Plasma endothelin levels during myocardial ischemia and reperfusion

Endothelin, an endothelium-derived vasoconstrictive peptide, has a strong potency of coronary artery constriction. However, the role of endogeneous endothelin under pathophysiological conditions has not yet been known. In this study, we examined plasma endothelin concentration in dogs with myocardial ischemia and reperfusion. Anesthetized open-chest dogs underwent either 45 minutes occlusion of the left anterior descending coronary artery followed by 3 hours reperfusion, or 4-10 hours of continuous occlusion. Plasma concentration of endothelin from the central vein was measured by the highly sensitive enzyme-immunoassay. Plasma endothelin concentration increased 2.2-fold with the peak level at 60 minutes after release of the ligated artery, but occlusion per se caused no remarkable change. These data suggest that reperfusion of the occluded artery might be needed to increase the plasma concentration of endothelin in case of myocardial infarction.     

Actin is oxidized during myocardial ischemia

Exposure of isolated rat hearts to 30 min global ischemia followed by 60 min reperfusion resulted in a significant 80% increase (p <.05) in actin content of carbonyl groups, which was associated with significant depression (p <.05) of postischemic contractile function. This result supports the hypothesis that one mechanism of postischemic contractile dysfunction may be oxidation of contractile proteins.     

Changes in tissue levels of carnitine and other metabolites during myocardial ischemia and anoxia

The induction of ischemia in the open chest dog, or anoxia in the perfused rat heart, causes dramatic changes in the tissue levels of free acyl carnitine and related metabolites. During the early phase of ischemia or anoxia the tissue levels of free carnitine decline, while acetyl carnitine rapidly increases. These changes are accompanied by elevation in long-chain acyl carnitine, long-chain acyl CoA, and lactate and by decreases in acetyl CoA, CoA, ATP, and creatine phosphate. As the degree of ischemia becomes more severe, carnitine appears to be lost from the myocardium. A scheme is presented which relates carnitine-linked mitochondrial metabolism to the activity of carnitine acyl transferase, ANT, carnitine/acyl carnitine translocase, creatine phosphokinase, and pyruvate dehydrogenase. It is suggested that the conversion of carnitine to acyl carnitine during the onset of ischemia may play an important role, by virtue of its effect on these enzymes, in the regulation of metabolism during the early or reversible phase of ischemia.     

Antioxidant vitamin levels and glutathione peroxidase activity during ischemia/reperfusion in myocardial infarction

The consequences of increased oxidative stress, measured as the level of malondialdehyde (MDA) during ischemia/reperfusion, were studied in 48 patients in the acute phase of myocardial infarction (AMI) and a control group (21 blood donors). The serum levels of alpha-tocopherol and beta-carotene were followed. Immediately after the treatment onset the level of alpha-tocopherol started to decrease, reaching a plateau after 24 h. The consumption of beta-carotene was delayed by 90 min. Steady decline was detected during the whole time interval studied (48 h). Glutathione peroxidase (GPx) activity, as a representative of antioxidant enzymes, was estimated in whole blood. The influx of oxygenated blood was accompanied by a stimulation of GPx activity, which reached its maximum at the time of completed reperfusion. When comparing the AMI patients with the control group, the levels of MDA were found significantly increased, which indicates that oxidative stress is already increased during ischemia. Lower antioxidant levels found in the patients might either already be the result of vitamin consumption during ischemia or be a manifestation of their susceptibility to AMI. Monitored consumption of alpha-tocopherol and beta-carotene during reperfusion indicated that in the case of patients, whose level of antioxidant vitamins is below the threshold limit, a further substantial decrease of antioxidant vitamins during reperfusion could enhance the oxidative damage of the myocardium.     

Specificity of autonomic influences on cardiac responses during myocardial ischemia.

A possible role of the autonomic nervous system in the left ventricular response to acute regional myocardial ischemia was sought in conscious dogs instrumented for measurement of left ventricular pressure, internal diameter, and aortic flow. Ischemia produced by occluding the left circumflex coronary artery caused tachycardia and reduced contractility. Changes during control occlusions were compared with those during occlusion.s after beta-adrenergic blockade, parasympathetic blockade, and combined sympathetic and parasymphatetic blockade. Beta-blockade did reduce the tachycardia and slightly reduced left ventricular diameter changes in response to coronary occlusion. Results obtained in animals following surgical cardiac sympathectomy indicated reduced tachycardia and no effects on other parameters. The principal effect of parasympathetic blockade was to augment the increase in end diastolic diameter during occlusion Right atrial pacing indicated this change was due to higher initial heart rates. Combined parasympathetic and sympathetic blockade did not alter inotropic responses to coronary occlusion. Results indicated that inotropic support due to changes in activity in autonomic nerves is not increased during acute occlusion of the left circumflex coronary artery.     

Persistence of gap junction communication during myocardial ischemia

During myocardial ischemia, severe ATP depletion induces rigor contracture followed by intracellular Ca2+ concentration ([Ca2+]i) rise and progressive impairment of gap junction (GJ)-mediated electrical coupling. Our objective was to investigate whether chemical coupling through GJ allows propagation of rigor in cardiomyocytes and whether it persists after rigor development. In end-to-end connected adult rat cardiomyocytes submitted to simulated ischemia the interval between rigor onset was 3.7 +/- 0.7 s, and subsequent [Ca2+]i rise was virtually identical in both cells, whereas in nonconnected cell pairs the interval was 71 +/- 12 s and the rate of [Ca2+]i rise was highly variable. The GJ blocker 18alpha-glycyrrhetinic acid increased the interval between rigor onset and the differences in [Ca(2+)]i between connected cells. Transfer of Lucifer yellow demonstrated GJ permeability 10 min after rigor onset in connected cell pairs, and 30 min after rigor onset in isolated rat hearts submitted to nonflow ischemia but was abolished after 2 h of ischemia. GJ-mediated communication allows propagation of rigor in ischemic myocytes and persists after rigor development despite acidosis and increased [Ca2+]i.     

AMPK mediates autophagy during myocardial ischemia in vivo

We have recently shown that autophagy is induced by ischemia and reperfusion in the mouse heart in vivo. Ischemia stimulates autophagy through an AMP activated protein kinase (AMPK)-dependent mechanism, whereas reperfusion after ischemia stimulates autophagy through a Beclin 1-dependent, but AMPK-independent, mechanism. Autophagy plays distinct roles during ischemia and reperfusion: autophagy may be protective during ischemia, whereas it may be detrimental during reperfusion. We will discuss the role of AMPK in mediating autophagy during myocardial ischemia in vivo.     

Inhibition of eicosanoid-mediated coronary constriction during myocardial ischemia

Thromboxane A2 and cysteinyl leukotrienes are highly effective microvessel constrictors in normally perfused myocardium. Their release during acute coronary thrombosis might augment myocardial underperfusion. The constrictor action of these substances could be modified substantially, however, by concomitant myocardial ischemia. We compared the effects of the two eicosanoid constrictors in normally perfused and ischemic myocardium of 24 open-chest, pentobarbital-anesthetized pigs. Left anterior descending coronary flow was measured after intracoronary bolus injections of the stable thromboxane A2 analog U46619 (1-10 micrograms) or leukotriene D4 (LTD4, 1-10 micrograms). Each dose was given before and during myocardial ischemia induced by a snare adjusted to produce 63 +/- 2% decrease in coronary flow for 10 min. Marked dose-independent inhibition of eicosanoid-induced coronary flow decrease occurred during ischemia. With 10 micrograms U46619, coronary flow decrease in the unoccluded state (25 +/- 2 from 55 +/- 4 ml/min pretreatment baseline) was virtually eliminated during snare occlusion (1 +/- 1 from 21 +/- 3 ml/min pretreatment baseline, P less than 0.001). Similar results occurred with LTD4. Distal coronary pressure during ischemia indicated a lack of microvessel responsiveness to the eicosanoids rather than a buffering of resistance change by the snare. U46619 and LTD4 did induce transient, small reductions in regional shortening fraction during ischemia. Our data suggest that eicosanoid-induced constriction of myocardial resistance vessels is not a likely complication of acute coronary thrombosis. However, eicosanoids could depress residual contractility in moderately ischemic regions.     

Non-linear dynamic analysis of the cardiac rhythm during transient myocardial ischemia.

Coronary artery occlusions related to myocardial ischemia drive cardiac control system reactions that may lead to heart failure. The purpose of this study was to assess the autonomic nervous system (ANS) response during prolonged percutaneous transluminal coronary angioplasty (PTCA). Continuous ECG data were acquired from 50 patients before and during PTCA, with occlusions in the left anterior descending, left circumflex or right coronary artery. Heart rate variability (HRV) was analyzed for 3-min segments of the R-R interval signal obtained from ECG data. The ANS behavior was evaluated by HRV analysis using fractal-like indices. The fractal scalar exponent alpha(1) and power-law slope beta decreased considerably during PTCA. This indicates that significant reactions of autonomic control of the heart rate occurred during coronary artery occlusions, with a reduction in complexity of the ANS.     

Biphasic changes in phospholipid hydroperoxide levels during renal ischemia/reperfusion.

The involvement of lipid peroxidation in renal ischemia/reperfusion was explored by measuring changes in the cortical content of specific primary lipid hydroperoxides (using chemluminescent detection with HPLC) following ischemia and reperfusion and by correlating the changes in hydroperoxide content with measurements of renal blood flow. Phosphatidylcholine and phosphatidylethanolamine hydroperoxide concentrations were significantly lowered during 30 or 60 min of ischemia (to levels less than 50% of control at 60 min). Following 30 min of renal ischemia, reperfusion resulted in a rebound of phospholipid hydroperoxide tissue content to levels higher than controls. Increased phospholipid hydroperoxide formation was not, however, observed in response to reperfusion following long-term (60 min) ischemia. In separate animals it was demonstrated that following 30 min ischemia and reperfusion, renal blood flow recovers to about 65% of control in 1 h. In contrast, following 60 min ischemia and reperfusion, the renal blood flow remains more highly impaired (less than 25% recovery for periods up to 24 h). These results imply that phospholipid hydroperoxides are produced and accumulate in the kidneys under normal aerobic conditions and that lipid peroxidative activity increases during renal ischemia/reperfusion to an extent dependent on the degree of local blood perfusion.     

Cardiac outflow of amino acids and purines during myocardial ischemia and reperfusion.

A novel application of microdialysis was studied, in which myocardial outflow of amino acids and purines was monitored by intravasal microdialysis in the myocardial venous outflow during ischemia and reperfusion. Microdialysis catheters were introduced into the great cardiac vein, pulmonary artery, and external jugular vein in 20 anesthetized pigs. The left anterior descending artery was occluded in four groups of pigs for 0, 10, 15, and 60 min. Ischemia was followed by 120 min of reperfusion. Microdialysis samples were analyzed for taurine, aspartate, glutamate, hypoxanthine, inosine, and guanosine. Myocardial infarction developed when ischemia exceeded 10 min. Taurine, aspartate, inosine, and guanosine increased early in the great cardiac vein during ischemia. We found the outflow patterns of amino acids and purines to be graded in response to different lengths of ischemia. In this study we have demonstrated a graded outflow of amino acids and purines in response to ischemia and a positive correlation between infarct size and myocardial outflow of amino acids and purines. This could be of value in a clinical setting to quantify the extent of myocardial damage.     

Proteasome mediates removal of proteins oxidized during myocardial ischemia

Numerous proteins are known to be lost following myocardial ischemia/reperfusion yet little is known about the mediating proteinases. This study examines the hypothesis that proteasome plays a significant role in the removal of proteins oxidized during myocardial ischemia. Proteasome was inhibited by perfusing isolated rat hearts with buffer containing lactacystin, 2 micromol/L, for 10 min, which resulted in 51 and 42% decreases in 20S and 26S proteasome activities that persisted for a minimum of 90 min. Lactacystin pretreatment had minor effects on postischemic recovery of isolated hearts exposed to 30 min global ischemia and 60 min reperfusion. Protein carbonyl content of lactacystin-pretreated ischemic hearts was significantly (P < 0.05) increased. One band with approximate molecular mass of 50 kDa is known to contain oxidized actin. Actin degradation was quantitated by analysis of 3-methylhistidine which was significantly (P < 0.05) decreased by 15% following 30 min ischemia and 60 min reperfusion. Pretreatment of ischemic hearts with lactacystin prevented much of the loss (-6.5%) of 3-methylhistidine. Probing immunoprecipitated actin with an antibody specific for ubiquitin revealed no bands containing ubiquitinated homologues of this protein. These observations support the conclusion that proteasome mediates removal of some of the proteins oxidized during myocardial ischemia/reperfusion, and that at least oxidized actin is removed by the 20S proteasome.     

Early activation of IKKbeta during in vivo myocardial ischemia

We have demonstrated that in vitro brief ischemia activates nuclear factor (NF)-kappaB in rat myocardium. We report in vivo ischemia-reperfusion (I/R)-induced NF-kappaB activation, IkappaB kinase -beta (IKKbeta) activity, and IkappaBalpha phosphorylation and degradation in rat myocardium. Rat hearts were subjected to occlusion of the coronary artery for up to 45 min or occlusion for 15 min followed by reperfusion for up to 3 h. Cytoplasmic and nuclear proteins were isolated from ischemic and nonischemic areas of each heart. NF-kappaB activation was increased in the ischemic area (680%) after 10 min of ischemia and in the nonischemic area (350%) after 15 min of ischemia and remained elevated during prolonged ischemia and reperfusion. IKKbeta activity was markedly increased in ischemic (1,800%) and nonischemic (860%) areas, and phosphorylated IkappaBalpha levels were significantly elevated in ischemic (180%) and nonischemic (280%) areas at 5 min of ischemia and further increased after reperfusion. IkappaBalpha levels were decreased in the ischemic (45%) and nonischemic (36%) areas after 10 min of ischemia and remained low in the ischemic area during prolonged ischemia and reperfusion. The results suggest that in vivo I/R rapidly induces IKKbeta activity and increases IkappaBalpha phosphorylation and degradation, resulting in NF-kappaB activation in the myocardium.     

Free fatty acid metabolism during myocardial ischemia and reperfusion

Long chain free fatty acids (FFA) are the preferred metabolic substrates of myocardium under aerobic conditions. However, under ischemic conditions long chain FFA have been shown to be harmful both clinically and experimentally. Serum levels of free fatty acids frequently are elevated in patients with myocardial ischemia. The proposed mechanisms of the detrimental effects of free fatty acids include: (1) accumulation of toxic intermediates of fatty acid metabolism, such as long chain acyl-CoA thioesters and long chain acylcarnitines, (2) inhibition of glucose utilization, particularly glycolysis, during ischemia and/or reperfusion, and (3) uncoupling of oxidative metabolism from electron transfer. The relative importance of these mechanisms remains controversial. The primary site of FFA-induced injury appears to be the sarcolemmal and intracellular membranes and their associated enzymes. Inhibitors of free fatty acid metabolism have been shown experimentally to decrease the size of myocardial infarction and lessen postischemic cardiac dysfunction in animal models of regional and global ischemia. The mechanism by which FFA inhibitors improve cardiac function in the postischemic heart is controversial. Whether the effects are dependent on decreased levels of long chain intermediates and/or enhancement of glucose utilization is under investigation. Manipulation of myocardial fatty acid metabolism may prove beneficial in the treatment of myocardial ischemia, particularly during situations of controlled ischemia and reperfusion, such as percutaneous transluminal coronary angioplasty and coronary artery bypass grafting. (Mol Cell Biochem 166: 85-94, 1997)     

Reactive oxygen species generated during myocardial ischemia enable energetic recovery during reperfusion

We studied the differences between the functional and bioenergetic effects of antioxidants (AOX) administered before or after myocardial ischemia. Sprague-Dawley rat hearts were perfused with a modified Krebs-Henseleit solution and bubbled with 95% O(2)-5% CO(2). The protocol consisted of 10 min of baseline perfusion, 20 min of global ischemia, and 30 min of reperfusion. An AOX, either 1,2-dihydroxybenzene-3,5-disulfonate (Tiron), a superoxide scavenger, or N-acetyl-L-cysteine, was infused during either baseline or reperfusion. An additional group received deferoxamine as a bolus before ischemia. Hearts were freeze-clamped at baseline, at end of ischemia, and at end of reperfusion for analysis of high-energy phosphates. All AOX, when given before ischemia, inhibited recovery of ATP compared with controls. Both Tiron and deferoxamine also inhibited recovery of phosphocreatine. AOX given before ischemia decreased the efficiency of contraction during reperfusion compared with controls. All of the changes in energetics and efficiency brought on by preischemic AOX treatment could be blocked by a preconditioning stimulus. This suggests that reactive oxygen species, which are generated during ischemia, enhance bioenergetic recovery by increasing the efficiency of contraction.