Cardioplegic Strategies for Calcium Control
Low Ca2+, High Mg2+, Citrate, or Na+/H+ Exchange Inhibitor HOE-642
Background—Ca2+ overload plays an important role in the pathogenesis of cardioplegic ischemia-reperfusion injury. The standard technique to control Ca2+ overload has been to reduce Ca2+ in the cardioplegic solution (CP). Recent reports suggest that Na+/H+ exchange inhibitors can also prevent Ca2+ overload. We compared 4 crystalloid CPs that might minimize Ca2+ overload in comparison with standard Mg2+-containing CP: (1) low Ca2+ CP (0.25 mmol/L), (2) citrate CP/normal Mg2+ (1 mmol/L Mg2+), (3) citrate CP/high Mg2+ (9 mmol/L Mg2+), and (4) the addition of the Na+/H+ exchange inhibitor HOE-642 (Cariporide). We also tested the effect of citrate titration in vitro on the level of free Ca2+ and Mg2+ in CPs.
Methods and Results—Isolated working rat heart preparations were perfused with oxygenated Krebs-Henseleit buffer and subjected to 60 minutes of 37°C arrest and reperfusion with CPs with different Ca2+ concentrations. Cardiac performance, including aortic flow (AF), was measured before and after ischemia. Myocardial high-energy phosphates were measured after reperfusion. The in vitro addition of citrate to CP (2%, 21 mmol/L) produced parallel reductions in Mg2+ and Ca2+. Because only Ca2+ was required to be low, the further addition of Mg2+ increased free Mg2+, but the highest level achieved was 9 mmol/L. Citrate CP significantly impaired postischemic function (AF 58.3±2.5% without citrate versus 41.6±3% for citrate with normal Mg2+, P<0.05, versus 22.4±6.2% for citrate with high Mg2+, P<0.05). Low-Ca2+ CP (0.25 mmol/L Ca2+) significantly improved the recovery of postischemic function in comparison with standard CP (1.0 mmol/L Ca2+) (AF 47.6±1.7% versus 58.3±2.5%, P<0.05). The addition of HOE-642 (1 μmol/L) to CP significantly improved postischemia function (47.6±1.7% without HOE-642 versus 62.4±1.7% with HOE-642, P<0.05). Postischemia cardiac high-energy phosphate levels were unaffected by Ca2+ manipulation.
Conclusions—(1) A lowered Ca2+ concentration in CP is beneficial in Mg2+-containing cardioplegia. (2) The use of citrate to chelate Ca2+ is detrimental in the crystalloid-perfused isolated working rat heart, especially with high Mg2+. (3) The mechanism of citrate action is complex, and its use limits precise simultaneous control of Ca2+ and Mg2+. (4) HOE-642 in CP is as efficacious in preservation of the ischemic myocardium as is the direct reduction in Ca2+.
Despite the dramatic improvement in myocardial protection during cardiac surgery with the use of cardioplegia, ischemia-reperfusion–induced cardiac dysfunction remains a significant contributing factor to reduced postoperative recovery. A major contributing factor to ischemia-reperfusion injury is Ca2+ overload of the cardiac myocyte,1 especially in the aged heart, in which Ca2+ homeostasis is prone to impairment.2 Reduction in Ca2+ overload has been shown to improve recovery after ischemia-reperfusion injury in various animal models.3 Various strategies have been advocated specifically to prevent or reduce Ca2+ overload: (1) lowering of the Ca2+ content of the cardioplegic solution (CP) or reperfusion solution,4 (2) Ca2+ antagonism by Ca2+ channel blockers or Mg2+,5 (3) reduction in Ca2+ by citrate,6 and (4) the use of Na+/H+ exchange inhibitors.7 8
The aim of the present study was to compare the efficacy of these approaches in an isolated working rat heart model of cardioplegic arrest and reperfusion through the use of a clinically relevant K+-Mg2+ CP. Citrate as a chelator of divalent ions acts to lower both Mg2+ and Ca2+. This double action of citrate had not been previously investigated in CPs. Thus, before we compared the individual effects of varied concentrations of Ca2+ and citrate in Mg2+-containing CPs, we examined in vitro the complex interactions of citrate with Mg2+ and Ca2+ in crystalloid and blood cardioplegia.
Animal Preparation and Experimental Time Course
Male Sprague-Dawley rats weighing 250 to 350 g were anesthetized with halothane and heparinized, and the hearts were rapidly excised. The aorta was cannulated, and the hearts were mounted on an isolated heart perfusion apparatus. Hearts were perfused in Langendorff mode at a pressure of 100 cm H2O with modified Krebs-Henseleit bicarbonate buffer (KHB) that contains (in mmol/L) NaCl 118, KCl 4.7, CaCl2 1.25, MgSO4 1.2, KH2PO4 1.2, glucose 11, and NaHCO3 25, equilibrated with 95% O2/5% CO2 at 37°C. During perfusion, the pulmonary artery was incised, the left atrium was cannulated, and perfusion was converted to the working mode with left atrial pressure set at 17 cm H2O and afterload set at 90 cm H2O. All animals received humane care according to the Code of Practice of the National Heart and Medical Research of Australia, and methods were approved by the institutional ethics committee.
After control perfusion in working heart mode for 15 minutes, cardiac function was measured; then, induction CP (23 mmol/L K+) was infused at 37°C for 2 minutes to produce cardiac arrest. The hearts were subjected to global ischemia for 60 minutes at 37°C. In addition, during global ischemia, maintenance CP (13 mmol/L K+) was reinfused for 2 minutes every 20 minutes. Sixty minutes after the initial induction of ischemia, induction CP was infused as terminal cardioplegia for 2 minutes. Hearts were then reperfused in Langendorff mode for 15 minutes at a pressure of 100 cm H2O, followed by working perfusion for 20 minutes. At the end of the 20-minute working reperfusion, cardiac performance was measured, and hearts were freeze-clamped for the measurement of myocardial high-energy phosphates and lactate.
Measurement of Cardiac Function
Aortic flow (AF), coronary flow, aortic pressure, and heart rate were determined at 5-minute intervals during working perfusion before and after ischemia. AF was measured with a flowmeter in the aortic column, and coronary flow was measured with timed volumetric collections. Steady-state baseline (control) function was measured after a 15-minute perfusion in working heart mode. Functional recovery of preischemic performance was expressed as a percent of baseline values.
Tissue Sampling and Biochemical Analysis
Hearts were frozen in liquid nitrogen at the end of the postreperfusion working period. Frozen ventricular tissue was homogenized with 1 mol/L perchloric acid. The extract concentrations of ATP, ADP, ATP, phosphocreatine, and lactate were measured through enzymatic methods9 with an automated spectrophotometer (CobasBio; Roche).
Ion-selective electrodes in a Stat Profile Ultra blood analysis system (Nova Biomedical) were used to measure Ca2+ and Mg2+ concentrations in CPs. Progressive amounts of citrate were added to the standard induction CP, and the changes in Ca2+ and Mg2+ were monitored. Additional Mg2+ (MgCl2) was added (16 mmol/L plus 5, 10, or 15 mmol/L Mg2+) to the standard CP to return the Mg2+ concentration to 16 mmol/L. The highest achievable level of free Mg2+ was 9 mmol/L (15 mmol/L addition). Because further additions made the solution hyperosmolar without increasing free Mg2+, additions of >15 mmol/L were not used in the isolated working heart study. All solutions were adjusted for constant pH (7.35 to 7.4) and osmolality (340 mOsmol/L induction, 327 mOsmol/L maintenance). Osmolality was measured according to the freezing point depression method.
Experimental Groups and CPs
To simulate the preparation of clinical blood CP based on St Thomas’ Hospital No. 2 formulation, the crystalloid CP used as the standard solution in the present study was diluted with KHB 4:1 as in our clinical practice, where this same Alfred Hospital cardioplegic concentrate (Alfred Hospital Pharmacy) is diluted 4:1 with blood. KHB simulated the basic ionic composition of blood particularly for Ca2+ (1.0 mmol/L) (Table 1⇓). The standard CP contained the same high concentration of Mg2+ as in St Thomas’ Hospital No. 2 solution: 16 mmol/L.10
We then designed 4 modified CP solutions: (1) low-Ca2+ CP, (2) citrate CP with normal Mg2+, (3) citrate CP with high Mg2+, and (4) HOE-642 CP. All CP solutions were oxygenated with 95% O2/5% CO2 at 37°C. The concentration of Ca2+ in low-Ca2+ CP was set at 0.25 mmol/L. In the citrate CP, 21 mmol/L citrate (final concentration) as citrate phosphate solution was added to reduce Ca2+ to 0.25 mmol/L. The composition of the citrate phosphate solution (in mmol/L) was sodium citrate 894, citric acid monohydrate 156, and sodium phosphate 161. This is similar in composition to acid citrate phosphate solution but 10 times more concentrated. MgCl2 was added to adjust Mg2+ to 1.0 mmol/L for the normal physiological level or high to 9 mmol/L (the highest level of Mg2+ possible with this level of citrate). The Na+/H+ exchange inhibitor HOE-642 was added to standard CP at a concentration (1 μmol/L) known to be effective in cardioplegia.7 Two types of each of the 4 solutions were prepared with differing K+ concentrations: 1 for induction (23 mmol/L) and 1 for maintenance (13 mmol/L), as shown in Table 1⇑. Sodium content was adjusted to maintain constant osmolarity. All solutions were adjusted for constant pH (7.35 to 7.4) and osmolarity (340 mOsmol/L induction, 327 mOsmol/L maintenance) and were infused for 2 minutes at a pressure of 60 cm H2O.
Results are expressed as mean±SEM. In the 2-group comparisons (see Table 3⇓), the unpaired t test was used. In the 3-group comparisons (see Tables 4⇓ and 5⇓), 1-way ANOVA was used, followed by Student-Newman-Keuls multiple comparisons test when the variance was equal or Dunn’s multiple comparison test when the variance was unequal. A probability value of P<0.05 was accepted as statistically significant.
The citrate titration in KHB-based cardioplegia showed a steady decrease in the Ca2+ concentration with the progressive addition of citrate in vitro (Figure 1A⇓). The fall in the concentration of Ca2+ was paralleled by a fall in Mg2+ (Figure 1B⇓). At a concentration of 21 mmol/L citrate (2%), there was a decrease in Ca2+ from 1.35 to 0.25 mmol/L and a concomitant decrease in Mg2+ from 14.7 to 3.2 mmol/L. To restore Mg2+ toward the desirable cardioplegic concentration of 16 mmol/L,11 we used several different amounts of added Mg2+: (Mg2+ “spikes”): 5, 10, and 15 mmol/L Mg2+ (as magnesium chloride) to 1 L CP. The Mg2+ spikes increased Mg2+ toward the precitrate values. In the presence of 21 mmol/L citrate, the largest Mg2+ spike (15 mmol/L) increased Mg2+ from 3.2 to 9 mmol/L (Figure 1B⇓). Notably, due to competition for chelation by citrate, each addition of Mg2+ caused a concomitant increase in Ca2+. The addition of the 15 mmol/L Mg2+ spike increased Ca2+ from 0.25 to 0.5 mmol/L (Figure 1A⇓). Further additions of Mg2+ were not made because the solution would become hyperosmolar without an effective rise in Mg2+. To determine whether these interactions were similar in blood cardioplegia, we performed similar titrations in samples of our standard blood cardioplegia taken during cardiac surgery. The composition of our blood CPs, based on St Thomas’ No. 2 solution, were as follows: the induction solution (in mmol/L) consisted of Na+ 153, K+ 21, Mg2+ 16, aspartate 4, and lignocaine 0.7, and the maintenance solution consisted of Na+ 154, K+ 12, Mg2+ 16, aspartate 4, and lignocaine 0.18. The progressive addition of acid citrate phosphate solution to a concentration of 2% induced steady decreases in both Ca2+ (Figure 2A⇓) and Mg2+ (Figure 2B⇓) in a similar pattern to that seen in KHB-based cardioplegia.
Reduction of Baseline Ca2+ in CP
There were no significant differences in baseline functional parameters between the groups (Table 2⇓). This made it possible to express recovery as a percentage of baseline for all of the between-group comparisons.
Low baseline Ca2+ CP (0.25 mmol/L Ca2+) was associated with significantly greater recovery of cardiac function in terms of AF and cardiac output than was standard CP (1 mmol/L Ca2+) (Table 3⇓, Figure 3A⇓). However, the lowered Ca2+ produced no significant change in high-energy phosphate and lactate concentrations after reperfusion (Table 3⇓).
In CP that contained citrate (21 mmol/L, 2%), Mg2+ was elevated, with further Mg2+ addition to approach, as close as possible, the level in standard cardioplegia (only 9 mmol/L was possible). This high-Mg2+ CP showed less recovery of AF and cardiac output than did low-Ca2+ CP (Table 4⇓, Figure 3B⇑). Myocardial ATP, CP, and lactate concentrations at the end of reperfusion did not differ significantly between these groups (Table 4⇓).
To test whether the decreased recovery after citrate CP was due to an adverse interaction between high Mg2+ (31 mmol/L total, 9 mmol/L ionized) and citrate, a second citrate CP was tested in which Mg2+ was adjusted to the approximate level apparent in serum (1 mmol/L). This low-Ca2+/“normal”-Mg2+ citrate CP produced greater postischemia recovery than high-Mg2+ citrate CP, but recovery remained less than that seen with the low-Ca2+ CP (Figure 3B⇑, Table 3⇑). High-energy phosphates measured in the second citrate CP group were similar to levels after high-Mg2+ citrate CP (Table 4⇑).
HOE-642 cardioplegia was associated with recovery of function that was at the same elevated level as that seen with low baseline Ca2+ CP and significantly greater than that with standard CP (Figure 3C⇑, Table 5⇓). High-energy phosphate levels did not differ significantly between these groups (Table 5⇓).
The aim of the present study was to improve the efficacy of a standard, clinical K+/Mg2+-based CP through modifications designed to reduce intracellular Ca2+ overload due to ischemia-reperfusion of the myocardium. Because our isolated rat heart model precluded the use of blood, our clinical concentrated CP was diluted 4:1 in KHB, which has an ionic composition similar to that of blood. The protective effects of K+ and Mg2+ in CPs10 have been well demonstrated. These components are in common use clinically, so we did not reinvestigate them. We compared 3 strategies to control intracellular Ca2+ that have been shown to be effective in various experimental models: (1) direct lowering of Ca2+ in the baseline CP; although this is the simplest approach, this is possible only for crystalloid cardioplegia; (2) chelation of Ca2+ with citrate; this is complicated by the Mg2+-lowering effect of citrate, so preliminary work was required to assess the interactions of citrate, Ca2+, and Mg2+, but this approach proved to be very complex and too difficult to allow precise control over free Ca2+ and Mg2+; and (3) the addition to the CP of the new Na+/H+ exchange inhibitor HOE-6427 ; Na+/H+ exchange inhibitors have shown promise in reducing intracellular Ca2+ overload during ischemia-reperfusion.12 These agents have the advantage of acting via a mechanism that does not involve Mg2+ and thus can simply be added to Mg2+-blood cardioplegia.
In the present study, lowering of the Ca2+ concentration from 1 to 0.25 mmol/L in cardioplegia significantly improved functional recovery after normothermic cardioplegic arrest, which supports previous results after hypothermic cardioplegia.13 A similar benefit was observed with HOE-642 cardioplegia as shown previously after 35 minutes of global ischemia.7 In the present study, HOE-642 cardioplegia was effective in improving recovery after 60 minutes of global ischemia. However, the addition of citrate to standard cardioplegia in this model was associated with very low recovery (Figure 3⇑). A reduction in Mg2+ in citrate cardioplegia to normal serum levels doubled recovery rates, but this was still below that associated with the low-Ca2+ cardioplegia and no greater than that for standard normocalcemic cardioplegia. This finding was surprising because citrate is widely used clinically in blood cardioplegia to reduce Ca2+ concentrations.11 However, citrate has seldom been directly examined as a variable in CPs. We began our study by first examining the free Ca2+ and Mg2+ in titration experiments with citrate addition to our standard CP.
Ca2+ Overload in Ischemia-Reperfusion
Although Ca2+ enters the cell via voltage-gated L-type Ca2+ channels during the action potential, it is now evident that the mechanism of Ca2+ entry during ischemia-reperfusion is the Na+/Ca2+ antiporter.4 During ischemia, protons accumulate in the cell due to the buildup of lactic acid and the breakdown of ATP. Protons exchange with Na+, causing an increase in intracellular Na+. Excess intracellular Na+ then leaves the cell via the Na+/Ca2+ exchanger, resulting in intracellular Ca2+ overload. The 2 main strategies for reducing ischemia-reperfusion–induced Ca2+ overload were compared in this study: (1) reduction or chelation of extracellular Ca2+ and (2) Na+/H+ exchange inhibition.
Lowering Extracellular Ca2+
Tani and Neely4 reported optimal recovery of contractile function in rat heart after the reduction in extracellular Ca2+ to 0.15 mmol/L during postischemia reperfusion. In a study of crystalloid-perfused rat hearts, Robinson and Harwood13 found that recovery after cardioplegic arrest with St Thomas’ solution was maximized by reducing Ca2+ in the solution from 1.2 to 0.6 mmol/L. The current concept of multidose citrate-containing blood cardioplegia11 is based mainly on a single previous study of the effect of citrate added to the 28°C reperfusate after 1 hour of topical (noncardioplegic) hypothermic arrest in dogs.6 The optimal concentration of citrate as citrate-phosphate-dextrose (USP) was ≈3.7%, and Ca2+ was not measured in this study. This concentration of citrate corresponds to the 4.5% concentration of citrate-phosphate-dextrose recommended for inclusion in warm induction and warm reperfusate cardioplegia.11 It is further recommended that sufficient citrate be added to cardioplegia and reperfusate to lower Ca2+ to 0.5 to 0.6 mmol/L under hypothermic conditions and to 0.15 to 0.25 mmol/L under normothermic conditions.11
In contrast to Follette et al,6 in a different model, the isolated working heart rat heart at normothermia, in the present study, we were unable to demonstrate a protective effect by lowering Ca2+ in crystalloid cardioplegia with citrate. Notably, if citrate was combined with high Mg2+ (16 mmol/L+15 mmol/L addition, but only 9 mmol/L was Mg2+), there was a marked depression of cardiac recovery. This may be explained by excessive anti-Ca2+ effects (ie, low Ca2+ due to citrate combined with Ca2+ antagonism by Mg2+). Mg2+ has been described as a “natural Ca2+ antagonist” because it limits Ca2+ entry via competition for sarcolemmal Ca2+ channels.10 14 Mg2+ has been shown to reduce Ca2+ release from the sarcoplasmic reticulum,15 decrease postischemia cellular Mg2+ loss, and limit mitochondrial Ca2+ accumulation.5
It is possible that citrate itself exerted a negative effect on postischemia myocardial recovery in this preparation. Citrate at a concentration ranging from 1 to 20 mmol/L has been shown to have a negative inotropic action in a number of animal heart preparations in vitro.16 17 The citrate concentration used in our citrate-induced, low-Ca2+ cardioplegia (21 mmol/L) was ≈10 times greater than the minimum concentration shown to have a sustained myocardial depressant action.16 This is, however, of a similar magnitude to the concentration recommended for clinical cardioplegia of ≈4.5% (≈42 mmol/L11 ). It should be noted that these previous studies with citrate were conducted in the presence of steady-state citrate elevations, whereas in our study, citrate had been washed out for 30 to 35 minutes before function measurements were made. It has been shown that citrate specifically decreases myocardial contraction and Ca2+ current (ICa) through a direct effect on Ca2+ channels rather than through a Ca2+ chelation effect per se.17 It is possible that the adverse effect of citrate on postischemia recovery in the present study included some sustained direct interaction with sarcolemmal membrane sites. It is unclear whether these effects may also include complex interactions with Mg2+-dependent effectors. Indeed, the complexity of the interactions with cell membranes17 and free ions therefore warrants more direct study of citrate cardioplegia in blood-perfused animal models. Because we used a crystalloid perfusion buffer and cardioplegia in the present study, the unexpected effect of citrate cardioplegia should not be interpreted before the role of citrate cardioplegia is also examined in blood-perfused preparations. In light of the current incomplete definition of the specific mechanisms of action by citrate in cardioplegia, another approach to ionic control during ischemia-reperfusion may be useful in the interim.
Effect of Na+/H+ Exchange Inhibition During Cardioplegia
The regulation of intracellular pH by Na+/H+ exchange is perturbed by ischemia due to augmented H+ production, thus triggering events that may contribute to ischemia-reperfusion injury.18 Myocardial ischemia produces intracellular acidosis, and the pH gradient augments Na+/H+ exchange, causing excess Na+ to enter the cytosol. High intracellular Na+ in turn causes increased exchange with Ca2+ via the Na+/Ca2+ exchanger, so intracellular Ca2+ accumulates during ischemia-reperfusion.18 Moreover, reperfusion activates protein kinase C,19 which is also implicated in the stimulation of Na+/H+ exchange,20 thus further augmenting intracellular Ca2+ accumulation. The Na+/H+ exchange inhibitors HOE-642 (Cariporide) and HOE-694 have major advantages over their predecessor amiloride: (1) increased potency of sarcolemmal Na+/H+ exchanger inhibition21 and (2) no apparent effect on cardiac contractile function.22 HOE-642 was designed to afford greater potency and specificity for the inhibition of the Na+/H+ exchanger (sarcolemmal isoform subtype 1) than HOE 694.18 22
In the present study, the addition of 1 μmol/L HOE-642 to CP enhanced the recovery of postischemia function by the same extent as low-Ca2+ CP. In a study with nuclear MRI, amiloride has been directly shown to prevent the accumulation of myocardial cytosolic free Ca2+ and Na+ during cardiac ischemia and reperfusion.12 HOE-694 has also been shown to prevent intracellular Ca2+ overload and to improve postischemia contractile function.23 HOE-642 has been shown to directly limit infarct size after 30 minutes of coronary occlusion in rabbits.24 Although we did not measure intracellular pH, Na+, Ca2+, or markers of ischemic injury in the present study, we believe that the improved postischemia cardiac performance may relate to a prevention of intracellular Ca2+ overload during ischemia-reperfusion via these mechanisms.
The efficacious use of HOE-642 in cardioplegia in our experimental model supports the findings of Shipolini et al.7 The findings of the multicenter GUARDIAN clinical trial of HOE-642 in 11 590 patients at high risk for cardiac cell death were recently presented at the 48th Scientific Sessions of the American College of Cardiology.25 High-risk unstable angina patients underwent PTCA or CABG. Only in surgical patients was the primary end point (rate of death or myocardial infarction at 36 days postintervention) significantly reduced by HOE-642 (120 mg TID 2 to 7 days pretreatment) compared with placebo (HOE-642 12.8% versus placebo 16.7%, P=0.03, n=2918).25 These data suggest that the most promising use of HOE-642 may be the setting of CABG.
Although the reduction of Ca2+ in CP affords additional protection compared with standard CP during crystalloid buffer perfusion, it is difficult to directly reduce Ca2+ in blood CP. In the use of citrate to reduce Ca2+ concentration, citrate effectively chelated Mg2+ and Ca2+ in our CP solutions; moreover, even after washout, the combined anti-Ca2+ effect of citrate and high Mg2+ was detrimental to postischemia pump function. Precise control over free Ca2+ and Mg2+ was not possible due to the complex chelation properties of citrate. On this basis, we would not add citrate to our Mg2+-containing CP; however, our present crystalloid buffer perfusion model may not be considered physiological or directly transposable to humans. Therefore, future work should examine the action of citrate cardioplegia in blood-perfused preparations. Because Ca2+ control in the cardiac surgical setting is critical, we advocate additional study of a highly specific and less complex, alternative approach: that of Na+/H+ exchanger inhibition during ischemia with HOE-642 as an additive to blood cardioplegia.
This work was supported in part by a grant from the National Health and Medical Research Council of Australia. We thank Dr Michael Wadsley, Department of Chemistry, Monash University, for detailed discussions on citrate/cation interactions.
- Copyright © 2000 by American Heart Association
Steenbergen C, Fralix TA, Murphy E. Role of increased cytosolic free calcium concentration in myocardial ischemic injury. Basic Res Cardiol. 1993;88:456–470.
McCully JD, Tsukube T, Ataka K, et al. Myocardial cytosolic calcium accumulation during ischemia/reperfusion: the effects of aging and cardioplegia. J Card Surg. 1994;9:449–452.
Chen RH. The scientific basis for hypocalcemic cardioplegia and reperfusion in cardiac surgery. Ann Thorac Surg. 1996;62:910–914.
Tani M, Neely JR. Mechanisms of reduced reperfusion injury by low Ca2+ and/or high K+. Am J Physiol. 1990;258:H1025–H1031.
Jynge P, Falck G. High magnesium improves the postischaemic recovery of cardiac function. Cardiovasc Res. 1995;29:439.
Follette DM, Fey K, Buckberg GD, et al. Reducing postischemic damage by temporary modification of perfusate calcium, potassium, pH, and osmolarity. J Thorac Cardiovasc Surg. 1981;82:221–238.
Shipolini AR, Galinanes M, Edmondson SJ, et al. Na+/H+ exchanger inhibitor HOE-642 improves cardioplegic myocardial preservation under both normoxic and hypoxic conditions. Circulation. 1997;96(suppl II):II-266–II-273.
Levitsky J, Gurell D, Frishman WH. Sodium ion/hydrogen ion exchange inhibition: a new pharmacologic approach to myocardial ischemia and reperfusion injury. J Clin Pharmacol. 1998;38:887–897.
Bergmeyer HU, ed. Methods of Enzymatic Analysis. Vol 4. 2nd ed. New York, NY: Academic Press; 1974.
Hearse DJ, Stewart DA, Braimbridge MV. Myocardial protection during ischemic cardiac arrest: the importance of magnesium in cardioplegic infusates. J Thorac Cardiovasc Surg. 1978;75:877–885.
Buckberg GD, Beyersdorf F, Allen BS, et al. Integrated myocardial management: background and initial application. J Card Surg. 1995;10:68–89.
Murphy E, Perlman M, London RE, et al. Amiloride delays the ischemia-induced rise in cytosolic free calcium. Circ Res. 1991;68:1250–1258.
Robinson LA, Harwood DL. Lowering the calcium concentration in St Thomas’ Hospital cardioplegic solution improves protection during hypothermic ischemia. J Thorac Cardiovasc Surg. 1991;101:314–325.
Headrick JP, McKirdy JC, Willis RJ. Functional and metabolic effects of extracellular magnesium in normoxic and ischemic myocardium. Am J Physiol. 1998;275:H917–H929.
Terada H, Hayashi H, Noda N, et al. Effects of Mg2+ on Ca2+ waves and Ca2+ transients of rat ventricular myocytes. Am J Physiol. 1996;270:H907–H914.
Rebekya IM, Yeh T, Hanan SA, et al. Altered contractile response in neonatal myocardium to citrate-phosphate-dextrose infusion. Circulation. 1990;82(suppl. IV):IV-367–IV-370.
Bers DM, Hryshko LV, Harrison SM, et al. Citrate decreases contraction and Ca current in cardiac muscle independent of its buffering action. Am J Physiol. 1991;260:C900–C909.
Avkiran M. Rational basis for use of sodium-hydrogen exchange inhibitors in myocardial ischemia. Am J Cardiol. 1999;83:10G–18G.
Otani H, Prasad MR, Engelman RM, et al. Enhanced phosphodiesteratic breakdown and turnover of phosphoinositides during reperfusion of ischemic rat heart. Circ Res. 1988;63:930–936.
Rehring TF, Shapiro JI, Cain BS, et al. Mechanisms of pH preservation during global ischemia in preconditioned rat heart: roles for PKC and NHE. Am J Physiol. 1998;275:H805–H813.
Loh SH, Sun B, Vaughan JR. Effect of Hoe694, a novel Na+-H+ exchange inhibitor, on intracellular pH regulation in the guinea-pig ventricular myocyte. Br J Pharmacol. 1996;118:1905–1912.
Scholz W, Albus U, Counillon L, et al. Protective effects of HOE642, a selective Na+-H+ exchange subtype 1 inhibitor, on cardiac ischaemia and reperfusion. Cardiovasc Res. 1995;29:260–268.
Hendrikx M, Mubagwa K, Verdonck F, et al. New Na+-H+ exchange inhibitor HOE694 improves postischemic function and high-energy phosphate resynthesis and reduces Ca2+ overload in isolated perfused rabbit heart. Circulation. 1994;89:2787–2798.
Miura T, Ogawa T, Suzuki K, et al. Infarct size limitation by a new Na+-H+ exchange inhibitor HOE694: difference from preconditioning in the role of protein kinase C. J Am Coll Cardiol. 1997;29:693–701.
GUARDIAN Trial (GUARD during Ischaemia Against Necrosis). Clin Cardiol. 1999;22:371.