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Am J Physiol Cell Physiol 284: C1123-C1132, 2003. First published January 8, 2003; doi:10.1152/ajpcell.00148.2002
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Vol. 284, Issue 5, C1123-C1132, May 2003

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.

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; black-triangle). Mean intracellular Na+ (meq/kg dry wt) is plotted vs. minutes.

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.

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; black-triangle). Mean [Ca]i (nM) is plotted vs. minutes (*P < 0.05 between age groups).

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.

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 (black-triangle). Inhibition of intracellular Na+ accumulation apparently diminishes energy consumption during hypoxia (*P < 0.05 vs. hypertonic; dagger P < 0.05 vs. isotonic).

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 beta  = {[NH4]o × 10 exp(pHo - pHi)}/ Delta  pHi. Please see text for explanation.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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<UP><SUB>3</SUB><SUP>−</SUP></UP> 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 (beta  = Delta  [NH4]i/ Delta  pHi = [NH4]i/ Delta  pHi = {[NH4]o × 10 exp (pHo - pHi)}/ Delta  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 Delta  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<UP><SUB>3</SUB><SUP>−</SUP></UP>-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<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange may serve to perform a greater portion of newborn heart pH regulation than in adults (37) under HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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