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Am J Physiol Heart Circ Physiol 287: H1072-H1080, 2004. First published April 22, 2004; doi:10.1152/ajpheart.00133.2004
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Left ventricular mechanoenergetics after hyperpolarized cardioplegic arrest by nicorandil and after depolarized cardioplegic arrest by KCl

Shuichi Kobayashi,1 Yoshiro Yoshikawa,2 Susumu Sakata,1 Chikako Takenaka,1 Hiroji Hagihara,1,2 Yoshimi Ohga,1 Takehisa Abe,2 Shigeki Taniguchi,2 and Miyako Takaki1

Departments of 1Physiology II and 2Surgery III, Nara Medical University, Kashihara, Nara 634-8521, Japan

Submitted 9 February 2004 ; accepted in final form 21 April 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
We hypothesized that there are no differences in left ventricular (LV) mechanoenergetics between after hyperpolarized cardioplegic arrest by nicorandil (nicorandil arrest) and after depolarized one by high potassium chloride (KCl arrest). The aim of the present study was to test this hypothesis using LV curved end-systolic pressure-volume relation (ESPVR) and linear pressure-volume area (PVA)-myocardial oxygen consumption per beat (VO2) relation. All hearts underwent 30 min global ischemia (30°C) after infusion of 5 ml of cardioplegia. Cardioplegia consisted of either 30 mmol/l KCl (7 hearts) or nicorandil (100 µmol/l) in Tyrode solution (6 hearts). After a 30-min blood reperfusion, ESPVR and VO2-PVA relation were assessed again. Mean end-systolic pressure (ESPmLVV) and mean PVA at midrange LV volume (PVAmLVV) significantly (P < 0.05) decreased to 79.1 ± 13.4% and 85.4 ± 17.1% of control after KCl arrest and to 85.3 ± 14.8% and 86.4 ± 16.9% of control after nicorandil arrest. There were no significant differences in both decreases of mean ESPmLVV and PVAmLVV between each arrest. The slopes of VO2-PVA relations were also unchanged after each arrest. There was a significant (P < 0.005) difference in the decreases of mean VO2 intercepts of VO2-PVA relations between post-KCl arrest (73.9 ± 8.2% of control) and post-nicorandil arrest (99.2 ± 10.1% of control), however. Proteolysis of {alpha}-fodrin due to Ca2+ overload was significantly marked after KCl arrest. The present results indicate that the total calcium handling in excitation-contraction coupling is transiently impaired after KCl arrest, whereas it is unchanged after nicorandil arrest. This suggests the possibility that nicorandil is a better cardioplegia than KCl.

excitation-contraction coupling; {alpha}-fodrin; left ventricle; myocardial oxygen consumption

HIGH KCl cardioplegia has been the golden standard for myocardial protection during cardiac surgery for >25 yr, but patients undergoing surgery almost always have some postoperative cardiac dysfunction (2). One factor may be the depolarizing nature of KCl, which results in continuing transmembrane fluxes and metabolism, even during hypothermic ischemia (2). Cardioplegic KCl depolarizes the resting membrane potential to about –50 ~ –40 mV and inactivates the fast voltage-activated Na+ channels, resulting in diastolic arrest. The reversal potential of the Na+/Ca2+ exchanger is –50 mV, and thus at this resting membrane potential a net influx of Ca2+ may occur particularly if the intracellular Na+ concentration is high as a result of Na+ pump inhibition, hypothermia, or ischemia. This will lead to intracellular Ca2+ overloading and contribute to ventricular dysfunction associated with cardiac operations (2, 5). Hyperpolarized cardioplegic arrest induced by ATP-sensitive K+ (KATP) channel openers has been considered superior to depolarized cardioplegic arrest (2, 5, 9, 15). KATP channel openers shorten the action potential duration and hyperpolarize resting membrane potential to the equilibrium potential for potassium. Although it is unknown whether the hyperpolarization will be maintained during ischemia (2), at this membrane potential, the fast sodium channels are not activated and the heart will arrest on diastole.

On the other hand, there are some reports showing no differences in cardioprotective effects between KCl and KATP channel opener arrest (10, 13). Furthermore, the limitation of KATP channel opener cardioplegia due to increased myocardial O2 consumption on immediately after reperfusion has been proposed (11, 12). This may be related to reparative processes of viable myocytes or to a higher O2 debt generated during ischemia (11, 12). Detailed analysis of energy utilization after hyperpolarized cardioplegic arrest, however, has not been performed yet.

We have recently reported a linear relation between myocardial O2 consumption per beat (VO2) and systolic pressure-volume area (PVA) in the rat left ventricle (LV) of the blood-perfused whole heart preparation, similar to that observed in canine hearts (22), from a curved end-systolic pressure-volume relation (ESPVR) (3, 4, 20). The VO2 intercept of the linear VO2-PVA relation is mainly composed of VO2 for Ca2+ handling in the excitation-contraction coupling, which is primarily consumed by sarcoplasmic reticulum Ca2+ ATPase, and basal metabolism (3, 4), like in canine hearts (16, 19). We hypothesized that there are no differences in LV mechanoenergetics between after hyperpolarized cardioplegic arrest by nicorandil (nicorandil arrest) and after depolarized one by high KCl arrest. The aim of the present study was to test this hypothesis using the framework of LV ESPVR-PVA-VO2 relationship (1, 17, 22, 24).


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
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The investigation conformed to the protocols in the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Pub. No. 85-23, Revised 1996).

Surgical preparation. Experiments were performed on 16 excised, cross-circulated rat heart preparations, as reported previously (3, 4, 25). In each experiment, two retired breeder male crj:Wistar rats weighing 603 ± 42 g (32–40 wk of age), purchased from Charles River (Yokohama, Japan), were anesthetized with pentobarbital sodium (50 mg/kg ip) and used as blood supplier and metabolic supporter rats, respectively. All rats were heparinized (1,000 units iv). The beating heart was excised from other retired rats without interruption of coronary perfusion and supported by cross-circulation with the metabolic supporter rat, as previously reported in detail (3). The excised heart was maintained at 37°C.

A thin latex balloon (balloon material volume, 0.08 ml) fitted into the LV was connected to a pressure transducer (Life Kit DX-312, Nihon Kohden; Tokyo, Japan) and a 0.5-ml precision glass syringe with fine scales (minimum scale: 0.005 ml). The maximum unstretched balloon volume was below ~0.30 ml. Thus LV volume (LVV) was changed and measured by adjusting the intraballoon water volume with the syringe in 0.05-ml steps between 0.08 and 0.23 or 0.26 ml. Systolic unstressed volume (V0) was determined by filling the balloon to the level where peak isovolumic pressure and, hence, PVA (see Data analysis) were zero. The sum of intraballoon water volume and balloon material volume (0.08 ml) was used as an initial estimate of V0. This procedure was repeated during different LVV-loading runs. V0 was then finally determined as the volume-axis intercept of the best-fit ESPVR. We obtained the best-fit ESPVR with Eq. 1 (see Table 1) by means of the least-squares method (Delta-Graph, DeltaPoint; Monterey, CA) on a personal computer (1, 3, 4, 17, 24, 25). Correlation coefficients of the best-fit ESPVRs were >0.99 (Table 1).


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  		Table 1. Variables of LV mechanics

 
A midrange LVV (mLVV) corresponded to 0.16 [=(0.23 – V0)/2 + V0] (n = 12) or 0.17 [=(0.26 – V0)/2 + V0] ml (n = 1) as previously reported (24, 25). The value of mLVV normalized by LV mass to 1 g ranged between 0.12 and 0.17 ml/g in 13 hearts. Mean of mLVV was 0.16 (±0.01) ml/g in seven hearts in the prearrest KCl cardioplegia (pre-KCl) group and 0.14 (± 0.01) ml/g in six hearts in the prearrest nicorandil cardioplegia (pre-nicorandil) group. Mean of systolic unstressed volume (V0) normalized by LV mass to 1 g was 0.08 (± 0.01) ml/g in pre-KCl group and 0.07 (± 0.01) ml/g in the pre-nicornadil group (Table 1).

The LV epicardial ECG was recorded, and the heart rate was constantly maintained at 300 beats/min by electrical pacing of the right atrium (Table 1). The pacing rate was adjusted to avoid causing incomplete relaxation or arrhythmia. The systemic arterial blood pressure of the supporter rat served as the coronary perfusion pressure (100~130 mmHg). Arterial pH, PO2, and PCO2 of the supporter rat were maintained within their physiological ranges with supplemental O2 and sodium bicarbonate.

Oxygen consumption. Myocardial O2 consumption was obtained as the product of coronary flow and coronary arteriovenous O2 content difference (3, 25). Total coronary blood flow was continuously measured with an electromagnetic flowmeter (model MFV-3100, Nihon Kohden; Tokyo, Japan) placed in the middle of the coronary venous drainage tubing from the right ventricle. LV thebesian flow was negligible. The coronary arteriovenous O2 content difference was continuously measured by passing all of the arterial and venous cross-circulation blood through the cuvettes of a custom-made arteriovenous O2 content difference analyzer (model PWA-200S, Shoe Technica; Chiba, Japan), as previously reported in detail (3, 25). The mean concentration of hemoglobin in the perfused blood was 14.2 ± 0.5 g/dl.

As shown previously (1, 3, 4, 17, 24, 25), the VO2-PVA relationship was linear in the rat LV. Its slope represents the O2 cost of PVA, and its VO2 intercept represents the PVA-independent VO2. The PVA-independent VO2 is composed of VO2 in excitation-contraction coupling and basal metabolism (17, 21). The right ventricle was kept collapsed by continuous hydrostatic drainage of the coronary venous return so that the right ventricular PVA and, hence, PVA-dependent VO2 were assumed to be negligible (3, 25). The right ventricular PVA-independent VO2 (1, 3, 4, 17, 24, 25) was subtracted from the total VO2 to yield LV VO2. The LV (including the septum) and the right ventricle were weighed for normalization of LVV (ml/LV 1 g). They were 1.04 ± 0.08 and 0.32 ± 0.03 g (n = 7) in the KCl group and 1.13 ± 0.11 and 0.34 ± 0.04 g (n = 6) in the nicorandil group. There were no significant differences in LV and right ventricular weights between the two rat groups.

Experiment protocol. LV pressure (LVP) and myocardial O2 consumption were measured simultaneously (vol-run). Cardioplegia consisted of either 30 mmol/l KCl [this concentration was determined by previous studies (9, 13, 15) and corresponded to that usually used for cardiac surgery in our department] (7 hearts) or nicorandil (100 µmol/l; this concentration was determined from Refs. 6, 10, and 15) (6 hearts) in Tyrode solution. All hearts underwent 30 min of global ischemia and were arrested (30°C) after infusion of 5 ml of cardioplegia (arrested within 30 s after KCl:K group; arrested within 3 min after nicorandil: nicorandil group) and were then immersed in a Tyrode and heparin-containing solution to prevent from air suction and blood coagulation. Immediately after onset of reperfusion, the cardioplegia was removed for 1.5 min. After 30 min reperfusion with blood, the different volume-loading run was performed again (postarrest vol-run), as shown in Fig. 1.



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  		Fig. 1. Experimental protocol. CP, cardioplegia; vol-run, different volume-loading run.

 
Simultaneous aortic blood sampling from the coronary perfusion tubing from the carotid artery of the supporter rat and venous blood sampling from the venous return tubing from the right ventricle of the excised heart were performed for lactate analysis before prearrest vol-run, immediately after reperfusion, and before postarrest vol-run, as shown in Fig. 1. The difference of the lactate concentration between aortic and venous blood reflected the net difference in the excised heart.

To measure basal metabolic O2 consumption, cardiac arrest was induced by infusing 1.0 M KCl solution into the coronary perfusion tubing at a constant rate (in each three heart in the control group, KCl group, and nicorandil group) that was adjusted to abolish electrical excitation under monitoring ventricular electrocardiograms but not to generate any KCl-induced constrictions of coronary vessels, as previously reported (1, 4, 17, 24). VO2 and PVA data were obtained by minimal volume loading to avoid volume-loading effects, if any, on VO2 data.

In each steady state, data were sampled at 500 Hz for 2 s, and the sampling was usually repeated three times at intervals of 0.5~1 min.

Data analysis. We attempted to fit experimentally obtained LV pressure-volume data to the exponential equations to obtain ESPVRs (see Table 1) and end-diastolic pressure-volume relationships (EDPVRs) and thus determine PVA by the same method as described previously in detail (1, 3, 4, 17, 24, 25).

On the basis of our previous proposal (14, 20, 25), we obtained a best-fit ESPVR, and calculated ESP at mLVV (ESPmLVV) and PVAmLVV to assess LV mechanical work and energetics in the two rat groups.

PAGE and immunoblotting of 145- and 150-kDa fragments and intact {alpha}-fodrin. To evaluate whether each heart after KCl or nicorandil arrest experiences a transient Ca2+ overloading state associated with inactivation of the L-type Ca2+ channel (24), we examined the proteolysis from 240-kDa {alpha}-fodrin to the 145- and 150-kDa fragments induced by calpain (26).

LV myocardium from each heart was frozen and stored at –80°C after the mechanoenergetic studies. The frozen hearts were homogenized in the sucrose-Tris-EGTA buffer and then centrifuged at 1,000 g for 10 min. The supernatants were centrifuged at 100,000 g for 60 min at 4°C. The 100,000-g centrifuged pellets were cellular membrane fractions (1, 17, 24). The membrane proteins (40 µg/lane) were subjected to SDS-PAGE by the method of Laemmli (8), followed by immunoblotting according to the method of Towbin et al. (23) with modifications (26). The blots were blocked with 4% Block Ace (Dainippon Pharmaceutical; Osaka, Japan) and then incubated with 1,000-fold diluted antibody against anti-{alpha}-fodrin (Biohit Genex; Helsinki, Finland) for 1.5 h at room temperature. An ECL Western blotting detection kit (Amersham Bioscience; Piscataway, NJ) visualized the protein. The film was scanned with a scanner, and the intensity of the bands was calculated by NIH Image analysis (1, 17, 24). Each value of the calculated area was finally expressed by the relative value of the 145- and 150-kDa fragments to that of 240-kDa {alpha}-fodrin.

Statistics. Comparison of paired and unpaired individual values was performed by paired and unpaired t-test, respectively. Multiple comparisons were performed by one-way or repeated-measures ANOVA with post hoc Bonferroni's test or Fisher's protected least-significant difference method. A value of P < 0.05 was considered statistically significant. All data are expressed as the means ± SD.


   RESULTS
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 ABSTRACT
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DISCUSSION
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LV mechanoenergetics after KCl arrest and after nicorandil arrest. Figure 2 showed a representative set of ESPVRs and EDPVRs, and VO2-PVA relations post-KCl arrest (Fig. 2, A and B) and post-nicorandil arrest (Fig. 2, C and D). Curvilinear ESPVR post-KCl arrest markedly shifted downward, but curvilinear EDPVR did not change at all (Fig. 2A). The linear VO2-PVA relation shifted downward without changes in slope (Fig. 2B). On the other hand, ESPVR post-nicorandil arrest moderately shifted downward, and EDPVR did not change at all (Fig. 2C). Although PVA value at each LVV post-nicorandil arrest was smaller than that pre-nicorandil arrest, the VO2-PVA relations could be superimposed; neither slope nor VO2 intercept changed (Fig. 2D).



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  		Fig. 2. Left ventricular (LV) pressure-volume data and linear relationships of myocardial O2 consumption per beat (VO2)-systolic pressure-volume area (PVA) pre- and post-KCl arrest (K) or nicorandil arrest (N). A: LV pressure and volume data during different volume-loading runs between 0.08 and 0.21 ml/g pre-KCl ({circ}) and post-KCl ({blacksquare}). mLVV, midrange LV volume (0.16 ml/g). Curved end-systolic pressure-volume relationship (ESPVR) shifted downward. Curved end-diastolic PVR (EDVPR) did not change. ESPmLVV, ESP at mLVV. PVAmLVV, systolic pressure-volume area at mLVV (hatched area). B: linear VO2-PVA relations of pre-KCl ({circ}) and post-KCl ({blacksquare}). C: LV pressure and volume data during different volume-loading runs between 0.06 and 0.19 ml/g pre- ({circ}) and post-nicorandil ({blacksquare}). D: linear VO2-PVA relations of pre- ({circ}) and post-nicorandil ({blacksquare}). –, vectors a–c.

 
Mean ESPmLVV (0.16 ± 0.01 ml/g) was significantly decreased from 115.9 ± 15.6 mmHg pre-KCl arrest to 90.4 ± 13.3 mmHg post-KCl arrest. Mean ESPmLVV (0.14 ± 0.01 ml/g) was moderately decreased after nicorandil arrest [not significant (NS)] (119.6 ± 38.5 vs. 103.9 ± 45.2 mmHg) (Table 1 and Fig. 3). Mean PVAmLVV significantly (P < 0.05) decreased from 5.5 ± 1.3 to 4.6 ± 1.0 mmHg·ml·beat–1·g–1 post-KCl arrest. Mean PVAmLVV post-nicorandil arrest (4.9 ± 1.7 vs. 4.3 ± 1.9 mmHg·ml·beat–1·g–1) was only moderately decreased (Table 1 and Fig. 3).



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  		Fig. 3. Mean ESPmLVV and mean PVAmLVV pre- and post-KCl arrest (K) or nicorandil arrest (N). A: mean ESPmLVV pre- and post-KCl (n = 7). B: mean PVAmLVV pre- and post-KCl (n = 7). C: mean ESPmLVV pre- and post-nicorandil (n = 6). D: mean PVAmLVV pre- and post-nicorandil (n = 6). NS, not significant. *P < 0.05 by paired t-test.

 
Nevertheless, there were no significant differences in the decreases of mean ESPmLVV (79.1 ± 13.4% vs. 85.3 ± 14.8% of control) and mean PVAmLVV (85.4 ± 17.0% vs. 86.4 ± 16.9% of control) between post-KCl arrest and post-nicorandil arrest.

Mean slopes of VO2-PVA linear relations were unchanged both after KCl and after nicorandil arrests (Table 2 and Fig. 4, A and B). Thus PVA-dependent VO2 at mLVV did not change post-KCl arrest (Fig. 5A) and post-nicorandil arrest (Fig. 5B). Mean VO2 intercept (PVA-independent VO2) of VO2-PVA linear relation was significantly decreased from 0.26 ± 0.10 to 0.19 ± 0.08 µl O2·beat–1·g–1 post-KCl arrest (73.9 ± 8.2% of the control) but was unchanged (0.28 ± 0.16 vs. 0.26 ± 0.14 µl O2·beat–1·g–1) after nicorandil arrest (99.2 ± 10.1% of the control) (Table 2 and Fig. 4, C and D). There was a significant (P < 0.005) difference in the decreases of mean VO2 intercepts between post-KCl and nicorandil arrests. Mean mechanically unloaded VO2 (actually observed) was also significantly decreased post-KCl arrest, but was unchanged after nicorandil arrest (Table 2). Basal metabolic O2 consumption did not change after 60 min of reperfusion post-KCl and nicorandil arrests compared with that in control hearts with no treatment (Fig. 5C). Therefore, the decrease in VO2 intercept is attributable to the decrease in VO2 utilized in excitation-contraction coupling, i.e., decreased VO2 for Ca2+ handling.


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  		Table 2. Variables of LV energetics

 


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  		Fig. 4. Mean slopes and VO2 intercepts of VO2-PVA relations of pre- and post-KCl or pre- and post-nicorandil arrest (N). A: mean slopes pre- and post-KCl (n = 7). B: mean slopes pre- and post-nicorandil (n = 6). C: mean VO2 intercepts pre- and post-KCl (n = 7). D: mean VO2 intercepts pre- and post-nicorandil. *P < 0.01 by paired t-test.

 


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  		Fig. 5. Mean PVA-dependent VO2 at mLVV (PVAmLVV-dependent VO2) pre- and post-KCl or pre- and post-nicorandil arrest and basal metabolic O2 consumption in control, post-KCl, and post-nicorandil. A: mean PVAmLVV-dependent VO2 pre- and post-KCl. B: mean PVAmLVV pre- and post-nicorandil (n = 6). C: basal metabolic O2 consumption in control (n = 3), post-KCl arrest (n = 3), post-nicorandil arrest (n = 3). NS, not significant by paired t-test (A and B) and one-way ANOVA (C).

 
Time courses of PVA-independent VO2 (VO2 intercept) immediately after KCl and nicorandil arrests were compared for 30 min after reperfusion, as shown in Fig. 6A. There were no significant differences in the time courses of VO2 intercept during 30-min reperfusions post-KCl and nicorandil arrests. Figure 6B indicates that the time courses of coronary flow during reperfusions post-KCl and nicorandil arrests at unloaded conditions (PVA = 0). There were no significant differences in the time courses of coronary flow between post-KCl and post-nicorandil arrests.



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  		Fig. 6. Time courses of mean PVA-independent VO2 (A) and coronary flow (B) at unloaded conditions immediately after reperfusion post-KCl and post-nicorandil arrests. {blacktriangleup}, after KCl arrest; {bullet}, after nicorandil arrest.

 
During blood perfusion, no lactate production was observed in both KCl (–0.27 ± 0.41 mmol/l) and nicorandil groups (–0.63 ± 1.09 mmol/l). Immediately after reperfusion post-KCl arrest, however, lactate production (2.69 ± 2.21 mmol/l; n = 7) was observed and it was significantly (P < 0.05 by unpaired t-test) larger than that (0.22 ± 0.71 mmol/l; n = 6) immediately after reperfusion post-nicorandil arrest.

A previous report (24) has already revealed that the downward shift of VO2 intercept without change in slope of VO2-PVA linear relation is causally related to proteolysis of a cytoskeletal protein, 240-kDa {alpha}-fodrin due to activation of calpain by Ca2+ overload. To investigate whether the state of Ca2+ overload transiently occurs after KCl arrest or after nicorandil arrest, proteolysis of 240-kDa {alpha}-fodrin was examined.

Figure 7A showed each two representative set of immunoblottings of 240-kDa {alpha}-fodrin, and 145- and 150-kDa products of {alpha}-fodrin in control (n = 5), KCl (n = 7), and nicorandil (n = 6) groups. The mean ratio of 145- and 150-kDa products to 240-kDa {alpha}-fodrin (% of proteolysis of {alpha}-fodrin) in the KCl group (post-KCl) was significantly (P < 0.005 by one-way ANOVA with Bonferroni's post hoc analysis) larger than that in control, but not in the nicorandil group (post-nicorandil) (Fig. 7B).



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  		Fig. 7. Western blot analysis of 150- and 145-KDa products and intact membrane {alpha}-fodrin post-KCl arrest (K) or nicorandil (N) arrest. A: two each respresentative set of 150- and 145-kDa products and intact {alpha}-fodrin (240 kDa) in control hearts (C) and post-KCl (K) and post-nicorandil (N) hearts. B: mean ratio of 150- and 145-KDa products to 240-kDa {alpha}-fodrin (% of proteolysis of {alpha}-fodrin) in control hearts (n = 5), and post-KCl (n = 7), and post-nicorandil (n = 6) hearts. *P < 0.005 vs. control by ANOVA with Bonferroni's post hoc analysis.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
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 DISCUSSION
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 REFERENCES
 
Both KCl and nicorandil caused cardiac arrest and thus could lead the heart to the energy-saving state. This could bring about the cardioprotective effect. If so, no difference in the cardioprotective effect would be observed between KCl and nicorandil. Indeed, previous studies (10, 13) have reported that there are no differences in cardioprotective effects between KCl and nicorandil cardioplegia. In the present study, however, following depolarized cardioplegic arrest by KCl, the ESPVR markedly shifted downward and the VO2-PVA relationship also shifted downward with unchanged its slope. After hyperpolarized cardioplegic arrest by nicorandil, the ESPVR moderately shifted downward but the VO2-PVA relationship was unchanged. The most marked differences after depolarized cardioplegic arrest by KCl were the significant decrease in VO2 intercept (see Fig. 4). The VO2 intercept, i.e., PVA-independent VO2, is composed of total Ca2+ handling VO2 in excitation-contraction coupling and basal metabolic VO2. In the present study, basal metabolic O2 consumption post-high-KCl arrest was unchanged, indicating that VO2 for total Ca2+ handling in excitation-contraction coupling decreased post-KCl arrest. This result suggests the possibility that total Ca2+ handling in excitation-contraction coupling is transiently impaired post-KCl arrest, at least for 1 h.

Indeed, Ca2+ overload must have occurred, mediated through reverse mode of Na+/Ca2+ exchanger. Cardioplegic KCl depolarizes the resting membrane potential to about –50 ~ –40 mV and inactivates the fast voltage-activated Na+ channels, resulting in diastolic arrest. At this resting membrane potential, the reverse mode of the Na+/Ca2+ exchanger must have been activated because the reversal potential of Na+/Ca2+ exchanger is –50 mV. Furthermore, high concentration of the intracellular Na+ accumulated during ischemia must have accelerated the reverse mode of the Na+/Ca2+ exchanger activity (18). Consequently, the increase in Ca2+ influx would cause the state of Ca2+ overload in the myocardium. Indeed, the state of transient Ca2+ overload in the hearts of the KCl group was evidenced by a cytoskeleton protein, {alpha}-fodrin proteolysis like in the transiently Ca2+-overloaded hearts made by high Ca2+ infusion into the coronary artery without ischemia and acidosis (24). Although O2 wasting for Ca2+ handling has occurred during formation of high Ca2-induced Ca2+ overloading state, it was transient. Finally, at a Ca2+-overloaded state, VO2 intercept significantly decreased without changes in slope of the VO2-PVA relation and basal metabolism (24). This indicated that the Ca2+ overload caused depressed the total Ca2+ handling in the excitation-contraction coupling. {alpha}-Fodrin proteolysis is tightly related to the decrease in VO2 intercept without changes in slope of the VO2-PVA relation (24). This is in the present case of post KCl arrest.

On the other hand, nicorandil is a KATP channel opener and thus causes a K+ outward current, resulting in a shortening of the duration of action potentials. This will decrease the Ca2+ influx and consequently the heart in the nicorandil group will not cause the state of Ca2+ overload even after ischemia. This was evidenced by much less proteolysis of {alpha}-fodrin. The present result after nicorandil arrest suggests the possibility that total Ca2+ handling in excitation-contraction coupling is unchanged, at least for 1 h.

VO2 when PVA is not zero is composed of basal metabolism and PVA-dependent VO2 consumed for cross-bridge cycling and PVA-independent VO2 consumed for total Ca2+ handling in excitation-contraction coupling. After nicorandil arrest, each PVA was decreased due to suppressed cross-bridge cycling, resulting in decreased PVA-dependent VO2 without decrease in PVA-independent VO2 (VO2 for the total the Ca2+ handling) (see vector a in Fig. 2D). However, after KCl arrest, each PVA was decreased due to suppressed cross-bridge cycling and impairment of total Ca2+ handling in excitation-contraction coupling, resulting in decreased PVA-dependent VO2 (vector a) with significant decrease in PVA-independent VO2 (VO2 for the total Ca2+ handling) (vector b) (see vector c = vector a + vector b in Fig. 2B). The impairment of the total the Ca2+ handling after KCl arrest seems to be not so marked, resulting in no significant differences in the decreases of mean ESPmLVV and mean PVAmLVV between post-KCl arrest and post-nicorandil arrest.

A significant increase in lactate production was observed immediately after reperfusion in the KCl group, but almost no lactate production was observed immediately after reperfusion in the nicorandil group. During the ischemic period after KCl arrest, the reverse mode of the Na+/Ca2+ exchanger must have been activated, thereby increasing energy demand for uptake and extrusion of Ca2+ (22, 24), although the differences in the time courses of PVA-independent VO2 during 30-min reperfusion between after KCl and after nicorandil arrests could not be observed (see Fig. 6A). This is attributable to significantly larger amount of lactate production immediately after reperfusion in the KCl group. This metabolic effect also could attenuate the cardioprotective action of KCl cardioplegia.

Although it has been reported (6, 12) that nicorandil cardioplegia increases the coronary flow after reperfusion and this leads a better cardioprotective effect, no significant difference was found in the present study between time courses of the coronary flow during the reperfusions in the KCl and nicorandil groups. This might be due to a sufficient O2-carrying capacity of blood despite the higher concentration of lactate production immediately after KCl arrest.

Taken together with the present results, KCl cardioplegia transiently impairs total Ca2+ handling in excitation-contraction coupling due to transient cytosolic Ca2+ overload, whereas nicorandil cardioplegia does not at all.

Although this blood-perfused model offers a closer approximation to the clinical scenario than a crystalloid perfused one, care should be taken in extrapolating results from the present study to the clinical setting, as shown in a blood-perfused, parabiotic, isolated rabbit heart model (6, 7, 9, 15). To determine the clinical feasibility of nicorandil cardioplegia, in vivo studies are clearly needed.

Nevertheless, the present results suggested that hyperpolarized arrest with a KATP channel opener, such as nicorandil, represent an attractive alternative to traditional cardioplegic methods, such as depolarized arrest with KCl.


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 GRANTS
REFERENCES
 
This work was partly supported by Grant-in-Aid for Encouragement of Young Scientists (B) 14770016 (to C. Takenaka) from the Ministry of Education, Science, Sports and Culture of Japan.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. Takaki, Dept. of Physiology II, Nara Medical Univ., 840 Shijo-cho, Kashihara, Nara 634-8521, Japan (E-mail: mtakaki{at}naramed-u.ac.jp).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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