Age-related differences in Na+-dependent
Ca2+ accumulation in rabbit hearts exposed to hypoxia
and acidification
S. E.
Anderson,
H.
Liu,
H. S.
Ho,
E. J.
Lewis, and
P. M.
Cala
Department of Human Physiology, University of California,
Davis, California 95616-8644
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ABSTRACT |
In this study, we test the hypothesis
that in newborn hearts (as in adults) hypoxia and acidification
stimulate increased Na+ uptake, in part via pH-regulatory
Na+/H+ exchange. Resulting increases in
intracellular Na+ (Nai) alter the force driving
the Na+/Ca2+ exchanger and lead to increased
intracellular Ca2+. NMR spectroscopy measured
Nai and cytosolic Ca2+ concentration
([Ca2+]i) and pH (pHi) in
isolated, Langendorff-perfused 4- to 7-day-old rabbit hearts. After
Na+/K+ ATPase inhibition, hypoxic hearts gained
Na+, whereas normoxic controls did not [19 ± 3.4 to
139 ± 14.6 vs. 22 ± 1.9 to 22 ± 2.5 (SE) meq/kg dry
wt, respectively]. In normoxic hearts acidified using the
NH4Cl prepulse, pHi fell rapidly and recovered,
whereas Nai rose from 31 ± 18.2 to 117.7 ± 20.5 meq/kg dry wt. Both protocols caused increases in [Ca]i;
however, [Ca]i increased less in newborn hearts than in
adults (P < 0.05). Increases in Nai and
[Ca]i were inhibited by the
Na+/H+ exchange inhibitor
methylisobutylamiloride (MIA, 40 µM; P < 0.05), as
well as by increasing perfusate osmolarity (+30 mosM) immediately before and during hypoxia (P < 0.05). The data support
the hypothesis that in newborn hearts, like adults, increases in
Nai and [Ca]i during hypoxia and after
normoxic acidification are in large part the result of increased uptake
via Na+/H+ and Na+/Ca2+
exchange, respectively. However, for similar hypoxia and acidification protocols, this increase in [Ca]i is less in newborn than
adult hearts.
newborn heart; intracellular Na+, Ca2+, and
pH
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INTRODUCTION |
MANY STUDIES HAVE SHOWN
the newborn heart is less susceptible to hypoxia-induced
dysfunction and damage than the adult. For example, mechanical
function, high energy phosphates, and enzyme release are altered less
by hypoxia in the newborn heart than the adult (23, 24,
39). However, no unifying hypothesis for the mechanisms of
hypoxic injury, much less its age-related variations, has been
accepted. We and others have reported results obtained from studies of
adult and newborn hearts consistent with the general hypothesis that
hypoxia/ischemia stimulates pH-regulatory Na+/H+ exchange, which increases net
Na+ uptake and thereby leads to reduction of the
transmembrane Na+ gradient and, consequently, increases
Ca2+ uptake via Na+/Ca2+ exchange
(2, 4, 30, 46, 47). This hypothesis states that increased
Na+ uptake is the first step toward
hypoxic/ischemic injury in that it gives rise to increased
intracellular Na+ (Nai), Ca2+
(Cai), and ATP consumption (30).
If our hypothesis is correct, age-related differences in response to
myocardial hypoxia are likely to be the result of age-related differences in Na+-dependent Ca2+ accumulation.
Given the scenario described above, this could arise from differences
in proton production and/or ion transport through the
Na+/H+ and/or Na+/Ca2+
exchangers. Here, we report the results of testing the general hypothesis in newborn hearts and compare the results with those from
the adult. Newborn hearts were exposed to hypoxia or NH4Cl washout (9) to stimulate pH-regulatory
Na+/H+ exchange under hypoxic and normoxic
conditions, respectively. Nai, Cai, and
intracellular pH (pHi), as well as high-energy phosphates, were measured using NMR. To our knowledge, this is the first report including measurement of all three ions in intact newborn hearts during
hypoxia and after normoxic acidification.
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METHODS |
General.
The methods used were modified from those previously reported (1,
4, 22). New Zealand White rabbits (newborn, 4-7 days;
adult, 11-14 wk) were anesthetized with pentobarbital sodium (35-65 mg/kg) and heparinized (1,000 USP units/kg). Hearts were removed and perfused at a constant rate (9-10 ml/min for newborns; 27-29 ml/min for adults) at 23-25°C. Control perfusate
contained (in mM): 133 NaCl, 4.75 KCl, 1.25 MgCl2, 1.82 CaCl2, 25 NaHCO3, (or 20 HEPES, 8 NaOH), and
11.1 dextrose. 23Na, 19F, and 31P
NMR were used to measure Nai, Cai,
pHi, and high-energy phosphates, respectively. To measure
Nai, 15 mM dysprosium triethylenetetraminehexaacetic acid
(DyTTHA) was substituted iso-osmotically for NaCl in the perfusate, and
Ca2+ was added to reach a perfusate concentration of
1.8-2 mM as measured by Ca2+ electrode. To measure
Cai, hearts were loaded during the control interval
(30-40 min) with perfusate containing the acetoxymethyl ester of
5-fluoro-1,
2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (FBAPTA) at 2.5 µM for newborns or 5 µM for adults
(26). FBAPTA was then washed out of the extracellular
space with control solution for 15 min before measurement of
Cai. Perfusates were titrated to pH 7.35-7.45 and
equilibrated with 95% O2-5% CO2 or 95%
N2-5% CO2 (100% O2 or 100%
N2 with HEPES) for normoxic and hypoxic conditions,
respectively. The latter provided a PO2 at the
aorta of 20 ± 1 torr during hypoxic perfusion. To discriminate between changes in dissipative Na+ uptake and active
Na+ extrusion, Na+ efflux via
Na+/K+-ATPase was inhibited by removal of KCl
from the perfusate (osmotic substitution with sucrose) (4,
45).
Because the precipitating event for the observed responses is
hypothesized to be a decrease in pHi, we also used normoxic acidification to test the hypothesis. Normoxic acidification was achieved using the NH4Cl prepulse (9), which
consisted of 1) 10-15 min of control perfusion,
2) 40 min of perfusion with perfusate to which 20 mM
NH4Cl was added, 3) 5 min of perfusion with
K+-free perfusate to which 20 mM NH4Cl was
added, 4) 30 min of K+-free perfusion without
NH4Cl, and 5) 30-40 min of perfusion with normal K+ control perfusate.
To identify the pathway responsible for changes in Na+
uptake, methylisobutylamiloride (MIA, 40µM), a known inhibitor of
pH-regulatory Na+/H+ exchange
(27), was added during hypoxia or NH4Cl
washout. Hypertonic perfusion has also been shown to diminish
Na+ accumulation during hypoxia (22). To test
for this effect in newborn hearts, another set of experiments was
conducted in which 30 mosM of sucrose was added to all perfusates,
beginning with the K+-free portions of the hypoxic and
NH4Cl washout protocols.
After perfusions were complete, hearts were weighed wet and dried to
constant weight (at least 48 h) at 65°C to determine dry weight.
NMR spectroscopy.
23Na and 31P experiments were conducted using a
Bruker AMX400 spectrometer, and 19F experiments were
conducted using a GE Omega 300 horizontal bore system.
23Na, 19F, and 31P spectra were
generated from the summed free induction decays of 1,000, 1,500, and
148 excitation pulses (90°, 45°, and 60°) using 2K-, 2K-, and
4K-word data files and ±4,000-, ±5,000-, and ±4,000-Hz sweep widths,
respectively. For all nuclei, data files were collected over 5-min
intervals. To improve the signal-to-noise ratio for 19F
measurement of Cai, two 5-min 19F files were
added together. Data are represented in time as corresponding to the
midpoint of the appropriate 5- or 10-min acquisition interval. Please
note also that lines connecting data points are not meant to imply that
the measured variable follows a linear path from point to point.
Nai content (in meq/kg dry wt) was calculated from the
calibrated area under the unshifted peak of the 23Na
spectra after subtracting the extracellular peak (4, 31). [Ca]i in nanomoles per liter of cell water was calculated
as the product of the ratio of the areas of the Ca2+-bound
and Ca2+-free peaks in the FBAPTA spectrum and the 500 nM
Ca2+-FBAPTA dissociation constant (26). Using
calibrated areas for newborn and adult 19F spectra and
assuming cytosolic volume is 2.5 l/kg dry wt (2), the
total FBAPTA in the cytosol (bound + free) was calculated as
27.5 ± 8.0 µM in newborn hearts and 69.5 ± 13.1 µM in
adult hearts. The pHi was determined from the chemical
shift of the inorganic phosphate (Pi) resonance [with
reference to control phosphocreatine (PCr)] calibrated at 25°C
(1). High-energy phosphates are reported as a percentage
of baseline peak intensity (30).
Statistics.
Results are reported as means ± SE unless otherwise
indicated. Two-factor analysis of variance (ANOVA) with repeated
measures on one factor (time) was used to test for differences among
treatment and age groups. The Tukey multiple comparison test was used
to identify significant differences between treatments and age groups when differences among groups were significant (17). The
Tukey test was used to compare both full data sets, as well as data for
specific time points, and significant differences for the latter
are indicated by asterisks in the figures. For all comparisons, differences were considered significant at P < 0.05.
Validation of Nai measurement.
It has been suggested that the method employed to measure
Nai using DyTTHA as a shift reagent is inferior to
the more recently developed method using thulium
1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(methylene phosphonate) (TmDOTP) (11). To address this issue, two
series of experiments were performed using the hypoxia protocol. One used 4 mM TmDOTP, and the other used 15 mM DyTTHA to shift the extracellular Na+ resonance. In Fig.
1, the data indicated by open squares
depict Nai during K+-free hypoxia measured
using DyTTHA and analyzed by reversing the spectra and subtracting the
extracellular Na+ peak (4, 31). (This method
was used for all Nai measurements in this study except
those described immediately following for Fig. 1.) The data depicted by
the closed circles and closed squares were acquired from another four
hearts exposed to the same hypoxic conditions but using TmDOTP to shift
the extracellular Na+ resonance. The data depicted by the
closed squares were analyzed using the reverse and subtract method
(4, 31), whereas the data depicted by the closed circles
were analyzed using NMR1 software (New Methods Research, Syracuse, NY)
to deconvolute the Na+ spectra. The sample size required to
"prove" that these three sets of data are not different prohibits
statistically testing that hypothesis. Nevertheless, it is apparent
that neither the method used to alter the extracellular Na+
resonance frequency (DyTTHA or TmDOTP) nor the method of analysis has a
significant effect on the measured change in Nai (reverse and subtract vs. NMR1; P = 0.967 by ANOVA for the two
methods of analyses of TmDOTP data). Thus we find no benefit in using TmDOTP. Given the decrease in arterial blood pressure caused by TmDOTP
in vivo (8), we prefer using DyTTHA, which has no
measurable effect on blood pressure in vivo (7).

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Fig. 1.
Comparison of hypoxia-induced Na+ uptake in
newborn hearts measured with 2 different shift reagents: dysprosium
triethylenetetraminehexaacetic acid (DyTTHA) and thulium
1,4,7,10- tetraazacyclododecane-1,4,7,10-tetrakis(methylene
phosphonate) (TmDOTP). Intracellular Na+ is plotted
vs. minutes of K+-free hypoxic perfusion using DyTTHA
( ) or TmDOTP ( ) to shift the
extracellular Na+ peak. Data depicted by closed and open
squares were analyzed by subtracting the extracellular Na+
peak from the spectrum. For comparison, the TmDOTP spectra were also
analyzed using NMR1 deconvolution ( ). Please see text
for explanation. In all figures, number of experiments are
given in parentheses.
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RESULTS |
Hypoxia stimulates Na+ uptake.
Our hypothesis predicts that during hypoxia, decreased pHi
will stimulate Na+/H+ exchange (functioning in
a pH-regulatory mode), resulting in increased Na+ uptake
(4, 12). To measure Na+ uptake,
Na+ efflux via the Na+/K+ pump must
be quantified. To achieve this, we measured Nai
accumulation under conditions in which the Na+ pump was
allowed to function (normal K+ perfusion) and in which
Na+ efflux via the Na+ pump was inhibited by
K+-free perfusion (4, 45). Figure
2 shows the results of experiments comparing Nai in newborn hearts during normoxic and hypoxic
K+-free perfusion. After 52.5 min of K+-free
perfusion, Nai had increased from 19 ± 3.4 to
139 ± 14.6 meq/kg dry wt during hypoxia but did not change
measurably under normoxic conditions (from 22 ± 1.9 to 22 ± 2.5 meq/kg dry wt). Because these experiments were conducted while
Na+ efflux was inhibited by K+-free perfusion,
the data unequivocally demonstrate that hypoxia stimulates an increase
in Na+ uptake. (Please see DISCUSSION for
further explanation.)

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Fig. 2.
Isolated newborn heart intracellular Na+
(means ± SE) during hypoxic ( ) and normoxic
( ) K+-free perfusion. Mean intracellular
Na+ (meq/kg dry wt) is plotted vs. minutes. (In all
figures, signal-averaged NMR data are represented by points plotted at
the time corresponding to the midpoint of the interval over which the
data were acquired.) After 52.5 min of hypoxic perfusion, mean
intracellular Na+ is more than 6 times greater than after
normoxic perfusion.
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Figure 3 shows a comparison of
Nai in newborn and adult hearts during hypoxic
K+-free perfusion, as well as the effect of the
Na+/H+ inhibitor MIA (40 µM)
(27) on Nai in newborn hearts for the same
perfusion protocol. Although an increase in Nai occurs
under hypoxic conditions in both adults and neonates, there is no
measurable difference between age groups. Additionally, Na+
uptake is inhibited in both adult and newborn hearts when the Na+/H+ exchange inhibitor MIA is added to the
hypoxic perfusate (adult data not shown).

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Fig. 3.
Hypoxic Na+ uptake is not measurably
different in perfused newborn ( ) and adult
( ) hearts, and in newborns, like adults, uptake is
inhibited by the Na+/H+ exchange inhibitor
methylisobutylamiloride (40 µM MIA; ). Mean
intracellular Na+ (meq/kg dry wt) is plotted vs. minutes.
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Decreased pHi stimulates
Na+ uptake and proton efflux.
Nai was also measured after decreasing pHi
under normoxic conditions. Figure
4A shows that in newborn
hearts NH4Cl (20 mM) washout results in rapid acidification
(pHi falls from 7.23 ± 0.12 to 6.55 ± 0.24),
which is followed by regulation of pH back to 7.25 ± 0.14 within
20 min. Figure 4B shows that while pHi is being
regulated in the newborn heart, Na+ uptake is increased
(Nai increases from 31 ± 18.2 to 117.7 ± 20.5 meq/kg dry), with Nai reaching a plateau as pHi
returns to the control level. Furthermore, addition of the
Na+/H+ exchange inhibitor MIA (40 µM) to the
perfusate used to washout NH4Cl prevents both pH regulation
and Na+ uptake (data not shown). Finally, Fig. 4,
A and B, shows that after NH4
washout, for a given proton load, there is no measurable difference
between age groups with regard to excursions in pHi and
Nai.

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Fig. 4.
Intracellular acidification (20 mM NH4Cl washout) under
normoxic conditions stimulates pH regulation and Na+
uptake. Mean intracellular pH (A) and intracellular
Na+ (meq/kg dry wt) (B) are plotted vs. minutes.
Changes in intracellular pH and Na+ after NH4Cl
washout are not measurably different in adult ( ) and
newborn ( ) hearts. Na+ uptake appears to
plateau as intracellular pH returns to control, supporting the
hypothesis that uptake is via pH-regulatory
Na+/H+ exchange.
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Hypoxia and normoxic acidification stimulate increases in
[Ca]i.
Figure 5A shows changes in
[Ca]i in newborn and adult hearts exposed to
K+-free hypoxic perfusion. In addition, these data
illustrate that increases in [Ca]i in newborn hearts
during K+-free hypoxic perfusion are inhibited by the
Na+/H+ exchange inhibitor MIA (40 µM).
Finally, Fig. 5A also provides evidence that during hypoxia,
increases in [Ca]i are less in newborn than adult hearts
(P < 0.05 by ANOVA).

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Fig. 5.
Intracellular Ca2+ ([Ca]i) increases more
in adult ( ) than newborn ( ) hearts
during hypoxia (A) and after normoxic acidification
(B) (20 mM NH4Cl washout). Additionally, in
newborn (like adult) hearts, increases in [Ca]i during
hypoxia are inhibited by the Na+/H+ exchange
inhibitor MIA (40 µM; ). Mean [Ca]i
(nM) is plotted vs. minutes (*P < 0.05 between age
groups).
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Changes in [Ca]i were also measured after acidifying the
hearts under normoxic conditions. The data in Fig. 5B
demonstrate that exposure to normoxic acidification (20 mM
NH4Cl prepulse) increases [Ca]i in newborn
hearts and that for the similar acidification conditions (see Fig.
4A), [Ca]i increases less in newborn hearts than adults (P < 0.05 by ANOVA).
The effects of hypertonic perfusion on intracellular
Na+ and
Ca2+.
To further test the general hypothesis, and in particular to test the
assertion that pH-regulatory Na+/H+ exchange is
the pathway responsible for increased Na+ uptake during
hypoxia, hypertonic perfusion was used to inhibit hypoxia- and
acidification-induced Na+ uptake (12, 13, 22).
(Please see DISCUSSION for further explanation of this
hypothesis.)
Figure 6, A and B, show the effect of
hyperosmotic perfusion on Nai and [Ca]i,
respectively, in neonate hearts exposed to hypoxia. (Neonate data from
Figs. 2 and 5A are included for comparison.) compared with
isotonic, hypertonic perfusion significantly decreased Nai
during hypoxia and reoxygenation (P < 0.05).
Similarly, hypertonic perfusion diminished increases in
[Ca]i during hypoxia (P < 0.05). Note
also that the recovery of Nai and [Ca]i
toward control when K+ and O2 are replaced
provides further evidence for the dependence of [Ca]i on
Nai.

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Fig. 6.
In
newborn hearts, Na+ uptake and Ca2+
accumulation during hypoxic K+-free perfusion are inhibited
by hypertonic solution (P < 0.05 by ANOVA for repeated
measures). Mean intracellular Na+ (meq/kg dry wt)
(A) and [Ca]i (nM) (B) are plotted
vs. minutes (*P < 0.05 for individual time intervals).
Hypertonic perfusion is achieved by addition of 30 mosM of sucrose to
the perfusate 5 min before and during hypoxic perfusion.
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Inhibition of Na+ accumulation
preserves high-energy phosphates and diminishes changes in coronary
resistance during hypoxia.
Figure 7 demonstrates the effects of MIA and hypertonic
perfusion on pHi and high-energy phosphates during hypoxia.
Significant differences for individual time points are indicated by the
symbols described in Fig. 7 (P < 0.05), but the most
salient features are summarized as follows. Figure 7A shows
that when Na+ accumulation is inhibited by MIA (see
Fig. 3), pHi decreases more during hypoxia than without MIA
(P < 0.05). The general hypothesis predicts that
inhibiting increases in Nai and [Ca]i during
hypoxia (Figs. 3, 5A, and 6, A and B),
would decrease ATP consumption. This hypothesis is supported by the
results depicted in Fig. 7, B-D, where MIA
and hypertonic perfusion are shown to limit depletion of ATP and PCr
and limit accumulation of Pi (P < 0.05 in
each case).

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Fig. 7.
The Na+/H+ exchange inhibitor MIA (40 µM)
increases, and 30 mM hypertonic perfusion decreases, the change in
intracellular pH (pHi), otherwise observed during hypoxic
perfusion. Both interventions decrease Na+ uptake and
[Ca]i during hypoxia and likewise decrease associated
changes in high-energy phosphates. pHi and high-energy
phosphates [ATP, phosphocreatine (PCr), and inorganic phosphate
(Pi) as a percentage of baseline] are plotted vs. minutes
with ( ) and without ( ) MIA and after
hypertonic perfusion ( ). Inhibition of intracellular
Na+ accumulation apparently diminishes energy consumption
during hypoxia (*P < 0.05 vs. hypertonic;
P < 0.05 vs. isotonic).
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In addition to the effects on Na+, H+,
Ca2+, and high-energy phosphates discussed above, MIA and
hypertonic inhibition of Na+ accumulation both limit
increases in perfusion pressure otherwise observed during
K+-free hypoxic perfusion. That is, in newborn hearts under
constant flow conditions during 60 min of K+-free hypoxic
perfusion, perfusion pressure increases 89% in isotonic controls, 23%
in hearts treated with 40 µM MIA, and 40% in hearts treated with 30 mosM hypertonic perfusion (from 44.9 ± 3.1 to 84.9 ± 7.1 mmHg, 42.0 ± 4.0 to 51.7 ± 1.8 mmHg and 44.8 ± 1.6 to
62.5 ± 7.0 mmHg, respectively). These data thus demonstrate that
inhibiting Na+, and thereby [Cai],
accumulation diminishes hypoxia-induced increases in coronary
resistance, the latter indicating decreased
hypoxia/ischemia-induced myocardial damage (28).
Finally, to better characterize the pathway responsible for the
observed changes in Nai and pHi, we measured
pHi during the NH4Cl washout protocol with and
without 40 µM MIA. The results are shown in Fig. 8, which
demonstrates that MIA prevents pHi regulation under these
conditions. (Again, please see DISCUSSION for further
interpretation of this result.)

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Fig. 8.
Na+/H+ inhibition prevents pH
regulation after 20 mM NH4Cl washout. pHi is
plotted vs. minutes with ( ) and without
( ) 40 µM MIA. During the first 10 min after
NH4Cl washout, MIA has no measurable effect on
pHi, allowing calculation of buffer capacity as = {[NH4]o × 10 exp(pHo pHi)}/ pHi. Please
see text for explanation.
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DISCUSSION |
The H+,
Na+, Ca2+
paradigm: hypoxia and the control of pHi.
In newborn rabbit hearts, as in adults, hypoxia stimulates an increase
in cell Na+ uptake, which is accompanied by an increase in
[Ca]i (Figs. 2 and 5A). Similarly, after
normoxic acidification, Nai increases with a time course
similar to that of pHi and net Na+ uptake
ceases when pHi has returned to its control value (~7.25) (Fig. 4). Increased Na+ uptake after normoxic acidification
is also accompanied by an increase in [Ca]i (Fig.
5B). The Na+/H+ exchange inhibitor
MIA inhibits increases in [Na]i and [Ca]i during hypoxia (Figs. 3 and 5A). MIA also limits
Na+ uptake (data not shown) and pHi recovery
after normoxic acidification (Fig. 8). Inhibition of Na+
accumulation by hypertonic perfusion (Fig.
6A) is also associated with a
reduction in the Cai accumulation during hypoxia (Fig. 6B). Parallel increases in Nai and
[Ca]i during hypoxia and after normoxic acidification are
reversed when Na+/K+ ATPase function is
restored and Nai is allowed to return toward control,
providing further evidence that the changes in [Ca]i are
secondary to changes in Nai (Figs. 5 and 6). Thus, as
previously demonstrated in the adult heart, these results are all
consistent with the hypothesis that myocardial hypoxic/ischemic injury
is in part a result of intracellular proton accumulation, which
stimulates pH-regulatory ([H]i-activated)
Na+/H+ exchange, which increases net
Na+ uptake, reduces the transmembrane Na+
gradient, and thereby increases Ca2+ uptake via
Na+/Ca2+ exchange (4, 12, 22, 30, 35, 36,
40, 47, 48).
Na+ and
Ca2+ accumulation in the adult and
neonate heart.
Our results demonstrate that under both hypoxic and acidotic
conditions, the increase in [Ca]i is less in the newborn
heart than in the adult (Fig. 5, A and B) . Given
the accepted notion that cell injury is a result of increases in
[Ca]i (46), this is consistent with the
well-documented finding that the newborn heart is resistant to hypoxic
injury (23, 24, 39). The mechanism responsible for this
age-related difference in Cai accumulation, however,
remains to be investigated. One simple explanation is that differences
in Ca2+ are the result of differences in
[Na]i and its effect on Na+/Ca2+
exchange. On the basis of Na+ uptake per dry weight, our
data show no significant difference between age groups. Thus our
Nai data appear to be inconsistent with a previous report
by Seguchi et al. (44) showing that the Na+/H+ exchange rate is higher in newborn
hearts than adults during hypoxic respiratory acidosis. Seguchi et al.,
however, drew their conclusions from the amiloride sensitivity of
changes in developed tension and 22Na uptake in sarcolemmal
vesicles via unidentified pathways. On the other hand, our results are
consistent with a more recent report from Nakanishi et al.
(37), which concludes, based on ethylisopropylamiloride
sensitivity of pH changes, that there is no difference between
Na+/H+ exchange rates in newborn and adult
hearts after NH4Cl washout.
The age-related differences we find in Cai accumulation
might at first be considered inconsistent with previous reports that Na+/Ca2+ exchange activity of newborn
myocardial sarcolemma is greater than that of adult in rabbit hearts
(5, 50) and not different than that of adult in dog hearts
(21). However, for a number of reasons, results from
sarcolemmal preparations cannot be directly compared with those
acquired from the intact perfused organ. First, transport activity in
vesicles is unlikely to be modulated by transduction pathways, which
function under more physiological conditions in intact cells.
Therefore, the number and activity of transporters in vesicles isolated
from cells may be largely irrelevant to flux that occurs in the hypoxic
organ. Second, the activity of the Na+/Ca2+
exchanger in vesicles is commonly determined in media which have high
Na+ (100-140 mM). This is much greater than
physiological [Na]i and likely to be saturating for the
transporter. That being the case, meaningful comparisons with cells
whose [Na]i may be near or below the
K1/2 for Na+/Ca2+
exchange (41) may not be possible. A recent study using
the whole cell patch-clamp method demonstrated that
Na+/Ca2+ exchange current in cardiac myocytes
isolated from newborn rabbit hearts is greater than that measured from
cells isolated from adult rabbit hearts (6). Although it
was not explicitly stated, it is assumed that these studies were
conducted under normoxic conditions, making it difficult to compare the
results with our findings during hypoxia and after acidification.
Finally, at least one study of Na+ transport proteins
(Na+/K+ pumps) has demonstrated that increased
numbers of transporters may be associated with a decrease in net
transport function (42). Thus, even though the capacity of
the Na+/Ca2+ exchanger in newborn myocardium
may exceed that of adults under resting or saturating conditions, our
data do not support the interpretation that there is greater net
Ca2+ transport via Na+/Ca2+
exchange in intact newborn rabbit hypoxic or acidotic myocardium than
in adult. On the contrary, our data are consistent with decreased net
Ca2+ transport via Na+/Ca2+
exchange in newborn relative to the adult under the conditions tested.
Contributions of other Ca2+ transport pathways, however,
remain to be investigated.
It will be noted that FBAPTA, as a Ca2+ buffer, is likely
to create artifacts in our [Ca]i measurements.
Nevertheless, when the artifacts and limitations of FBAPTA are compared
with other techniques (32), we conclude that
19F NMR remains the best method for measuring mean
[Ca]i in the intact organ. That is, other methods for
measuring [Ca]i (as opposed to Cai content)
are limited to use in isolated cells or cells on the surface of a
tissue that are accessible using microelectrode or optical techniques.
We argue that we may reasonably draw conclusions from our data for the
following reasons. Even though FBAPTA will have a large capacity for
buffering Cai in our experiments, its primary effect will
be to slow or diminish changes in [Ca]i away from its
Kd (500 nM). More specifically, because the
response time of FBAPTA to changes in [Ca]i is
conservatively estimated to be on the order of 20 ms (32)
and we report time-averaged values for [Ca]i acquired
over 10-min intervals, the [Ca]i values we report are
inaccurate to the extent that the mean value of [Ca]i is
biased toward the Kd. This means that under
control conditions, our estimates of [Ca]i may be
somewhat high, but as the mean value of [Ca]i rises past
500 nM, our measurements provide an underestimate. Furthermore, because
the buffer capacity of FBAPTA decreases as [Ca]i moves
away from 500 nM, changes in [Ca]i at low and,
especially, high [Ca]i will be measured with less
artifact due to FBAPTA buffering of Cai. In addition,
because the effects of hypoxia and normoxic acidification on
Nai and [Ca]i are measured during
K+-free perfusion (cells are depolarized and asystolic),
the effects of FBAPTA on myocardial contractility are minimized.
Finally, and most importantly, because our conclusions are based on
statistical assessment of differences in [Ca]i between
groups and treatments that develop over time from the same baseline,
our conclusions do not depend on the effects of FBAPTA on
[Ca]i (25). In other words, with regard to
[Ca]i, our conclusions are based only on differences
between measured values of [Ca]i and not on the
actual values of [Ca]i.
Effects of hypertonicity on pH-regulatory
Na+/H+
exchange.
Hypertonic perfusion has previously been shown to inhibit pH-regulatory
Na+/H+ exchange in Amphiuma red
blood cells (12, 13) and to limit hypoxia-induced
increases in Nai and [Ca]i in adult rabbit
hearts (22). The results presented in this study similarly
show that in the newborn heart hypertonic perfusion initiated before,
and continued during, hypoxia decreases Na+ accumulation
and [Ca]i compared with isotonic perfusion. The relative
decrease in Na+ uptake is similar to that previously
reported for adult hearts (22) and consistent with the
hypothesis that hypertonic solutions decrease the response of
Na+/H+ exchange to decreased intracellular pH.
Although the mechanism responsible for the decrease in Na+
accumulation under hypertonic conditions remains obscure, the effect of
hypertonic perfusion on [Ca]i is consistent with the hypothesis that hypoxia-induced increases in [Ca]i are
Na+ dependent in the newborn heart.
Figure 7A, however, suggests a
corollary or alternative explanation for the effect of hypertonic
perfusion on Na+ uptake during hypoxia. In this case, the
initiation of hypertonic perfusion 5 min before beginning hypoxic
perfusion is shown to increase pHi compared with isotonic
perfusion. Because our hypothesis states that increased
[H]i provides the stimulus for
Na+/H+ exchange during hypoxia, the data shown
in the top curve of Fig. 7A suggest that hypertonic
perfusion would decrease the stimulus for
Na+/H+ exchange during hypoxia. This
interpretation is also consistent with Fig. 6, A and
B. That is, if hypertonic perfusion increases pHi secondary to a volume regulatory response (13,
22), the relatively higher pHi during hypoxia (Fig.
7A) will attenuate Hi-induced
Na+/H+ exchange and result in less
Na+ and, therefore, Ca2+ uptake.
Energy cost of stimulating pH-regulatory
Na+/H+
exchange.
The results summarized for perfusion pressure and in Fig. 7 are
consistent with previous reports that inhibition of pH-regulatory Na+/H+ exchange preserves high-energy
phosphates and function in myocardium and cardiac myocytes exposed to
hypoxia/ischemia (4, 30, 35, 36, 40). As such,
they also support the hypothesis that increased Na+
and, therefore, Ca2+ uptake resulting from increased
pH-regulatory Na+/H+ exchange are central to
hypoxic/ischemic injury (30, 46). More
specifically, the data presented for perfusion pressure and in Fig. 7
suggest that both pharmacological as well as hypertonic inhibition of
Na+ accumulation protect the myocardium from the changes
otherwise observed during hypoxia. Further insight into the metabolic
cost of increasing Na+ uptake can be gained from
experiments conducted when Nai was measured during hypoxia
with normal K+ in the perfusate (data not shown). Again, in
newborn rabbit hearts, when Na+-K+-ATPase was
not inhibited by K+-free perfusion, there was no measurable
change in Nai after 52.5 min of hypoxic perfusion (from
21 ± 16 to 27 ± 18 meq/kg dry weight; n = 3). (Na+ uptake during hypoxic perfusion with normal
K+ is essentially the same as that shown in Fig. 3 for
hypoxic K+-free perfusion with MIA.) In comparison, when
Na+-K+-ATPase was inhibited by
K+-free perfusion during hypoxia (Figs. 2 and 3),
Nai rose from 19 ± 3.4 to 139 ± 14.6 meq/kg dry
wt. Here, the difference between Nai measured with normal
K+ and K+-free perfusates represents the amount
of Na+ leaving the cells due to
Na+-K+-ATPase activity during hypoxia. In other
words, in order for Nai to remain unchanged during normal
K+ hypoxic perfusion, the Na+ extrusion
(Na+-K+ pump) rate must actually increase to
match the increase in uptake shown in Figure 2. Thus the data
demonstrate that, contrary to historical opinion, increases in
Nai observed during myocardial hypoxia are not the result
of decreased Na+/K+ pump rate but, instead, as
observed during ischemia (2, 30), are the result
of a relatively larger increase in uptake rate compared with a
measurable but smaller increase in Na+/K+ pump
rate. As a result, the hypoxia-induced increase in Na+
uptake will increase the rate of ATP consumption by
Na+-K+-ATPase and likely increase the rate and
magnitude of ATP depletion. However, because most of the experiments
reported in this study were conducted using K+-free
perfusion to inhibit the Na+-K+-ATPase, the
observed effects of limiting Na+ uptake on ATP depletion
are more likely to stem from limiting Na+- and
Ca2+-dependent ATP consumption other than
Na+-K+-ATPase. Inhibition of Na+
uptake may also diminish effects of Nai and Cai
accumulation on mitochondria (19, 29), which could limit
ATP depletion by marginally increasing ATP production.
To reiterate, our comparisons of Na+ uptake during normoxic
and hypoxic conditions have been completed under conditions of K+-free perfusion to measure Na+ uptake
directly in the absence of Na+ efflux via the
Na+-K+ pump. Furthermore, the perfusions were
performed at ~23°C in order decrease the rate of change in
Nai and [Ca]i and thereby increase the
sensitivity of the NMR measurements (increased number of acquisitions
per unit change in Nai and [Ca]i), while at
the same time limiting irreversible changes in cell membrane ion
transport. These procedures undoubtedly decreased the heart's
consumption of energy for contraction, but they have allowed us to
assess the relative rates of Na+ uptake and efflux via
Na+-K+-ATPase under normoxic and hypoxic conditions. Our
previous studies of adult hearts demonstrated that K+-free
perfusion completely inhibits Na+ efflux via the
Na+-K+ pump (4). Similar studies
using ouabain and K+-free superfusion with chick heart
cells to assess the role of Na+ uptake in cell swelling
have led the authors to conclusions similar to ours, that
Ca2+ uptake after Na+-K+ pump
inhibition is via Na+/Ca2+ exchange and that
"swelling during ischemic injury may not result from
Na+/K+ pump failure alone" (45).
It could be further argued that because our studies were conducted
under asystolic conditions, metabolism (including proton production)
associated with muscle contraction is minimized and, therefore, the
changes in high-energy phosphates that occur in response to MIA and
hypertonic perfusion reflect changes in metabolism resulting from
changes in Na+ uptake in the absence of changes in
contractility. We have considered this to be a reasonable compromise
between minimizing uncontrolled variables and using a physiologically
relevant model to the extent that the changes in Nai and
[Ca]i that we measure are reversible (and therefore
mediated by cell membranes that maintain near normal Na+
and Ca2+ transport function) and predictably consistent
with those reported for other preparations at a variety of
temperatures, including body temperature (4, 25, 26, 36, 40, 47,
48). It will also be noted that during 55-min normoxic
K+-free perfusion, Nai appeared to be unchanged
in newborn hearts, whereas we have previously shown in adult hearts
that Nai increases nominally by 120% under the same
conditions (4). The difference between these groups is,
however, not significant (P = 0.981 by ANOVA),
reflecting the difficulty of measuring small changes in Nai
using notoriously insensitive NMR.
It will be further noted that we have used 40 µM MIA to inhibit
Na+/H+ exchange in these studies. This dosage
was chosen because it was the lowest dose at which we were unable to
measure Na+ uptake during 1 h of hypoxic
K+-free perfusion. That is, the criteria for inhibition of
Na+/H+ exchange is based on Na+
uptake measured under physiological extracellular Na+
conditions (33) rather than changes in pHi or
Na+ flux measured under less than physiological
Na+ conditions (15, 18, 37, 48). Controversy
remains concerning the effect of amiloride and its analogs on
Na+, and Na+-dependent, transport in cardiac
myocytes. Although the lack of specificity of the amiloride analogs is
well documented (43), the potency of analogs such as MIA
and ethylisopropylamiloride (EIPA) for Na+/H+
exchange inhibition is well accepted (34). Thus, at the
concentration used in this study, the major effect of MIA is likely to
be inhibition of Na+/H+ exchange. We cannot,
however, rule out an effect of MIA on noninactivating Na+
channels and Na+/Ca2+ exchange, which have also
been implicated under the conditions of this study. For example, based
on the fact that EIPA inhibits veratridine-induced hypercontracture,
Haigney et al. (20) concluded that EIPA inhibits
noninactivating Na+ channels, and similar conclusions were
reached by others using a whole cell patch-clamp technique
(14). On the other hand, Frelin et al. (15)
reported that in chick cardiac cells, Na+/Ca2+
exchange is not affected by the most active inhibitors of
Na+/H+ exchange (including MIA) at
concentrations <1 mM, whereas Garcia et al. (16) reported
that, in porcine cardiac sarcolemmal vesicles, these same
amiloride analogs competitively inhibit binding of L-type
Ca2+ channel inhibitors and, by inference, will themselves
inhibit L-type Ca2+ channels. Although we have not
determined the specificity of MIA in newborn myocardium, our results
remain consistent with the interpretation that the response we measure
is the result of inhibiting Na+-dependent Ca2+ accumulation.
Mechanisms of newborn resistance to hypoxic cell injury.
Although the mechanisms responsible for the newborn heart's apparent
resistance to hypoxia (23, 24, 39) and acidosis (38) remain unclear, our data can be used to address at
least two current explanations. First, it has been hypothesized that the proton load or the total number of protons added to the
intracellular solution during hypoxia or acidosis is less in the
newborn than the adult heart. This could be the result of less proton
production and/or greater proton buffering in the newborn heart.
Second, the newborn heart may have Na+-independent
pH-regulatory transport systems that are not active in the adult, e.g.,
there may be age-related differences in
Cl
/HCO
exchange (37).
Either one, or the combination, of these scenarios would result in less
stimulation of pH-regulatory Na+/H+ exchange
and, therefore, less Na+-dependent Ca2+ uptake.
The results of the NH4 prepulse experiment most directly
address these questions because H+ delivery or initial
proton load will be the same for both age groups. That is, from the
Henderson-Hasselbalch equation, we calculate that when pHi,
pHo, and [NH4]o are the same in
both age groups before NH4 washout,
[NH4]i is the same and, therefore,
intracellular [H] added by NH4 washout will be the same.
Figure 4 shows no measurable difference between age groups in the
initial acidification after NH4Cl washout. If no
pH-regulatory transport has occurred during this interval, the data
would indicate that there is no measurable age-related difference in
the heart cell's "intrinsic" or fixed buffer capacity. However,
this is only true if there are no active pH-regulatory processes
functioning (10). Figure 8
addresses this issue, illustrating that the
Na+/H+ exchange inhibitor MIA has no measurable
effect on pHi during the first 10 min of NH4
washout [NH4 washout should be complete in <2 min
(3)]. Thereafter, pHi continues to fall,
presumably the result of continued proton production, while
Na+/H+ exchange remains inhibited. Previous
experiments (data not shown) similarly demonstrated the same response
to Na+/H+ inhibition in the adult heart
(4). The buffer capacity calculated from the data shown
in Fig. 4 (
=
[NH4]i/
pHi = [NH4]i/
pHi = {[NH4]o × 10 exp
(pHo
pHi)}/
pHi, where o
and i refer to extra- and intracellular compartments, respectively,
[NH4]o is the concentration of
NH4 in the perfusate used before washout, and
pHi is the change in
pHi measured during the first 10 min after washout) is 36.7 meq/l pH unit for adult hearts and 43.16 meq/l pH unit for newborn
hearts. These values are not significantly different, similar
to those previously measured in guinea pig papillary muscle, isolated
ferret hearts, and sheep Purkinje fibers (10, 48, 49), and
somewhat higher than isolated myocytes (10). Thus our
results do not support reports that the "intrinsic" buffer capacity
of newborn hearts is greater than that of adults (44). Our
results showing that the Na+/H+ exchange
inhibitor MIA completely inhibits pH regulation in nominally HCO
-free, HEPES-buffered perfusate are consistent
with previous reports from adult and newborn hearts (18,
37) that suggest that Na+/H+ exchange
mediates a major portion of pH recovery after normoxic acidification.
We have not, however, tested whether
Cl
/HCO
exchange may serve to perform a
greater portion of newborn heart pH regulation than in adults (37) under HCO
-buffered conditions.
Conclusions.
The data presented here are consistent with the hypothesis that newborn
hearts, like adult, respond to hypoxia and normoxic acidification with
an increase in pH-regulatory Na+/H+ exchange,
which leads to increased Na+ uptake, collapse of the
transmembrane Na+ gradient, and, consequently, increased
uptake and accumulation of Ca2+ via
Na+/Ca2+ exchange. The data also demonstrate
that under similar conditions of hypoxia and acidification, while
age-related differences in Na+ uptake are not significant,
[Ca]i is significantly less in newborn than adult hearts.
 |
ACKNOWLEDGEMENTS |
This work was done during the tenure of a research fellowship from
the American Heart Association, California Affiliate, and was supported
by the University of California Medical Center Hibbard Williams Fund
and by National Heart, Lung, and Blood Institute Grants HL-21179 and
HL-56681. NMR spectrometer expense was funded in part by National
Institutes of Health Grant RR-02511 and National Science Foundation
Grant PCM-8417289.
 |
FOOTNOTES |
Address for reprint requests and other correspondence:
S. E. Anderson, Dept. of Human Physiology, Univ. of
California, One Shields Ave., Davis, California 95616-8644 (E-mail: seanderson{at}ucdavis.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.
First published January 8, 2002;10.1152/ajpcell.00148.2002
Received 3 April 2002; accepted in final form 23 December 2002.
 |
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