INTRODUCTION

Top Abstract Introduction Materials & Methods Results Discussion References

REPERFUSION of isolated mammalian hearts with a Ca²⁺-containing solution after a short Ca²⁺-free period at 37°C results in irreversible cell damage: the Ca²⁺ paradox (34). The damage consists of extensive ultrastructural membrane lesions, massive release of intracellular constituents, and depletion of high-energy phosphates. A massive Ca²⁺ influx during the Ca²⁺ repletion phase is thought to be responsible for this damage (18). However, disagreement exists about what makes the heart susceptible to the Ca²⁺ paradox during the Ca²⁺-free period and about the route of entry of Ca²⁺ during Ca²⁺ repletion (1, 9, 11, 26, 28).

One theory implies that the intensity of the Ca²⁺ paradox is primarily determined by entry of Na⁺ through the Ca²⁺ channels during Ca²⁺ depletion and a subsequent influx of Ca²⁺ into the cytosol via the Na⁺/Ca²⁺ exchange mechanism during Ca²⁺ repletion (10). Most experimental evidence for this theory has been obtained from studies in which combined Ca²⁺ and Mg²⁺ depletion was used or a chelating agent was present in the Ca²⁺-free perfusate. In a previous study, using ²³Na nuclear magnetic resonance (NMR), we have shown that a rise in intracellular Na⁺ concentration ([Na⁺]_i) indeed occurs during Ca²⁺- and Mg²⁺-free (Ca²⁺/Mg²⁺-free) perfusion (31). However, this rise in [Na⁺]_i is not a prerequisite for the Ca²⁺ paradox to occur, because after Ca²⁺-free perfusion a full Ca²⁺ paradox was observed during Ca²⁺ repletion without a rise in [Na⁺]_i during the previous Ca²⁺-free period (30).

Another theory states that the primary event during Ca²⁺ repletion is Ca²⁺ entry through the Ca²⁺ channels, causing a contracture-mediated disruption of intercalated disk junctions, which are weakened during Ca²⁺ depletion, and a massive secondary influx of Ca²⁺ (15). According to the first theory, a rise in [Na⁺]_i during Ca²⁺ depletion is essential for the Ca²⁺ paradox to occur, whereas the second theory assumes that mechanical factors during Ca²⁺ repletion are involved.

Hypothermia is one of the most effective ways to protect the heart against Ca²⁺ paradox damage (3, 5). A study with isolated ferret ventricular trabeculae has shown that, in the presence of ethylene glycol-bis(-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), intracellular Na⁺ activity does increase during hypothermic Ca²⁺-free perfusion, albeit less than under normothermic conditions (29). In the present study we used ²³Na NMR to measure [Na⁺]_i in isolated rat hearts during Ca²⁺- or Ca²⁺/Mg²⁺-free perfusion under hypothermic conditions to study further the possible involvement of [Na⁺]_i in the origin of the Ca²⁺ paradox. The results are compared with those obtained under normothermic (37°C) Ca²⁺-free conditions.

	MATERIALS AND METHODS

Top Abstract Introduction Materials & Methods Results Discussion References

Heart preparation. Male Wistar rats (300-400 g) were anesthetized with diethyl ether and heparinized (250 IU iv). The hearts were rapidly excised and cooled in ice-cold perfusate. Within 2 min, the aorta was cannulated and perfusion was started at a constant pressure of 100 cmH₂O at 37°C. The perfusion apparatus was equipped with four reservoirs and perfusion lines, which allowed immediate switching of perfusates and temperatures. The hearts were placed in a 20-mm NMR tube together with a reference capillary. The glass tube was lowered into the magnet, and the heart was positioned in the center of a Helmholtz coil. To allow a rapid temperature change in the glass tube from a warm perfusate to a cold perfusate and vice versa, the effluent was removed from the bottom of the tube at a speed slightly below coronary flow rate, leaving the heart submerged. Excess flow was aspirated. The animal experiments were approved by the Committee for Animal Experiments of the Faculty of Medicine of the University of Utrecht.

Perfusion solutions. All perfusion fluids were filtered (0.8 µm; Schleicher and Schuell, Dassel, Germany) before use and saturated with 95% O₂-5% CO₂, which resulted in a final perfusate pH of 7.27 ± 0.05 at 20°C and 7.38 ± 0.05 at 37°C at an effective pressure of 100 cmH₂O. The standard perfusate contained (in mM) 148.0 Na⁺, 4.7 K⁺, 1.3 Ca²⁺, 1.0 Mg²⁺, 128.3 Cl, 24.0 , and 11.0 glucose. No correction was made for the small changes in osmolarity when Ca²⁺ or both Ca²⁺ and Mg²⁺ were omitted from the standard perfusate. For ²³Na NMR measurements, the standard perfusate was modified by inclusion of the shift reagent thulium(III) 1,4,7,10-tetraazacyclododecane-N,N',N",N'"-tetra(methylene-phosphonate) (TmDOTP⁵) in the perfusion fluid. To this end, Na₃H₂TmDOTP (Magnetic Resonance Solutions, Dallas, TX) was dissolved in H₂O and subsequently added to the standard perfusate to reach a concentration of 3.5 mM. To maintain a [Na⁺] of 148 mM, less NaCl was used when the shift reagent was present in the perfusates. To correct for the Ca²⁺ affinity of the shift reagent, the Ca²⁺ concentration ([Ca²⁺]) in the standard perfusate was increased to 3.42 mM, which resulted in a final free [Ca²⁺] of 0.85 mM, as measured with a Ca²⁺-sensitive electrode (Radiometer, Copenhagen, Denmark). A further increase in [Ca²⁺] would have caused precipitation problems. In perfusates in which Ca²⁺ or both Ca²⁺ and Mg²⁺ were omitted, the final concentration of shift reagent was reduced to 1.5 mM TmDOTP⁵ because the omission of Ca²⁺ resulted in sufficient shift of the extracellular Na⁺ signal. After each experiment, TmDOTP⁵ was recovered according to Pike et al. (24) and reused.

NMR measurements. ²³Na NMR spectra were recorded on a Bruker MSL 200 spectrometer at 52.9 MHz. The spectrometer was equipped with a 4.7-T vertical, 150-mm room temperature bore magnet and a multinuclear 20-mm NMR probe. Magnetic field homogeneity was optimized using the ²³Na free induction decay (FID). For each ²³Na spectrum, 256 FIDs were accumulated after 90° pulses with a repetition time of 0.21 s, using 2,048 data points and 5-kHz spectral width. FIDs were Fourier transformed after Lorentzian and Gaussian multiplication. After polynomial baseline correction, the intracellular and reference ²³Na signals were quantified by integration. All quantification was performed relative to the peak area of the reference signal. The reference solution contained (in mM) 250 Na⁺, 0 Ca²⁺, and 5 TmDOTP⁵, and its pH was adjusted to 12.0 at 20°C. The reference capillary contained a known amount of this solution. NMR visibility of all ²³Na signals was assumed to be 100% (30).

Experimental protocol. In Fig. 1, a schematic representation of the protocols is shown. After a stabilization period of 20 min, which was used to optimize instrumental settings, standard perfusate was replaced by perfusate containing shift reagent [time (t) = 20 min]. Minor adjustments to field homogeneity and coil tuning were completed within 10 min. Subsequently, one control spectrum was obtained in 1 min. For the next 10 min, the heart was perfused at either 37°C (Ca²⁺ paradox group) or 20°C (hypothermia groups I and II) with standard perfusate (n = 6 hearts for all groups). Perfusion in the hypothermia groups with hypothermic standard perfusate was performed to be sure that the heart had completely cooled down before switching to a hypothermic Ca²⁺-free or Ca²⁺/Mg²⁺-free perfusate. Subsequently, the heart was perfused with either a Ca²⁺-free perfusate at 37°C (Ca²⁺ paradox group), a Ca²⁺-free perfusate at 20°C (hypothermia group I), or a Ca²⁺/Mg²⁺-free perfusate at 20°C (hypothermia group II) for 20 min, followed by 30 min of Ca²⁺ repletion with standard perfusate at 37°C in all groups. In this period, fifty 1-min ²³Na spectra were collected.

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Schematic representation of the 3 protocols. After an equilibration period, control perfusion with standard perfusate (S) containing shift reagent was started (time = 20 min). Shift reagent was present in perfusate between 20 min and 50 min. Creatine kinase (CK) release (and mechanical performance) was assessed in separate series of experiments. Gray areas represent hypothermia (hypothermia groups I and II: 20°C; hypothermia groups III and IV: 25°C). In hypothermia groups III and IV, no 23Na nuclear magnetic resonance (NMR) experiments were performed. For further explanation see MATERIALS AND METHODS.

Statistical analysis. Results are presented as means ± SD. One-way analysis of variance was used to analyze the data. If significant differences between the groups were found, a post hoc pairwise comparison was performed using Bonferroni's method (14). A test result with a P value of <0.05 was considered significant. The rate of rise of intracellular Na⁺ was determined by linear regression.

	RESULTS

Top Abstract Introduction Materials & Methods Results Discussion References

Figure 2 shows 1-min ²³Na NMR spectra of an isolated rat heart during control perfusion (Fig. 2A), after a 20 min Ca²⁺-free perfusion (Fig. 2B) at 37°C, and after a subsequent 30-min Ca²⁺ repletion period (Fig. 2C). The extracellular Na⁺ resonances had to be clipped to display the intracellular and reference Na⁺ signals properly. It can be seen that no clear difference in the intracellular Na⁺ signal intensity was present after 20 min of Ca²⁺-free perfusion. The chemical shift of the extracellular Na⁺ resonance had changed during Ca²⁺-free perfusion (despite the reduction in concentration of shift reagent), because no interaction of the shift reagent with Ca²⁺ occurred in this situation. During Ca²⁺ repletion, the intracellular Na⁺ resonance collapsed and a broad Na⁺ resonance remained (Fig. 2C), most likely because of entry of the shift reagent into the cells after rupture of the sarcolemma, which is one of the characteristics of the Ca²⁺ paradox (33). In this situation it was impossible to determine [Na⁺]_i.

View larger version (12K): [in this window] [in a new window]   Fig. 2.   23Na NMR spectra of an isolated rat heart during Ca2+ paradox protocol. A: control perfusion (37°C). B: 20-min Ca2+-free perfusion (37°C). C: 30-min Ca2+ repletion with standard perfusate (37°C). Peak assignment: 1, reference; 2, extracellular Na+; 3, intracellular Na+. PPM, parts per million.

Figure 3 shows spectra of an isolated rat heart obtained during the protocol of hypothermia group II. Figure 3A shows a spectrum during control perfusion at 37°C, Fig. 3B after 20 min of Ca²⁺/Mg²⁺-free perfusion at 20°C, and Fig. 3C after 30 min of Ca²⁺ repletion at 37°C. The intracellular Na⁺ signal intensity is larger in Fig. 3B than in Fig. 3, A and C. On switching to a hypothermic perfusate, the reference peak shifted downfield. Line widths of the peaks also increased. During the protocol of hypothermia group I comparable spectra were obtained, except that there was no clear increase in the intracellular Na⁺ signal intensity after 20 min of Ca²⁺-free perfusion at 20°C.

View larger version (13K): [in this window] [in a new window]   Fig. 3.   23Na NMR spectra of an isolated rat heart during hypothermia group II protocol. A: control perfusion (37°C). B: 20-min Ca2+/Mg2+-free perfusion (20°C). C: 30-min Ca2+ repletion with standard perfusate (37°C). Peak assignment: 1, reference; 2, extracellular Na+; 3, intracellular Na+.

Figure 4 shows the mean [Na⁺]_i obtained from the NMR spectra in hypothermia groups I and II and in the Ca²⁺ paradox group. In the Ca²⁺ paradox group, [Na⁺]_i was 12.1 ± 2.5 mM during control perfusion and 13.6 ± 2.0 mM [not significant (NS) vs. control] after 20 min of Ca²⁺-free perfusion. [Na⁺]_i in hypothermia group I was 10.4 ± 1.0 mM during control perfusion, 12.0 ± 2.7 mM (NS vs. control) after 20 min of Ca²⁺ depletion at 20°C, and 8.6 ± 1.3 mM (P < 0.05 vs. control) after a subsequent 30-min period of Ca²⁺ repletion at 37°C. In hypothermia group II, [Na⁺]_i was 11.9 ± 1.2 mM during control perfusion, rose to 26.9 ± 5.8 mM (P < 0.001 vs. control) after 20 min of Ca²⁺/Mg²⁺-free perfusion at 20°C with an average rate of 0.89 ± 0.03 mM/min, and started to decline rapidly after 2 min of Ca²⁺ repletion at 37°C, to reach a value of 8.5 ± 1.7 mM (P < 0.01 vs. control) after 30 min of Ca²⁺ repletion. After 20 min of Ca²⁺ depletion, [Na⁺]_i in the Ca²⁺/Mg²⁺-free hypothermia group was significantly higher than in the Ca²⁺-free groups (P < 0.001).

View larger version (26K): [in this window] [in a new window]   Fig. 4.   Intracellular Na+ concentration ([Na+]i) in isolated rat hearts during 20-min Ca2+-free perfusion at 37°C (), 20-min Ca2+-free perfusion at 20°C (), or 20-min Ca2+/Mg2+-free perfusion at 20°C () and a subsequent 30-min period of Ca2+ repletion with standard perfusate at 37°C (n = 6 hearts, means ± SD). In normothermia group, [Na+]i could not be determined during Ca2+ repletion because of occurrence of a Ca2+ paradox.

Figure 5 shows the cumulative CK release in the five groups during 30 min of Ca²⁺ repletion at 37°C with standard perfusate. Total CK release was 3,290 ± 234 IU · 30 min¹ · g dry wt¹ in the Ca²⁺ paradox group, 484 ± 61 IU · 30 min¹ · g dry wt¹ in hypothermia group I, 271 ± 101 IU · 30 min¹ · g dry wt¹ in hypothermia group II, 2,409 ± 800 IU · 30 min¹ · g dry wt¹ in hypothermia group III, and 2,404 ± 469 IU · 30 min¹ · g dry wt¹ in hypothermia group IV. Total CK release in the Ca²⁺ paradox group was significantly higher than in hypothermia groups I and II (P < 0.001) and hypothermia groups III and IV (P < 0.01). No significant differences existed between hypothermia groups I and II or between groups III and IV. CK release in the 20°C groups was significantly less than in the 25°C groups (P < 0.001).

View larger version (20K): [in this window] [in a new window]   Fig. 5.   Cumulative CK release during 30-min Ca2+ repletion (37°C) with standard perfusate after 20-min Ca2+-free perfusion at 37°C (), 20-min Ca2+-free perfusion at 20°C (), 20-min Ca2+/Mg2+-free perfusion at 20°C (), 20-min Ca2+-free perfusion at 25°C (), or 20-min Ca2+/Mg2+-free perfusion at 25°C () (n = 6 hearts, means ± SD). For clarity, only 1 error bar is depicted.

Table 1 shows the RPP of hearts in the five groups. On switching from standard perfusate to perfusate containing shift reagent, the hearts showed a decrease in RPP (because of a decrease in LVDP), which was the result of the lower free [Ca²⁺] (0.85 mM instead of 1.3 mM). The hearts in the Ca²⁺ paradox group showed no mechanical recovery during Ca²⁺ repletion. Recovery of RPP at 30 min of Ca²⁺ repletion with standard perfusate containing shift reagent was 73% in hypothermia group I and 45% in hypothermia group II compared with the RPP during control perfusion with shift reagent. After a subsequent 5-min Ca²⁺ repletion period with standard perfusate without shift reagent, the RPP was 66% in hypothermia group I and 43% in hypothermia group II compared with the RPP during control perfusion without shift reagent. Recovery in hypothermia group I was different from recovery in hypothermia group II (P < 0.05). Obviously, recovery in hypothermia groups I and II was different from recovery in the Ca²⁺ paradox group (P < 0.001). In most hearts of hypothermia groups III and IV there was some mechanical activity during Ca²⁺ repletion. However, RPPs were difficult to calculate because the hearts contracted very irregularly.

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

The present results show that despite a rise in [Na⁺]_i during Ca²⁺ depletion a full Ca²⁺ paradox did not occur during normothermic Ca²⁺ repletion after a 20-min period of Ca²⁺/Mg²⁺-free perfusion at 20°C. CK release was >10 times less than under full Ca²⁺ paradox conditions. Mechanical recovery in the 20°C Ca²⁺/Mg²⁺-free group was 44%, whereas in the Ca²⁺ paradox group no mechanical recovery at all was observed. These results provide further evidence that [Na⁺]_i is not involved in the origin of the Ca²⁺ paradox. The small CK release during Ca²⁺ repletion in the 20°C Ca²⁺/Mg²⁺-free group, indicating relatively minor cell damage, was not a consequence of an increased [Na⁺]_i during Ca²⁺ depletion but was caused by incomplete protection by hypothermia, because a similar CK release was observed in the 20°C Ca²⁺-free group.

To exclude the possibility that some protective effect of hypothermia at 20°C may have uncoupled the tentative action of an elevated [Na⁺]_i in the occurrence of the Ca²⁺ paradox, experiments were also performed at 25°C. In a previous study (5), we demonstrated that 20 min of Ca²⁺ depletion at 25°C predisposes rat hearts to a submaximal Ca²⁺ paradox. No ²³Na NMR experiments were performed in the 25°C groups, but it is reasonable to assume that 20 min of Ca²⁺/Mg²⁺-free perfusion at 25°C will increase [Na⁺]_i to a value >26.9 mM (at 20°C; this study) but <50.7 mM (at 37°C; Ref. 31). CK release during normothermic Ca²⁺ repletion after Ca²⁺/Mg²⁺-free perfusion was not different from that after Ca²⁺-free perfusion at 25°C, indicating that an elevated [Na⁺]_i during Ca²⁺ depletion was unable to increase the extent of a submaximal Ca²⁺ paradox on Ca²⁺ repletion.

In 1984, Chapman et al. (10) stated that the extent of cell damage on Ca²⁺ repletion closely correlates with a rise in [Na⁺]_i during Ca²⁺ depletion. These authors developed the hypothesis that the intensity of the Ca²⁺ paradox is primarily determined by Na⁺ entry through the Ca²⁺ channels during Ca²⁺ depletion and influx of Ca²⁺ into the cytosol via Na⁺/Ca²⁺ exchange during Ca²⁺ repletion. Other groups have also associated a rise in [Na⁺]_i during Ca²⁺ depletion with the Ca²⁺ paradox (16, 17, 19, 25). However, the experimental evidence for this notion has been obtained in the absence or at low levels of extracellular Mg²⁺ or in the presence of chelators, which may lower Mg²⁺ concentration. In previous studies using ²³Na NMR, we did not observe an increase in [Na⁺]_i during 30 min of Ca²⁺-free perfusion (30, 31), whereas a full Ca²⁺ paradox could be evoked on Ca²⁺ repletion. [Na⁺]_i only increased when not only Ca²⁺ but also Mg²⁺ was omitted from the perfusate (31). Verapamil prevented this increase in [Na⁺]_i, indicating that when both Ca²⁺ and Mg²⁺ are absent in the perfusate, Na⁺ enters the cell through the L-type Ca²⁺ channels. In the present study using the shift reagent TmDOTP⁵, we have also confirmed our earlier observations that [Na⁺]_i does not increase significantly during Ca²⁺ depletion, although a full Ca²⁺ paradox occurred on readmission with Ca²⁺.

Several other studies have also questioned the involvement of [Na⁺]_i in Ca²⁺ paradox damage. Nayler et al. (22) studied the effect of Na⁺ loading on the gain in Ca²⁺ and the loss of myoglobin during the Ca²⁺ paradox. They found that raising cell Na⁺ did not alter the degree or rate of Ca²⁺ gain or myoglobin release during Ca²⁺ repletion after >2 min of Ca²⁺-free perfusion. In 1987, Busselen (6) showed that myoglobin release during the Ca²⁺ paradox did not depend on the Na⁺ gradient across the sarcolemma. This author used sucrose, Li⁺, or choline⁺ to replace Na⁺ during Ca²⁺ depletion. Protection against the Ca²⁺ paradox only occurred when sucrose was used to replace Na⁺. It was suggested that sucrose (as opposed to Na⁺ or other cations) attenuates the displacement of Ca²⁺ from critical binding sites during Ca²⁺ depletion. Another explanation could be that high concentrations of sucrose counteract an oncotic pressure gradient across the sarcolemma, resulting from an increase in membrane permeability after Ca²⁺ readmission, and consequently prevent lysis of the myocytes (23). Diederichs (13) demonstrated that Na⁺/Ca²⁺ exchange was thermodynamically unlikely to elevate intracellular [Ca²⁺] to critical values during Ca²⁺ repletion. Finally, Bakker et al. (4) used lidocaine to inhibit Na⁺ entry through the Na⁺ channels during Ca²⁺ depletion. They used laser microprobe mass analysis to analyze the intracellular Ca²⁺ content. Their results showed no protection of lidocaine on ultrastructural damage and massive Ca²⁺ loading of the mitochondria during Ca²⁺ repletion.

According to Suleiman and Chapman (29), the protection against the Ca²⁺ paradox afforded by hypothermia during Ca²⁺ depletion can be explained by the lower rate of rise of intracellular Na⁺ activity at lower temperatures. We also observed a lower rate of rise of [Na⁺]_i during hypothermic Ca²⁺/Mg²⁺-free perfusion (20°C) than under normothermic conditions. The average rate of rise during Ca²⁺/Mg²⁺-free perfusion at 20°C amounted to 0.89 mM/min at 20°C and 2.32 mM/min at 37°C (31). However, the increase in Na⁺ during Ca²⁺/Mg²⁺-free perfusion at 20°C was considerable compared with the absence of a significant rise during normothermic Ca²⁺-free perfusion. Therefore, a lower rate of rise of Na_i activity cannot explain the protective effect of hypothermia.

Experimental evidence supporting the theory that weakening of intercalated disk junctions during Ca²⁺ depletion predisposes the heart to the Ca²⁺ paradox is scarce. It has been suggested that an intercellular adherens junction-specific cell adhesion molecule is involved in intercellular adhesion. In cultured chick lens cells that were incubated in an EGTA-containing, Mg²⁺-free, low-Ca²⁺ buffer, it was demonstrated that essentially all adherens junctions were cleaved after a 30-s incubation period (32). Ultrastructural analysis of isolated rat hearts showed progressive separation of intercalated disks between 3 and 5 min of Ca²⁺-free perfusion (33), coinciding with the occurrence of a full Ca²⁺ paradox on Ca²⁺ repletion after 4 min of Ca²⁺ depletion (5). It has been proposed that any event that delays extraction of Ca²⁺ from the intercalated disks, such as hypothermia, attenuates development of the Ca²⁺ paradox (1).

The inclusion of shift reagents in the perfusate allows us to discriminate between intracellular and extracellular Na⁺ resonances obtained with ²³Na NMR spectroscopy. Shift reagents interact with Na⁺ but do not cross intact cell membranes and have little or no effect on hemodynamic and metabolic parameters (20). Compared with another widely used shift reagent [dysprosium(III) triethylenetetramine hexaacetate (DyTTHA³); Ref. 12], TmDOTP⁵ has distinct advantages. First, less shift reagent is necessary to obtain the same chemical shift (7). Second, line widths of resonances are less broadened. One disadvantage is that TmDOTP⁵ interacts more strongly with Ca²⁺, which may lead to the formation of precipitates. Therefore, we corrected only partially for this interaction by increasing [Ca²⁺] in the perfusate to 3.42 mM, which resulted in a free [Ca²⁺] of 0.85 mM. This lower free [Ca²⁺] and the presence of the shift reagent had no protective effect on the Ca²⁺ paradox, because total CK release in the present study was similar to the CK release measured in the absence of shift reagents and a free [Ca²⁺] of 1.3 mM (27). The appearance of the broad Na⁺ resonance, which occurred just after the beginning of Ca²⁺ repletion in the Ca²⁺ paradox group, is most likely caused by mixing of intra- and extracellular compartments through membrane lesions (33). In this situation it was impossible to determine [Na⁺]_i. The limited myocardial cell damage in hypothermia groups I and II may explain why the calculated [Na⁺]_i (Fig. 4) returned to a lower value after 30 min of Ca²⁺ repletion than during control perfusion. During Ca²⁺ repletion with standard perfusate in hypothermia group II, the decrease in [Na⁺]_i started only after 2 min. This delay could be the result of a slow reactivation of the Na⁺ extrusion mechanisms during this "warming-up" period from perfusion at 20°C to perfusion at 37°C.

Although this study provides further evidence that [Na⁺]_i is not involved in the origin of the Ca²⁺ paradox, it cannot entirely be excluded that Na⁺/Ca²⁺ exchange during Ca²⁺ repletion still occurs as a result of local accumulation of Na⁺ close to the inner side of the membrane in a fuzzy space during Ca²⁺ depletion (8). In ²³Na NMR studies of isolated hearts, only information about the bulk [Na⁺]_i is provided, and no regional differences can be measured. Further research is necessary to exclude this possibility.