A thrombocyte-induced myocardial dysfunction in the ischemic and reperfused guinea pig heart is mediated by reactive oxygen species

In recent investigations, we could demonstrate that thrombocytes are able to contribute to ischemia- and reperfusion-induced injury of the heart. The aim of the current study was to investigate whether reactive oxygen species are responsible for induction of myocardial dysfunction under these conditions. Isolated, perfused, and pressure-volume work–performing guinea pig hearts were exposed to a 30-min low-flow ischemia (1 ml/min) and were reperfused (5 ml/min). Washed, homologous blood platelets were administered as a 1-min bolus (20,000 per microliter of perfusion buffer), either during the 15th minute of ischemia or in the first or fifth minute of reperfusion in the presence of thrombin (0.3 U/ml perfusion buffer)). The radical scavengers superoxide dismutase (SOD; 10 U/ml perfusate) and catalase (30 U/ml perfusate) were added during ischemia or in the first or fifth minute of reperfusion, respectively. Intracoronary platelet retention (in percentage of platelets applied) and recovery of EHW (postischemic EHW in percentage of preischemic EHW) were quantified. Ischemic and reperfused hearts with time-matched application of platelets but without administration of SOD or catalase served as controls. Interestingly, both administration of SOD during ischemia and in reperfusion significantly improved recovery of EHW (88.4 ± 2%, 82.6 ± 1%, and 90 ± 3%, respectively) as compared with the case of controls (56.2 ± 3%, 42 ± 2%, and 75 ± 2%, respectively). Platelet retention, however, was not significantly influenced by administration of SOD during ischemia or reperfusion (26 ± 2%, 31 ± 2%, and 26 ± 2%) compared with controls (30.5 ± 3%, 33 ± 2%, and 22 ± 3%, respectively). Coadministration of catalase, on the other hand, exhibited some cardioprotective potential only in the first minute of reperfusion (recovery, 61% ± 4%) as compared with the case of control (42 ± 2%). We conclude that thrombocytes under conditions of ischemia and reperfusion are able to induce a myocardial dysfunction mediated by reactive oxygen species. Superoxide seems to play a major role in this respect.     

Acetaminophen in the post-ischemia reperfused myocardium

Acetaminophen was administered acutely at the onset of reperfusion after 20 min of low-flow, global myocardial ischemia in isolated, perfused guinea pig hearts (Langendorff) to evaluate its influence in the postischemia, reperfused myocardium. Similarly prepared hearts were treated with vehicle or with uric acid (another phenol for comparison). Functionally, acetaminophen-treated hearts (0.35 mM) achieved significantly greater recovery during reperfusion. For example, left ventricular developed pressures at 40 min reperfusion were 38 +/- 3, 27 +/- 3, and 20 +/- 2 in the presence of acetaminophen (P < 0.05, relative to the other two groups), vehicle, and uric acid, respectively. Coronary perfusion pressures and calculated coronary vascular resistances, in the acetaminophen-treated hearts, were significantly lower at the same time (e.g., coronary perfusion pressures in the three groups, respectively, were 40 +/- 2 [P < 0.05], 51 +/- 3, and 65 +/- 12 mm Hg). Under baseline, control conditions, creatine kinase ranged from 12-15 units/liter in the three groups. It increased to 35-40 units/liter (P < 0.05) during ischemia but was significantly reduced by acetaminophen during reperfusion (e.g., 5.3 +/- 0.8 units/liter at 40 min). Oxidant-mediated chemiluminescence in all three treatment groups during baseline conditions and ischemia was similar (i.e., approximately 1.5-2.0 min for peak luminescence to reach its half maximal value). It took significantly more time during reperfusion for the oxidation of luminol in the presence of acetaminophen (>20 min, P < 0.05) than in its absence (3-8 min in uric acid- and vehicle-treated hearts). These results suggest that administration of acetaminophen (0.35 mM), at the onset of reperfusion, provides anti-oxidant-mediated cardioprotection in the postischemia, reperfused myocardium.     

Mechanical function and fatty acid oxidation in the neonatal pig heart with ischemia and reperfusion

We investigated mechanical function and exogenous fatty acid oxidation in neonatal pig hearts subjected to ischemia, followed by reperfusion. Isolated, isovolumically-beating hearts, from pigs 12 h to 2 days of age, were perfused with an erythrocyte-enriched (hematocrit approximately 15%) solution (37 degrees C). All hearts were studied for 30 min. with a perfusion pressure of 60 mmHg (pre-ischemia). One group of hearts (low-flow ischemia, N = 12) was then perfused for 30 min. with a perfusion pressure of approximately 12 mmHg. In the other group (no-flow ischemic arrest, N = 9), the perfusion pressure was zero for 30 min. Following ischemia in both groups, the perfusion pressure was restored to 60 mmHg for 40 min. (reperfusion). Pre-ischemia parameters for all hearts averaged: left ventricular peak systolic pressure, 99.0 +/- 2.0 mmHg; end diastolic pressure, 1.9 +/- 0.2 mmHg; coronary flow, 3.4 +/- 0.1 ml/min per g; myocardial oxygen consumption, 56.6 +/- 1.6 microliter/min per g and fatty acid oxidation, 33.4 +/- 1.4 nmol/min per g. During low-flow ischemia, hearts released lactate, and the corresponding parameters decreased to: 30.7 +/- 0.9 mmHg; 1.2 +/- 0.3 mmHg; 0.8 +/- 0.1 ml/min per g; 26.6 +/- 2.3 microliters/min per g and 12.9 +/- 1.1 nmol/min per g, respectively. Early in reperfusion in both groups, all parameters, except for fatty acid oxidation, exceeded pre-ischemia values, before recovering to near pre-ischemia values. Late in reperfusion, however, rates of fatty acid oxidation exceeded pre-ischemia rates by approximately 60%. Thus, the neonatal pig heart demonstrated similar recovery following 30 min of low-flow ischemia or no-flow ischemic arrest.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chronically administered acetaminophen and the ischemia/reperfused myocardium

Male and female Hartley strain guinea pigs weighing 280 +/- 10 g were given acetaminophen-treated water ad libitum for 10 days. Sham-treated control animals were given similar quantities of untreated tap water (vehicle-treated control group). On Day 10, hearts were extracted, instrumented, and exposed to an ischemia (low-flow, 20 min)/reperfusion protocol. Our objective was to compare and contrast ventricular function, coronary circulation, and selected biochemical and histological indices in the two treatment groups. Left ventricular developed pressure in the early minutes of reperfusion was significantly greater in the presence of acetaminophen, e.g., at 1 min, 40 +/- 4 vs 21 +/- 3 mmHg (P < 0.05). Coronary perfusion pressure was significantly less from 3 to 40 min of reperfusion in the presence of acetaminophen. Creatine kinase release in vehicle-treated hearts rose from 42 +/- 14 (baseline) to 78 +/- 25 units/liter by the end of ischemia. Corresponding values in acetaminophen-treated hearts were 36 +/- 8 and 44 +/- 14 units/liter. Acetaminophen significantly (P < 0.05) attenuated release of creatine kinase. Chemiluminescence, an indicator of the in vitro production of peroxynitrite via the in vivo release of superoxide and nitric oxide, was also significantly attenuated by acetaminophen. Electron microscopy indicated a well-preserved myofibrillar ultrastructure in the postischemic myocardium of acetaminophen-treated hearts relative to vehicle-treated hearts (e.g., few signs of contraction bands, little or no evidence of swollen mitochondria, and well-defined light and dark bands in sarcomeres with acetaminophen; opposite with vehicle). We conclude that chronic administration of acetaminophen provides cardioprotection to the postischemic, reperfused rodent myocardium.     

Acetaminophen-mediated cardioprotection via inhibition of the mitochondrial permeability transition pore-induced apoptotic pathway

Our laboratory has previously reported that acetaminophen confers functional cardioprotection following cardiac insult, including ischemia/reperfusion, hypoxia/reoxygenation, and exogenous peroxynitrite administration. In the present study, we further examined the mechanism of acetaminophen-mediated cardioprotection following ischemia/reperfusion injury. Langendorff-perfused guinea pig hearts were exposed to acute treatment with acetaminophen (0.35 mM) or vehicle beginning at 15 min of a 30-min baseline stabilization period. Low-flow global myocardial ischemia was subsequently induced for 30 min followed by 60 min of reperfusion. At the completion of reperfusion, hearts were homogenized and separated into cytosolic and mitochondrial fractions. Mitochondrial swelling and mitochondrial cytochromec release were assessed and found to be significantly and completely reduced in acetaminophen- vs. vehicle-treated hearts following reperfusion. In a separate group of hearts, ventricular myocytes were isolated and subjected to fluorescence-activated cell sorting. Acetaminophen-treated hearts showed a significant decrease in late stage apoptotic myocytes compared with vehicle-treated hearts following injury (58 +/- 1 vs. 81 +/- 5%, respectively). These data, together with electron micrograph analysis, suggest that acetaminophen mediates cardioprotection, in part, via inhibition of the mitochondrial permeability transition pore and subsequent apoptotic pathway.     

Effect of various reperfusion regimens on the recovery of myocardial contractile function after total ischemia

Isolated guinea pig hearts were subjected to 25-min total ischemia at 37 degrees C followed by 30-min reperfusion. The product of the left ventricular isovolumic systolic pressure and heart rate representing the cardiac work index was restored to 33 +/- 5% of initial value and diastolic pressure (DP) remained substantially elevated by 47 +/- 9 mm Hg if reperfusion was resumed with initial rate 10 ml/min. The gradual restoration of perfusion rate initiating from 2 or 4 ml/min was performed in other series, and was associated with slower but higher recovery of cardiac work and lower DP by the end of reperfusion. The similar result was observed when reperfusion was resumed with initial rate but a modified solution was used for first 5 min. In which Ca++ content was reduced while K+ and Mg++ elevated. In this case final recovery of cardiac work was 59 +/- 2% and DP completely returned to initial level. It is suggested that optimal reperfusion mode should be associated with slower work recovery.     

Cardiac preconditioning with 4-h, 17 degrees C ischemia reduces [Ca(2+)](i) load and damage in part via K(ATP) channel opening

Brief ischemia before normothermic ischemia protects hearts against reperfusion injury (ischemic preconditioning, IPC), but it is unclear whether it protects against long-term moderate hypothermic ischemia. We explored in isolated guinea pig hearts 1) the influence of two 2-min periods of normothermic ischemia before 4 h, 17 degrees C hypothermic ischemia on cardiac cytosolic [Ca(2+)], mechanical and metabolic function, and infarct size, and 2) the potential role of K(ATP) channels in eliciting cardioprotection. We found that IPC before 4 h moderate hypothermia improved myocardial perfusion, contractility, and relaxation during normothermic reperfusion. Protection was associated with markedly reduced diastolic [Ca(2+)] loading throughout both hypothermic storage and reperfusion. Global infarct size was markedly reduced from 36 +/- 2 (SE)% to 15 +/- 1% with IPC. Bracketing ischemic pulses with 200 microM 5-hydroxydecanoic acid or 10 microM glibenclamide increased infarct size to 28 +/- 3% and 26 +/- 4%, respectively. These results suggest that brief ischemia before long-term hypothermic storage adds to the cardioprotective effects of hypothermia and that this is associated with decreased cytosolic [Ca(2+)] loading and enhanced ATP-sensitive K channel opening.     

Effects of genetic deletion of angiotensin-(1-7) receptor Mas on cardiac function during ischemia/reperfusion in the isolated perfused mouse heart

In this study we investigated the role of Mas on cardiac function during ischemia/reperfusion in isolated perfused mouse heart. Following a stabilization period of 30 min, hearts from WT and Mas KO mice were subjected to global ischemia. After 20 min of ischemia, the flow was restarted and the hearts were reperfused for 30 min. An additional group of WT mice was perfused with solution containing the Ang-(1-7) receptor Mas antagonist A-779. Isolated heart of Mas KO and WT treated with A-779 presented an increase in the perfusion pressure in the baseline period. This difference increased with 5 min of reperfusion reaching similar values to baseline period at the end of the reperfusion. Isolated hearts of Mas KO and WT treated with A-779 also presented a decreased systolic tension, +/-dT/dt, and HR. Upon global ischemia WT hearts showed a significant decrease in systolic tension and an increase in diastolic tension. During reperfusion an increase in systolic and diastolic tension was observed in WT mice. Deletion or blockade of Mas markedly attenuated these changes in isolated hearts. These results indicate that Mas plays an important role in cardiac function during ischemia/reperfusion which is in keeping with the cardiac and coronary effects previously described for Ang-(1-7).     

Myocardial triglyceride turnover during reperfusion of isolated rat hearts subjected to a transient period of global ischemia.

Triglyceride turnover in reperfused/ischemic rat hearts was investigated. Hearts were initially perfused under aerobic conditions for a 1-h "pulse" perfusion with 1.2 mM [1-14C]palmitate to label the endogenous lipid pools, followed by a 30-min period of no-flow ischemia or a 10-min period of retrograde perfusion (control). Hearts were then reperfused under aerobic conditions with buffer containing 1.2 mM [9,10-3H]palmitate. All buffers contained 11 mM glucose and 500 microunits/ml insulin. Rates of endogenous triglyceride lipolysis and synthesis were measured during reperfusion, whereas rates of exogenous palmitate oxidation were measured both prior to ischemia and during reperfusion following ischemia. During reperfusion of ischemic hearts, a 20% increase in exogenous fatty acid oxidation rates was seen compared with pre-ischemic rates. Despite an initial burst of endogenous fatty acid oxidation, no acceleration of steady state endogenous triglyceride lipolysis was seen compared with their nonischemic hearts. In contrast, a significant increase in triglyceride synthesis was observed. Triglyceride turnover was also measured in a series of hearts reperfused following ischemia in the absence of exogenous fatty acids. A significant enhancement of functional recovery was seen compared with hearts reperfused with 1.2 mM palmitate. In addition, a significant increase in fatty acid oxidation from endogenous triglyceride lipolysis was observed. We conclude that the heart quickly recovers its ability to oxidize exogenous fatty acids during reperfusion and that although triglyceride lipolysis is not accelerated during reperfusion of ischemic hearts in the presence of 1.2 mM palmitate, a significant increase in triglyceride synthesis does occur.     

Reversibility of ultrastructural, contractile function and Ca2+ transport changes in guinea pig hearts after global ischemia

To understand the subcellular basis of contractile failure due to ischemia-reperfusion injury, effects of 20, 60, and 90 min of global ischemia followed by 30 min of reperfusion were examined in isolated guinea pig hearts. Cardiac ultrastructure and function as well as Ca2+ transport abilities of both mitochondrial and microsomal fractions were determined in control, ischemic, and reperfused hearts. Hearts were unable to generate any contractile force after 20 min of ischemia and showed a 75% recovery upon reperfusion. However, there were no significant changes in the subcellular Ca2+ transport in the 20-min ischemic or reperfused hearts. When hearts were made ischemic for 60 and 90 min, the recovery of contractile force on reperfusion was 50 and 7%, respectively. There was a progressive decrease in mitochondrial and microsomal Ca2+ binding and uptake activities after 60 and 90 min of ischemia; these changes were evident at various times of incubation period and at different concentrations of Ca2+. Mitochondrial Ca2+ transport changes were only partially reversible upon reperfusion after 60 and 90 min of ischemia, whereas the microsomal Ca2+ binding, uptake and Ca2+ ATPase activities deteriorated further upon reperfusion of the 90-min ischemic hearts. Ultrastructural changes increased with the duration of the ischemic insult and reperfusion injury was extensive in the 90-min ischemic hearts. These data show that the lack of recovery of contractile function upon reperfusion after a prolonged ischemic insult was accompanied by defects in sarcoplasmic reticulum Ca2+ transporting properties and structural damage.     

Effect of gradual reperfusion on the restoration of the pump function and energy stores of the heart after ischemia

The contractile and pump function of guinea pig hearts was evaluated 40 min after total normothermic ischemia and 30 min reperfusion. A half of the hearts was reperfused with rapid mode restoration of the preischemic coronary flow ("sudden" reperfusion, SR), while the other half was reperfused with gradual mode restoration (GR) of coronary flow by 2 ml/min each 4 min till the initial level. The cardiac output and external work after SR constituted 49 and 28 of initial values, while after GR-87 and 71%, respectively. A distinct rise in minimal diastolic left ventricular pressure in the former group indicates a deteriorated filling of the heart. The total pool of adenine nucleotides and ATP content remained almost unchanged after GR: while after SR they decreased to 61 and 54% of the initial level, and myocardial lactate content was three times higher. The beneficial effect of GR seems to be due to better preservation or restoration of ATP content and more profound relaxation of the heart.     

Effects of endotoxin on neutrophil-mediated I/R injury in isolated perfused rat hearts

With the use of a syngeneic model, we demonstrate that rat polymorphonuclear neutrophils (PMNs) exacerbate ischemia-reperfusion injury in the isolated rat heart. However, PMNs (19 x 10(6) cells) from lipopolysaccharide (LPS)-treated rats (LPS-PMNs; 100 mg/kg administered 7 h before exsanguination) induce less reperfusion injury in the isolated heart. Average recovery of left ventricular developed pressure after 20 min of ischemia and 60 min of reperfusion was 51 +/- 4% in hearts receiving PMNs from saline-treated control rats (saline-PMNs) versus 78 +/- 2% in hearts receiving LPS-PMNs. Ischemic hearts reperfused with LPS-PMNs recovered to the same extent as did hearts reperfused with Krebs buffer only. LPS-PMNs and saline-PMNs showed no difference in basal or phorbol ester-induced superoxide production. Whereas twice the number of LPS-PMNs was positive for nitroblue tetrazolium, the percent positive for L-selectin, a receptor integral in PMN-adhesion to endothelium, was 50% less in LPS-PMNs than in controls. After reperfusion, three-fourths of the saline-PMNs remained within the hearts, whereas only one-fourth of LPS-PMNs were trapped. These data suggest that PMNs from LPS-treated rats do not exacerbate ischemia-reperfusion injury as do control PMNs, possibly, due to impaired PMN adhesion to endothelium as a result of decreased L-selectin receptors.     

O2 delivery and redox state are determinants of compartment-specific reactive O2 species in myocardial reperfusion

Reperfusion of the ischemic myocardium leads to a burst of reactive O(2) species (ROS), which is a primary determinant of postischemic myocardial dysfunction. We tested the hypothesis that early O(2) delivery and the cellular redox state modulate the initial myocardial ROS production at reperfusion. Isolated buffer-perfused rat hearts were loaded with the fluorophores dihydrofluorescein or Amplex red to detect intracellular and extracellular ROS formation using surface fluorometry at the left ventricular wall. Hearts were made globally ischemic for 20 min and then reperfused with either 95% or 20% O(2)-saturated perfusate. The same protocol was repeated in hearts loaded with dihydrofluorescein and perfused with either 20 or 5 mM glucose-buffered solution to determine relative changes in NADH and FAD. Myocardial O(2) delivery during the first 5 min of reperfusion was 84.7 +/- 4.2 ml O(2)/min with 20% O(2)-saturated buffer and 354.4 +/- 22.8 ml O(2)/min with 95% O(2) (n = 8/group, P < 0.001). The fluorescein signal (intracellular ROS) was significantly increased in hearts reperfused with 95% O(2) compared with 20% O(2). However, the resorufin signal (extracellular ROS) was significantly increased with 20% O(2) compared with 95% O(2) during reperfusion. Perfusion of hearts with 20 mM glucose reduced the (.)NADH during ischemia (P < 0.001) and the (.)ROS at reperfusion (P < 0.001) compared with 5.5 mM-perfused glucose hearts. In conclusion, initial O(2) delivery to the ischemic myocardium modulates a compartment-specific ROS response at reperfusion such that high O(2) delivery promotes intracellular ROS and low O(2) delivery promotes extracellular ROS. The redox state that develops during ischemia appears to be an important precursor for reperfusion ROS production.     

Coronary and myocardial effects of acetaminophen: protection during ischemia-reperfusion

Acetaminophen is a phenol with antioxidant properties, but little is known about its actions on the mammalian myocardium and coronary circulation. We studied isolated, perfused guinea pig hearts, and tested the hypothesis that acetaminophen-treated hearts would be protected during ischemia-reperfusion. Acetaminophen concentrations in the range of 0.3-0.6 mmol/l caused modest but significant (P < 0.05) coronary vasoconstriction and positive inotropy. The effects were more brisk during constant pressure perfusion than during constant flow. During 20 min of low-flow, global myocardial ischemia and 40 min of reperfusion, hearts treated with acetaminophen retained or recovered a greater percentage of left ventricular function than hearts treated with vehicle. Myofibrillar ultrastructure appeared to be preserved in the reperfused myocardium with acetaminophen. By using chemiluminescence and spin-trap methodologies, we investigated acetaminophen-mediated antioxidant mechanisms to help explain the cardioprotection. The burst of hydroxyl radicals seen between 0 and 10 min of reperfusion was significantly attenuated (P < 0.05) by acetaminophen but not by vehicle. The 3-morpholinosydnominine (SIN-1) generation of peroxynitrite and its oxidative interaction with luminol to produce blue light during ischemia-reperfusion was also blocked by acetaminophen. Our results show that acetaminophen provides significant functional and structural protection to the ischemic-reperfused myocardium, and the mechanism of cardioprotection seems to involve attenuation of the production of both hydroxyl radicals and peroxynitrite.     

VEGFR1 (Flt-1+/-) gene knockout leads to the disruption of VEGF-mediated signaling through the nitric oxide/heme oxygenase pathway in ischemic preconditioned myocardium

This report demonstrates that mice deficient in Flt-1 failed to establish ischemic preconditioning (PC)-mediated cardioprotection in isolated working buffer-perfused ischemic/reperfused (I/R) hearts compared to wild type (WT) subjected to the same PC protocol. WT and Flt-1+/- mice were divided into four groups: (1) WT I/R, (2) WT + PC, (3) Flt-1+/- I/R, and (4) Flt-1+/- + PC. Group 1 and 3 mice were subjected to 30 min of ischemia followed by 2 h of reperfusion and group 2 and 4 mice were subjected to four episodes of 4-min global ischemia followed by 6 min of reperfusion before ischemia/reperfusion. For both wild-type and Flt-1+/- mice, the postischemic functional recovery for the hearts was lower than the baseline, but the recovery for the knockout mice was less compared to the WT mice even in preconditioning. The myocardial infarction and apoptosis were higher in Flt-1+/- compared to wild-type I/R. Flt-1+/- KO mice demonstrated pronounced inhibition of the expression of iNOS, p-AKT & p-eNOS. Significant inhibition of STAT3 & CREB were also observed along with the inhibition of HO-1 mRNA. Results demonstrate that Flt-1+/- mouse hearts are more susceptible to ischemia/reperfusion injury and also document that preconditioning is not as effective as found in WT and therefore suggest the importance of VEGF/Flt-1 signaling in ischemic/reperfused myocardium.     

Different tolerance to hypothermia and rewarming of isolated rat and guinea pig hearts.

We examined the effect of hypothermia and rewarming on myocardial function and calcium control in Langendorff-perfused hearts from rat and guinea pig. Both rat and guinea pig hearts demonstrated a rise in myocardial calcium ([Ca]total) in response to hypothermic perfusion (40 min, 10 degrees C), which was accompanied by an increase in left ventricular end diastolic pressure (LVEDP). The elevation in [Ca]total was severalfold higher in guinea pig than in rat hearts, reaching 12.9 +/- 0.8 and 3.1 +/- 0.6 micromol.g dry wt-1, respectively. The rise in LVEDP, however, was comparable in the two species: 62.5 +/- 2.5 (guinea pig) and 52.5 +/- 5.1 mm Hg (rat). Following rewarming, [Ca]total remained elevated in guinea pig, whereas a moderate decline in [Ca]total was observed in the rat (13.6 +/- 1.9 and 2.2 +/- 0.3 micromol.g dry wt-1, respectively). Posthypothermic values of LVEDP were also significantly higher in guinea pig compared to rat hearts (42.5 +/- 6.8 vs 20.5 +/- 5.1 mm Hg, P < 0.027). Furthermore, whereas rat hearts demonstrated a 78 +/- 7% recovery of left ventricular developed pressure, there was only a 15 +/- 7% recovery in guinea pig hearts. Measurements of tissue levels of high energy phosphates and glycogen utilization indicated a higher metabolic requirement in guinea pig than in rat hearts in order to oppose the hypothermia-induced calcium load. Thus, we conclude that isolated guinea pig hearts are more sensitive to a hypothermic insult than rat hearts.     

Dichloroacetate improves cardiac efficiency after ischemia independent of changes in mitochondrial proton leak

Dichloroacetate (DCA) is a pyruvate dehydrogenase activator that increases cardiac efficiency during reperfusion of ischemic hearts. We determined whether DCA increases efficiency of mitochondrial ATP production by measuring proton leak in mitochondria from isolated working rat hearts subjected to 30 min of ischemia and 60 min of reperfusion. In untreated hearts, cardiac work and efficiency decreased during reperfusion to 26% and 40% of preischemic values, respectively. Membrane potential was significantly lower in mitochondria from reperfused (175.6 +/- 2.2 mV) versus aerobic (185.8 +/- 3.1 mV) hearts. DCA (1 mM added at reperfusion) improved recovery of cardiac work (1.9-fold) and efficiency (1.5-fold) but had no effect on mitochondrial membrane potential (170.6 +/- 2.9 mV). At the maximal attainable membrane potential, O(2) consumption (nmol O(2) x mg(-1) x min(-1)) did not differ between untreated or DCA-treated hearts (128.3 +/- 7.5 and 120.6 +/- 7.6, respectively) but was significantly greater than aerobic hearts (76.6 +/- 7.6). During reperfusion, DCA increased glucose oxidation 2.5-fold and decreased H(+) production from glucose metabolism to 53% of untreated hearts. Because H(+) production decreases cardiac efficiency, we suggest that DCA increases cardiac efficiency during reperfusion of ischemic hearts by increasing the efficiency of ATP use and not by increasing the efficiency of ATP production.     

Ischemic preconditioning and superoxide dismutase protect against endothelial dysfunction and endothelium glycocalyx disruption in the postischemic guinea-pig hearts

The effect of ischemic preconditioning and superoxide dismutase (SOD) on endothelial glycocalyx and endothelium-dependent vasodilation in the postischemic isolated guinea-pig hearts was examined. Seven groups of hearts were used: group 1 underwent sham aerobic perfusion; group 2 was subjected to 40 min global ischemia without reperfusion; group 3, 40 min ischemia followed by 40 min reperfusion; group 4 was preconditioned with three cycles of 5 min global ischemia followed by 5 min of reperfusion (IPC), prior to 40 min ischemia; group 5 was subjected to IPC prior to standard ischemia/reperfusion; group 6 underwent standard ischemia/reperfusion and SOD infusion (150 U/ml) was begun 5 min before 40 min ischemia and continued during the initial 5 min of the reperfusion period; group 7 was subjected to 80 min aerobic perfusion with NO-synthase inhibitor, L-NAME, to produce a model of endothelial dysfunction independent from the ischemia/reperfusion. Coronary flow responses to acetylcholine (ACh) and sodium nitroprusside (SNP) were used as measures of endothelium-dependent and endothelium-independent vascular function, respectively. Reduction in coronary flow caused by NO-synthase inhibitor, L-NAME, served as a measure of a basal endothelium-dependent vasodilator tone. After completion of each experimental protocol, the hearts were stained with ruthenium red or lanthanum chloride for electron microscopy evaluation of the endothelial glycocalyx. While ischemia led only to a slightly flocculent appearance of the glycocalyx, in ischemia/reperfused hearts the glycocalyx was disrupted, suggesting that it is the reperfusion injury which leads to the glycocalyx injury. Moreover, the coronary flow responses to ACh and L-NAME were impaired, while the responses to SNP were unchanged in the ischemia/reperfused hearts. The disruption of the glycocalyx and the deterioration of ACh and L-NAME responses was prevented by IPC. In addition, the alterations in the glycocalyx and the impairment of ACh responses were prevented by SOD. The glycocalyx appeared to be not changed in the hearts subjected to 80 min aerobic perfusion with L-NAME. In conclusion: (1) the impairment of the endothelium-dependent coronary vasodilation is paralleled by the endothelial glycocalyx disruption in the postischemic guinea-pig hearts; (2) both these changes are prevented by SOD, suggesting the role of free radicals in the mechanism of their development; (3) both changes are prevented by IPC. We hypothesize, therefore, that alterations in the glycocalyx contribute to the mechanism of the endothelial dysfunction in the postischemic hearts.     

The delta-opioid receptor agonist DADLE at reperfusion protects the heart through activation of pro-survival kinases via EGF receptor transactivation

The specific delta-opioid receptor agonist [D-Ala(2)-D-Leu(5)]enkephalin (DADLE) protects against infarction in the heart when given before ischemia. In rabbit, this protection leads to phosphorylation of the pro-survival kinases Akt and extracellular signal-regulated kinase (ERK) and is dependent on transactivation of the epidermal growth factor receptor (EGFR). DADLE reportedly protects rat hearts at reperfusion. We therefore tested whether DADLE at reperfusion could protect isolated rabbit hearts subjected to 30 min of regional ischemia and 120 min of reperfusion and whether this protection is dependent on Akt, ERK, and EGFR. DADLE (40 nM) was infused for 1 h starting 5 min before reperfusion and reduced infarct size from 31.0 +/- 2.3% in the control group to 14.6 +/- 1.6% (P = 0.01). This protection was abolished by cotreatment of the metalloproteinase inhibitor (MPI) and the EGFR inhibitor AG1478. In contrast, 20 nM DADLE, although known to be protective before ischemia, failed to protect. Western blotting revealed that DADLE's protection was correlated to increase in phosphorylation of the kinases Akt and ERK1 and -2 in reperfused hearts (2.5 +/- 0.5, 1.6 +/- 0.2, and 2.3 +/- 0.7-fold of baseline levels, P < 0.05 vs. control). The DADLE-dependent increases in Akt and ERK1/2 phosphorylation were abolished by either MPI or AG1478, confirming a signaling through the EGFR pathway. Additionally, DADLE treatment increased phosphorylation of EGFR (1.4 +/- 0.2-fold, P = 0.03 vs. control). Thus the delta-opioid agonist DADLE protects rabbit hearts at reperfusion through activation of the pro-survival kinases Akt and ERK and is dependent on the transactivation of the EGFR.     

缺血预处理合并低温及晶体停搏液对兔未成熟心肌的保护作用

本文拟探讨缺血预处理(ischemic preconditioning,IP)合并低温及晶体停搏液对幼兔的离体心脏是否具有心肌保护作用.采用Langendorff离体心脏灌注模型,灌注液为Krebs-Henseleit液(K-H液).取3～4周龄幼兔心脏,在第一部分实验中分为Con、IP1、IP2、IP3组(n=6),分别给予0、1、2、3次IP,其后各组心脏均在20℃低温下停灌2 h,37℃常温下再灌注30 min.在第二部分实验中分为SConI、SCon2、SCon3、SIPl、SIP2、SIP3组(n=8),其中SIPl、SIP2、SIP3组给予2次IP后灌注St.Tho-mas Ⅱ晶体停搏液(CCS)使心脏停搏,然后分别使心脏在32℃、25℃、20℃下停灌30、90和120min,其后各组均在37℃再灌注30 min.SConl,SCon2,SCon3三组则不给予IP,继续灌注20min后灌注CCS使心脏停搏,然后分别在32℃、25℃、20℃下停灌30、90和120 min,其后各组均在37℃再灌注30 min.以Maclab/4 s生理实验系统记录平衡末、缺血前、再灌注后1、3、5、10、20、30 min时心率(HR)、左心室发展压(LVDP)以及左心室内压上升及下降最大速率(±dp/dtmax),测定再灌注末心肌组织中ATP和丙二醛(MDA)的含量,以及超氧化物歧化酶(SOD)的活性.在20℃低温停灌且停灌期间不给予CCS时,再灌注末IP2组LVDP×HR、+dp/dtmax和-dp/dtmax的恢复率分别为96%±21%、101%±19%和84%±15%,显著高于Con组和IP3组(P＜0.01,P＜0.05);心肌组织的ATP含量亦高于Con组(P＜0.01).在不同低温停灌且停灌期间给予CCS时,再灌注末SIP1、SIP2组的LVDP×HR、+dp/dtmax分别恢复到87%±14%、99%±26%(P＜0.05,vs SConl group)和87%±16%、102%±20%(P＜0.05,vs SCon2 group);心肌组织的ATP含量均分别显著高于SCon1组和SCon2组(P＜0.05),心肌组织MDA含量亦分别低于SCon1组和SCon2组(P＜0.05).上述结果提示,IP对在20℃低温停灌的兔未成熟心脏具有一定的心肌保护作用,2次IP的保护效应优于1次或3次IP.在停灌期间应用CCS,IP的心肌保护作用随停灌期间低温程度的升高而减弱.