Relation between high energy phosphate and lethal injury in myocardial ischemia in the dog.

The relationship between progressive depletion of high energy phosphate and the onset of lethal cell injury in ischemic myocardium following coronary occlusion has been evaluated. Myocardial ischemia was induced by proximal occlusion of the circumflex coronary artery for 15, 30, 40, or 60 minutes. Cell injury in the severely ischemic posterior papillary muscle (PP) was evaluated by electron microscopy and by measuring the capacity of slices of the injured PP to maintain electrolytes, resynthesize high energy phosphate, and exclude inulin during in vitro incubation. ATP content in the ischemic myocardium decreased to 35%, 9%, 7%, and 5% of control values after 15, 30, 40, and 60 minutes of ischemia, respectively, and was associated with a corresponding depletion of total adenine nucleotides. The loss of 65% of the ATP after 15 minutes of ischemia (reversible injury) was associated with only minimal ultrastructural changes and no significant defects of electrolytes in incubated slices. However, the depletion of over 90% of the ATP after 40 minutes of ischemia (irreversible injury) was associated with significant fine structural changes and markedly altered cell volume regulation. The results suggest a close relationship between the marked depletion of high energy phsophates and the development of lethal injury in acutely ischemic myocardium.     

Ischemic tissue injury.

The subendocardial to subepicardial gradient in the severity of ischemia following acute coronary occlusion is described. The effects of mild, moderate, and severe ischemia on cell structure and function are compared in summary form, and special attention is given to the effects of severe ischemia on myocardial cells. The characteristics of reversible and irreversible ischemic injury are defined in biologic terms. The failure of cell volume regulation in cells which have entered an irreversible state of ischemic injury is demonstrated by the use of free-hand slices in vitro. Irreversibility is associated with structural defects in the plasma membrane and is reflected in an increased slice inulin-diffusible space, increased slice H2O and Na+ content, and failure of the tissue to maintain the high K+ and Mg2+ levels characteristic of normal left ventricular myocardium. Defective cell membrane function is an early feature of irreversible ischemic injury and may be a primary event in the genesis of the irreversible state.     

Abnormal myocardial fluid retention as an early manifestation of ischemic injury.

Fifty-seven isolated, blood perfused, continuously weighed canine hearts have been utilized to study the development of abnormal myocardial fluid retention during early myocardial ischemic injury. Inflatable balloon catheters were positioned around the left anterior descending coronary arteries (LAD) of 54 hearts or the proximal left circumflex coronary arteries of three hearts for study of the following intervals of coronary occlusion: a) 10 minutes followed by 20 minutes of reflow, b) 40 minutes followed by either no reflow or by 20 minutes of reflow, and c) 60 minutes without reflow. After 60 minutes of fixed coronary occlusion, histologic and ultrastructural examination revealed mild swelling of many ischemic cardiac muscle cells in the absence of interstitial edema, cardiac weight gain, and obvious structural defects in cell membrane integrity. After 40 minutes of coronary occlusion and 20 minutes of reflow, significant cardiac weight gain occurred in association with characteristic alterations in the ischemic region, including widespread interstitial edema and focal vascular congestion and hemorrhage and swelling of cardiac muscle cells. Focal structural defects in cell membrane integrity were also noted. The development of abnormal myocardial fluid retention after 40 minutes of LAD occlusion occurred in association with a significant reduction in sodium-potassium-ATPase activity in the ischemic area, but with no significant alteration in either creatine phosphokinase or citrate synthase activity in the same region. Despite the abnormal myocardial fluid retention in these hearts, it was possible pharmacologically to vasodilate coronary vessels with adenosine and nitroglycerin infusion to maintain a consistently high coronary flow following release of the coronary occlusion after 40 minutes and to even exceed initial hyperemic flow values following release of the occlusion when adenosine and nitroglycerin infusion was delayed until 15 minutes after reflow. Thus, the data indicate that impaired cell volume regulation and interstitial fluid accumulation and focal structural defects in cell membrane integrity are early manifestations of ischemic injury followed by reflow, but fail to establish a major role for the abnormal fluid retention in altering coronary blood flow prior to the development of extensive myocardial necrosis. In contrast, fixed coronary occlusion for 60 minutes results in mild intracellular swelling but no significant interstitial edema and no obvious structural defects in cell membrane integrity.     

Focal brain ischemia in rat: acute changes in brain tissue T1 reflect acute increase in brain tissue water content

Several recent studies have reported changes of brain tissue T(1) in ischemic models during the first minutes after occlusion of the middle cerebral artery (MCA). In order to assess whether these tissue T(1) changes are related to an increase in tissue water content, we performed T(1) (7 T) and tissue water content measurements in a rat model (n = 10, Sprague-Dawley) of focal cerebral ischemia (intraluminal occlusion model). The tissue water content was determined using a gravimetric technique. The animals were divided into two groups: an ischemic group, with an effective MCA occlusion (n = 6) and a control group, with animals having undergone sham surgery but no MCA occlusion (n = 4). In the ipsilateral cortex, the tissue water content was 81.1 +/- 0.7% at 2 h 15 min following ischemic insult (contralateral value: 79.3 +/- 0.5%). Concomitantly, the tissue T(1) in the ipsilateral cortex was 2062 +/- 60 ms at ischemia onset + 1 h (contralateral 1811 +/- 28 ms) and 2100 +/- 38 ms at ischemia onset + 2 h (contralateral 1807 +/- 18 ms). The tissue T(1) and tissue water content values measured in the contralateral area do not differ from the values obtained in the control group. A significant T(1) increase is observed at ischemia onset + 1 h (+ 14%) and ischemia onset (+ 2 h) + 16%, together with a significant increase in tissue water content (+ 2.3%). This suggests that there is an increase in tissue water content concomitant with cell swelling during the first hours of ischemia.     

Verapamil in two reperfusion models of myocardial infarction. Temporary protection of severely ischemic myocardium without limitation of ultimate infarct size

The ability of verapamil to protect severely ischemic myocardium was assessed in dogs using 40 minutes of temporary coronary occlusion. Reperfusion was established for 4 days after which infarcts were sized histologically. Untreated dogs developed subendocardial infarcts (the more moderately ischemic subepicardial region being salvaged by reperfusion). Pretreatment with verapamil reduced the size of these subendocardial infarcts from 34 +/- 8 to 8 +/- 3% of the ischemic circumflex vascular bed at risk (identified by postmortem perfusion of the previously occluded and unoccluded arteries with different dyes). Thus, verapamil prevented cell death in the severely ischemic subendocardial region for the 40-minute test period. In a second study to establish whether verapamil could delay cell death for a longer period of time in the less severely ischemic subepicardial region, a 3-hour period of coronary occlusion was used. This period of occlusion caused infarcts averaging 60 +/- 6% of the ischemic area at risk in untreated dogs. Dogs treated with verapamil 15 minutes postocclusion and throughout the remaining 165 minutes of the 3-hour test period had no limitation of infarct size (53 +/- 3% of the area at risk). In this 3-hour study, the effect of variation in collateral blood flow on infarct size was evaluated by plotting infarct size versus subepicardial collateral flow. Verapamil neither improved collateral flow nor altered the relationship between infarct size and baseline collateral flow. Thus, pretreatment with verapamil prevented necrosis of severely ischemic myocytes, when reperfusion was established at 40 minutes, but failed to prevent necrosis of moderately ischemic myocardium and thus failed to limit infarct size when the period of coronary occlusion was prolonged to 3 hours and treatment was started 15 minutes after the onset of ischemia.     

Detection of early myocardial infarction in formalin-fixed, paraffin-embedded tissue

Whether the early infarct area in formalin-fixed, paraffin-embedded tissue could be delineated by the immunohistochemical method using myoglobin-antibody was studied in 23 pig hearts without collateral circulation. Five hearts were examined at 20 minutes, 2 hours, 4 hours, and 6 hours after occlusion of the distal one third of the left anterior descending coronary artery, respectively. Three pigs were killed 24 hours after occlusion. Heart rate and aortic pressure before and after occlusion did not change in any groups. The subepicardial and subendocardial regional blood flows were reduced to almost zero in all hearts after occlusion (0.88 +/- 0.10 to 0.02 +/- 0.02 mL/g/min). Slight myoglobin defects in the ischemic tissue were noted in the five pigs examined 2 hours after occlusion and definite myoglobin defects were detected in all pigs examined at 4, 6, and 24 hours after occlusion. Nitrotetrazorium blue stain of myocardial tissue before formalin fixation showed slight demarcation of the ischemic area at 4 hours after occlusion and definite demarcation at 6 and 24 hours after occlusion. Slight demarcation was noted at 2 hours after occlusion in Masson trichrome stain and 4 hours after occlusion in the hematoxylin-eosin stain. However, definite demarcation of the ischemic area was seen in Masson trichrome stain only at 24 hours after occlusion and was not noted in hematoxylin-eosin stain even at 24 hours after occlusion. Our previous electron microscopic study revealed that, in the pig heart, irreversible cellular damage was transmurally seen at two hours after occlusion of the coronary artery. Therefore, a definite myoglobin defect reflects irreversible cellular damage such as infarction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Myocardial Calcium and Magnesium in Acute Ischemic Injury

The effect of ischemic injury on calcium and magnesium distribution in dog myocardial cells was investigated in tissue damaged by occlusion of the circumflex branch of the left coronary artery for 60 minutes or for 40 minutes followed by 20 minutes of reperfusion of the damaged tissue by arterial blood. No significant change in the concentration of these cations was noted in permanently ischemic, irreversibly injured myocardial cells, but tissue calcium was markedly increased in cells killed by an episode of transient ischemia. Tissue water and sodium also were increased and magnesium was decreased significantly in the transient ischemia model. Investigation of the localization of the increased Ca^-- by cellular fractionation and chemical analysis as well as by electron miscroscopy and microincineration showed that much of it was localized in dense bodies within the mitochondria. Within the intramitochondrial dense bodies, the calcium appeared to be a precipitate of an as yet undefined form of calcium phosphate.     

Overestimation of myocardial infarct size by histologic measurement in a model of occlusion followed by reperfusion.

We studied 32 transverse left ventricular slices of myocardium from 16 pigs after 45 to 100 minutes of coronary artery occlusion followed by 180 minutes of reperfusion. Infarct area for each slice was determined as follows: (1) grossly, by triphenyl tetrazolium chloride staining of each slice, and (2) microscopically, by complete histologic sectioning of the triphenyl tetrazolium chloride-stained surface of each slice. Planimetry of necrotic and nonnecrotic areas was performed from tracings and photographs of triphenyl tetrazolium chloride-stained slices and from actual histologic sections. When triphenyl tetrazolium chloride and histologic measurements were compared, necrotic tissue area had decreased 11.4% +/- 15.0% (2.59 +/- 1.04 vs 2.09 +/- 0.86 cm2). Nonnecrotic tissue area decreased 20.6% +/- 24.0% (8.31 +/- 3.79 vs 5.16 +/- 2.73 cm2). In this model of ischemia followed by reperfusion, with fixation and processing, viable tissue shrank almost twice as much as necrotic tissue. This differential shrinkage introduces an error resulting in overestimation of infarct size by histologic quantitation.     

Role of platelet-activating factor in the reperfusion injury of rabbit ischemic heart.

This study shows that the administration of the PAF receptor antagonist SDZ 63.675 (5 mg/kg body weight) before reperfusion significantly reduced the hematologic and hemodynamic alterations, as well as the size of necrotic area in rabbits subjected to 40 minutes of coronary occlusion and reperfusion. Pretreatment with SDZ 63.675 prevented the reduction of platelet counts in the blood obtained from the right ventricle (86.6 +/- 2.8% of the control preischemia value) and the transient bradycardia (85.0 +/- 2.8%), the systemic hypotension (58.0 +/- 2.8%), and the increase in right ventricular pressure (125.0 +/- 3.6%) that were evident in the first minutes of reperfusion in untreated control rabbits. Two as well as 24 hours after reperfusion, the infarct size, judged by staining with tetrazolium, was significantly reduced in rabbits treated with SDZ 63.675 (infarct size in control animals, 66.0 +/- 2.9% and 63.46 +/- 2.09% of the risk region at 2 or 24 hours, respectively, compared with 38.9 +/- 5.2% and 37.11 +/- 2.44% of the risk region at 2 and 24 hours in rabbits treated with SDZ 63.675). This result was confirmed by histologic examination of cardiac tissue 24 hours after reperfusion. In addition, SDZ 63.675 markedly reduced the accumulation of 111In-oxine-labeled platelets that occurs 15 minutes after reperfusion in the central ischemic area of the heart and in the lungs. These results suggest that PAF plays a role in the evolution of myocardial injury observed during reperfusion.     

Distribution of cytosolic and mitochondrial aspartate aminotransferase in normal, ischemic, and necrotic myocardium. An immunohistochemical study

We utilized immunoperoxidase methods to study the distribution of both cytosolic or soluble(s) and mitochondrial (m) aspartate aminotransferase (AspAT) in normal, ischemic, and necrotic myocardium. Human myocardium was obtained from autopsy (n = 9) and surgery (n = 6). Cardiac tissue from 26 dogs with experimental myocardial infarction induced by closed-chest balloon occlusion and four dogs with myocardial ischemia without necrosis induced by a 50% reduction in left main coronary artery flow for 3 hours were studied. Duration of occlusion was 45 minutes (n = 1), 3 hours (n = 11), 5 to 6 hours (n = 10), or 15 to 24 hours (n = 4). Highly purified m- and s-AspAT and specific antibodies were prepared in our laboratory. In all cases, control experiments were performed. Microscopically normal human and dog myocardium uniformly stained for m- and s-AspAT. Necrotic myocardium from patients with infarcts showed markedly reduced immunostaining. In those dogs with myocardial necrosis, all dogs with coronary occlusion of 5 to 24 hours, and eight of 11 dogs with 3-hour occlusions, immunostaining was significantly reduced for both s- and m-AspAT in regions confirmed to be necrotic by triphenyl tetrazolium chloride and hematoxylin and eosin staining. Myocardial necrosis was confirmed in the 3-hour infarcts by electron microscopy. In the four dogs with a 50% reduction in left main flow for 3 hours, and one dog with a 45-minute coronary occlusion, ischemia was demonstrated by glycogen loss in period acid-Schiff-stained sections but there was no evidence of necrosis by electron microscopy or triphenyl tetrazolium chloride staining and there was no loss of immunostaining evident for s- or m-AspAT. Thus, s- and m-AspAT were visualized in normal and ischemic myocardium with decreased staining in necrotic tissue using immunoperoxidase techniques. Loss of both s- and m-AspAT can be demonstrated in human myocardium and in experimental canine myocardium as early as 3 hours after coronary occlusion and appears to be specific for irreversible myocardial injury. No depletion of isoenzyme can be detected by immunohistochemical techniques in tissue that is ischemic but not necrotic. Furthermore, by these immunoperoxidase techniques, loss of s- and m-AspAT from necrotic myocardium appears to be simultaneous.     

Ceramide is involved in triggering of cardiomyocyte apoptosis induced by ischemia and reperfusion.

Involvement of ceramide signaling in the initiation of apoptosis induction in myocardial cells by in vitro and in vivo ischemia and reperfusion was analyzed. Synthetic cell permeable C2-ceramide induced apoptotic death of rat neonatal cardiomyocytes in vitro. In vitro ischemia (oxygen/serum/glucose deprivation) led to a progressive accumulation of ceramide in cardiomyocytes. After 16 hours of simulated in vitro reperfusion (readdition of oxygen, serum and glucose), the level of ceramide in surviving cells was found to have returned to baseline, whereas, levels in nonadherent dead cells remained high. In the rat heart left coronary artery occlusion model, ischemia with the subsequent reperfusion, but not ischemia alone, induced apoptosis in myocardial cells as demonstrated by DNA electrophoresis and measurement of soluble chromatin degradation products. The content of ceramide in ischemic area was elevated to 155% baseline levels at 30 minutes, and to 330% after 210 minutes of ischemia. Ischemia (30 minutes) followed by reperfusion (180 minutes) increased the ceramide level to 250% in the ischemic area. The combination of results obtained in both in vitro and animal models demonstrate for the first time that ceramide signaling can be involved in ischemia/reperfusion death of myocardial cells.     

Experimental infarct size as a function of the amount of myocardium at risk.

Occlusion of the same coronary artery at the same anatomic site in a group of dogs results in significant variation in the size of the resultant infarcts. The present study shows that much of this variation can be explained by differences in the pattern of distribution of the occluded coronary artery. The relationship between the anatomic size of an occluded coronary bed and the the amount of necrosis resulting from 40 minutes of proximal circumflex coronary (LCC) occlusion and 3 days of reperfusion was determined in randomly selected dogs using an in vitro latex coronary injection technique. The amount of necrosis, calculated from serial histologic sections taken through the posterior papillary muscle (PP), correlated closely with the size of the occluded coronary bed: 5 dogs with small LCC beds (26 +/- 2%) had 10 +/- 3% PP necrosis; 5 dogs with medium-sized LCC beds (37 +/- 2%) had 33 +/- 1% necrosis; and 7 dogs with large LCC beds (42 +/- 1%) had 51 +/- 2% necrosis. The results show that much of the variability in infarct size among dogs subjected to coronary artery occlusion at the same anatomic site is a function of differences in ischemic bed size. Grouping dogs by occluded coronary bed size may improve the resolution of experimental studies testing the effect of various therapies on infarct size.     

Contribution of catechol O-methyltransferase to the removal of accumulated interstitial catecholamines evoked by myocardial ischemia

Catechol O-methyltransferase (COMT) plays an important role for clearance of high catecholamine levels. Although myocardial ischemia evokes similar excessive catecholamine accumulation, it is uncertain whether COMT activity is involved in the removal of accumulated catecholamines evoked by myocardial ischemia. We examined how COMT activity affects myocardial catecholamine levels during myocardial ischemia and reperfusion. We implanted a dialysis probe into the left ventricular myocardial free wall and measured dialysate catecholamines levels in anesthetized rabbits. Dialysate catecholamine levels served as an index of myocardial interstitial catecholamine levels. We introduced myocardial ischemia by 60 min occlusion of the main coronary artery. The ischemia-induced dialysate catecholamines levels were compared with and without the pretreatment with entacapone (COMT inhibitor, 10 mg/kg, i.p.). Acute myocardial ischemia progressively increased dialysate catecholamine levels. Acute myocardial ischemia increased dialysate norepinephrine (NE) levels (20,453+/-7186 pg/ml), epinephrine (EPI) levels (1724+/-706 pg/ml), and dopamine (DA) levels (1807+/-800 pg/ml) at the last 15 min of coronary occlusion. Inhibition of COMT activity by entacapone augmented the ischemia-induced NE levels (54,306+/-6618 pg/ml), EPI levels (2681+/-567 pg/ml), and DA (3551+/-710 pg/ml) levels at the last 15 min of coronary occlusion. Myocardial ischemia evoked NE, EPI, and DA accumulation in the myocardial interstitial space. The inhibition of COMT activity augmented these increments in NE, EPI, and DA. These data suggest that cardiac COMT activity influences on the removal of accumulated catecholamine during myocardial ischemia.     

Intramural PCO2: a reliable index of the severity of myocardial ischemic injury.

The present study was performed to evaluate the usefulness of changes in intramural oxygen (PmO2) and carbon dioxide (PmCO2) tensions shortly after coronary artery occlusion as indices of the severity of myocardial ischemic injury. In 14 open-chest, anesthetized dogs, a 60-min coronary artery occlusion was performed, during which PmO2 and PMCO2 were measured continuously with a mass spectrometer. Regional myocardial blood flow (RMBF) adjacent to the mass spectrometer probes was measured by the xenon-127 washout technique both before and 30 min after coronary artery occlusion. At the end of 60 min of occlusion, the dogs were killed, and biopsies for histological examination of 1-micron-thick sections were obtained from the tissue surrounding each mass spectometer probe. The decline in PmO2 during the 60-min occlusion bore no relationship either to the severity of ischemic injury as assessed by histological examination, or to the reduction of RMBF. In contrast, the magnitude of rise in PmCO2 during the 60 min of occlusion corresponded closely to both the severity of injury assessed histologically and the reduction of RMBF.     

Extracellular space in some isolated tissues

1. The spaces occupied by isotopically labelled inulin, polyethylene glycol, mol. wt. 4000 (PEG 4000), polyethylene glycol, mol. wt. 1000 (PEG 1000) and sucrose in metabolizing mammalian kidney and liver slices and in toad bladder epithelial cell preparations incubated in vitro have been examined.

2. In slices of mammalian tissue, and in homogenized liver, it proved impossible to extract inulin completely from tissue which had been dried. However, inulin was recovered as completely from both dried and undried toad bladder epithelial cells scraped from hemibladders incubated in vitro.

3. PEG 4000 occupied a space in all preparations similar to that from which inulin was extracted in dried tissue.

4. PEG 1000 and sucrose entered cellular water in mammalian slices, but PEG 1000 occupied a similar space to inulin in toad bladder epithelial cell preparations.

6. It is concluded that inulin enters cellular water in mammalian slices from which after drying of the slices it cannot be extracted. It thus rather fortuitously provides a measure of extracellular water under these conditions. In preparations of toad bladder epithelial cells inulin seems to be a satisfactory extracellular marker. PEG 4000, which did not appear to enter cellular water also allows a reasonable estimate of extracellular water. PEG 1000 is a suitable extracellular marker for toad bladder epithelial cell preparations but not for mammalian slices. Sucrose entered cellular water in both slices and toad bladder epithelial cells and is not a satisfactory extracellular marker in these tissues.

     

Kinetics of Calcium Accumulation in Acute Myocardial Ischemic Injury

The effect of ischemic injury on calcium uptake by dog myocardial cells was investigated in tissue damaged by transient or permanent occlusion of the circumflex branch of the left coronary artery. Tracer doses of ⁴⁵CaCl₂ were given at selected intervals before or after occlusion, and tissue uptake was measured in damaged and control left ventricular myocardium. No significant uptake of ⁴⁵Ca occurred after 60 minutes of ischemia produced by permanent occlusion of a coronary artery. However, 40 minutes of ischemia followed by 10 minutes of arterial reflow resulted in an 18-fold increase in Ca uptake in the injured tissue. Tissue ⁴⁵Ca increased linearly up through 10 minutes of arterial reflow but did not increase further with an additional 10 minutes of reflow. Myocardium reversibly injured by 10 minutes of ischemia followed by 20 minutes of arterial reflow did not accumulate excess ⁴⁵Ca. Calcium uptake is assumed to be an active process associated with mitochondrial accumulation of calcium into dense intramitochondrial granules of calcium phosphate. The uptake is a feature of irreversible cellular injury, but occurs only when arterial blood flow is present. The mechanism of the uptake has not been established. It appears to be related to defects in cellular permeability or mitochondrial function.     

Myocardial infarction in the baboon: regional function and the collateral circulation.

Occlusion of the anterior descending coronary artery was produced in sedated baboons 7-15 days after implantation of a micromanometer and ultrasonic crystals for measurement of regional left ventricular dimensions in ischemic, marginal, and control segments. One minute after coronary occlusion (CO), ischemic segments exhibited a marked systolic bulge with wall thinning, and percent systolic shortening of marginal segments decreased. Over the ensuing weeks, there was a progressive increase of end-diastolic lengths in marginal and ischemic segments, whereas systolic shortening in these segments did not improve significantly. Control segments did not change. In control baboons, the coronary collateral index was 55 +/-25 (SE) compared to 560 +/- 74 in normal dogs. One month after CO, the collateral index was 543 +/- 144 in baboons compared to 6,685 +/- 716 in dogs, regions of normal tissue were seen in the infarct (14.2 +/- 2% of left ventricular mass). Minimal coronary collateral development in the baboon provides a likely explanation for differences from the dog in regional functional responses and in the character of the infarct.     

Determinants of hemorrhagic infarcts. Histologic observations from experiments involving coronary occlusion, coronary reperfusion, and reocclusion.

Quantification of intramyocardial hemorrhage was performed in 69 pigs submitted to various protocols of coronary artery occlusion and reperfusion. The study groups include 1) permanent occlusion; 2) reperfusion after periods of coronary occlusion of 30, 45, 60, 90, and 120 minutes; 3) reperfusion with diltiazem and with 4) methoxamine after a 60-minute occlusion period; and 5) permanent reocclusion after a 30-minute period of reperfusion. Red blood cell counts were directly assessed by visual examination of histologic slices of myocardium and in a subgroup of animals by counts of red blood cells labeled with 99m-technetium pertechnetate. Hemorrhage occurs in infarcts reperfused after a duration of 45 minutes or more of coronary occlusion and after a period of reperfusion maintained for at least 30 minutes. Red blood cell counts were maximal in the mid portions of transmural sections of the infarcts, with decreasing values toward epicardium and endocardium. Diltiazem decreased total red blood cell counts, whereas methoxamine increased it and also caused subendocardial hemorrhage. The most powerful predictors of the severity of hemorrhage after sustained reperfusion were infarct size and higher blood pressure.     

Changes in ultrasonic attenuation indicative of early myocardial ischemic injury.

This study was designed to determine whether quantitative alterations in ultrasonic attenuation are associated with myocardial changes occurring after acute ischemic injury. Five hundred seventeen regions of myocardium from 41 dogs were studied in vitro at five intervals after coronary occlusion: 15 min, 1 h, 6 h, 24 h, 3 days, and 6 wk. Quantitative indices of ultrasonic attenuation were determined from the measured frequency dependence of the ultrasonic attenuation coefficient characterizing each myocardial region over the range 2-10 MHz. Independent definition of regions of ischemic injury was provided by either creatine kinase depletion or colloidal carbon dye distribution. Results of this study indicate that ischemic myocardial regions investigated 15 min to 24 h after coronary occlusion demonstrated ultrasonic attenuation significantly decreased from nonischemic regions (P less than 0.05). In contrast, ultrasonic attenuation was significantly increased in zones of ischemia or infarction investigated at 3 days and 6 wk after coronary occlusion (P less than 0.05 and P less than 0.01, respectively). These results indicate that altered attenuation of transmitted ultrasound by myocardium in vitro is an early manifestation of ischemia.     

Ischemic preconditioning modulates mitochondrial respiration, irrespective of the employed signal transduction pathway

We tested in the in vivo rat heart the hypothesis that although ischemic preconditioning can employ different signal transduction pathways, these pathways converge ultimately at the level of the mitochondrial respiratory chain. Infarct size produced by a 60-min coronary artery occlusion (69%+/-2% of the area at risk) was limited by a preceding 15-min coronary occlusion (48%+/-4%). Cardioprotection by this stimulus was triggered by adenosine receptor stimulation, which was followed by protein kinase C and tyrosine kinase activation and then mitochondrial K(+)(ATP)-channel opening. In contrast, cardioprotection by 3 cycles of 3-min coronary occlusions (infarct size 27%+/-5% of the area at risk) involved the release of reactive oxygen species, which was followed by protein kinase C and tyrosine kinase activation, but was independent of adenosine receptor stimulation and K(+)(ATP)-channel activation. However, both pathways decreased respiratory control index (RCI; state-3/state-2, using succinate as complex-II substrate) from 3.1+/-0.2 in mitochondria from sham-treated hearts to 2.4+/-0.2 and 2.5+/-0.1 in hearts subjected to a single 15-min and triple 3-min coronary occlusions, respectively (both P<0.05). The decreases in RCI were due to an increase in state-2 respiration, whereas state-3 respiration was unchanged. Abolition of cardioprotection by blockade of either signal transduction pathway was paralleled by a concomitant abolition of mitochondrial uncoupling. These observations are consistent with the concept that mild mitochondrial uncoupling contributes to infarct size limitation by various ischemic preconditioning stimuli, despite using different signal transduction pathways. In conclusion, in the in vivo rat heart, different ischemic preconditioning (IPC) stimuli can activate highly different signal transduction pathways, which seem to converge at the level of the mitochondria where they increase state-2 respiration.