MAGNETIC RESONANCE I N MEDICINE 28,249-263 ( 1992)

Sodium Ion Transport in Rat Hearts during Cold Ischemic Storage: 23Naand 31PNMR Study* NADIRASKENASY,ANTONIO VIVI, MARIATASSINI, AND GILNAVON School of Chemistry, Tel Aviv University Ramat Aviv 69978, Israel Received August 29, 1991; revised February 18, 1992; accepted February 18, 1992 The success of heart transplantation is limited by the negative correlation between the length of the cold ischemic storage period and the quality of functional recovery. We use 23Na, 3'P NMR spectroscopy, and hemodynamic parameters to describe temperature-dependent changes in sodium influx and the concentration of phosphorus highenergy compounds during different storage periods. Perfusion with Krebs-Henseleit solutions containing Dy( TTHA)'- permitted discrimination of intra- and extracellular sodium during cold ischemic storage. The 23NaNMR visibilities under the acquisition and processing parameters used in our experiments were 40 f 4% for the intracellular compartment and 97 k 11% for the extracellular compartment. A t 4"C, the intracellular Na+ accumulation exceeded that observed at 15 and 22°C. The ATP and PCr depletion rates were much lower at 4°C and the left ventricular contractility was higher after longer periods of storage, as the storage temperature decreased. The intracellular Na+ concentration cannot serve as a marker for the postischemic recovery probability. The relative activity of the N a / K ATPase pumps is not correlated with the preservation success. However, intracellular sodium ion accumulation is a major factor in the time lag of the reperfusion recovery. o 1992 Academic Press, Inc. INTRODUCTION

The search for techniques of hypothermic preservation of the heart during prolonged interventions and storage for transplantation has focused mainly on correlations with hemodynamic parameters. The goal of these studies is to delineate the myocardial processes responsible for the irreversible injury during cold ischemia (1). Numerous attempts have been made to describe the morphologic ( 2 ) ,metabolic, and electrolytic status (3-7) of the myocardium during ischemia. Most of the techniques used have two main limitations: the preparation of the tissue precludes its further use in the experiment and the tissue compartments are not readily distinguished. NMR spectroscopy offers a noninvasive approach to the study of ischemia and preservation that circumvents these limitations. 31PNMR spectroscopy enables dynamic monitoring of metabolic changes, such as the energetic status and intracellular pH (8-1 3 ) . The transport of sodium ions across the sarcolemmal membrane is central

i

* Th's work was supported by grants from the National Council for Research and Development, Israel, Gesells aft fur Strahlen und Umweltforschung, Munchen, JXG, and Israel-United States Binational Science Foundation. A.V. and M.T. were supported in part by Centro Nazionale delle Ricerche, Rome, Italy. 249

0740-3 194192 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproductionin any form reserved.

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ASKENASY ET AL.

to the physiological processes that occur during ischemia ( 5 - 7), and variations in the intracellular concentration of this ion affects several cellular functions and properties: the activity of ionic pumps is involved in the dissipation of high-energy phosphates; accumulation of sodium ions is a major cause of edema ( 4 ) ; sodium ion transport affects membrane potential and all voltage-dependent processes; the intracellular concentration of sodium ions influences the transport of other ions like H + , K t , C1-, and Ca2+,through the various exchange and cotransport mechanisms. The introduction of extracellular cationic shift reagents enables discrimination of the compartmental distribution of the sodium ion ( 14, 1 5 ) . One effective shift reagent is dysprosium triethylene tetraamine hexaacetate (DyTTHA) ( 1 5 ) , which has a relatively low affinity for the bivalent cations such as calcium and magnesium, thus it is presumed to have minor influences on the physiological function of the heart (16, 18). The present study investigates the compartmental distribution of the sodium ion during hypothermic ischemic preservation. It examines the compartmental NMR visibility of the sodium ion and evaluates whether intracellular Na+ can serve as a marker of heart recovery from hypothermic ischemic preservation. METHODS

Solutions The basic perfusion and preservation solution was a modified physiologic KrebsHenseleit (KH) solution without Na2H2EDTA.It contained 122 mMNaC1, 5.9 m M KCl, 1.2 m M MgClz, 1.75 m M CaC12, 23 m M NaHC03, and 10 m M glucose. Two Krebs-Henseleit solutions containing 10 m M of the shift reagents, Na3Dy(TTHA) 3NaCl (KH-SR) or its diamagnetic analog Na3La(TTHA) 3NaCl ( KH-La), were used. The sodium ion concentration in both solutions was corrected to 145 m M by lowering the NaCl content. The concentration of CaC12 was increased to 3 m M to compensate for the calcium fraction bound by the shift reagents ( 17, 19). The free calcium ion concentrations, determined by a specific electrode, were 1.75 k 0.05 m M ( calibration was performed with solutions containing 1.2 mMmagnesium). The solutions were oxygenated during perfusion by fine bubbling with a mixture of 95% oxygen and 5% carbon dioxide to a final pH of 7.4. The mannitol solution contained 270 m M d-mannitol and 10 m M shift reagent Tris3Dy(TTHA) 3Tris (mannitol-SR) at 3 10 mosm.

-

-

-

Experimental Protocols Male Sprague-Dawley rats weighing 220-300 g were anesthetized by intraperitoneal injection of sodium pentobarbital (30 mg/ kg). The heart was excised through a bilateral thoracotomy and arrest was induced by immersion in Krebs-Henseleit solution at 4"C, containing 1000 units of heparin sulfate. Following aortic cannulation, the heart was perfused in a Langendorff system of retrograde coronary perfusion for 30 min at 37 "C, during which all hearts presented steady-state hemodynamic parameters for at least 10 min. The hearts were then perfused for 4 min with KH-SR. Ischemic arrest was achieved by perfusing 4 ml KH-SR solution at 4°C for 20 s while the heart was

SODIUM TRANSPORT IN ISCHEMIC RAT HEARTS

25 1

bathed in cold (4°C) isotonic mannitol-SR solution. All hearts were at rest within 10 s from initiation of the cold perfusion. The hearts were preserved in a 350-ml flask containing either the KH-SR or KHLa solutions. The ischemic periods were 8 h of preservation at 4"C, 5 h at 15"C,and 2 h at 22°C. 23NaNMR measurements were performed while the heart was immersed in mannitol-SR solution; the average period of mannitol bathing for each heart was about 8%of the total preservation time. For the ouabain experiments the same protocol was applied and the perfusion solution contained 0.1 m M ouabain (20, 21). All solutions, instruments, and glassware were precooled to the appropriate temperature of the experiment. A Teflon rosette mounted on the aortic cannula positioned the heart in the center of the NMR tube, preventing it from bumping the tube walls during the exchange of bathing solutions, and stabilized the intraventricular balloon catheter. Reperfusion consisted of simultaneous warm perfusion and bathing in 37 "C solution. Hemodynamic parameters were monitored during the initial perfusion and reperfusion periods by a latex balloon introduced into the left ventricle through an incision at the base of the left auricle. Data were recorded on a Gould 402 recorder equipped with a pressure amplifier. The balloon was left deflated in situ throughout the ischemic period. Pacing was performed by a Devices Implants LD external stimulator, at rates of 240-300 bpm (excitation potential 4V).

Visibility Experiments The intraventricular balloon was inflated with about 0.2 ml of distilled water in order to decrease the extracellular sodium signal. Following the cold arrest the hearts were kept in a KH-SR solution for 2 h, allowing the increase of intracellular sodium ion concentration. Then the hearts were bathed in a mannitol-SR solution for sodium washout from the extracellular compartment. The intracellular Na' content depended directly on the period of storage in sodiumfree solution. The mannitol solution was exchanged every 3 h. Immediately after the last 23Na-NMRmeasurement was recorded, the hearts were dried at 80°C for 24 h in 20 ml ceramic cups and ashed in an oven at 700°C. Active conflagration of the tissue was avoided by gradually increasing the temperature by 100°C per hour. The tissue residue was dissolved with 0.5 ml HNO, 3.5 M and then diluted to a final volume of 5 ml with 0.5 m M HNO,; 1.5-ml samples were introduced into glass bulbs with dimensions similar to those of the hearts for the NMR measurements. A similar procedure was employed for estimation of the 23Navisibility of the extracellular compartment; the periods of extracellular Na 'washout were shorter, for measurement of high extracellular Na' concentrations.

NMR Spectroscopy A Bruker AM 360 WB spectrometer equipped with a 20-mm broad band probe operating at 95.245 MHz was used for 23Nameasurements. The temperature of the probe was adjusted and stabilized at the preservation temperature, and fluctuations were within 1"C. A thermocouple was present in the bathing solution of the measured hearts at all times. 23NaNMR spectra were collections of 600 transients, acquired

*

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ASKENASY ET AL..

with 90" pulses and an acquisition time of 0.26 s. The FID was multiplied by a Gaussian-Lorentzian function before Fourier transformation. Integrals of the lines were measured by the relative weights of paper of the appropriate signals. An external reference of 50 m M NaCl and 10 m M Na,Dy(TPP)2 was used throughout all the experiments. The reference solution was contained in a 6-mm-diameter glass bulb, mounted on a Teflon pedicle, and placed at the bottom of the tube at a distance of about 2 mm from the heart. The shift reagent Dy( TPP)T7 caused an upfield shift of the sodium ion peak of the reference (14). The area of the reference throughout the ischemic periods had fluctuations less than 10%.The reference was calibrated several times during each experiment against a glass bulb with diameter similar to that of the heart; the bulb was filled with a NaCl solution of known volume and concentration. 3'P measurements were performed at 145.7 MHz with a 20-mm selective probe. Each spectrum was a collection of 400 transients during perfusion and 800 transients during ischemia. The acquisition parameters were 40" rf pulses, acquisition time 0.28 s, and relaxation delay 1.0 s. Spectra were collected with 4K data points of a 7350Hz spectral width. Quantitation of the peak integrals was performed with two references, as described for the 23Nameasurements. The external reference was ethylenediamine NNN'W-tetramethylene phosphonate ( EDTMP) (T. Kushnir and G. Navon, unpublished data). It was calibrated against a solution of 2 m M KH2P04, 145 m M NaCl, and 0.06 m M NaGd( EDTA). The integrals of the phosphorous metabolites were corrected by their saturation factors which were determined by comparing spectra of the hearts using short relaxation delays ( 1.O s) and long relaxation delays (8.0 s at 4°C and 12.0 s at 37°C). Collecting these spectra by alternating acquisition of the short and long relaxation delays eliminated the possibility of concentration changes of the metabolites. Intracellular pH values were calculated from the chemical-shift differences between the Pi and the PCr signals at the various temperatures, using the formula given recently by Kost (22). RESULTS

NMR visibility of Na+ in the intra- and extracellular compartments was measured as described under Methods. Typical 23Naspectra used for the visibility measurements are given in Fig. 1. Figure 1a is a spectrum taken after 2 h of storage at 4°C in KHSR solution, during which the intracellular Nat peak increased; the heart was then immersed in a 4°C mannitol-SR solution. Figure 1b is the spectrum obtained from the same heart after 6 h of storage in mannitol-SR, during which the extracellular sodium peak disappeared. This heart was ashed, dissolved in acid, and the 23NaNMR spectrum of the acid solution was measured using the same external reference. The residual peak of sodium in the mannitol bath was not included in the integral since it remained in the solution when the heart was removed for ashing. The areas of the intracellular Na' peaks of the hearts were compared with the integral values of the sodium contents following ashing of the tissue. The visibility was calculated assuming 100%sodium visibility of the acid solutions of the ashed hearts. The value obtained for the visibility of the intracellular sodium at 4°C was 40 14%. There was no correlation between the concentration of sodium in tissue and the NMR visibility. This was evidenced by the fact that the results (Table 1 ) were evenly scattered around the

253

SODIUM TRANSPORT IN ISCHEMIC RAT HEARTS

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FIG. 1. 23Naspectra used for the determination of the sodium ion visibility:out, Z3Nafrom the surrounding mannitol-SR; ex, Z3Nafrom the extracellular compartment; in, 23Nafrom the intracellular compartment. (a) Z3Naspectrum of a heart after 2 h of ischemia in KH-SR at 4°C. ( b ) 23Naspectrum of the same heart after an additional 6 h of storage mannitol-SR at 4°C. (c) "Na spectrum of a heart stored for 2 h in KHSR and 3 h in mannitol-SR. FIG.2. Sequential "Na spectra of a heart stored in KH-SR at 4°C:. Spectra were recorded while the heart was immersed in mannitol-SR.

value of 40% over the entire range of sodium concentrations from 59 to 149 pmol/ grams dry weight of the heart (gdw). The quantitative determination of the intracellular Na+ enabled calculation of the extracellular visibility. Figure 1c represents an experiment like Fig. Ib, but some of the extracellular Na' was retained by shorter periods of bathing in mannitol-SR solution. The visibility of this extracellular Na+ was calculated using the expression visibility,,(%)

=

100 X Zex/(Is- (Zi,,/0.4)),

where Zi, and I,, are the areas of intra- and extracellular sodium signals measured in tissue and Z, is the integral in the solution of the ashed hearts. The value of the NMR visibility of the extracellular Na+ was 97 f 1 1% (Table 2, the error is the standard deviation). Since these results depend on our measured values of 40 f 4% visibility of the intracellular sodium, the error in the calculated extracellular visibility is somewhat

254

ASKENASY ET AL. TABLE 1 NMR Visibility of Intracellular Na' Experiment No.

"a']

intracellular pmol/gdw"

1

Visibility intracellular %

59 64 74 75 19 94 117 120 121 133 137 149

2 3 4 5 6 7 8 9 10 11 12

40 38 34 39 40 33 46 31 46 41 41 42 mean

gdw

=

=

40 f 4

gram dry weight.

larger. It should be noted that our definition of the extracellularcompartment includes the interstitium, the vasculature, and the heart chambers, the volume of which was partially occupied by the left ventricular balloon. The relative contributions of these spaces to the spectral peak were not estimated. In all the experiments described below, the values of 40 and 97% were used for the NMR visibilities of the intracellular and extracellular compartments, respectively. Perfusion with DY(TTHA)~-was camed out according to Pike et al. ( 1 7 ) . In general, the shift reagent was well tolerated by the beating hearts. Hearts exposed to

TABLE 2 NMR Visibility of Extracellular Naf Experiment No.

"a+] intracellular pmol/gdw

"a+] extracellular pmol/gdw

Visibility extracellular %

13 14 15 16 17 18 19 20

30 56 58 66 68 83 119 132

57 53 56 16 45 12 63 79

109 86 103 92 100 115 84 90 mean

=

97 f 11

255

SODIUM TRANSPORT IN ISCHEMIC RAT HEARTS

KH-SR solution ( n = 56) showed a slight decrease of the spontaneous rate (from 255 20 to 220 ? 25 bpm), similar to that observed by Pike et a1 ( 17). At the same time, they showed an increase in contractility force from 139 4 15 to 155 18 mmHg. No effect of the shift reagent on the contractility force was observed when the hearts were paced at a constant rate. Paced hearts exhibited a short period of instability following the switch of solutions without and with SR, which stabilized after about 20 s and then reached the previous level of contractility. The amount of Ca2+in the KH-SR solution was adjusted to obtain the same free calcium concentration as that in the KH solution. Perfusion of hearts with shift reagent for longer periods of time or higher concentrations ( 15 mM) did not improve the quality of the 23Naspectrum. The sequential plots presented in Fig. 2 illustrate the Na+ transport during ischemic preservation at 4°C in KH-SR solution. The 23Naspectra are composed of an unshifted peak corresponding to the intracellular Na+, a broad downfield shifted peak representing the extracellular Na+, and a sharp peak shifted further to lower field representing the Na+ in the mannitol-SR bath. The assignment was confirmed by measurements of the chemical shifts of the KH-SR solution in glass bulbs, and those of sodium and mannitol-SR solutions in the outer tube. The extracellular peak is composed of the interstitial and vascular compartments, which are not resolved. This insufficient resolution limits the possibility of dealing with these two compartments separately, in a quantitative manner. The broad signal of the extracellular sodium that looks like a conglomerate of resonances must be a result of a combination of different ratios of SR to sodium within the various compartments and the effect of the bulk susceptibility variation within the tissue which is amplified by the presence of shift reagent. Figure 3 illustrates the compartmental distribution of the sodium ion in hearts preserved at different temperatures in a medium containing 145 m M sodium. The standard deviation reflects mainly the variability between individual hearts within each of the experimental groups. The experimental error was much lower than the physiological variability. Figure 4 compares the intracellular sodium influx at the three temperatures. The biphasic time course of the Na+ influx of hearts preserved for 8 h at 4°C is composed at the first approximation by the rates: 1.45 0.17 pmol/(gdw X min) and 0.17 t 0.05 pmol/( gdw X min) for 0- 1 and 2-7 h, respectively. At 15"C, the Na+ influx rates were 0.71 ? 0.04 and 0.21 k 0.02 pmol/(gdw X min) for 0-1 and 1-5 h, respectively. At 22"C, the initial Na+ concentration was 24 5 pmol/ gdw and the influx rates were 0.69 -t 0.06 and 0.28 :t 0.04 pmol/(gdw X min) for 0-1 and 1-2 h of storage, respectively (Fig. 4). In experiments at 22°C with 0.1 m M ouabain in the perfusion and storage solutions the initial intracellular Nai concentration was 42 4 pmol/gdw, and the sodium influx rates were 0.36 0.06 and 0.53 T 0.06 pmol/ (gdw X min) for 0- 1 and 1-2 h, respectively. The time courses of the sodium content in the extracellular compartment at the three temperatures (Fig. 3 ) appear at first glance to be mirror images of the time courses in the intracellular compartment, indicating that trans-sarcolemmal influx is the dominant process. However, there is a substantial exchange between the extracellular compartment and the surrounding solution evidenced by the increase in sodium content in the mannitol solution during spectra acquisition of consecutive measurements. In our experimental protocol, both ventricles were in contact with the sur-

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256

ASKENASY ET AL.

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120 240 Ti me ( m i n )

120

240

Time (mtn)

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480

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TIME (rnin)

FIG. 3. Extracellular and intracellular sodium ion time courses at different storage temperatures. The expressed standard deviations represent the variability within the experimental groups. The filled circles in the top plot represent the group preserved at 22°C and perfused and stored with KH-SR containing 0.1 m M ouabain. FIG.4. A summary of intracellular Na+ influxes at different temperatures of hearts stored in KH-SR.

rounding bath: the right side through an incision in the pulmonary artery which released the coronary perfusate, and the left side through an incision made at the base of the auricle for introduction of the intraventricular balloon. This leakage is substantially slower at the lower temperature, which may explain the higher extracellular sodium content observed at 4°C relative to that at 15 and 22°C where the exchange is faster during the NMR measurements. The extent of interstitial Na+ loss to the surrounding mannitol bath was estimated by an experiment where no repeated immersion in mannitol was done. Two hearts were stored at each temperature in KH-SR solution and the sodium content was measured once, 15 min before reperfusion. The intracellular sodium values were within the variability range of the hearts in all the temperature groups. While the extracellular

257

SODIUM TRANSPORT IN ISCHEMIC RAT HEARTS

values at 4 and 15°C were within the experimental error of the normal protocol, the values at 22°C were higher by 47 2 12%, indicating a substantial loss of sodium to the surrounding bath during cyclic immersion in mannitol-SR solution. The changes in the overall Na' content of the whole hearts were as follows: (a) at 4°C the content increased by 10 2% during the period 4-8 h; (b) at 15°C the content increased by 7 2 2% after 2 h and 23 +- 5% after 5 h; ( c ) at 22°C the content decreased by 18 k 3% after 1 h and remained stable during the second hour. The results show a gain in the sodium content caused by the influx into the intracellular compartment, a partial replenishment of the extracellular sodium from the surrounding KH solution, and at 22°C a loss during the NMR measurements when the hearts were immersed in mannitol-SR instead of KH-SR. Figure 5 illustrates "P spectra of a heart stored at 4°C in KH-La solution for 8 h. The protocol was the same as for the 23Na experiment except DY(TTHA)~-was replaced by La(TTHA)3-. The use of the diamagnetic analog of Dy( TTHA)3- gave conditions similar to those with the SR without the broadening effects of the paramagnetic Dy( 111). The depletion of high-energy compounds throughout the ischemic periods of storage are shown in Fig. 6. The depletion curve of the peak at 6 = 18.83 ppm representing the PATP and that at 6 = -5.26 ppm representing the ATP and PADP were parallel throughout the storage period, indicating no accumulation of ADP. A phosphomonoester peak appeared at the same time, presumably part of it representing the accumulation of AMP. The levels of the high-energy phosphorus compounds are given in absolute values, conforming to the calibration procedure as described in the expenmental section. In accordance with previous studies ( 2 6 - N ) , we found that during cold storage PCr and ATP gradually decayed to low values at the end of the ischemic period. During normothermic reperfusion there was only a partial recovery of the ATP levels. The pH rapidly became acidic in all the experimental groups at the three temperatures, but recovered rapidly during reperfusion to near preischemic values. The hemodynamic recovery parameters of the hypothermically preserved hearts are listed in Table 3; hearts that failed to recover (NR in the table) were not included. Control of the heart rates permitted a more accurate evaluation of myocardial performance independent of the storage damage caused to the excitatory and conductive systems. Hearts allowed to resume spontaneous left ventricular activity before the initiation of pacing had faster recoveries and reached their steady-state function earlier. This was true for both fast (240-300 bpm) and intermediate ( 100- 150 bpm) stimulation rates. Following 4°C storage, the recorded pressure of the hearts before they resumed function was in the range of 40-60 mmHg, with the diastolic pressure during contraction taken as zero. This high-pressure state was less pronounced at higher temperatures. When temperature of the myocard was measured during the storage and reperfusion periods with a needle thermocouple, the temperature of the anterior wall of the left ventricle was found to be 0.5-1°C higher than that of the surrounding bath. During the exchange of solutions, the temperature rose no more than 2 2 0.5"C for less than 10 s. At reperfusion, hearts stored at all the temperatures reached 37°C within 1.5 k

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258

ASKENASY ET AL.

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Fie. 5. Sequential ,'P spectra of a heart during perfusion, ischemia at 4°C for 8 h, and reperfusion. The shift reagent was exchanged for its diamagnetic form, La( TTHA)-3. Spectra recorded during perfusion and reperfusion are accumulations of 400 transients; those recorded during ischemia are accumulations of 800 transients. A, reference; B, phosphomonoesters; C, inorganic phosphate; D, phosphocreatine; E, F, G , ATP. Fie. 6 . High-energy phosphorus compounds and intracellular pH monitored by 3'P NMR spectroscopy at storage temperature of 4, 15, and 22°C. The solution contained the diamagnetic analog ofthe shift reagent, Na,La(TTHA). Integrals were corrected by the saturation factors at the three temperatures and were referred to an external reference for quantitative conversion. The expressed standard deviations represent the variability within the experimental groups.

0.5 min. These hearts were excluded from the results because of the mechanical damage caused by implantation of the thermocouple. DISCUSSION

The quantitative conversion of 23Na NMR integrals to sodium contents requires knowledge of the NMR visibilities of sodium in the different tissue compartments. In

259

SODIUM TRANSPORT IN ISCHEMIC RAT HEARTS TABLE 3 Hemdynamic Recovery % Spontaneous

Recovery time (min)

Temp. "C

Time h

n

NR

Paced LVP%

LVP

HR

Initiation LV activity

Steady state

22°C 15°C

2 5 8

12 12 12

2 2 0

48k28 52114 69 k 2 6

53228 59k19 7 5 ? 33

87 f 10 75*15 75 + 16

3+2 15+6 826

9* 3 23k 9 3 7 + 12

4°C

Note. n, number ofexperiments; NR, number of hearts that did not recover; LVP, left ventricular pressure (percentage of preischemic value); HR, heart rate (percentage of preischemic value).

principle, sodium ions are 100%visible. But, NMR visibility has to be measured for each experimental system because of the multiexponential decay of the transverse magnetization and the occurence of broad components that merge with the baseline and are reduced by the delay between the pulse and the beginning of the acquisition. Our results indicated 40 k 4% visibility of the intracellular sodium, and 97 k 1 1% visibility in the extracellular compartment. While these values might be specific to our experimental conditions, they are directly applicable for the conversion of NMR integrals to molar amounts in the present work. Previous reports on intra- and extracellular 23NaNMR visibility in excised rat hearts gave conflicting results. Pike et al. ( 1 7 ) estimated the intracellular Na' visibility to be lower than 20%, based on assumptions of intracellular Na+ concentrations and volumes at physiological temperature. Fossel and Hoefeler ( 2 3 )and Jelicks and Gupta ( 2 5 )on the other hand, reported NMR visibility close to 100%for both the intracellular and extracellular Na+ in excised frog and rat hearts and in mammalian myocites ( 2 4 ) .The discrepancies between these authors might be due to assumed volumes and sodium ion concentrations in the myocard based on different experimental conditions ( 1 7 )and/or excess intraventricular sodium that was not taken into account ( 2 5 ) .In our work we made no assumptions of unmeasured parameters: a single signal originating solely from the intracellular sodium was obtained by extensive washing of the hearts with mannitol-SR solution. The absolute visibility of the intracellular sodium was obtained by comparison of the tissue signal to the sodium signal of the tissue extract contained in a glass bulb with a spatial configuration similar to that of the heart. During global ischemia at 4"C, we found a net influx of sodium at a mean rate of 1.9 pmol/(gdw X min) during the first 30 min, which decreased continuously to 0.17 pmol/(gdw X min) after 2 h of storage. This decrease was expected in view of the decrease of the sodium ion concentration gradient across the sarcolemmal membrane. The equilibrium of the sodium ions in the ischemic myocardium is determined by several ionic fluxes: passive diffusion influx, channel mediated influx, cotransport systems, ion exchangers, and ion extrusion by active transport systems ( 3 2 ) . The diffusion fluxes are generally determined by the membranal permeability constant and the intercompartmental gradient. The transmembranal sodium gradient is, in

260

ASKENASY ET AL..

fact, the main driving force for the passive inward translocation of the sodium ion. For constant permeability indices, the gradient shapes the time course of intracellular accumulation. A quantitative estimation of the ionic gradient, which should be expressed in units of molar concentrations, requires knowledge of the volumes of the various compartments throughout the storage period. Figure 3, which represents the intra and extracellular Na+ curves for each experimental temperature, illustrates the decrease in the intercompartmental gradient and its rate. At 4°C the active pumps responsible for Na+ extrusion operate at very low rates, which means that the net changes are determined mainly by the passive transport. However, a detailed analysis of the various influx pathways and driving forces is not feasible with the limited information presented here. Increasing the temperature decreases the initial influx rate: from 1.9 pmol/(gdw X min) at 4°C to 0.71 pmol/(gdw X min) at 15°C and 0.69 pmol/(gdw X min) at 22°C (during the first 30 min, Fig. 4). It is not reasonable to assume that the passive ionic transport has a negative temperature dependence; rather, an opposing process might be more active at higher temperatures. One such process is an active efflux caused by Na/K ATPase, which is known to be temperature sensitive (33, 3 4 ) . The activity of this pump was demonstrated by introduction of ouabain, its specific inhibitor. While previous studies reported the insensitivity of rat hearts to ouabain ( 2 0 ) ,intracellular sodium concentration increased in our experiments at an ouabain concentration of 0.1 mM. Ouabain affected the sodium levels during normothermic perfusion, but not the influx rate during ischemia. Thus ouabain's influence appears to be limited to the period when the heart has not yet reached the final low temperature ( 3 3 ) .This observation suggeststhat at 22"C, temperature inhibition of the Na/K ATPase pumps is stronger than ouabain inhibition. Our results indicate that temperature affects both the active and passive transport systems in the cellular membrane. At lower temperatures we observe mainly the passive transport; at higher temperatures the passive transport is expected to be faster, but the energy-dependent pumps are more active as well. Thus, the net influx during the initial 30 min, which drops from about 1.9 pmol/ (gdw X min) at 4°C to 0.71 pmol/(gdw X min) at 15"C, changes insignificantly to 0.69 pmol/(gdw X min) at 22°C. It should be noted that the use of initial influx rates had the advantage that the ATP reserves were not yet depleted. The decrease in the net sodium ion influx rate between 4 and 15°C reflects a sharp increase in the activity of the pumps, exceeding that of the passive transport. On the other hand, the lack of change between 15 and 22°C means that the increased activities of the sodium pumps and the passive transport compensate each other. These results are in line with previous studies on rabbit kidney slices ( 3 3 ) and rabbit and toad hearts ( 3 4 ) , which show a steep temperature dependence of the ATPase activity below 18°C associated with the change in the fluidity of the myocardial membrane ( 3 4 ) . The differences in the success of long-term preservation of different organs was attributed to the differential activity and temperature dependence of the Na/ K ATPase pumps ( 5 ) . In a comparative study of the electrolytic changes in certain tissues during 1 h of perfusion at 10°C in plasma, the net gain of Na+ in hearts was 180%,in livers 1609'0, and in kidneys only 60% of the initial content ( 5 ) . It was assumed that the capability of the pumps to maintain the ionic equilibrium was responsible for the

SODIUM TRANSPORT IN ISCHEMIC RAT HEARTS

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preservation success of kidneys in a physiologic solution. In our results the presumed critical role of the pumps in successful storage was not so evident. At the lowest temperature (4"C), the concentration of intracellular sodium at reperfusion time was 209 -+ 25 pmol/gdw, while at 15°C the concentration was 114 -+ 15 pmol/gdw, and at 22°C it was 77 f 6 pmollgdw. These Na+ values and the overall Na' gain of the whole organ did not correlate with the hemodynamic outcome of storage. Moreover, intracellular Na+ concentrations were not correlated with functional recovery on an individual basis within the experimental groups. These results indicate that sodium ion accumulation is not the dominant factor determining ischemic injury. The hemodynamic parameters show a better recovery of the hearts stored at lower temperatures, in agreement with previous studies of long-term storage (30, 31 ). The recovery following 4°C storage for 8 h was better than the recovery for shorter periods of storage at higher temperatures. For very short periods of cardiac arrest, Jamieson (35)recommended 1O-2O0C, based on 15-min experiments performed by Hearse et al. (36). In the absence of adequate preservation solutions to handle the specific cold metabolism of the heart for long periods of time, hypothermia should be considered a delay procedure of ischemic injury. Besides the left ventricular pressure recovery differences between the experimental groups, two additional hemodynamic parameters were significant: the high diastolic pressure during reperfusion before contraction was recorded and the period of time required to reach steady-state function (Table 3). The diastolic tonicity was in the range of 40-60 mmHg following 4°C storage, and less at higher temperatures. Similar, high diastolic pressures were reported to occur at 37"C, when the Na/K ATPase pumps were inhibited by low extracellular potassium concentrations, ouabain (37, 38),or perfusion with low sodium concentrations ( 3 9 ) .Under our experimental conditions, the pumps were inhibited by low temperatures. Storage at 4°C caused a marked delay in the time until the hearts reached steady-state myocardial activity. The myocard reached 37°C after about 1.5 min of reperfusion, and thus the delay is not due to the equilibration of temperature. The diastolic tonicity was attributed in a previous work ( 4 0 ) to increased intracellular calcium ion concentration caused by a change in the calcium homeostasis following partial inhibition of the Na/ K ATPase pumps. An increased rate of calcium influx is assumed to be responsible for the reperfusion behavior in our experiments as well. Not only were the Na/K ATPase pumps inhibited, but the high intracellular Na+ concentrations could have caused an early reperfusion influx of Ca2+through the Na+/Ca2+exchange system. This exchanger was shown to be a voltage-dependent process ( 4 0 ) with the stoichiometry of exchange of 3 Na+ per each Ca2+(41) under physiological conditions. Although this ratio was measured for sodium ion influx, this system also proved to be reversible in direction at high intracellular Na+ concentrations ( 4 2 ) , as was the case in our experiments (calcium ion influx). It is reasonable to assume that preservation solutions that limit the accumulation of intracellular sodium will assist in the recovery process during reperfusion. In conclusion, 23NaNMR spectroscopy using shift reagents can monitor the sodium ion translocation across the sarcolemmal membrane, and 31PNMR spectroscopy can analyze high-energy phosphorous compounds. The 23NaNMR visibilities of the heart compartments are 40% for intracellular and 97% for extracellular. Intracellular influx

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of sodium during ischemia is an important parameter for the assessment of ischemic damage. The intracellular concentration is not correlated with the reperfusion recovery probability, indicating that the presence of sodium in the cytosol is not the dominant factor for ischemic injury. Its concentration modulates the reperfusion hemodynamics, probably affecting the calcium ion homeostasis. The best preservation is achieved at 4°C. The finding that the Na/K ATPase pumps are inactive implies that this mechanism is not directly involved in the determination of cold preservation success. Further studies are required to clarify the physiological meaning of these experimental observations and their application to the composition of storage solutions. REFERENCES

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