Nonischemic Control Hearts Research Articles

Abstract 14959: Mitochondrial Transplantation in Pediatric and Neonatal Porcine Hearts in a Model of DCD Heart Transplantation

Introduction: Heart transplantation in pediatric and neonatal patients is limited by the donor pool. Donation after circulatory death (DCD) could potentially expand the donor pool by offering an additional source of cardiac allografts. In this study, we investigated the efficacy of mitochondrial transplantation to enhance myocardial function in pediatric and neonatal DCD hearts. Hypothesis: Autologous mitochondrial transplantation enhances the viability and function of pediatric and neonatal DCD donor hearts. Methods: Circulatory death was induced by extubation in neonatal (25 - 50g heart weight, representing 4-16 month human) and pediatric (68 - 98g heart weight, representing 4-6 year human) swine model. Hearts were subjected to 20 min of warm ischemia and 10 min of cold cardioplegic arrest and then harvested. The aorta and pulmonary artery were cannulated, inferior and superior venae cavae ligated and a balloon was inserted in the left ventricle. The hearts were then mounted on the ex situ perfusion device. After 15 minutes of reperfusion, hearts received either vehicle alone ([VEH], 10 mL n = 6) or vehicle containing autologous mitochondria ([MT] 5x10 9 in 10 mL; n = 6). A Sham non-ischemic group (n = 4) did not undergo warm ischemia, mimicking donor after brain death (DBD). Hearts were machine perfused for 2 hours unloaded and 2 hours of loaded conditions. Results: Following 4 hours of reperfusion, neonatal and pediatric DCD hearts receiving VEH showed significantly decreased myocardial function and viability as compared to Sham non-ischemic control hearts. In contrast, in both neonatal and pediatric DCD hearts receiving MT, LVDP, dP/dt max, and fractional shortening were significantly increased (p < 0.001 for each) and were equal or better than that observed in Sham non-ischemic hearts. Infarct size was significantly decreased in both neonatal and pediatric DCD hearts receiving MT as compared to VEH (p < 0.001). Conclusions: Mitochondrial transplantation provides for significantly enhanced preservation of myocardial function and viability in neonatal and pediatric DCD hearts that is equivalent to or better than that observed in DBD hearts. Mitochondrial transplantation provides a possible option to expand the pediatric and neonatal heart donor pool.

Calcium sensitisation impairs diastolic relaxation in post-ischaemic myocardium: implications for the use of Ca2+ sensitising inotropes after cardiac surgery☆

Calcium sensitising inotropes are increasingly being used in cardiac surgical patients. Theoretically, increasing contractile protein sensitivity to Ca(2+) prevents the Ca(2+) elevation associated arrhythmogenicity and potentiates the inotropic effect of catecholamines. On the other hand, we hypothesised that Ca(2+) sensitisation exacerbates post-ischaemic myocardial stunning by impairing diastolic relaxation, which might have deleterious effects in postoperative cardiac surgical patients. In an isolated rabbit heart model, 45 min normothermic ischaemia with potassium-induced cardioplegic arrest was followed by 120 min reperfusion. Isovolumetric left ventricular (LV) function and myocardial oxygen consumption (MvO(2)) were measured, and cytosolic Ca(2+) was monitored by rhod-2 surface spectrofluorometry. During reperfusion, ORG 30029 (250 microM) and levosimendan (0.5 microM) were used as Ca(2+) sensitisers (ORG, n=6, Levo, n=6), Ca(2+) de-sensitisation was induced with butanedione-monoxime (5mM, BDM, n=6), and dopamine (20 nM) served as a representative catecholamine (n=6). To counteract the PDE III inhibiting properties of ORG and Levo, IGF-1 (0.1 microM) and parathyroid hormone (0.05 microM) were used. As expected, ischaemia/reperfusion induced moderate cytosolic calcium overload. Dopamine increased LV contractility and MvO(2) by augmenting the amplitude of the Ca(2+) transient, but relaxation was unchanged due to faster diastolic Ca(2+) removal. Dopamine-induced Ca(2+) handling was unchanged after uncoupling the Mg-ATPase with BDM, and MvO2 decreased in proportion with the reduced LV mechanical work load. ORG improved contractility without apparent effects on Ca(2+) handling, and MvO(2) remained constant despite increased contractile work. Conversely, ORG induced a rightward shift of the diastolic pressure-volume relationship in post-ischaemic hearts (diastolic pressure at 0.8 ml balloon volume 14.3+/-5 mmHg, p=0.01 vs control), but not in non-ischaemic control hearts. With levosimendan, the Ca(2+) sensitising effects were less pronounced (7.6+/-3 mmHg, p=0.4 vs control). By counteracting the PDE inhibiting effects of ORG and Levo using parathyroid hormone and IGF-1, the negative lusotropic effects of Ca(2+) sensitisation were unmasked. Calcium sensitisation improves systolic function and energetic efficiency. However, Ca(2+) sensitisers should be used with caution during post-ischaemic reperfusion, as they may exacerbate myocardial stunning and thus impair cardiac output.

Ischemic Preconditioning Protects Mitochondrial Respiratory Function In Complex I With Decreased Free Radical Production

Mitochondrial respiratory complexes are damaged by ischemia reperfusion injury. Oxygen free radicals (OFR) may be involved in the cardioprotective mechanism of ischemic preconditioning (IPC). The purpose of this study was to determine the effects of IPC on mitochondrial respiratory complex I activity and OFR production.Rat hearts (n=6/group) were subjected to either A) 30 minutes (min) of equilibration (EQ), 30 min of ischemia (I), and 30 min of reperfusion (R) (CONTROL) or B) 10 min of EQ, three 5 min episodes of IPC, 30 min I, and 30 min of R (IPC group). A third set of non‐ischemic control hearts were also studied (NIC). Mitochondria were isolated at end reperfusion. Respiratory complex I activity was assessed by polarography using specific substrates and inhibitors for complex I in the presence of ADP. Complex I OFR production was measured by Electron Paramagnetic Resonance (EPR) spectrometry. Complex I substrates were glutamate and malate (4.7 mM) and the inhibitor was rotenone (188 nM). Data is expressed as mean±SEM.Ischemia reperfusion impairs mitochondrial complex I activity and increases complex I OFR production. IPC preserves mitochondrial respiratory complex I activity and decreases mitochondrial free radical generation at end reperfusion.

Expression of vascular endothelial growth factor and receptor tyrosine kinases in cardiac ischemia/reperfusion injury

Vascular endothelial growth factor (VEGF) expression is regulated by hypoxia and cytokines, including insulin-like growth factor (IGF)-1. We examined the influence of ischemia/reperfusion (I/R) on IGF-1, VEGF, fetal liver kinase (flk-1), fms-like tyrosine kinase-1 (flt-1), and laminin using an isolated hemoperfused working porcine heart model of acute ischemia (2 h) and reperfusion (4 h). Twenty-three porcine hearts were randomized into the following groups: five nonischemic control hearts (Group C), five I/R hearts with occlusion of the ramus circumflexus; three I/R hearts treated with quinaprilat, a potent angiotensin-converting enzyme (ACE) inhibitor (Group Q); five I/R hearts treated with angiotensin I (Group Ang I), and 5 I/R hearts treated with Ang I and quinaprilat (Group QA). Compared to C, VEGF mRNA and protein contents were significantly increased in I/R and Ang I hearts. flk-1 and flt-1 were increased in I/R (2.2-/1.95-fold) and further elevated by Ang I (3.2-/3.4-fold) compared with C. Quinaprilat application attenuated the amount of VEGF significantly and of flk-1 slightly but not that of flt-1. In contrast, IGF-1 and IGF-1 receptor (IGF-1R) proteins were elevated in I/R hearts (3-/1.4-fold vs. C) and further increased in the presence of Q. These findings were accompanied by an elevation of laminin mRNA and protein levels. Moreover, we observed an increase in collagen Type IV and chondroitin sulfate content in I/R (2.9-/1.4-fold) and Ang I (3.5-/1.5-fold) hearts. Quinaprilat significantly reduced laminin and chondroitin sulfate proteins. These data suggest that the VEGF/VEGF receptor and IGF-1-IGF-1R systems are activated by I/R. The benefits of ACE inhibition in attenuation of cardiac remodeling may be mediated by IGF-1.

Regulation of ventricular fibrillation by heme oxygenase in ischemic/reperfused hearts.

We have assessed the relationship between reperfusion-induced ventricular fibrillation (VF) and heme oxygenase (HO) mRNA expression using northern blotting, reverse transcription-polymerase chain reaction (RT-PCR), and enzyme activity in isolated working ischemic/reperfused rat hearts. Isolated hearts were subjected to 30 min of global ischemia followed by 120 min of reperfusion. Upon reperfusion with VF, cardiac function was registered (n = 6 in each group), and HO mRNAs and enzyme activities were measured at the end of reperfusion in hearts that showed VF or did not develop VF. The expression of HO-1 mRNA (about fourfold) was observed in ischemic/reperfused nonfibrillated myocardium in comparison with the nonischemic control hearts. In those hearts when VF was developed, the expression of HO-1 mRNA was not observed in comparison with the nonischemic control myocardium. The results measured by RT-PCR and enzyme analysis support the data obtained by northern blotting. In additional studies, we decided to approach the question from a different angle. Thus, the purpose of our work was also to study the role of HO expression and enzyme activity in electrically fibrillated hearts without the ischemic/reperfused protocol. To simulate the period of 10 min of reperfusion-induced VF, hearts were electrically fibrillated, then defibrillated, and perfused for an additional 110 min, and HO-1 mRNA expression and enzyme activities were determined. Thus, electrically induced VF resulted in about 60%, 60%, and 70% reduction in HO-1 mRNA expression, RT-PCR signal intensity, and enzyme activity, respectively, compared with the nonfibrillated ischemic/reperfused group. In conclusion, our data provide evidence that the development of reperfusion-induced VF inhibits HO-1 mRNA expression and enzyme activity in both electrically fibrillated myocardium and ischemic/reperfused fibrillated hearts. The results clearly show that HO-1 mRNA expression and enzyme activity were increased in ischemic/reperfused nonfibrillated myocardium, suggesting that interventions that are able to increase HO-1 mRNA expression and enzyme activity may prevent the development of VF.

Measurement of 1,2-diacylglycerol and ceramide in hearts subjected to ischemic preconditioning

An accumulation of recent evidence suggests that the mechanism in ischemic preconditioning (IPC) may involve the activation of protein kinase C (PKC) regulatory pathway. In this study, we examined whether the content of 1,2-diacylglycerol (1,2-DAG) and ceramide, which are intracellular second messengers regulating PKC activity, change during IPC in isolated perfused rat hearts, and whether the observed change in 1,2-DAG is accompanied with alteration in its fatty acid composition. Hearts subjected to IPC, consisting of 5-min transient global ischemia followed by 5-min reperfusion, presented a significant functional recovery during subsequent 40-min reperfusion following 40-min global ischemia compared with non-preconditioned hearts. An increase in 1,2-DAG content was observed in hearts subjected to 5-min transient ischemia compared with non-ischemic control hearts, however this was not seen in hearts harvested after 5-min reperfusion following 5-min ischemia. While fatty acid composition in 1,2-DAG was virtually unchanged in hearts subjected to 5-min ischemia, saturated 1,2-DAG decreased and monounsaturated/polyunsaturated 1,2-DAG increased in hearts reperfused for 5-min following 5-min ischemia compared with the non-ischemic control hearts. Ceramide mass did not change significantly, suggesting that the contribution of ceramide may be small in IPC. These data are in concert with the hypothesis that 1,2-DAG is a second messenger in IPC and the changes in fatty acid composition of 1,2-DAG may add new insight concerning signal transduction pathway in IPC.

Trace Amounts of Albumin Protect Against Ischemia and Reperfusion Injury in Isolated Rat Hearts

Albumin is used to provide colloid osmotic pressure in some resuscitation and organ preservation protocols. These solutions are expensive and carry the risks of using high concentrations of blood products. Used as a carrier of drugs and substrates, the concentration of albumin present in perfusates may be considerably lower in experimental ischemia. The present study examined if trace amounts of albumin (0.0004%) reduce injury from ischemia and reperfusion in isolated rat hearts. Hearts were perfused by the Langendorff technique (60 mmHg) with an intraventricular balloon. Zero-flow ischemia (20 min, 37°C) was followed by reperfusion (35 min, 37°C). Recovery of contractile function during reperfusion was significantly improved by the presence of fatty acid-free bovine serum albumin (BSA) (22 290±1280 mmHg/min, pressure–rate product) or rat serum albumin (RSA) (21 095±2836 mmHg/min) compared with Krebs–Henseleit buffer with no albumin (KHB) (9660±2324 mmHg/min). Release of lactate dehydrogenase activity, formation of tissue edema and accumulation of tissue malonyldialdehyde were significantly reduced in hearts receiving BSA or RSA compared with KHB alone. These parameters were not altered by the presence of albumin in non-ischemic control hearts or in the pre-ischemic values of the hearts subjected to ischemia and reperfusion. Development of ischemic contracture with an extended period of ischemia (27 min) was not altered by the presence of BSA, suggesting that protection observed with albumin occurred during reperfusion, rather than during ischemia. Reperfusion following 45 min of ischemia with bovine serum albumin resulted in similar myocardial injury to hearts that were reperfused following 20 min of ischemia without bovine serum albumin. Thus, trace amounts of albumin provide significant reduction in myocardial injury from ishemia and reperfusion, probably via antioxidant mechanisms.

Fucoidin reduces coronary microvascular leukocyte accumulation early in reperfusion

Background. Leukocytes rapidly accumulate in the heart early in reperfusion after ischemia, contributing to reperfusion injury. The purpose of this study was to determine whether treatment with the selectin blocker fucoidin (FCN) would attenuate early leukocyte retention in coronary venules and capillaries during low flow reperfusion.Methods. Isolated rat hearts subjected to 30 minutes of 37°C, no-flow ischemia were initially reperfused with blood containing labeled leukocytes, followed by reperfusion with a Krebs red cell solution. The deposition of leukocytes in coronary capillaries and venules was observed using intravital microscopy. Three groups were studied: nonischemic control hearts, untreated postischemic hearts reperfused at low flow, and postischemic hearts reperfused at low flow, where both the hearts and the blood reperfusate were pretreated with FCN (0.36 mg/mL blood).Results. In the ischemia-reperfusion group, we observed a rapid and significant increase in leukocyte accumulation in both capillaries and venules. Treatment with FCN significantly reduced the leukocyte accumulation in both capillaries and venules (p < 0.05). In addition, FCN significantly reduced the persistence of leukostasis in both capillaries and venules, indicating that FCN affected a transient adhesion process.Conclusions. These results suggest that the selectin family of leukocyte–endothelial cell adhesion proteins mediates the initial retention of leukocytes in both coronary capillaries and venules during reperfusion. Selectin blockade may be effective in reducing the contribution of leukocytes to early reperfusion injury.

Effects of dichloroacetate on mechanical recovery and oxidation of physiologic substrates after ischemia and reperfusion in the isolated heart.

The effects of dichloroacetate (DCA) on fatty acid oxidation and flux through pyruvate dehydrogenase (PDH) were studied in ischemic, reperfused myocardium supplied with glucose, long-chain fatty acids, lactate, pyruvate, and acetoacetate. The oxidation rates of all substrates were determined by combined 13C nuclear magnetic resonance (NMR) spectroscopy and oxygen-consumption measurements, and PDH flux was assessed by lactate plus pyruvate oxidation. In nonischemic control hearts, DCA increased PDH flux more than eightfold (from 0.68 +/- 0.28 to 5.81 +/- 1.16 micromol/min/g dry weight; n = 8 each group; p < 0.05) and significantly inhibited the oxidation of acetoacetate and fatty acids. DCA also improved mechanical recovery after 30 min of ischemia plus 30 min of reperfusion but did not significantly increase PDH flux measured at the end of the reperfusion period (1.35 +/- 0.42 micromol/min/g dry weight) compared with untreated ischemic hearts (0.87 +/- 0.28 micromol/min/g dry weight; n = 8 each group; p = NS). Although DCA had a modest effect on functional recovery in the reperfused myocardium, this beneficial effect was not associated with either marked stimulation of PDH flux or inhibition of fatty acid oxidation.

Breakdown and release of myofilament proteins during ischemia and ischemia/reperfusion in rat hearts: identification of degradation products and effects on the pCa-force relation.

Our objective in experiments reported here was to identify myofilament proteins of rat hearts either lost or degraded by cardiac ischemia (15- or 60-minute duration) with and without 45 minutes of reperfusion. We correlated these changes with alterations in myofilament sensitivity to Ca2+ and maximum force generation. Protein degradation and loss were assessed by high-performance liquid chromatography, SDS-PAGE, Western blotting analysis, and amino acid sequencing. Compared with nonischemic control hearts, bundles of skinned fibers from hearts subjected to ischemia alone demonstrated a decrease in maximum force generation and an increase in sensitivity to Ca2+. These changes in function were increased with the duration of the ischemia and with reperfusion. With increasing duration of ischemia, there was an increased loss and degradation of myofibrillar alpha-actinin and troponin I (TnI) at its C-terminus. Alpha-actinin and TnI were most susceptible to ischemia, but with 60 minutes of ischemia/reperfusion, there was also degradation of myosin light chain-1 (MLC1) involving a clip of residues 1 to 19. The MLC1 degradation product was detected in the reperfusion effluent (along with troponin T, tropomyosin, and alpha-actinin) but not in the tissue with 60 minutes of ischemia with no reperfusion. Moreover, with ischemia the following proteins became associated with the myofibrils: GAPDH and proteins of the mitochondrial ATP synthase complex. Our results provide new evidence regarding the mechanism by which ischemia/reperfusion causes myocardial injury and support the hypothesis that an important element in the injury is altered activity and structure of the myofilaments.

Low-flow reperfusion after myocardial ischemia enhances leukocyte accumulation in coronary microcirculation.

During early reperfusion after myocardial ischemia, the mechanisms responsible for leukocyte accumulation in the heart are unclear. We examined the effects of reducing coronary blood flow during reperfusion on leukocyte accumulation in coronary capillaries and postcapillary venules. Isolated rat hearts were perfused for 30 min and then subjected to 30 min of 37 degrees C, no-flow ischemia. The deposition of fluorescently labeled leukocytes was observed directly in coronary capillaries and venules using intravital microscopy after 5, 20, and 35 min of reperfusion. Blood cell velocity was measured in venules after 5 min of reperfusion (R5), and shear rate (s-1) was calculated. Four groups were studied: nonischemic control (NIC) hearts and postischemic hearts reperfused at full flow (I/R100) and at 50 and 10% of full flow (I/R50 and I/R10, respectively). In I/R100 hearts, there was a significant increase in leukocyte trapping in capillaries compared with the NIC group (R5: 5.7 +/- 0.6 vs. 2.0 +/- 0.4 leukocytes/capillary field, respectively; P < 0.05). However, the increase in leukocyte adhesion to venules was not statistically significant compared with NIC (R5: 3.2 +/- 0.4 vs. 1.5 +/- 0.6 leukocytes/100-micron venule, respectively; P < 0.2). In I/R50 hearts, a further increase in leukocyte accumulation occurred in the capillaries but not in the venules. However, in I/R10 hearts, there was a statistically significant increase in both capillaries (R5: 9.2 +/- 0.8; P < 0.05) and venules (R5: 4.4 +/- 0.5; P < 0.05). When leukocyte margination in coronary venules was examined as a function of venular shear rate, a significant correlation (r = 0.99, P < 0.05) was found. These results suggest that, after ischemia, a reduction in reflow enhances leukocyte trapping in capillaries and that leukocyte adhesion in venules is inversely related to shear rate. Enhanced leukocyte accumulation may in turn increase the leukocyte contribution to early reperfusion injury in the heart.

Increased Sarcolemmal Glucose Transporter Abundance in Myocardial Ischemia

Many clinical and laboratory studies suggest that an increase in glucose uptake and metabolism by ischemic myocardium helps protect myocardial cells from irreversible injury. We have examined whether increased sarcolemmal abundance of cardiomyocyte glucose transporters plays a role in this adaptive response. We have shown that acute myocardial ischemia in perfused rat hearts results in increased sarcolemmal abundance of the major glucose transporter, GLUT4, by causing translocation of GLUT4 molecules from an intracellular compartment to the sarcolemma. In nonischemic control hearts only 18 ± 2.8% of GLUT4 molecules were on the sarcolemma whereas in ischemic hearts this increased to 41 ± 9.3%. Insulin also caused translocation of GLUT4 molecules to the sarcolemma, and resulted in 61 ± 2.6% of GLUT4 molecules on the sarcolemma. The combination of ischemia and insulin did not result in additive increases in sarcolemmal GLUT4 abundance. In more persistent or chronic ischemia, the other major myocardial glucose transporter, GLUT1, appears to play an important role. The mRNA for this transporter, which is constitutively expressed on cardiomyocyte sarcolemma, was increased 2.0-fold in regions of hibernating myocardium in humans with coronary heart disease as well as in persistently hypoxic rat neonatal cardiomyocytes in primary culture. In neither of these conditions was GLUT4 mRNA expression increased. Thus, acute myocardial ischemia increases sarcolemmal glucose transporter abundance mainly by translocating previously synthesized GLUT4 molecules from an intracellular compartment, whereas more chronic ischemia also increases GLUT1 abundance via enhanced mRNA expression. Increased GLUT1 and GLUT4 abundance may participate in the augmented glucose uptake of ischemic myocardium and therefore may help protect ischemic myocardium from irreversible injury.

Restoration of endothelium-dependent vasodilation after reperfusion injury by tetrahydrobiopterin.

A deficit in the endothelial production of nitric oxide (NO) is associated with the sequelae of reperfusion injury. Because endothelial NO synthesis depends on the cofactor tetra-hydrobiopterin (BH4), we hypothesized that depletion of this cofactor underlies the reduction of endothelium-dependent dilation in reperfusion injury. After occlusion of the left anterior descending coronary artery of a pig for 60 minutes followed by 90 minutes of reperfusion (ischemia/reperfusion), hearts were removed and the arterioles were isolated, cannulated, pressurized, and placed on an inverted microscope stage. Dose responses to the endothelium-independent dilator sodium nitroprusside and the endothelium-dependent dilators serotonin, A23187, and substance P were obtained under control conditions, after incubation with sepiapterin (intracellularly converted to BH4) or synthetic BH4 6-methyltetrahydropterin (MH4), and again after their washout. After ischemia/reperfusion, sodium nitroprusside maximally dilated arterioles (99 +/- 3%), whereas relaxation to serotonin, A23187, and substance P was significantly reduced (19 +/- 9%, 44 +/- 9%, and 54 +/- 8%, respectively). During incubation with sepiapterin (1 mumol/L) or MH4 (10 mumol/L), endothelium-dependent dilation was significantly enhanced (P < .05), whereas the response to sodium nitroprusside was unaltered. After washout, the vasodilatory responses were not significantly different from the initial ischemia/reperfusion responses. Sepiapterin and MH4 did not affect vasodilatory responses in vessels obtained from nonischemic control hearts. As after ischemia/reperfusion, incubation of control vessels with 2,4-diamino-6-hydroxypyrimidine, an inhibitor of GTP cyclohydrolase I, decreased endothelium-dependent vasodilation, which was restored in the presence of sepiapterin or MH4. These data indicate that exogenous administration of sepiapterin or MH4 restores the response to endothelium-dependent vasodilators in pig coronary arterioles after ischemia/ reperfusion. We therefore conclude that ischemia/reperfusion alters the availability or production of BH4, which contributes to blunted endothelial nitroxidergic vasodilation.

Inhomogeneous disappearance of myofilament-related cytoskeletal proteins in stunned myocardium of guinea pig.

The decrease in Ca2+ responsiveness of myofilaments in stunned myocardium implies that there may be structural changes in proteins composing the contractile machinery. To elucidate the lesion in stunned myocardium, isolated guinea pig hearts were subjected to global ischemia at 37 degrees C and reperfused. SDS-PAGE revealed that the contents of desmin, alpha-actinin, and spectrin decreased in the myofibrillar fraction isolated from hearts reperfused after 60-minute ischemia compared with nonischemic control hearts. To examine the change of cytoskeletal proteins in stunned myocardium, immunohistochemical studies with antibodies against these proteins were performed after 15 minutes of ischemia. In stunned myocardium, the staining was largely intact, but there were some lesions where desmin was not stained and alpha-actinin and spectrin were only weakly identified. The percentage of normally stained areas in the myocardium (percent stained area), quantified by image processing, was significantly lower in stunned myocardium (79.6 +/- 3.6%, mean +/- SEM) than in nonischemic control myocardium (96.5 +/- 0.7%). Percent recovery of developed pressure significantly correlated with percent stained area (r = .82, P < .001). In hearts subjected to 15-minute ischemia but not reperfused, or in hearts reperfused with Ca(2+)-free solution after 15-minute ischemia, staining by the antibodies remained intact, suggesting that the change of the cytoskeletal proteins is mediated by Ca2+ overload during reperfusion. In hearts treated with the protease inhibitor leupeptin (50 mumol/L) or calpain inhibitor I (100 mumol/L), both developed pressure and staining were well preserved. These results indicate that contractile dysfunction in stunned myocardium has a strong correlation with the disappearance of cytoskeletal proteins that may be mediated by a Ca(2+)-dependent intracellular protease activated during reperfusion. The disruption of cytoskeletal proteins is a possible mechanism for stunning, although it may be a secondary effect of protease activation.

Prolonged neonatal myocardial preservation with a highly buffered low-calcium solution

Optimal methods of myocardial preservation remain controversial in the neonate. This study compared prolonged hypothermic storage of neonatal hearts with modified University of Wisconsin solution (group I) with a solution formulated to promote anaerobic glycolysis by providing proton buffering with histidine (100 mmol/L) and exogenous glucose and insulin (group II). Hearts from piglets aged 3 to 5 days were given a single dose of either solution ( n = 6 each), subjected to 20 hours of global ischemia at 4° C, and reperfused with an erythrocyte-enhanced perfusate in an isovolumic Langendorff preparation. After 1 hour of reperfusion, in comparison with hearts preserved with University of Wisconsin solution, those in group II demonstrated (mean ± standard error of the mean) greater left ventricular developed pressure (101 ± 7 versus 62 ± 9 mm Hg, p < 0.01) and the first derivative of left ventricular pressure (816 ± 23 versus 614 ± 69 mm Hg · sec -1, p < 0.05). Diastolic indices were reduced to a similar degree in the two groups. Myocardial oxygen consumption was significantly greater (38.8 ± 2.4 versus 11.8 ± 2.4 μl oxygen · min · g -1, p < 0.01) in group II hearts. Group I hearts vasoconstricted (6% increase in coronary vascular resistance) in response to an intracoronary infusion of acetylcholine (20 nmol·min -1); in contrast, acetylcholine produced coronary dilation in group II hearts (5% decrease in coronary resistance, p < 0.02) that was similar to that observed in nonischemic control hearts. These results demonstrate enhanced preservation of myocardial contractility, oxidative metabolism, and vascular function in neonatal hearts provided by a solution designed to buffer protons and promote anaerobic glycolysis during ischemia. (J THORAC CARDIOVASC SURG 1994;108:772-9)

Ultrastructural demonstration of peroxidative activity and peroxidation in ischaemic and ischaemic-reperfused rabbit hearts

The aim was to characterise subcellular histochemical evidence of the involvement of peroxidation and peroxidases in myocardial reperfusion injury. The histochemical technique involved the use of 3,3'-diaminobenzidine (DAB), which reacts with peroxides and proteins with peroxidase activity to form an electron dense polymer. Isolated rabbit hearts were perfused (Langendorff method) for 30 min with oxygenated physiological saline solution. Some were subjected to 30 min of normothermic global ischaemia, with or without 30 min reperfusion. Non-ischaemic control hearts were perfused continuously for 90 min. Hearts were fixed with glutaraldehyde and cut into 100-150 microns sections that were incubated for 1 h in buffered DAB (1 mg.ml-1) with or without added KCN or H2O2. They were processed further for transmission electron microscopy. Planimetry was done on micrographs taken from random fields (approximately 500 photos). The total amount of DAB polymer in non-ischaemic control heart sections incubated with DAB alone occupied 1.19(SEM 0.44) micron 2 x 1000 micron-2 total cell area. For ischaemic-nonreperfused hearts, the value was 2.32(0.90) micron 2 x 1000 micron-2 (p = 0.223 v control); DAB occupied 7.49(1.42) micron 2 x 1000 micron-2 in ischaemic-reperfused hearts (p = 0.001 v control). DAB positive staining of mitochondria and lipid droplets, but not of peroxisomes, was significantly increased in reperfused hearts compared with non-ischaemic controls. Reperfusion, but not ischaemia, was associated with increased DAB staining. This suggests a reperfusion induced increase in myocyte peroxidation. Increased staining may be due to the actions of haem proteins with peroxidase activity on peroxidized lipid.

Epinephrine-stimulated contractile and metabolic reserve in postischemic rat myocardium

Recovery of contractile function and of fatty acid oxidation may be delayed in viable postischemic myocardium. To determine whether a metabolic reserve is preserved after reperfusion of reversibly injured myocardium, we studied the effect of epinephrine on myocardial fatty acid oxidation in isolated rat hearts perfused retrogradely with erythrocyte enriched buffer containing albumin 0.4 mM, palmitate 0.4 mM, and glucose 11 mM. Hearts were subjected to 60 min of low-flow ischemia (5% of control flow) followed by 60 min of reperfusion. Five minutes following the onset of reperfusion, developed left ventricular pressure (DLVP) and oxidation of palmitate were reduced to 53% (p less than 0.01) and 46% (p less than 0.01), respectively, of values measured in nonischemic control hearts. Subsequently, DLVP and oxidation of palmitate gradually recovered to 78% (NS) and 91% (NS) by 60 min of reperfusion. Epinephrine 5.10(-1) M elicited an immediate stimulation of both contractile function and palmitate oxidation. Early after reperfusion stimulated DLVP and palmitate oxidation were still lower compared to values measured in control hearts exposed to the same concentration of epinephrine. Later than 15 min after the onset of reperfusion the response of DLVP and of palmitate oxidation to epinephrine no longer differed between control and reperfused hearts. These results indicate that viable postischemic myocardium exhibits a remarkable oxidative metabolic reserve. The observation provides further evidence for the view that impairment of myocardial energy production is not responsible for contractile dysfunction early after reperfusion.

Cerium chloride as a histochemical marker of hydrogen peroxide in reperfused ischemic hearts

Hydrogen peroxide (H 2O 2) has been implicated in cardiac damage due to ischemia and reperfusion. We adapted an electron microscopic, histochemical method for demonstrating H 2O 2 produced by isolated cells to isolated, buffer-perfused rabbit hearts. The method involves formation of an electron-dense precipitate when H 2O 2 reacts with cerium chloride (CeCl 3). We perfused hearts retrograde via the aorta with well-oxygenated bicarbonate-buffered solution, followed by one in which bicarbonate was replaced with imidazole (IPSS) to prevent precipitation of bicarbonate and CeCl 3. Some hearts were made globally ischemic (30 min, 37°C), reperfused 5 min with well-oxygenated IPSS containing 1 m m CeCl 3, then processed for electron microscopy. Others were perfused with IPSS containing catalase (300 U/ml) or albumin before ischemia and upon reperfusion, followed by CeCl 3 administration. Nonischemic control hearts perfused with IPSS (± catalase) were also studied. Electron micrographs were assessed visually and by computer for precipitate localization and amount. There was abundant precipitate on the luminal face of the coronary vascular endothelium in ischemic-reperfused, cerium-treated hearts, including those treated with albumin. There was significantly less in reperfused catalase-treated or nonischemic control hearts. X-ray microbeam analysis of the endothelial precipitate indicated the presence of Ce. This appears to be the first visual demonstration of a CeCl 3-H 2O 2-dependent reaction product in intact isolated ischemic hearts. The data indicate that at the time of reperfusion some H 2O 2 is accessible to the vascular space, and that its amount can be reduced by perfused catalase. Further modifications this technique may be useful for assessing the sites and pathways by which H 2O 2 is generated by hearts or other buffer-perfused organs subjected to stresses such as ischemia or hypoxia.

The effect of reserpine and guanethidine on carbohydrate metabolism in ischaemic rat myocardium

We have investigated the role of endogenous catecholamines in myocardial carbohydrate metabolism in isolated, perfused rat hearts after left coronary artery occlusion for 30 min. A significant decrease in ATP and glycogen, and an increase in glucose-6-phosphate (G-6-P) content in the ischaemic myocardium was obtained. After depletion of the cardiac noradrenaline stores by reserpine or guanethidine pretreatment the increase in the G-6-P levels was very markedly enhanced, and the myocardial glycogen content of non-ischaemic control hearts was significantly increased by reserpine. However, the amount of glycogen broken down during the ischaemia in pretreated animals was similar to that in the ischaemic myocardium from control animals, and the decrease in the myocardial ATP was not altered by reserpine or guanethidine. Thus the well known release of noradrenaline during myocardial ischaemia is not an essential prerequisite for the activation of the ischaemic breakdown of glycogen. Rather, it is of importance for later steps in anaerobic carbohydrate metabolism, probably for the activation of phosphofructokinase, as suggested by the large ischaemic accumulation of G-6-P in noradrenaline depleted hearts.

Platelet induced aggravation of acute ischaemia in an isolated rabbit heart model

In isolated coronary ligated rabbit hearts the effects of washed human platelets on the size of the epicardial ischaemic area and on recovery from ischaemia during 60 min of reperfusion was evaluated by endogenous NADH-surface-fluorescence photography. Infusion of washed human platelets to non-ischaemic control hearts produced a decrease in left ventricular pressure to 64(3)% of control (n = 4), no change in global coronary flow rate, no retention of platelets (recovery 108(9)%), and no alteration in basal NADH-fluorescence. When platelets were infused into coronary ligated hearts, however, the size of the epicardial ischaemic area was significantly enhanced to 127(8)% (n = 5) of control. Moreover, this increase in size was negatively correlated with the recovery of platelets in the coronary effluent: the lower the recovery rate the larger the ischaemic area (y = 253-1.44x, r = -0.855, n = 15, p less than 0.001). When platelet infusion was stopped after 30 min the enlargement of the ischaemic area seemed to be reversible. When the coronary ligature was released, however, and the ischaemic myocardium reperfused the recovery from ischaemia was retarded in the platelet treated hearts as shown by functional (left ventricular pressure and flow) and metabolic (NADH-fluorescence) indices. Thus it is directly shown for the first time in an isolated in vitro heart preparation that platelets aggravate myocardial ischaemia.