Role of Intracellular Na+ Kinetics in Preconditioned Rat Heart
Abstract—To elucidate the role of intracellular Na+ kinetics in the mechanism for ischemic preconditioning (IPC), we measured intracellular Na+ concentration ([Na+]i) using 23Na–magnetic resonance spectroscopy in isolated rat hearts. IPC significantly delayed the initial [Na+]i increase (d[Na+]i/dt) compared with non-IPC control, resulting in attenuation of Na+ accumulation (Δ[Na+]i) during 27 minutes of ischemia with better functional recovery. [Na+]i in IPC, but not in control, recovered to preischemic level during a 6-minute reperfusion. The Na+-H+ exchange inhibitor further suppressed d[Na+]i/dt in both control and IPC hearts with concomitant improvement of functional recovery, suggesting little contribution to the mechanism of IPC. The mitochondrial ATP-sensitive K+ (mito KATP) channel activator diazoxide (30 μmol/L) completely mimicked both [Na+]i kinetics and functional recovery in IPC without any additive effects to IPC. The mito KATP channel blocker 5-hydroxydecanoic acid (100 μmol/L) lost protective effect as well as the attenuation of d[Na+]i/dt and [Na+]i recovery induced by diazoxide. However, 5-hydroxydecanoic acid also lost IPC-induced protection, but incompletely abolished the alteration of d[Na+]i/dt and the [Na+]i recovery. The Na+/K+-ATPase inhibitor ouabain (200 μmol/L) did not change d[Na+]i/dt in non-IPC hearts, but it abolished the IPC- or diazoxide-induced reduction of d[Na+]i/dt and the [Na+]i recovery, whereas IPC followed by ouabain treatment showed partial functional recovery with smaller Δ[Na+]i than other ouabain groups. In conclusion, alteration of Na+ kinetics by preserving Na+ efflux via Na+/K+-ATPase mediated by mito KATP channel activation mainly contributes to functional protection in IPC hearts. The contribution of mito KATP channel–independent pathway relating to Na+ kinetics including reduced Na+ influx is limited in functional protection of IPC.
We have previously shown that intracellular Na+ accumulation during ischemia is the substrate for reperfusion injury, and the recovery kinetics during reperfusion, which is coupled with Ca2+ influx, also determines the degree of injury.1 2 A phenomenon in which brief episodes of ischemia and reperfusion before a prolonged ischemia reduces postischemic injury3 4 5 has been well recognized as ischemic preconditioning (IPC). It has been demonstrated that IPC attenuates Na+ accumulation and Ca2+ overload during ischemia and reperfusion.6 The reduced Na+-H+ exchange (NHE) activity was suggested as a contributor to less Ca2+ influx during reperfusion in IPC hearts.6 7 However, the role of NHE in the mechanism of IPC has been recently questioned.8 9 10 In contrast, the mitochondrial ATP-sensitive K+ (mito KATP) channel has been focused on as a critical mediator of the mechanism; the activation of this channel exerts cardioprotection,11 12 and the protective effect of IPC was abolished by the treatment with the mito KATP channel blocker.13 However, the downstream pathway after the channel activation has not been elucidated. Furthermore, it has been demonstrated that the inhibition of Na+/K+-ATPase also abolished the infarct size–limiting effect induced by IPC.14 Although these candidates are considered to modify intracellular Na+ kinetics that is critically coupled with the subsequent Ca2+ overload, the details of the change in Na+ kinetics are not clear.
To elucidate the mechanism for the alteration in intracellular Na+ kinetics induced by IPC, we measured intracellular Na+ concentration during ischemia and reperfusion in IPC rat hearts. In particular, we characterized the role of NHE and Na+/K+-ATPase in the Na+ kinetics of IPC hearts and the contribution of the mito KATP channel to the mechanism for IPC.
Materials and Methods
Isolated Rat Heart Preparation
The whole-heart preparation was described previously.1 Briefly, hearts were excised from male Sprague-Dawley rats (body weight, 400 to 450 g; Nihon-Dobutsu, Osaka, Japan) anesthetized with pentobarbital sodium (50 mg/kg IP; Abbott Laboratories), and the hearts were heparinized. After excision, the aorta was cannulated for Langendorff perfusion with modified HEPES buffer (in mmol/L) NaCl 108, KCl 5, MgCl2 1, HEPES 5, CaCl2 2, sodium acetate 20, glucose 10. The pH was adjusted to 7.40 at 37°C, and the solution was bubbled continuously with 100% O2. Heart rate was maintained at 300 bpm by right ventricular pacing. A latex balloon tied to the end of a polyethylene tube was passed into the left ventricle through the mitral valve, and it was connected to pressure transducer (SPB-101, San-ei Electric). Coronary flow rate was controlled by a peristaltic pump and was initially adjusted so that coronary pressure equaled 75 to 85 mm Hg, after which the flow rate was kept constant throughout the experiment except during global ischemia. All experiments were performed with the approval of the Animal Care and Use Committee of Osaka University Medical School.
Nuclear Magnetic Resonance Spectroscopy (MRS) Measurements
To measure intracellular Na+ concentration ([Na+]i), we acquired 23Na-MRS spectra obtained on a Bruker AMX-400wb spectrometer; the resonance frequency for 23Na was 105.843 MHz, as described previously.1 15 16 17 Two hundred fifty-six free induction decays were collected into 1 23Na- spectrum; it took 90 seconds to obtain 1 spectrum. To distinguish intra- and extracellular 23Na-nuclear magnetic resonance signals, the perfusate with the following composition (in mmol/L) was used for 23Na-MRS measurement: NaCl 18, KCl 5, MgCl2 1, CaCl2 2, HEPES 5, glucose 10, sodium acetate 20, and dysprosium triethylenetetraminehexaacetic acid [Na3Dy(TTHA) · 3NaCl] 15 as a shift reagent (the solution was supplemented with CaCl2 1.5 to compensate for the binding to Dy(TTHA)3−). The bathing solution contained mannitol 150, HEPES 5, KCl 5, MgCl2 1, CaCl2 2, tris(hydroxymethyl)aminomethane 15, (Tris)3Dy(TTHA) · 3Tris, at a pH of 7.4. The reference filled in the left ventricular balloon was prepared from dysprosium tripolyphosphate (Na7Dy(PPP)2 · 3NaCl). The areas of intracellular Na+ peak in the 23Na-MRS spectrum were measured by using planimetry, normalized by the peak for the reference, and corrected with the measured weight of each heart, resulting in the intracellular concentration in units of micromoles per gram wet weight ([Na+]i).
IPC protocol consisted of 4 cycles of 5 minutes of ischemia separated by 10 minutes of reflow. For 23Na-MRS measurements, the perfusate was switched from a standard one to one containing a shift reagent and was started with bathing after IPC. Two 23Na-MRS spectra were acquired over 3 minutes before ischemia, and the hearts were subjected to zero-flow global ischemia at 37°C for 27 minutes. The hearts were then reperfused for 30 minutes. 23Na-MRS spectra were acquired during ischemia and during the initial 6 minutes of reperfusion. The perfusate was switched back to the standard after 6 minutes.
To elucidate the role of NHE in intracellular Na+ kinetics during ischemia and reperfusion, some of the hearts were pretreated with a NHE inhibitor, EIPA [5-(N-ethyl-N-isopropyl) amiloride, 1 μmol/L, Research Biochemicals]. The administration of EIPA was started 10 minutes before the 27 minutes of ischemia, and continued for 10 minutes after reperfusion. In IPC hearts, EIPA was applied during the last reperfusion after the 4th ischemic episode. To elucidate the contribution of the mito KATP channel, an activator of this channel, diazoxide (30 μmol/L, Sigma Chemical) or an inhibitor, 5-hydroxydecanoic acid (5HD, 100 μmol/L, Research Biochemicals), was administered for 10 minutes (including washout for last 1 minute) before the 27 minutes of ischemia. Some hearts were treated with diazoxide for 10 minutes followed by washout for 10 minutes before ischemia. 5HD was applied between IPC and subsequent ischemia. Furthermore, 5HD was also administered during the IPC cycle in some hearts. To determine the role of Na+/K+-ATPase, an inhibitor of this enzyme, ouabain (200 μmol/L, Sigma Chemical), was applied for 3 minutes before ischemia and 6 minutes after reperfusion. These protocols were summarized in Figure 1⇓. These drugs, except 5HD and ouabain, were dissolved in DMSO before addition into the perfusate. The final concentration of DMSO was less than 0.04%.
Data were presented as mean±SEM. Statistical analysis was performed by using ANOVA except comparison between control and IPC hearts, which was performed with unpaired t test. P<0.05 was considered significant.
Alteration of Na+ Kinetics in IPC Hearts
Functional recovery was significantly better in IPC hearts (protocol 2 in Figure 1⇑, 55.9±4.2%, n=7) than in control hearts (protocol 1, 15.6±5.4%, n=6, P<0.05). During 27 minutes of ischemia at 37°C, [Na+]i in control hearts increased to 508.0±10.2% of preischemic level, whereas IPC slightly but significantly attenuated Na+ accumulation as shown in Figure 2A⇓ (359.3±14.9%, P<0.0001). The degree of Na+ accumulation and its relation to functional recovery were equivalent to those in the hearts subjected to 21 minutes of ischemia without IPC (Figure 2⇓ in Reference 11 ). Figure 2⇓ suggests that the Na+ kinetics in IPC hearts is characterized by the delay of Na+ accumulation in the early phase of ischemia. Thus, we calculated the initial [Na+]i increase rate (d[Na+]i/dt) as the index of the Na+ kinetics during early ischemia. As shown in Figure 2B⇓, d[Na+]i/dt (0.27±0.02 μmol/g wet weight/min), which was calculated from the data during the initial 15 minutes of ischemia, in IPC hearts was significantly less than that in control (0.35±0.01 μmol/g wet weight/min, P<0.05). In contrast, the increment rate of [Na+]i during the late phase of ischemia (18 to 27 minutes) was not significantly different between IPC and control (0.21±0.02 versus 0.22±0.01, P=0.62). After 6 minutes of reperfusion, the recovery of [Na+]i was not completed in the control hearts, whereas [Na+]i in the IPC hearts rapidly recovered to preischemic levels (Figure 2A⇓).
Contribution of NHE to the Mechanism of IPC
EIPA (1 μmol/L) significantly decreased d[Na+]i/dt both in non-IPC and IPC hearts (protocols 3 and 4, Figure 2A⇑), resulting in the amelioration of accumulated Na+ during ischemia (non-IPC, 289.0±18.4%, n=5, P<0.01 versus control; IPC, 276.6±22.7%, n=5, P<0.05). EIPA had no additional effect on IPC on the Na+ recovery during reperfusion. [Na+]i at the end of ischemia was not significantly different between EIPA-treated non-IPC and EIPA-treated IPC hearts (P>0.05). Functional recovery was significantly better in the EIPA-treated hearts than non-treated hearts (P<0.05, Figure 3⇓) even after IPC. These results suggest that the contribution of NHE to the mechanism of IPC is limited.
Contribution of Mito KATP Channel to the Mechanism of IPC
A potent activator of mito KATP channel, diazoxide (30 μmol/L), was administered for 10 minutes before the 27 minutes of ischemia both in non-IPC (protocol 5 in Figure 1⇑) and IPC hearts (protocol 6). Diazoxide delayed the initial increase in [Na+]i (0.25±0.02 μmol/g wet weight/min, n=5, P<0.05 versus control; P>0.05 versus IPC in Figure 2B⇑) and attenuated Na+ accumulation during ischemia in non-IPC hearts (Figure 2A⇑), as IPC did. After 6 minutes of reperfusion, Na+ recovered completely to preischemic levels in diazoxide-treated hearts as in IPC hearts. Diazoxide improved functional recovery, which was almost equivalent with that in IPC hearts (P<0.05 versus control, P>0.05 versus IPC, Figure 3⇑). In addition, the administration of diazoxide followed by 10 minutes of washout before ischemia (protocol 7 in Figure 1⇑), which mimicked protocol 2, also induced the protection [DIAZO(E) in Figure 3⇑] with altering [Na+]i kinetics (Figure 2B⇑). Thus, mito KATP channel activation completely mimicked [Na+]i kinetics obtained by IPC. Furthermore, administration of diazoxide had no additional effects to IPC on [Na+]i kinetics and functional recovery (P>0.05 versus IPC and diazoxide-treated, Figures 2⇑ and 3⇑).
Next, we confirmed whether the alteration of [Na+]i kinetics and protection mentioned above were caused by activation of the mito KATP channel. When the mito KATP channel was blocked by 5HD (100 μmol/L) in control hearts (protocol 8), it did not change d[Na+]i/dt (0.39±0.01 μmol/g wet weight/min, n=4, P>0.05 versus control), and Na+ remained elevated after 6 minutes of reperfusion (Figure 4⇓). 5HD abolished the protective effect induced by IPC (protocol 9) or diazoxide (protocol 10); functional recovery in IPC or diazoxide-treated hearts simultaneously exposed to 5HD was not significantly different compared with control (P>0.05, Figure 4C⇓). 5HD reversed the delayed increase in [Na+]i (n=5, P>0.05 versus control, Figure 4B⇓) obtained by diazoxide treatment, and [Na+]i was elevated after reperfusion (Figure 4A⇓). This complement effect was also observed in the early diazoxide-treatment group (protocol 11, Figures 4B⇓ and 4C⇓). In contrast, when 5HD was applied through IPC (protocol 9), it abolished the IPC-induced change in [Na+]i kinetics during ischemia (n=5, P>0.05 versus IPC and diazoxide-treated, Figures 4A⇓ and 4B⇓), but the kinetics during reperfusion was only partially aggravated. 5HD treatment after the IPC cycle (protocol 12) did not reverse the IPC effects except functional recovery (Figures 4B⇓ and 4C⇓).
Role of Na+/K+-ATPase in the Mechanism of Na+ Kinetics in IPC
The delayed increase in [Na+]i during ischemia induced by IPC or mito KATP channel activation may be attributed to the preservation of Na+-extruding activity. To elucidate the involvement of Na+/K+-ATPase activity in the change in [Na+]i kinetics in IPC, the ATPase inhibitor ouabain (200 μmol/L) was administered to non-IPC (protocol 13 in Figure 1⇑), diazoxide-treated (protocol 14), or IPC hearts (protocol 15). As shown in Figure 5B⇓, d[Na+]i/dt was almost identical among the 3 groups (control + ouabain: 0.39±0.02, n=5; IPC + ouabain: 0.35±0.03, n=5; diazoxide + ouabain: 0.44±0.03, n=5; P>0.05) and not significantly different compared with that in untreated, non-IPC hearts (P>0.05). Furthermore, the Na+ recovery was not completed in ouabain-treated hearts, whereas the Na+ recovery in IPC + ouabain hearts was better than that in other hearts (P<0.05). Function was recovered only in ouabain-treated IPC hearts (28.8±8.5%; P<0.05 versus control + ouabain [2.3±1.0%], diazoxide + ouabain [4.7±1.8%], and untreated IPC hearts). This indicates that preservation of Na+/K+-ATPase activity is necessary to exert mito KATP channel–mediated changes in [Na+]i kinetics, whereas factors other than Na+/K+-ATPase activity are also required for the functional protection induced by IPC.
Intracellular Na+ Kinetics During Ischemia and Reperfusion in IPC Hearts
The present results demonstrate that IPC slightly but significantly attenuates the initial Na+ accumulation during ischemia and completed the [Na+]i recovery after 6 minutes of reperfusion compared with that in non-IPC hearts. In particular, when the recovery process was assessed by the time constant in the regression with %Δ[Na+]i=(100−α)exp(−t/τ)+α,1 time constants (τ) were not significantly different between control (1.09±0.20 minutes) and IPC hearts (1.30±0.17 minutes, P>0.05), but the irreversible accumulation (α) was significantly smaller in IPC hearts (1.03±0.80 versus 37.3±3.88 minutes in control, P<0.05). This indicates that the number of the irreversibly injured myocytes is significantly reduced in IPC hearts.
It was reported that IPC stimulates Na+ accumulation during ischemia.18 Although this was detected by 23Na-MRS, the appropriate methods to improve the resolution between intra- and extracellular Na+ peaks were not applied. In contrast, we carefully measured to assess [Na+]i by 23Na-MRS with a shift reagent. In the present study, the reference, which was adjacent to the heart and simultaneously measured, and the bathing solution to wash out Na+-containing perfusate were applied to compensate weak resolution between intra- and extracellular signals. These methods improved resolution for reliable quantification of [Na+]i. Finally, our results indicate that Na+ accumulation during ischemia is attenuated in IPC hearts, and it is consistent with the report by Steenbergen et al6 that applied another shift reagent.
The degree of Na+ accumulation during the 27 minutes of ischemia and the functional recovery after reperfusion in IPC hearts was equivalent to that in the 21-minute ischemia group (see Figure 2⇑ in Reference 11 ). This reduction in IPC hearts was characterized by the delay of Na+ increase during the early phase of ischemia. Thus, we applied the initial [Na+]i increase rate (d[Na+]i/dt) as an index of Na+ kinetics during ischemia. However, it has been reported that Na+ efflux activity via Na+/K+-ATPase is decreased by the duration of ischemia,19 20 and Na+ recovery kinetics during reperfusion after prolonged ischemia (ie, 27 minutes) is not completed.21 Furthermore, as we have previously shown, the Na+ kinetics during reperfusion as well as “substrate” Na+ accumulation during ischemia is an important factor when determining the degree of reperfusion injury.1 It is reasonable that the complete [Na+]i recovery in IPC hearts also contributes to better functional recovery. Thus, we focused on both d[Na+]i/dt during the early ischemia and the [Na+]i recovery kinetics after reperfusion as the main indexes of major determinants for functional recovery. [Na+]i kinetics during ischemia and reperfusion is regulated by a balance between Na+ influx and efflux across the cell membrane. We focused on NHE activity as the Na+ influx pathway and Na+/K+-ATPase activity as the efflux pathway during ischemia and reperfusion in IPC hearts.
Contribution of NHE to Na+ Kinetics in IPC Hearts
NHE activity is critically involved in the mechanism of ischemia/reperfusion injury. NHE activation induces Na+ accumulation during ischemia by compensating to extrude H+, resulting in subsequent Ca2+ influx via Na+-Ca2+ exchange after reperfusion.1 2 15 22 The inhibition of NHE improves functional recovery and prevents the incidence of arrhythmias.22 23 24 The attenuation of Ca2+ overload has been demonstrated in IPC hearts.6 Thus, the reduced NHE activity has been proposed as a main mechanism of IPC.6 The present study demonstrated that the administration of the NHE inhibitor attenuated Na+ accumulation during ischemia both in non-IPC and in IPC hearts and exerted additive protection on functional protection. Although EIPA reduced Na+ accumulation during ischemia at almost the same level in non-IPC and IPC hearts, the functional recovery in EIPA-treated IPC hearts was significantly higher than that in EIPA-treated non-IPC hearts. This implies that the contribution of the NHE activity to the mechanism of IPC is very limited, and it strongly supports that the mechanism of NHE inhibitor–induced protection is different from that of IPC.10 24 25 Furthermore, the reduced Na+ accumulation during ischemia per se is not the sole determinant of functional recovery in IPC hearts.
Mito KATP Channel Activation Mimics Na+ Kinetics in IPC Hearts
The contribution of mito KATP channel to the mechanism of IPC has been strongly suggested from the results using KATP channel openers or inhibitors.11 26 Furthermore, in the present results, diazoxide completely mimicked [Na+]i kinetics observed in IPC hearts. Both this similarity and no additive effect in diazoxide-treated IPC hearts strongly support that the mito KATP channel is the central mediator of IPC.27 28
Mito KATP channel blockade by 5HD abolished the protection induced by either IPC or diazoxide treatment as demonstrated previously.13 5HD reversed the delay of the Na+ increase during ischemia induced by diazoxide, and [Na+]i remained elevated after reperfusion. This reversibility by 5HD was also observed in the early treatment of diazoxide (protocols 7 and 11), suggesting that the mito KATP channel is not only a trigger but also an inducer of diazoxide-mediated protection. In contrast to diazoxide, 5HD abolished IPC-induced alteration of [Na+]i kinetics during ischemia and reperfusion as well as functional protection. However, this reversibility was partial for Na+ recovery after reperfusion. Furthermore, when 5HD was administered after the IPC cycle (protocol 12), no reversibility was observed although functional recovery was abolished. These results indicate that there was the dissociation between [Na+]i kinetics and functional recovery only in the IPC hearts treated with 5HD (see Figure⇑ in online data supplement, available at http://www. circresaha.org), suggesting the existence of mito KATP channel-independent pathways to alter [Na+]i kinetics in IPC hearts. Because this mito KATP channel-independent pathway is not the major contributor to IPC-mediated protection as we have indicated in the first part of this study, functional protection was not achieved in 5HD-treated IPC hearts even when [Na+]i kinetics was almost consistent with non-treated IPC hearts.
Mitochondrial dysfunction has been considered one of the mechanisms for reperfusion injury.29 The mito KATP channel modulates mitochondrial function,30 and the activation of the mito KATP channel protects the myocardium against ischemia through maintaining intramitochondrial Ca2+ homeostasis, ie, enhancement of Ca2+ release from and reduction of Ca2+ uptake into mitochondria.31 Thus, preserved mitochondrial function by mito KATP channel activation28 may be necessary to achieve better functional recovery after reperfusion. However, the downstreams after the mito KATP channel activation is still unclear. Especially, it has not been elucidated how the mito KATP channel contributes to the alteration of [Na+]i kinetics.
Contribution of Na+/K+-ATPase to Na+ Kinetics in IPC Hearts
The Na+/K+-ATPase activity is important for extruding Na+ accumulated during the initial phase of ischemia and after reperfusion.32 33 Inhibition of Na+/K+-ATPase reduced the infarct size–limiting effect by IPC,14 suggesting the important role of the ATPase. Functional interaction between the sarcolemmal KATP channel and Na+/K+-ATPase has been reported.34 35 There is the possibility that the mito KATP channel interacts with Na+/K+-ATPase because the interaction between actin microfilament and the sarcolemmal KATP channel36 or mito KATP channel has been demonstrated.37 Because free radicals can alter the activity of Na+/K+-ATPase,38 the alterations activated by the mito KATP channel39 may enhance the enzyme activity leading to [Na+]i kinetics in IPC hearts.
The delayed increase in Na+ during the early ischemia and the complete recovery during reperfusion observed in IPC and diazoxide-treated hearts can be attributed to the prevention of ischemia-induced dysfunction of Na+/K+-ATPase. Inhibition of Na+/K+-ATPase abolished the beneficial change in [Na+]i kinetics induced by IPC and diazoxide-treated hearts. However, the amount of Na+ accumulation during ischemia and at 6 minutes of reperfusion was significantly smaller in ouabain-treated IPC hearts than in other ouabain-treated hearts. Furthermore, function was not recovered in the ouabain-treated hearts except IPC hearts. This indicates that the prevention of ischemia-induced reduction of Na+ efflux via Na+/K+-ATPase resulted from the activation of the mito KATP channel. Preserved Na+/K+-ATPase activity reflects the delayed [Na+]i increase during early ischemia and the complete [Na+]i recovery during reperfusion and contributes to functional protection in IPC hearts. However, the mito KATP channel–independent pathway, including the reduced Na+ influx, although the contribution may be limited, is required for both the change in Na+ kinetics and functional protection in IPC hearts.
Mechanism for the Change in Na+ Kinetics in IPC Hearts
The experiments using 5HD and ouabain suggest the mechanism for the dissociation between Na+ kinetics and functional protection in IPC hearts followed by 5HD (see Figure⇑ in online data supplement, available at http://www.circresaha.org). Although the intracellular Na+ kinetics was almost identical in non-treated IPC hearts and IPC hearts followed by 5HD, these underlying mechanisms may be different. When the prevention of ischemia-induced reduction of Na+ efflux was blocked by 5HD, the reduced Na+ influx and the other mito KATP channel–independent pathway contribute to the alteration of Na+ kinetics in IPC hearts followed by 5HD. But the inhibition of Na+/K+-ATPase suggests that the Na+ extrusion during reperfusion is mainly mediated in turn by Na+-Ca2+ exchange, leading to Ca2+ overload and resulting in the lack of functional protection in these groups with 5HD. Thus, even when the Na+ kinetics is consistent, the different underlying mechanisms for the alteration of Na+ kinetics lead to the dissociation observed in 5HD-treated hearts.
In conclusion, the alteration of Na+ kinetics by preserving Na+ efflux via Na+/K+-ATPase mediated by mito KATP channel activation mainly contributes to functional protection in IPC hearts. The contribution of the mito KATP channel–independent pathway relating with Na+ kinetics, including reduced Na+ influx, is limited in functional protection of IPC. >
This work is partly supported by the research grants for Cardiovascular Disease (11C-1) from the Ministry of Health and Welfare of Japan (to H.K.) and that from the Japan Society for the Promotion of Science (JSPS, to K.I.). K.I. is a research fellow of JSPS. We thank Yasuo Katsuki for the support of nuclear magnetic resonance facilities and Katsuji Hashimoto (Osaka National Hospital), and Shinji Hasegawa (Osaka University) for their suggestions. We also thank Yuka Tamai for laboratory assistance.
Original received August 4, 2000; resubmission received March 13, 2001; revised resubmission received April 18, 2001; accepted April 27, 2001.
Presented in part at the 72nd Scientific Sessions of the American Heart Association, Atlanta, Ga, November 7–10, 1999, and published in abstract form (Circulation. 1999;100[suppl I]:I-343) and at the 65th Annual Scientific Meeting of the Japanese Circulation Society, Kyoto, Japan, March 25–27, 2001, and published in abstract form (Jpn Circ J. 2001;65[suppl 1-A]:84).
- © 2001 American Heart Association, Inc.
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