Endothelin A and B receptor antagonist bosentan reduces postischemic myocardial injury in the rat: critical timing of administration

The purpose of this study was to investigate the effects of bosentan, a mixed endothelin receptor A and B subtype antagonist, on myocardial ischemia-reperfusion injury and to explore the influence of the timing of bosentan administration on its cardioprotective effects. Adult rat hearts were perfused by the Langendorff technique with Krebs-Henseleit solution (KH) at a constant flow rate at 10 mL/min. Global myocardial ischemia was induced by stopping KH perfusion for 40 min, and this was followed by 60 min of reperfusion. Hearts were randomized to 1 of 3 experimental groups (n = 7 each): untreated control; treatment with bosentan 1 micromol/L 10 min prior to, during 40 min global ischemia, and for 15 min of reperfusion (BOS); or treatment with bosentan 1 micromol/L after 15 min of reperfusion (BOS-R). We observed that BOS-R, but not the BOS treatment regimen, significantly reduced the release of cardiac-specific creatine kinase and postischemic myocardial infarct size (P < 0.05 vs. control) without affecting myocardial contractility. Left ventricular developed pressure in the BOS group was significantly (P < 0.01) lower than that in the control group throughout reperfusion. It is concluded that pharmacologically delayed antagonism of endothelin-1 during reperfusion attenuates postischemic myocardial injury. Endothelin-1 antagonist application during early reperfusion may exacerbate postischemic myocardial dysfunction.     

Dose-dependent protection of cardiac function by propofol during ischemia and early reperfusion in rats: effects on 15-F2t-isoprostane formation

We examined the effects of propofol (2,6-diisopropylphenol) on functional recovery and 15-F2t-isoprostane generation during ischemia-reperfusion in Langendorff-perfused rat hearts. Before the induction of 40 min of global ischemia, hearts were perfused (10 min) with propofol at 5 (lo-P) or 12 microg/mL (hi-P) in saline or with saline only (control). During ischemia, saline, lo-P, or hi-P was perfused through the aorta at 60 microL/min. During the first 15 min of reperfusion, propofol (5 or 12 microg/mL) was continued, followed by perfusion with 5 microg/mL propofol for 75 min in both propofol-treated groups. After 90 min of reperfusion (Rep-90), heart tissues were harvested for assessment of antioxidant status. In hi-P, we observed increased latency to and greater reduction of ischemic contracture relative to the lo-P or control groups. 15-F2t-Isoprostane concentrations increased during ischemia and were significantly lower in hi-P and lo-P than in control (P < 0.01). At Rep-90, myocardial functional recovery was greater in both propofol-treated groups relative to control, and it correlated positively with tissue antioxidant capacity preservation. Tissue antioxidant capacity was better preserved in hi-P than in lo-P treatment (P < 0.05). We conclude that oxidant injury occurs during ischemia and reperfusion, and propofol provides dose-dependent protection primarily by enhancing tissue antioxidant capacity and reducing lipid peroxidation.     

Intrinsic A(1) adenosine receptor activation during ischemia or reperfusion improves recovery in mouse hearts

We assessed the role of A(1) adenosine receptor (A(1)AR) activation by endogenous adenosine in the modulation of ischemic contracture and postischemic recovery in Langendorff-perfused mouse hearts subjected to 20 min of total ischemia and 30 min of reperfusion. In control hearts, the rate-pressure product (RPP) and first derivative of pressure development over time (+dP/dt) recovered to 57 +/- 3 and 58 +/- 3% of preischemia, respectively. Diastolic pressure remained elevated at 20 +/- 2 mmHg (compared with 3 +/- 1 mmHg preischemia). Interstitial adenosine, assessed by microdialysis, rose from approximately 0.3 to 1.9 microM during ischemia compared with approximately 15 microM in rat heart. Nonetheless, these levels will near maximally activate A(1)ARs on the basis of effects of exogenous adenosine and 2-chloroadenosine. Neither A(1)AR blockade with 200 nM 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) during the ischemic period alone nor A(1)AR activation with 50 nM N(6)-cyclopentyladenosine altered rapidity or extent of ischemic contracture. However, ischemic DPCPX treatment significantly depressed postischemic recovery of RPP and +dP/dt (44 +/- 3 and 40 +/- 4% of preischemia, respectively). DPCPX treatment during the reperfusion period alone also reduced recovery of RPP and +dP/dt (to 44 +/- 2 and 47 +/- 2% of preischemia, respectively). These data indicate that 1) interstitial adenosine is lower in mouse versus rat myocardium during ischemia, 2) A(1)AR activation by endogenous adenosine or exogenous agonists does not modify ischemic contracture in murine myocardium, 3) A(1)AR activation by endogenous adenosine during ischemia attenuates postischemic stunning, and 4) A(1)AR activation by endogenous adenosine during the reperfusion period also improves postischemic contractile recovery.     

The effect of an adenosine and lidocaine intravenous infusion on myocardial high-energy phosphates and pH during regional ischemia in the rat model in vivo

We have previously shown that an intravenous infusion of adenosine and lidocaine (AL) solution protects against death and severe arrhythmias and reduces infarct size in the in vivo rat model of regional ischemia. The aim of this study was to examine the relative changes of myocardial high-energy phosphates (ATP and PCr) and pH in the left ventricle during ischemia-reperfusion using 31P NMR in AL-treated rats (n = 7) and controls (n = 6). The AL solution (A: 305 microg.(kg body mass)-1.min-1; L: 608 microg.(kg body mass)-1.min-1) was administered intravenously 5 min before and during 30 min coronary artery ligation. Two controls died from ventricular fibrillation; no deaths were recorded in AL-treated rats. In controls that survived, ATP fell to 73% +/- 29% of baseline by 30 min ischemia and decreased further to 68% +/- 28% during reperfusion followed by a sharp recovery at the end of the reperfusion period. AL-treated rats maintained relatively constant ATP throughout ischemia and reperfusion ranging from 95% +/- 6% to 121% +/- 10% of baseline. Owing to increased variability in controls, these results were not found to be significant. In contrast, control [PCr] was significantly reduced in controls compared with AL-treated rats during ischemia at 10 min (68% +/- 7% vs. 99% +/- 6%), at 15 min (68% +/- 10% vs. 93% +/- 2%), and at 20 min (67% +/- 15% vs. 103% +/- 5%) and during reperfusion at 10 min (56% +/- 22% vs. 99% +/- 7%), at 15 min (60% +/- 10% vs. 98% +/- 7%), and at 35 min (63% +/- 14% vs. 120% +/- 11%) (p < 0.05). Interestingly, changes in intramyocardial pH between each group were not significantly different during ischemia and fell by about 1 pH unit to 6.6. During reperfusion, pH in AL-treated rats recovered to baseline in 5 min but not in controls, which recovered to only around pH 7.1. There was no significant difference in the heart rate, mean arterial pressure, and rate-pressure product between the controls and AL treatment during ischemia and reperfusion. We conclude that AL cardioprotection appears to be associated with the preservation of myocardial high-energy phosphates, downregulation of the heart at the expense of a high acid-load during ischemia, and with a rapid recovery of myocardial pH during reperfusion.     

Triiodothyronine concomitantly inhibits calcium overload and postischemic myocardial stunning in diabetic rats.

Acute effects of triiodothyronine (T3) on postischemic myocardial stunning and intracellular Ca2+ contents were studied in the isolated working hearts of streptozotocin-induced diabetic rats and age-matched controls. After two weeks of diabetes, serum T3 and T4 levels were decreased to 62.5% and 33.9% of control values. Basal preischemic cardiac performance did not differ between diabetic and control rats. In contrast, during reperfusion after 20-min ischemia, diabetic rats exhibited an impaired recovery of heart rate (at 30-min reperfusion 57.5% of baseline vs. control 88.5%), left ventricular (LV) systolic pressure (44.1% vs. 89.5%), and cardiac work (23.1% vs. 66.0%). When 1 and 100 nM T3 was added before ischemia, heart rate was recovered to 77.2% and 81.8% of baseline, LV systolic pressure to 68.3% and 81.9%, and cardiac work to 50.8% and 59.0%, respectively. Diabetic rat hearts showed a higher Ca2+ content in the basal state and a further increase after reperfusion (4.96+/-1.17 vs. control 3.78+/-0.48 micromol/g, p<0.01). In diabetic hearts, H+ release was decreased after reperfusion (5.24+/-2.21 vs. 8.70+/-1.41 mmol/min/g, p<0.05). T3 administration caused a decrease in the postischemic Ca2+ accumulation (lnM T3 4.66+/-0.41 and 100 nM T3 3.58+/-0.36) and recovered the H+ release (lnM T3 16.2+/-3.9 and 100 nM T3 11.6+/-0.9). T3 did not alter myocardial O2 consumption. Results suggest that diabetic rat hearts are vulnerable to postischemic stunning, and T3 protects the myocardial stunning possibly via inhibiting Ca2+ overload.     

Is reduced SERCA2a expression detrimental or beneficial to postischemic cardiac function and injury? Evidence from heterozygous SERCA2a knockout mice

Recent studies have demonstrated that increased expression of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) 2a improves myocardial contractility and Ca2+ handling at baseline and in disease conditions, including myocardial ischemia-reperfusion (I/R). Conversely, it has also been reported that pharmacological inhibition of SERCA might improve postischemic function in stunned hearts or in isolated myocardium following I/R. The goal of this study was to test how decreases in SERCA pump level/activity affect cardiac function following I/R. To address this question, we used a heterozygous SERCA2a knockout (SERCA2a+/-) mouse model with decreased SERCA pump levels and studied the effect of myocardial stunning (20-min ischemia followed by reperfusion) and infarction (30-min ischemia followed by reperfusion) following 60-min reperfusion. Our results demonstrate that postischemic myocardial relaxation was significantly impaired in SERCA2a+/- hearts with both stunning and infarction protocols. Interestingly, postischemic recovery of contractile function was comparable in SERCA2a+/- and wild-type hearts subjected to stunning. In contrast, following 30-min ischemia, postischemic contractile function was reduced in SERCA2a+/- hearts with significantly larger infarction. Rhod-2 spectrofluorometry revealed significantly higher diastolic intracellular Ca2+ in SERCA2a+/- hearts compared with wild-type hearts. Both at 30-min ischemia and 2-min reperfusion, intracellular Ca2+ levels were significantly higher in SERCA2a+/- hearts. Electron paramagnetic resonance spin trapping showed a similar extent of postischemic free-radical generation in both strains. These data provide direct evidence that functional SERCA2a level, independent of oxidative stress, is crucial for postischemic myocardial function and salvage during I/R.     

Limiting sarcolemmal Na+ entry during resuscitation from ventricular fibrillation prevents excess mitochondrial Ca2+ accumulation and attenuates myocardial injury.

BACKGROUND: intracellular Na+ accumulation during ischemia and reperfusion leads to cytosolic Ca2+ overload through reverse-mode operation of the sarcolemmal Na+ -Ca2+ exchanger. Cytosolic Ca2+ accumulation promotes mitochondrial Ca2+ (Ca2+ m) overload, leading to mitochondrial injury. We investigated whether limiting sarcolemmal Na+ entry during resuscitation from ventricular fibrillation (VF) attenuates Ca2+ m overload and lessens myocardial dysfunction in a rat model of VF and closed-chest resuscitation. METHODS: hearts were harvested from 10 groups of 6 rats each representing baseline, 15 min of untreated VF, 15 min of VF with chest compression given for the last 5 min (VF/CC), and 60 min postresuscitation (PR). VF/CC and PR included four groups each randomized to receive before starting chest compression the new NHE-1 inhibitor AVE4454B (1.0 mg/kg), the Na+ channel blocker lidocaine (5.0 mg/kg), their combination, or vehicle control. The left ventricle was processed for intracellular Na+ and Ca2+ m measurements. RESULTS: limiting sarcolemmal Na+ entry attenuated cytosolic Na+ increase during VF/CC and the PR phase and prevented Ca2+ m overload yielding levels that corresponded to 77% and 71% of control hearts at VF/CC and PR, without differences among specific Na+ -limiting interventions. Limiting sarcolemmal Na+ entry attenuated reductions in left ventricular compliance during VF and prompted higher mean aortic pressure (110 +/- 7 vs. 95 +/- 11 mmHg, P < 0.001) and higher cardiac work index (159 +/- 34 vs. 126 +/- 29 g x m x min(-1) x kg(-1), P < 0.05) with lesser increases in circulating cardiac troponin I at 60 min PR. CONCLUSIONS: Na+ -limiting interventions prevented excess Ca2+ m accumulation induced by ischemia and reperfusion and ameliorated myocardial injury and dysfunction.     

Postconditioning fails to improve no reflow or alter infarct size in an open-chest rabbit model of myocardial ischemia-reperfusion

Postconditioning (PoC) with brief intermittent ischemia after myocardial reperfusion has been shown to lessen some elements of postischemic injury including arrhythmias and, in some studies, the size of myocardial infarction. We hypothesized that PoC could improve reflow to the risk zone after reperfusion. Anesthetized, open-chest rabbits were subjected to 30 min of coronary artery occlusion followed by 3 h of reperfusion. In protocol 1, rabbits were randomly assigned to the control group (n = 10, no further intervention after reperfusion) or to the PoC group, which consisted of four cycles of 30-s reocclusions with 30 s of reperfusion in between starting at 30 s after the initial reperfusion (4 x 30/30, n = 10). In protocol 2, rabbits were assigned to the control group (n = 7) or the PoC group, which received PoC consisting of four cycles of 60-s intervals of ischemia and reperfusion starting at 30 s after the initial reperfusion (4 x 60/60, n = 7). No reflow was determined by injecting thioflavine S (a fluorescent marker of capillary perfusion), risk zone by blue dye, and infarct size by triphenyltetrazolium chloride. In protocol 1, there were no statistical differences in hemodynamics, ischemic risk zone, or infarct size (35 +/- 6% of the risk zone in the PoC group vs. 29 +/- 4% in the control group, P = 0.38) between the groups. Similarly, in protocol 2, PoC failed to reduce infarct size compared with the control group (45 +/- 4% of the risk zone in the PoC group vs. 42 +/- 6% in the control group, P = 0.75). There was a strong correlation in both protocols between the size of the necrotic zone and the portion of the necrotic zone that contained an area of no reflow. However, PoC did not affect this relationship. PoC did not reduce infarct size in this model, nor did it reduce the extent of the anatomic zone of no reflow, suggesting that this intervention may not impact postreperfusion microvascular damage due to ischemia.     

Inhibition of myocardial injury by ischemic postconditioning during reperfusion: comparison with ischemic preconditioning

Ischemic preconditioning (Pre-con) is an adaptive response triggered by a brief ischemia applied before a prolonged coronary occlusion. We tested the hypothesis that repetitive ischemia applied during early reperfusion, i.e., postconditioning (Post-con), is cardio-protective by attenuating reperfusion injury. In anesthetized open-chest dogs, the left anterior descending artery (LAD) was occluded for 60 min and reperfused for 3 h. In controls (n = 10), there was no intervention. In Pre-con (n = 9), the LAD was occluded for 5 min and reperfused for 10 min before the prolonged occlusion. In Post-con (n = 10), at the start of reperfusion, three cycles of 30-s reperfusion and 30-s LAD reocclusion preceded the 3 h of reperfusion. Infarct size was significantly less in the Pre-con (15 +/- 2%, P < 0.05) and Post-con (14 +/- 2%, P < 0.05) groups compared with controls (25 +/- 3%). Tissue edema (% water content) in the area at risk was comparably reduced in Pre-con (78.3 +/- 1.2, P < 0.05) and Post-con (79.7 +/- 0.6, P < 0.05) versus controls (81.5 +/- 0.4). Polymorphonuclear neutrophil (PMN) accumulation (myeloperoxidase activity, Deltaabsorbance.min-1.g tissue-1) in the area at risk myocardium was comparably reduced in Post-con (10.8 +/- 5.5, P < 0.05) and Pre-con (13.4 +/- 3.4, P < 0.05) versus controls (47.4 +/- 15.3). Basal endothelial function measured by PMN adherence to postischemic LAD endothelium (PMNs/mm2) was comparably attenuated by Post-con and Pre-con (15 +/- 0.6 and 12 +/- 0.6, P < 0.05) versus controls (37 +/- 1.5), consistent with reduced expression of P-selectin on coronary vascular endothelium in Post-con and Pre-con. Endothelial function assessed by the maximal vasodilator response of postischemic LAD to acetylcholine was significantly greater in Post-con (104 +/- 6%, P < 0.05) and Pre-con (109 +/- 5%, P < 0.05) versus controls (71 +/- 8%). Plasma malondialdehyde (microM/ml), a product of lipid peroxidation, was significantly less at 1 h of reperfusion in Post-con (2.2 +/- 0.2, P < 0.05) versus controls (3.2 +/- 0.3) associated with a decrease in superoxide levels revealed by dihydroethidium staining in the myocardial area at risk. These data suggest that Post-con is as effective as Pre-con in reducing infarct size and preserving endothelial function. Post-con may be clinically applicable in coronary interventions, coronary artery bypass surgery, organ transplantation, and peripheral revascularization where reperfusion injury is expressed.     

Glutathione disulfide as an index of oxidative stress during postischemic reperfusion in isolated rat hearts

The objectives of this study were to determine 1) whether reactive oxygen species generated upon postischemic reperfusion lead to oxidative stress in rat hearts, and 2) whether an exogenous prooxidant present in the early phase of reperfusion causes additional injury. Isolated buffer-perfused rat hearts were subjected to 30 min of hypothermic no-flow ischemia followed by 30 min of reperfusion. Increased myocardial content of glutathione disulfide (GSSG) and increased active transport of GSSG were used as indices of oxidative stress. To impose a prooxidant load, cumene hydroperoxide (20 M) was administered during the first 10 min of reperfusion to a separate group of postischemic hearts. Reperfusion after 30 min of hypothermic ischemia resulted in a recovery of myocardial ATP from 28% at end-ischemia to 50–60%, a release of 5% of total myocardial LDH, and an almost complete recovery of both coronary flow rate and left ventricular developed pressure. After 5 and 30 min of reperfusion, neither myocardial content of GSSG nor active transport of GSSG were increased. These indices were increased, however, if cumene hydroperoxide was administered during early reperfusion. After stopping the administration of cumene hydroperoxide, myocardial GSSG content returned to control values and GSH content increased, indicating an unimpaired glutathione reductase reaction. Despite the induction of oxidative stress, reperfusion with cumene hydroperoxide did not cause additional metabolic, structural, or functional injury when compared to reperfusion without cumene hydroperoxide. We conclude that reactive oxygen species generated upon postischemic reperfusion did not lead to oxidative stress in isolated rat hearts. Moreover, even a superimposed prooxidant load during early reperfusion did not cause additional injury.     

Effects of ischemic preconditioning and adenosine pretreatment on myocardial function and energetics in a clinically relevant model

Preconditioning (PC) is a potential approach to myocardial protection. We hypothesize that brief ischemia or adenosine given prior to an extended period of warm ischemia may prevent myocardial stunning by altering myocardial metabolism. Using a global ischemia model, 19 dogs were subjected to no PC(control), two episodes of ischemia (2 min of global ischemia followed by 3 min of reperfusion) (IPC), or 30 min of pulmonary artery adenosine infusion (AP), to a maximum of 350 microg/kg/min, followed by 20 min of global warm ischemia on cardiopulmonary bypass. Left ventricular pressure-volume loops and myocardial oxygen consumption (MVO(2)) were measured at baseline and after 60 min of reperfusion, on right heart bypass. All data were compared between baseline and reperfusion. Load independent left ventricular function, defined as preload recruitable stroke work (PRSW), decreased in control and IPC groups (72+/-7%, 71+/-12%, respectively). AP blunted the decrease in PRSW (45+/-9%, p<.05 compared to control). Myocardial energetic conversion efficiency, defined as the slope of the MVO(2)-Stroke work relationship was not significantly changed for controls (2.17+/-0.47 to 1.84+/-0.68) and IPC (2.99+/-0.45 to 2.16+/-0.65), but was for AP (1.16+/-0.88 to 5.71+/-1.66, p<0.04). IPC did not prevent ventricular stunning or alter myocardial energetics. AP reduced ventricular stunning but resulted in worsened myocardial energy efficiency. The benefits to ventricular function of the adenosine pretreatment protocol used in this study were only possible at a cost of higher metabolic requirements.     

Glycogen turnover and anaplerosis in preconditioned rat hearts

Using (13)C NMR, we tested the hypothesis that protection by preconditioning is associated with reduced glycogenolysis during ischemia. Preconditioned rat hearts showed improved postischemic function and reduced ischemic damage relative to ischemic controls after 30 min stop-flow ischemia and 30 min reperfusion (contractility: 30+/-10 vs. 2+/-2%; creatine kinase release: 41+/-4 vs. 83+/-15 U/g; both P<0.05). Preconditioning decreased preischemic [(13)C]glycogen by 24% (a 10% decrease in total glycogen), and delayed ischemic [(13)C]glycogen consumption by 5-10 min, reducing ischemic glycogenolysis without changing acidosis relative to controls. Upon reperfusion, glycogen synthesis resumed only after preconditioning. Glutamate (13)C-isotopomer analysis showed recovery of Krebs cycle activity with higher anaplerosis than before ischemia (23+/-4 vs. 11+/-3%, P<0.05), but in controls reperfusion failed to restore flux. Compared to control, preconditioning before 20 min ischemia increased contractility (86+/-10 vs. 29+/-14%, P<0.05) and restored preischemic anaplerosis (13+/-3 vs. 39+/-9%, P<0.05). Preconditioning is associated with reduced glycogenolysis early during ischemia. However, protection does not rely on major variations in intracellular pH, as proposed earlier. Our isotopomer data suggest that preconditioning accelerates metabolic and functional recovery during reperfusion by more efficient/active replenishment of the depleted Krebs cycle.     

Na(+)/H(+) exchange subtype 1 inhibition reduces endothelial dysfunction in vessels from stunned myocardium

Myocardial ischemia and reperfusion cause myocyte and vascular dysfunction, frequently termed "stunning." We hypothesized that inhibiting the Na(+)/H(+) exchanger subtype 1 isoform (NHE(1)) during ischemia and reperfusion limits myocardial and coronary microvascular stunning. Anesthetized rats completed 2 x 10-min coronary artery occlusions separated by 5-min of reperfusion, followed by 15 or 60 min of reperfusion. Vehicle (saline) or the NHE(1) inhibitor cariporide (HOE-642) was administered 15 min before ischemia and was continued throughout each protocol. After reperfusion, hearts were excised, and the reactivity of resistance arteries (internal diameter, approximately 120 microm) was assessed. The first derivative of left ventricular (LV) pressure, LV developed pressure, and LV systolic wall thickening were depressed (P < 0.05) similarly in vehicle- and cariporide-treated rats during ischemia and after 15 or 60 min of reperfusion compared with sham-operated animals that were not exposed to ischemia (i.e., controls). In vessels obtained after 15 min of reperfusion, the maximal response to acetylcholine-induced relaxation (10(-8)-10(-4) M) was blunted (P < 0.05) in vessels from vehicle- (approximately 35%) and cariporide-treated rats (approximately 55%) compared with controls (approximately 85%). However, the percent relaxation to acetylcholine was greater (P < 0.05) in cariporide-treated rats compared with vehicle-treated rats. Maximal contractile responses to endothelin-1 (10(-11)-10(-7) M) were increased (P < 0.05) similarly in vehicle- and cariporide-treated rats compared with controls. Relaxation to sodium nitroprusside (10(-4) M) was not different among groups. Results were similar in vessels obtained from animals after 60 min of reperfusion. These findings suggest that NHE(1) inhibition before coronary occlusion lessens ischemia-induced microvascular dysfunction for 15-60 min after reperfusion but does not alter myocardial contractile function in the area at risk.     

Effects of adenosine and acadesine on interstitial nucleosides and myocardial stunning in the pig.

5-Amino-4-imidazolecarboxamide riboside (AICAr) or acadesine has been proposed to exert cardioprotection by enhancing adenosine production in ischemic myocardium. However, there are conflicting reports on acadesine's effects in ischemic myocardium and few studies in which myocardial adenosine levels have been measured. The purpose of this study was to determine whether acadesine increases interstitial fluid adenosine levels and attenuates myocardial stunning or potentiates the effects of adenosine in the intact pig. In pentobarbital-anesthetized pigs, myocardial stunning was induced by 10 min left anterior descending coronary artery occlusion and 90 min reperfusion. Regional ventricular function was assessed by measuring systolic wall thickening, and interstitial nucleosides were estimated by cardiac microdialysis. Control hearts were compared with hearts treated with acadesine, adenosine, and adenosine plus acadesine. Adenosine pretreatment (100 microg x kg(-1) x min(-1), intracoronary) immediately prior to ischemia increased interstitial adenosine levels 9-fold and improved postischemic functional recovery from a control value of 17.6 +/- 4.1% to 43.6 +/- 3.4% of preischemic systolic wall thickening. In contrast, acadesine (20 mg/kg i.v. bolus 10 min prior to ischemia + 0.5 mg x kg (-1) x min(-1), i.v. infusion through 60 min reperfusion) had no effect on interstitial fluid adenosine levels or the recovery of regional function (21.5 +/- 5.9% recovery), nor were the functional effects of adenosine potentiated by acadesine. These findings indicate that acadesine does not enhance myocardial adenosine levels, attenuate myocardial stunning, or potentiate the cardioprotective effects of adenosine in the pig.     

Apomorphine prevents myocardial ischemia/reperfusion-induced oxidative stress in the rat heart

This study examined the hypothesis that low-concentration apomorphine improves postischemic hemodynamic and mitochondrial function in the isolated rat heart model by attenuating oxidation of myocardial proteins. Control and apomorphine-treated hearts were subjected to 35 min of perfusion, 25 min of normothermic global ischemia, and 60 min of reperfusion. Apomorphine (2 microM) was introduced into the perfusate for 20 min starting from the onset of reperfusion. Apomorphine significantly (p <.05) improved postischemic hemodynamic function: work index of the heart (product of LVDP and heart rate) was twice as high in apomorphine-treated hearts compared to controls at the end of reperfusion (p <.01). After isolation of cardiac mitochondria, the respiratory control ratio (RCR) was calculated from the oxygen consumption rate of State 3 and State 4 respiration. Apomorphine significantly improved postischemic RCR (87% of preischemic value vs. 39% in control, p <.05). Using an immunoblot technique, carbonyl content of multiple unidentified myocardial proteins (mitochondrial and nonmitochondrial) was observed to be elevated after global ischemia and reperfusion. Apomorphine significantly attenuated the increased protein oxidation at the end of reperfusion. These results support the conclusion that apomorphine is capable of preventing ischemia/reperfusion-induced oxidative stress and thereby attenuating myocardial protein oxidation and preserving mitochondrial respiration function.     

Trolox C, a lipid-soluble membrane protective agent, attenuates myocardial injury from ischemia and reperfusion

The lipophilic antioxidant Trolox C, a vitamin E analog, was administered to isolated, buffer-perfused rabbit hearts subjected to 25 min of global stop-flow ischemia and 30 min of reperfusion. In six hearts, Trolox C (200 microM) was infused for 15 min immediately prior to ischemia and for the first 15 min of reperfusion. Six control hearts received only vehicle. Gas chromatography analysis confirmed that effective myocardial levels of Trolox were attained. At 30 min reperfusion, the recovery of left ventricular developed pressure was 56 +/- 3% of baseline in control hearts versus 70 +/- 4% in Trolox-treated hearts (p < .01). There was also significant improvement in recovery of Trolox-treated hearts in diastolic pressure and both maximum and minimum values of the first derivative of left ventricular pressure (dP/dt). Creatine phosphokinase release into the coronary effluent at 30 min of reperfusion was 16.5 +/- 8.4 IU/min in untreated and 6.3 +/- 1.0 IU/min (p < .05) in Trolox-treated hearts. Thus Trolox C, a lipophilic antioxidant, attenuated myocardial injury during stop-flow ischemia and reperfusion.     

Heat pretreatment differentially affects cardiac fatty acid accumulation during ischemia and postischemic reperfusion

We investigated whether the cardioprotection induced by heat stress (HS) pretreatment is associated with mitigation of phospholipid degradation during the ischemic and/or postischemic period. The hearts, isolated from control rats and from heat-pretreated rats (42 degrees C for 15 min) either 30 min (HS0.5-h) or 24 h (HS24-h) earlier, were subjected to 45 min of no-flow ischemia, followed by 45 min of reperfusion. Unesterified arachidonic acid (AA) accumulation was taken as a measure for phospholipid degradation. Significantly improved postischemic ventricular functional recovery was only found in the HS24-h group. During ischemia, AA accumulated comparably in control and both HS groups. During reperfusion in control and HS0.5-h hearts, AA further accumulated (control hearts from 82 +/- 33 to 109 +/- 51 nmol/g dry wt, not significant; HS-0.5h hearts from 52 +/- 22 to 120 +/- 53 nmol/g dry wt; P < 0.05). In contrast, AA was lower at the end of the reperfusion phase in HS24-h hearts than at the end of the preceding ischemic period (74 +/- 18 vs. 46 +/- 23 nmol/g dry wt; P < 0.05). Thus accelerated reperfusion-induced degradation of phospholipids in control hearts is completely absent in HS24-h hearts. Furthermore, the lack of functional improvement in HS0.5-h hearts is also associated with a lack of beneficial effect on lipid homeostasis. Therefore, it is proposed that enhanced membrane stability during reperfusion is a key mediator in the heat-induced cardioprotection.     

Cerebral Phospholipid Content and Na+, K+-ATPase Activity During Ischemia and Postischemic Reperfusion in the Mongolian Gerbil

Using bilateral carotid artery occlusion in adult gerbils we examined the effects of ischemia and ischemia/reperfusion on cerebral phospholipid content and Na+,K+-ATPase (EC 3.6.1.3) activity. In contrast to the large changes in phospholipid content and membrane-bound enzyme activity that have been observed in liver and heart tissues, we observed relatively small changes in the cerebral content of total phospholipid, phosphatidylcholine (PC), phosphatidylserine (PS), and phosphatidylethanolamine (PE) following ischemic intervals of up to 240 min. Following 15 min of ischemia the cerebral content of sphingomyelin (SM) was decreased to less than 50% of control values but returned to near-normal levels with longer ischemic periods. Significant decreases in the cerebral content of phosphatidylinositol (PI) and phosphatidic acid (PA) were observed following shorter intervals of ischemia (15-45 min). Na+,K+-ATPase activity of cerebral homogenates prepared from the brains of gerbils subjected to 30-240 min of ischemia was decreased but significantly different from control activity only after 30 min of ischemia (-29%, p less than or equal to 0.05). With the exception of PS, reperfusion for 60 min following 60 min of ischemia resulted in marked increases in cerebral phospholipid content with PC, SM, PI, and PA levels exceeding and PE levels equal to preischemic values. Longer periods of reperfusion (180 min) resulted in decreases in cerebral phospholipid content toward (PC, SM, PI, and PA) or below (PE) preischemic levels. In contrast, the cerebral content of PS significantly decreased during reperfusion (-51% at 60 min, p less than or equal to 0.05) and remained below preischemic values even after 180 min of reperfusion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Myoelectric bowel activity in ischemia/reperfusion damage. Role of sensory neurons.

The present knowledge indicates that afferent sensory neurons (C-fibres) play an important role in the relationship between intestinal myoelectric activity (IMA) and blood flow (LDBF). The aim of this study was to evaluate the role of C-fibers in myoelectric activity of small intestine during its ischemia and reperfusion. A neurotoxin-capsaicin (CAP) was used to induce functional ablation of afferent sensory neurons. Experiments were performed on 6 groups of anesthetized rats. In the I, II, III group of rats IMA and LDBF were recorded during 100% ischemia induced by AMA 15, 30 and 60 min total occlusion and during 60 min reperfusion period. In group V and VI, IMA and LDBF were registered after intrajejunal placement of 1% CAP. In group IV we measured effects of intraluminal instillation of CAP alone. Intraluminal placement of CAP induced an early increase in slow wave amplitude SWA and slow wave frequency SWF by 35+/-11% and 19+/-10% (p<0.05) with the subsequent decrease in both by 25+/-6 and 24+/-8% (p<0.05) respectively. Short 15 min lasting ischemia induced by 100% occlusion of AMA evoked only a slight increase of SWA. During reperfusion period SWA and SWF returned to the baseline values after 15 min. Total 30 min occlusion decreased SWA and SWF by 25+/-9 and 24+/-6% (p<0.05) respectively. During reperfusion period recovery of IMA parameters to preocclusion values were slower. Intestinal hyperemia was smaller than in previous group. After 60 min lasting intestinal ischemia SWA and SWF were decreased by 58+/-7 and 40+/-6% (p<0.01) respectively. There was no return of IMA parameters to control values. These data demonstrated that intestinal ischemia induces typical changes in the bowel myoelectric activity. These changes possess their own electrical characteristics which can be used in clinical practice for evaluation of the degree ischemically-induced intestinal injury. Capsaicin pretreatment significantly decreased SWA and SWF and LDBF in comparison with those observed in group II and III during 30 and 60 min occlusion and reperfusion period. We conclude that afferent neurons C activated during mesenteric ischemia/reperfusion play an important role in protecting ischemic bowel viability.     

Recombinant human, active site-blocked factor VIIa reduces infarct size and no-reflow phenomenon in rabbits

Oxygen free radicals induce de novo synthesis of tissue factor (TF), the initiator of the extrinsic pathway of coagulation, within the coronary vasculature during postischemic reperfusion. In the present study we wanted to assess whether TF expression might cause myocardial injury during postischemic reperfusion. Anesthetized rabbits underwent 30 min of coronary occlusion followed by 5.5 h of reperfusion. At reperfusion the animals received 1) saline (n = 8), 2) human recombinant, active site-blocked activated factor VII (FVIIai, 1 mg/kg, n = 8), or 3) human recombinant activated FVII (FVIIa, 1 mg/kg, n = 8). FVIIai binds to TF as native FVII, but with the active site blocked it inhibits TF procoagulant activity. The area at risk of infarction (AR), the infarct size (IS), and the no-reflow area (NR) were determined at the end of the experiment. FVIIai resulted in a significant reduction in IS and NR with respect to control animals (28.1 +/- 11.3 and 11.1 +/- 6.1% of AR vs. 59.8 +/- 12.8 and 24.4 +/- 2.7% of AR, respectively, P < 0.01), whereas FVIIa resulted in a significant increase in IS and NR to 80.1 +/- 13. 1 and 61.9 +/- 13.8% of AR, respectively (P < 0.01). In conclusion, TF-mediated activation of the extrinsic coagulation pathway makes an important contribution to myocardial injury during postischemic reperfusion.