would not disappear under the condition of metabolic inhibition during the reperfusion period. We found no difference between the 4.8 mM KCl group and the 10.0 mM KCl group in terms of the post-ischemic recovery under the condition of substrate-free (glucose-free), mitochondrial inhibition (CN⁻), or hypoxia (lack of oxygen) during the first 10 min of reperfusion. The results of metabolic inhibition suggest that the cardioprotective effects of high potassium reperfusion are likely to be supported by aerobic metabolism during reoxygenation (an ATP production process requiring consumption of oxygen and substrate) rather than by energy saving during cardiac arrest (an ATP saving process in non-beating hearts).

In conclusion, we found a temperature and extracellular potassium dependence of Na⁺/K⁺ pump activity, an ATP-producing process-dependent cardioprotective effect of high potassium reperfusion, and a detrimental effect of Na⁺/K⁺ pump inhibition during reperfusion during post-ischemic recovery. These results support the hypothesis that enhanced Na⁺/K⁺ pump activity during reperfusion may be important for myocardial protection, which may be, at least in part, related to the effect of normothermic reperfusion with high potassium cardioplegia.

☆

This work was supported by a Grant-in-Aid for Scientific Research (C) from the Ministry of Education, Science, Sports and Culture, Japan, and by a Hokkaido Heart Association Research grant.