Cardiovascular Research 61 (2004) 522 – 529
www.elsevier.com/locate/cardiores
Limited effects of post-ischemic NHE blockade on [Na+]i and pHi
in rat hearts explain its lack of cardioprotection
Michiel Ten Hove a,1, Cees J.A. Van Echteld b,*
a
b
Interuniversity Cardiology Institute of the Netherlands, The Netherlands
Department of Cardiology, Heart Lung Center Utrecht, University Medical Center, Room G02.523, P.O. Box 85500, 3508 GA Utrecht, The Netherlands
Received 26 February 2003; received in revised form 4 July 2003; accepted 26 July 2003
Time for primary review 27 days
Abstract
Objective: Na+/H+ exchanger (NHE) blockade fails as reperfusion therapy in patients with acute myocardial infarction. In experimental
studies, the reports on the efficacy of NHE blockade only during reperfusion are inconsistent. Differences in the severity of ischemia and in
drug delivery may explain these inconsistencies. Little is known about the primary goal of post-ischemic NHE blockade, i.e. reduction of
Na+i overload. Methods: Isolated rat hearts were subjected either to 60 min of low flow (0.2 ml/min) ischemia or 25 min of zero flow
ischemia. Hearts were reperfused with or without the selective NHE blocker cariporide added to the perfusate. [Na+]i and pHi were measured
with simultaneous 23Na and 31P NMR spectroscopy. Results: After 60 min of low flow ischemia [Na+]i had risen to 424 F 14% of baseline
and pHi was 6.36 F 0.03. After low flow ischemia [Na+]i and pHi recovered similarly in treated and untreated hearts. Recovery of the rate
pressure product (RPP) was poorly in both groups. After 25 min of zero flow ischemia [Na+]i had risen to 279 F 7% of baseline and pHi was
6.12 F 0.02. NHE blockade after zero flow ischemia caused [Na+]i to decrease during the first 30 s of reperfusion, followed by a partial and
transient rise during the second 30 s. Untreated hearts showed a very small rise in [Na+]i during the first minute. pHi recovered 30 s slower in
cariporide treated hearts than in untreated hearts ( p < 0.05). No effect of cariporide on RPP could be detected since RPP recovered fully in
untreated hearts. The end diastolic pressure, however, was increased during reperfusion to a similar extent in both groups. Conclusion: The
lack of cardioprotection under these specific conditions of zero flow and low flow ischemia can be explained by the fact that NHE blockade
only resulted in a small and transient effect on [Na+]i and pHi.
D 2003 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.
Keywords: Na/H-exchanger; NMR; Ischemia; Reperfusion; Intra/extracellular ions
1. Introduction
In patients with an acute myocardial infarction (MI)
blockade of Na+/H+ exchanger (NHE) at the onset of
reperfusion failed to improve clinical outcome [1]. In a
study with a small sample size, post-ischemic administration
of the NHE blocker cariporide did result in reduced infarct
sizes [2]. In contrast, in a large-scale trial with the NHE
blocker eniporide, post-ischemic administration did not
reduce infarct size [3]. As recently discussed [1], the
discrepancy between both studies is most likely the result
* Corresponding author. Tel.: +31-30-2507065; fax: +31-30-2522879.
E-mail address: [email protected] (C.J.A. Van Echteld).
1
Present address: Department of Cardiovascular Medicine, Wellcome
Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive,
Oxford OX3 7BN, UK.
of a chance finding in the study with cariporide due to the
small sample size.
This raises the question why NHE blockade fails as
reperfusion therapy. As recently reviewed [4], many experimental studies show that blocking the NHE during ischemia is cardioprotective but reports on the efficacy of
blocking the NHE only during reperfusion are inconsistent.
In different species and models post-ischemic NHE blockade has been found to be protective [5– 9] but other studies
showed no effect [10 – 12]. However, when any residual
flow is present during ischemia several studies report
consistently that post-ischemic NHE blockade is ineffective
[13,14]. Therefore, the efficacy of NHE blockade only
during reperfusion may well depend on the nature of the
ischemia, i.e. with or without residual flow.
Although the [Na+]i is quickly reduced upon reperfusion
after ischemia [15] it is likely that recovery of ischemic
0008-6363/$ - see front matter D 2003 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.cardiores.2003.07.005
M. Ten Hove, C.J.A. Van Echteld / Cardiovascular Research 61 (2004) 522–529
acidosis mediates Na+ influx through the NHE. This NHE
mediated Na+ influx may well be enhanced compared to the
ischemic period due to the restored extracellular pH, resulting in a large transmembrane proton gradient. Therefore, it
is likely that blocking the NHE upon reperfusion will reduce
Na+ influx and thereby reversed Na+/Ca2 + exchange. However, the effect on the [Na+]i of NHE blockade with specific
NHE blockers like cariporide (HOE 642) during reperfusion
only never has been studied.
The purpose of this study was to find possible explanations for the failure of post-ischemic NHE blockade to
offer cardioprotection in animal models, which may help to
explain the poor clinical outcome in patients with acute MI
treated with NHE blockers. The efficacy of post-ischemic
NHE blockade may depend on the nature of the ischemia,
which may vary within the group of patients with an MI.
Therefore, we tested the effect of post-ischemic NHE
blockade in isolated rat hearts subjected to either zero flow
ischemia or low flow ischemia. To block the NHE, cariporide was administered immediately upon reperfusion
(zero flow) or from the last 5 min of ischemia onwards
(low flow). Simultaneous 31P and 23Na NMR spectroscopy
was used to measure the pHi, Phosphocreatine (PCr),
adenosine triphosphate (ATP), inorganic phosphate (Pi)
and [Na+]i, respectively.
2. Methods
2.1. Heart perfusion
Twenty four Male Wistar rats were anesthetized with
diethylether and hearts were perfused according to Langendorff as described before [16]. Briefly, hearts were perfused at
37 jC and a perfusion pressure of 73.5 mm Hg. Contractility
523
was 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 LVDP) was used as an index of cardiac contractility. The
investigation conforms with the Guide for the Care and Use
of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).
All perfusion fluids were saturated with 95% O2/5% CO2,
resulting in a final perfusate pH of 7.35 F 0.05. The standard
perfusate contained (in mmol/l) Na+ 148.0; K+ 4.7; Ca2 + 1.3;
Mg2 + 1.0; Cl 133.3; HCO3 24.0; glucose 11.0. To discriminate between intra- and extracellular Na+, 3.5 mmol/
l TmDOTP5 was used as a shift reagent, necessitating a
lower [Ca2 +]free of 0.85 mmol/l.
2.2. Experimental protocols
Two sets of experiments were performed. After stabilization, hearts were monitored during 5 min of control
perfusion. Thereafter, hearts were either subjected to 25
min of zero flow ischemia or to 60 min of low flow
ischemia (0.2 ml/min). In both cases hearts were reperfused for 30 min with a constant pressure. Hearts were
reperfused with or without 3 AM cariporide (HOE 642)
added to the perfusate. In the low flow group the drug was
also administered during the last 5 min of low flow. To
maintain gas composition of the perfusate all perfusion
lines were surrounded by water gassed with 95% O2/5%
CO2 to avoid gas leakage. Furthermore, during low flow
hearts were perfused through a perfusion line with a small
diameter (0.38 mm) to achieve a high flow velocity. After
the protocol hearts were dried to determine dry weights.
Intracellular volume was assumed to be 2.45 ml/g dry
weight [17].
Fig. 1. [Na+]i during 60 min low flow (0.2 ml/min) ischemia and 30 min reperfusion; hearts were untreated (solid diamonds) or treated with 3 AM cariporide
from the last 5 min of low flow ischemia onwards (open squares) (n = 6 for both groups); black bar indicates period of drug administration.
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M. Ten Hove, C.J.A. Van Echteld / Cardiovascular Research 61 (2004) 522–529
PCr, ATP and Pi were quantified using a time domain
fitting routine (AMARES), as described before [16]. To
quantify the speed of pHi recovery upon reperfusion the
onset of pHi recovery was analyzed by determination of
the time point at which pHi was increased by more then
0.1. We calculated the intracellular pH buffering capacity
at the end of 25 min zero flow ischemia, using the
intrinsic intracellular buffer capacity, bi [18], and we
calculated the buffering capacity of the accumulated
intracellular Pi and the CO2 dependent buffering capacity
assuming no CO2 leakage nor CO2 formation during zero
flow ischemia. To estimate the Na+ influx via the NHE
Fig. 2. pHi during 60 min low flow (0.2 ml/min) ischemia and 30 min
reperfusion with a temporal resolution of 5 min (A) and 30 s (B); hearts
were untreated (solid diamonds) or treated with 3 AM cariporide from the
last 5 min of low flow ischemia onwards (open squares) (n = 6 for both
groups); black bar indicates period of drug administration.
2.3. NMR methods
As described before [16], 23Na and 31P NMR spectra
were recorded simultaneously at 105.9 and 162.0 MHz,
respectively, on a three channel Bruker Avance DRX400
spectrometer, equipped with a 9.4 T magnet, a dual tuned
probehead and two digital receivers. Thirty-second 23Na
NMR spectra were obtained by accumulation of 144
consecutive free induction decays (FIDs) using 90j pulses
and a 210-ms interpulse delay. Thirty-second 31P spectra
were acquired by adding 12 FIDs using 90j pulses and a
2.5-s interpulse delay. Parameters were quantified with a
temporal resolution of 30 seconds, 1 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.
2.4. Calculations and statistics
pHi values were calculated from the chemical shift of
the inorganic phosphate (Pi), as described before [16].
Fig. 3. Phosphocreatine (PCr) (A), adenosine triphosphate (ATP) (B) and
inorganic phosphate (Pi) (C) during 60 min low flow (0.2 ml/min) ischemia
and 30 min reperfusion; hearts were untreated (solid diamonds) or treated
with 3 AM cariporide from the last 5 min of low flow ischemia onwards
(open squares) (n = 6 for both groups); black bar indicates period of drug
administration.
M. Ten Hove, C.J.A. Van Echteld / Cardiovascular Research 61 (2004) 522–529
525
during the first 30 s of reperfusion after 25 min of zero
flow ischemia, we assumed that the difference in [Na+]i
between treated and untreated hearts was solely the result
of the absence of NHE activity in the cariporide treated
hearts. Results are presented as mean F S.E. Data were
analyzed by oneway analysis of variance (ANOVA) with
repeated measurements or with a Student’s t-test, when
appropriate.
3. Results
Baseline parameters were identical in all groups. Average
intracellular concentrations (mmol/l) amounted to 9.8 F 0.4
for Na+, 19.2 F 1.2 for PCr and 14.8 F 0.7 for ATP.
3.1. Low flow ischemia
3.1.1. Intracellular Na+
During low flow ischemia the [Na+]i increased immediately and constantly in both groups (Fig. 1). The rate of
rise was similar to the one found in hearts subjected to
zero flow ischemia. After 60 min of low flow ischemia
[Na+]i had risen to 424 F 14% of baseline. Upon reperfusion the [Na+]i decreased partially in both untreated and
treated hearts (NS).
Fig. 5. [Na+]i during 25 min zero flow ischemia and 30 min reperfusion
with a temporal resolution of 1 min (A) and 30 s (B); hearts were untreated
(solid diamonds) or treated with 3 AM cariporide immediately upon
reperfusion (open squares) (n = 6 for both groups); black bar indicates
period of drug administration.
3.1.2. Intracellular pH
During low flow ischemia the pHi slowly decreased to
6.25 after 20 min in both groups (Fig. 2A). During the last 40
min of low flow ischemia, the pHi gradually increased to
6.36. During reperfusion 31P spectra of all hearts showed split
Pi peaks, indicating heterogeneous pHi recovery. This most
likely represents heterogeneous reperfusion. Well perfused
areas will show a recovery of the pHi, whereas poorly
reperfused areas will not. The well reperfused areas showed
a quick and complete recovery in both groups (NS) (Fig. 2B).
Poorly reperfused areas in both groups showed no recovery of
acidosis at all. Since the cariporide is only certainly present in
well reperfused areas, only the most alkaline peak is represented in pH figures.
Fig. 4. Rate pressure product (RPP) (A) and enddiastolic pressure (EDP)
(B) before and after 60 min low flow (0.2 ml/min) ischemia; hearts were
untreated (solid diamonds) or treated with 3 AM cariporide from the last 5
min of low flow ischemia onwards (open squares) (n = 6 for both groups).
3.1.3. Energy related phosphates
PCr rapidly declined during ischemia followed by a fast
partial recovery during reperfusion in both groups (Fig. 3A).
ATP gradually decreased during ischemia and poorly recovered during reperfusion in both groups (Fig. 3B). Pi accumulated during ischemia followed by a fast reduction during
reperfusion in both groups (Fig. 3C). Pi peaks were split in
all hearts during reperfusion. In the cariporide treated hearts
the peak representing the lowest pHi was relatively larger
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decrease gradually after 1 min reaching 140% of baseline
values after 30 min of reperfusion. In hearts treated with
cariporide the [Na+]i showed an immediate fast decrease
during the first 30 s of reperfusion (Fig. 5B), followed by a
transient increase during the second 30 s. Thereafter [Na+]i
gradually decreased to a similar level as found in untreated
hearts (Fig. 5A).
3.2.2. Intracellular pH
During zero flow ischemia the pHi rapidly decreased in
both groups to 6.1 after 25 min (Fig. 6A). pHi recovery
differed, in contrast to the low flow experiments, between
both groups. As shown in Fig. 6B, upon reperfusion the
pHi in untreated hearts rapidly increased from the second
period of 30 s onwards, whereas the pHi in cariporide
Fig. 6. pHi during 25 min zero flow ischemia and 30 min reperfusion with a
temporal resolution of 1 min (A) and 30 s (B); hearts were untreated (solid
diamonds) or treated with 3 AM cariporide immediately upon reperfusion
(open squares) (n = 6 for both groups); black bar indicates period of drug
administration.
than in the untreated hearts (data not shown). Data on PCr,
ATP and Pi showed no significant differences between both
groups.
3.1.4. Contractile performance and coronary flow
During reperfusion the RPP recovered only partially in
both groups (Fig. 4A). The recovery of the RPP tended to be
better in untreated hearts but there were no significant differences. During reperfusion, heart rate did not follow in all
hearts the pacing signal. During ischemia all hearts went into
contracture. Characteristics of contracture did not differ
between groups. As shown in Fig. 4B, the LVEDP increased
upon reperfusion, followed by a gradual decrease in both
groups (NS). During reperfusion the coronary flow was
58 F 5% and 60 F 4% of baseline in untreated and cariporide
treated hearts, respectively (NS).
3.2. Zero flow ischemia
3.2.1. Intracellular Na+
During zero flow ischemia the [Na+]i showed an immediate and constant increase as observed before [16] (Fig. 5A).
Upon reperfusion the [Na+]i in untreated hearts started to
Fig. 7. Phosphocreatine (PCr) (A), adenosine triphosphate (ATP) (B) and
inorganic phosphate (Pi) (C) during 25 min zero flow ischemia and 30 min
reperfusion; hearts were untreated (solid diamonds) or treated with 3 AM
cariporide immediately upon reperfusion (open squares) (n = 6 for both
groups); black bar indicates period of drug administration.
M. Ten Hove, C.J.A. Van Echteld / Cardiovascular Research 61 (2004) 522–529
treated hearts increased rapidly from the third period of 30
s onwards ( p < 0.01). In both groups pHi recovered completely within 5 min.
3.2.3. Energy related phosphates
PCr decreased quickly during zero flow ischemia followed by a fast recovery during reperfusion in both groups
(Fig. 7A). ATP gradually decreased during zero flow ischemia followed by a partial recovery during reperfusion (Fig.
7B). Pi accumulated during zero flow ischemia followed by
a fast reduction during reperfusion (Fig. 7C). Data on PCr,
ATP and Pi showed no significant differences between both
groups.
3.2.4. Contractile performance and coronary flow
The RPP collapsed immediately during ischemia and
completely recovered in both groups during reperfusion
(NS) (Fig. 8A). During reperfusion, heart rate did not
follow in all hearts the pacing signal. During ischemia all
hearts went into contracture. Characteristics of contracture
did not differ between groups. As shown in Fig. 8B, the
LVEDP rapidly increased upon reperfusion followed by a
gradual decrease in both groups (NS). During reperfusion
the coronary flow was 81 F 7% and 75 F 6% of baseline
in untreated and cariporide treated hearts, respectively (NS).
Fig. 8. Rate pressure product (RPP) (A) and enddiastolic pressure (EDP)
(B) before and after 25 min zero flow ischemia; hearts were untreated (solid
diamonds) or treated with 3 AM cariporide immediately upon reperfusion
(open squares) (n = 6 for both groups).
527
4. Discussion
To elucidate why NHE blockade fails as reperfusion
therapy this study shows, for the first time, the effect of
post-ischemic NHE blockade on its primary goal, i.e.
prevention of [Na+]i overload, with a specific NHE blocker.
After 60 min of low flow, we did not find any significant
effect of post-ischemic NHE blockade on either of the
measured parameters. After 25 min of zero flow ischemia
there was only a small effect on the decrease in [Na+]i, and a
small delay in pHi recovery.
The different effects of NHE blockade after zero flow
ischemia and after low flow ischemia may be explained by
the difference in ischemic pHi. During low flow the acidosis
is less severe, [H+]i,free was 1.8 times higher after 25 min of
zero flow ischemia than after 60 min of low flow ischemia.
In addition, the transsarcolemmal Na+ gradient is smaller
after 60 min of low flow ischemia, since [Na+]i is higher
than after 25 min of zero flow ischemia. Therefore, it is
likely that the NHE will mediate much less Na+ influx after
low flow ischemia than after zero flow ischemia under these
conditions. Since we found no effect of NHE blockade after
low flow ischemia on [Na+]i and pHi one would not expect a
reduction in reversed Na+/Ca2 + exchange.
These results therefore explain the lack of an acute
cardioprotective effect after a period of ischemia with
residual flow, found in this study as well as in others
[13,14], in spite of the fact that the residual coronary flow
during ischemia was very small (1 –2%).
In the low flow groups, cariporide treated hearts showed
a trend towards a worse recovery of the RPP than untreated
hearts. This effect, however, was not significant ( p = 0.18).
It could be related to areas in the heart with a low pHi due to
NHE blockade. As mentioned, we found multiplicity in the
Pi peaks during reperfusion. In the cariporide treated hearts
the Pi peak representing a low pHi was relatively larger than
in the untreated hearts, in spite of a slightly higher coronary
flow. In well reperfused areas the pHi recovers quickly, also
in the presence of cariporide. It could be, however, that in
poorly reperfused areas NHE blockade impairs pHi recovery. This would have a negative inotropic effect.
Although not the purpose of this study, we found,
surprisingly, that the rate of rise in [Na+]i in hearts subjected
to low flow ischemia was the same as in hearts subjected to
zero flow ischemia, in spite of the difference in ischemic
pHi.
Our results only provide limited information on the
cardioprotective effect of NHE blockade after a period of
ischemia without residual flow since the untreated hearts
showed a complete recovery of the RPP. Cariporide, however, did not prevent the post-ischemic elevation of the
LVEDP, indicating only limited or no cardioprotection in
this situation. This, as well as the inconsistent data in the
literature can be explained by our data on [Na+]i and on pHi.
On both parameters there was an effect, which proves that
cariporide is active immediately, but the effect was only
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M. Ten Hove, C.J.A. Van Echteld / Cardiovascular Research 61 (2004) 522–529
transient. The initial faster decrease in [Na+]i was partly
cancelled out during the second period of 30 s and the delay
in pHi recovery was only 30 seconds. NHE blockade is
thought to be cardioprotective via reduced reversed Na+/
Ca2 + exchange [4]. This effect could result from reduced
Na+ influx as well as from delayed pHi recovery, since a low
pHi may inhibit the Na/Ca2 + exchanger [19 – 21]. Our data
show that the reduction in Na+ influx is only limited and
transient. This mechanism would therefore prevent only a
little reversed Na+/Ca2 + exchange. Furthermore, the effect
on the [Na+]i is mainly present during the first 30 s when the
pHi is still low, further abolishing the effect of the transiently lower [Na+]i.
The immediate faster reduction in [Na+]i, followed by a
transient increase has been found before in our laboratory
with the less specific NHE blocker EIPA [15]. In that same
study it was shown that the Na+/K+ ATPase is immediately
active upon reperfusion. The initial faster reduction in [Na+]i
can therefore be explained by Na+ efflux through the Na+/
K+ ATPase and the absence of additional Na+ influx via
Na+/H+ exchange. The concomitant rise in [Na+]i was
explained by Na+ influx via the Na+/HCO3 co-transporter,
suggesting that post-ischemic NHE blockade may be more
effective when accompanied by simultaneous blockade of
Na+/HCO3 co-transport.
This explains the data on [Na+]i but appears in conflict
with the data on pHi. In untreated hearts the pHi does not
rise substantially during the first 30 s. This is in conflict with
H+ efflux via the NHE. Also the rise in [Na+]i during the
second period of 30 s of reperfusion in cariporide treated
hearts is not accompanied by any change in pHi, which is in
apparent conflict with enhanced Na+/HCO3 co-transport.
The delay in pHi recovery compared to the Na+ influx may
be explained by the fact that the pHi is buffered, whereas the
[Na+]i is not. Within the assumptions mentioned in the
methods, we calculated that during the first 30 s of reperfusion Na+/H+ exchange transports 4.5 Amol Na+ per ml
cytosol in 30 s. This implies that the maximum increase
in pHi as a result of Na+/H+ exchange during the first 30 s of
reperfusion was 0.06, which is very close to the value of
0.05 we found in untreated hearts with our 31P data.
In the cariporide treated hearts [Na+]i decreased during
the first 30 seconds of reperfusion by 4.3 Amol/ml cytosol.
During the second period of 30 s [Na+]i increased by 2.5
Amol/ml cytosol. Therefore the nett Na+ influx is increased
by 6.8 Amol/ml cytosol. When this difference would be
solely the result of enhanced Na+/HCO3 co-transport the
maximum increase in pHi during the second period of 30 s
of reperfusion would be 0.09, assuming that all Na+/HCO3
co-transport was electroneutral, whereas we found an increase of 0.05. However, when all Na+/HCO3 co-transport
is electrogenic with a stochiometry of 1:2 for Na+:HCO3 the
maximum increase in pHi would be 0.18. Therefore, the
increase in the [Na+]i during the second period of 30 s of
reperfusion in the cariporide group can only partially be
explained by Na+/HCO3 co-transport.
An additional explanation for the delay in pHi recovery
compared to the Na+ influx may be intracellular heterogeneity. The [Na+]i signal in the 23Na spectrum arises from the
total Na+i. The pHi signal is determined by the chemical
shift of the Pi signal. An immediate subsarcolemmal decrease in [Na+]i will result in an immediate decrease in the
Na+i signal. The chemical shift of Pi, however, may be
compromised by a heterogeneous distribution of H+ and of
Pi in the cell. When protons are transported over the
sarcolemma the subsarcolemmal [H+] decreases followed
by a subcellular redistribution of protons. Therefore, the
bulk Pi may experience a somewhat delayed decrease in
[H+] compared to the subsarcolemmal areas.
Although the small effects of cariporide administration
on [Na+]i and pHi indicate NHE inhibition, the blockade
could be incomplete. However, previously we have shown
that in this same model the same concentration of cariporide
effectively inhibits ischemic Na+i overload [16]. Furthermore, the idea that the NHE is inhibited is supported by the
finding that the effect on [Na+]i is similar as the one found
previously using EIPA [15].
Our findings add to the ongoing debate on NHE activity
during ischemia and reperfusion [9,22,23]. As recently
reviewed [24], only studies using the fluorescent indicator
SBFI to study [Na+]i find a limited rise in [Na+]i during
ischemia and a fast and steep increase immediately upon
reperfusion, whereas all 23Na NMR spectroscopy studies
report a substantial increase in [Na+]i during ischemia
similar to the one found in this study [15,16,25 – 29].
Avkiran et al. [23] convincingly argumented against the
idea that the NHE is inhibited during ischemia, to which our
previous results lend further support [16]. The spectral
characteristics of SBFI and other fluorescent indicators like
indo-1 are affected by pHi and intracellular protein environment [30,31], which change during ischemia and reperfusion. Most studies using SBFI in intact hearts, however,
use pH calibration in a protein free aqueous solution [9,30]
or do not correct for pH changes at all [6,32]. When pHi is
normalized data on [Na+]i measured by NMR or SBFI can
be remarkably similar. For example, Varadarajan et al. [32]
reported a [Na+]i of f 24 mM after 3 min of reperfusion
following 30 min of global ischemia in isolated guinea pig
hearts, which is comparible to what we found in rat hearts at
that timepoint [16]. In that same article it was stated that
their [Na+]i measurements during ischemia may underestimate the actual [Na+]i by more than 50% during ischemia
due to intracellular acidosis.
In conclusion, our data explain the limited protection of
post-ischemic NHE blockade as reflected by the inconsistency in the literature as well as the lack of efficacy of postischemic NHE blockade found in a large scale clinical trial.
After a period of zero flow ischemia there is only a transient
faster decrease in the [Na+]i and the recovery of the pHi is
delayed only by 30 s. After a period of ischemia with
residual flow there is no effect at all on the [Na+]i and the
pHi, probably due to the less severe acidosis.
M. Ten Hove, C.J.A. Van Echteld / Cardiovascular Research 61 (2004) 522–529
Acknowledgements
MtH was supported by The Netherlands Heart Foundation (Grant 98.141). Equipment was financed by The
Netherlands Organization for Scientific Research (NWO,
Grant 902-16-202), the Royal Netherlands Academy for
Arts and Sciences (KNAW, Grant D96.695) and the
Interuniversity Cardiology Institute of the Netherlands.
Cariporide was kindly provided by Aventis Pharma GmbH
(Frankfurt am Main, Germany).
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