Multi-dose Crystalloid Cardioplegia Preserves Intracellular Sodium Homeostasis in Myocardium

J Mol Cell Cardiol 31, 1643–1651 (1999) Article No. jmcc.1999.1002, available online at http://www.idealibrary.com on

Multi-dose Crystalloid Cardioplegia Preserves Intracellular Sodium Homeostasis in Myocardium Victor D. Schepkin1,2, Isaac O. Choy3, Thomas F. Budinger1, J. Nilas Young3 and William M. DeCampli4 1

Center for Functional Imaging, Lawrence Berkeley National Lab, Berkeley, CA, 2Biomedical Magnetic Resonance Lab, University of Illionois at Urbana-Champaign, Urbana, IL, 3Division of Cardiothoracic Surgery, Children’s Hospital Oakland, Oakland, CA, 4Department of Pediatric Cardiac Surgery, Beth Israel Medical Center, Newark, NJ, USA (Received 16 November 1998, accepted in revised form 28 May 1999) V. D. S, I. O. C, T. F. B, J. N. Y  W. M. DC. Multi-dose Crystalloid Cardioplegia Preserves Intracellular Sodium Homeostasis in Myocardium. Journal of Molecular and Cellular Cardiology (1999) 31, 1643–1651. The goal of this study was to assess the effect of multi-dose St Thomas’ cardioplegia on intracellular sodium homeostasis in a rat heart model. A new magnetic resonance method was applied which enable us to detect intracellular Na changes without chemical shift reagents. Three groups of isolated rat hearts were subjected to 51 min of ischemia and 51 min of reperfusion at 37°C: Group 1—three infusions of St Thomas’ cardioplegia every 17 min for 2 min (n=7); Group 2—single-dose infusion of cardioplegia at the beginning of stop-flow ischemia (n=8); and Group 3—clamp ischemia (n=3) without cardioplegia administration. Performance of the heart was assessed by rate–pressure product relative to the pre-ischemic level (RPP). An NMR method was applied which continuously detects the Nai concentration in the heart, using the ability of bound sodium to exhibit triple-quantum transitions and the growth of the corresponding signal when sodium ions pass from extracellular to intracellular space. Clamp ischemia without cardioplegia and 50 min of reperfusion left the heart dysfunctional, with Nai growth from the pre-ischemic level of 13.9±1.2 m to 34.9±1.3 m and 73.9±1.9 m at the end of ischemia and reperfusion, respectively. During single-dose cardioplegia the corresponding values for Nai were 30.2±1 m and 48.5±1.7 m (RPP=29%). Multiple infusions of cardioplegic solution resulted in a remarkable preservation of the heart’s intracellular Na concentration with a non-significant increase in Nai during ischemia and only 16.7±1 m, (P=0.01), after subsequent reperfusion (RPP=85%). The time course of Nai changes in the rat heart model demonstrates a prominent potential of multi-dose St Thomas’ cardioplegia in preserving intracellular sodium homeostasis at 37°C. The growth of Nai concentration during ischemia, as an indicator of the viability of the myocytes, can have a prognostic value for the heart’s performance during reperfusion.  1999 Academic Press K W: Intracellular sodium; Cardioplegia; NMR; Triple-quantum filtering.

Introduction Administration of a crystalloid cardioplegic solution is an effective means for myocardial preservation during heart surgery (Buckberg, 1995; Gebhard et al., 1995; Cleveland et al., 1996). Multi-dose cardioplegia (CP), first introduced in 1976 (Nelson

et al., 1976), enhances the recovery of cardiac function after surgically-induced ischemia. The goal of the present study was to investigate whether improvement in functional recovery provided by multi-dose CP as compared to a single-dose CP is correlated with maintenance of sodium homeostasis during ischemia.

Please address all correspondence to: Victor D. Schepkin, Biomedical Magnetic Resonance Lab, University of Illinois at UrbanaChampaign, Urbana, IL 61801, USA.

0022–2828/99/091643+09 $30.00/0

 1999 Academic Press

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A quantitative relationship between Nai concentration and the triple-quantum filtered (TQF) Na magnetic resonance (MR) signal has been reported recently in a rodent heart during continuous perfusion of extracellular space (Schepkin et al., 1998a, b). The method utilizes the property of bound sodium to exhibit multiple quantum transitions under the influence of the electric field of the macromolecule (Pekar et al., 1987; Payne et al., 1990; Rooney and Springer, 1991; Jelicks and Gupta, 1994; Navon et al., 1994; Dizon et al., 1998). Unlike previous NMR methods (Pike et al., 1985; Malloy et al., 1990; Miller et al., 1991; Navon et al., 1994; Ingwall et al., 1996; Radford et al., 1998; Van Emous et al., 1998; Winter et al., 1998) this method does not use chemical shift reagents. Our choice of the TQF NMR is crucial because the high concentration of Mg2+ ions (16 m) in St Thomas’ cardioplegia may reduce chemical shift for extracellular Na (Pike et al., 1985) making it impossible to detect the Nai in the heart. Validation of the TQF NMR technique during cardioplegic arrest has been carried out by applying a different means of Nai detection. Sucrose–histidine wash-out of extracellular space in combination with NMR measurements of residual Na signals gave us the possibility to distinguish sodium changes in the extracellular and intracellular compartments. It has been demonstrated (Schepkin et al., 1996; Choy et al., 1997) that at the beginning of ischemia a single-dose cardioplegia may delay the Na triplequantum filtered (TQF) NMR signal growth, which suggests a corresponding delay in Nai increase. In this study, the experiment was modified by using multi-dose cardioplegia, and we now present the quantitative results of Nai measurements, which evidence the efficiency of cardioplegia in preserving of intracellular Na.

Materials and Methods Male Sprague–Dawley rat (250–300 g) hearts were excised rapidly and attached to a Langendorff system. Perfusion and reperfusion were performed using modified Krebs–Henseleit (K–H) solution (m) at 37°C: 118.5 NaCl, 25.0 NaHCO3, 4.9 KCl, 1.2 KH2PO4, 1.2 MgSO4, 1.2 CaCl2, 11.1 -glucose, oxygenated with 95% O2/5% CO2 (pH 7.4). The rat heart was suspended in the air in the temperaturecontrolled chamber at 37°C. An additional waterjacketed reservoir was used to supply warm, oxygenated cardioplegic solution. Cardioplegic arrest was performed by perfusion of warm (37°C) oxygenated St Thomas’ cardioplegic solution (m): 110

NaCl, 10 NaHCO3, 16 KCl, 1.2 CaCl2, 16 MgCl2 at pH 7.8. A warm supply of the CP was ensured by flushing the infusion line with warm CP solution immediately before each cardioplegia intervention. Eighteen rat hearts were divided into three groups and subjected to 51 min of ischemia and 51 min of reperfusion. In the first two groups (1 and 2), ischemia was achieved by infusion of St Thomas’ cardioplegia. In group 1, after a stabilization period of >30 min, the cardioplegia was introduced three times for 2 min, where each infusion was followed by a 15-min stop-flow interval (n=7); in group 2, there was only a single-dose infusion of cardioplegia for 2 min at the beginning of stop-flow ischemia (n=8). Group 3 served as a reference, where a clamp ischemia (n=3) was introduced without administration of cardioplegia. Performance of the heart was controlled by monitoring the heart rate and LVDP pressure product (RPP) relative to the pre-ischemic level. We used a non-circulating perfusion system. Perfusate was evacuated through the tubing connected to the bottom of the NMR coil container. The wash-out of the extracellular Na from the rat heart was performed using a specialized procedure (Alto and Dhalla, 1979; Tani and Neely, 1989). Ten milliliters of the ice-cold wash-out solution (350 m sucrose, 5 m histidine, pH 7.4) were perfused through the heart for 1–2 min. The time dependence of the loss of the Na NMR signal during wash-out consisted of two exponential functions, with the amplitude of its slow component corresponding to the Nai NMR signal detected by using chemical shift reagent. Altogether this proved that the applied wash-out procedure is sufficient to eliminate the extracellular Na (Schepkin et al., 1998a). Intracellular sodium changes were detected on the NMR imaging system (2.35 T) using TQF pulse sequence 90°φ−s/2−180°φ+a−s/2−90°φ+a−d−90°e where s=4.4 ms, d=40 ls (Schepkin et al., 1996). Repetition time of the whole pulse sequence was 310 ms. Each observation point used 192 acquisitions and required 1.5 min. Na TQF NMR peak intensities were used to assess the effects of interventions.

Intracellular Na measurement The linear relationship between sodium concentration [Nai] and Na TQF NMR signal found in

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Figure 1 Time course of intracellular Na concentration in the rat heart before intervention, during multi-dose cardioplegia and after reperfusion in comparison to single-dose cardioplegia and clamp ischemia. The rate–pressure product recovery of the heart (relative to the pre-ischemic level) at the end of reperfusion were 85% (multi-dose CP), 29% (single-dose CP) and 2% for clamp ischemia.

a continuously perfused rat heart model (Schepkin et al., 1998a,b) can be written as: [Nai](m)=[Nai]0+A1×(S/S0−1),

(1)

where A1 is a calibration constant, which is equal to 45 m. S is the Na TOF NMR peak intensity signal and S0 is its pre-ischemic level. The preischemic intracellular Na concentration [Nai]0 is 13.9 m. Equation (1) can be rewritten in the form

S/S0=K1×[Nai]+K2×[Naex],

(2)

where [Naex] is extracellular Na concentration, K1= 22.2×10−3 m−1 and K2=4.8×10−3 m−1. The ratio K1/K2 (=4.6) is an indication of the higher per ion yield of the TQF signal from intracellular Na than from extracellular Na. During stop-flow conditions, there is no perfusion of extracellular space, so the increase of Nai produces an equal decrease in extracellular Na content. There is also a large contraction of extracellular

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heart volume and some decrease in intracellular volume as well (Clarke et al., 1994; Askenasy and Navon, 1997). The difference in TQF sensitivity between intra- and extracellular Na makes possible the detection in Nai changes. It can be shown that in these conditions the intracellular Na concentration is [Nai](m)=[Nai]0+A1×

(S/S0−C) [1−(K2×Vi)/(K1×Vex)] (3)

where Vi and Vex are intra- and extracellular volumes; C=1 during clamp ischemia or 0.73 during cardioplegic arrest. The C value of 0.73 represents the initial change in the Na TQF signal, detected after infusion of cardioplegia and will be addressed later in the Discussion section. The value A2=A1/ (1−K2×Vi/K1×Vex)=54±5 m for the ratio Vi/ Vex in the range of 0.5–1. So the effect of ischemic changes in ratio Vi/Vex is dramatically attenuated by a factor of K1/K2=4.6 times. During stop-flow conditions in the rat heart, the larger value of A2 indicates only >20% less sensitivity of the TQF signal to intracellular Na changes in comparison to the perfused heart, which consequently has an unchanged value of [Naex]. Results of all intracellular Na measurements are presented as mean±SEM.

Results The time courses of Nai changes in the rat hearts before and during three different ischemic interventions with subsequent reperfusion periods are presented in Figure 1. Clamp ischemia gave the largest increase in intracellular Na concentration. The increase started almost immediately and resulted in Nai concentration of 34.9±1.3 m at the end of ischemia. Increases in intracellular Na concentration continued during reperfusion and reached a level of 73.9±1.7 m at the end of reperfusion with diminished RPP recovery of the rat heart of 2±2% (n=3). Several effects of single-dose CP can be seen in comparison to clamp ischemia (Fig. 1). First, the delay of >15 min in the starting point of the intracellular Na increase was observed, which corresponds to a previously noticed period of no changes in NMR TQF signal in similar conditions (Schepkin et al., 1996). After 50 min of cardioplegic arrest the intracellular sodium concentration was only 30.2±1 m (P=0.001). During reperfusion the rate of intracellular Na increase was

substantially reduced with a corresponding Na concentration of 48.5±1.7 m at the end of reperfusion. The heart rate pressure product recovery was accordingly better than for clamp ischemia (29±7%, n=8). Application of second and third infusions of CP at 17-min intervals dramatically extended the period of no change in Nai (Fig. 1). Fifty minutes of ischemia resulted in a non-significant increase of intracellular Na (14.3±1 m). At the end of reperfusion, the intracellular Na content was only slightly different from the pre-ischemic level (16.7±1 m, P=0.01). There was a marked RPP recovery of the heart at the end of reperfusion of 85±7% (n=7). The time course of the Na TQF NMR signal during multi-dose cardioplegic arrest is presented in Figure 2. The Na TQF signal immediately decreased by >27% after infusion of CP; this was followed by a period >50 min of no growth in the signal. There was restoration of the pre-ischemic TQF NMR signal after reperfusion. The above changes in the signal represent the decrease in extracellular sodium contribution to the TQF signal. The interpretation of this decrease was validated using a wash-out technique as an independent method of Nai detection which was performed during the first 15 min after CP arrest (Fig. 3). The sucrose–histidine wash-out of the extracellular space produced the same intensity of residual Na NMR signal observed before and after cardioplegic arrest. Two wash-outs were performed on each heart in both sets of experiments. In the first set, the first wash-out was accomplished during normal perfusion and the second was done after CP infusion (Fig. 3(a), n=2). In the second set of experiments, the first wash-out was completed immediately after CP arrest and the second wash-out was done during the following reperfusion (Fig. 3(b), n=2). All experiments produced the same residual Na signal of >11.6% (relative to the Na NMR signal from normally perfused hearts) corresponding to unchanged intracellular Na content (Schepkin et al., 1998a).

Discussion The results of this study represent, to our knowledge, the first observation of the effects of multidose cardioplegic solution on Nai in the whole rat heart. They also demonstrate the feasibility of measuring intracellular Na without chemical interventions. The observed preservation of intracellular sodium concentration yields insights into

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Figure 2 Time course of Na TQF NMR signal from a rat heart during multi-dose cardioplegic arrest. Sharp decrease in the TQF signal represents the effect of cardioplegia on extracellular sodium contribution to the total Na TQF NMR signal.

the mechanism of action of St Thomas’ CP, which may also be common to other types of cardioplegia. Differences between cardioplegic arrest and clamp ischemia can be seen during the first 15 min of ischemia. At the end of the subsequent 15 min of stop-flow ischemia a large difference in Nai accumulation was detected between these two interventions. Cardioplegic arrest gave dramatically less Nai of >15 m, while after clamp ischemia Nai was substantially more >26 m. A short delay of >3–5 min before apparent irreversible structural damage during clamp

normo-thermic ischemia is a well-known phenomena (Reimer and Jennings, 1992; Miura, 1995). The observed delay of >15 min before the intracellular Na rise which commences during single-dose CP arrest is an indication of the existence of the additional conservation period produced by cardioplegia (Fig. 1). One may expect this delay because of the known ability of CP to induce diastolic cardiac arrest, which decreases metabolic demands and decreases oxygen consumption by a factor of ten relative to stop-flow ischemia (Buckberg et al., 1977; Gebhard et al., 1995). At the same

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Figure 3 Validation of intracellular Na stability during first 15 min after application of cardioplegia: (A) wash-outs performed at normal perfusion and after CP; (B) wash-outs accomplished after CP and during reperfusion. The residual Na NMR signals after wash-out of extracellular space represent the unchanged intracellular Na content in a rat heart.

Multi-dose Cardioplegia

time, the stimulation of Na efflux through the Na/ K pump by the high potassium content (16 m) in the cardioplegic solution (Ko et al., 1995) may be another important mechanism of intracellular sodium preservation. The decreased concentration of Na in the extracellular space, created by cardioplegia, can decrease Na influx and may also be part of the mechanism of preventing the Nai growth (Stinner et al., 1989). A similar delay in myocardial intracellular Na overload was also detected in isolated guinea-pig hearts (Decking et al., 1998). A newly synthesized inhibitor of voltage-gated Na+ channels was applied. This accelerated the process of ceasing the electro-cardiac activity (similar to cardioplegia) and delayed intracellular Na growth for 12 min, after which the accumulation of Nai was identical to the control ischemic heart. The delay in intracellular Na increase produced by single-dose CP was extended effectively by multidose CP, which correlates with the cardioprotective effects of multi-dose cardioplegia (Buckberg, 1995). The cardioplegia solution used in this study did not contain glucose, implying that the CP infusions were capable of maintaining Nai level without directly supplying an energy substrate. Stabilization of Nai homeostasis during multi-dose cardioplegia corresponds to a superior recovery of the heart compared to other interventions applied in this study. Some experimental factors can also affect the outcome of multi-dose cardioplegic arrest. Auxiliary oxygen supply and wash-out of the extracellular space are occurring with each infusion of CP, and these conditions may augment the protective effects of cardioplegia. For all three ischemic interventions recovery of the heart is enhanced when Nai concentration during ischemia resembles the pre-ischemic level (Fig. 1). The ischemic intracellular Na accumulation may be a predictive factor for the extent of cardiac recovery upon reperfusion. A strong predictive possibility of Nai accumulation throughout the hypoxic period was also observed in isolated rat heart undergoing ventricular fibrillation (Neubauer et al., 1992; Pike et al., 1995). The wash-out technique is an established method of Nai detection (Alto and Dhalla, 1979; Tani and Neely, 1989). In our study, we applied it in combination with an NMR assessment of sodium content, which allowed us to monitor immediately the results of wash-out and, therefore, to perform a double intervention on the same heart. The observed unchanged residual signal before and after CP arrest indicates that there was no immediate

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change of intracellular Na concentration due to the infusion of cardioplegia. The sharp decrease in the Na TQF signal during CP arrest (Fig. 2), as confirmed in the validation experiments (Fig. 3), is due to the change of the extracellular sodium contribution to the TQF signal. The main part of this decrease is determined by the lower sodium content in cardioplegia (120 m) than in the KH solution (143.5 m) and by the presence of 16 m magnesium, which may compete with sodium for its binding sites (Bleam et al., 1980; Timonin et al., 1991; Schepkin et al., 1996). The time course of Na TQF signal during multidose CP and rat heart recovery are comparable to single-dose CP arrest at 21°C (Schepkin et al., 1996), thus suggesting that multiple CP at 37°C for 50 min may be as efficient in a rat heart preservation as single-dose cardioplegia at 21°C. Destabilization of Nai during ischemia is only a part of the multiple ischemic processes which contribute to the final dysfunction of the heart (Tani and Neely, 1989; Kloner et al., 1998; Meldrum, 1998). These include calcium dyshomeostasis, free radical and local inflammatory monokine production. However, the positive effects of sodium channel blockers (Snabaitis et al., 1997) and the correlation between the concentration of Nai and cardiac function shown in the present study demonstrate a strong relationship of Na transport with the outcome of ischemia.

Conclusion Multi-dose modified St Thomas’ cardioplegia maintains Nai homeostasis during 50 min of normothermic ischemia in an isolated rat heart. The observed intracellular Na during ischemia and reperfusion inversely correlated with the functional recovery of the rat heart. The accumulation of intracellular Na during ischemia can be an effective predictive factor for cardiac functional recovery following reperfusion. Na TQF NMR is able to monitor intracellular Na changes and has a potential to detect early cell response to pharmaceutical treatment and to assist in the development of the cardioprotective strategies.

Acknowledgement This work was supported in part by the National Institutes of Health (NIH-P41-RR05964).

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