AJP - Heart Watch the video to learn how APS reaches out to developing nations.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 281: H2398-H2409, 2001;
0363-6135/01 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (45)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by An, J.
Right arrow Articles by Stowe, D. F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by An, J.
Right arrow Articles by Stowe, D. F.
Vol. 281, Issue 6, H2398-H2409, December 2001

Blocking Na+/H+ exchange reduces [Na+]i and [Ca2+]i load after ischemia and improves function in intact hearts

Jianzhong An, Srinivasan G. Varadarajan, Amadou Camara, Qun Chen, Enis Novalija, Garrett J. Gross, and David F. Stowe

Anesthesiology Research Laboratories, Departments of Anesthesiology, Physiology, and Pharmacology and Toxicology, and Cardiovascular Research Center, The Medical College of Wisconsin, Milwaukee 53226; and Research Service, Veterans Affairs Medical Center, Milwaukee, Wisconsin 53295


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We determined in intact hearts whether inhibition of Na+/H+ exchange (NHE) decreases intracellular Na+ and Ca2+ during ischemia and reperfusion, improves function during reperfusion, and reduces infarct size. Guinea pig isolated hearts were perfused with Krebs-Ringer solution at 37°C. Left ventricular (LV) free wall intracellular Na+ concentration ([Na+]i) and intracellular Ca2+ concentration ([Ca2+]i) were measured using fluorescence dyes. Hearts were exposed to 30 min of ischemia with or without 10 µM of benzamide (BIIB-513), a selective NHE-1 inhibitor, infused for 10 min just before ischemia or for 10 min immediately on reperfusion. At 2 min of reperfusion, BIIB-513 given before ischemia decreased peak increases in [Na+]i and [Ca2+]i, respectively, from 2.5 and 2.3 times (controls) to 1.6 and 1.3 times preischemia values. At 30 min of reperfusion, BIIB-513 increased systolic-diastolic LV pressure (LVP) from 49 ± 2% (controls) to 80 ± 2% of preischemia values. BIIB-513 reduced ventricular fibrillation by 54% and reduced infarct size from 64 ± 1% to 20 ± 3%. First derivative of the LVP, O2 consumption, and cardiac efficiency were also improved by BIIB-513. Similar results were obtained with BIIB-513 given on reperfusion. These data show that Na+ loading is a marker of reperfusion injury in intact hearts in that inhibiting NHE reduces Na+ and Ca2+ loading during reperfusion while improving function. These results clearly implicate the ionic basis by which inhibiting NHE protects the guinea pig intact heart from ischemia-reperfusion injury.

cardiac reperfusion injury; infarction; contractility; relaxation


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

ACCUMULATION OF MYOPLASMIC Ca2+ during reperfusion after ischemia in intact hearts is associated with depressed myocardial relaxation and contractile function (1, 41, 43, 46). Systolic Ca2+ loading immediately after ischemia is believed to be caused, in large part, by Na+/H+ exchange (NHE) triggered primarily on initial reperfusion by the higher intracellular H+ due to anaerobic metabolism (20, 43). NHE allows Na+ entry in exchange for extrusion of excess protons; in tandem, excess Na+ is thought to be extruded in exchange for Ca2+ entry via slowed or reverse-mode Na+/Ca+ exchange (NCE) (18, 21, 22, 28, 43, 44). Inhibiting NHE may be an important prophylactic measure to improve cardiac function on reperfusion after pathological or induced cardiac ischemia. It is unclear whether NHE activity is low during ischemia and high only at the onset of reperfusion. Our main hypothesis was that inhibiting NHE lowers intracellular Na+ concentration ([Na+]i) and cytosolic Ca2+ concentration ([Ca2+]c) loading on reperfusion and that it reduces infarct size and attenuates depression of myocardial mechanical and metabolic function in isolated guinea pig hearts. To test this, two intracellular fluorescent indicators, sodium benzofuran isophthalate (SBFI) and indo 1, were used to measure [Na+]i and [Ca+]c, respectively, as frequently as every minute before, during, and after 30 min of global ischemia in isolated guinea pig hearts. Our aim was to verify the ionic basis for tandem NHE and NCE activities during ischemia-reperfusion injury in the intact heart. A corollary hypothesis was that the heart is protected equally by NHE inhibition initiated during reperfusion as well as before ischemia. To examine this, a selective NHE isoform 1 inhibitor, benzamide (BIIB-513), was given before ischemia or just before reperfusion.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Langendorff Isolated Heart Preparation and Measurements

The investigation conformed to the Guide for the Care and Use of Laboratory Animals (National Institutes of Health No. 85-23, 1996). Prior approval was obtained from the Medical College of Wisconsin animal studies committee. Our methods have been described in detail previously (1, 8, 39, 41, 46). Several individual control experiments in the present study were used in averaged data of a previous study (46). Ketamine (30 mg) and heparin (1,000 units) were injected intraperitoneally into 52 albino English short-haired guinea pigs (250-300 g). When the animals were unresponsive to noxious stimulation, they were decapitated (~15 min). After thoracotomy, inferior and superior vena cavae were cut, and the aorta was cannulated distal to the aortic valve. Each heart was immediately perfused via the aortic root (at 55 mmHg with a cold, oxygenated Krebs-Ringer solution equilibrated with 97% O2 - 3% CO2; pH 7.39 ± 0.01, PO2 560 ± 10 mmHg) and was then rapidly excised. Krebs-Ringer solution contained (in mM) 137 Na+, 5 K+, 1.2 Mg2+, 2.5 Ca2+, 134 Cl-, 15.5 HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, 1.2 H2PO<UP><SUB>4</SUB><SUP>−</SUP></UP>, 11.5 glucose, 2 pyruvate, 16 mannitol, 0.05 EDTA, and 0.1 probenecid and 5 U/l insulin. Perfusate and bath temperature was maintained at 37.2 ± 0.1°C.

Left ventricular (LV) pressure (LVP) was measured isovolumetrically with a saline-filled latex balloon inserted into the LV through the mitral valve from a cut in the left atrium. Balloon volume was adjusted to maintain a diastolic LVP of 0 mmHg during the initial control period so that any increase in diastolic LVP reflected an increase in LV wall stiffness or diastolic contracture. Pairs of bipolar electrodes were placed in the right atrial appendage and right ventricular free wall to monitor spontaneous heart rate (HR) and atrioventricular conduction time. The occurrence and number of ventricular fibrillations (VF) per heart were recorded during 60 min of reperfusion. If VF did not convert spontaneously to sinus rhythm after 30 s, 100 µg of lidocaine was injected into the aortic cannula. Coronary flow (aortic inflow, CF) was measured at constant temperature and constant perfusion pressure (55 mmHg) by a self-calibrating, in-line, ultrasonic flowmeter (model T106X, Transonic; Ithaca, NY) placed directly into the aortic inflow line.

Coronary arterial and coronary effluent Na+, K+, Ca2+, PO2, pH, and PCO2 were measured off-line with an intermittently self-calibrating gas analyzer system. Coronary sinus effluent was collected after placing a small catheter into the right ventricle through the pulmonary artery after ligating both vena cavae. Coronary sinus venous PO2 tension was also measured continuously on-line with an O2 Clark-type electrode. Percent O2 extraction (%O2E) was calculated as 100 · (PaO2 - PvO2/PaO2); myocardial O2 consumption (MVO2) as CF/g · (PaO2 - PvO2) · 24 µl O2/ml at 760 mmHg; and cardiac efficiency as systolic-diastolic LVP · HR/MVO2, where PaO2 and PvO2 are arterial and venous PO2, respectively.

Measurement of Ca2+ Transients in Intact Hearts

Our methods for measuring [Ca2+] in intact hearts have been previously published (1, 8, 39, 41, 46). Experiments were carried out in a light-shielded Faraday cage. The distal end of a trifurcated Silica fiber-optic cable was placed against the LV epicardial surface through a hole in the bath, and a rubber O ring was placed between the ferrule and the heart to reduce cardiac motion at the contact point of the fiber optic tip. Background autofluorescence (AF) was determined for each heart after initial perfusion and equilibration at 37°C. Hearts were loaded with indo 1-AM at 25 ± 0.6°C for 20-30 min with 165 ml of a recirculated, modified Krebs-Ringer solution containing 6 µM indo 1-AM (Sigma; St. Louis, MO). Indo 1-AM was initially dissolved in 1 ml of dimethyl sulfoxide containing 16% (wt/vol) Pluronic I-127 (Sigma) and diluted to 165 ml with modified Krebs-Ringer solution. Loading was stopped when the fluorescence (F) intensity of the 385-nm signal was increased 7- to 10-fold. Residual indo 1-AM was washed out by perfusing the heart with standard perfusate for at least another 20 min and then each heart was rewarmed to 37.2 ± 0.1°C before the study was initiated. The perfusate contained 100 µM probenecid to retard leakage of indo 1. Loading and washout of indo 1 reduces LVP due to its vehicle and to intracellular Ca2+ buffering by indo 1 per se.

The LV region of the heart was excited via one limb of the fiber-optic cable with light filtered through a 360 ± 16-nm (bandwidth) monochromator. To avoid bleaching of indo 1, the arc lamp shutter was opened only for 2.5-s recordings. F emissions were collected by fibers of the remaining two limbs of the fiber-optic cable with one limb filtered at 385 (390 ± 5) nm and the other at 456 (460 ± 5) nm. Photomultiplier tube output settings for F emissions at 385 and 456 nm (F385 and F456) were set at 525 and 385 mV, respectively, to optimize recordings of physiological concentrations of Ca2+. F385 and F456 transients were recorded using a modified luminescence spectrophotometer (SLM Aminco-Bowman II, Spectronic Instruments; Urbana, IL). At each sampling interval, F385, F456, F385/F456, and LVP were recorded digitally over 7 to 8 cardiac cycles every 8 ms for 2.5 s. Each experiment was composed of 40 to 50 recordings. Data were stored (computer software OS/2 version 4, IBM; Armonk, NY) for background correction and conversion of F data to [Ca2+] off-line (Matlab, The Math Works; Natick, MA, and Excel, Microsoft; Redmond, WA). Using this ratiometric method applied to our model, F385 and F456 both declined over time but remained at least fivefold greater than background after 3 h at 37°C; however, the F385/F456 ratio was unchanged over this time in the absence of ischemia. Our methods for correcting background AF and noncytosolic [Ca2+] have been detailed previously (1, 8, 39, 41, 46).

Measurement of Intracellular Free Na+ in Intact Hearts

Loading of SBFI fluorescent indicator. The Na+ - binding fluorescent dye SBFI has been used to follow changes in myocyte [Na+] in isolated hearts (27, 45-47). This ratiometric technique for Na+ is similar to that for indo 1 except that the F ratio is obtained by alternating the excitation wavelength from 340 to 380 nm and collecting and dividing the emitted light at 530 nm (19, 24, 27, 45-47). Our method to calibrate [Na+] from SBFI in isolated hearts has been published previously (46). The same trifurcated fiberoptic system was used in the same manner as for indo 1, but Na+ (series A) and Ca2+ (series B) measurements were performed in separate hearts because a change in filters and software was necessary. Each of 17 hearts was loaded for ~30 min with 6 µM SBFI at 25°C until the emitted signal after loading was between 6 to 10 times the baseline signal. SBFI fluorescence gradually declined over time, but the F340/F380 ratio remained relatively unchanged. To assure that the F ratio (R) was being calculated only from the component of the heart's F produced by SBFI, in 11 additional hearts only the SBFI vehicle was loaded and washed out before initiating the same ischemia-reperfusion protocol. Corrections were made for the average basal AF, measured before SBFI loading, and for the change in AF that occurs during ischemia and reperfusion at each measurement point (time = t). R was calculated from the formula
R<IT>=</IT>(F<SUP><IT>t</IT></SUP><SUB>340</SUB><IT>−</IT>AF<SUP><IT>t</IT></SUP><SUB>340</SUB>)<IT>&cjs0823;  </IT>(F<SUP><IT>t</IT></SUP><SUB>380</SUB><IT>−</IT>AF<SUP><IT>t</IT></SUP><SUB>380</SUB>)
F385 and F456 indo 1 Ca2+ transients, or F340 and F380 SBFI Na+ signals, LVP, and first derivative of LVP (LV dP/dt) were displayed simultaneously on a computer screen and stored digitally using proprietary software on an IBM OS/2 system. After correction for tissue AF over time, with or without ischemia and reperfusion, and quenching of the cytosolic Ca2+ compartment to quantify the noncytosolic Ca2+ compartment, the signals were calibrated to nanomolars [Ca2+] or millimolars [Na+] using algorithms developed by our group. LVP and raw metabolic data were recorded (PowerLab, ADInstruments; Castle Hills, Australia) and, with the Na+ or Ca2+ transient signals, were later analyzed using our programs written on Matlab and Microsoft Excel software. At the end of 70 min of reperfusion, whole hearts were removed and stained by incubating them at 37°C for 20-30 min in 1% 2,3,5-triphenyltetrazolium chloride in 0.1 M phosphate buffer adjusted to pH 7.4. Hearts were then stored in 10% formaldehyde for later analysis. Infarcted tissue (unstained) was dissected carefully from noninfarcted tissue (red stained) and weighed so that percent infarct size was expressed as the weight of infarcted tissue divided by total tissue weight times 100.

Protocol

BIIB-513, benzamide {N-(aminominomethyl)-4[4-(2-furanylcarbonyl)-1-piperazinyl]-3-(methylsulfonyl), methanesulfonate} is 4 × 10-3 more selective for NHE-1 than NHE-3; binding to the Na+ site 1 is 0% and to the Na+ site 2 is 18.7% at 10 µM; BIIB-513 does not bind to Ca2+ channels (9). BIIB-513 is eight times more potent than cariporide (HOE-642) and the BIIB-513 IC50 is 0.25 µM; the half-life of BIIB-513 is ~60 min at a plasma concentration of 10 µM (9).

There were five primary study groups, each with identical protocols in which mechanical and metabolic function was measured. Each heart was subjected to 30 min of no-flow global ischemia at 37°C and 70 min of reperfusion. [Na+]i (series A) was measured in two groups, control (n = 8) and pre-BIIB-513 (n = 9), where 10 µM of BIIB-513 was infused for 10 min up to the cessation of flow (onset of ischemia). Phasic [Ca2+]c (series B) was measured in two other groups, control (n = 12) and pre-BIIB-513 (n = 13). BIIB-513 was trapped in the heart during ischemia until the unbound BIIB-513 was washed out on drug-free reperfusion. In a final group (series C), post-BIIB-513 (n = 10), 10 µM BIIB-513 was infused immediately on reperfusion for 10 min and phasic [Ca2+]c was measured. To ensure that BIIB-513 was present immediately on reperfusion, the drug was infused slowly (1 ml over 15 s) from the aortic cannula into the heart at 5 mmHg, 60 s before the onset of reperfusion at 55 mmHg.

Each of these groups was backed by a number of calibration studies, time controls, and AF controls as detailed above. Initial control measurements (time 0) were obtained 30 min after fluorescent dye or vehicle washout. Recordings were obtained every 1-10 min at 2.1 mM ionized extracellular [Ca2+]. In series B and C hearts (and nonischemic time controls), 100 µM MnCl2 was infused at the end of each experiment to quench cytosolic Ca2+ transients. Quenching does not alter phasic LVP. Nonischemic time control data are not given in the text.

All data were expressed as means ± SE. Within-group data for a given variable were compared with a preischemia control period (at 40 min) by Duncan's comparison of means tests whenever univariate ANOVA for repeated measures tests were significant (Super ANOVA 1.11 software for Macintosh; Abacus Concepts, Berkeley, CA). Between-group data for BIIB-513 compared with nontreated controls were compared at each time point by two-way ANOVA and Student-Newman-Keuls means comparison tests. Significance of incidence of VF was analyzed by Fisher's exact test. Series A results (Na experiments) were not directly compared with series B or C results (Ca experiments) but had identical protocols except for the fluorescent dye utilized. Differences among means were considered statistically significant when P <=  0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

BIIB-513 had no apparent effects on function or metabolism when given before ischemia. HR before ischemia averaged 247 ± 4 beats/min and was not different between the two Na groups (control and pre-BIIB-513), the two Ca groups (control and pre-BIIB-513), and the post-BIIB-513 Ca group at 30 and 70 min of reperfusion (112 and 152 min, respectively). On reperfusion after ischemia, HR averaged 242 ± 4 beats/min and there were no differences among the groups. Atrioventricular conduction time averaged 67 ± 2 ms before as well as after 30 and 70 min of ischemia with no significant differences among the five groups. The incidence (%) and median number of VF per heart in the control Na (series A) and control Ca (series B), respectively, were 100% and 5 and 100% and 4. In BIIB-513-treated groups, %VF incidence and median VFs/heart were 88% and 2 for pre-BIIB-513 Na (series A), 71% and 2 for pre-BIIB-513 Ca (series B), and 44% and 1 for post-BIIB-513 Ca (series C). The median VFs/heart was lower in each BIIB-513-treated group compared with control (P < 0.05). Infarct size weight as a percentage of total heart weight was control 64 ± 1% (series A and B), pre-BIIB-513 20 ± 3% (series A), pre-BIIB-513 21 ± 3% (series B), and post-BIIB-513 23 ± 2% (series C) (P < 0.01 each BIIB-513 group vs. control group).

Figure 1 displays LVP and Ca2+ concentration ([Ca2+]) tracings (converted from raw Ca2+ transients) obtained before and after ischemia in the presence or absence of BIIB-513 given before ischemia. BIIB-513 treatment reduced systolic [Ca2+] and diastolic LVP and enhanced systolic LVP on reperfusion after ischemia. Figure 2 plots temporal changes in [Na+]i and systolic and diastolic [Ca2+] before, during, and after 30 min of global ischemia. Figure 2A shows that [Na+] rose during ischemia in the control group but not after preischemia treatment with 10 µM BIIB-513. The peak rise in [Na+] during reperfusion after control and after pre-BIIB-513, respectively, was 2.5 and 1.6 times the preischemia values, which is 40% smaller after pre-BIIB-513. [Na+] decreased steadily during reperfusion in both groups, but returned to preischemia levels in the pre-BIIB-513 group. Figure 2B shows that [Ca2+] transients remained during the first 10 min of ischemia, whereas diastolic [Ca2+] increased during ischemia in both groups. The peak rise in systolic [Ca2+] during reperfusion after control and after pre-BIIB-513, respectively, was 2.3 and 1.3 times the preischemia values, which is 44% less after BIIB-513. Systolic and diastolic [Ca2+] decreased steadily during reperfusion in both groups, but systolic [Ca2+] returned to preischemia levels more quickly and completely in the pre-BIIB-513 group; diastolic [Ca2+] returned to preischemia values during reperfusion. MnCl2, given to quench cytosolic compartment F, obliterated phasic [Ca2+] and decreased apparent [Ca2+].


View larger version (32K):
[in this window]
[in a new window]
  		Fig. 1.   Tracings of individual calibrated cytosolic Ca2+ concentration ([Ca2+]) (solid line) and left ventricular (LV) pressure transients (dotted line) before ischemia (ISCH), during ISCH, and at 2 and 30 min of reperfusion (RP), with prior benzamide (BIIB-513) and without (CON) treatment from two hearts. [Ca2+] is corrected for autofluorescence background and noncytosolic [Ca2+]. CON and BIIB-513 tracings before and during 2-min ISCH are not different. Notice that [Ca2+] transients are reduced but continued during the first several minutes of ISCH. On reperfusion at 2- and 30-min pre-BIIB-513 treatment improved LV pressure and reduced [Ca2+] loading.



View larger version (24K):
[in this window]
[in a new window]
  		Fig. 2.   Intracellular Na2+ concentration ([Na+]) (A) and systolic and diastolic [Ca2+] (B) before, during, and after 30 min of global ISCH in the absence (CON) and presence of 10 µM BIIB-513 infused 10 min before ISCH and washed out on reperfusion. A: [Na+] rose during ISCH in CON but not after BIIB-513, peak [Na+] on reperfusion rose less after BIIB-513 than after CON, and [Na+] returned to pre-ISCH levels only after BIIB-513. B: peak systolic [Ca2+] rose less after BIIB-513 than after CON; systolic and diastolic [Ca2+] decreased during reperfusion, but systolic [Ca2+] returned to pre-ISCH levels only after BIIB-513; and diastolic [Ca2+] returned to pre-ISCH values during reperfusion after CON and BIIB-513. MnCl2 was used to quench cytosolic [Ca2+]. Not all statistical notations are displayed in the figures. See text for details.

Figure 3 shows that contracture (diastolic LVP) occurred in each group during ischemia. On reperfusion, systolic-diastolic LVP returned to 81 ± 4% in the Na series (A) and 80 ± 2% in the Ca series (B) of preischemia values after pre-BIIB-513 compared with 47 ± 2% (A) and 52 ± 2% (B) in control groups. In data not displayed, the time to onset of diastolic contracture was 25 ± 1 min in controls (series A and B) and 28 ± 1 min (P < 0.05 vs. control) after pre-BIIB-513 (series A and B). Maximal contracture pressure at the end of 30 min of ischemia was 11 ± 4 mmHg in controls and 3 ± 1 mmHg after pre-BIIB-513 (P < 0.05 vs. control). Figure 4 shows that during reperfusion peak LV dP/dtmax returned to 71 ± 3% (A) and 89 ± 2% (B) of preischemia values after pre-BIIB-513 compared with 34 ± 3% (A) and 39 ± 2% (B) in control. Peak LV dP/dtmin returned to 67 ± 2% (A) and 81 ± 2% (B) of preischemia values after pre-BIIB-513 treatment compared with 38 ± 2% (A) and 50 ± 2% (B) in control.


View larger version (27K):
[in this window]
[in a new window]
  		Fig. 3.   Systolic and diastolic LV pressure before, during, and after ISCH in two Na+ groups (A) and two Ca2+ groups (B). Diastolic LV pressure (contracture) occurred during ISCH. During reperfusion, systolic LV pressure returned to pre-ISCH levels only after BIIB-513 (A and B); diastolic LV pressure decreased during reperfusion but remained elevated (A and B). Details in text.



View larger version (26K):
[in this window]
[in a new window]
  		Fig. 4.   Peak positive and negative first derivative of LV pressure (LV dP/dt) before, during, and after ISCH in two Na+ groups (A) and two Ca2+ groups (B). During reperfusion, peak LV dP/dtmax and peak LV dP/dtmin improved toward pre-ISCH values more after BIIB-513 (A and B).

Figure 5 shows that cardiac efficiency (HR · systolic-diastolic LVP/MVO2) returned to 100 ± 6% (A) and 100 ± 5% (B) of preischemia values (pre-BIIB-513) compared with 52 ± 4% (A) and 60 ± 4% (B) (control). In the post-BIIB-513 group, cardiac efficiency returned to 90 ± 5% of the preischemia value (data not displayed). CF (Fig. 5) remained decreased in each group during reperfusion but was slightly higher in pre-BIIB-513 groups. In the post-BIIB-513 group (data not displayed) CF was 8.5 ± 0.3 before ischemia and 6.6 ± 0.4 ml · g-1 · min-1 at 60 min of reperfusion.


View larger version (29K):
[in this window]
[in a new window]
  		Fig. 5.   Cardiac efficiency (eff) and coronary flow before, during, and after ISCH in two Na+ groups (A) and two Ca2+ groups (B). Cardiac efficiency (HR · systolic-diastolic LV pressure/MVO2, in units of beats · mmHg · 0.1 µl O2 · g-1 · min-1) returned to pre-ISCH values during reperfusion only after BIIB-513 (A and B). Coronary flow remained decreased in each group during reperfusion.

Figure 6 shows changes in systolic and diastolic [Ca2+] and LVP when BIIB-513 was given immediately on reperfusion (post-BIIB-513, series C) compared with control (series B). Figure 6A shows that [Ca2+] transients remained during the first 10 min of ischemia while diastolic [Ca2+] increased during ischemia in both groups. The peak rise in systolic [Ca2+] during reperfusion in control and post-BIIB-513 hearts, respectively, was 2.3 and 1.6 times the preischemia values, which was 32% less after post-BIIB-513 treatment. Systolic and diastolic [Ca2+] decreased steadily during reperfusion in both groups, but systolic [Ca2+] returned to preischemia levels more quickly and completely in the post-BIIB-513 group; diastolic [Ca2+] returned to preischemia values during reperfusion. Figure 6B shows that contracture (diastolic LVP) similarly occurred in this group during ischemia. At 30 min of reperfusion, systolic-diastolic LVP returned to 90 ± 3% of preischemia values after post-BIIB-513 treatment compared with 52 ± 2% (Fig. 6B) in control.


View larger version (29K):
[in this window]
[in a new window]
  		Fig. 6.   Systolic and diastolic [Ca2+] (A) and LV pressure (B) before, during, and after 30 min of global ISCH in the absence (CON) and presence of 10 µM BIIB-513 infused for 10 min immediately on reperfusion (post-BIIB-513). CON is the same control group shown in Figs. 2B and 3B. A: peak systolic [Ca2+] rose less after post-BIIB-513 treatment than after CON; systolic and diastolic [Ca2+] decreased during reperfusion, but systolic [Ca2+] returned to pre-ISCH levels only after BIIB-513; and diastolic [Ca2+] returned to pre-ISCH values during reperfusion in both CON and BIIB-513 groups. B: during reperfusion, systolic LV pressure returned to pre-ISCH levels only after BIIB-513; diastolic LV pressure decreased during reperfusion but remained elevated.

In data not displayed, the time to onset of diastolic contracture in post-BIIB-513-treated hearts was 26 ± 1 compared with 25 ± 1 min in controls (series A and B) hearts (NS, P > 0.1 vs. control). Maximal contracture pressure at the end of 30 min of ischemia was 11 ± 4 mmHg in control and 8 ± 1 mmHg after pre-BIIB-513 (P > 0.05 vs. control). MVO2 (µl O2 · g-1 · min-1) was not different (P > 0.05) between groups before ischemia in control, 97 ± 8 (series A) and 90 ± 6 (series B); pre-BIIB-513, 98 ± 5 (series A) and 86 ± 5 (series B); and post-BIIB-513, 101 ± 9 (series C) groups. After 30 min of reperfusion, MVO2 was lower in control hearts [65 ± 8 (series A) and 59 ± 5 (series B) (P < 0.05)] than in pre-BIIB-513-treated hearts [85 ± 5 (series A) and 86 ± 6 (series B)], or post-BIIB-513-treated hearts [88 ± 5 (series C)] (P < 0.05).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

This study performed in guinea pig intact hearts shows clearly that inhibiting NHE before ischemia reduces Na+ loading during ischemia and reperfusion and concomitantly reduces systolic Ca2+ loading on reperfusion while effectively decreasing infarct size, reducing dysrhythmias, and enhancing postischemic mechanical and metabolic function. Moreover, inhibiting NHE just before and for 10 min after reperfusion also similarly reduces Ca2+ loading and improves function. We (46) reported recently that Na+ accumulates moderately during ischemia and peaks during early reperfusion as do systolic, diastolic, and mitochondrial [Ca2+]. In the present study, we have established the link between Na+ accumulation during ischemia and Na+ and phasic Ca2+ loading during reperfusion in intact hearts as evidenced by reduced postischemic accumulation of Na+ and Ca2+ after inhibiting NHE. Our results indicate that cytosolic Na+ and Ca2+ loading are comarkers of ischemia and reperfusion injury and clearly implicate NHE and slowed or reverse-mode NCE as being largely responsible for mediating the large increase in [Ca2+] due to an increase in [Na+] on reperfusion after cardiac injury. In addition, the results imply that NHE activity is probably highest during initial reperfusion because inhibiting NHE just before reperfusion was as effective as inhibiting NHE before ischemia.

Changes in Na+ and Ca2+ With Cardiac Ischemia and Reperfusion

Increased intracellular H+ concentration ([H+]i) during ischemia-reperfusion is believed to activate NHE (7, 13, 20, 23, 27, 42, 43, 47). The higher NHE activity on reperfusion (23, 27, 47) causes enhanced Na+ influx and secondarily promotes Ca2+ influx via slowed forward-mode (Ca2+ exit) or enhanced reverse-mode (Ca2+ entry) operation of NCE (18, 21, 22, 38, 43, 44). Most evidence in favor of the above cascade of ionic events has been provided by studies employing variably selective drugs that block NHE to protect the heart or isolated myocytes from dysfunction and damage after true or simulated ischemia-reperfusion (4, 9-11, 15, 25-27, 32, 34-36, 40, 47, 49). In only a few of these studies (27, 42, 47) were the intracellular concentrations of H+, Na+, or Ca2+ actually measured.

Methods to measures of changes in [Na+]i during ischemia-reperfusion show considerable variation. Total intracellular Na from myocardial biopsies was found to increase by 40% after 10 min of ischemia (43); One Na NMR study (29) reported increases in Na of up to +230%, whereas another study reported only a 20% increase after 10 min of ischemia (14). Methodological factors may explain the divergent results (12). SBFI is a reliable and proven Na+ indicator (24) in single myocardial cells (6, 19), and, more recently, in intact isolated rat (27, 45, 47) and guinea pig (46) hearts. Park et al. (27) used SBFI in rat isolated hearts and showed that [Na+]i was unchanged during 10 min of ischemia but was increased by 6 to 8 mM above baseline on initial reperfusion; [H+]i increased by -1 ± 0.1 pH units during ischemia but rapidly returned to control on reperfusion. Our results in guinea pig isolated hearts using SBFI are qualitatively similar, but we additionally observed a moderate (30%) increase in [Na+] during ischemia and a larger increase in [Na+] during reperfusion. The free Ca2+ indicator indo 1 is a very suitable probe to ratiometrically measure phasic [Ca2+] in intact hearts (2, 3). We have observed higher [Ca2+] during hypothermia (39), and on reperfusion after long hypothermic (41) and short normothermic ischemia (1, 46).

NHE and NCE Activities With Cardiac Ischemia and Reperfusion

Myocardial ischemia results in intracellular acidosis because of a lack of oxygen atoms to accept electrons during oxidation of NADH to NAD+. The failure of cellular respiration and oxidative phosphorylation of ADP to ATP leads to accumulation of NADH and lactate by anaerobic metabolism. Na+ pump activity is reduced or absent so this reduces the Na+ (and K+) gradient causing cell depolarization (14). Myoplasmic and mitochondrial Ca2+ overload (46) during early reperfusion is believed to contribute, along with release of reactive O2 species, to depressed myocardial function as well as to cause necrosis of irreversibly injured (infarcted) cells (43). Myoplasmic Ca2+ loading after ischemia may impair myocardial relaxation and contractile function by a direct action on the myofilament apparatus or by effects mediated via mitochondria Ca2+ overloading (46).

The study by Park et al. (27) together with the present study strongly implicate the ionic basis for both NHE and NCE during ischemia-reperfusion in intact hearts. In the former study, the nonspecific NHE and NCE inhibitor methylisobutyl amiloride (MIA) was given for 10 min before ischemia and shown to greatly decrease the rise in [Na+] and the rate of pHi increase on reperfusion in isolated rat hearts. NHE can be activated by other means. Park et al. (27) also found that a lactate-induced decrease in pHi increased [Na+]i, an effect blocked by MIA. In the present study pretreatment with a more selective NHE inhibitor, BIIB-513, also greatly decreased the rise in [Na+]i on reperfusion; in addition, the marked increase in systolic [Ca2+] during reperfusion was reduced by BIIB-513. The present study and that of Park et al. (27), and a more recent study by the same group (47) using HOE-642, point to concomitant and immediate activation of NHE and reverse-mode NCE on initial reperfusion. The present study demonstrates that Na+ and Ca2+ accumulate during ischemia-reperfusion in guinea pig hearts and that inhibiting NHE activity during ischemia and reperfusion blocks this accumulation.

Timing of Inhibition of NHE During Cardiac Ischemia and Reperfusion

It is well understood from ischemia and hypoxia studies that the proper ionic balance of Ca2+, Na+, and H+ requires maintenance of Na+ and Ca2+ pumps and functional NCE (14, 18, 21, 28, 34, 38). What is not well understood is the relative activity of NHE before, during, and after ischemia. Inhibiting NHE by itself had no effects on [Na+], [Ca2+], and cardiac function before ischemia, so it is likely that NHE is either relatively inactive during normal aerobic perfusion or its activity to extrude H+ and take up Na+ is balanced by other regulators of [H+]. NHE activity is also likely quite low during ischemia compared with reperfusion because inhibiting NHE does not appreciably alter the fall in pHi or alter [Na+]i during cardiac ischemia (27). Intracellular acidosis is thought to protect vital contractile processes during ischemia and early reperfusion. Metabolic inhibition of isolated myocytes can increase [Na+] both by NHE and by suppressing Na+ extrusion via the Na+ pump (34). However, the mechanism of NHE inhibition during ischemia is not evident. Moreover, the study by Park et al. (27) does not support the original hypothesis by Lazdunski et al. (20): that the concomitantly lowered pHo during ischemia inhibits NHE. It has been suggested that inhibition of NHE activity during ischemia is due to 1) a rapidly inactivating exchanger during ischemia due to the low pHi, 2) extrusion of protons generated by ischemia by the Na+-dependent Na+/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and/or lactate/H+ symporter, or 3) a coupling of influx of Cl- and efflux of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> through the Na+ independent HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/Cl- exchanger (30, 37).

In contrast to during ischemia, NHE is probably very rapidly activated on initial reperfusion (23, 27, 30, 47). The increase in Na+ entry via NHE on reperfusion probably initiates excess Ca2+ entry via NCE (22, 38, 43, 44, 48) either by reducing Ca2+ efflux or by increasing Ca2+ influx during the plateau phase of the action potential. Excess Ca2+ entry via NCE coupled to NHE also likely triggers excess ryanodine-sensitive Ca2+-induced Ca2+ release from the sarcoplasmic reticulum (18, 22), causing a dissociation in the normal relationship between intracellular Na+ and Ca2+, but the importance of this has been questioned (38).

Much of the controversy over NHE activities during ischemia versus reperfusion is derived from studies using NHE inhibitors. Many of these studies (10, 11, 19, 23, 27, 30, 47) strongly support cardioprotection by NHE inhibitors given only on reperfusion, but others (4, 15, 26) do not. Our ex vivo studies agree with the contention that NHE is more active on reperfusion than during ischemia, because inhibiting NHE after ischemia reduced Ca2+ loading and improved function as well as did inhibiting NHE during ischemia and presumably at initial reperfusion. Differences in species, heart models, blood versus artificial perfusate, and precise timing of NHE administration may account for the diversity of findings. We speculate that Na+ accumulated during ischemia results from reduced Na+ pump activity but concede that NHE during ischemia could have an effect to reduce Na+ accumulation. The much greater Na+ accumulated on reperfusion was immediately attenuated by inhibiting NHE activity either before or after ischemia; this effect was coupled to reduced [Ca2+] suggesting reduced forward- mode NCE.

Functional Effects of NHE Inhibition During Ischemia and Reperfusion

Extracellular-imposed intracellular acidosis, retarded correction of pHo (16, 17, 20, 31), and pharmacological inhibition of NHE activity on reperfusion have each proven beneficial in reducing cardiac infarct size or improving myocardial reperfusion function. Various NHE inhibitors have exhibited protective effects against hypoxic (19), hypothermic (25, 40), and normothermic ischemia-reperfusion injury (9-11, 15, 23, 26, 27, 32, 35, 36, 42, 47, 49). But it is most important that the inhibitor selected to block NHE is specific and sensitive for this exchanger with minimal effects on Na+ and Ca2+ channels. In this regard, BIIB-513 (9) is a markedly more selective and potent than earlier drugs. Methylsulfonyl-piperidinobenzoyl guanidine (HOE-694), another more specific inhibitor of NHE than amiloride, a mixed function drug, protected isolated cardiomyocytes against hypercontracture on reoxygenation after hypoxia (19), especially when Na+ pump activity was critically reduced. HOE-694 protected rat hearts against reperfusion dysrhythmias (49). HOE-642 protected hearts against reperfusion dysfunction after ischemia (35, 42, 47) while it reduced Ca2+ overload and prolonged intracellular acidosis (42). The NHE inhibitor EMD-85131 (HCl salt of eniporide) given before 60 min of ischemia or before reperfusion of the left anterior descending artery reduced infarct size as a percentage of area at risk after 3 h of reperfusion (11). We observed that the NHE inhibitor eniporide, which has a profile like BIIB-513, reduced infarct size and improved function on reperfusion when given before as well as after 6 h of cardioplegic storage at 3°C (40).

In summary, Na+ accumulation during ischemia, and particularly on reperfusion, likely accounts for the initial increase in cytosolic and mitochondrial Ca2+ excess (46) and cardiac dysfunction. Our results support the hypothesis that Na+ overload is an essential cause of Ca2+ overload and cardiac dysfunction during early reperfusion because preventing Na+ loading is temporally associated with reduced cytoplasmic Ca2+ loading and reduced reperfusion dysfunction and damage. Therapeutic interventions that secondarily reduce Na+ and Ca2+ loading, e.g., NHE inhibitors, should be quite beneficial to attenuate reperfusion injury. Intervention may be as successful if applied before ischemia or during the first moment of reperfusion.

Possible Limitations

We (1) have shown that it is unlikely that the rapid increase in [Na+] and diastolic [Ca2+] on initial reperfusion comes from nonviable cells releasing their contents into the extracellular fluid during ischemia. In the guinea pig heart (1.5 g) washout (flow 10 ml/min) is likely complete within 30 s of reperfusion and [Na+] and [Ca2+] remained elevated up to 30 min in controls. Moreover, subepicardial LV tissue at the location of the fiberoptic probe was not infarcted, so the recordings are most likely from living cells. Measurements of [Ca2+]i and [Na+]i apply only to cells surviving reperfusion because nonviable cells are permeable to large molecules. Finally, [Ca2+] was phasic even on initial reperfusion and so must represent [Ca2+]c.

[H+]i was not measured, so the effect of NHE inhibition on altering pHi could not be assessed. It was necessary to conduct the Na+ and Ca2+ studies in different series of hearts, albeit with identical protocols, so changes in [Na+] and [Ca2+] were not measured simultaneously. The contribution of oxygen reactive species to reperfusion contractile dysfunction was not assessed. Factors such as leukocyte migration and activation of complement are also involved in reperfusion injury. The important role of the endothelium and vasculature in mediating ischemia-reperfusion injury must also be acknowledged. We have published additional limitations using these methodologies (1, 8, 39, 41, 46).


    ACKNOWLEDGEMENTS

The authors thank Samhita Rhodes for assistance with programming software and Anita Tredeau and Steve Contney for administrative assistance.


    FOOTNOTES

The research was supported in part by National Institutes of Health Grants HLBI-58691 and GM-8204-06, American Heart Association Grant 0020503Z, and Research Service, US Department of Veterans Affairs. BIIB-513 was a gift from Dr. Juergen Daemmgen, Boerhinger Ingelheim, Pharma KG, Biberach, Germany.

Portions of this work have appeared in abstract form: Anesth Analg 88: SCA49, 1999; Anesthesiology 91: A711, 1999; and FASEB J 14: A422, 2000.

Address for reprint requests and other correspondence: D. F. Stowe, M4280, 8701 Watertown Plank Road, The Medical College of Wisconsin, Milwaukee Regional Medical Center, Milwaukee, WI 53226 (E-mail: dfstowe{at}mcw.edu).

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.

Received 23 January 2001; accepted in final form 13 August 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   An, J, Varadarajan SG, Novalija E, and Stowe DF. Ischemic and anesthetic preconditioning reduces cytosolic [Ca2+] and improves Ca2+ responses in intact hearts. Am J Physiol Heart Circ Physiol 281: H1508-H1523, 2001[Abstract/Free Full Text].

2.   Brandes, R, Figueredo VM, Camacho SA, Baker AJ, and Weiner MW. Quantitation of cytosolic [Ca2+] in whole perfused rat hearts using indo 1 fluorometry. Biophys J 65: 1973-1982, 1993[Abstract/Free Full Text].

3.   Brandes, R, Figueredo VM, Camacho SA, Baker AJ, and Weiner MW. Investigation of factors affecting fluorometric quantitation of cytosolic [Ca2+] in perfused hearts. Biophys J 65: 1983-1993, 1993[Abstract/Free Full Text].

4.   Bugge, E, and Ytrehus K. Inhibition of sodium-hydrogen exchange reduces infarct size in the isolated rat heart: a protective additive to ischaemic preconditioning. Cardiovasc Res 29: 269-274, 1995[ISI][Medline].

5.   Chapman, RA. Sodium/calcium exchange and intracellular calcium buffering in ferret myocardium: an ion-selective microelectrode study. J Physiol (Lond) 373: 163-179, 1986[Abstract/Free Full Text].

6.   Donoso, P, Mill JG, O'Neill SC, and Eisner DA. Fluorescence measurements of cytoplasmic and mitochondrial sodium concentration in rat ventricular myocytes. J Physiol (Lond) 448: 493-509, 1992[Abstract/Free Full Text].

7.   Fliegel, L, and Frohlich O. The Na+/H+ exchanger: an update on structure, regulation, and cardiac physiology. Biochem J 296: 273-285, 1993.

8.   Fujita, S, Smart SC, and Stowe DF. Enhanced myofilament Ca2+ sensitivity by delta-2 opioid agonist deltorphin in intact guinea pig hearts. J Mol Cell Cardiol 32: 1647-1659, 2000[ISI][Medline].

9.   Gumina, RJ, Buerger E, Eickmeier C, Moore J, Daemmgen J, and Gross GJ. Inhibition of the Na+/H+ exchanger confers greater cardioprotection against 90 min of myocardial ischemia than ischemic preconditioning in dogs. Circulation 100: 2519-2526, 1999[Abstract/Free Full Text].

10.   Gumina, RJ, Daemmgen J, and Gross GJ. Inhibition of the Na+/H+ exchanger attenuates phase 1b ischemic arrhythmias and reperfusion-induced ventricular fibrillation. Eur J Pharmacol 396: 119-124, 2000[ISI][Medline].

11.   Gumina, RJ, Mizumura T, Beier N, Schelling P, Schultz JJ, and Gross GJ. A new sodium/hydrogen exchange inhibitor, EMD 85131, limits infarct size in dogs when administered before or after coronary artery occlusion. J Pharmacol Exp Ther 286: 175-183, 1998[Abstract/Free Full Text].

12.   Ingwall, JS. How high does intracellular sodium rise during acute myocardial ischemia? A view from NMR spectroscopy. Cardiovasc Res 29: 279, 1995[ISI][Medline].

13.   Karmazyn, M. Role of sodium-hydrogen exchange in mediating myocardial ischemia and reperfusion injury. In: Mechanism and Therapeutic Implications. The Na/H Exchanger, edited by Fliegel L.. Heidelberg, Germany: Springer, 1996, p. 189-215.

14.   Kleber, AG. Resting membrane potential, extracellular potassium activity, and intracellular sodium activity during acute global ischemia in isolated perfused guinea pig hearts. Circ Res 52: 442-450, 1983[Abstract/Free Full Text].

15.   Klein, HH, Pich S, Bohle RM, Wollenweber J, and Nebendahl K. Myocardial protection by Na+-H+ exchange inhibition in ischemic, reperfused porcine hearts. Circulation 92: 912-917, 1995[Abstract/Free Full Text].

16.   Kitakaze, M, Takashima S, Funaya H, Minamino T, Node K, Shinozaki Y, Mori H, and Hori M. Temporary acidosis during reperfusion limits myocardial infarct size in dogs. Am J Physiol Heart Circ Physiol 272: H2071-H2078, 1997[Abstract/Free Full Text].

17.   Kitakaze, M, Weisfeldt ML, and Marban E. Acidosis during early reperfusion prevents myocardial stunning in perfused hearts. J Clin Invest 82: 920-927, 1988.

18.   Kohomoto, O, Levi AJ, and Bridge JH. Relation between reverse sodium-calcium exchange and sarcoplasmic reticulum calcium release in guinea pig ventricular cells. Circ Res 74: 500-554, 1994.

19.   Ladilov, YV, Siegmund B, and Piper HM. Protection of reoxygenated cardiomyocytes against hypercontracture by inhibition of Na/H exchange. Am J Physiol Heart Circ Physiol 268: H1531-H1539, 1995[Abstract/Free Full Text].

20.   Lazdunski, M, Frelin C, and Vigne P. The sodium/hydrogen exchange system in cardiac cells: its biochemical and pharmacological properties and its role in regulating internal concentrations of sodium and internal pH. J Mol Cell Cardiol 17: 1029-1042, 1985[ISI][Medline].

21.   Lee, JA, and Allen DG. Mechanisms of acute ischemic contractile failure of the heart: role of intracellular calcium. J Clin Invest 88: 361-367, 1991.

22.   Litwin, SE, and Bridge JH. Enhanced Na+/Ca2+ exchange in the infarcted heart. Implications for excitation-contraction coupling. Circ Res 81: 1083-1093, 1997[Abstract/Free Full Text].

23.   Maddaford, TG, and Pierce GN. Myocardial dysfunction is associated with activation of Na+/H+ exchange immediately during reperfusion. Am J Physiol Heart Circ Physiol 273: H2232-H2239, 1997[Abstract/Free Full Text].

24.   Minta, A, and Tsien RY. Fluorescent indicators for cytosolic sodium. J Biol Chem 264: 19449-19457, 1989[Abstract/Free Full Text].

25.   Myers, ML, and Karmazyn M. Improved cardiac function after prolonged hypothermic ischemia with the Na+/H+ exchange inhibitor HOE 694. Ann Thorac Surg 61: 1400-1406, 1996[Abstract/Free Full Text].

26.   Myers, ML, Mathur S, Li GH, and Karmazyn M. Sodium-hydrogen exchange inhibitors improve postischaemic recovery of function in the perfused rabbit heart. Cardiovasc Res 29: 209-214, 1995[ISI][Medline].

27.   Park, CO, Xiao XH, and Allen DG. Changes in intracellular Na+ and pH in rat heart during ischemia: role of Na+/H+ exchanger. Am J Physiol Heart Circ Physiol 276: H1581-H1590, 1999[Abstract/Free Full Text].

28.   Pierce, GN, and Czubryt MP. The contribution of ionic imbalance to ischemia/reperfusion injury. J Mol Cell Cardiol 27: 53-63, 1995[ISI][Medline].

29.   Pike, MM, Kitakaze M, and Marban E. 23Na NMR measurements of intracellular sodium in intact perfused ferret hearts during ischemia and reperfusion. Am J Physiol Heart Circ Physiol 259: H1767-H1773, 1990[Abstract/Free Full Text].

30.   Piper, HM, Balser C, Ladilov YV, Schaefer M, Siegmund B, Ruiz-Meana M, and Garcia-Dorado D. The role of Na+/H+ exchange in ischemia-reperfusion. Basic Res Cardiol 91: 191-202, 1996[ISI][Medline].

31.   Prigent, KL, Lagadic-Gossmann DL, and Feuvray D. Modulation by pHo and intracellular Ca2+ of Na+-H+ exchange in diabetic rat isolated ventricular myocytes. Circ Res 80: 253-260, 1997[Abstract/Free Full Text].

32.   Sack Mohri, M S, Schwarz ER, Arras M, Schaper J, Ballagi-Pordany G, Scholz W, Lang HJ, Scholkens BA, and Schaper W. Effects of a new Na/H antiporter inhibitor on postischemic reperfusion in pig heart. J Cardiovasc Pharmacol 23: 72-78, 1994[ISI][Medline].

33.   Sage, SO, Rink TJ, and Mahaut-Smith MP. Resting and ADP-evoked changes in cytoplasmic free sodium concentration in human platelets loaded with the indicator SBFI. J Physiol (Lond) 441: 559-573, 1991[Abstract/Free Full Text].

34.   Satoh, H, Hayashi H, Katoh H, Terada H, and Kobayashi A. Na+/H+ and Na+/Ca2+ exchange in regulation of [Na+]i and [Ca2+]i during metabolic inhibition. Am J Physiol Heart Circ Physiol 268: H1239-H1248, 1995[Abstract/Free Full Text].

35.   Scholz, W, Albus U, Counillon L, Goegelein H, Lang HJ, Linz W, Weichert A, and Scholkens BA. Protective effects of HOE 642, a selective sodium-hydrogen exchange subtype 1 inhibitor, on cardiac ischaemia and reperfusion. Cardiovasc Res 29: 260-268, 1995[ISI][Medline].

36.   Shimada, Y, Hearse DJ, and Avkiran M. Impact of extracellular buffer composition on cardioprotective efficacy of Na+/H+ exchanger inhibitors. Am J Physiol Heart Circ Physiol 270: H692-H700, 1996[Abstract/Free Full Text].

37.   Siegmund, B, Ladilov YV, and Piper HM. Importance of sodium for recovery of calcium control in reoxygenated cardiomyocytes. Am J Physiol Heart Circ Physiol 267: H506-H513, 1994[Abstract/Free Full Text].

38.   Sipido, KR, Mases M, and Van de Werf F. Low efficiency of Ca entry through the Na+/Ca2+ exchanger as trigger for Ca2+ release from the sarcoplasmic reticulum. A comparison between L-type Ca2+ current and reverse-mode Na+/Ca2+ exchange. Circ Res 81: 1034-1044, 1997[Abstract/Free Full Text].

39.   Stowe, DF, Fujita S, An JZ, Paulsen RA, Varadarajan SG, and Smart SC. Modulation of myocardial function and Ca2+ sensitivity by moderate hypothermia in guinea pig isolated hearts. Am J Physiol Heart Circ Physiol 277: H2321-H2332, 1999[Abstract/Free Full Text].

40.  Stowe DF, Heisner JS, An JZ, Camara A, Varadarajan SG, Novalija E, Chen Q, and Schelling P. Inhibition of Na+/H+ exchange-1 isoform protects hearts reperfused after six hour cardioplegic cold storage. J Heart Lung Transplant. In press.

41.   Stowe, DF, Varadarajan SG, An JZ, and Smart SC. Reduced cytosolic Ca2+ loading and improved cardiac function after cardioplegic cold storage of guinea pig isolated hearts. Circulation 102: 1172-1177, 2000[Abstract/Free Full Text].

42.   Stroemer, H, De Groot MCH, Horn M, Faul C, Leupold A, Morgan JP, Scholz W, and Neubauer S. Na+/H+ exchange inhibition with HOE 642 improves postischemic recovery due to attenuation of Ca2+ overload and prolonged acidosis on reperfusion. Circulation 101: 2749-2755, 2000[Abstract/Free Full Text].

43.   Tani, M. Mechanisms of Ca2+ overload in reperfused ischemic myocardium. Annu Rev Physiol 52: 543-559, 1990[ISI][Medline].

44.   Terracciano, CM, and MacLeod KT. Effects of acidosis on Na+/Ca2+ exchange and consequences for relaxation in guinea pig cardiac myocytes. Am J Physiol Heart Circ Physiol 267: H477-H487, 1994[Abstract/Free Full Text].

45.   Turvey, SE, and Allen DG. Changes in myoplasmic sodium concentration during exposure to lactate in perfused rat heart. Cardiovasc Res 28: 987-993, 1994[Abstract/Free Full Text].

46.   Varadarajan, SG, An JZ, Novalija E, Smart SC, and Stowe DF. Altered [Na+], compartmental [Ca2+], and NADH with dysfunction during ischemia reperfusion in intact hearts. Am J Physiol Heart Circ Physiol 280: H280-H293, 2001[Abstract/Free Full Text].

47.   Xiao, XH, and Allen DG. Activity of the Na+/H+ exchanger is critical to reperfusion damage and preconditioning in the isolated rat heart. Cardiovasc Res 48: 244-253, 2000[Abstract/Free Full Text].

48.   Yao, A, Matsue H, Spitzer KW, Bridge JH, and Barry WH. Sarcoplasmic reticulum and Na+/Ca2+ exchanger function during early and later relaxation in ventricular myocytes. Am J Physiol Heart Circ Physiol 273: H2765-H2773, 1997[Abstract/Free Full Text].

49.   Yasutake, M, Ibuki C, Hearse DJ, and Avkiran M. Na+/H+ exchanger and reperfusion arrhythmias: protection by intracoronary infusion of a novel inhibitor. Am J Physiol Heart Circ Physiol 267: H2430-H2440, 1994[Abstract/Free Full Text].

Am J Physiol Heart Circ Physiol 281(6):H2398-H2409
0363-6135/01 $5.00 Copyright © 2001 the American Physiological Society


This article has been cited by other articles:


Home page
 J. Appl. Physiol.Home page
S. E. McAllister, M. A. Moses, K. Jindal, H. Ashrafpour, N. J. Cahoon, N. Huang, P. C. Neligan, C. R. Forrest, J. E. Lipa, and C. Y. Pang
Na+/H+ exchange inhibitor cariporide attenuates skeletal muscle infarction when administered before ischemia or reperfusion
J Appl Physiol, January 1, 2009; 106(1): 20 - 28.
[Abstract] [Full Text] [PDF]

Home page
 J. Appl. Physiol.Home page
C. D. Garciarena, C. I. Caldiz, M. V. Correa, G. R. Schinella, S. M. Mosca, G. E. Chiappe de Cingolani, H. E. Cingolani, and I. L. Ennis
Na+/H+ exchanger-1 inhibitors decrease myocardial superoxide production via direct mitochondrial action
J Appl Physiol, December 1, 2008; 105(6): 1706 - 1713.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
Q. Chen, S. Moghaddas, C. L. Hoppel, and E. J. Lesnefsky
Ischemic defects in the electron transport chain increase the production of reactive oxygen species from isolated rat heart mitochondria
Am J Physiol Cell Physiol, February 1, 2008; 294(2): C460 - C466.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
M. Aldakkak, D. F. Stowe, Q. Chen, E. J. Lesnefsky, and A. K.S. Camara
Inhibited mitochondrial respiration by amobarbital during cardiac ischaemia improves redox state and reduces matrix Ca2+ overload and ROS release
Cardiovasc Res, January 15, 2008; 77(2): 406 - 415.
[Abstract] [Full Text] [PDF]

Home page
 Anesth. Analg.Home page
J. An, Z. J. Bosnjak, and M. T. Jiang
Myocardial Protection by Isoflurane Preconditioning Preserves Ca2+ Cycling Proteins Independent of Sarcolemmal and Mitochondrial KATP Channels
Anesth. Analg., November 1, 2007; 105(5): 1207 - 1213.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
A. Kanaan, R. M. Douglas, S. L. Alper, W. F. Boron, and G. G. Haddad
Effect of chronic elevated carbon dioxide on the expression of acid-base transporters in the neonatal and adult mouse
Am J Physiol Regulatory Integrative Comp Physiol, September 1, 2007; 293(3): R1294 - R1302.
[Abstract] [Full Text] [PDF]

Home page