Download Mechanisms of pHi Recovery After Global Ischemia in the Perfused

993
Mechanisms of pHi Recovery After Global
Ischemia in the Perfused Heart
Jamie
I.
Vandenberg, James C. Metcalfe, and Andrew A. Grace
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A Na'-HCO3- coinflux carrier and the Na+-H+ antiport have both been shown to contribute to recovery
from intracellular acidosis in cardiac tissue. We have investigated the participation of these mechanisms
as well as metabolite (lactate and C02) washout in the recovery of pH; after myocardial ischemia.
Isovolumic ferret hearts were Langendorfl-perfused with either HCO3-buffered or nominally HCO3-free
(HEPES-buffered) medium at 30°C. pHi was estimated from the chemical shift of the 31P-nuclear magnetic
resonance signal of intracellular P04-, and net H' efflux rates were calculated at pH; 6.80. The H' efflux
rate during reperfusion, after 10 minutes of global ischemia, was 15.5 +1.9 mmol * I-l min' (n=10) in
hearts perfused with HC03-buffered medium and 8.2± 1.5 mmol Il * min-1 (n=9, p<0.01) in hearts
perfused with HEPES-buffered medium. HCO3- influx, assessed either by inhibition by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (20 ,uM) or by initially perfusing hearts with HEPES-buffered medium
but reperfusing with HCO3-buffered medium, accounted for 3.5-4.9 mmol * I1 min', and CO2 efflux
accounted for 3.8-6.2 mmol * 1-l. min' of the additional H' efflux in HCO3--buffered medium.
H+-coupled lactate efflux, measured by NAI)-linked spectrophotometric assay and inhibited by the
sarcolemmal monocarboxylate transport inhibitor 4,4'-dibenzamidostilbene-2,2'-disulfonate (0.25 mM),
contributed 3.7-6.2 mmol * 1- min'l. H' efflux via the 5-(N-ethyl-N-isopropyl)amiloride-sensitive
Na+-HW antiport was 1.0-2.9 mmol* I-1' min'. pHi recovery after ischemia is therefore principally
mediated by metabolite (lactate and C02) washout. Na+-coupled acid extrusion contributed approximately
35% of total H' efflux in this system. However, the associated Na+ entry ("5 mmol * -* min-1) may
contribute to Ca2+ overload after reperfusion. (Circulation Research 1993;72:993-1003)
KEY WoRDs * pH; * ischemia * reperfusion * nuclear magnetic resonance spectroscopy
he deleterious effects of ischemia on the mechanical and electrical activity of the heart are
well described.1-3 These effects are reversible
if reperfusion is initiated within a few minutes; however, it may take several days before normal function is
regained.4'5 Although the role of intracellular acidosis
in ischemic contractile failure has been extensively
studied,3'6'7 the mechanisms of pHi recovery after
reperfusion and the relation between pHi recovery and
restoration of cardiac contraction have not been
established.
Regulation of pHi in cardiac tissue is mediated by
intracellular buffers and several membrane transport
processes, including the ClW-HCO3 exchanger,8 the
Na+-H+ antiport,9 and a recently described acid extrusion mechanism that is both Na+ and HCO3- dependent.'0-13 However, the regulation of pHi during ischemia and reperfusion may not be mediated by the same
mechanisms as in well-perfused cardiac preparations.'4
T
From the Department of Biochemistry, University of Cambridge (UK).
Supported by a grant from the British Heart Foundation. J.I.V.
was supported by a Sir Robert Menzies Memorial Scholarship in
Medicine and a Sydney University Medical Foundation Travelling
Fellowship. A.A.G. was supported by a British Heart Foundation
Clinical Scientist Award.
Address for correspondence: Jamie I. Vandenberg, University
of Cambridge, Department of Biochemistry, Tennis Court Road,
Cambridge CB2 lQW, UK.
Received July 22, 1992; accepted January 14, 1993.
For example, the hydrolysis of phosphocreatine (PCr)
and accumulation of inorganic phosphate (Pi) will alter
the buffering capacity of the heart,14'15 and the accumulation of CO2 and lactate may, when washed out during
reperfusion, contribute to pHi recovery.16-20
In this study, we report on the mechanisms of pHi
recovery after global ischemia in the Langendorffperfused ferret heart and correlate the activity of
these mechanisms with recovery of cardiac contraction. The role of H'-coupled lactate efflux has been
estimated by assaying lactate in the myocardial effluent during reperfusion and by using 4,4'-dibenzamidostilbene-2,2'-disulfonate (DBDS) to inhibit lactate
efflux.20 22 The contribution of the Na+-H antiport
has been assessed using the high-affinity Na+-H+
antiport inhibitor 5-(N-ethyl-N-isopropyl)amiloride
(EIPA).23 The role of HCO3--dependent mechanisms
has been investigated by comparing pHi recovery after
ischemia in hearts perfused with nominally HCO3-free medium with recovery in hearts perfused with
HCO3--buffered medium and by using 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) as an inhibitor of HCO3- transport mechanisms.24 From these
protocols, the relative contributions of metabolite
washout (lactate and C02) and Na+-coupled acid
extrusion (Na+-H+ exchange and Na+-HCO3- coinflux)
to the recovery of pHi after global ischemia have been
estimated, and the relation between recovery of pH,
and contractile function is considered.
994
Circulation Research Vol 72, No D May 1993
TABLE 1. Experimental Protocols
Mechanisms potentially active
after reperfusion
N4a _H'
Lactate
CO2
HCO,efflux
efflux
influx
e)xchange
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Hearts perfused with HCOf-buffered medium
Control
+
+±
+DBDS*
--h
4
+ EIPAt
+
+
+
+ DIDSt
+
+
±DDS+EIPA (after DIDS)
Hearts perfused with HEPES-buffered medium
Control
+
+DBDS
+
+EIPA
+
+DBDS+ElPA
Hearts perfused with HEPES- or HCO3--buffered medium before ischemia
and reperfused with HCO3- or HEPES-buffered medium, respectively
HCO3 to HEPES
+
+
±t
HEPES to HCO3+
+
DBDS, 4,4'-dibenzamidostilbene-2,2'-disulfonate; EIPA, 5-(N-ethyl-N-isopropyl)amiloride; DIDS, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid; +, active; -, inactive; -+, partially active.
*DBDS (0.25 mM) inhibits sarcolemmal monocarboxylate transport.21'22
tEIPA (1 gM) inhibits Na+-H+ exchange.23
tDIDS (20 ^rM) inhibits anion exchangers including HCO3 influx mechanisms.24
Materials and Methods
Heart Preparation
The preparation and monitoring of Langendorffperfused ferret hearts have been described previously.13,25 Briefly, male ferrets (n = 43), 3-12 months of age,
were anesthetized with pentobarbitone (250 mg* kg`1
i.p.) and heparinized (2,000 units i.p.). The heart was
excised and immediately arrested in ice-cold HCO3-buffered medium (see below). The aorta was cannulated, and perfusion was commenced with HCO3-buffered medium at 30°C. Perfusion was at constant
flow (5-6 ml* g` min-1) using a peristaltic pump
(Watson-Marlow 502S) without recirculation of the
perfusate at any stage. This resulted in an initial perfusion pressure of 50-90 mm Hg; perfusion pressure was
monitored with a pressure transducer (Statham P23XL)
connected via polyethylene tubing to a side arm on the
aortic cannula. Isovolumic left ventricular developed
pressure (LVDP, calculated by subtracting end-diastolic
from systolic pressure) was monitored using a salinefilled latex balloon inserted in the left ventricular cavity.
The initial left ventricular end-diastolic pressure was
adjusted to 5-10 mm Hg. Pressures were recorded on a
four-channel chart recorder (Gould 2400S), and LVDP
was calculated by subtracting end-diastolic pressure
from systolic pressure. The atrioventricular node was
crushed to abolish atrioventricular conduction. Agarfilled polyethylene electrodes were sutured to the right
ventricular epicardium, and hearts were paced at 1.0 Hz
by use of square-wave stimuli of 10-msec duration at
twice threshold voltage.
Solutions
The two perfusion media used were as follows: 1)
HCO3 -buffered medium containing (mM) NaCl 119,
NaHCO3 25, KCl 4, KH2PO4 1.2. MgCl, 1.0, CaClI 1.8,
and glucose 10, equilibrated with 95% 02-5% CO2 at
30°C (pH 7.42-7.44), and 2) nominally HCO3 -free
HEPES-buffered medium containing (mM) NaHCO3 0,
HEPES 5, and sodium gluconate 22, equilibrated with
100% 02 and pH-adjusted to 7.42-7.44 at 30°C with 2N
NaOH. Sodium gluconate was added to HEPES-buffered media to maintain chloride and sodium at the same
concentrations in both perfusion media. All perfusion
media reagents were analytical reagent grade.
EIPA (a gift from Hoechst AG, Germany) was dissolved in dimethyl sulfoxide, 2-mM stock concentration,
and added to the perfusate to achieve a final concentration of 1 ,gM. In the Langendorff-perfused ferret heart,
this dose of EIPA reduces flux through the Na+-H+
antiport by >90%.13 DIDS (Sigma Chemical Co., Poole,
UK) was added directly to media to a final concentration
of 20 ,uM, a dose that has previously been estimated to
reduce flux via HCO3--dependent acid extrusion mechanisms by 70-80%.13 DBDS (a generous gift from Dr.
Robert Poole, Department of Biochemistry, University of
Bristol, UK) was dissolved in dimethyl sulfoxide: methanol
(4:1), 50-mM stock concentration, and added to the
perfusate to a final concentration of 0.25 mM. This
concentration of DBDS causes > 10-fold reduction in the
initial rate of transport of L-lactate (0.5 mM) across the
erythrocyte plasmalemma21 and 50-80% inhibition of
lactate transport across the cardiac sarcolemma of rat and
guinea pig ventricular myocytes.22 The perfusion media
and pharmacological agents used to assess the relative
contributions of CO2 efflux, lactate efflux, Na+-H+ exchange, and HCO3- influx to net H' efflux after reperfusion are summarized in Table 1.
Hearts were perfused initially with HCO3--buffered
medium for 30-60 minutes until LVDP was stable. Perfusion was then switched to either HEPES-buffered or
HCO3--buffered Pi-free medium (KH2PO4 replaced with
Vandenberg et al pHi Recovery After Ischemia
995
an equimolar concentration of KCl) and allowed to equilibrate for a further 30 minutes before experimental interventions. In control hearts, perfusion with Pi-free medium
for up to 4 hours had no significant effect on LVDP, pHi,
PCr, or ATP levels. During pharmacological interventions, hearts were equilibrated with perfusion medium
containing the relevant drug for 2-10 minutes before
ischemia and for the first 5 minutes of reperfusion. The
time required for changes of perfusion media was determined with methylene orange and took =1.5 minutes to
complete.
to incomplete relaxation of magnetization at high pulsing frequencies.25 Saturation factors (mean ± SEM)
were determined from eight experiments by comparing
areas of resonances in fully relaxed spectra obtained
using an interpulse delay of 15 seconds with the corresponding areas when the interpulse delay was 0.9 seconds. The saturation factors used were as follows: Pi,
1.3±0.3; PCr, 1.4+0.1; and ,B-ATP, 1.1+0.2.
Ischemia Procedure
following formula:
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Hearts were made globally ischemic by switching off
the peristaltic pump and clamping the perfusion inflow
line for 10 minutes. Circulation of water through the
water jacket and probe head was continued during
ischemia to ensure that the temperature of the heart
was maintained at 30°C. A second ischemic episode was
induced, 60-90 minutes after the first episode, only in
those hearts in which metabolic and functional parameters had returned to preischemic values within 60
minutes.26 In a separate series of experiments, hearts
were perfused in the probe head outside the magnet
and made ischemic using the above procedure, except
that immediately before reperfusion the heart was removed from the probe head so that the myocardial
effluent could be collected during the first 2 minutes of
reperfusion. Effluents were collected in Eppendorf
tubes and frozen immediately, or perchloric acid was
added at a final concentration of =1 M to precipitate
DBDS, which is insoluble at low pH. After centrifugation, excess perchlorate was precipitated by neutralizing
the supernatant with K2C03.27 Supernatants from the
neutralized samples were stored at -20°C, and all
samples were later analyzed for lactate by an NAD+linked spectrophotometric assay.28 Phosphate content
of effluents was analyzed by a nuclear magnetic resonance (NMR) spectroscopic assay.29
NMR Spectroscopy
In well-perfused hearts, the signal from intracellular Pi
is very small, making it an insensitive indicator for measurement of pH1.13 However, during ischemia and the first
1.5-2 minutes of reperfusion, the accumulated intracellular Pi permits rapid sequential estimation of pHi from the
chemical shift between the PCr and Pi resonances. pHi
values were obtained from the chemical shift of the Pi
resonance using a standard calibration curve.30
Hearts were housed in a purpose-built dual-tuned
(31PP1H) probe head3' and placed in a Bruker AM400
wide-bore spectrometer. 3MP-NMR spectra were recorded at 162 MHz with 400 pulses delivered at 0.9second intervals. Control spectra, acquired 5 minutes
before ischemia, were averaged from 300 transients.
During ischemia and the first 2.5 minutes of reperfusion, 16-32 transients were averaged for each spectrum.
For pH, measurements, NMR spectra were processed
with a gaussian broadening factor of 0.02 and a line
broadening of -40 Hz to enhance resolution. For
assessment of bioenergetic status, NMR spectra were
processed with a line broadening of 20 Hz. Relative
phosphate metabolite concentrations were determined
3MP-NMR resonances after correcting for the differential saturation of resonances due
from the areas of the
Data Analysis
The net proton efflux rate (JH) during reperfusion was
calculated (in millimoles per liter per minute) by the
JH=18tr*dpHi!dt
where tot is total H' buffering capacity. We have
previously estimated the intrinsic H' buffering capacity
of the Langendorff-perfused ferret heart to be 37-42
mmol * 11 at pH, 6.85-6.95 and the buffering capacity
due to HCO3-/C02 (I3co2) to be -14 mmol .1 -.13 During ischemia and reperfusion, the hydrolysis and resynthesis of PCr will also contribute to H' buffering, which
we have estimated to be -8 mmol - 1', assuming 8090% hydrolysis of 15-20 mmol PCr and a pKa for Pi of
=6.8.3,14,25 Therefore, the values for 8to used in this
study were 62 mmol. 1 1 for hearts perfused with
HCO3--buffered medium and 48 mmol . 11 for hearts
perfused with HEPES-buffered medium. Values for
dpHi/dt were calculated from the slope of a linear
least-squares regression line fitted to pHi recovery data.
All results are quoted as mean±SEM. Statistical comparisons use the two-tailed unpaired Student's t test,32
with significance being taken at p<0.05.
Results
Effects of 10 Minutes of Ischemia
Ischemia leads to rapid cessation of oxidative phosphorylation. The concentration of ATP is initially maintained by a compensatory acceleration of anaerobic
glycolysis and hydrolysis of PCr, accompanied by accumulation of Pi (see Figure la). After reperfusion, PCr
was resynthesized, reaching 90% of the preischemic
value within 1.5 minutes, and there was a corresponding
decrease in Pi to preischemic levels in 1.5-2 minutes. The
changes in phosphate metabolite concentrations during
ischemia and reperfusion were similar in all hearts
described in this study and are illustrated for hearts
perfused with HCO3--buffered medium in Figure 2a.
The chemical shift of the Pi resonance (control position
indicated by dashed line, Figure la) indicates the fall in
pHi during 10 minutes of ischemia and its subsequent
recovery during reperfusion. The corresponding time
course of change in pH, obtained from the spectra illustrated in Figure la is shown in Figure lb. In hearts
perfused with HCO3 -buffered medium, there was no
significant change in pHi from the control value of
7.15±0.02 (n=10) during the first 2 minutes of ischemia;
thereafter, there was a monotonic fall in pH. to 6.73+0.03
(see Figure 2b). During the first 15 seconds of reperfusion,
there was no significant recovery of pHi. Subsequently,
pH, increased approximately linearly (dashed line, Figure
2b) at a rate of 0.25±0.03 pH units. minm. The rate of pHi
recovery appeared to slow after the first 1-1.5 minutes, but
accurate pHi determination during this period was not
Circulation Research Vol 72, No 5 May 1993
996
a)
a)
400-
I
d k\~J
'--J' <~
,J~
~
~
mins
reperfusion
O
+5
c
0hC
OD
CD CO
1~vv~j~#
tP
j
+ 2 mins
o
reperfusion
30
20
10
40
Time (mins)
end
ischemb
b)
7.2
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pHj 7.0
pre-ischemia
10
-20
-10
0
Chemical shift (ppm)
i0i
6.8
1
b)
7.2'
a*a.
Time (min)
7.0
pH i
15
10
5
0
c)
a
aa am
6.8-
5
0
a
10
LVDP
15
Time (min)
(%)
C)
150
LVDP
(mmHg)
Reperfusion
lsd"mia |
0
510
(mins)
FIGURE 1. Effects of 10 minutes of ischemia and 5 minutes of
reperfusion with HCO3--buffered medium at 30°C on pHi and
left ventricular developed pressure (LVDP) in the Langendorffperfused ferret heart. Panel a: Serial 31P nuclear magnetic
resonance spectra obtained at the times indicated. The peaks in
the spectra correspond to (from left to right) intracellular
inorganic phosphate (P,; dashed line at 5.03 ppm indicates a pH,
of 717), phosphocreatine, and the y-, a-, and a-phosphates of
ATP. Spectra are averages of 300 transients (preischemia and
+5 minutes of reperfusion) or 16-32 transients (during ischemia
and the first 2.5 minutes of reperfusion). Panel b: Plot of pH,
during 10 minutes of ischemia and the first 5 minutes of
Time
reperfusion; measurements were obtained from the chemical
shift of the Pi peak. Panel c: LVDP recording during the first 5
minutes of ischemia and first 5 minutes of reperfusion.
always possible because of the small Pi signal. Measurements of pHi from spectra acquired 2.5-7.5 minutes after
reperfusion indicated that pHi had returned to preisch-
emic levels in all hearts.
0
15
10
20
30
40
(mins)
FIGURE 2. Graphs showing effects of 10 minutes ofischemia
and subsequent reperfusion on hearts perfused with HCO3-buffered medium (mean +SEM, n=10). Panel a: Changes in
phosphate metabolite concentrations. PCr, phosphocreatine;
P,, inorganic phosphate. Panel b: Changes in pH. Panel c:
Changes in left ventricular developed pressure (LVDP). The
dashed line in panel b illustrates the slope from which dpHi/dt
Time
was
calculated.
During the first minute of ischemia, LVDP declined to
-50% of the preischemic value and then declined more
slowly, with contraction ceasing after -5 minutes (see
Figure lc). After reperfusion, there was an initially rapid
partial recovery of LVDP to 80% after 2 minutes. LVDP
subsequently recovered more slowly and did not return to
the preischemic value for 30-60 minutes (see Figure 2c).
pHi Recovery After Reperfusion
Contribution of lactate efflux. Lactate efflux during the
first 2 minutes in hearts perfused outside the magnet
Vandenberg et al pH; Recovery After Ischemia
a ugntuu
I
E 3:
1;
0.14
0.0
0-30
30-60
60-90
Time (secs)
FIGURE 3. Bar graph showing the effect of 4,4'-dibenzamidostilbene-2,2'-disulfonate (DBDS) on lactate efflux during
the first 90 seconds of reperfusion in hearts perfused with
HEPES-buffered medium (n=4, both groups).
(see "Materials and Methods") was 5.4±+1.0 gmol g
wt-1 (n=5) in hearts reperfused with HCO3-buffered medium and 4.1±0.5 ,umol g wet wt-1 (n=5)
in hearts reperfused with HEPES-buffered medium.
These values represent total lactate efflux, i.e., lactate
that has accumulated in the extracellular space during
ischemia in addition to lactate leaving cells during
reperfusion. In hearts perfused with HEPES-buffered
medium, the presence of 0.25 mM DBDS reduced
lactate efflux after reperfusion by 45% during the first
30 seconds and by 65-70% during the subsequent 60
seconds (see Figure 3). Perfusion of hearts placed in the
NMR spectrometer with either HCO3--buffered or
HEPES-buffered medium containing DBDS also
caused a slowing of pHi recovery (see Figure 4 and
Table 2), consistent with H'-coupled lactate efflux
contributing to acid extrusion after reperfusion.
Contribution of Na+-H+ antiport. To test whether the
residual pH, recovery seen in hearts perfused with
HEPES-buffered medium with lactate efflux inhibited
by DBDS was due to Na+-HW exchange, hearts were
perfused with medium containing 0.25 mM DBDS and 1
,uM EIPA for 2 minutes before ischemia and the first 5
minutes of reperfusion. A typical series of NMR spectra
obtained from a heart during blockade of the Na+-H+
antiport and inhibition of lactate efflux is illustrated in
Figure 5. During ischemia, PCr decreased and subse-
wet
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6.9
*
1
BICARB
BICARB
+
*
DBDS
HEPES
pHi
fT
HEPES
DBDS
+
6.6
10
11
Time (mins)
FIGURE 4. Graph showing the effect of 4,4'-dibenzamido-
stilbene-2,2'-disulfonate (DBDS, 0.25 mM)
on
pHI,
recovery
during the first minute of reperfusion in hearts perfused with
HCO3- (BICARB) -buffered (alone [a] and with DBDS [z])
or HEPES-buffered (alone [*1 and with DBDS [0]) medium.
997
quently recovered during reperfusion with corresponding changes in Pi (compare with Figure la). The Pi
resonance shift during ischemia indicated a fall in pHi
from 6.98 (dashed line in Figure 5) to 6.59. During
reperfusion, the chemical shift of the Pi resonance did
not alter, indicating that pHi had not recovered in this
experiment. Simultaneous inhibition of lactate efflux
and Na+-H+ exchange slowed pHi recovery from
0.17+0.02 pH units * min' (hearts perfused with
HEPES-buffered medium) to 0.03±+ 0.02 pH
units. min' (see Figure 6 and Table 2). The contribution of the Na+-H+ antiport to recovery of pHi with
lactate efflux mechanisms intact was assessed by adding
EIPA to HCO3-- or HEPES-buffered medium for 2
minutes before ischemia and during the first 5 minutes
of reperfusion. EIPA (1 ,M) caused a small reduction
in the rates of pHi recovery under both conditions
(hearts perfused with HCO3--buffered medium,
0.1<p<0.2; hearts perfused with HEPES-buffered medium, 0.05<p<0.1; see Table 2).
Contribution of external HCO3 -dependent mechanisms. In three experiments, hearts were perfused with
HCO3--buffered medium containing 20 ,uM DIDS for
2-10 minutes before ischemia and during 5 minutes of
reperfusion. In the presence of DIDS, pHi fell from
7.07+0.03 to 6.64+0.04 during ischemia, and after
reperfusion, pH, recovered at an initial rate of
0.17±0.03 pH units min`1 (p<0.05 compared with
initial rate of pHi recovery in the absence of DIDS). In
a second series of experiments, five hearts were perfused with HEPES-buffered medium before ischemia
but reperfused with HCO3--buffered medium. After 10
minutes of ischemia in HEPES-buffered medium, there
should be minimal accumulation of CO2 and therefore
minimal CO2 efflux after reperfusion. However, reperfusion with HCO3--buffered medium will permit HCO3influx to contribute to pH, recovery. By use of this
protocol, illustrated in Figure 7, pHi fell from 6.98 ±0.03
to 6.64±0.02 during ischemia. Reperfusion with
HCO3--buffered medium caused an immediate further
decrease in pHi to 6.62+0.02, consistent with an initial
influx of CO2, before pHi recovered at 0.19±0.02 pH
units . min' (see Table 2).
Contribution of CO2 effiux. The contribution of CO2
efflux was assessed by the inhibition of lactate efflux by use
of DBDS, Na+-H+ exchange by use of EIPA, and HCO3influx by preperfusion with 20 ,uM DIDS for 30 minutes in
hearts perfused with HCO3--buffered medium.13 The isothiocyanate group on DIDS reacts with free amino
groups,21,24 such that DIDS will precipitate with EIPA if
they are mixed in solution. DIDS binds covalently to
HCO3- transporters, causing irreversible inhibition of
HCO3- transport.24 Simultaneous inhibition of HCO3transport and Na+-H+ exchange can therefore be achieved
by preperfusion of hearts with DIDS, followed by addition
of EIPA13 or amiloride.1142 During simultaneous inhibition of lactate efflux, Na+-H+ exchange, and HCO3- influx,
pHi recovery after reperfusion was reduced to 0.11±0.03
pH units . min' (p<0.05 compared with hearts perfused
with HCO3- medium).
An indirect estimate of the potential contribution of
CO2 efflux to pHi recovery was also obtained using the
reverse perfusion sequence to that described above for
estimating HCO3- influx. That is, hearts were perfused
with HCO3--buffered medium before ischemia but reper-
998
Circulation Research Vol 72, No 5 May 1993
TABLE 2. Changes in pHi During Ischemia and Reperfusion
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n
Perfusion medium
Hearts reperfused with HCO,--buffered medium
10
HCO34
HCO3- +DBDS
5
HCO3-+EIPA
3
HCO- +DIDS
5
HCO3 +DBDS+EIPA (after DIDS)
Hearts reperfused with HEPES-buffered medium
9
HEPES
6
HEPES+DBDS
7
HEPES+EIPA
5
HEPES+EIPA+DBDS
Hearts perfused with HEPES- or HCO3--buffered
medium before ischemia and reperfused with HCO3- or
HEPES-buffered medium, respectively
4
HCO3- to HEPESt
5
HEPES to HCO31§
Preischemic
pH,
End-ischemic
dpHi/dt
pHi
(at pHi 6.80)
7.15+0.02
7.13+0.01
6.73+0.04
0.25+0.02
0.19-+-0.02
0.20 -'-0.03
0.17±0.03
0.11±0.03*
6.75+0.05
6.77+0.01
JH
(at pHi 6.80)
15 5
11. 8
.4
.2
7.12+0.01
7.07±0.03*
7.09+0.02
6.64+0.04
7.07±0.02
7.09±0.01
6.98±0.02*
6.97±0.02*
6.64±0.03
6.67±0.03
6.62±0.02
6.58±0.03
0.17±0.03
0.06±0.01*
0.13±0.02
0.03±0.02,
2.9 0.6
6.2±0.9
1.4± 108
7.15±0.02
6.98±0.04
6.70±0.0W-
0.25±0.02
0.19±0.02
12±1.0
12±1 .1
6.72+0.02
6.64+0.02
7+1,8
10.6-1.9
6.8±I.8*
8.2±1.
JH, net H+ efflux rate; DBDS, 4,4'-dibenzamidostilbene-2,2'-disulfonate; EIPA, 5-(N-ethyl-N-isopropyl)amiloride; DIDS, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. Values are mean±SEM. JH was calculated by multiplying the measured dpHj/dt by the calculated total
H+ buffering capacity (see text for discussion).
*p<0.05 compared with control in each group (Student's unpaired t test).
tpHi did not recover to 6.80 during the first 2 minutes of reperfusion; therefore, the dpH./dt reported is that calculated during 1-2 minutes
of reperfusion.
tFor these groups, total H+ buffering capacity was assumed to be the same as that in hearts perfused with HEPES-buffered medium (see
text for discussion).
§For these groups, total H' buffering capacity was assumed to be the same as that in hearts perfused with HCO3--buffered medium (see
text for discussion).
fused with HEPES-buffered medium, which prevented
HCO3- influx during reperfusion but still permitted CO2
efflux. In this series of experiments, pH- fell from
7.15+0.02 to 6.70±0.03 (n=4) during ischemia and sub-
J
't
%
,
+ 120 secs
reperfusion
f,'
sequently recovered at 0.25 ±0.03 pH units minm after
reperfusion with HEPES-buffered medium.
H2PO4- efflux. To test the hypothesis that Na+H2P04- efflux contributes to net H' efflux during reperfusion, Pi was assayed in effluents. In control hearts
(n =5) equilibrated with Pi-free buffer for 60 minutes, Pi
efflux was <0.1 ,umol* g wet wt-'. min-l. Pi in effluent
collected during the first 2 minutes of reperfusion in
hearts ranged from <0.1 ,mol * g wet wt` (n =5) to 0.3
+ 75 secs
reperfusion
7.1
j
+
HEPES
S
IA
t/\~~~~~~~~~
a+3
s e +3 s
reperfusion
6.9.
pH1
itJn
e
j
ischemia
6.7.
HEPES
+ EIPA
+ DBDS
1
i
pre-ischemia
10
0
-10
-20
Chemical shift (ppm)
FIGURE 5. 31P nuclear magnetic resonance spectra obtained
from a heart perfused with HEPES-buffered medium with
lactate efflux inhibited by 4,4'-dibenzamidostilbene-2,2'-disulfonate (0.25 mM) and the Na+-H+ antiport inhibited by
5-(N-ethyl-N-isopropyl)amiloride (1 ,uM). Peak assignments
are as indicated for Figure la. The dashed line (at 4.79 ppm)
in the enlarged section (from 6 to 3 ppm) indicates a pHi of
6.98.
r: c;
u.;
.lI
V-
0
Reperfusion
5
10
15
Time (mins)
FIGURE 6. Time course of averaged changes in pHi during
10 minutes of ischemia followed by reperfusion of hearts with
HEPES-buffered medium (a) or HEPES-buffered medium
plus 4,4'-dibenzamidostilbene-2,2'-disulfonate (DBDS, 0.25
mM) plus 5-(N-ethyl-N-isopropyl)amiloride (EIPA, 1 gM)
(w).
Vandenberg et al pHi Recovery After Ischemia
999
7.2~
a
.G
1
* BICARB
1
7.0O
BICARB+DIDS
* HEPES
a
pH1
81
i
-J
A
6.8-
eperfusionj|
6.6
iWSEA
0
5
10.0
HCO3-
10
12.5
15.0
Time (mins)
15
Time (mins)
FIGURE 7. Time course of averaged changes in pHi in five
hearts perfused with HEPES-buffered medium before ischemia followed by reperfusion with HCO3--buffered medium.
This protocol was used to estimate the contribution of HCO3influx to the recovery of pH, (see text for details).
C
a.
* HEPES
o HEPES+DBDS
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
A
gmol * g wet wt- . The corresponding H' efflux rate of
<0.26 mmol I`. min-1 is small compared with the
contributions from the other mechanisms (see below).
Relative Contributions of Lactate Efflux,
Na+-H+ Exchange, HCO3- Influx, and CO2
Efflux to pH, Recovery
The changes in pHi during ischemia and reperfusion
for all hearts included in this study are shown in Table
2. Where possible, net H' efflux rates have been calculated (as described in "Materials and Methods") at a
pHi of 6.80 rather than at the pHi immediately after
reperfusion. Although the values for buffering capacity
were obtained at slightly higher pHi,13 there should not
be a significant discrepancy over this small range of pH,
values.12 The H' efflux rates are shown in the final
column of Table 2. The relative contributions of the
different acid extrusion mechanisms to net H' efflux
were estimated by comparing the differences in H'
efflux rates in the presence or absence of inhibitors of
each mechanism. CO2 and lactate efflux each accounted
for 30-50% of net H' efflux at pHi 6.80, whereas
Na+-H+ exchange and HCO3- influx each contributed
10-30% (see Table 2).
Recovery of LVDP After Reperfusion
During the first 5 minutes of reperfusion, LVDP
recovered to 30-80% of the preischemic LVDP (see
Figure 8 and Table 3). The most striking differences
occurred between hearts perfused with HCO3--buffered
medium (±EIPA or DBDS) and those perfused with
HEPES-buffered medium (±EIPA or DBDS). For example, in HCO3--buffered medium, LVDP recovered to
79±3% (n=10) after 2 minutes of reperfusion compared with 32±2% (n=9) at the same time point in
HEPES-buffered medium.
In hearts perfused with HEPES-buffered medium in
the presence of DBDS and EIPA, there was a significant recovery of LVDP to 29+2% (n=5) of the preischemic value after 2 minutes of reperfusion, in the absence of any significant recovery of pHi (see Figure 6).
During the same period, however, there was recovery of
PCr and Pi to preischemic levels (see Figure 5). LVDP
HEPES+DBDS+EIPA
0*6
10.0
12.5
15.0
Time (mins)
FIGURE 8. Graphs showing left ventricular developed pressure (LVDP) plotted against time. BICARB, HCO3-; DIDS,
4,4'-diisothiocyanostilbene-2,2'-disulfonic acid; DBDS, 4,4'-
dibenzamidostilbene-2,2'-disulfonate; EIPA, 5-(N-ethyl-Nisopropyl)amiloride. Recovery of LVDP (expressed as percentage of preischemic level) during the first 5 minutes of
reperfusion is shown for the experimental groups indicated:
hearts reperfused with BICARB-buffered medium (alone [i]
and with DIDS [A]) and hearts reperfused with HEPESbuffered medium (alone [of, with DBDS [w:], and with
DBDS plus EIPA [A]).
did not reach preischemic levels during the first 5
minutes of reperfusion in any experimental group, despite the recovery of pHi and restoration of PCr and Pi
to preischemic levels during this time. Full recovery of
LVDP took 30-90 minutes.
Discussion
Metabolic Effects of Ischemia
In the experiments reported here, [ATP]i remained
unaltered from the preischemic value after 10 minutes
of global ischemia. Therefore, it is very likely that the
hearts have suffered only mild and reversible damage
(see Reference 2 for review). In addition, there was no
splitting of the Pi resonance observed in these experiments, which suggests that there was no substantial
inhomogeneity with respect to pHi within the globally
ischemic myocardium, a phenomenon that has been
observed after more prolonged periods of ischemia.17
pHi During Ischemia and Reperfusion
During the first 1-2 minutes of ischemia, pH, remained
unaltered from the preischemic pHi in all experiments
before falling =0.4 pH units after 10 minutes of ischemia.
This pH, fall is less than has been reported from in vivo
studies and in isolated hearts perfused at 37°C (0.6-1.0 pH
units after 10 minutes).16,33 This may be explained by the
1000
Circulation Research Vol 72, No S May 1993
TABLE 3. Changes in Left Ventricular Developed Pressure During Reperfusion After 10 Minutes of Global
Ischemia
Perfusion medium
Hearts reperfused with HCO,--buffered medium
HCOHCO3- +DBDS
HCO0-- +EIPA
HCO,- +DIDS
HC03-- +DBDS+EIPA (after DIDS)
Hearts reperfused with HEPES-buffered medium
HEPES
HEPES+DBDS
HEPES+EIPA
n
10
4
LVDP (% of preischemic level)
2-Minute REP
5-Minute REP
30-Minute REP
3
5
79+3
66±5
90±2
66+6
73+10
59±7
61+ 11
90+3
91±4
91+2
81±6
92±9
9
6
7
5
32±2
39±2
30±5
29±2
51±3
50±4
31±4
29+3
93±4
100±6
74±6
87+3
Hearts perfused with HEPES- or HC03--buffered
medium before ischemia and reperfused with HCO -or HEPES-buffered medium, respectively
HCO3 to HEPES
4
HEPES to HCO35
100±7
66+5
90±+8
74+2
61 +4
115+6
HEPES+EIPA+DBDS
78+3
76±3
76+2
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
LVDP, left ventricular developed pressure; REP, reperfusion; DBDS, 4,4'-dibenzamidostilbene-2,2'-disulfonate:
EIPA, 5-(N-ethyl-N-isopropyl)amiloride; DIDS, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. Values are
mean±SEM and are expressed as percentage of preischemic level in each group.
slower rates of ATP utilization and anaerobic glycolysis
during ischemia at 30°C. Most groups have seen a
monophasic fall in pHi during early ischemia,3334 but
others have noted that the fall in pH; is slower in the first
minute than subsequently,'6 and during hypoxia a transient alkalosis has been described.25 The discrepancy in
these observations has been attributed to differences in
the relative rates of proton production by ATP hydrolysis'4
and breakdown of PCr, which absorbs protons and produces Pi, which increases the buffering capacity of the
heart.'1425
Buffering Capacity After Reperfusion
To compare H' efflux rates under different perfusion
conditions, it is necessary to consider both the rate of
pHi recovery as well as the buffering capacity.35 The
value for 3itot (62 mM in hearts perfused with HCO3-buffered medium) was obtained from the sum of intrinsic buffering capacity (:40 mmol 1- 1),13 l3CO2 (14
mmol 1 1),13 and biochemical buffering due primarily to
accumulation of p,14 (-8 mmol I1 1). The fot value used
in our study is similar to that obtained by Wolfe et all5
(~58 mmol 1' between pHi 6.4 and 6.9) in the ischemic rat myocardium. These values were derived from
hearts exposed to an acid load (i.e., either using an
NH4Cl prepulsel3 or lactic acid accumulation during
ischemia15). During reperfusion, however, the ability of
the heart to resist a rise in pHi will not necessarily be the
same as its ability to resist a fall in pHi.
Intrinsic buffering capacity primarily reflects the
presence of histidyl residues of intracellular proteins.35
We have assumed that after a brief episode of ischemia
this has not changed, and so we used the value derived
from the well-perfused ferret heart, i.e., 40 mM.13
During reperfusion, the ability of intracellular CO2/
HCO3- to resist a rise in pHi (i.e., via the dissociation of
H2C03 to H' and HCO3) is dependent on the presence
of a constant extracellular C02.3 This is similar to the
situation in the well-perfused heart, and so we have
used the value derived during normal perfusion, i.e.,
14 mM. 13 This possibly represents an underestimate of
the 13co2 during reperfusion, because the [HCO3-]' in the
heart at end ischemia is likely to be higher at a given pH,
than the value in the well-perfused heart (see discussion
in Reference 15). Nevertheless, our estimate of the
contribution of C02/HCO3- (23% of I,3to) is similar to
that estimated by Wolfe et al'5 during ischemia in the
rat heart (21% at pHi 6.9). The [HCO3-1i estimated in
cardiac tissue superfused with HEPES-buffered medium is =0.5 mM.36 At this concentration, /3co2 would be
small (< 1.2 mM); therefore, we have ignored this value
in our estimate of A3Oto in hearts reperfused with HEPESbuffered medium. The contribution of PCr hydrolysis
and Pi accumulation was based on estimates of concentrations of these metabolites at the time of reperfusion
and shortly thereafter.3.25 Because we did not find any
significant differences in phosphate levels in hearts
perfused with different media, we used the same value
for all hearts. During ischemia, lactate will contribute to
3otot and its contribution will be greater than would be
suggested by the low pK. of lactate (=3.8), because
lactate is distributed in the intracellular as well as
extracellular space.15 However, during reperfusion the
ability of lactate to resist increases in pHi would be
dependent on the presence of a constant extracellular
lactate, which is not the case; therefore, its contribution
to /,, will be negligible during reperfusion.
Recovery of pH, After Reperfusion
Contribution of lactate effiux. Conversion of lactate
efflux (in micromoles per gram wet weight) to a corresponding H' efflux (in millimoles per liter intracellular
fluid) was calculated by assuming that the stoichiometry
of H+-lactate efflux is 1:135 and that the intracellular fluid
volume is 0.43 ml * g wet wt-.37 We have also taken into
account that total lactate efflux includes intracellular
Vandenberg et al pH; Recovery After Ischemia
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
lactate that is washed out during reperfusion as well as
lactate that accumulated in the extracellular space during
ischemia, which Wolfe et al15 measured to be 15-50% of
total lactate efflux. In hearts perfused with HCO3-buffered medium, lactate efflux during the first 2 minutes
corresponded to a net H' efflux of 6.4-10.8 mmol l
intracellular fluid-', and in hearts perfused with HEPESbuffered medium, the value was 4.8-8.2 mmol 1 intracellular fluid-l. If the rate of lactate efflux was constant
during this time, then the corresponding H' efflux rates
would be =4.3 and =3.3 mmol I`- min-1 in hearts
perfused with HCO3-- and HEPES-buffered media, respectively. The data presented in Figure 3 suggest that
the rate of lactate efflux was probably constant for the
first minute of reperfusion but subsequently decreased.
The above estimates of the contribution of H+-coupled
lactate efflux to pHi recovery (3.3-4.3 mmol * I`. min-m )
are therefore probably underestimates.
Lactate efflux is inhibited by a number of compounds,
including cinnamic acid derivatives such as a-cyano-4hydroxycinnamate19,38 and stilbene derivatives such as
DBDS.21,22 DBDS does not cross the sarcolemmal membrane and therefore has the advantage that it will not
affect mitochondrial monocarboxylate uptake while inhibiting lactate efflux.22 In our experiments, DBDS (0.25
mM) reduced lactate efflux by 45% during the first 30
seconds and 65-70% during the subsequent 60 seconds
after reperfusion. The lactate assayed in the first 30
seconds will include lactate washed out from both the
extracellular and intracellular spaces. Although DBDS
will slow the rate of lactate efflux during ischemia over
a 10-minute period, one would still anticipate a substantial accumulation in the extracellular space. Therefore,
it is likely that the latter values (65-70%) are a better
estimate of the extent of inhibition of lactate efflux by
0.25 mM DBDS after reperfusion. This is intermediate
between the values estimated by Wang et a122 for
inhibition of sarcolemmal lactate transport by DBDS in
rat and guinea pig ventricular myocytes (50% and 80%,
respectively). Incomplete inhibition of lactate efflux by
DBDS could be due to simple diffusion of undissociated
lactic acid or to the presence of other lactate transporters that are not inhibited by DBDS, as appears to be the
case in the cardiac sarcolemma of rats and guinea pig.22
The contribution of lactate efflux to pHi recovery is
therefore likely to be P80% of net H' efflux in hearts
reperfused with HEPES-buffered medium and '-40% in
hearts reperfused with HCO3--buffered medium.
Contribution of Na+-H+ antiport. EIPA (1 ,uM) slowed
net H' efflux by P2 mmol I.l*min-l. This dose of
EIPA has been shown to cause significant inhibition of
the Na+-H+ antiport in the ferret heart after acid
loading using the NH4C1 prepulse technique.13 Furthermore, in hearts perfused with HEPES-buffered medium
containing DBDS to inhibit lactate efflux, blockade of
the Na+-H+ antiport almost completely abolished pH1
recovery (see Figure 7). The residual pHi recovery
under these conditions may be explained in a number of
ways. First, there is likely to have been incomplete
inhibition of lactate effilux (as discussed above). Second,
oxidation of lactate trapped within the cell would also
remove protons. Third, although these hearts are nominally HCO3- free in cardiac tissue perfused with
HEPES-buffered medium, [HCO3- ] has been estimated
to be =0.5 mM,36 and if all this HCO3- reacted with H'
1001
and was then washed out as CO2 during the first minute
of reperfusion, it would contribute 0.5 mmol * I 1 min'
to total net H' efflux.
HCO3--Dependent pHi recovery. In hearts perfused with
HCO3--buffered medium, pHi recovery was significantly
faster than in hearts perfused with HEPES-buffered medium (p<0.01). The faster rate of lactate efflux in hearts
perfused with HCO3--buffered medium can account for
only 10-15% of the additional net H' efflux compared
with hearts perfused with HEPES-buffered medium,
which suggests that CO2 efflux and/or HCO3- influx contribute to pHi recovery. It is important to separate the
relevative contributions made by CO2 efflux and HCO3influx, because the HCO3- influx mechanism is Na+ dependentll-'3 and thus may contribute to Na+ overload
after reperfusion.39
Inhibition of HCO3 influx by use of DIDS (20 ,uM)
reduced net H' efflux by 4.9 mmol * 1 * min '. However, DIDS also partially inhibits lactate transport21"22;
therefore, some of the effect of DIDS on net H' efflux
may have been mediated by inhibition of lactate efflux.
When hearts were preperfused with DIDS and then
perfused with EIPA and DBDS, pHi recovery after
reperfusion, which may be attributed predominantly to
CO2 efflux, was 6.2+2.6 mmol. l-1 min '. However,
this value is likely to be an overestimate because of the
incomplete inhibition of other H' extrusion mechanisms (see above).
Estimates of HCO3 influx and CO2 efflux were also
obtained from hearts perfused with either HEPES or
HCO3--buffered medium before ischemia but reperfused
with HCO3-- and HEPES-buffered media, respectively
(see Figure 7 and Tables 1 and 2). These estimates are
associated with uncertainties that are due to the time
taken to change perfusion media and also to the additional
acid load or washout caused by changes in Pco2 when
switching between HEPES- and HCO3--buffered media.1240 This latter problem has to an extent been taken
into account by including the presence or absence of
C02/HCO3- during reperfusion in the calculation of Ax.
For example, in hearts reperfused with HCO3--buffered
medium, CO2 influx at the point of reperfusion would
resist rises in pHi, thereby increasing the effective buffering capacity of the heart. Estimates of the relative contributions of CO2 efflux and HCO3- influx from the different
protocols discussed above (3.5-6.2 and 3.5-4.9
mmol l -*min-', respectively) would suggest that CO2
efflux and HCO3- influx are of similar importance, each
contributing 20-25% to total H' efflux.
Under the conditions of our experiments, pH, recovery after a brief episode of ischemia is principally
mediated by washout of metabolites, specifically lactate
and CO2 (see Figure 9), as suggested by Bailey et al.17
However, we have also shown that Na+-H+ exchange
and HCO3- influx may contribute as much as 20-35% of
net H' efflux. At 30°C, the rate of H' efflux is slower
than at 37°C (data not shown), but in four experiments
performed at 37°C, the relative rates of H' efflux in
hearts perfused with HEPES- or HCO3--buffered medium were similar to those at 30°C. Therefore, it is likely
that at 37°C the relative contributions to H' efflux of the
four mechanisms described above will be similar to that
in our study. Extrapolating our results to reperfusion
after prolonged ischemia is more difficult. For example,
ATP depletion may have differential effects on the
1002
Circulation Research Vol 72, No 5 May 1993
HCO3Na+
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
FIGURE 9. Schema for recovery of pHi after ischemia in the
mammalian heart. Thick lines indicate major H' efflux
pathways (metabolite washout). Cl--HCO3- exchange
(dashed line) will be inoperative at low pH18 and thus will not
contribute significantly to pHi recovery after reperfusion.
active H' extrusion mechanisms (Na+-H+ exchange and
Na+-HCO3- coinflux) compared with passive H' extrusion mechanisms (lactate and CO2 efflux).
Ischemic Contractile Failure
LVDP has been shown in many studies to be determined by multiple factors,294' including pH.42 In our
experiments, the fall in LVDP during the first 1-2 minutes
occurred before there was a significant fall in pH, (Figures
1 and 2). The accumulation of Pi during this initial period
(Figure 2a) could probably account for 20-30% of the
observed fall in LVDP.3,29 In the isovolumic Langendorffperfused heart, there is evidence to suggest that vascular
collapse contributes to early contractile failure during
ischemia4'; this could explain the fall in LVDP seen during
the first 30-60 seconds of ischemia in our experiments.
Our results are therefore Consistent with the hypothesis of
Lee and Allen3 that the sequence of events leading to
ischemic contractile failure is vascular collapse followed by
Pi accumulation, with a significant contribution from pH,
only occurring later.
Recovery of LVDP After Reperfusion
In hearts perfused with HEPES-buffered medium with
lactate efflux and Na+-H+ exchange inhibited, there was
partial recovery of LVDP despite minimal pH, recovery
(see Figure 9). This demonstrates that factors other than
pHi, such as vascular refilling4' and decreasing p, 29 also
contribute to LVDP recovery. In hearts perfused with
HCO3--buffered medium (+EIPA, DIDS, and DBDS),
there was an initially rapid recovery of LVDP (reaching
>60% of the preischemic level within 2 minutes in all
groups), which suggests that pHi recovery due to CO2
efflux contributes to the initial recovery of LVDP. It is
difficult to estimate the possible contributions of other
acid extrusion mechanisms to the initial recovery of LVDP
from our data. For example, EIPA caused a slowing of
LVDP recovery in hearts perfused with HEPES-buffered
medium but augnented the initial recovery of LVDP in
hearts perfused with HCO3--buffered medium. Conversely, inhibition of lactate efflux augmented the initial
recovery of LVDP in hearts perfused with HEPES-buffered medium but slowed recovery of LVDP in hearts
perfused with HCO3--buffered medium. Simultaneous
measurement of [Na+]i and [Ca`], in addition to pHi and
P, will be required to determine the relative contributions
of these mechanisms to the recovery of LVDP during
reperfusion.
In hearts under all experimental conditions, recovery of
LVDP was never complete before 30 minutes of reperfusion, despite the recovery of pHi and high-energy phosphates after 5 minutes. This phenomenon, termed stunning, has been extensively studied.' In 1985, Lazdunski et
al43 hypothesized that, during reperfusion, activation of
the Na+-H+ antiport would lead to Na` overload and
consequently Ca>2 overload, the putative mediator of
stunning (see Reference 5 for review). Although our
results show that H' efflux during reperfusion is principally mediated via lactate and CO2 efflux, there is a
contribution from Na+-coupled net HI- efflux (Na+-HCO3
coinflux and Na+-H+ exchange). A small increase in [Na+],
will lead to a significant rise in [Ca2+]i because of the 3:1
Na+: Ca> stoichiometry of the Na+-Ca' exchanger44
unless there is a compensatory increase in Na+ efflux via
Na+ ,K+-ATPase. In our experiments, Na+ influx, coupled
to acid extrusion, during reperfusion was -5 mmol *l(sum of Na+-H+ exchange and Na+-HCO33 coinflux).
During normal perfusion, Na+ efflux via Na+,K+-ATPase
is proportional to [Na+]i and will rise from 0.4 mmol l
(at a control value for [Na+]i of -6 mM) to 4 mmol . p at
[Na+]i of -12 mM (calculated using the computer program HEART, version 3.6, Oxsoft Ltd., Oxford, UK44). Pike
et a145 showed that [Na+]i increases by 5-100% after 10
minutes of ischemia in the Langendorif-perfused ferret
heart, which would be consistent with an increase from -'6
to :12 mM. Assuming that Na+,K+-ATPase has not been
inhibited, then these rough calculations would suggest that
after reperfusion the myocyte may be able to extrude Na`
as rapidly as it is entering. However, even if Na+ influx
linked to acid extrusion does not contribute to a further
rise in [Na+Ji, it will cause [Nat], and hence [Ca 2,i to
remain elevated during early reperfusion.
Many previous studies have shown that inhibitors of
Na+-H+ exchange have beneficial effects on the recovery of cardiac contractility and normalization of [Ca 2+]
after myocardial ischemia (e.g., see References 46-48)5
thus suggesting that Na+-coupled acid extrusion may
contribute significantly to [Ca>], overload after reperfusion. Although no studies have specifically looked at
the effects of inhibition of Na+-HCO3- influx on the
recovery of contractile function and normalization of
[Ca]2+i after reperfusion, our results suggest that this
may be a more significant route for Na+ entry after
reperfusion. Unfortunately, DIDS would not be an
appropriate inhibitor to use in such studies, because it
has nonspecific deleterious effects on contraction if used
in high doses for long periods.13
Conclusions
After brief episodes of ischemia, the recovery of pH,
is principally mediated by metabolite washout, i.e.,
lactate and CO2. Nevertheless, Na+-coupled acid extrusion does contribute to net H+ efflux, with Na+-HCO3coinflux likely to be more important than Na`-H+
exchange. The associated Na+ entry may contribute to a
Vandenberg et al pH; Recovery After Ischemia
rise in [Na']i and therefore Ca2' overload via Na+-Ca2'
exchange after reperfusion.
Acknowledgments
We wish to thank Dr. Andrew Halestrap and Dr. Robert
Poole for many valuable discussions, criticism of the manuscript, and providing the DBDS; Ms. Glenna Bett for assistance with the computer simulations; Dr. Martyn Plummer for
his helpful advice on statistical analysis; and the Department
of Clinical Biochemistry, Addenbrookes Hospital, Cambridge,
UK, for performing the lactate assays. The computer program
HEART may be obtained from Oxsoft Ltd., 49 Old Road,
Oxford OX3 7JZ, UK.
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J I Vandenberg, J C Metcalfe and A A Grace
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Circ Res. 1993;72:993-1003
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