Custodiol HTK versus Plegisol: in-vitro comparison with the use of immature (H9C2) and mature (HCM) cardiomyocytes cultures.

BACKGROUND: Although cardioplegia is used since the '70s of the last century, debate on cardioprotection during cardio-surgical procedures is still actual. The selection of a particular method depends mainly on the preferences and experience of a specific center or even surgeon. Crystalloid cardioplegia is an aqueous ion solution similar to intracellular (Custodiol HTK) or extracellular (Plegisol) fluid. The potensional clinical advantages of relatively new idea of cardioplegia solution based on intracellular composition (Custodiol HTK) justifies futher research, but only a few used cultured cells in laboratory conditions. METHODS: In this study, the authors sought to compare Custodiol HTK with Plegisol cardioplegia solutions using an in-vitro model simulating cardioplegic arrest. The efficacy of myocardial protection during ischemia was investigated with susceptible indicators like the appearance of the deleterious effect of reactive oxygen species and oxidative stress markers. Immersed human cardiomyocytes and rat cardiomyoblasts H9C2 in cardioplegia for 4 h were examined for expression of oxidative stress markers (MnSOD, iNOS, HSP27), cardioplegic solutions cytotoxicity, and peroxidation damage of the cell's lipids and proteins. All tests were performed after 0.5 h, 1 h, 2 h, and 4 h of incubation in identical physical and biological conditions, which is difficult to achieve in clinical trials. RESULTS: The lower cytotoxicity index performed on matured cells of human cardiomyocytes and highest dehydrogenase level showed after incubation with Custodiol HTK. This did not apply to tests on immature cells H9C2. Custodiol HTK induced significantly stronger iNOS expression. The decrease of HSP27 concentration has been instantaneous and maintained troughout the study only in both cultures incubated with Custodiol HTK. The other tests: lipid peroxidation, carbonyl groups concentration and MnSOD expression show no clear superiority evidence of used cardioplegic solutions. CONCLUSIONS: Considering proceeded examinations on cultured cardiomyocytes, Custodiol HTK appears to be safer than Plegisol.

Adipose stem cell secretome markedly improves rodent heart and human induced pluripotent stem cell-derived cardiomyocyte recovery from cardioplegic transport solution exposure.

Heart transplantation is a life-saving therapy for end-stage organ failure. Organ deterioration during transportation limits storage to 4 hours, limiting hearts available. Approaches ameliorating organ damage could increase the number of hearts acceptable for transplantation. Prior studies show that adipose-derived stem/stromal cell secretome (ASC-S) rescues tissues from postischemic damage in vivo. This study tested whether ASC-S preserved the function of mouse hearts and human induced pluripotent stem cell-derived cardiomyocytes (iCM) exposed to organ transportation and transplantation conditions. Hearts were subjected to cold University of Wisconsin (UW) cardioplegic solution ± ASC-S for 6 hours followed by analysis using the Langendorff technique. In parallel, the effects of ASC-S on the recovery of iCM from UW solution were examined when provided either during or after cold cardioplegia. Exposure of hearts and iCM to UW deteriorated contractile activity and caused cell apoptosis, worsening in iCM as a function of exposure time; these were ameliorated by augmenting with ASC-S. Silencing of superoxide dismutase 3 and catalase expression prior to secretome generation compromised the ASC-S cardiomyocyte-protective effects. In this study, a novel in vitro iCM model was developed to complement a rodent heart model in assessing efficacy of approaches to improve cardiac preservation. ASC-S displays strong cardioprotective activity on iCM either with or following cold cardioplegia. This effect is associated with ASC-S-mediated cellular clearance of reactive oxygen species. The effect of ASC-S on the temporal recovery of iCM function supports the possibility of lengthening heart storage by augmenting cardioplegic transport solution with ASC-S, expanding the pool of hearts for transplantation.

Cyclosporine A as a Cardioprotective Agent During Donor Heart Retrieval, Storage, or Transportation: Benefits and Limitations.

BACKGROUND: Storage of donor hearts in cardioplegic solutions supplemented with conditioning agents activating endogenous mitochondrial protective signaling enhanced their postreperfusion recovery. The present study investigates the role of timing and duration of cardiac exposure to cyclosporine A (CsA), another putative mitochondrial protectant, on cardiac functional recovery and potential mechanisms of CsA action in an isolated working rat heart model of donor heart retrieval and storage. METHODS: After measurement of baseline function, hearts were arrested and stored for 6 hours at 4°C in either Celsior alone or Celsior + CsA (0.2 µM), then reperfused for 45 minutes in Krebs solution, when functional recovery was assessed. Two additional groups of Celsior-alone stored hearts were exposed to 0.2 µM CsA for the initial 15 minutes (nonworking period) or the full 45-minute period of reperfusion. Coronary effluent was collected pre- and poststorage for assessment of lactate dehydrogenase release. Tissue samples were collected at the end of each study for immunoblotting and histological studies. RESULTS: CsA supplementation during cold storage or the first 15-minute reperfusion significantly improved functional recovery and significantly increased phospho-AMPKαThr172 and phospho-ULK-1Ser757. Hearts exposed to CsA for 45 minutes at reperfusion recovered poorly with no phospho-AMP-activated protein kinase α activation, decreased phospho-eNOSSer633, and decreased mitochondrial cytochrome c content with increased lactate dehydrogenase release. CONCLUSIONS: Inclusion of CsA during cold storage is cardioprotective. Effects of CsA addition to the perfusate during reperfusion were time dependent, with benefits at 15 minutes but not 45 minutes of reperfusion. The toxic effect with the presence of CsA for the full 45-minute reperfusion is associated with impaired mitochondrial integrity and decreased eNOS phosphorylation.

How to avoid severe coronary vasoconstriction in potassium induced cardioplegia.

OBJECTIVES: The aim of this study was to investigate how high K+ concentrations can be safely used in cardioplegic solutions without causing severe coronary artery vasocontraction. DESIGN: Twenty-four 50 kg pigs were used. The distal part of the left anterior descending coronary artery was cut into ring segments and transferred into organ baths with Krebs solution bubbled with 95% O2 and 5% CO2. K+ concentrations between 16 and 127 mM were used to induce vasocontractions at 37, 22, 15, and 8 °C. Mg2+ (0-20 mM) were used to attenuate K+ induced vasocontractions. RESULTS: K+-Krebs solution 127 mM at 37 °C induced a strong, sustained vasocontraction defined as 100%. The contractions induced by 16, 23, 30 and 127 mM K+ were: 7.7, 38, 72 and 100% at 37 °C; 1.7, 7.4, 21 and 65% at 22 °C; 1, 6.6, 15 and 33% at 15 °C; 0.6, 2.1, 6 and 14% at 8 °C, respectively. Mg2+ reduced the K+-induced contraction at 37 °C in a concentration-dependent way and Mg2+ at 8 mM practically eliminated the risk for severe vasocontraction. CONCLUSIONS: Hypothermia (8 °C) abolishes coronary contraction induced by K+-cardioplegic solutions. In normothermic cardioplegia 8 mM Mg2+ prevents vasoconstriction.

Is warm or cold Calafiore blood cardioplegia better? Hemodynamic, metabolic, and electron microscopic differences.

BACKGROUND: Controversy exists as to whether warm or cold Calafiore blood cardioplegia (BCP) is better for cardiac preservation. Therefore, we compared hemodynamic performance, myocardial metabolism, and ultrastructural preservation in rat hearts after application of cold or warm BCP. MATERIALS AND METHODS: The hearts of 24 male Wistar rats were excised and inserted into a blood perfused isolated heart apparatus, and after a stabilization period of 30 minutes, either cold (4°C) or warm (36°C) Calafiore BCP was administered during an aortic clamping time of 90 minutes (12 rats each). Hearts clamped without BCP and hearts immediately excised in anesthesia served as worst case and no damage controls, respectively (n=3 each). During reperfusion, functional hemodynamic parameters were recorded in BCP groups, and myocardial oxygen consumption and lactate production were calculated. After perfusion fixation, the hearts of three rats in each group were investigated for cellular edema and mitochondrial damage by morphometry using transmission electron microscopy. RESULTS: Cardiac function after BCP application during aortic clamping showed a slightly better recovery with warm than with cold Calafiore BCP as indicated by higher left ventricular developed pressure (warm 97% of baseline, cold 68% of baseline) after warm BCP. Other hemodynamic parameters and coronary flow were not different between warm and cold BCP. Myocardial oxygen consumption and lactate production were similar under warm and cold conditions. Electron microscopy showed typical signs of ischemia in the ischemia group without BCP. Mitochondrial ultrastructure was well preserved in both BCP groups, but cellular edema was more pronounced with cold than with warm BCP. The qualitative analysis was confirmed by the morphometric cellular edema index and the volume-to-surface ratio of the mitochondria. CONCLUSION: Only mild differences were observed between warm and cold BCP in rats with respect to cardiac function, metabolism, and tissue preservation after aortic clamping. However, a small tendency toward better postischemic recovery was observed with warm BCP.

Activated protein C in the cardioplegic solution on a porcine model of coronary ischemia-reperfusion has deleterious hemodynamic effects.

PURPOSE: In reperfusion injury activation of coagulation and inflammation contribute to organ dysfunction. Activated protein C (APC) exhibits anticoagulant and anti-inflammatory properties in models of reperfusion injury. We hypothesized that APC could be cardioprotective after ischemia and cardiopulmonary bypass (CPB). METHODS: 20 pigs, undergoing 120 min of CPB and aortic cross-clamping, were randomized to receive 1 mg of human APC or placebo to the last cardioplegic solution given 15 min before declamping to the systemic circulation. After aortic declamping the heart was supported by continuing CPB for 30 min followed by 30 min surveillance. Thrombin-antithrombin complexes, neutrophil L-selectin expression in blood and myeloperoxidase activity (MPO) of myocardial biopsies were measured. RESULTS: There was no indication of APC-induced increased bleeding. Thrombin levels were significantly lower in the APC group than in the placebo group and so were the rates of thrombin formation during the first 3 min of reperfusion and between 10 and 30 min after declamping. There were no differences in MPO or in the proportion of L-selectin (+) to L-selectin (-) neutrophils between groups. Significant systolic hypotension in the APC group was observed at 30 and 45 min compared with the placebo group which associated with the increased mortality observed in the APC group (p = 0.019). CONCLUSIONS: Human APC in cardioplegic solution during CPB in pigs, decreased reperfusion induced thrombin formation with no associated bleeding. No anti-inflammatory effects of human APC were seen. However, in this setting, APC caused hemodynamic deterioration. The observed phenomenon could be explained by systolic hypotension potentially produced by the release of vasoactive substances generated by the APC activation of PARs in the endothelium.

Role of the sarcolemmal adenosine triphosphate-sensitive potassium channel in hyperkalemic cardioplegia-induced myocyte swelling and reduced contractility.

BACKGROUND: Hyperkalemic cardioplegia (Plegisol) has been shown to result in myocyte swelling and reduced contractility. We have demonstrated the elimination of these detrimental effects by the addition of an adenosine triphosphate-sensitive K+ (KATP) channel opener. To examine whether the mitochondrial or sarcolemmal KATP channel might be involved, volume and contractility in isolated myocytes from wild-type mice and mice lacking the sarcolemmal KATP channel (Kir6.2-/-) were evaluated. METHODS: Myocytes were perfused for 20 minutes each with control 37 degrees C Tyrode's solution, test solution, and then control solution. Test solutions were (n = 10 per group) either 9 degrees C Plegisol or 9 degrees C Plegisol with 100 micromol/L of diazoxide, a putative mitochondrial-specific KATP channel opener. Cell volume and contractility were measured by digital video microscopy at baseline and during the test solution and reexposure periods. RESULTS: Myocytes from wild-type mice, perfused with 9 degrees C Plegisol, demonstrated significant cell swelling (11.2% +/- 0.4%; p < 0.01) and diminished contractility (32.5% +/- 9.6% reduction in percent shortening, 47.2% +/- 10.1% reduction in peak velocity of shortening, and 52.0% +/- 8.8% reduction in peak velocity of relengthening; p < 0.05) versus baseline. Cell swelling and diminished contractility were significantly reduced by the addition of diazoxide. In Kir6.2-/- myocytes, Plegisol caused a greatly reduced level of cell swelling (3.2% +/- 0.1%; p < 0.01), and this was unaffected by diazoxide. Contractility was unchanged in Kir6.2-/- myocytes after Plegisol. CONCLUSIONS: The sarcolemmal KATP channel appears necessary for exaggerated cell swelling and reduced contractility to occur after hyperkalemic cardioplegia in mouse myocytes.

Hyperkalemic cardioplegia-induced myocyte swelling and contractile dysfunction: prevention by diazoxide.

BACKGROUND: Hyperkalemic cardioplegia (9 degrees C) results in significant myocyte swelling and reduced contractility, representing a possible mechanism of myocardial stunning. Adenosine triphosphate-sensitive potassium channel (KATP) openers have been shown to ameliorate stunning. This study evaluated the hypothesis that a KATP opener would prevent hyperkalemic cardioplegia-induced myocyte swelling and reduced contractility. METHODS: Isolated rabbit myocytes were perfused with 37 degrees C Tyrode's solution for 20 minutes, followed by test solution (9 degrees C or 37 degrees C) including control Tyrode's, Tyrode's + 100 micromol/L diazoxide (KATP opener), St. Thomas's solution; or 9 degrees C St. Thomas's + 100 micromol/L diazoxide or St. Thomas's + 100 micromol/L diazoxide + 20 micromol/L HMR1098 or 50 micromol/L 5-hydroxydeconoate (KATP blockers) for 20 minutes (n = 8 per group). Myocytes were then reexposed to 37 degrees C Tyrode's solution for 20 minutes. Volume and contractility were measured by videomicroscopy and video-based edge detection, respectively. RESULTS: St. Thomas's solution (9 degrees C) caused significant myocyte swelling and associated reduced contractility (p < 0.05). The addition of diazoxide abolished myocyte swelling (p < 0.0001), and eliminated the associated reduced contractility (p < 0.05). Findings were unchanged by the addition of HMR 1098 and 5-hydroxydeconoate. CONCLUSIONS: Diazoxide prevented myocyte swelling and reduced contractility secondary to hyperkalemic cardioplegia, and this was unchanged by the addition of either KATP channel blocker. Prevention of myocyte swelling was associated with improved contractility, consistent with the hypothesis that myocyte swelling may be a mechanism of myocardial stunning. Diazoxide may play a role in myocyte volume homeostasis by means of a mechanism separate from opening the KATP channel.

Endothelial cell injury induced by preservation solutions: a confocal microscopy study.

BACKGROUND: We evaluated the effects of standard preservation solutions on cultured human greater saphenous vein endothelial cells. METHODS: Endothelial cells (eight strains) were preincubated for 6 or 24 hours at 4 degrees C in Celsior, Euro-Collins, St. Thomas Hospital II, and University of Wisconsin solutions, reincubated in warm oxygenated culture medium 199, and observed up to 48 hours. Culture viability was assessed through cell counting and confocal microscopy of calcein loaded cells. RESULTS: Incubation in both Euro-Collins and St. Thomas, but not in Celsior or University of Wisconsin solutions, caused significant cells losses and diffuse morphological damages characterized by solution-specific distinctive alterations. Injury caused by 6-hour, but not by 24-hour treatment, was reversible. CONCLUSIONS: The incubation with Celsior and University of Wisconsin solutions substantially preserved endothelial viability and proliferative capability. Conversely, a prolonged incubation in either Euro-Collins or St. Thomas solutions caused severe and potentially irreversible damage referable to the induction of, respectively, apoptotic or necrotic changes.

Preconditioning improves cardioplegia-related coronary microvascular smooth muscle hypercontractility: role of KATP channels.

OBJECTIVES: The effect of preconditioning before hyperkalemic cardioplegia on the coronary smooth muscle remains to be elucidated. We tested the hypothesis that hypoxic preconditioning could protect coronary smooth muscle against subsequent hyperkalemic cardioplegia-induced coronary vasospasm and that this preconditioning effect could be mediated by K(ATP) channels. METHODS: Rat coronary arterioles (endothelium-denuded) were studied in a pressurized, no-flow, normothermic state. Simultaneous monitoring of luminal diameter and intracellular calcium concentration of vascular smooth muscle loaded with fura-2 was made with microscopic image analysis. All vessels were subjected to 60 minutes of hypoxic hyperkalemic cardioplegia (K(+) = 25.0 mmol/L) and were then reperfused. Six groups were studied: (1) controls, no precardioplegic intervention; (2) preconditioning, achieved with 10 minutes of hypoxia (PO2 < 30 mm Hg) and 10 minutes of reoxygenation; (3) preconditioning plus glibenclamide (10 micromol/L), achieved with 10 minutes of preconditioning in the presence of K(ATP) channel blocker glibenclamide; (4) pretreatment with K(ATP) channel opener pinacidil (100 micromol/L); (5) pretreatment with pinacidil (100 micromol/L) plus glibenclamide (10 micromol/L); and (6) pretreatment with glibenclamide (10 micromol/L) alone. RESULTS: Hypoxic preconditioning significantly (P <.01) reduced hyperkalemic cardioplegia-induced intracellular calcium concentration accumulation and prevented the hypercontractility during and after hyperkalemic cardioplegia compared with control vessels. Pinacidil provided effective microvascular protection similar to hypoxic preconditioning. These vasoprotective effects of preconditioning were significantly antagonized in glibenclamide-treated vessels. CONCLUSIONS: Hypoxic preconditioning can prevent coronary microvascular hypercontractility during and after subsequent cardioplegia by a K(ATP ) channel mechanism that regulates intracellular calcium concentration of the vascular smooth muscle.

Age-related effects of St Thomas' Hospital cardioplegic solution on isolated cardiomyocyte cell volume.

OBJECTIVES: We tested the hypothesis that neonatal cells are more sensitive to cardioplegia-induced cell swelling than more mature cells and spontaneous swelling in the absence of ischemia can be prevented by cardioplegia with a physiologic KCl product. METHODS: Cell volumes of isolated ventricular myocytes from neonatal (3-5 days), intermediate (10-13 days), and adult (>6 weeks) rabbits were measured by digital video microscopy. After equilibration in 37 degrees C physiologic solution, cells were suprafused with 37 degrees C or 9 degrees C St Thomas' Hospital solution (standard or low Cl(-)) or 9 degrees C physiologic solution followed by reperfusion with 37 degrees C physiologic solution. RESULTS: Neonatal cells swelled 16.2% +/- 1.8% (P <.01) in 37 degrees C St Thomas' Hospital solution and recovered during reperfusion, whereas more mature cells maintained constant volume. In contrast, 9 degrees C St Thomas' Hospital solution caused significant age-dependent swelling (neonatal, 16.8% +/- 1.5%; intermediate, 8.6% +/- 2.1%; adult, 5.6% +/- 1.1%). In contrast to more mature cells, neonatal cells remained significantly edematous throughout reperfusion (8.1% +/- 1.5%). Swelling was not due to hypothermia because 9 degrees C physiologic solution did not affect volume. Lowering the KCl product of St Thomas' Hospital solution by partially replacing Cl(-) with an impermeant anion prevented cellular edema in all groups. CONCLUSION: In the absence of ischemia, neonatal cells were more sensitive to cardioplegia-induced cellular edema than more mature cells, and edema observed in all groups was avoided by decreasing the KCl product of St Thomas' Hospital solution to the physiologic range. Differences in cell volume regulation may explain the sensitivity of neonatal hearts to hyperkalemic cardioplegic arrest and suggest novel approaches to improving myocardial protection.

Human albumin enriched St. Thomas Hospital cardioplegic solution increases reperfusion injury in isolated perfused rat hearts.

In open heart surgery it is very important to protect the heart during the ischaemic period in terms of mortality and morbidity. Many different cardioplegic solutions are in clinical use without being tested experimentally. In this study we intended to investigate the effects of albumin addition to St. Thomas Hospital cardioplegic solution on cardiac protection to ischaemia. Rat hearts were isolated and perfused in Langendorff apparatus (n = 6 for each group). After the stabilisation period, the hearts in the control group were arrested with St. Thomas Hospital cardioplegic solution for 3 min then subjected to 30 min of global ischaemia in cardioplegic solution, this is followed by reperfusion for 10 min. In albumin groups, the experimental protocol was the same but 2.25%, 4.5% or 9% human albumin was added to the cardioplegic solution. All of the hearts were compared for their pre-ischaemic and post-ischaemic contractility, heart rate, coronary flow, LDH and CK enzyme leakage, and wet/dry weight ratio values. The contraction, heart rate (P < 0.01 for both), and coronary flow (only for the 9% albumin group, P < 0.05) values in the albumin group were less than the control group during the reperfusion period. There was no difference between groups in LDH, and CK leakage, and wet/dry weight ratio. The circulation of ischaemic hearts in the albumin group were diminished, possibly due to protein precipitation. This condition negatively affected the performance of the heart. The fact that there is no difference in enzyme leakage and wet/dry weight ratio, indicates that this event is not irreversible.

Validity of a model of cultured myocardial cells for assessment of cardioplegia.

Myocardial protection is usually studied in vitro on perfused heart preparations, but never directly on cultured cardiomyocytes. We evaluated a model of cultured newborn rat cardiomyocytes to study both the cytotoxicity and the protective effect against chemical hypoxia of three cardioplegic solutions (St Thomas' I, Bretschneider, St Thomas' II) under normothermic (37 degrees C) and hypothermic (4 degrees C) conditions. Cytotoxicity was evaluated in 50% and 100% concentrations of the cardioplegic solutions with incubation times from 90 to 360 min. Myocardial protection was studied in 50% cardioplegic solution with metabolic inhibitors. Immediate and late viabilities, after 24 h of recovery in the medium, were evaluated by simultaneous staining with fluorescein diacetate and propidium iodide. At 37 degrees C, the 50% concentration of the three cardioplegic solutions did not modify cell viability. At 37 degrees C, with 360 min of incubation, the 100% concentration of the St Thomas' I and Bretschneider solutions diminished immediate viability (mean +/- SD; medium 87% +/- 2%; St Thomas' I 58% +/- 5%; Bretschneider 37% +/- 8%; St Thomas' II 89% +/- 3%) as well as late viability (medium 69% +/- 2%; St Thomas' I 32% +/- 3%; Bretschneider 24% +/- 7%; St Thomas' II 65% +/- 4%). At 4 degrees C, immediate and late viabilities were unaffected by cardioplegic solutions. At 37 degrees C, after 360 min incubation time, metabolic inhibitors diminished immediate viability to 29% +/- 1% and late viability to zero. None of the three cardioplegic solutions used at 50% concentration prevented this effect. At 4 degrees C, immediate viability was not significantly affected by metabolic inhibitors (73% +/- 10%), but the use of Bretschneider cardioplegic solution seemed to be detrimental (53% +/- 9%). On the other hand, recovery phase after pretreatment with metabolic inhibitors with or without cardioplegic solutions for 360 min significantly diminished late viability (medium 63% +/- 7%; metabolic inhibitors 17% +/- 8%; St Thomas' I 17% +/- 6%; Bretschneider 8% +/- 6%; St Thomas' II 15% +/- 3%) and again cardioplegia was inefficient. In conclusion, in this in vitro model for the study of cardioplegic solutions, only pure concentrations of the St Thomas' I and Bretschneider solutions under normothermic conditions were cytotoxic. The well-known protective effects of hypothermia against ischemia and reperfusion injury were both reproduced. Therefore, and even though cardioplegia failed to have any protective effect, probably owing to a severe metabolic inhibition, this model may be useful for studying myocardial protection.

Iatrogenic myocardial edema: increased diastolic compliance and time course of resolution in vivo.

BACKGROUND: Perfusion-induced edema reduces diastolic compliance in isolated hearts, but this effect and the time for edema to resolve after blood reperfusion have not been defined in large animals. METHODS: Edema was induced by coronary perfusion with Plegisol (750 mL, 289 mOsm/L) during a 1-minute aortic occlusion in 6 pigs. This was followed by whole blood reperfusion, inotropic support, and circulatory assistance until sinus rhythm and contractile function were restored. A control group (n = 6) was treated similarly, with 1 minute of electrically induced ventricular fibrillation and no coronary perfusion. Recorded data included electrocardiogram, left ventricular pressure and conductance, aortic flow, and two-dimensional echocardiography. Preload reduction by vena caval occlusion was used to define systolic and diastolic properties. Data were recorded at baseline and at 15-minute intervals for 90 minutes after reperfusion. RESULTS: In the edema group, average left ventricular mass (132 +/- 7 [standard error of the mean] versus 106 +/- 4 g) and ventricular stiffness constant (0.15 +/- 0.02 versus 0.05 +/- 0.01) increased after Plegisol versus baseline (p < 0.05), returning to normal after 45 minutes of reperfusion. In controls, mass (118 +/- 6 versus 116 +/- 4 g) and ventricular stiffness (0.06 +/- 0.01 versus 0.05 +/- 0.01) did not change significantly. There was no significant change in systolic function. Myocardial water content at the end of the study was not different for the two groups. CONCLUSIONS: Crystalloid-induced edema and diastolic stiffness resolve after 45 minutes in pigs. This suggests that edema caused solely by cardioplegia during cardiac operations should not cause significant perioperative ventricular dysfunction.

Loss of endothelium-dependent vasodilatation and nitric oxide release after myocardial protection with University of Wisconsin solution.

University of Wisconsin solution has proved to be a superior form of cardioplegia for cardiac transplantation, demonstrating better functional recovery than that provided by extracellular crystalloid solutions. Furthermore, experimental data have suggested a role for University of Wisconsin solution in protection of the neonatal heart during operations for congenital heart defects. However, significant concerns have been raised regarding potential endothelial injury from the high potassium concentration contained in University of Wisconsin solution that could affect its safety and thus its clinical application. Fourteen neonatal (aged 1 to 3 days) piglet hearts were harvested and supported on an isolated, blood-perfused circuit. Endothelium-dependent vasodilatation was measured by bradykinin (10(-6) mol/L) infusion and nitric oxide release was determined. Endothelium-independent vasodilatation was then induced by sodium nitroprusside (10(-6) mol/L) infusion. A 2-hour period of cold cardioplegic arrest was instituted with multidose University of Wisconsin solution (group 1, n = 7) or blood cardioplegia (group 2, n = 7). After reperfusion and stabilization, another stimulation with bradykinin and nitroprusside was carried out and nitric oxide was again measured. After 2 hours of arrest with University of Wisconsin solution, there was a near-complete loss of vasodilatation in response to bradykinin infusion; coronary blood flow reached 245% of baseline before arrest versus only 117% of baseline after arrest (p = 0.0011). This correlated with an inability of the endothelium to release nitric oxide (96 +/- 30 nmol/min before arrest versus -32 +/- 9 nmol/min after arrest, p = 0.0039. In group 2, the vasodilatory response to bradykinin was preserved after arrest and reperfusion; 265% of baseline before arrest versus 222% of baseline after arrest. These results demonstrate a loss of endothelium-dependent vasodilatation after multidose University of Wisconsin cardioplegia caused by the inability of the endothelium to release nitric oxide. In contrast, blood cardioplegia does not result in impaired endothelial function.

Cardioplegia alters porcine coronary endothelial cell growth and responses to aggregating platelets.

Experiments were designed to determine the viability of endothelial cells and their responses to products released by aggregating platelets following single flush perfusion of the coronary arteries with cardioplegic solutions used for cardiac transplantation. Porcine coronary arteries were perfused with crystalloid (Plegisol) or blood cardioplegic solutions; nonperfused hearts placed in 0.9% saline were used as controls. Immediately following perfusion and after 5-hour storage of the hearts in the same cardioplegic solution, rings were cut from the right coronary arteries and suspended in organ chambers for the measurement of isometric force. In some rings the endothelium was removed deliberately. The left circumflex coronary arteries were flushed with collagenase and the harvested endothelial cells were plated for cell culture. Left anterior descending coronary arteries were perfusion fixed with glutaraldehyde for scanning electron microscopy. In the organ chamber experiments, aggregating platelets and adenosine diphosphate caused relaxations in rings with endothelium. These relaxations were reduced immediately following crystalloid cardioplegia and were restored following 5-hour storage. Serotonin caused contractions in all rings. Rings without endothelium were more sensitive than rings with endothelium to the amine; this difference was augmented following 5-hour storage of the heart. Significantly fewer foci of endothelial cells grew in culture following 5-hour storage of the hearts in crystalloid cardioplegic solution compared to control (p < 0.05). There were no anatomical differences identified among groups by scanning electron microscopy. These results suggest that crystalloid cardioplegia alters the responses of coronary arteries to substances released by aggregating platelets and reduces the ability of endothelial cells to replicate. Such changes may contribute to altered vascular resistance following reperfusion of transplanted hearts and potentially to later structural changes in the coronary arteries.

Calcium content of St. Thomas' II cardioplegic solution damages ischemic immature myocardium.

Clinical application of hypothermic pharmacologic cardioplegia in pediatric cardiac surgery is less than satisfactory, despite its well known benefits in adults. Protection of the ischemic immature rabbit heart with hypothermia alone is better than with hypothermic St. Thomas' II cardioplegic solution. Control of cellular calcium is a critical component of cardioplegic protection. We determined whether the existing calcium content of St. Thomas' II solution (1.2 mmol/L) is responsible for suboptimal protection of the ischemic immature rabbit heart. Modified hypothermic St. Thomas' II solutions (calcium content, 0 to 2.4 mmol/L) were compared with hypothermic Krebs bicarbonate buffer in protecting ischemic immature (7- to 10-day-old) hearts. Hearts (n = 6 per group) underwent aerobic "working" perfusion with Krebs buffer, and cardiac function was measured. The hearts were then arrested with a 3-minute infusion of either cold (14 degrees C) Krebs buffer (1.8 mmol calcium/L) as hypothermia alone or cold St. Thomas' II solution before 6 hours of hypothermic (14 degrees C) global ischemia. Hearts were reperfused, and postischemic enzyme leakage and recovery of function were measured. A bell-shaped dose-response profile for calcium was observed for recovery of aortic flow but not for creatine kinase leakage, with improved protection at lower calcium concentrations. Optimal myocardial protection occurred at a calcium content of 0.3 mmol/L, which was better than with hypothermia alone and standard St. Thomas' II solution. We conclude that the existing calcium content of St. Thomas' II solution is responsible, in part, for its damaging effect on the ischemic immature rabbit heart.

Detrimental effects of multidose hypothermic cardioplegia in the neonatal heart: the role of the frequency of cardioplegic infusions.

In the neonatal rabbit heart, multidose crystalloid cardioplegia is protective against normothermic ischemia, but its beneficial effects are lost under hypothermia. In order to determine the relationship between myocardial protection and the number of cardioplegic infusions administered during the ischemic period, we examined the effects of an increasing number of infusions on postischemic recovery at three temperatures (37 degrees, 20 degrees, or 10 degrees C). Isolated working hearts from rabbits aged 7-10 days were perfused aerobically (37 degrees C) for 20 min before infusion of St. Thomas' Hospital cardioplegic solution at the selected temperature. At each temperature, the cardioplegic solution was given either as a single 2-min infusion (single-dose) or as repeated 2-min infusions (multidose) at various intervals. Following the ischemic period, hearts were reperfused (15 min Langendorff, 20 min working) before assessment of the recovery of function. Ischemic durations (selected to result in approximately 55%-70% recovery in the single-dose group at each temperature) were 1, 10, or 18 h at 37 degrees, 20 degrees, and 10 degrees C. At 37 degrees C, there was a positive correlation between postischemic recovery and the number of infusions during the ischemic period. However, at 20 degrees or 10 degrees C the relationship was reversed and recovery was depressed with increasing number of infusions.(ABSTRACT TRUNCATED AT 250 WORDS)

Endothelial cell toxicity of solid-organ preservation solutions.

Endothelial cell damage caused by myocardial cardioplegic solutions (Bretschneider HTK and St. Thomas' Hospital No. 2) or renal and hepatic cold storage solutions (modified Collins and University of Wisconsin solution) was assessed in monolayer cultures of adult human venous endothelial cells at 4 degrees to 10 degrees C with phase-contrast microscopy. St. Thomas' Hospital solution caused the cells to contract, resulting in disruption of monolayer integrity and opening of intercellular gaps, and resulted in a 24-hour postexposure survival of 51.0% +/- 2.4%. Bretschneider HTK solution altered cellular morphology less and produced the best postexposure survival (80.2% +/- 2.6%; p less than 0.001). Although morphology was altered the least with University of Wisconsin solution, postexposure survival with this solution, which was similar to that with modified Collins solution, was superior to that with St. Thomas' (p less than 0.01) but inferior to that with Bretschneider HTK (p less than 0.05). The superior protection provided by Bretschneider HTK was due to its additives histidine, tryptophan, and KH-2-oxygluterate (p less than 0.005), and to its low chloride content (p less than 0.005). Furthermore, modifying St. Thomas' solution by decreasing its chloride content improved cell survival to 71.2% +/- 2.3% (p less than 0.001). Normothermic (37 degrees C) exposure to Bretschneider HTK, modified Collins, and University of Wisconsin solution was cytotoxic, whereas normothermic exposure to St. Thomas' cardioplegia was not. In conclusion, the preservation solution that is the least harmful to endothelial cells at hypothermia is Bretschneider HTK cardioplegic solution.