© 1997 by European Society of Cardiology
Copyright © 1997, European Society of Cardiology
Short-term inhibition of the Na–H exchanger limits acidosis and reduces ischemic injury in the rat heart
Division of Cardiovascular Medicine, University of California Davis, TB 172, Davis, CA 95616, USA
* Corresponding author. Tel. +1 (916) 752-0717; fax: +1 (916) 752-3264.
Received 21 June 1996; accepted 9 January 1997
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Abstract |
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 Top Abstract 1 Introduction2 Methods3 Results4 DiscussionReferences |
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Introduction: Pharmacologic inhibition of the Na–H exchanger prior to and during ischemia has been shown to protect the ischemic heart by reducing Na–H exchange. However, pH regulation in the ischemic heart is primarily mediated by other pH regulatory mechanisms, such as metabolite efflux and sodium-coupled HCO3– influx, which may compensate for a reduction in Na–H exchange by increasing proton efflux. We hypothesized that short-term pharmacologic inhibition of the Na–H exchanger would result in increases in other compensatory pH regulatory mechanisms and thereby limit acidosis during ischemia and reduce ischemic injury. Methods: In order to test this hypothesis, we exposed isolated perfused rat hearts to ethylisopropylamiloride (EIPA, 3 µM) for 40 min, followed by 10 min of EIPA-free perfusate and 30 min of global ischemia (termed CTL/EIPA hearts). The effects of this intervention were compared to hearts perfused with either glucose alone (CTL) or EIPA 3 µM for 10 min before ischemia (EIPA). Ischemic injury was measured using creatine kinase (CK) release on reperfusion, while pH and metabolic effects were measured using 31P nuclear magnetic resonance spectroscopy. The effect of this intervention on recovery from an acid load was assessed using an NH4Cl pre-pulse in bicarbonate-containing Krebs-Henseleit as well as HEPES buffer. Results: Both CTL/EIPA and EIPA hearts had marked reduction in ischemic injury (CK control 1191±116 IU/g dry weight; CTL/EIPA 406±42 IU/gdw; EIPA 333±78 IU/gdw), as well as significantly reduced end-diastolic pressure on reperfusion. Intracellular pH was higher in the CTL/EIPA hearts (end-ischemic pH = 6.34±0.05) compared to either control (5.86±0.02) or EIPA hearts (6.01±0.02), while pH recovery on reperfusion was markedly slowed in the CTL/EIPA hearts. CTL/EIPA hearts had rapid ATP depletion during ischemia, but PCr recovery comparable to EIPA hearts. Acidification on exposure to NH4Cl was increased in the presence of HEPES, but pH recovery was not altered by short-term exposure to EIPA. Conclusions: These data show that short-term inhibition of the Na–H exchanger altered pH regulation in the ischemic heart, resulting in reduced acidosis and slow pH recovery on reperfusion, coupled with reduction in ischemic injury and end-diastolic pressure on reperfusion. These findings are consistent with short-term exposure to EIPA accelerating ATP depletion during ischemia, as well as limiting proton efflux during reperfusion.
KEYWORDS Na+/H+ exchange; Myocardial ischemia; pH; Ethylisopropylamiloride; Rat, heart
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1 Introduction |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
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Myocardial ischemia results in numerous perturbations which can cause ischemic and reperfusion injury [1]. These perturbations include factors such as energy depletion [2, 3], loss of membrane integrity [4], formation of free radicals [5], and calcium overload [6]. Reduced intracellular acidification has frequently been associated with interventions that reduce ischemic injury due to global ischemia and reperfusion [7, 8]. These interventions, including ischemic preconditioning, inhibition of glycolysis with pyruvate, and inhibition of oxidative phosphorylation with cyanide [8], have generally been thought to reduce proton production during ischemia with resultant lowering in intracellular sodium and calcium loading during ischemia and on reperfusion. Recent experiments in this laboratory have suggested that, at least in ischemic preconditioning, reduced intracellular acidosis may also result from enhanced proton efflux from the ischemic cell as well as reduced proton generation [9].
Studies in renal epithelial cells and isolated hearts have also suggested that the activity of sarcolemmal proton transport mechanisms can be up-regulated by factors such as acidosis and moderate ischemia [10, 11]. Since there is evidence that the Na–H exchanger has a role in maintenance of intracellular pH under normal perfusion conditions [12], we postulated that short-term inhibition of the exchanger would result in a compensatory increase in other pH regulatory systems, thus limiting acidosis during subsequent ischemia. Therefore, we employed short-term inhibition of the Na–H exchanger with the inhibitor ethylisopropylamiloride (EIPA, 3 µM) in an isolated rat heart model of global ischemia, with measurements of ischemic injury (creatine kinase release) using an enzymatic assay and phosphorus metabolites and pH using 31P nuclear magnetic resonance spectroscopy. Short-term inhibition of the Na–H exchanger was achieved by exposing the non-ischemic heart to EIPA for 40 min, followed by 10 min of washout. These hearts are abbreviated as CTL/EIPA. Control experiments were performed in hearts perfused with standard Krebs-Henseleit buffer with and without constant infusion of EIPA (to inhibit the Na–H exchanger during ischemia and reperfusion). To determine whether short-term exposure to EIPA altered recovery from an acid load in the absence of ischemia, the pH response to an acid load (NH4Cl) was measured in CTL and CTL/EIPA hearts in both bicarbonate-containing (Krebs-Henseleit) and bicarbonate-free (HEPES) buffers.
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2 Methods |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
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2.1 General methods
All experiments were performed with the approval of the University of California Davis Animal Research Committee.
2.1.1 Isolated heart model
Experiments were performed using an isovolumic isolated perfused rat heart preparation. Male Sprague-Dawley rats (approx. 350 g) were pre-treated with heparin (1000 U i.p.), followed by sodium pentobarbital (65 mg/kg i.p.). After deep anesthesia was achieved as determined by the absence of a foot reflex, the heart was rapidly excised and placed into iced saline. The arrested heart was retrograde perfused through the aorta within 2 min. The left ventricular developed pressure (LVDP) was determined using a latex balloon in the left ventricle with high-pressure tubing connected to a pressure transducer. Perfusion pressure (PP) was also monitored using high-pressure tubing off the perfusion line. Hemodynamic measurements were recorded on a 4-channel Gould Windowgraf Recorder (Gould Inc., Valley View, OH). The heart was perfused using an accurate roller pump at a flow rate of 12.5 ml/min (Rainin Instrument Co, Woburn, MA). The perfusate consisted of (in mM/l) NaCl 118, KCl 4, CaCl2 1.2, MgCl2 1, NaHCO3 25, with the substrate being 11 mM glucose. The perfusion apparatus was tightly temperature-controlled to maintain heart temperature at 37±0.5°C under all conditions. Oxygenation of the perfusate was provided by thin-walled silicone tubing immediately proximal to the heart.
2.1.2 Creatine kinase
Creatine kinase (CK) was measured from timed 5 min collections of the effluent for 60 min of reperfusion following the ischemic period. Each 5 min collection was analyzed in duplicate using established spectrophotometric methods [13]. Total integrated CK activity over the reperfusion period was calculated for each heart and corrected for the dry weight of the heart. CK release was expressed in International Units/g dry weight. As previously shown, [14]the integral of the CK release is a representative measure of ischemic injury and infarction.
2.1.3 NMR spectroscopy
All NMR spectroscopy was performed on a Bruker AMX 400 vertical bore spectrometer using a switchable 20 mm probe tuned for 31P. 31P NMR spectroscopy was performed using 228 acquisitions of a 60-degree pulse and 1.6 s interpulse delay, with spectra processed using an exponential multiplication of 20 Hz and Fourier-transformed [15]. Intracellular pH was calculated from the chemical shift between the Pi and PCr resonance using a titration curve established in this facility [16]. Metabolite intensities were obtained using Lorentzian deconvolution software on the spectrometer.
2.1.4 NH4Cl pre-pulse
In order to determine whether the ability of the cell to extrude protons was altered in the CTL/EIPA hearts, pH recovery was measured in control and CTL/EIPA hearts using exposure to NH4Cl [17]. The NH4Cl load occurred at the time at which ischemia would have been initiated in the parallel ischemia experiments. Both control and CTL/EIPA hearts were exposed to 20 mM NH4Cl for 10 min in a K+-free buffer that contained 100 µM ouabain (to inhibit sodium transport via Na–K ATPase). After this 10 min exposure to NH4Cl, hearts were perfused for 30 min with K+-free/ouabain buffer not containing NH4Cl (NH4Cl washout). Since bicarbonate-dependent pH regulatory mechanisms have been shown to contribute significantly to pH recovery [18], the NH4Cl pre-pulse experiments were also performed in the presence of bicarbonate-free HEPES buffer in place of Krebs-Henseleit. These experiments were therefore able to distinguish any direct effects of short-term exposure to EIPA on both bicarbonate-dependent and bicarbonate-independent mechanisms of pH recovery. Finally, since the HPLC measurements of EIPA in the hearts demonstrated a residual tissue EIPA concentration of 1.4 µM, 3 additional hearts were studied using that concentration of EIPA. Intracellular pH was measured with 5 min time resolution using 31P NMR as described above and in Ref. [9].
2.1.5 EIPA concentrations
In order to determine whether any residual EIPA remained in the tissue after short-term exposure to EIPA, tissue from hearts corresponding to the 3 groups in the ischemic protocol (CTL, CTL/EIPA and EIPA, n=3 in each group) were freeze-clamped and assayed for EIPA concentrations using high-performance liquid chromatography. Briefly, following the specific protocols in the preparation period (Fig. 1), hearts were freeze-clamped using aluminum tongs dipped in liquid N2. Approximately 200 mg samples were homogenized in 2 ml K2CO3, centrifuged for 10 min, and the supernatant mixed with 5 ml ethylacetate. The organic component was incubated at 50°C under N2 and the residual powder dissolved in 1% HCl–methanol–H2O. Extracted samples (100 µl) were injected into a Gilson UV Detector (Model 116) using a standard reversed-phase (C18) column with detection at 365 nm [19]. Concentrations were derived from a standard curve established using 2–200 ng of EIPA.
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2.1.6 Statistical methods
Data were analyzed using INSTAT (GraphPad, San Diego, CA) software operating on an IBM-compatible personal computer. Differences between groups at a given time point were assessed using ANOVA with subsequent Student-Newman-Keuls if ANOVA showed significance. Differences between 2 groups were assessed using the Mann-Whitney non-parametric test. A P-value of less than 0.05 was used to test significance. All data are expressed as mean±s.e.m.
2.2 Specific protocols
2.2.1 Global ischemia
All hearts had 30 min of global no-flow ischemia and 60 min of reperfusion with Krebs-Henseleit buffer containing glucose 11 mM as the sole substrate without any EIPA. Control hearts (n=6) were perfused for 60 min prior to ischemia with standard buffer (Fig. 1). CTL/EIPA hearts (n=6) were perfused with standard buffer for 10 min, followed by 40 min of buffer containing EIPA (3 µM). A 10 min period of perfusion of buffer without EIPA to allow washout of the inhibitor was then followed by global ischemia. EIPA hearts (n=4) had 50 min of perfusion with standard buffer, followed by 10 min of buffer containing EIPA (3 µM) and then global ischemia without EIPA washout. These hearts were studied in order to determine the direct effects of EIPA. All hearts were reperfused with Krebs-Henseleit buffer without EIPA.
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3 Results |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
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3.1 Ischemic injury
Compared to control hearts, both CTL/EIPA hearts and EIPA hearts had significantly reduced creatine kinase (CK) release on reperfusion (CTL 1191±116 IU/g dry weight;, CTL/EIPA 406±42 IU/gdw; EIPA 333±78 IU/gdw).
3.2 Functional recovery
3.2.1 Left-ventricular developed pressure
Left ventricular developed pressure under baseline conditions was highest in the CTL hearts (CTL 110±3; CTL/EIPA 89±7; EIPA 94±10 cmH2O), although there was no statistically significant difference between the CTL hearts and those exposed to EIPA. CTL hearts had poor recovery on reperfusion, with all hearts developing <20 cmH2O on reperfusion. CTL/EIPA hearts had intermediate recovery after reperfusion, with an overall developed pressure of 34±10 cmH2O at the first reperfusion period in the CTL/EIPA hearts (Fig. 2). EIPA hearts had excellent functional recovery, with initial developed pressure on reperfusion being 84±6 cmH2O; this degree of function was maintained throughout reperfusion. These differences in developed pressure between groups in recovery persisted throughout the reperfusion period.
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3.2.2 Left ventricular end-diastolic pressure
In contrast to the minimal benefit on systolic function, CTL/EIPA hearts showed substantial improvement in end-diastolic pressure on reperfusion (Fig. 3). While control hearts had an approximate 35 cm H2O increase in end-diastolic pressure on reperfusion, end-diastolic pressure fell in CTL/EIPA hearts despite increases in end-diastolic pressure during ischemia equivalent to control hearts. EIPA hearts also had low diastolic pressures on reperfusion, which were not significantly different from the CTL/EIPA hearts. While the end-diastolic pressure after 30 min of ischemia was equivalent in the CTL/EIPA, control and EIPA hearts, the temporal response of the rise in end-diastolic pressure during ischemia was distinct between the groups. The increase in end-diastolic pressure was similar in the control and CTL/EIPA hearts during ischemia, although the increase in end-diastolic pressure plateaued after 15 min of ischemia. In contrast, EIPA hearts maintained normal end-diastolic pressure for the first 15 min of ischemia, with the end-diastolic pressure only increasing significantly in the latter half of ischemia.
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3.3 Intracellular pH
Short-term exposure to EIPA did not alter intracellular pH prior to ischemia. CTL/EIPA hearts had a significantly higher pH during global ischemia than control hearts (Fig. 4), with end-ischemic pH being 6.34±0.05 in the CTL/EIPA hearts versus 5.88±0.02 in the control hearts (P<0.001). Not only was the degree of acidification during the first 10 min less in the CTL/EIPA compared to the CTL hearts, but acidification in the CTL/EIPA hearts stopped after 15 min of ischemia. Acidification in the EIPA hearts was intermediate between the CTL and CTL/EIPA hearts.
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The return of intracellular pH to baseline values on reperfusion was rapid (within 5 min) in the control hearts, but was significantly slowed in the CTL/EIPA hearts, with intracellular pH only returning to baseline levels by the 15 min reperfusion point. Recovery of pH on reperfusion was intermediate in the EIPA hearts compared to control and CTL/EIPA hearts.
3.4 Metabolites
3.4.1 Phosphocreatine (PCr)
All hearts had rapid losses of PCr at the onset of global ischemia and partial return of PCr levels on reperfusion, although the level of phosphocreatine after 5 min of ischemia was higher in the EIPA than either the control or CTL/EIPA hearts (Fig. 5). PCr returned to 36±17% of baseline on reperfusion in control hearts, while CTL/EIPA hearts had recovery to 68±8% of baseline; this level of recovery persisted throughout reperfusion with only gradual loss of PCr. Similarly, EIPA hearts had excellent recovery of PCr on reperfusion which was identical to the CTL/EIPA hearts.
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3.4.2 Adenosine triphosphate (ATP)
ATP levels were maintained in the first 5 min of ischemia in all groups (Fig. 6). However, subsequent ATP depletion was more rapid in the CTL/EIPA hearts than either the CTL or EIPA hearts, such that ATP in the CTL/EIPA hearts was not observed after 15 min of ischemia. Reperfusion resulted in low levels of ATP in the control and CTL/EIPA hearts (approx. 20% of control). EIPA hearts, in contrast, had significantly greater early recovery of ATP levels on reperfusion (33±4% of baseline, P<0.05 vs. control and CTL/EIPA), although the levels of ATP slowly fell as reperfusion continued.
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3.4.3 EIPA concentrations
HPLC measurement of control hearts did not show any detectable EIPA, while the CTL/EIPA hearts had a calculated EIPA concentration of 1.44±0.28 µM. Thus, despite the 10 min washout period, there was evidence of residual EIPA in these hearts.
3.4.4 NH4Cl pre-pulse
Exposure to 20 mM NH4Cl resulted in acidification of control hearts to 6.72±0.01 and recovery to baseline pH within 10 min (Fig. 7). The 5 min data points are shown, fitted with a spline function. CTL/EIPA hearts acidified to a significantly lower pH than the CTL hearts after NH4Cl exposure and washout (nadir pH = 6.44±0.03) and had a greater initial rate of pH recovery. However, the CTL/EIPA hearts did not recover to normal pH within the experimental period. Although the different degrees of acid loading preclude direct comparison of immediate pH recovery between these groups, comparison of the slope of pH recovery at identical pH allows a qualitative assessment of proton extrusion. At pH 6.73, the slope of 
pH/t was similar in the CTL and CTL/EIPA hearts, suggesting that early pH recovery was not impaired in the CTL/EIPA hearts. As seen in Fig. 7, the pH response to an acid load in hearts perfused with 1.4 µM EIPA (the measured concentration in the CTL/EIPA hearts) was characterized by greater acidosis and delayed recovery compared to the CTL/EIPA hearts.
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), CTL/EIPA (
) and EIPA 1.4 µM hearts (
) (n=3 in each group) in the presence of bicarbonate-containing KH buffer. NH4Cl washout occurred at time 0. The data are fitted with a spline function. Initial pH (time –5 and 0) was similar in all groups. CTL/EIPA and EIPA 1.4 µM hearts acidified more than the CTL hearts following removal of NH4Cl. While initial pH recovery from the nadir was rapid in the CTL/EIPA hearts, pH recovery was incomplete even after 15 min. EIPA 1.4 µM hearts had markedly delayed acidification and recovery when compared to the CTL/EIPA hearts. *P<0.001 CTL vs. EIPA 1.4; +P<0.01 CTL/EIPA vs. EIPA 1.4; P<0.001 vs. CTL; #P<0.001 CTL/EIPA vs. CTL and EIPA 1.4.Exposure to NH4Cl in the presence of HEPES buffer increased acidification on NH4Cl washout in both CTL and CTL/EIPA hearts (pH nadirs = 6.50±0.02 and 6.18±0.01, respectively). Despite these changes in acid loading, the rate of pH recovery at equal pH was not significantly altered in CTL and CTL/EIPA hearts in the presence of HEPES.
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4 Discussion |
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4.1 Acidification during ischemia—ATP production and utilization
One of the important findings of the current study is that the protective effect of short-term inhibition of the Na–H exchanger prior to global ischemia was associated with more rapid ATP depletion, yet less intracellular acidosis, during ischemia. Acidification during ischemia results from the balance of proton production (primarily from anaerobic glycolysis and ATP hydrolysis) [20]and proton utilization, either by intracellular buffering mechanisms [21]or proton efflux pathways [22]. The evidence from the current experiments suggests that the reduced acidification during ischemia in the CTL/EIPA hearts resulted from changes in ATP depletion (production and/or utilization) rather than changes in proton efflux. This is based on the similar rapid depletion of ATP and limitation of acidosis in the CTL/EIPA hearts as previously observed in glycogen-depleted pyruvate hearts [7]and hearts exposed to cyanide [8], conditions in which ATP production is impaired. In addition, the critical role of ATP hydrolysis in the generation of protons is further supported by the absence of any further acidification when ATP was depleted (Figs. 4 and 6
). The relationship between ATP and pH in the EIPA hearts suggests an opposite effect of EIPA when present during ischemia (EIPA hearts). While one previous study showed that EIPA did not alter acidification during ischemia [12], the current findings of slower ATP depletion are consistent with recent findings in neonatal rabbit hearts showing both preservation of ATP and limitation of acidosis when EIPA was present during ischemia [23]. Preservation of ATP during ischemia may result from greater anaerobic production of ATP and/or reduced hydrolysis, effects which cannot be distinguished by these experiments. However, the current findings are similar to those of preconditioned hearts [7, 24]in which both the rate of ATP depletion and the degree of acidification during ischemia were limited. Thus, in summary, the CTL/EIPA hearts had markedly limited acidification likely due to rapid ATP depletion and loss of ATP available for hydrolysis, while the EIPA hearts had less significant limitation of acidosis consistent with a reduction in the rate of ATP hydrolysis. However, a direct effect of EIPA on proton efflux pathways apart from the Na–H exchanger cannot be excluded as a cause of differences in intracellular pH during ischemia.
4.2 Recovery from an acid load (NH4Cl pre-pulse)
In the current experiments, increased proton efflux via the Na–H exchanger as a mechanism of the limitation of acidosis during ischemia in the CTL/EIPA hearts was not supported by changes in proton efflux when non-ischemic hearts were given an exogenous acid load (NH4Cl). In these pre-pulse experiments, there were no significant changes in the rate of pH recovery at equal pH with either bicarbonate-containing or bicarbonate-free buffers following an acid load, suggesting no changes in either the Na–H exchanger or bicarbonate-dependent mechanisms. However, the pre-pulse experiments did show a lower nadir pH in the CTL/EIPA compared to CTL hearts during acid loading, consistent with an effect of EIPA on influx of NH4+ or proton efflux during washout. Comparison between these pre-pulse experiments and the pH measurements during ischemia must be tempered since pH recovery in the pre-pulse experiments was made under non-ischemic conditions and different mechanisms may have been operative.
4.3 pH recovery on reperfusion
The rate of intracellular pH recovery on reperfusion following global ischemia is primarily mediated by lactate and CO2 efflux, with smaller contributions by sodium-coupled H+ efflux (the Na–H exchanger) and HCO3– influx [25]. Consistent with previous experiments [12, 26], pH recovery following ischemia in the EIPA hearts was slower than in control hearts, presumably because proton efflux via the exchanger was directly inhibited. In contrast, pH recovery was markedly slowed on reperfusion from ischemia in the CTL/EIPA hearts. Since pH recovery from ischemia is primarily dependent on metabolite washout and Na+-coupled HCO3– influx [25, 27, 28], the slow pH recovery in the CTL/EIPA hearts may, in part, be related to reduced production of lactate during ischemia and, consequently, less lactate-coupled efflux of protons. Although not directly measured in the current experiments, the reduction in acidosis during ischemia, as well as the rapid depletion of ATP, is consistent with less lactate production [29, 30]. However, the markedly slower pH recovery on reperfusion has not been seen in previous experiments with only limitation of acidosis during ischemia [7, 8], suggesting additional mechanisms beyond a reduction in lactate production. Since direct inhibition of the lactate carrier using 
-cyano-4-hydroxycinnamate slowed pH recovery from ischemia in the isolated rat heart [31], inhibition of the lactate-H+ co-transporter as a secondary result of short-term inhibition of the Na–H exchanger is a possible mechanism for the marked slowing in pH recovery observed in the current experiments. This postulate is also consistent with the findings of the pre-pulse experiments, since lactate efflux is not important in pH recovery from an acid load under non-ischemic conditions [18].
The slower recovery and more prolonged acidosis after reperfusion may also be, in part, a mechanism for the lower ischemic injury in the CTL/EIPA hearts. Since H+ inhibits the Na–Ca exchanger and the slow calcium channels [32], as well as reducing binding of calcium to cellular and intracellular sites [33], prolonged intracellular acidosis may have limited calcium entry in the CTL/EIPA hearts.
Finally, the low reperfusion end-diastolic pressures in the CTL/EIPA hearts, as well as in previously reported pyruvate-perfused hearts [7], despite rapid ATP depletion during ischemia, does not support the postulate that rapid ATP depletion results in contracture. Rather, while these data suggest that ATP depletion during ischemia is an important and direct modulator of intracellular pH (Figs. 3 and 5), interventions that alter cation transport can markedly alter the hemodynamic effects of ischemia independent of ATP and pH.
4.4 Limitations
These experiments employed short-term inhibition of the Na–H exchanger using 3 µM EIPA for 40 min with a 10 min washout period prior to ischemia. This concentration was chosen because previous studies demonstrated inhibition of the Na–H exchanger at this concentration without significant hemodynamic effects [9, 12]. Thus, other effects may be expected at different concentrations, different exposures, or different washout periods. Similarly, transference of these findings to more prolonged oral treatment with pharmacologic peaks and troughs is complex. Therefore, studies with longer dosing are required to determine if similar changes in pH regulation are observed.
While these data are consistent with secondary effects of short-term Na–H exchanger inhibition, a residual direct effect of EIPA, or other direct effects of EIPA on sodium channels [34], may have been present. Direct measurement of EIPA concentrations in CTL/EIPA hearts showed that almost 50% of the original concentration of drug was present in the tissue. However, the functional consequence of the residual EIPA was very different from constant perfusion with EIPA, as evidenced by (a) the markedly different response to an acid load between CTL/EIPA hearts and hearts perfused with an equivalent concentration of EIPA (Fig. 7), (b) the directionally opposite effects on ATP depletion during ischemia in the EIPA hearts (Fig. 6) and (c) the marked different effect on acidification during ischemia and the rate of pH recovery on reperfusion compared to the other groups. Thus, while residual inhibition of the Na–H exchanger was likely in the CTL/EIPA hearts, this effect was either minimal compared to the effect of 3 µM EIPA and/or superseded by secondary effects of Na–H exchanger inhibition during the 40 min exposure period and subsequent EIPA-free perfusion.
4.5 Conclusions
These experiments show that short-term inhibition of the Na–H exchanger altered pH regulation in response to ischemia, with marked limitation of acidosis and slowed recovery of pH on reperfusion. These changes in pH regulation were associated with rapid ATP depletion, reduced ischemic injury, and lower end-diastolic pressures compared to control hearts, and were distinct from changes observed with direct inhibition of the Na–H exchanger during ischemia using EIPA. Although the mechanisms for these effects are unknown, the changes in pH are consistent with reduced proton production during ischemia and slower proton efflux on reperfusion. These findings show that short-term treatment with EIPA is protective, but may have multiple effects under conditions of myocardial ischemia that are different from direct inhibitory effects of EIPA. These effects may be important under conditions of intermittent dosing with oral Na–H inhibitors in human subjects prior to ischemia.
Time for primary review 31 days.
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Acknowledgements |
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Supported by grants from the California Affiliate of the American Heart Association (SS) and the American Diabetes Association (RR). The authors wish to thank LiFeng Wang, M.S., for her technical assistance in performing the creatine kinase assays, P. Richard Vulliet, Ph.D., and Christopher Barton for performing the EIPA tissue assays.
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