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Am J Physiol Heart Circ Physiol 288: H287-H292, 2005. First published August 19, 2004; doi:10.1152/ajpheart.01102.2003
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Relative contributions of Na+/H+ exchange and Na+/HCO3– cotransport to ischemic Nai+ overload in isolated rat hearts

Michiel Ten Hove,1 Marcel G. J. Nederhoff,1 and Cees J. A. Van Echteld2

1Interuniversity Cardiology Institute of the Netherlands and 2Department of Cardiology, Heart Lung Center Utrecht, University Medical Center, Utrecht, The Netherlands

Submitted 24 November 2003 ; accepted in final form 11 August 2004


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The Na+/H+ exchanger (NHE) and/or the Na+/HCO3– cotransporter (NBC) were blocked during ischemia in isolated rat hearts. Intracellular Na+ concentration ([Na+]i), intracellular pH (pHi), and energy-related phosphates were measured by using simultaneous 23Na and 31P NMR spectroscopy. Hearts were subjected to 30 min of global ischemia and 30 min of reperfusion. Cariporide (3 µM) or HCO3–-free HEPES buffer was used, respectively, to block NHE, NBC, or both. End-ischemic [Na+]i was 320 ± 18% of baseline in HCO3–-perfused, untreated hearts, 184 ± 6% of baseline when NHE was blocked, 253 ± 19% of baseline when NBC was blocked, and 154 ± 6% of baseline when both NHE and NBC were blocked. End-ischemic pHi was 6.09 ± 0.06 in HCO3–-perfused, untreated hearts, 5.85 ± 0.02 when NHE was blocked, 5.81 ± 0.05 when NBC was blocked, and 5.70 ± 0.01 when both NHE and NBC were blocked. NHE blockade was cardioprotective, but NBC blockade and combined blockade were not, the latter likely due to a reduction in coronary flow, because omission of HCO3– under conditions of NHE blockade severely impaired coronary flow. Combined blockade of NHE and NBC conserved intracellular H+ load during reperfusion and led to massive Na+ influx when blockades were lifted. Without blockade, both NHE and NBC mediate acid-equivalent efflux in exchange for Na+ influx during ischemia, NHE much more than NBC. Blockade of either one does not affect the other.

Na+/H+ exchanger; cariporide; Na+-HCO3– cotransporter; 23Na nuclear magnetic resonance spectroscopy; ischemia

PREVENTION OF ISCHEMIC INTRACELLULAR (Nai+) overload by blocking the Na+/H+ exchanger (NHE) or the Na+ channel during ischemia is cardioprotective (3, 25, 27). This protection is thought to be mediated via reduced reversed Na+/Ca2+ exchange and concomitant reduced postischemic Ca2+ overload (3, 24). The Na+/HCO3– cotransporter (NBC) and NHE are both Na+-influx-mediating, intracellular pH (pHi)-regulating transporters. The effects of simultaneous blockade of NBC and NHE on ischemic Nai+ overload in whole hearts are unknown.

When the pHi drops below physiological values, at least two acid-equivalent efflux-mediating transporters are activated: NHE and NBC (11, 12, 15, 17). Two other pHi-regulating mechanisms, Cl–/HCO3– exchange and Cl–/OH– exchange, mediate acid-equivalent influx and are thought to be activated when the pHi becomes alkaline. Therefore, these latter two are unlikely to play a role during ischemia and reperfusion. In cardiac tissue, both electroneutral (16, 22) and electrogenic (1, 5, 10) NBC has been reported, the latter with a stoichiometry of 1:2 for Na+:HCO3– (1).

Several studies report on the role of HCO3– transport during ischemia in isolated cells (18, 19). Isolated cells, however, form a limited model for ischemia, especially with respect to HCO3–-dependent buffering. Because of the large extracellular space, the extracellular cation concentrations, including the extracellular pH, are only in a very limited way affected by transsarcolemmal cation transport. Furthermore, produced CO2 can freely escape, having an alkaline effect. An isolated heart, especially during zero-flow ischemia, forms a "closed" model. As in vivo, cations and CO2 cannot, or can only to a very limited extent, leave the heart. This has important implications for Nai+ and intracellular Ca2+ loading as well as for intracellular buffering and pHi.

Studies on isolated hearts indicate that blocking NBC does not protect the heart against ischemic damage but does enhance the efficacy of NHE blockade (4, 6, 21), with the exception of one study that used antibodies and in which NBC blockade was found to offer protection (10). Although reports consistently indicate delayed postischemic pHi recovery when NBC and/or NHE are blocked, data on ischemic pHi under conditions of NBC and NHE blockade are conflicting (4, 6). Therefore, the existing data on pHi do not provide a full insight into ischemic NBC and NHE activity. To understand the interaction of NBC and NHE during ischemia, it is important to know the effect of both individual pathways on pHi and Nai+ concentration ([Na+]i). Possibly, the heart compensates for the diminished H+ efflux during NHE blockade via activation of NBC. If so, the reduction in ischemic [Na+]i overload by NHE blockade would be compromised by additional Na+ influx via NBC. Therefore, combined blockade could result in a reduction in Na+ greater than a summation of blocking either one, making combined blockade an interesting cardioprotective option. On the other hand, blocking both H+ efflux routes could lead to a more severe and prolonged acidosis. A more severe acidosis could be detrimental, whereas a somewhat prolonged acidosis could be cardioprotective via reduced reversed Na+/Ca2+ exchange, because a low pHi inhibits the Na+/Ca2+ exchanger (7, 8, 20). To study the effects of blocking both NHE and NBC, we measured the [Na+]i and pHi in isolated perfused rat hearts with simultaneous 23Na and 31P NMR spectroscopy, respectively. Hearts were subjected to 30 min of global ischemia followed by 30 min of reperfusion. To block NHE, cariporide (3 µM) was administered. To block NBC, hearts were perfused with a HCO3–-free, HEPES-buffered medium.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Heart preparation. Male Wistar rats (398 ± 13 g) were anesthetized with diethyl ether and heparinized. Hearts were excised and perfused according to Langendorff as described previously (25). Briefly, hearts were perfused at 37°C and a perfusion pressure of 73.5 mmHg. Left ventricular developed pressure (LVDP) and end-diastolic pressure (LVEDP) were assessed by an intraventricular balloon, and coronary flow was measured continuously. Hearts were paced at a constant voltage and at a frequency of 5 Hz throughout the protocol. The rate-pressure product (RPP; heart rate x LVDP) was used as an index of cardiac contractility. HCO3–-buffered perfusion fluids were saturated with 95% O2-5% CO2, resulting in a final perfusate pH of 7.35 ± 0.05. HCO3–-free HEPES-buffered perfusion fluids were saturated with 100% O2, and pH was set to 7.35 ± 0.05 at 37°C with NaOH. The standard HCO3–-buffered perfusate contained (in mM) 148.0 Na+, 4.7 K+, 1.3 Ca2+, 1.0 Mg2+, 133.3 Cl–, 24.0 HCO3–, and 11.0 glucose. The standard HEPES-buffered perfusate contained (in mM) 148.0 Na+, 4.7 K+, 1.3 Ca2+, 1.0 Mg2+, 154.3 Cl–, 5.0 HEPES, and 11.0 glucose. To discriminate between intra- and extracellular Na+, 3.5 mM TmDOTP5- was used as a shift reagent, necessitating a higher total Ca2+ concentration ([Ca2+]total) of 3.42 mM, resulting in a lower free Ca2+ concentration ([Ca2+]free) of 0.85 mM. Switching from the standard perfusate to perfusate containing shift reagent resulted in a reduction of LVDP of ~45% due to the lower [Ca2+]free. The animal experiments were approved by the Committee for Animal Experiments of the Faculty of Medicine of Utrecht University.

Experimental protocols. Hearts were assigned to four different groups (Fig. 1). In group I, hearts were perfused with HCO3– buffer and monitored during 10 min of control perfusion, followed by 30 min of global zero-flow ischemia and 30 min of reperfusion. In group II, hearts were perfused with HCO3– buffer and monitored during 5 min of control perfusion. Thereafter, hearts were perfused for 5 min with cariporide (3 µM) added to the perfusate, followed by 30 min of global zero-flow ischemia and 30 min of reperfusion with cariporide added to the perfusate. In group III, hearts were perfused with HEPES buffer and monitored during 10 min of control perfusion, followed by 30 min of global zero-flow ischemia and 30 min of reperfusion. In group IV, hearts were perfused with HEPES buffer and monitored during 5 min of control perfusion. Thereafter, hearts were perfused for 5 min with cariporide (3 µM) added to the perfusate, followed by 30 min of global zero-flow ischemia and 10 min of reperfusion with cariporide added to the HEPES perfusate. Subsequently, perfusate was switched to cariporide-free HCO3– buffer and hearts were perfused for another 20 min. The latter switch was made because pilot experiments had revealed that the coronary flow was severely impaired in hearts perfused with HEPES buffer containing cariporide, possibly preventing contractile recovery. The switch was made to investigate whether the effect on coronary flow was reversible.



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  		Fig. 1. Schematic representation of the experimental protocol. NHE, Na+/H+ exchanger.

 
After termination of the protocol, hearts were dried at 80°C for at least 48 h before determination of the dry weights. Intracellular volume was assumed to be 2.45 ml/g dry weight (2).

NMR methods. 23Na and 31P NMR spectra were recorded simultaneously at 105.9 MHz and 162.0 MHz, respectively, on a three-channel Bruker Avance DRX400 spectrometer equipped with a 9.4-T magnet, a dual tuned probe head, and two digital receivers, as described previously (25). Thirty-second 23Na NMR spectra were obtained by accumulation of 144 consecutive free induction decays (FIDs) using 90° pulses and a 210-ms interpulse delay. Thirty-second 31P spectra were acquired by adding 12 FIDs using 90° pulses and a 2.5-s interpulse delay. Parameters were quantified with a temporal resolution of 1 min or 5 min. Spectra were added accordingly. 23Na and 31P signals were quantified with respect to the signal intensity of a reference solution in a glass capillary containing known amounts of Na+ and methylene diphosphonate. 23Na NMR spectra were quantified by integration after a Gaussian and Lorentzian multiplication and a polynomial baseline correction had been performed. NMR visibility of all Na+ signals was assumed to be 1.0 (26). pHi values were calculated from the chemical shift of the Pi peak by using the equation pH = 6.72 – log({delta} – 5.66)/(3.27 – {delta}) (13). Zero parts per million were assigned to creatine phosphate (PCr). PCr, ATP, phosphomonoesters (PME), and Pi were quantified by using a time-domain-fitting routine (AMARES) from jMRUI (14). Data were corrected for partial saturation as before (25).

Statistics. Results are presented as means ± SE. Data were analyzed by Student's t-test, one-way ANOVA, or ANOVA with repeated measurements, as appropriate. If significant differences were observed, groups were compared at relevant time points by using the ANOVA mean squares within, adjusted for multiple comparisons according to Tukey's procedure when required (28). Data were separately analyzed for control perfusion, ischemia, first 10 min of reperfusion, and last 20 min of reperfusion.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Contractile performance and coronary flow. Hearts perfused with HCO3– buffer had, under control conditions, a higher LVDP than hearts perfused with HEPES buffer (P < 0.05). The LVDP during the first 5 min of the protocol was 45.4 ± 2.3 and 37.3 ± 2.1 mmHg in HCO3– buffer- and HEPES buffer-perfused hearts, respectively. Administration of cariporide did not affect the RPP (Fig. 2), the LVEDP (Fig. 3), or the coronary flow (Fig. 4) in hearts perfused with HCO3– buffer. In hearts perfused with HEPES buffer, however, administration of cariporide resulted in a reduction of the coronary flow from 49.4 ± 1.0 to 21.5 ± 0.4 ml·min–1·g dry weight–1 (P < 0.001 vs. baseline) (Fig. 4) and of the RPP to 61.1 ± 3.9% of baseline values (P < 0.01 vs. all other groups; Fig. 2) after 5 min. During ischemia, all hearts went into contracture. Characteristics of the contracture are shown in Table 1. During reperfusion, heart rate did not follow the pacing signal in all hearts. During reperfusion, the RPP showed a complete recovery in group II (Fig. 2) (P < 0.001 vs. all other groups). In all other groups, recovery was absent or very poor (Fig. 2). In HCO3–-perfused hearts, cariporide partly prevented the increase in LVEDP during reperfusion (Fig. 3) (P < 0.05). In HEPES-perfused hearts, cariporide prevented an ongoing increase in LVEDP during the first 10 min of reperfusion (Fig. 3) (P < 0.05). Thereafter, when perfusate was switched to cariporide-free HCO3– buffer, the LVEDP roughly doubled in 10 min and then stabilized. During the last 20 min of reperfusion, the LVEDP was significantly higher in the HEPES groups than in the HCO3– groups (P < 0.05). During reperfusion, the coronary flow was, as shown in Fig. 4, reduced in all groups but mainly in the HEPES groups (P < 0.01). In groups III and IV, the average coronary flow during reperfusion was only 43 ± 5% and 39 ± 2% of baseline values, respectively. In group IV, switching to cariporide-free HCO3– buffer did not restore the coronary flow (Fig. 4).



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  		Fig. 2. Rate-pressure product (RPP) before and after 30 min of global ischemia. Hearts were perfused with HCO3– buffer without (group I) or with (group II) the NHE blocker cariporide (3 µM) added to the perfusate or with a HCO3–-free HEPES buffer without (group III) or with (group IV) cariporide (3 µM) added to the perfusate (n = 6 for all groups). Cariporide was administered from 5 min before ischemia onward. In group IV, hearts were perfused with cariporide-free HCO3– buffer during the last 20 min of reperfusion, as indicated by the arrow and the change in symbols. *P < 0.01 vs. all other groups.

 


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  		Fig. 3. Left ventricular end-diastolic pressure (EDP) before and after 30 min of global ischemia. Hearts were perfused with HCO3– buffer without (group I; P < 0.05 vs. groups III and IV during last 20 min) or with (group II; P < 0.05 vs. groups III and IV during last 20 min) the NHE blocker cariporide (3 µM) added to the perfusate or with a HCO3–-free HEPES buffer without (group III) or with (group IV) cariporide (3 µM) added to the perfusate (n = 6 for all groups). Cariporide was administered from 5 min before ischemia onward. In group IV, hearts were perfused with cariporide-free HCO3– buffer during the last 20 min of reperfusion, as indicated by the arrow and the change in symbols. *P < 0.05 vs. group I; §P < 0.05 vs. group III; {dagger}P < 0.05 vs. group IV.

 


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  		Fig. 4. Coronary flow before and after 30 min of global ischemia. Hearts were perfused with HCO3– buffer without (group I) or with (group II) the NHE blocker cariporide (3 µM) added to the perfusate or with a HCO3–-free HEPES buffer without (group III) or with (group IV) cariporide (3 µM) added to the perfusate (n = 6 for all groups). Cariporide was administered from 5 min before ischemia onward. In group IV, hearts were perfused with cariporide-free HCO3– buffer during the last 20 min of reperfusion, as indicated by the arrow and the change in symbols. *P < 0.05 vs. groups I and II; {ddagger}P < 0.001 vs. baseline.

 

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  		Table 1. Characteristics of contracture

 
Nai+. Baseline [Na+]i amounted to 9.8 ± 0.5 mM and 10.4 ± 0.7 mM in HCO3–- and HEPES-buffered hearts, respectively (no significant difference). Administration of cariporide did not affect [Na+]i in either HCO3– buffer- or HEPES buffer-perfused hearts (Fig. 5). During ischemia, [Na+]i in group I linearly increased to 320 ± 18% of baseline after 30 min of ischemia (Fig. 5). Administration of cariporide (group II) substantially reduced this increase during the entire ischemic period (P < 0.001), resulting in an end-ischemic [Na+]i of 184 ± 6% of baseline (Fig. 5). In group III, [Na+]i also increased during the entire ischemic period but to a lesser extent than in group I (Fig. 5) (P < 0.02). End-ischemic [Na+]i in group III was 253 ± 19% of baseline. Also in HEPES buffer-perfused hearts, cariporide largely reduced ischemic Na+i accumulation. End-ischemic [Na+]i in group IV was 154 ± 6% of baseline, which was significantly lower than in all other groups (P < 0.01). The rates of rise in [Na+]i during the last 20 min of ischemia are presented in Table 2.



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  		Fig. 5. Intracellular Na+ concentration ([Na+]i) during ischemia and reperfusion. Hearts were perfused with HCO3– buffer without (group I) or with (group II) the NHE blocker cariporide (3 µM) added to the perfusate or with a HCO3–-free HEPES buffer without (group III) or with (group IV) cariporide (3 µM) added to the perfusate (n = 6 for all groups). Cariporide was administered from 5 min before ischemia onward. In group IV, hearts were perfused with cariporide-free HCO3– buffer during the last 20 min of reperfusion, as indicated by the arrow and the change in symbols. Indexes of significance were omitted for reasons of clarity. End-ischemic [Na+]i was 320 ± 18% of baseline in group I, 184 ± 6% of baseline in group II (P < 0.001 vs. group I), 253 ± 19% of baseline in group III (P < 0.02 vs. group I), and 154 ± 6% of baseline when both NHE and NBC were blocked (P < 0.01 vs. all other groups).

 

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  		Table 2. Rate of rise in [Na+]i during the last 20 minutes of ischemia

 
In group I, [Na+]i started to decrease immediately upon reperfusion (Fig. 5). In group II, [Na+]i showed a small decrease (Fig. 5). In group III, [Na+]i showed no recovery during reperfusion (Fig. 5). In group IV, [Na+]i slightly decreased until perfusion was switched to cariporide-free HCO3– buffer. The latter switch resulted in a rise in [Na+]i, which stabilized after ~12 min (Fig. 5).

pHi. Under control conditions, pHi was lower in HEPES buffer-perfused hearts (6.97 ± 0.01) than in HCO3– buffer-perfused hearts (7.05 ± 0.01) (P < 0.01). Administration of cariporide did not affect pHi in either HCO3– buffer- or HEPES buffer-perfused hearts (Fig. 6). During ischemia, pHi rapidly decreased in all groups. pHi stabilized at 6.09 ± 0.06 in group I (P < 0.01 vs. all other groups), 5.85 ± 0.02 in group II, 5.81 ± 0.05 in group III, and 5.70 ± 0.01 in group IV during the last 10 min of ischemia.



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  		Fig. 6. Intracellular pH (pHi) during ischemia and reperfusion. Hearts were perfused with HCO3– buffer without (group I) or with (group II) the NHE blocker cariporide (3 µM) added to the perfusate or with a HCO3–-free HEPES buffer without (group III) or with (group IV) cariporide (3 µM) added to the perfusate (n = 6 for all groups). Cariporide was administered from 5 min before ischemia onward. In group IV, hearts were perfused with cariporide-free HCO3– buffer during the last 20 min of reperfusion, as indicated by the arrow and the change in symbols. Indexes of significance were omitted for reasons of clarity.

 
Upon reperfusion, pHi rapidly recovered in group I (Fig. 6). Cariporide (group II) slightly delayed pHi recovery (no significant difference). In group III, pHi recovery was more severely impaired and delayed (no significant difference). In group IV, pHi showed only very limited recovery until perfusion was switched to cariporide-free HCO3– buffer, which resulted in a gradual recovery of pHi (P < 0.01 vs. all other groups) (Fig. 6).

Energy-related phosphates. Baseline PCr concentration was 14.0 ± 1.9 mM for HCO3– buffer-perfused hearts and 14.1 ± 1.9 mM for HEPES buffer-perfused hearts (no significant difference). Baseline ATP concentration was 12.1 ± 1.5 mM for HCO3– buffer-perfused hearts and also 12.1 ± 1.5 mM for HEPES buffer-perfused hearts (no significant difference). During ischemia, PCr rapidly decreased to undetectable levels and ATP gradually decreased to zero in all groups. Pi accumulated in all groups, but the accumulation was highest in group I (P < 0.01 vs. group IV). PME also accumulated in all groups, but the accumulation was highest in group IV (P < 0.05 vs. groups I and II). During reperfusion, PCr partially recovered in all groups, with recovery being strongest in both HCO3– groups (P < 0.05), which is in line with data on postischemic coronary flow. During the last 10 min of reperfusion, PCr concentration was 68 ± 8% of baseline in group I (P < 0.05 vs. groups III and IV), 57 ± 3% of baseline in group II (P < 0.05 vs. group IV), 30 ± 5% of baseline in group III, and 30 ± 5% of baseline in group IV. ATP recovered partially in all groups but recovered more rapidly in both HCO3– groups than in both HEPES groups (P < 0.01). During reperfusion, Pi and PME resonances overlapped, especially in both HEPES groups, possibly due to pHi heterogeneity, making quantification during reperfusion impossible.


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Although many studies report on the role of NHE and NBC, the effects of blocking both mechanisms on Na+ transport during ischemia and reperfusion has never been studied in an isolated heart model. Our data show that perfusion with a HCO3–-free buffer limits ischemic Nai+ overload but is not cardioprotective. This lack of cardioprotection may be related to the limited nature of the reduction as well as to the lower ischemic pHi and lower postischemic coronary flow we found. This lack of cardioprotection has been found before (4, 6, 21), also when hearts were perfused with a constant flow.

The reduced ischemic Nai+ overload and the lower ischemic pHi in HCO3–-free buffer-perfused hearts can be explained by the prevention of Na+ influx via NBC. The finding that ischemic pHi was lower in HEPES buffer-perfused hearts than in HCO3– buffer-perfused hearts is in line with one previous finding (4) but in contrast with another (6).

When hearts were perfused with a HCO3– buffer, NHE blockade with cariporide substantially reduced ischemic Nai+ overload and was cardioprotective, as we and others have shown before (9, 25). This reduced ischemic Na+ overload in cariporide-treated hearts can be explained by prevention of Na+ influx via NHE. The >100% recovery of the RPP in these hearts may be explained by the higher [Na+]i during reperfusion resulting in enhanced contractility via reduced Na+/Ca2+ exchanger-mediated Ca2+ efflux, much like the ouabain effect. This is in contrast to group I, which showed a higher [Na+]i during reperfusion but in which the ischemic [Na+]i was much higher, resulting in detrimental Ca2+ overload and preventing contractile recovery during reperfusion. We found a significantly lower ischemic pHi after NHE blockade. Previously, we (25) and others (9, 23) have found a similar, yet not significant, trend, although in one study a significantly lower ischemic pHi after NHE blockade with EIPA was reported (4). The lower ischemic pHi also indicates inhibition of NHE.

Our 23Na data show that NHE mediates more Na+ influx during ischemia than NBC. This is in line with the finding of Leem et al. (12) showing that NHE mediates more acid-equivalent efflux than NBC when pHi drops below 6.9.

Ischemic NHE blockade under HCO3–-free conditions reduced ischemic Nai+ overload by 75%, which is more than in any of the other studied groups. The residual 25% will at least partially be mediated by the Na+ channel (27). Although the difference in end-ischemic [Na+]i between combined NHE plus NBC blockade and NHE blockade alone is small, it could be just enough to prevent reversed Na+/Ca2+ exchange. The reduction in the rate of rise of [Na+]i by blocking both NHE and NBC is slightly (0.6% of baseline/min) smaller than the summation of the reductions caused by blocking either one. This opposes the idea that blocking either one of the two pHi-regulating mechanisms during ischemia results in enhanced activity of the other one, suggesting that NHE and NBC activity are regulated independently. Our data on pHi indicate that both NHE and NBC contribute to diminishing ischemic acidosis. Also, in our pHi data, we found that the effect of blocking both Na+-dependent pHi regulators appears to be smaller than the summation of the effect of blocking either one.

Omission of HCO3– in conditions of NHE blockade severely impaired coronary flow. This has also been found before (6) and is in line with findings from Shimada et al. (21), who found in a constant-flow model that administration of cariporide in HEPES buffer-perfused hearts resulted in an increase in vascular resistance. The flow reduction of >50% before ischemia when both NHE and NBC were blocked resulted in a concurrent reduction of RPP. However, none of the other measured parameters, i.e., Nai+, pHi, and high-energy phosphates, which are all very sensitive to ischemia, changed during this period, most likely due to this concurrent change in RPP.

During reperfusion, the coronary flow in the HEPES and cariporide groups did not recover and stabilized at the low preischemic level, explaining the lack of postischemic contractile recovery in this group, because in constant-flow models cariporide was even more cardioprotective under HCO3–-free conditions than when hearts were perfused with a HCO3– buffer (21). Because in our study HCO3–-free buffer was used to block NBC, it does not provide information about whether the reduction in coronary flow is due to NBC blockade or due to the lack of HCO3–. To evaluate the potential cardioprotection that NBC and NHE blockade could offer when a truly selective NBC inhibitor would be used, further research is required. Unfortunately, such an inhibitor is currently unavailable. Khandoudi et al. (10) showed that NBC blockade with polyclonal antibodies reduced ischemic injury, which indicates that a selective inhibitor could have cardioprotective properties. The specificity of these polyclonal antibodies, however, was not studied, and because the experiments were not performed in the presence of an NHE inhibitor, partial NHE blockade cannot be ruled out.

Upon reperfusion, pHi, [Na+]i, and LVEDP only slightly changed in cariporide-treated, HEPES buffer-perfused hearts. A similar effect on pHi has been found before (6) and is similar to the severely impaired restoration of pHi under conditions of NBC and NHE blockade found in isolated cardiomyocytes (19). When, however, in our experiments the blockades of the pHi-regulating mechanisms were lifted after 10 min, pHi recovered and [Na+]i and the LVEDP increased. These findings can be explained by sustained intracellular H+ load due to the lack of H+ extrusion mechanisms during the first 10 min of reperfusion. Thereafter, protons are extruded by NHE and NBC, resulting in massive Na+ influx. This posttreatment Nai+ overload may result in reversed Na+/Ca2+ exchange, explaining the elevation in LVEDP. These findings imply a limitation in the cardioprotective potential of preventing ischemic Na+ overload. Under these experimental conditions, blockade of NHE and NBC prevents H+ efflux in exchange for Na+ influx, but this process leading to Nai+ overload and concomitant intracellular Ca2+ overload is only postponed. After the blockades are removed the damage occurs after all, reflected in the elevation of the LVEDP.

In conclusion, we found that blockade of NBC with HCO3–-free HEPES buffer reduces ischemic Nai+ overload but is not cardioprotective. Blocking NHE with cariporide results in a much larger reduction in ischemic Nai+ overload and is cardioprotective. Blockade of both NBC and NHE results in a reduction of ischemic Nai+ overload larger than blocking either one of these two but smaller than a summation of the effects of NBC and NHE blockade, respectively. When both NBC and NHE were blocked, however, coronary flow was severely impaired, preventing postischemic contractile recovery. In these experiments, a sustained intracellular H+ load during reperfusion resulted in massive Na+ influx via NHE and/or NBC when perfusion was switched to cariporide-free HCO3– buffer.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
M. Ten Hove was supported by The Netherlands Heart Foundation Grant 98.141. Equipment was financed by The Netherlands Organization for Scientific Research Grant 902-16-202, Royal Netherlands Academy for Arts and Sciences Grant D96.695, and the Interuniversity Cardiology Institute of The Netherlands.


   ACKNOWLEDGMENTS
 
Cariporide was kindly provided by Aventis Pharma (Frankfurt am Main, Germany).

Present address of M. Ten Hove: Dept. of Cardiovascular Medicine, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Dr., Oxford OX3 7BN, UK.


   FOOTNOTES
 

Address for reprint requests and other correspondence: C. J. A. van Echteld, Dept. of Cardiology, Rm. G02.523, Univ. Medical Center, P.O. Box 85500, 3508 GA Utrecht, The Netherlands (E-mail: c.j.a.vanechteld{at}hli.azu.nl)

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
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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