Structural basis for selective recognition of oligosaccharides by DC-SIGN and DC-SIGNR.

Dendritic cell specific intracellular adhesion molecule-3 (ICAM-3) grabbing nonintegrin (DC-SIGN), a C-type lectin present on the surface of dendritic cells, mediates the initial interaction of dendritic cells with T cells by binding to ICAM-3. DC-SIGN and DC-SIGNR, a related receptor found on the endothelium of liver sinusoids, placental capillaries, and lymph nodes, bind to oligosaccharides that are present on the envelope of human immunodeficiency virus (HIV), an interaction that strongly promotes viral infection of T cells. Crystal structures of carbohydrate-recognition domains of DC-SIGN and of DC-SIGNR bound to oligosaccharide, in combination with binding studies, reveal that these receptors selectively recognize endogenous high-mannose oligosaccharides and may represent a new avenue for developing HIV prophylactics.

Movement of sodium into human platelets.

Addition of ADP induces platelets in plasma to undergo shape change from a disc to a spiny sphere and to develope adhesiveness, i.e. to aggregate. The aggregation of human platelets by ADP is associated with a net uptake of Na+. The present experiments demonstrate that the induction of shape change by ADP in acidified or EGTA-treated plasma conditions which inhibit aggregation, is also associated with a movement of Na+ into platelets. When ADP-induced platelet shape change and aggregation is inhibited by prostaglandin E1 Na+ uptake is also blocked. Platelets aggregated by epinephrine do not take up Na+. In a manner analogous to the effect of ADP, polylysine also induces Na+ uptake during aggregation. Vasopressin, in a manner analogous to epinephrine, induces aggregation without Na+ uptake. The increase in platelet Na+ resulting from ouabain inhibition of Na+ efflux induces an increase in the aggregation response to ADP and to epinephrine.

Movement of sodium into human platelets induced by ADP.

1. In normal human platelets the concentrations of Na+ and K+ were 42.1 +/- 4.3 and 98.8 +/- 3.7 mequiv/l of platelet water respectively (mean +/- S.E. of 22 samples). 2. When platelet-rich plasma was incubated with 22Na+ at 37 degrees C for 2-3 h an increase in platelet Na+ concentration was found which was significant after 210 min. Platelet K+ concentration did not change significantly. The platelet 22Na+ radioactivity increased faster than did the total Na+ suggesting a Na+o-Na+ exchange process in unactivated platelets. 3. Addition of ADP to platelet-rich plasma resulted in platelet aggregation and a rapid rise (within seconds) in 22Na+-radioactivity within the platelets and after 300 s this increase diminished toward control levels. 4. Under the same experimental conditions, ADP did not bring about an increase of 36Cl- in the platelets. 5. Ouabain (10-(6) M) added to platelet-rich plasma induced an increase in Na+ concentration and 22Na+ radioactivity in the platelets, as well as a decrease in K+ concentration. ADP produced a further increase in 22Na+, which did not return toward control values, in the presence of ouabain. 6. The association of an increase in 22Na+ but not of 36Cl- accompanying aggregation by ADP suggests a selective mechanism for the movement of Na+ into platelets rather than a movement of NaCl together with water under an osmotic gradient.

Increased membrane-bound calcium in platelets of hypertensive patients.

The fluorescent indicator chlortetracycline was used to estimate membrane-bound calcium in mild, untreated hypertensive patients (n = 39) and normotensive controls (n = 42). All participants were black. After incubation with chlortetracycline, platelet-rich plasma was centrifuged into a pellet and fluorescence was measured with a microspectrofluorometer. At an interval of 45 minutes mean fluorescence values were 11% higher in the hypertensive than in the normotensive group (567 +/- 95 vs. 512 +/- 100 counts/sec, p less than 0.02). With both groups of participants combined, a correlation of borderline statistical significance was noted between diastolic blood pressure and chlortetracycline fluorescence (r = 0.213, p = 0.056). In parallel experiments, sodium and potassium concentrations were measured in red blood cells. Intracellular sodium was also significantly higher in the hypertensive group (p less than 0.01). These data indicate that the total cell burden of calcium is increased in the platelets of hypertensive individuals, possibly a result of abnormal cell metabolism of calcium, and further suggest that circulating platelets in hypertensive individuals may be in a hyperaggregable state.

ADP induced depolarization of human platelet membrane potential.

The transmembrane potential of human blood platelets suspended in plasma was investigated by studying the distribution of a radiolabeled permeant ion [14C] thiocyanate. The membrane potential of resting platelets was found to be -54.50 mV +/- 9.23 S.D. with a range of -39 to -76 mV (n = 27). The possibility that platelet activation alters membrane potential or that changes in membrane potential serve as an activation trigger was investigated. Stimulation by ADP (10 microM) resulted in a significant (p less than 0.05) depolarization of the membrane potential. Preincubation with 6 mM EGTA failed to inhibit ADP-induced depolarization even though EGTA effectively prevented primary and secondary aggregation but not shape change. Preincubation with PGE1 inhibited shape change, aggregation, and the ADP-induced depolarization. No significant change in membrane potential was observed following stimulation by epinephrine (50 microM). These results suggest that the initial interaction of ADP and its receptor may involve an inward positive current which can be determined by thiocyanate distribution.

Platelet Ca2+ homeostasis: Na+-Ca2+ exchange in plasma membrane vesicles.

A vesicular plasma membrane-enriched fraction obtained from human platelets exhibited 45Ca2+ uptake in exchange for intravesicular Na+. The rate of Ca2+ uptake was linear up to 4 sec. The apparent Km for Ca2+ was 22 microM and the Vmax 280 pmol/mg/sec. Ca2+ efflux from Ca2+ loaded vesicles was obtained upon dilution into a NaCl but not a KCl medium. The extent of Ca2+ uptake was monotonically increased as the pH increased from 6 to 9. Na+-Ca2+ exchange was shown to be electrogenic. Ca2+ uptake was distinguished from binding by the induction of Ca2+ release after A23187 addition. These findings support a role for Na+-Ca2+ exchange in human platelet Ca2+ transport.

Efficacy of crystalloid cardioplegic solutions in patients undergoing myocardial revascularization. Effect of infusion route and regional wall motion on preservation of adenine nucleotide stores.

The effect of varying the mode of cardioplegic delivery and the presence of regional wall motion abnormalities on myocardial protection by crystalloid cardioplegic solutions was assessed in 68 patients undergoing coronary artery bypass grafting. Serial transmural biopsy specimens from the left ventricular apex were assayed for adenosine triphosphate. All patients had more than 75% stenosis of the left anterior descending coronary artery. They were prospectively randomized into Groups I and II to receive (I) all cardioplegic solution infused via the aortic root or (II) reinfusions of cardioplegic solution given both centrally and through the completed distal left anterior descending anastomosis. Patients were also stratified as to the presence of normal (N) or impaired (Ab) apicoanterior regional wall motion. Inadequate delivery of cardioplegia during ischemia in Group I was manifested by a 41% (p less than 0.01) depletion of adenosine triphosphate stores in abnormally contracting myocardium distal to the left anterior descending stenosis that was not repleted after restoration of coronary flow and a 27% (p less than 0.05) decline in ATP stores during reperfusion in myocardium with normal preoperative wall motion. In contrast, nucleotide stores were preserved at preischemic levels throughout ischemia and reperfusion in Group II regardless of preoperative wall motion. Preservation of ATP did not correlate with duration of ischemia, highest recorded septal temperature, or volume of cardioplegic solution infused. Two patients in each group had a new perioperative infarction. However, 38% of patients in Group IAb required transient inotropic support versus 5% in Group IIAb (p less than 0.05). These data emphasize that reinfusion of cardioplegic solutions distal to coronary obstructions is mandatory for optimal myocardial protection during coronary revascularization.

Redistribution of myocardial calcium during ischemia. Relationship to onset of contracture.

Cytosolic calcium accumulation has been proposed as a mediator for the pathologic changes that occur during myocardial ischemia. Whether the rise in cytosolic calcium is a result of influx or redistribution from internal stores has not been elucidated. Isolated retroperfused rabbit hearts were subjected to ischemia at 37 degrees C. The distribution of calcium between cytosol and internal membrane stores and the relationship between cytosolic calcium and the onset of left ventricular contracture were investigated. One group of hearts was loaded with the fluorescent calcium probe Fura 2-AM to measure cytosolic calcium and a second group with chlortetracycline to indicate changes in membrane-bound calcium. After the onset of ischemia there is a rise in cytosolic calcium, at least in part attributable to redistribution of calcium from intraorganellar sites to cytosol. The release of membrane-bound calcium and rise in cytosolic calcium preceded the onset of irreversible ischemic injury, that is, contracture.

Beneficial metabolic effect of nucleoside augmentation on reperfusion injury following cardioplegic arrest.

Restoration of coronary flow after hyperkalemic cardioplegic arrest (HCA) is associated with a unique metabolic reperfusion injury (RI) manifested by declining nucleotide stores despite their end-ischemic preservation. Prevention of this RI by exogenous provision of adenosine with or without inhibition of adenosine's major catabolic enzyme was assessed in 27 dogs subjected to 60 minutes of HCA. The effect of aortic root infusion of 40 mg/kg of adenosine in addition to adenosine deaminase inhibition by 10 mg/kg of EHNA (group 2) initiated during 60 minutes of reperfusion on myocardial adenosine triphosphate (ATP) and creatine phosphate (CP) stores and coronary blood flow (CBF) were compared to animals having adenosine infusion alone (group 3) or controls (group 1). Although ATP levels were preserved at the end of HCA in all groups, adenosine infusion with or without EHNA prevented the significant 23 percent decline in ATP stores incurred during unmodified reperfusion (p less than 0.01, group 1). The CP stores decreased (p less than 0.05, all groups) during arrest, but were restored to preischemic levels during reperfusion. When measured 60 minutes after aortic unclamping, CBF was 312 percent of preischemic flow in group 3 (p less than 0.01), only 170 percent in group 2 (p less than 0.05), and unchanged in controls (group 1). The data indicate that provision of adenosine as a nucleotide precursor prevents the metabolic RI following HCA. In addition, inhibition of adenosine catabolism is not necessary for this salutary effect, nor is adenosine's efficacy solely mediated by augmentation of CBF.

Pulsatile reperfusion does not modify global myocardial ischemic injury.

In an attempt to arbitrate the reputed clinical efficacy of pulsatile flow during reperfusion in minimizing ischemic injury, 32 mongrel dogs supported by normothermic cardiopulmonary bypass were subjected to 30 minutes (Groups IC and IP) or 60 minutes (Groups IIC and IIP) of global myocardial ischemia. The effect of pulsatile flow (P) initiated during 30 minutes of reperfusion on the recovery of myocardial adenosine triphosphate (ATP) and creatine phosphate (CP) stores, coronary blood flow, and myocardial water content (MWC) was compared to the effect of linear reperfusion (C) in another group of animals. ATP stores, which significantly decreased to 43% and 53% of preischemic levels (Groups IC and IP, respectively, p less than 0.01) and 36% and 31% of control values (Groups IIC and IIP, respectively. p less than 0.001), did not increase with either pulsatile or linear reperfusion. CP stores, depleted 97% during ischemia in all groups, returned to preischemic levels regardless of the mode of reperfusion flow. Coronary blood flow measured 30 minutes after aortic unclamping was not significantly different from control flow in any group. MWC significantly decreased during ischemia from 80.5% +/- 0.8% to 76.5% +/- 1.1% in Group IC and from 81.8% +/- 1.2% to 76.8% +/- 0.8% in Groups IP (p less than 0.05) and returned to preischemic levels with reperfusion. However, following 60 minutes of ischemia, pulsatile reperfusion prevented the significant increase in MWC that accrued after linear reperfusion (80.7% +/- 1.5% to 84.0% +/- 0.7%, p less than 0.05). These data indicate that pulsatile reperfusion initiated after an ischemic injury that results in a 50% or greater depletion of myocardial ATP stores does not restore myocardial nucleotide levels or enhance coronary blood flow, although the pathological increase in MWC may be avoided.

Optimal intraoperative protection of myocardium distal to coronary stenoses.

Metabolic evidence of improved delivery of cardioplegic solutions by adjuvant use of nitroglycerin (NTG) and reinfusing these solutions distal to an obstructed coronary artery was sought in 40 dogs subjected to cold cardioplegic arrest. The left anterior descending coronary artery was occluded prior to initiating arrest by intra-aortic root infusion. Cardioplegic solution was reinfused with the left anterior descending occluded throughout ischemia (Group I), or with this artery reopened, to simulate a completed distal anastomosis (Group II). Serial biopsy specimens of the left ventricular apex were assayed for adenosine triphosphate and creatine phosphate, while specimens from the posterior left ventricular wall served as controls. Regional myocardial temperatures were recorded throughout ischemia. Half of the hearts in each group received 300 micrograms of nitroglycerin in the cardioplegic solution. Adenosine triphosphate was preserved in myocardium distal to a patent coronary artery whether nitroglycerin was added to the cardioplegic solution or not (control, control + NTG). Moreover, nitroglycerin did not prevent the 26% to 34% (p less than 0.05) decline in adenosine triphosphate levels when the left anterior descending remained obstructed throughout ischemia (Group I, I + NTG). However, opening the left anterior descending for reinfusion of cardioplegic solution allowed adenosine triphosphate to be preserved at end-ischemia (Group II, II + NTG). In addition, the metabolic reperfusion injury manifested by a 37% (p less than 0.01) decline in adenosine triphosphate levels after aortic unclamping (Group II) was obviated when nitroglycerin was added to the cardioplegic solution delivered in this manner (II + NTG). The depletion of cardioplegic solution stores during ischemia was more severe in the experimental groups than in controls (p less than 0.05). These metabolic changes did not correlate with regional myocardial temperature gradients. The data indicate that myocardium jeopardized by coronary stenoses can be preserved as well as myocardium supplied by a patent coronary artery by adjuvant use of nitroglycerin and varying the mode of delivery of the cardioplegic solution.

Developmental changes in reperfusion injury. Comparison of intracellular ion accumulation in ischemic and cardioplegic arrest.

Developmental differences in ischemic and potassium cardioplegic arrest were evaluated in newborn (birth to 7 day old) and adult (6 to 12 month old) New Zealand white rabbit hearts isolated and perfused by Langendorff's method. An extracellular space washout technique was used to measure intracellular sodium and calcium in the two age groups after ischemia alone, after normothermic and hypothermic cardioplegia, and after cardioplegia with reperfusion. Although the intracellular ionic contents of nonreperfused adult hearts after 30 and 40 minutes of ischemia were identical, there was a twofold elevation in intracellular sodium level after 40 minutes of ischemia in the newborn hearts. Adult hearts arrested by normothermic potassium cardioplegia demonstrated no alteration in the intracellular ionic content, whereas in the newborn hearts, potassium cardioplegia produced excess intracellular calcium loading before reperfusion, which was greater than that occurring with ischemia alone. When hypothermia (12 degrees C) was combined with cardioplegic arrest, a prereperfusion influx of sodium and calcium was not observed in the newborn hearts, and ionic reperfusion injury was blunted in both newborn and adult hearts. These studies demonstrate that the newborn heart is more susceptible than the adult to both ischemia and cardioplegia. This may be due to age-dependent differences in transmembrane passive diffusion, sodium/calcium exchange, or calcium slow channel properties and suggests alternative myocardial protective strategies for the newborn infant.

Myocardial energetics after thermally graded hyperkalemic crystalloid cardioplegic arrest.

Previous studies assessing the efficacy of myoprotective regimens have compared preischemic and postischemic myocardial oxygen consumption within a limited range of cardiac performance. However, recent data suggest that ischemia-induced perturbations in myocardial energetics may occur only when the left ventricle develops physiologic pressures. Therefore, in canine hearts supported by cardiopulmonary bypass, myocardial oxygen consumption (ml oxygen X 10(-2)/beat/100 gm left ventricular weight) was determined during incremental isovolumic pressure-volume loading before and 30 minutes after 2 hours of cardioplegic arrest. The ischemic insult was graded by maintaining myocardial temperature at 12 degrees C (Group I, n = 6), 20 degrees C (Group II, n = 7), or 28 degrees C (Group III, n = 6). Postischemic Starling curves were unchanged in Groups I and II but depressed 53% in Group III hearts (p less than 0.005). In Group I, postischemic myocardial oxygen consumption at specific peak developed pressures was similar to preischemic oxygen consumption. In contrast, postischemic Group II and III hearts consumed 39% more oxygen than preischemically when peak developed pressure exceeded 75 mm Hg (p less than 0.01). Postischemic hearts demonstrated reciprocal changes in arteriovenous oxygen content difference (24%, 30%, and 34% lower than preischemic values for Groups I, II, and III, respectively) and coronary blood flow (156%, 195%, and 192% higher than preischemic values for Groups I, II, and III, respectively). Only in Group II and III hearts did the increased coronary blood flow offset the defect in oxygen extraction such that myocardial oxygen consumption was increased. These data suggest that inefficient utilization of oxygen when the heart is developing physiologic pressures is a sensitive marker for myocardial injury after crystalloid cardioplegic arrest.

Age-related differences in cardiac susceptibility to ischemia/reperfusion injury. Response to deferoxamine.

Age-related differences in susceptibility to ischemia/reperfusion injury and the response to the iron chelator deferoxamine during reperfusion were studied in isolated nonworking rabbit hearts subjected to 30 or 40 minutes of ischemia at 37 degrees C followed by 30 minutes of reperfusion. In the experimental group, hearts received a bolus of deferoxamine just before the moment of reflow, followed by a continuous infusion during the first 10 minutes of reperfusion. Isovolumic systolic (peak developed pressure) and diastolic (diastolic pressure versus balloon volume relationship) function was assessed with an intracavity balloon and incremental volume changes. In separate groups of hearts, adenine nucleotide content (adenosine triphosphate, diphosphate, and monophosphate) was measured before ischemia, at end-ischemia, and 30 minutes after reperfusion. The cardiac function measurements showed that after 30 minutes of ischemia and 30 minutes of reperfusion, peak developed pressure in newborn hearts recovered to 89% +/- 5% of preischemic levels; this recovery was significantly better than that of adult hearts, which exhibited 67% +/- 6% (p less than 0.01) recovery. Deferoxamine significantly improved cardiac function only in adult hearts (p less than 0.01). However, after 40 minutes of ischemia and 30 minutes of reperfusion, peak developed pressure in newborn hearts was reduced to 61% +/- 3% and was not significantly better than that of adult hearts (54% +/- 5%). Deferoxamine significantly improved systolic function in both newborn and adult hearts (p less than 0.01) exposed to 40 minutes of ischemia. Myocardial adenosine triphosphate content fell markedly by the end of 30 and 40 minutes of ischemia in both groups. After 30 minutes of ischemia, newborn but not adult hearts were able to completely recover adenosine triphosphate content by 30 minutes of reperfusion. This advantage was lost after 40 minutes of ischemia. Deferoxamine had no effect on recovery of adenosine triphosphate content in any group. We conclude that (1) newborn hearts recover postischemic function and metabolism faster than adult hearts after shorter periods of ischemia; (2) this advantage is lost as the ischemic period is prolonged; (3) deferoxamine improved postischemic cardiac function after longer ischemic periods, in both age groups, but failed to improve the recovery of myocardial adenosine triphosphate content.

Developmental changes in reperfusion injury. A comparison of intracellular cation accumulation in the newborn, neonatal, and adult heart.

Intracellular sodium and calcium accumulation were measured after 30 and 40 minutes of ischemia and 30 minutes of reperfusion in newborn (0 to 7 day), neonatal, (14 to 21 day), and adult (4 to 6 month) New Zealand white rabbit heart preparations. Newborn hearts showed twofold and threefold elevations of calcium when exposed to 30 and 40 minutes of ischemia and reperfusion, respectively, whereas sodium increase was noted only in the 40 minute group. Conversely, adult hearts exhibited sodium elevation if exposed to ischemia for 30 minutes and calcium accumulation occurred only after 40 minutes of ischemia. There was no significant alteration in intracellular sodium and calcium levels after ischemic intervals of 30 and 40 minutes in the neonate. These studies demonstrate that the immature newborn heart is the most susceptible and the neonatal heart is the least susceptible to injury sustained by ischemia followed by reperfusion. These differences in susceptibility to ischemia may be due to age-dependent alterations of intramembrane ionic pumps and channels.

Myocardial protection in normal and hypoxically stressed neonatal hearts: the superiority of blood versus crystalloid cardioplegia.

OBJECTIVES: Blood cardioplegia predominates in the adult because it provides superior myocardial protection, especially in the ischemically stressed heart. However, the superiority of blood over crystalloid cardioplegia in the pediatric population is unproved. Furthermore, because many pediatric hearts undergo a preoperative stress such as hypoxia, it is important to compare the different methods of protection in both normal and hypoxic hearts. METHODS: Twenty neonatal piglets were supported by cardiopulmonary bypass and subjected to 70 minutes of cardioplegic arrest. Of 10 nonhypoxic hearts, five (group 1) were protected with blood cardioplegia and five (group 2) with crystalloid cardioplegia (St. Thomas' Hospital solution). Ten other piglets underwent 60 minutes of ventilator hypoxia (inspired oxygen concentration 8% to 10%) before cardioplegic arrest. Five (group 3) were then protected with blood cardioplegia and the other five (group 4) with crystalloid cardioplegia. Myocardial function was assessed by means of pressure volume loops and expressed as a percentage of control. Coronary vascular resistance was measured with each infusion of cardioplegic solution. RESULTS: No difference was noted between blood (group 1) or crystalloid cardioplegia (group 2) in nonhypoxic hearts regarding systolic function (end-systolic elastance 104% vs 103%), diastolic stiffness (156% vs 159%), preload recruitable stroke work (102% vs 101%), or myocardial tissue edema (78.9% vs 78.9%). Conversely, in hearts subjected to a hypoxic stress, blood cardioplegia (group 3) provided better protection than crystalloid cardioplegia (group 4) by preserving systolic function (end-systolic elastance 106% vs 40%; p < 0.05) and preload recruitable stroke work (103% vs 40%; p < 0.05); reducing diastolic stiffness (153% vs 240%; p < 0.05) and myocardial tissue edema (79.6% vs 80.1%); and preserving vascular function, as evidenced by unaltered coronary vascular resistance (p < 0.05). CONCLUSION: This study demonstrates that (1) blood or crystalloid cardioplegia is cardioprotective in hearts not compromised by preoperative hypoxia and (2) blood cardioplegia is superior to crystalloid cardioplegia in hearts subjected to the preoperative stress of acute hypoxia.

Prevention of the hypoxic reoxygenation injury with the use of a leukocyte-depleting filter.

OBJECTIVES: Recent studies have shown that an injury occurs when the hypoxic heart is suddenly reoxygenated (as occurs with cardiopulmonary bypass), resulting in myocardial depression, impaired oxygenation, and increased pulmonary vascular resistance. We hypothesize that this injury is, in part, due to oxygen-derived radicals produced by activated white cells and may therefore be ameliorated by limiting leukocytes in the bypass circuit. METHODS: Fifteen neonatal piglets underwent 60 minutes of ventilator hypoxia (inspired oxygen fraction 8% to 10%), followed by reoxygenation with cardiopulmonary bypass at an inspired oxygen fraction of 100% for 90 minutes. In nine piglets (group 1) our routine bypass circuit was used with no modifications, and in six piglets (group 2) a leukocyte-depleting filter (Pall BC-1; Pall Biomedical Products Corporation, Glencoe, N.Y.) was inserted in the arterial line to lower the neutrophil count. Six additional piglets underwent 90 minutes of bypass without hypoxia (cardiopulmonary bypass controls). Postbypass myocardial and pulmonary function was assessed by pressure volume loops, arterial/alveolar ratio, and pulmonary vascular resistance index. Results are expressed as a percentage of control. RESULTS: By comparison with group 1 piglets (reoxygenation without a filter), hypoxic piglets undergoing reoxygenation with a leukocyte-depleting filter (group 2) had improved myocardial systolic function (88% vs 52%; p < 0.05), diastolic compliance (175% vs 275%; p < 0.05), and preload recruitable stroke work (91% vs 54%; p < 0.05); had better preservation of the arterial/alveolar ratio (97% vs 74%; p < 0.05); and had less increase in pulmonary vascular resistance (229% vs 391%; p < 0.05). Furthermore, leukocyte filtration prevented adenosine triphosphate depletion or a change in tissue antioxidants. Conversely, unprotected piglets (group 1) exhibited lower levels of adenosine triphosphate and significant loss of tissue antioxidants. Indeed, the results in the leukocyte-filtered piglets (group 2) were nearly identical to those of piglets subjected to bypass without hypoxia (controls). CONCLUSIONS: (1) This study demonstrates that a major component of the injury that occurs when the hypoxic heart is abruptly reoxygenated is caused by oxygen radicals produced by white blood cells; (2) this injury can be prevented by a leukocyte-depleting filter; and (3) avoidance of this injury improves postbypass myocardial and pulmonary function. These data suggest that leukocyte depletion should be used routinely in all children undergoing operations for cyanotic heart disease or extracorporeal membrane oxygenation.

Myocardial protection in normal and hypoxically stressed neonatal hearts: the superiority of hypocalcemic versus normocalcemic blood cardioplegia.

OBJECTIVES: The ideal cardioplegic calcium (Ca+2) concentration in newborns continues to be debated. Most studies examining cardioplegia calcium concentrations have been done with a nonclinical model (i.e., isolated heart preparation), the results of which may not be clinically applicable, and they have not examined the effect of calcium concentration in a clinically relevant stressed (hypoxic) heart. METHODS: Twenty neonatal piglets 5 to 18 days old were placed on cardiopulmonary bypass, and their aortas were crossclamped for 70 minutes with hypocalcemic or normocalcemic multidose blood cardioplegic infusions. Group 1 (n = 5; low Ca+2, 0.2 to 0.4 mmol/L) and group 2 (n = 5; normal Ca+2, 1.0 to 1.3 mmol/L) were nonhypoxic (uninjured) hearts. Ten other piglets were first ventilated at an FiO2 of 8% to 10% (O2 saturation 65% to 70%) for 60 minutes (i.e., causing hypoxia) and then reoxygenated at an FiO2 of 100% with cardiopulmonary bypass, which produces a clinically relevant stress injury. They then underwent cardioplegic arrest (as described above) with a hypocalcemic (n = 5, group 3) or normocalcemic (n = 5, group 4) blood cardioplegic solution. Myocardial function was assessed with pressure volume loops and expressed as a percentage of control values. Coronary vascular resistance was measured during each cardioplegic infusion. All values were reported as the mean +/- standard error. RESULTS: In nonhypoxic hearts (groups 1 and 2), good myocardial protection was achieved at either concentration of cardioplegia calcium, as demonstrated by preservation of postbypass systolic function (104% vs 99% end-systolic elastance), minimally increased diastolic stiffness (152% vs 162%), no difference in myocardial water (78.9% vs 78.9%), and no change in adenosine triphosphate levels or coronary vascular resistance. Low-calcium blood cardioplegia solution repaired the hypoxic reoxygenation injury in stressed hearts (group 3), resulting in no statistical difference in myocardial function, coronary vascular resistance, or adenosine triphosphate levels compared with nonhypoxic hearts (groups 1 and 2). Conversely, when a normocalcemic cardioplegia solution was used in hypoxic hearts (group 4), there was marked reduction in postbypass systolic function (49% +/- 4% end-systolic elastance; p < 0.05), increased diastolic stiffness (276% +/- 9%; p < 0.05), increased myocardial water (80.1% +/- 0.2%; p < 0.05), rise in coronary vascular resistance (p < 0.05), and lower adenosine triphosphate levels compared with groups 1, 2, and 3. CONCLUSIONS: This study demonstrates that, in the clinically relevant, intact animal model, good myocardial protection is independent of cardioplegia calcium concentration in nonhypoxic (noninjured) hearts; hypoxic (stressed) hearts are extremely sensitive to the cardioplegic calcium concentration; and normocalcemic cardioplegia is detrimental to neonatal myocardium subjected to a preoperative hypoxic stress.

The relationship between calcium and magnesium in pediatric myocardial protection.

OBJECTIVE: We previously demonstrated that calcium can be harmful to the hypoxic neonatal heart. Despite the fact that magnesium inhibits membrane transport of calcium, few studies have examined whether magnesium can prevent the deleterious effects of calcium in cardioplegic solutions. METHODS: Twenty neonatal piglets (5 to 18 days old) underwent 60 minutes of ventilator hypoxia (inspired oxygen fraction 8% to 10%) followed by reoxygenation with the use of cardiopulmonary bypass before cardioplegic arrest to produce a clinically relevant hypoxic "stress" injury. The aorta was then crossclamped for 70 minutes with multidose blood cardioplegia. Ten piglets received a hypocalcemic (0.2 to 0.4 mmol/L) cardioplegic solution without (group 1, n = 5) or with magnesium (10 mEq/L) (group II, n = 5) supplementation. Ten other piglets were protected with a normocalcemic (1.0 to 1.2 mmol/L) cardioplegic solution without (group III, n = 5) or with magnesium (group IV, n = 5). Myocardial function was assessed by means of pressure volume loops and expressed as a percentage of control. Coronary vascular resistance was assessed during each cardioplegic infusion. RESULTS: Adding magnesium to a hypocalcemic cardioplegic solution (groups I and II) had no effect: Both groups had complete preservation of postbypass systolic function (end-systolic elastance 101% vs 104%) and preload recruitable stroke work (101% vs 102%), minimal increase in diastolic stiffness (159% vs 153%), and no difference in myocardial tissue edema (78.8% vs 78.9%) or coronary vascular resistance. Conversely, when a normocalcemic cardioplegic solution was administered without magnesium supplementation (group III), the results were markedly poorer than results obtained with magnesium supplementation (group IV). Without magnesium, there was a marked reduction in postbypass systolic function (end-systolic elastance 49% vs 101%; p < 0.05), increased diastolic stiffness (276% vs 162%; p < 0.05), decreased preload recruitable stroke work (53% vs 102%; p < 0.05), increased myocardial tissue edema (80.0% vs 78.9%; p < 0.05), and a rise in coronary vascular resistance (p < 0.05). Magnesium supplementation of the normocalcemic cardioplegic solution, by contrast, resulted in complete functional recovery. CONCLUSIONS: This study demonstrates that (1) magnesium does not alter the cardioprotective effects of a hypocalcemic cardioplegic solution, (2) a normocalcemic cardioplegic solution is detrimental to neonatal myocardium subjected to a previous hypoxic stress, and (3) magnesium supplementation of normocalcemic cardioplegic solutions prevents the deleterious effects of calcium.

Platelet deposition after surgically induced myocardial ischemia. An etiologic factor for reperfusion injury.

Despite meticulous adherence to presently known principles of myocardial preservation, reperfusion after aortic cross-clamping results in a unique injury manifested by decreasing high-energy phosphate levels and increased coronary resistance. We hypothesize that platelet deposition into the coronary microvasculature is a major factor in reperfusion injury. To differentiate platelet deposition due to subendocardial hemorrhage from deposition due to vascular entrapment, we infused 111In-labeled platelets together with 51Cr-labeled erythrocytes into 15 dogs that were on normothermic bypass and subjected to 60 minutes of global ischemia followed by 30 minutes of reperfusion. Platelet deposition is indicated only when the proportion of platelets to erythrocytes in tissue exceeds that measured by peripheral blood. Myocardial biopsy specimens were obtained after 10 minutes of bypass, 120 minutes of continuous bypass (Group I), and at the end of reperfusion after global ischemia (Group II). In five dogs (Group III), dipyridamole (1 mg/kg), an antiplatelet activation agent, was administered in the preischemic period. Platelet deposition was expressed as the number of radioactive-labeled platelets deposited per gram of tissue. Bypass for 120 minutes resulted in only a minimal increase in platelet deposition. However, normothermic ischemia followed by reperfusion resulted in over a twofold increase in platelet deposition compared to controls. Pretreatment with dipyridamole appeared to avoid platelet deposition. These data indicate that platelet deposition in the coronary microcirculation following surgically induced myocardial ischemia may be associated with reperfusion injury and that antiplatelet drugs after this sequence.