Postischemic Myocardium Research Articles

Induction of nuclear factor κB and activation protein 1 in postischemic myocardium

Ischemia/reperfusion induces nuclear factor κB (NF-κB) and AP-1 in rat hearts after 15 min of ischemia followed by reperfusion (R) for various periods of time (15 and 30 min, 1, 2, 3, 6, 12, and 24 h). Low levels of NF-κB and no signal for AP-1 were detected in shams and in non-ischemic tissue distant from the ischemic zone. In postischemic tissue, NF-κB levels increased biphasically with peak levels at 15 min and again at 3 h R. Immunoblotting showed minimal NF-κB p50 subunit at all times, with changes in p65 similar to EMSA results. Northern blots showed low p50 and increased p65 expression levels at both 2 and 3 h R. By contrast, AP-1 increased monophasically, with peak levels at 15 min R, which dropped steadily thereafter. These results indicate that NF-κB and AP-1 are differentially regulated during reperfusion, which may be a control mechanism for gene expression in reperfused myocardium.

The redox state of coenzyme Q 10 in mitochondrial respiratory chain and oxygen-derived free radical generation in cardiac cells

The aim of this study was to relate changes in the redox state of mitocondrial electron carriers to the ‘burst’ of oxyradicals in postischemic myocardium. The free radical EPR signals of control and re-oxygenated rat hearts were mainly due to coenzyme Q 10, the line width was 0.81 ± 0.02 mT, and the intensities (1.58±0.12) × 10 16 and (1.41±0.13) × 10 16 spins/g. The low-temperature spectra of oxygenated myocardium contained a predominant signal from a S3 FeS center and weak signals from N1b, N2, N3, N4 and S1 centers. Global ischemia caused cardinal changes in the redox state of the mitochondrial respiratory chain. The low-temperature EPR spectrum now contained intensive signals from most FeS centers. The amount of coenzyme Q 10 semiquinones decreased during global ischemia, but the content of flavosemiquinones increased. The line width of the signal of the ischemic heart was 1.28±0.03 mT, and its intensity corresponded (3.16±0.94) × 10 16 spins/g. The spin-trapping experiments with TEMPONE-H showed that the rate of oxyradical generation in isolated cardiomyocytes essentially increased after hypoxia or on adding rotenone and antimycin A. It became equal to 4.2±0.3, 8.2±0.6 and 7.1±0.5 nmol/min mg −1 mitochondrial protein, respectively. The maximal stimulatory effect was observed in the presence of both inhibitors. The addition of superoxide dismutase, but not catalase, suppressed the formation of oxyradicals.

Inhibition of glyceraldehyde-3-phosphate dehydrogenase in post-ischaemic myocardium.

Myocardial reperfusion following brief period of ischaemic is associated with prolonged, reversible periods of metabolic dysfunction. As the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is inhibited in vitro by reactive oxygen species, we hypothesized that production of reactive oxygen species during reperfusion would lead to inhibition of GAPDH in post-ischaemic myocardium. Anaesthetized closed-chest-dogs were subjected to 20 min balloon occlusion of the left anterior descending coronary artery. Biopsy samples were taken after 3 and 24 h of reperfusion, to determine the activity of GAPDH and the concentrations of glycolytic intermediates in post-ischaemic and remote, non-ischaemic territories. A significant reduction in GAPDH activity was observed in post-ischaemic relative to remote tissue after 3 h reperfusion (4.8 +/- 0.5 vs. 2.9 +/- 0.2 mumol/min/mg protein; P < 0.01). Western blotting revealed no reduction in the levels of GAPDH protein. Analysis of enzyme kinetics showed the loss of activity to be associated with decreased Vmax (5.9 +/- 0.5 vs. 3.2 +/- 0.2 mumol/min/mg protein; P < 0.01) with no significant change in the Km for glyceraldehyde-3-phosphate (GAP). Incubation of the inhibited enzyme under both mild and strong reducing conditions failed to reactivate the enzyme. The acute reduction in enzyme activity in post-ischaemic tissue was accompanied by regional differences in glycolytic intermediates, notably a twofold accumulation of GAP (P < 0.05), and a reduction in the glucose metabolic rate (GMR) determined by positron emission tomography and [18F]2-fluorodeoxyglucose. By 24 h reperfusion, no regional differences in GAPDH activity, reaction Vmax or Km, GAP concentrations or GMR were detectable. These results suggest that inhibition of GAPDH activity may represent an important point at which glycolysis is limited during reperfusion, and further, that the mechanisms of enzyme inhibition do not involve simple oxidation or S-thiolation of critical active site thiol groups.

Thoracic epidural anesthesia improves functional recovery from myocardial stunning in conscious dogs.

The effects of thoracic epidural anesthesia (TEA) on the contractile performance of ischemic and postischemic myocardium have not been well investigated. The purpose of this investigation was to examine the effects of TEA on severity and duration of myocardial stunning in an experimental model for sublethal acute myocardial ischemia. Seven dogs were chronically instrumented for measurement of heart rate (HR), left atrial (LAP), aortic and left ventricular pressure (LVP), LV dP/dtmax' and myocardial wall-thickening fraction (WTF). An occluder around the left anterior descending artery (LAD) allowed induction of reversible LAD ischemia. TEA was performed with lidocaine 4 mg/kg through a chronically implanted epidural catheter at the second thoracic level. Regional myocardial blood flow was determined with colored microspheres. Two experiments were performed in a cross-over design on separate days: Experiment 1, induction of 10 min of LAD ischemia without TEA; and Experiment 2, induction of 10 min of LAD ischemia with TEA. WTF was measured at baseline (BL) and predetermined time points until complete recovery from ischemic dysfunction occurred. LAD ischemia caused a significant decrease of LAD-WTF with (-28% +/- 5.1% versus BL) and without TEA (-15.5% +/- 5.3% versus BL). After 3 h of reperfusion, WTF as percent of BL values was significantly higher with TEA (P < 0.001). BL values of WTF were reached after 24 h with TEA and after more than 48 h without TEA (P < 0.05). There were no significant differences for mean arterial pressure (MAP), heart rate (HR), LVP, LAP, and LVdP/dtmax between the groups during ischemia and reperfusion. In nonischemic myocardium TEA caused an increase of subendocardial blood flow. During ischemia neither the subendocardial/subepicardial nor the occluded/ normal zone blood flow was affected by TEA. TEA attenuates myocardial stunning in conscious dogs. This finding is consistent with data regarding a reduction of infarct size due to TEA.

Antecedent ischemia reverses effects of adenosine on glycolysis and mechanical function of working hearts.

This study compared the effects of adenosine (Ado) on the coupling of glycolysis and glucose oxidation and on mechanical function in normal hearts and in hearts subjected to transient ischemia. Isolated working rat hearts were perfused with Krebs containing 1.2 mM palmitate and 100 microU/ml insulin. After 15 min of aerobic perfusion, hearts underwent either two cycles of 10 min of ischemia and 5 min of reperfusion (stressed) or 30 min of aerobic perfusion (control). After 45 min, hearts underwent either aerobic perfusion for 35 min (series A) or 30 min of ischemia and 30 min of reperfusion (series B). In series A, left ventricular minute work (LV work) was similar in control and stressed hearts and was not affected by Ado (500 microM) or N6-cyclohexyladenosine (CHA 0.5 microM). Ado reduced glycolysis by 49% in control hearts but increased glycolysis by 74% in stressed hearts. CHA inhibited glycolysis in both groups by 50 and 62%, respectively. In series B, LV work during reperfusion recovered to a similar extent in untreated control and stressed hearts. In control hearts, Ado reduced glycolysis by 50% while enhancing LV work to 81% of preischemic values. In stressed hearts, Ado increased glycolysis by 34% and depressed LV work to 9%, whereas CHA inhibited glycolysis by 53% and LV work to 91%. These data indicate that coupling of glycolysis to glucose oxidation is a key determinant of mechanical function of the postischemic myocardium. They also show that the metabolic and protective effects of Ado depend on the status of the heart before sustained ischemia.

Effect of trimetazidine on postischemic regional myocardial stunning in the halothane-anesthetized dog.

Trimetazidine (TMZ) has been described as a new antiischemic agent. Whereas its precise mechanism of action remains unknown, antioxidant properties and the ability to preserve high-energy phosphate metabolism have been reported. Accordingly, we studied whether TMZ may limit postischemic regional myocardial stunning (known to be caused by reactive oxygen species) and influence recruitment of contractile reserve by inotropic stimulation in a dog model, using halothane to maintain steady anesthesia throughout the experiment. Dogs were submitted to a 15-min coronary artery occlusion followed by reperfusion. The blinded protocol included a 3-day oral pretreatment (1 mg/kg/day), a bolus injection (0.5 mg/kg), followed by intravenous infusion (0.5 mg/h) initiated 15 min before coronary artery occlusion. Despite lower heart rate (HR) and significant reduction of lipid peroxidation in treated dogs, myocardial stunning and recruitment of contractile reserve by dobutamine infusion in the postischemic myocardium were not modified by TMZ. Adenine nucleotide pool in the postischemic myocardium was considerably reduced as compared with the nonischemic myocardium in both groups. Therefore, in halothane-anesthetized dogs, the antioxidant properties of TMZ were not sufficient to protect myocardium in terms of postischemic dysfunction after 15-min ischemia.

Carvedilol improves function and reduces infarct size in the feline myocardium by protecting against lethal reperfusion injury

This study examined the effect of carvedilol, a vasodilating β-adrenoceptor antagonist and antioxidant, on lethal reperfusion injury in feline hearts subjected to 40 min of regional ischaemia and 180 min of reperfusion. 30 open chest anaesthetized cats were randomized into three groups. A control group ( n = 10) was compared with a group given carvedilol before coronary artery occlusion ( n = 10) and a group given carvedilol immediately before and during early reperfusion ( n = 10). Regional myocardial function was measured by sonomicrometry. Infarct size was determined by staining the left ventricle with triphenyl tetrazolium chloride. Myocardial blood flow was measured by radiolabeled microspheres. Tissue levels of glutathione were measured after reperfusion. Infarct size was significantly reduced compared to control both when carvedilol was given before ischaemia (0.2 ± 0.1 vs. 17.6 ± 3.6%, P < 0.05) and when given immediately before reperfusion (3.7 ± 1.3 vs. 17.6 ± 3.6%, P < 0.05). Regional shortening improved significantly and the incidence of ventricular fibrillation during early reperfusion was reduced in both groups treated with carvedilol compared to control. Oxidized glutathione did not differ between groups in the post-ischaemic myocardium. This study supports that lethal reperfusion injury is a significant phenomenon. Furthermore, carvedilol reduces infarct size and reperfusion arrhythmias, and improves post-ischaemic regional myocardial function by protecting against both ischaemic and lethal reperfusion injury. The present study does not answer whether it is the non-selective β- or α 1-adrenoceptor antagonism, the antarrhytmic or the antioxidant actions of carvedilol that is responsible for the protective effect.

Cellular response to reperfused oxygen in the postischemic myocardium.

Perfused rat heart experiments focused on determining the critical O2 level in postischemic myocardium. After a 20-min global ischemia, reperfusion began with O2-saturated saline buffer reflowing at different rates (0.5-12 ml/min). The 1H nuclear magnetic resonance (NMR) signal of the Val E11 myoglobin (Mb) gave an index of the intracellular oxygenation, whereas the 31P-NMR spectra reflected the high-energy phosphate and pH status. At the same time, physiological monitors recorded both contractile function and O2 consumption. Biochemical analysis determined the lactate concentration. Within 6-12 min of reperfusion, the O2 reached a new steady state, which depended directly on the flow rate. Below 12 ml/min reflow, the postischemic O2 level was consistently lower than the corresponding control values. Phosphocreatine, P(i), pH, myocardial O2 consumption, and lactate formation rate exhibited a similar linear relationship with MbO2 saturation in both the control and postischemic myocardium. It appears that neither the cellular energy production nor the steep intracellular O2 gradient has changed substantially in the postischemic myocardium.

Myocardial ischemia and in vitro mitochondrial metabolic efficiency.

The purpose of this study was to evaluate the oxidative capacities and the rate of energy synthesis in isolated mitochondria extracted from normal and post-ischemic myocardium. Isolated rat hearts were perfused according to the working mode with a Krebs Heinseleit buffer containing glucose (11 mM), insulin (10 IU/l) and caprylic acid (25 microM). After a 15 min perfusion in normoxic conditions, the hearts were subjected to a 20 min local zero-flow ischemia followed by a 20 min reperfusion. During the perfusion, the aortic and coronary flows, the aortic pressure and the electrocardiogram were monitored. At the end of the reperfusion period, the non-ischemic and ischemic zones (NIZ and IZ, respectively) were separated and the mitochondria were harvested from each zone. The oxygen uptake and the rate of energy production of the NIZ and IZ mitochondria were then assessed with palmitoylcarnitine as substrate in 2 buffers differing in their free calcium concentration (0.041 and 0.150 microM). Ischemia provoked a 50% reduction of coronary and aortic flows. The reperfusion of the IZ allowed the partial recovery of coronary flow, but the aortic flow decreased beneath its ischemic value because of the occurrence of severe arrhythmias, stunning and probably hibernation. The IZ mitochondria displayed a lower rate of oxygen consumption, whatever the buffer free calcium concentration. Conversely, their rate of energy production was increased, indicating that their metabolic efficiency was improved as compared to NIZ mitochondria. This might be due to the mitochondrial calcium overload persisting during reperfusion, to the activation of the inner membrane Na+/Ca2+ exchange and to a significant mitochondrial swelling. On the other hand, the presence of an elevated free calcium concentration in the respiration buffer provoked some energy wasting characterized by a constant AMP production. This was attributed to some accumulation of acetate and the activation of the energy-consuming acetylCoA synthetase. In conclusion, ischemia and reperfusion did not alter the membrane integrity of the mitochondria but improved their metabolic efficiency. Nevertheless, these in vitro results can not reflect the mitochondrial function in the reperfused myocardium. The mitochondrial calcium overload reported to last during reperfusion in the cardiomyocytes might mimic the free calcium-induced reduction of metabolic efficiency observed in vitro in the present study. The resulting energy wasting might be responsible for the contractile abnormalities noticed in the reperfused myocardium.

Inhibition of glycolysis and enhanced mechanical function of working rat hearts as a result of adenosine A1 receptor stimulation during reperfusion following ischaemia.

1. This study examined effects of adenosine and selective adenosine A1 and A2 receptor agonists on glucose metabolism in rat isolated working hearts perfused under aerobic conditions and during reperfusion after 35 min of global no-flow ischaemia. 2. Hearts were perfused with a modified Krebs-Henseleit buffer containing 1.25 mM Ca2+, 11 mM glucose, 1.2 mM palmitate and insulin (100 muu ml-1), and paced at 280 beats min-1. Rates of glycolysis and glucose oxidation were measured from the quantitative production of 3H2O and 14CO2, respectively, from [5-3H/U-14C]-glucose. 3. Under aerobic conditions, adenosine (100 microM) and the adenosine A1 receptor agonist, N6-cyclohexyladenosine (CHA, 0.05 microM), inhibited glycolysis but had no effect on either glucose oxidation or mechanical function (as assessed by heart rate systolic pressure product). The improved coupling of glycolysis to glucose oxidation reduced the calculated rate of proton production from glucose metabolism. The adenosine A1 receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX 0.3 microM) did not alter glycolysis or glucose oxidation per se but completely antagonized the adenosine- and CHA-induced inhibition of glycolysis and proton production. 4. During aerobic reperfusion following ischaemia, CHA (0.05 microM) again inhibited glycolysis and proton production from glucose metabolism and had no effect on glucose oxidation. CHA also significantly enhanced the recovery of mechanical function. In contrast, the selective adenosine A2a receptor agonist, CGS-21680 (1.0 microM), exerted no metabolic or mechanical effects. Similar profiles of action were seen if these agonists were present during ischaemia and throughout reperfusion or when they were present only during reperfusion. 5. DPCPX (0.3 microM), added at reperfusion, antagonized the CHA-induced improvement in mechanical function. It also significantly depressed the recovery of mechanical function per se during reperfusion. Both the metabolic and mechanical effects of adenosine (100 microM) were antagonized by the nonselective A1/A2 antagonist, 8-sulphophenyltheophylline (100 microM). 6. These data demonstrate that inhibition of glycolysis and improved recovery of mechanical function during reperfusion of rat isolated hearts are mediated by an adenosine A1 receptor mechanism. Improved coupling of glycolysis and glucose oxidation during reperfusion may contribute to the enhanced recovery of mechanical function by decreasing proton production from glucose metabolism and the potential for intracellular Ca2+ accumulation, which if not corrected leads to mechanical dysfunction of the postischaemic myocardium.

Cardiopulmonary Support Mitigates the Effects of Experimental Myocardial Ischemia

The effectiveness of cardiopulmonary support (CPS) as a rescue method following failed angioplasty is unknown. The proximal left anterior descending (LAD) was occluded for 20 min in 21 dogs. Group 1 animals (n=15) were given CPS and group 2 animals (n=6) served as controls. During coronary occlusion, animals receiving CPS had increased mean arterial pressure (71+/- 12 vs 58+/-7 mm Hg), decreased left atrial pressure (3+/-3 vs 12+/-3 mm Hg), increased ischemic area blood flow (0.20+/-0.16 vs 0.02+/-0.04 mL/min/g) and myocardial oxygen consumption (0.014+/- 0.008 vs 0.003+/-0.006 mL O2/min/g), decreased remote area myocardial oxygen consumption (0.026+/-0.010 vs 0.091+/-0.047 mL O2/min/g), and an improved myocardial oxygen consumption index (0.60+/-0.33 vs 0.02+/-0.03) when compared with controls (p<0.05). During reperfusion (no CPS), group 1 animals had increased cardiac index (210+/-95 vs 117+/-46 mL/min/kg), renal blood flow (110+/-38% vs 53+/-45%), ischemic area blood flow (1.13+/-0.40 vs 0.58+/-0.27), and myocardial oxygen consumption (0.066+/-0.015 vs 0.032+/-0.018) when compared with controls (p<0.05). CPS improves oxidative metabolism in selective myocardial segments during coronary occlusion, promotes recovery of the postischemic myocardium, and results in improved peripheral circulation.

High energy phosphates in postischemic myocardium with reduced or normalized function

Mitochondrial damage and decreased levels of high energy phosphates are well-known findings in reversibly injured postischemic myocardium (=stunning). Using an isovolumetrically beating isolated rat heart preparation (Langendorff perfusion apparatus) we investigated the effect of postischemic positive inotropic stimulation on the myocardial high energy phosphate content. During postischemic functional steady state with moderately reduced left ventricular developed pressure (118.7 mmHg after 30 minutes of ischemia vs 136.1 mmHg preischemic), dopamine in a dose to reestablish preischemic function was administered for a 20-minute period. Data were compared with postischemic hearts without positive inotropic stimulation: ATP was 2.5 vs 2.5 (μmol/g ww; phosphocreatine was 5.5 vs 6.7 μmol/g ww. These findings indicate that reversibly injured postischemic myocardium can be stimulated for a prolonged period without loss of high-energy phosphates. Therefore, a failure of the energy-generating apparatus is not the reason for the reversible postischemic dysfunction.

Utilization of oxygen by the contractile apparatus is disturbed during reperfusion of post-ischaemic myocardium.

Post-ischaemic ventricular function remains depressed (= myocardial stunning) despite nearly normal coronary blood flow during reperfusion. In order to illuminate the causes of this phenomenon, we studied the relationship between ventricular function and myocardial oxygen consumption (MVO2tot) in experiments on 15 isolated rabbit hearts perfused with erythrocyte suspension (hct = 30%). Left ventricular systolic function was assessed by measuring aortic flow (ml.min-1), peak systolic pressure (LVPmax), dP/dtmax, and early relaxation in terms of dP/dtmin during control and 30 min after the onset of reperfusion, following 20 min global no-flow ischaemia. The pressure-volume area was calculated as a measure of total mechanical energy. The external mechanical efficiency (Eext) was assessed from stroke work and MVO2tot. Both contractile efficiency (Econ = inverse slope of the MVO2-PVA relationship) and MVO2 of the unloaded contracting heart (MVO2unl = basal MVO2 + MVO2 for excitation-contraction coupling) were calculated using pressure-volume area and MVO2tot. At matched heart rate (149 +/- 30 vs 147 +/- 31 min-1; mean +/- SD) and end-diastolic volume (1.3 +/- 0.2 ml), the systolic variables were significantly decreased in the stunned myocardium: aortic flow: 38 +/- 13 vs 9 +/- 11 ml.min-1, LVPmax: 112 +/- 19 vs 74 +/- 18 mmHg, and dP/dtmax: 1475 +/- 400 vs 1075 +/- 275 mmHg.s-1. Likewise, dP/dtmin was significantly impaired (-1275 +/- 250 vs -975 +/- 250). The decrease in pressure-volume area (570 +/- 280 vs 270 +/- 200 mmHg.ml.100 g-1) was not statistically significant. In contrast, both Eext (0.75 +/- 0.29 vs 0.18 +/- 0.26 arbitrary units) and Econ (31 +/- 18 vs 14 +/- 7%) were significantly decreased, whereas MVO2tot (40 +/- 9 vs 34 +/- 8 microliters.beat-1.100 g-1) and MVO2unl (26 +/- 9 vs 22 +/- 6 microliters.beat-1.100 g-1) were not. Ventricular function after brief episodes of ischaemia is decreased whereas MVO2tot is maintained, i.e. external efficiency is decreased. MVO2 for the unloaded contraction remained unchanged, indicating that MVO2 for excitation-contraction coupling is inappropriately high for the depressed contractile state. The decreased contractile efficiency indicates further that O2 utilization of the contractile apparatus is disturbed during reperfusion.

Norepinephrine-induced hypertension following cardiac arrest: Effects on myocardial oxygen use in a swine model: Ann. Emerg. Med. 1994; 24/5: 907–914

Study objective: Recent studies suggest that norepinephrine-induced hypertension early after cardiac arrest ameliorates cerebral hypoperfusion and improves neurologic outcome. The purpose of this study was to evaluate the effects of early norepinephrine-induced hypertension on postresuscitation myocardial blood flow and oxygen use. Design: Prospective, controlled laboratory study. Participants: Ten swine. Interventions: All animals underwent 10 minutes of ventricular fibrillation cardiac arrest followed by 5 minutes of low-flow cardiopulmonary bypass (10 mL/kg•min), norepinephrine (0.12 mg/kg), and defibrillation. Animals then were assigned to a hypertension group (mean aortic pressure, 95 mm Hg) or a control group (mean aortic pressure, 75 mm Hg) by titrating a norepinephrine infusion to attain the prescribed aortic pressure. Results: Myocardial blood flow, perfusion pressure, and oxygen metabolism were compared between groups at different times using analysis of variance with a post-hoc Tukey test. Groups had similar myocardial blood flow during ventricular fibrillation, total defibrillation energy, and time to restoration of spontaneous circulation. Fifteen minutes after restoration of spontaneous circulation, the hypertension group had significantly elevated myocardial blood flow, 965±314 mL/min•100g versus 325±67 mL/min•100g in the control group ( P 2 /min•100g versus 6.4±3.4 mL O 2 /min•100g ( P P Conclusion: In the early resuscitation period, increasing the norepinephrine dose to induced mild hypertension significantly increases oxygen use in the postischemic myocardium.

Blood cardioplegia enhanced with nitric oxide donor SPM-5185 counteracts postischemic endothelial and ventricular dysfunction

This study tested the hypothesis that enhancement of blood cardioplegia with the nitric oxide donor agent SPM-5185 inhibits postischemic left ventricular and coronary endothelial dysfunction. Eighteen anesthetized dogs supported by total vented bypass were subjected to 30 minutes of normothermic ischemia followed by 4° C multidose blood cardioplegia. Hearts received either standard blood cardioplegia (vehicle group; n = 6), blood cardioplegia with 1 μmol/L SPM-5185 (low-dose group; n = 6), or 10 μmol/L SPM-5185 (high-dose group; n = 6). After 60 minutes of cardioplegic arrest, the heart was reperfused for a total of 60 minutes, first in the beating empty state for 30 minutes and then after discontinuation of bypass for 30 minutes. Baseline and postischemic left ventricular function was assessed by the slope of the end-systolic pressure-volume (impedance catheter) relation. Postischemic end-systolic pressure-volume relation was depressed by 53.7% of preischemic values in the vehicle group (from 8.2 ± 1.0 to 3.8 ± 0.3 mm Hg/ml) and by 33.7% (from 9.2 ± 1.1 to 6.1 ± 0.5 mm Hg/ml) in the low-dose group. In contrast, there was complete postischemic functional recovery in the high-dose group (from 7.6 ± 1.1 to 7.2 ± 1.2 mm Hg/ml). In coronary arteries isolated from these hearts, endothelium-dependent maximal relaxation to acetylcholine was impaired by 27% in the vehicle group and by 18% in the low-dose group, whereas the high-dose group showed complete endothelium-dependent relaxation. Myeloperoxidase activity, an index of neutrophil accumulation in postischemic myocardium, was elevated in the vehicle and low-dose groups (3.36 ± 0.58 and 2.56 ± 0.68 U/100 mg tissue) but was significantly reduced in the high-dose group to 1.27 ± 0.45 U/100 mg tissue. We conclude that inclusion of 10 μmol/L nitric oxide donor SPM-5185 in blood cardioplegia improves postischemic ventricular performance and endothelial function in ischemically injured hearts, possibly via inhibition of neutrophil-mediated damage. (J T HORAC C ARDIOVASC S URG 1995;109:1146-54)

Relationship Between Intracellular Calcium and Contractile Force in Stunned Myocardium

To elucidate the abnormalities of excitation-contraction coupling in stunned myocardium, we measured [Ca2+]i and force in thin fura 2-loaded ventricular trabeculae from control or stunned (20 minutes ischemia followed by 20 minutes reflow at 37 degrees C) rat hearts. At any given [Ca2+]o, force development was significantly lower in the stunned trabeculae than in control trabeculae. In contrast, there was no difference in the amplitude of Ca2+ transients between the two groups. The steady state force-[Ca2+]i relationship, assessed by tetanization in the presence of ryanodine, revealed both a decrease in maximal Ca(2+)-activated force and an increase in the [Ca2+]i required for 50% activation in stunned trabeculae. Postischemic myocardium also exhibited an accelerated rate of diastolic relaxation that was not due to changes in the rate of Ca2+ transient decay. Destabilization of attached cross-bridges in a quantitative model of cardiac myofibrils accurately reproduced the salient systolic and diastolic features of the stunned phenotype, suggesting an abnormality of the thin filaments. In response to supraphysiological increases in [Ca2+]o, diastolic [Ca2+]i and diastolic tone increased much more in stunned trabeculae than in controls, with the frequent occurrence of aftercontractions. This novel experimental model lends further support to the hypothesis that the primary lesion of excitation-contraction coupling resides at the level of the contractile proteins. The finding of enhanced susceptibility to calcium overload helps to rationalize the functional deterioration of stunned myocardium during intense inotropic stimulation and additionally suggests that stunned myocardium may represent a favorable substrate for triggered arrhythmias.

Effects of intracoronary calcium chloride on the postischemic heart in pigs

Whether calcium chloride (CaCl2) should be used to reverse myocardial dysfunction during cardiac operations remains a controversial issue. Calcium chloride may reduce, rather than increase, myocardial contractility and may produce exaggerated vasoconstriction in postischemic vessels in which the endothelium has been damaged. These possibilities were investigated in an open-chest porcine model that allowed control of systemic hemodynamics. Incremental doses of CaCl2 (1, 3, and 10 mg/min) were infused directly into a coronary artery before and after 10 or 15 minutes of ischemia followed by 15 minutes of reperfusion. Calcium chloride increased regional contraction, coronary blood flow, and oxygen consumption before ischemia, whereas oxygen and lactate extraction were unchanged. After ischemia and reperfusion, contraction was impaired and lactate extraction was reduced, but a similar response to CaCl2 was observed. Contraction returned to baseline values promptly after CaCl2. Thus, CaCl2 exerts a positive inotropic effect both in normal and in postischemic myocardium. Calcium chloride does not cause direct coronary constriction nor does it worsen myocardial stunning after a short period of normothermic myocardial ischemia.

Pyruvate Dehydrogenase Influences Postischemic Heart Function

The pyruvate dehydrogenase (PDH) enzyme complex determines the extent of carbohydrate oxidation in the myocardium. PDH is in a largely inactive state during early reperfusion of postischemic myocardium. The resultant decrease in pyruvate oxidation in postischemic hearts has been documented with 13C nuclear magnetic resonance (NMR) spectroscopy. This study demonstrates that counteracting depressed pyruvate oxidation can enhance contractile recovery in the absence of increases in either glycolytic activity or glucose oxidation. The findings indicate that increased incorporation of carbon units from pyruvate into the intermediates of the oxidative pathways by PDH influences the metabolic efficiency and mechanical work of postischemic hearts. Isolated rabbit hearts were situated in an NMR magnet and perfused or reperfused (10 minutes of ischemia) with 2.5 mmol/L [3-13C]pyruvate as sole substrate to target PDH directly and bypass the glycolytic pathway. Hearts were observed with or without activation of PDH with dichloroacetate. Mechanical function and oxygen consumption (MVO2) were monitored. 13C and 31P NMR spectroscopy allowed observations of pyruvate oxidation and bioenergetic state in intact, functioning hearts. Metabolite content and 13C enrichment levels were then determined with in vitro NMR spectroscopy and biochemical assay. PDH activation did not affect performance of normal hearts. Postischemic hearts with augmented pyruvate oxidation (dichloroacetate-treated) sustained improved mechanical function throughout 40 minutes of reperfusion. Rate-pressure-product (RPP) increased from 8300 +/- 1800 (mean +/- SEM) in untreated postischemic hearts to 21,300 +/- 2400 in hearts treated with dichloroacetate (P < .05). Oxygen use per unit work [MVO2 multiplied by 10(4) divided by RPP] was improved from 1.50 +/- 0.13 to 1.14 +/- 0.11 (P < .05) without differences in high-energy phosphate content between treated and untreated hearts. Values of dP/dt were also consistently higher, by as much as 185%, during reperfusion with dichloroacetate. Postischemic hearts displayed reduced pyruvate oxidation from the incorporation of 13C into the tissue glutamate pool. With the tissue alanine level as a marker of 13C-enriched pyruvate availability in the cell, the ratio of labeled glutamate to alanine was only 58% of the control value during early reperfusion. With dichloroacetate, that ratio was 167% greater than that of untreated hearts (P < .05). By the end of the reperfusion period, the 13C enrichment of the tissue glutamate pool by pyruvate oxidation was elevated from dichloroacetate treatment (untreated, 62 +/- 7%; DCA-treated, 81 +/- 6%; P < .05), but glycogen content was similar in both groups and 13C enrichment of tissue alanine remained unchanged, near 60%, indicating no increases in glycolytic end-product formation. Metabolic reversal of contractile dysfunction was achieved in isolated hearts by counteracting depressed PDH activity in the postischemic myocardium. Improved cardiac performance did not result from, nor require, increased glycolysis secondary to the activation of PDH. Rather, restoring carbon flux through PDH alone was sufficient to improve mechanical work by postischemic hearts.

Transmural distribution of 2‐deoxyglucose uptake in normal and post‐ischemic canine myocardium

This investigation was performed to determine (i) whether 31P spatially localized 31P NMR spectroscopy could be utilized to determine the transmural distribution of 2-deoxyglucose (2DG) uptake in the in vivo canine heart and (ii) whether transmural 2DG uptake would be affected by a preceding ischemic insult. 2DG was infused and the accumulation of 2-deoxyglucose-6-phosphate (2DGP) was monitored (by means of spatially localized 31P NMR) in control hearts, in pharmacologically hyperperfused hearts, and in hearts subjected to four (5 min) occlusions of the left anterior descending coronary artery. Myocardial blood flow was measured with radioactive microspheres. In control hearts, subendocardial (ENDO) 2DGP contents were significantly higher than those in the subepicardium (EPI) being 3.8 +/- 0.3 and 2.2 +/- 0.2 mumol/g, respectively; the ENDO/EPI ratio of 2DGP was 1.70 +/- 0.21. During hyperperfusion blood flow increased approximately four-fold but 2DGP accumulation was not altered. ATP levels in post-ischemic myocardium were significantly decreased (ENDO more than EPI) and 2DGP accumulation in each layer was increased (p < 0.01 vs control); however, the ENDO/EPI ratio of 2DGP was not altered. 2DG infusion induced a marked elevation of blood insulin and norepinephrine levels. These data demonstrate that in the presence of high blood levels of 2DG and insulin: (i) 2DGP accumulation can be measured in the in vivo canine heart; (ii) in normal hearts 2DG uptake is more pronounced in the inner layers of the left ventricular wall (this transmural 2DG uptake gradient is not due to subendocardial hypoperfusion); and (iii) 2DG uptake is greater in the post-ischemic heart but the ENDO/EPI gradient of 2DG uptake is not altered indicating that the more severe ischemic insult in the subendocardium does not result in a disproportionate increase in 2DG uptake in that region of the myocardium. Although 2DG uptake patterns in this model most probably reflect those of glucose (at comparable glucose and insulin levels), quantitative extrapolations with regard to the rate of glucose uptake are not possible from the present data.

The phosphocreatine overshoot occurs independent of myocardial work

Although the exact mechanism(s) responsible for the phosphocreatine/ATP overshoot have not been completely elucidated, our data demonstrate that the overshoot does not stem from reduced myocardial work, and consequently, reduced utilization of phosphocreatine (PCr). Additionally, we highlight a basic difference in the physiologic responses of skeletal and cardial muscle to work demands. By understanding the bioenergetic derangements which accompany reperfusion injury, one may hope to better salvage post-ischemic myocardium.