External Heart Work Research Articles

Tenascin-C aggravates ventricular dilatation and angiotensin-converting enzyme activity after myocardial infarction in mice.

AimsTenascin‐C (TN‐C) is suggested to be detrimental in cardiac remodelling after myocardial infarction (MI). The aim of this study is to reveal the effects of TN‐C on extracellular matrix organization and its haemodynamic influence in an experimental mouse model of MI and in myocardial cell culture during hypoxic conditions.Methods and resultsMyocardial infarction was induced in TN‐C knockout (TN‐C KO) and wild‐type mice. Six weeks later, cardiac function was studied by magnetic resonance imaging and under isolated working heart conditions. Myocardial mRNA levels and immunoreactivity of TN‐C, TIMP‐1, TIMP‐3, and matrix metalloproteinase (MMP)‐9, as well as serum and tissue activities of angiotensin‐converting enzyme (ACE), were determined at 1 and 6 weeks after infarction. Cardiac output and external heart work were higher, while left ventricular wall stress and collagen expression were decreased (P < 0.05) in TN‐C KO mice as compared with age‐matched controls at 6 weeks after infarction. TIMP‐1 expression was down‐regulated at 1 and 6 weeks, and TIMP‐3 expression was up‐regulated at 1 week (P < 0.01) after infarction in knockout mice. MMP‐9 level was lower in TN‐C KO at 6 weeks after infarction (P < 0.05). TIMP‐3/MMP‐9 ratio was higher in knockout mice at 1 and 6 weeks after infarction (P < 0.01). ACE activity in the myocardial border zone (i.e. between scar and free wall) was significantly lower in knockout than in wild‐type mice 1 week after MI (P < 0.05).ConclusionsTenascin‐C expression is induced by hypoxia in association with ACE activity and MMP‐2 and MMP‐9 elevations, thereby promoting left ventricular dilatation after MI.

St Thomas' Hospital polarizing blood cardioplegia improves hemodynamic recovery in a porcine model of cardiopulmonary bypass

ObjectiveCardiac surgery demands highly effective cardioprotective regimens. We previously demonstrated improved cardioprotection with “polarized” compared with “depolarized” arrest. This study uses a clinically relevant porcine model of cardiopulmonary bypass to compare the efficacy of blood-based St Thomas' Hospital polarizing cardioplegia (STH-Pol-B) with blood-based St Thomas’ Hospital hyperkalemic cardioplegia (STH2-B). MethodsPigs were monitored and subjected to normothermic cardiopulmonary bypass, cardiac arrest via antegrade cold (4°C) blood cardioplegia (STH2-B, control group: n = 6 or STH-Pol-B, study group: n = 7), and global ischemia (60 minutes) followed by on-pump reperfusion (60 minutes) and subsequent off-pump reperfusion (90 minutes). At termination, tissue samples were taken for analysis of high-energy phosphates, ultrastructure, and microRNAs. The primary endpoint of this study was creatine kinase-muscle/brain release during reperfusion. ResultsCreatine kinase-muscle/brain was comparable in both groups. After pigs were weaned from cardiopulmonary bypass, hemodynamic parameters such as mean arterial pressure (P = .007), left ventricular systolic pressure (P < .001), external heart work (P = .012), stroke volume (P = .015), as well as dp/dtmax (P = .027), were improved with polarizing cardioplegia. Wedge pressure was significantly lower in the study group (P < .01). Energy charge was comparable between groups. MicroRNA-708-5p was significantly lower (P = .019) and microRNA-122 expression significantly (P = .046) greater in STH-Pol-B hearts. ConclusionsPolarized cardiac arrest offers similar myocardial protection and enhances functional recovery in a porcine model of cardiopulmonary bypass. Differential expression of microRNAs may indicate possible new ischemia–reperfusion markers. These results confirm the noninferiority and potential of polarized versus depolarized arrest.

A myocardial ischemia- and reperfusion-induced injury is mediated by reactive oxygen species released from blood platelets

In recent experimental studies, blood platelets have been found to exhibit some cardiodepressive effects in ischemic and reperfused guinea pig hearts independent of thrombus formation. These effects seemed to be mediated by reactive oxygen species (ROS). However, the source of these ROS – platelets or heart – remained still unknown. Isolated, buffer-perfused and pressure-volume work performing guinea pig hearts were exposed to a low-flow ischemia (1 ml/min) of 30 min duration and reperfused at a constant flow of 5 ml/min. Human thrombocytes were administered as 1 min bolus (20 000 thrombocytes/µl perfusion buffer) in the 15th min of ischemia or in the 1st or 5th min of reperfusion in the presence of thrombin (0.3 U/ml perfusion buffer). Recovery of external heart work (REHW) was expressed as ratio between postischemic and preischemic EHW in percent. Intracoronary platelet retention (RET) was quantified as percent of platelets applied. In a second set of experiments, thrombocytes were incubated with 10 µM of the irreversible NADPH oxidase blocker diphenyliodonium chloride and washed twice, thereafter, and administered according to the same protocol as described above. Hearts exposed to ischemia and reperfusion in the presence of thrombin but without application of platelets served as controls. Controls without application of platelets did not reveal a severe compromisation of myocardial function (REHW 85.5 ± 1%). However, addition of platelets during ischemia or in the 1st or 5th min of reperfusion led to a significant reduction of REHW as compared with controls (REHW 62.4 ± 6, 53.9 ± 3, 40.5 ± 3, respectively). Application of platelets pretreated with diphenyliodonium chloride did not reveal any cardiodepressive effects being significantly different from controls without platelet application. Moreover, treatment of platelets with diphenyliodonium chloride did not significantly decrease intracoronary platelet retention. In conclusion, these results demonstrate that cardiodepressive effects of human thrombocytes in ischemic and reperfused guinea pig hearts are mediated by ROS released from thrombocytes and not the heart.

New NO Donor S-NO-HSA improves cardioplegic solutions

Introduction: HTK is a commonly used cardioplegia to perform open heart surgery. Aim of this study is to evaluate the myocardial and endothelial protection of its experimental successor HTK-N in a model of an acute damaged rat heart. Subsequently, evaluation of S-NO-HSA addition to the cardioplegic solution will be performed to investigate the protective effects of the NO-Donor. Material and methods: Male Sprague Dawley rats underwent 60' LAD ligature and 120' in vivo reperfusion. Afterwards, they were randomly assigned for evaluation of hemodynamic parameters in an erythrocyte perfused isolated working heart during 45' baseline measurements, 60' of HTK [group 1; n=10], HTK-N [group 2; n=10], or S-NO-HSA enriched HTK-N [group 3; n=8] protected ice cold ischemia and 45' of postischemic reperfusion. Blood gas analyses were performed before and after global ischemia. Results: At similar infarct size, postischemic recovery of hemodynamic parameters showed minor improvements in the HTK-N group whereas it was significantly enhanced in the S-NO-HSA enriched HTK-N group: External Heart Work (HTK: 79±14% vs. HTK-N: 83±9% n.s.; HTK vs. HTK-N + S-NO-HSA: 99±12% p≤0.01), Cardiac Output (HTK: 82±13% vs. HTK-N: 87±9% n.s.; HTK vs. HTK-N + S-NO-HSA: 99±9% p≤0.01). Recovery of Coronary Flow was significantly improved in the HTK-N group (HTK: 83±17% vs. HTK-N: 99±16% p<0.05.), but further improvement was observed after S-NO-HSA addition (HTK vs. HTK-N + S-NO-HSA: 109±22% p≤0.01). Blood gas analyses showed significantly increased recovery of myocardial oxygen delivery (HTK: 81±13% vs. HTK-N: 96±9% p≤0.05; HTK vs. HTK-N + S-NO-HSA: 103±16% p≤0.05) in both groups whereas myocardial oxygen consumption (HTK: 92±7% vs. HTK-N: 97±16% n.s.; HTK vs. HTK-N + S-NO-HSA: 110±16% p≤0.05) was significantly enhanced only in the S-NO-HSA enriched group. Discussion: HTK-N improves myocardial function after protected ex vivo global ischemia compared to its clinical predecessor HTK. Further improvement was observed through S-NO-HSA enrichment. In addition, S-NO-HSA shows signs of enhanced endothelial protection. Hence, S-NO-HSA might be an interesting therapeutic option to further improve cardiac protection for acute damaged hearts.

Protection of the right ventricle from ischemia and reperfusion by preceding hypoxia

We have previously shown that 2 weeks of hypoxia protect the right ventricle of the rat heart from subsequent ischemia and reperfusion (I/R). In the present study, we examined the following: (1) Do shorter periods of hypoxia protect from subsequent I/R? (2) Does intermittent normoxia increase the cardioprotective effect? (3) Is hypoxia-inducible factor-1alpha (HIF-1alpha), erythropoietin (EPO), or vascular endothelial growth factor (VEGF) involved in the protective effects? Preischemic cardiac work was followed by global ischemia, reperfusion, and postischemic cardiac work (15 min each). External heart work was determined at the end of both work phases. Four groups of hearts were investigated: hearts from normoxic rats (n=8), hearts from rats after 24 h of continuous hypoxia (10.5% inspired oxygen, n=7), hearts from rats after 24 h hypoxia with a single intermission of 30 min normoxia (n=9), and hearts from rats after 24 h hypoxia and multiple intermissions of 30 min normoxia (n=7). Protein levels of HIF-1alpha and mRNA levels of EPO and VEGF were determined in right ventricular tissue of normoxic and hypoxic hearts. Postischemic right heart recovery was better in all three hypoxic groups compared with normoxic hearts (61.8 +/- 5.9%, 65.6 +/- 3.0%, and 75.7 +/- 2.6% vs. 46.0 +/- 3.9%, p < 0.01). Hypoxia with multiple normoxic intermissions further improved right heart recovery compared to continuous hypoxia (p < 0.05). HIF-1alpha protein levels were 80.3 +/- 2.5 pg/microg in normoxic hearts and 108.0 +/- 10.3 pg/microg in hypoxic hearts (p = 0.02). No differences in EPO and VEGF mRNA levels were found between normoxic and hypoxic hearts. Twenty-four hours of continuous hypoxia protect the isolated working right heart from subsequent ischemia and reperfusion. When preceding hypoxia is interrupted by multiple reoxygenation periods, there is a further significant increase in cardiac functional recovery. HIF-1alpha may be involved in the protective effect.

The new HTK-N46 cardioplegic solution provides superior myocardial protection during ischemia/reperfusion in failing rat hearts

Aims: Aim of this investigation is the evaluation of HTK-N46, a new cardioplegic solution, in comparison to its precursor Custodiol (HTK) in failing rat hearts. Methods: In male Sprague Dawley rats myocardial infarction (MI) was induced by LAD ligation. Six weeks after MI 14 animals were randomly assigned to two groups, HTK (n=7) and HTK-N46 (n=7), respectively, and evaluated on a red cell perfused isolated working heart during 60 minutes of ischemia and 45 minutes of reperfusion. Hemodynamic parameters (external heart work, EHW; coronary flow, CF) were recorded, biochemical (high energy phosphates, HEP) and histological evaluation performed. Mean±SD. Results: At similar fractional shortening (HTK, 17.65±2.55%; HTK-N46, 19.56±6.59%; ns) postischemic recovery of EHW was significantly improved in the HTK-N46 rats compared to HTK (Fig. 1). Recovery of CF tended to be higher in the HTK-N46 treated group. There was no difference in HEP content. Conclusion: In failing rat hearts, HTK-N46 shows superior cardioprotective properties according to postischemic hemodynamic recovery and biochemical markers compared to its precursor HTK. As the number of patients with reduced left ventricular function undergoing cardiac surgery is increasing, HTK-N46 may be an interesting alternative to currently used cardioplegic solutions in this high risk group of patients.

Attenuation of ischemia reperfusion injury in failing hearts by endothelin-a receptor blockade

Objective: Ischemia/reperfusion (I/R) injury due to cardioplegic arrest is a problem in patients with reduced LV function. We investigated the effect of chronic versus acute administration of the selective endothelin-A receptor antagonist TBC-3214Na during I/R in failing hearts. Methods: Male SD rats underwent coronary ligation. 3 days post infarction group 1 (n=11) was administered TBC-3214Na continuously with their drinking water, group 2 and 3 received placebo. 7 weeks post infarction hearts were evaluated on a blood perfused working heart during 60′ ischemia and 30′ reperfusion. In group 2 (n=10) TBC-3214Na and in group 3 placebo were added to cardioplegia during ischemia. Data: At similar infarct size postischemic recovery of cardiac output (group1: 91±10%, group2: 86±11% vs. placebo: 52±15%; p<0,05) and external heart work (group1: 90±10%, group2: 85±13% vs. placebo: 51±17%, p<0,05) was significantly enhanced in group 1+2 while recovery of coronary flow was only improved in group 2 (group2: 121±23% vs. group1: 75±13%, placebo: 64±15%, p<0,05). In group 2 myocardial oxygen delivery and high energy phosphates were enhanced accompanied by a lower lactate production, and transmission electron microscopy revealed less ultrastructural damage. Conclusion: Ultrastructural and biochemical evaluation indicates an improvement in capillary perfusion only under acute TBC-3214Na administration during ischemia/reperfusion resulting in a better cardiac function post ischemia.

Intracoronary formation and retention of micro aggregates of leukocytes and platelets contribute to postischemic myocardial dysfunction

Cardiac pump function and coronary regulation can be impaired after short-term ischemia. Recent studies with platelets (P) and neutrophils (PMN) yielded contradicting results about the "cellular" contribution to reperfusion injury. Isolated guinea pig hearts performing pressure-volume work were employed, external heart work (EHW), aortic flow (AF), coronary flow (CF) and heart rate (HR) serving as parameters of cardiac function. After global ischemia, human blood cells were given as bolus (1 min) during reperfusion (intracoronary hematocrit 7%). Expression of specific adhesion molecules (P: CD62P, CD41; PMN: integrin CD11b) was measured on cells before and after coronary passage (FACS analysis). Postischemic recovery of pump function was significantly reduced in hearts with blood cell application (EHW: -cells 54 +/- 14%, +cells 41 +/-12%, p <0.05). Coronary response to bradykinin and reactive hyperemia were not effected. The blood-cell dependent functional loss was partly reduced by blocking CD18 (anti-CD 18) and completely abrogated by blockage of CD41 (lamifiban). The expression of CD11b on PMN and monocytes (M) and CD62P on platelets was significantly reduced in the coronary effluent and a significant decrease of CD41 on leukocytes occurred during coronary passage after ischemia. Increases in CD41 on PMN in the presence of lamifiban demasked intracoronary formation of micro aggregates (P/PMN). These micro aggregates were visualized by light microscopy. Electron microscopy revealed no significant microvascular plugging. 1) A specifically blood-cell induced loss of myocardial pump function has been demonstrated after short-term ischemia. 2) CD41 (= GpIIbIIIa) on P is responsible for this cardiac reperfusion damage. 3) The effect is causally linked to the formation of micro aggregates between PMN and P, but seems attenuated in the presence of erythrocytes as compared to effects reported from experiments in which PMN and P were applied singly or co-perfused. 4) Intracoronary retention of PMN, M and platelet-leukocyte micro aggregates seems to be transient, as adherence was not confirmed by electron microscopy.

PMN/Platelets Coinfused in Guinea Pig Hearts Exposed to Low-Flow Ischemia Have no Additive Cardiodepressive Effect

Recent studies revealed an additive cardiodepressive effect of polymorphonuclear granulocytes (PMN) and thrombocytes in hearts exposed to a no-flow ischemia. To find out whether or not this is also true for isolated guinea pig hearts exposed to a low-flow ischemia, the current study was performed. PMN or thrombocytes, together or separately, were applied as a 1-min bolus (1,000/µl or 20,000/µl, respectively) during ischemia or in reperfusion in the presence of thrombin (0.3 U/ml perfusate). Recovery of external heart work and intracoronary cell retention were quantified in percent. Sole application of PMN or platelets during ischemia and reperfusion significantly compromised myocardial function, whereas coapplication of PMN and platelets did not exhibit any further cardiodepressive effect. Coapplication of cells almost prevented intracoronary platelet retention during ischemia and in reperfusion, as opposed to sole platelet application. Known blockers of endogenously released anti-platelet substances like nitric oxide, PGI₂ or adenosine did not mediate a further aggravation of myocardial dysfunction. The platelet-activating factor (PAF) antagonist WEB 2170 BS, however, significantly improved recovery of external heart work during ischemia and in reperfusion. This indicates that an additive cardiodepressive effect of PMN and platelets in working guinea pig hearts exposed to a low-flow ischemia, cannot be demonstrated, whereas PAF antagonists seem to be cardioprotective, under these conditions. Even addition of fibrinogen to the perfusate, did not show an additive cardiodepressive effect of coapplication of PMN and platelets.

Optimizing Ischemia/Reperfusion in the Failing Rat Heart—Improved Myocardial Protection With Acute ACE Inhibition

Whereas the number of patients with reduced left ventricular function after myocardial infarction who need revascularization is increasing, the operative outcome is still inadequate. Consequently, drugs that increase myocardial perfusion and decrease oxygen consumption of the remodeled myocardium are of particular interest to cardiac surgeons. Angiotensin-converting enzyme inhibitors (ACE-I) provide this pharmacologic profile. This study tests the hypothesis whether acute ACE inhibition during cardioplegic arrest improves outcome in failing rat hearts. Male Wistar rats (260+/-15 g) underwent coronary ligation. Ten weeks later the rats had developed heart failure (HF). Hearts were harvested and studied on a red cell-perfused working heart: 60 minutes of ischemia, protected by cold blood cardioplegia (CP) every 20 minutes, and 45 minutes of reperfusion. Rats were randomly assigned to 2 groups, 1 group receiving the ACE-I quinaprilat with CP (QuinaMI, n=11), and 1 group receiving CP only (MI, n=8). Hemodynamic recovery, high-energy phosphates (HEP), and morphometry were analyzed. Groups showed similar degrees of myocardial infarction (44+/-5 versus 39+/-4% of LVmass), LVEDP (5.0+/-1 versus 4+/-1 mm Hg) and no differences in baseline values such as external heart work (EHW) and coronary flow (CF). At the end of reperfusion, EHW and CF were significantly higher in QuinaMI than MI (P<0.05 and 0.01), LVEDP had returned to baseline in QuinaMI (P<0.01). HEP were significantly higher preserved in QuinaMI than MI (P<0.05). Acute ACE inhibition during CP improves postischemic systolic and diastolic function, coronary perfusion as well as HEP-levels in a rat model of HF. These results may have clinical impact on patients with HF undergoing cardiac surgery.

Human thrombocytes are able to induce a myocardial dysfunction in the ischemic and reperfused guinea pig heart mediated by free radicals-role of the GPIIb/IIIa-blocker tirofiban

In recent studies, we could demonstrate a myocardial dysfunction induced by homologous platelets in ischemic and reperfused guinea pig hearts. Aim of the current study was to find out whether or not this is a phenomenon specific for platelets isolated from guinea pigs and to further examine the mechanisms of a possible cardiodepressive effect of human platelets. Isolated guinea pig hearts were exposed to a 30 min low-flow ischemia (1 ml/min) and reperfused. Human thrombocytes were administered as bolus (20.000 thrombocytes/μl perfusion buffer) in the 15 th min of ischemia or in the 1 st or 5 th min of reperfusion in the presence of thrombin. Recovery of external heart work (REHW) and intracoronary platelet retention (RET) were quantified in percent. In additional experiments, the GPIIb/IIIa-blocker tirofiban (10 μg/ml perfusion buffer) or the radical scavenger superoxide dismutase (SOD-10 U/ml perfusion buffer) were added. Platelet application in the absence of tirofiban, either during ischemia (REHW 75.4 ± 4%, RET 22.2 ± 2%) or the 1st min (REHW 71.6 ± 1%, RET 31.2 ± 2%) or the 5th min of reperfusion (REHW 63.2 ± 4%, RET 40.5 ± 1%) led to a significant reduction of REHW and a significant increase of RET. The coapplication of tirofiban, on the other hand, prevented RET at all three times of platelet application (1.1 ± 1.7%, 0% or 2.1 ± 1.2%, respectively). An improvement of REHW, however, could only be noticed during ischemia (89 ± 2%), whereas coapplication of tirofiban in early (72.9 ± 3%) or in late reperfusion (74.6 ± 2%) did not lead to a significant increase of REHW. Coapplication of SOD, on the other hand, significantly improved REHW in early (88.1 ± 1) or late (95.9 ± 1) reperfusion but not during ischemia (83.5 ± 2). Corresponding to REHW, RET was changed significantly by coapplication of SOD during early (1 ± 2%) or late (0%) reperfusion but not during ischemia (21.1 ± 4%). We conclude that human thrombocytes are able to induce a myocardial dysfunction in ischemic and reperfused guinea pig hearts mediated by reactive oxygen species and independent of intracoronary platelet adhesion.

Quinaprilat during cardioplegic arrest in the rabbit to prevent ischemia-reperfusion injury

Objectives: This study evaluated intracardiac angiotensin-converting enzyme inhibition as an adjuvant to cardioplegia and examined its effects on hemodynamic, metabolic, and ultrastructural postischemic outcomes. Methods: The experiments were performed with an isolated, erythrocyte-perfused, rabbit working-heart model. The hearts excised from 29 adult New Zealand White rabbits (2950 ± 200 g) were randomly assigned to four groups. Two groups received quinaprilat (1 μg/mL), initiated either with cardioplegia (n = 7) or during reperfusion (n = 7). The third group received l-arginine (2 mmol/L) initiated with cardioplegia (n = 7). Eight hearts served as a control group. Forty minutes of preischemic perfusion were followed by 60 minutes of hypothermic arrest and 40 minutes of reperfusion. Results: All treatments substantially improved postischemic recovery of external heart work (62% ± 6%, 69% ± 3%, and 64% ± 5% in quinaprilat during cardioplegia, quinaprilat during reperfusion, and l-arginine groups, respectively, vs 35% ± 5% in control group, P <.001) with similarly increased external stroke work and cardiac output. When administered during ischemia, quinaprilat significantly improved recovery of coronary flow (70% ± 8%, P =.028 vs quinaprilat during reperfusion [49% ± 5%] and P =.023 vs control [48% ± 6%]). l-Arginine (55% ± 7%) showed no significant effect. Postischemic myocardial oxygen consumption remained low in treatment groups (4.6 ± 1.2 mL · min−1 · 100 g−1, 6.0 ± 2.2 mL · min−1 · 100 g−1, and 4.7 ± 1.6 mL · min−1 · 100 g−1 in quinaprilat during cardioplegia, quinaprilat during reperfusion, and l-arginine groups, respectively, vs 4.2 ± 0.8 mL · min−1 · 100 g−1 in control group), even though cardiac work was markedly increased. High-energy phosphates, which were consistently elevated in all treatment groups, showed a significant increase in adenosine triphosphate with quinaprilat during ischemia (2.24 ± 0.14 μmol/g vs 1.81 ± 0.12 μmol/g in control group, P =.040). Ultrastructural grading of mitochondrial damage revealed best preservation with quinaprilat during ischemia (100% [no damage], P =.001 vs control). Conclusion: These experimental findings have clinical relevance regarding prevention of postoperative myocardial stunning and low coronary reflow in patients undergoing heart surgery.J Thorac Cardiovasc Surg 2002;124:352-60

Polymorphonuclear granulocytes induce myocardial dysfunction during ischemia and in later reperfusion of hearts exposed to low-flow ischemia

Polymorphonuclear granulocytes (PMNs) are known to contribute to reperfusion injury of the heart. However, whether PMNs compromise myocardial function of hearts exposed to a low-flow ischemia has not been determined. Moreover, not much is known about deleterious effects of PMNs at different times during ischemia and reperfusion. Isolated, working guinea pig hearts were subjected to 30 min of low-flow ischemia and reperfusion. Homologous PMNs were applied as 1-min boluses in the presence of thrombin during either ischemia or the first or fifth minute of reperfusion, and postischemic recovery of external heart work (REHW) and intracoronary PMN retention (PMNR) were quantified. In further experiments, the radical scavenger superoxide dismutase (SOD) was added. Compared with controls without PMNs (REHW, 92.4%), application of PMNs led to a significant loss of myocardial function, which was detected at all three examination times. Moreover, intracoronary PMNR increased significantly in comparison with that of controls with hearts not exposed to ischemia or reperfusion. On the other hand, addition of SOD significantly increased REHW. Intracoronary PMNR was not significantly changed by coapplication of SOD. We conclude that thrombin-stimulated PMNs applied at different times during ischemia and reperfusion significantly impaired cardiac function in hearts exposed to a low-flow ischemia.

Aspirin, but not the more selective cyclooxygenase (COX)-2 inhibitors meloxicam and SC 58125, aggravates postischaemic cardiac dysfunction, independent of COX function

Inhibition of cyclooxygenase (COX) might favour non-enzymatic formation of cardiodepressive isoprostanes from arachidonic acid by radicals generated during reperfusion. This could explain deleterious effects of acetylsalicylic acid (ASA) on cardiac function. We examined the influence of COX inhibition on myocardial function after low-flow ischaemia and reperfusion, employing either ASA (100 micromol/l), the partially selective COX-2 inhibitor meloxicam (0.3 micromol/l and 3.0 micromol/l), or the highly selective COX-2 inhibitor SC 58125 (1.0 micromol/l and 3.0 microgmol/l). Isolated, buffer-perfused guinea pig hearts, performing pressure-volume work before and after consecutive low-flow ischaemia and reperfusion, were used for the study. Measurement of coronary and aortic flow, ejection time and heart rate served to calculate external heart work (EHW), before and after ischaemia. Additionally, release of prostacyclin and thromboxane A2, production of lactate, consumption of pyruvate and tissue concentration of the isoprostane 8-iso-PGF2alpha were measured. ASA significantly reduced recovery of EHW (46+/-18% vs. 82+/-15% for controls), whereas meloxicam and SC 58125 did not (64+/-15% and 74+/-13% recovery, respectively). Paradoxically, ASA increased reactive hyperaemia and consumption of pyruvate in the early reperfusion phase in comparison to all other groups, while lactate production did not differ. Prostacyclin production did not increase during reperfusion and was not significantly different between groups at any time point. In contrast, thromboxane A2 release increased about fivefold in the 2nd min of reperfusion under control conditions and in the presence of SC 58125, but was inhibited by ASA and by meloxicam in both concentrations. Isoprostane content of heart tissue was not detectably influenced under the mild reperfusion conditions used here. We conclude that ASA can aggravate postischaemic cardiac dysfunction, independent of COX inhibition. The deleterious effect in the present model might be due to uncoupling of mitochondrial oxidative phosphorylation rather than to direct effects of reduced eicosanoid release or radical induced formation of isoprostanes.

Effects of endothelium/leukocytes/platelet interaction on myocardial ischemia--reperfusion injury.

Platelet fibrinogen receptor (GPIIb/IIIa) antagonists clinically improve the effectiveness of thrombolysis or PTCA in treatment of acute myocardial infarction. 7E3Fab, the chimeric Fab fragment of a monoclonal GPIIb/IIIa antibody, reduces the incidence of death, reinfarction or restenosis in patients and may improve blood flow and regional wall motion in reperfused myocardium. Besides inhibition of platelet aggregation, 7E3Fab may block fibrinogen bridging between the polymorphonuclear neutrophil (PMN) adhesion molecule MAC-1 and platelet GP IIb/IIIa, thus attenuating interaction of platelets with PMN. Experimentally, the interaction of platelets with PMN exacerbated postischemic myocardial stunning. In our own studies in isolated guinea pig hearts, human PMN, platelets and fibrinogen where simultaneously infused during the initial reperfusion period after 15 min of global ischemia. FACS analysis of cells in the coronary effluant revealed that 7E3Fab reduced platelet GP IIb/IIIa expression to 10% of baseline. PMN-platelet aggregate formation in the coronary effluate was markedly reduced by 7E3Fab, parallel to a decrease of PMN-platelet aggregates found by in situ double fluorescence microscopy in the postischemic coronary vasculature. The inhibition of PMN-platelet aggregate formation by 7E3Fab treatment coincided with a significant improvement of external heart work, which suffered a 50% reduction after ischemia, reperfusion, and exposure to PMN, platelets and fibrinogen. Obviously, application of 7E3Fab inhibits formation and coronary retention of PMN-platelet aggregates in the postischemic, reperfused myocardium. This effect may contribute to the clinically observed beneficial effects of this adjuvant treatment after myocardial ischemia.

C7E3Fab reduces postischemic leukocyte-thrombocyte interaction mediated by fibrinogen. Implications for myocardial reperfusion injury.

Reperfusion injury after coronary occlusion is in part mediated by leukocyte activation and adhesion. Platelets may interact with polymorphonuclear granulocytes (PMNs), causing aggravated reperfusion injury. We studied whether c7E3Fab, a chimeric Fab fragment blocking platelet glycoprotein (GP) IIb/IIIa, decreases PMN-platelet-dependent myocardial dysfunction after ischemia. Isolated guinea pig hearts (n=5 per group) perfused at a constant flow of 5 mL/min were subjected to ischemia (15 minutes, 37 degrees C) and reperfusion. Human PMNs (10x10(6) cells, 3 mL), platelets (400x10(6), 3 mL), and fibrinogen (1 mg/mL) were infused for 3 minutes after 2 minutes of reperfusion, with or without c7E3Fab. Flow cytometry detected GPIIb/IIIa (platelets) and MAC-1 (aMbeta2, PMNs) as well as coaggregates of both in the effluent, whereas double-fluorescence microscopy visualized intracoronary PMN-platelet coaggregates. Postischemic recovery of pressure-volume work (12-cm H(2)O preload and 60-mm Hg afterload) was defined as the ratio of postischemic to preischemic external heart work (mean+/-SEM). c7E3Fab reduced platelet GPIIb/IIIa detection to 10% of controls, blocked a transcoronary MAC-1 increase (+25% without versus -23% with c7E3Fab), and inhibited PMN-platelet coaggregation in the effluent (49+/-12% without versus 17+/-2% with c7E3Fab) as well as in the hearts themselves (5.0+/-0.7/cm(2) without versus 1.2+/-0.3/cm(2) surface area with c7E3Fab). Postischemic recovery of external heart work (83+/-5% in cell-free hearts) declined to 46+/-4% after postischemic PMN-platelet infusion, but not in the presence of c7E3Fab (74+/-11%) or LPM19c (71+/-6%). We conclude that c7E3Fab inhibits formation of PMN-platelet aggregates during myocardial reperfusion, an effect that protects against PMN-platelet-dependent stunning.

β-Agonistic Bronchodilators: Comparison of Dose/Response in Working Rat Hearts

Different beta-agonists are compared with regard to their cardiodepressive side effects. The metaphenolic bronchodilators reproterol, salbutamol, fenoterol, and terbutaline were introduced at a dosage of 0.0005 micromol to a maximum of 10 micromol per gram of heart tissue into the isolated working rat heart under hypoxic conditions, and the response was observed during subsequent reoxygenation. As an index of external heart work, aortic flow was measured. Heart rate, coronary flow, and developed pressure were recorded. At the end of heart perfusion, mitochondria were isolated and analyzed for adenosine triphosphatase activity, adenosine triphosphate (ATP) synthesis, and membrane fluidity. Moreover, intact mitochondria and lipid peroxidation were investigated using a model system. Compared to controls, reproterol gave the most favorable results, with an increase of 25 to 30% of aortic flow during reoxygenation at a concentration of 10 micromol/g heart tissue. In contrast, both fenoterol and salbutamol at a concentration of 1 micromol/g heart tissue decreased aortic flow during reoxygenation, whereas terbutaline had a negative influence on aortic flow at 0.01 to 0.1 micromol/g heart tissue. Mitochondria of these hearts were isolated at the end of the experiment. Mitochondrial ATP synthesis was increased above controls at nearly all concentrations of reproterol. ATP synthesis was decreased at 1 micromol and 10 micromol fenoterol. As little as 0.0005 micromol terbutaline decreased ATP synthesis by 50%. In intact mitochondria, adenosine diphosphate (ADP) to oxygen ratios were found to be increased with terbutaline and fenoterol, indicating ADP consumption by myokinase activation. Lipid peroxidation was increased in a model system between concentrations of 0.002 micromol/mg and 0.04 micromol/mg phosphatidylcholine by fenoterol and terbutaline, whereas a decrease was noted with reproterol. Membrane fluidity was found increased after addition of reproterol, which supports the evidence of efficient ATP synthesis by this compound. Cardiodepressive side effects and greater toxicity of fenoterol and terbutaline were found under the conditions of our experiment. Salbutamol and, in particular, reproterol appear much better tolerated. In addition to partial beta-adrenergic agonism, reproterol may exert an inhibitory influence on adenosine receptor sites and phosphodiesterase, which could result in membrane stabilization by saving cyclic adenosine monophosphate or ATP.

Sevoflurane and isoflurane do not enhance the pre- and postischemic eicosanoid production in guinea pig hearts.

Eicosanoids and volatile anesthetics can influence cardiac reperfusion injury. Accordingly, we analyzed the effects of sevoflurane and isoflurane applied in clinically relevant concentrations on the myocardial production of prostacyclin and thromboxane A2 (TxA2) and on heart function. Isolated guinea pig hearts, perfused with crystalloid buffer, performed pressure-volume work. Between two working phases, hearts were subjected to 15 min of global ischemia followed by reperfusion. The hearts received no anesthetic, 1 minimum alveolar anesthetic concentration (MAC) isoflurane (1.2 vol%), or 0.5 and 1 MAC sevoflurane (1 vol% and 2 vol%), either only preischemically or pre- and postischemically. In additional groups, cyclooxygenase function was examined by an infusion of 1 microM arachidonic acid (AA) in the absence and presence of sevoflurane. The variables measured included the myocardial production of prostacyclin, TxA2 and lactate, consumption of pyruvate, coronary perfusion pressure, and the tissue level of isoprostane 8-iso-PGF2alpha. External heart work, determined pre- and postischemically, served to assess recovery of heart function. Volatile anesthetics had no impact on postischemic recovery of myocardial function (50%-60% recovery), perfusion pressure, lactate production, or isoprostane content. Release of prostacyclin and TxA2 was increased in the early reperfusion phase 5-8- and 2-4-fold, respectively, indicating enhanced AA liberation. Isoflurane and sevoflurane did not augment the eicosanoid release. Only 2 vol% sevoflurane applied during reperfusion prevented the increased eicosanoid formation in this phase. Infusion of AA increased prostacyclin production approximately 200-fold under all conditions, decreased pyruvate consumption irreversibly, and markedly attenuated postischemic heart work (25% recovery). None of these effects were mitigated by 2 vol% sevoflurane. In conclusion, only sevoflurane at 2 vol% attenuated the increased liberation of AA during reperfusion. Decreased eicosanoid formation had no effect on myocardial recovery in our experimental setting while excess AA was deleterious. Because eicosanoids influence intravascular platelet and leukocyte adhesion and activation, sevoflurane may have effects in reperfused tissues beyond those of isoflurane. In an isolated guinea pig heart model, myocardial eicosanoid release was not increased by isoflurane or sevoflurane, either before or after ischemia. Sevoflurane (2 vol%) but not isoflurane attenuated the increased release of eicosanoids during reperfusion.

Reduction of oxygen delivery during post-ischemic reperfusion protects the isolated guinea pig heart

To further characterise the influence of oxygen delivery during early reperfusion (first 5 min) in the isolated guinea pig heart, three modes of coronary reperfusion were chosen, differing with respect to reperfusion flow and arterial PO2. Isolated working guinea pig hearts underwent ischemia and reperfusion (15 min each). Reperfusion was at constant pressure (Group 1, 60 mmHg, n = 7) or at constant flow (Group 2, 5 ml/min, n = 7) with a PO2 of 600 mmHg. Group 3 (n = 8) was reperfused at 5 ml/min with a PO2 of 300 mmHg for 5 min and a PO2 of 600 mmHg thereafter. Lactate release and oxygen consumption were determined during reperfusion. Glutathione release served to assess myocardial oxidative stress. After ischemia, hearts in Group 1 (mean coronary flow 14.4 +/- 1.1 ml/min during the first 5 min of reperfusion) performed external heart work at 31 +/- 2% of the pre-ischemic level. Performance in Group 2 recovered to 50 +/- 3% and in Group 3 to 68 +/- 3%. Myocardial oxygen consumption during early reperfusion (2nd min) was lowest in Group 3 (1.9 micromol/min) and highest in Group 1 (8.3 micromol/min). No difference in lactate release was observed. Release of glutathione during the first 5 min of reperfusion was 43.8 +/- 7.9 nmol in Group 1, but only 3.6 +/- 0.7 in Group 2 (p < 0.05). In isolated guinea pig hearts, controlled oxygen delivery during post-ischemic reperfusion by both, reduction of coronary flow and PO2, improves recovery of pump function. The effect is accompanied by less oxidative stress, as indicated by lowered rates of glutathione release.

Sevoflurane and isoflurane protect the reperfused guinea pig heart by reducing postischemic adhesion of polymorphonuclear neutrophils.

Polymorphonuclear neutrophils (PMNs) contribute to reperfusion injury. Because volatile anesthetics can reduce PMN adhesion in the reperfused, nonworking heart, the authors analyzed whether this action of volatile anesthetics affects cardiac performance after ischemia and reperfusion and further clarified the underlying mechanism. Isolated guinea pig hearts perfused with crystalloid buffer and performing pressure-volume work were used. Hearts were subjected to 15 min global ischemia and 20 min reperfusion. In the intervention groups an intracoronary bolus of 3 x 10(6) PMNs was applied in the second min of reperfusion, either in the absence or presence of 0.5 or 1 minimum alveolar concentration sevoflurane or isoflurane. The number of sequestered PMNs was calculated from the difference between coronary input and output (coronary effluent) of PMNs. Performance of external heart work, determined pre- and postischemically, served as criterion for recovery of myocardial function. Additionally, the expression of the integrin CD11b on the cell surface of PMN was measured before and after coronary passage. Injection of PMN in the reperfusion phase, but not under nonischemic conditions, reduced recovery of external heart work significantly (from 55+/-7% to 19+/-11%). Addition of sevoflurane or isoflurane in concentrations of 0.5 and 1 minimum alveolar concentration to the perfusate reduced postischemic PMN adhesion from 36+/-8% to basal values (20+/-7%) and prevented decline of cardiac function. CD11b expression on PMNs increased significantly during postischemic coronary passage under control conditions. Again, both anesthetics in both concentrations inhibited that activation. Volatile anesthetics reduce PMN adhesion in the reperfused coronary system and thereby preserve cardiac function. Reduced expression of the adhesion molecule CD11b on PMNs in the presence of sevoflurane or isoflurane is, at least in part, responsible for the cardioprotective effect.