Radioimmunoassay for creatine kinase isoenzymes.

Creatine kinase has three isoenzymes designated MM, MB, and BB, with BB being the grain form and MM the muscle form. Antibodies to BB creatine kinase were obtained by immunization of rabbits with human BB creatine kinase. The antibodies demonstrated specificity for BB and MB creatine kinase (myocardial isoenzyme) but no cross-reactivity with MM creatine kinase. With the use of this antibody, a highly sensitive radioimmunoassay capable of measuring picomolar amounts of MB creatine kinase has been developed. Clinical application of this method should provide a sensitive and specific test for the diagnosis of myocardial infarction.

Clot-selective coronary thrombolysis with tissue-type plasminogen activator.

Coronary thrombolysis, an intervention that can abort the sequelae of acute myocardial infarction, was accomplished within 10 minutes in dogs by intravenous administration of clot-selective, tissue-type plasminogen activator. In addition to inducing clot lysis, this promising fibrinolytic agent restored intermediary metabolism and nutritional myocardial blood flow, detectable noninvasively with positron tomography, without inducing a systemic fibrinolytic state.

Mitochondrial dysfunction induced by fatty acid ethyl esters, myocardial metabolites of ethanol.

Mechanisms responsible for alcohol-induced heart muscle disease have been difficult to elucidate partly because of previously obscure, demonstrable cardiac metabolism of ethanol. Recently, fatty acid ethyl esters were identified in our laboratory and found to be myocardial metabolites of ethanol. In the present study, they have been shown to induce mitochondrial dysfunction. Incubation of isolated myocardial mitochondria with fatty acid ethyl esters led to a concentration-dependent reduction of the respiratory control ratio index of coupling of oxidative phosphorylation and decrement of maximal rate of oxygen consumption. Furthermore, fatty acid ethyl esters were demonstrated to bind to mitochondria in vitro, and, importantly, 72% of intracellularly synthesized ethyl esters were found to bind to mitochondria isolated from intact tissue incubated with ethanol. Protein binding of fatty acid ethyl esters was markedly less than that of fatty acids. Because uncoupling of mitochondrial oxidative phosphorylation correlated with the cleavage of fatty acid ethyl ester shown to be initially bound to mitochondria, with resultant generation of fatty acid, a potent uncoupler, in a locus in or near the mitochondrial membrane, fatty acid ethyl esters may contribute to a potentially toxic shuttle for fatty acid with transport from physiological intracellular binding sites to the mitochondrial membrane; direct effects of fatty acid ethyl esters may also be deleterious. Operation of this shuttle as a result of ethanol ingestion and subsequent accumulation of fatty acid ethyl esters may account for the impaired mitochondrial function and inefficient energy production associated with toxic effects of ethanol on the heart.

Dependence of metabolic and structural heterogeneity of cholesterol ester-rich very low density lipoproteins on the duration of cholesterol feeding in rabbits.

Cholesterol ester-rich (CER) VLDL accumulate rapidly in the plasma of rabbits fed cholesterol-enriched diets. However, the major loci of enhanced synthesis of subfractions of CER-VLDL, their interaction with macrophages, and their relative contribution to atherogenesis have not yet been elucidated. To determine whether anabolism is hepatic or intestinal, subfractions of CER-VLDL were characterized at selected intervals from day 0 to 60 of cholesterol feeding. Rate zonal ultracentrifugation of CER-VLDL from rabbits fed cholesterol for 4 and 60 d demonstrated an early increase of the proportion of cholesterol carried in the intestinally-derived fraction (designated as Fx-I) of VLDL compared with that in hepatically-derived particles (Fx-H). Quantification by size exclusion HPLC also demonstrated that Fx-I was a prominent CER-VLDL component at day 4, while Fx-H particles became increasingly prominent with further cholesterol feeding. At both 4 and 60 d Fx-I stimulated cholesterol esterification and intracellular cholesterol content in macrophages more than the corresponding Fx-H did. In fact, Fx-H harvested at 4 d produced no cholesterol ester deposition. In contrast, Fx-H harvested at 60 d markedly stimulated cholesterol esterification and intracellular cholesterol content. Thus, both compositional and metabolic characteristics of CER-VLDL changed as a function of the duration cholesterol feeding.

Mechanisms contributing to malignant dysrhythmias induced by ischemia in the cat.

Continuously recorded bipolar electrograms were obtained simultaneously from epi-, endo-, and mid-myocardial regions of the ischemic and normal zones of cat left ventricle in vivo after coronary occlusion, analyzed by computer, and compared to regional cyclic AMP levels. Regional cyclic AMP content was used as an index of the combined local effects of: (a) efferent sympathetic nerve discharge; (b) release of myocardial catecholamines due to ischemia; and (c) circulating catecholamines. Ischemia resulted in a progressive increase in pulse width and rise time and a decrease in rate of rise of voltage (dV/dt) of the local electrograms from ischemic zones reaching a maximum within 2.4+/-0.3 min (mean+/-SE) at the time of onset of severe ventricular dysrhythmias, all of which returned toward control before the cessation of the dysrhythmia (33.5+/-1.5 min after coronary occlusion). Increases in cyclic AMP in ischemic zones preceded corresponding increases in the frequency of premature ventricular complexes (PVCs). Propranolol inhibited the increases in cyclic AMP and reduced the frequency of PVCs in animals without ventricular fibrillation. In animals with ventricular fibrillation, cyclic AMP was significantly elevated in normal and ischemic zones compared to animals with PVCs only. Electrical induction of PVCs or ventricular fibrillation in ischemic and nonischemic hearts failed to increase cyclic AMP. The results suggest that the changes in regional adrenergic stimulation of the heart may contribute to perpetuation of ventricular dysrhythmia and the genesis of ventricular fibrillation early after the onset of myocardial ischemia.

Oxygen at physiological concentrations. A potential, paradoxical mediator of reperfusion injury to mitochondria induced by phosphate.

Cellular injury induced by reperfusion after myocardial ischemia is manifested by striking mitochondrial damage as well as other hallmarks such as contraction band necrosis. Calcium has been implicated as a mediator of irreversible cellular injury in several systems. To identify other potential mediators of the mitochondrial injury associated with reperfusion, interactions between inorganic phosphate, oxygen, and mitochondria harvested from rabbit hearts were evaluated in vitro. Mitochondria exhibited rapid inactivation of oxidative phosphorylation after preincubation at 25 degrees C when phosphate and oxygen were present. Inactivation was partially but not completely precluded by EDTA, EGTA, magnesium, diltiazem, or ruthenium red, results in concert with findings of others suggesting involvement of a deleterious influx of calcium into mitochondria; exogenous calcium enhanced inactivation. However, the present data indicate that inactivation is prevented by incubation of mitochondria in the absence of oxygen, and demonstrate for the first time that injury elicited by phosphate is dependent on oxygen at physiological concentrations either because calcium and/or phosphate influx is linked to aerobic metabolism or because oxygen exerts deleterious effects on mitochondria, which may render them particularly susceptible to calcium influx. Since intracellular inorganic phosphate concentration increases markedly with ischemia, reperfusion with oxygenated medium may paradoxically augment mitochondrial injury in this setting. Thus, in the presence of increased intracellular concentrations of calcium and phosphate induced by ischemia, subsequent reestablishment of physiological levels of intracellular oxygen tension may promote mitochondrial damage, which is known to increase with reperfusion.

Quantitative assessment of the extent of myocardial infarction in the conscious dog by means of analysis of serial changes in serum creatine phosphokinase activity.

This study was designed to develop a method for quantitative assessment of infarct size in the conscious animal based on serial changes of serum creatine phosphokinase (CPK) activity. From 11 experiments in which myocardial CPK was injected intravenously in conscious dogs, the average CPK distribution space and average CPK fractional disappearance rate from serum were found to be 11.4% of body weight and 0.48% min respectively. In other experiments, myocardial infarction was produced in 22 conscious dogs by constriction of a left coronary artery snare and serum CPK activity was determined at frequent intervals for 24 hr. Since myocardial CPK depletion reflects infarct size, infarct size was determined directly by analysis of myocardial CPK content in the same animals 24 hr after coronary artery occlusion. CPK released from the infarct was determined from observed changes in serum CPK activity analyzed according to a model taking into account the fraction of CPK released from an infarct and the rates of appearance and disappearance of CPK activity from serum. Infarct size was calculated on the basis of observed changes in serum CPK and compared to infarct size determined directly by analysis of myocardial CPK depletion. Agreement was close and results from all experiments fit the equation: [infarct size (g) determined from serum CPK] = 1.13 x [infarct size (g) determined from myocardial CPK] - 1.3, r = 0.96, n = 22. The method described is useful for accurate assessment of infarct size in the conscious animal and for detection of modification of infarct size produced by pharmacologic interventions.

Metabolism of very low density lipoproteins after cessation of cholesterol feeding in rabbits. A factor potentially contributing to the slow regression of atheromatous plaques.

Aortic atheromatous plaques regress slowly in cholesterol-fed rabbits that have been returned to normal laboratory diet. To delineate metabolic factors potentially responsible for persistence of atherosclerosis under these conditions, the physical, chemical, and metabolic characteristics were determined for lipoproteins of d less than 1.006 g/ml; such lipoproteins are thought to be the major determinant of progression of atherosclerotic lesions in cholesterol-fed rabbits. At the time of return to a normal laboratory diet regimen after 3 mo of feeding with cholesterol-enriched laboratory diet, plasma cholesterol concentrations were 2,275 +/- 252 mg/dl, mostly attributable to cholesteryl ester-rich very low density lipoproteins (VLDL). On the hypercholesterolemic diet, fractional catabolic rates of plasma clearance of 125I-labeled VLDL were reduced (0.011 +/- 0.002 vs. 0.151 +/- 0.015 h-1), but the total VLDL catabolic rate was increased considerably (17.1 +/- 2.2 vs. less than 1.2 +/- 0.4 mg of protein/kg X d), because of the expansion of the endogenous pool of cholesteryl ester-rich VLDL. The total catabolic rate of VLDL was maintained above estimated control values (5.8 +/- 0.7 mg protein/kg X d) even 10 wk after return of the rabbits to a normal chow regimen, an effect attributable to continued high rates of cholesteryl ester-rich VLDL synthesis in liver. Accumulation of cholesteryl ester-rich VLDL into aortic tissue persisted at a high rate. Thus the persistence of aortic atheromatous lesions after cessation of cholesterol feeding was attributable in part to continued high rates of hepatic production of cholesteryl ester-rich VLDL and its persistent delivery into the aortic wall.

Alpha adrenergic contributions to dysrhythmia during myocardial ischemia and reperfusion in cats.

Alpha compared to beta adrenergic contributions to dysrhythmias induced by left anterior descending coronary occlusion and by reperfusion were assessed in chloralose-anesthetized cats (n = 96). Alpha receptor blockade with either phentolamine or prazosin significantly reduced the number of premature ventricular complexes during coronary reperfusion (321 +/- 62-14 +/- 10 premature ventricular complexes, P less than 0.001), abolished early ventricular fibrillation (from 25% in controls to 0%), and prevented the increase in idioventricular rate seen with coronary reperfusion. However, beta-receptor blockade was without effect. Ventricular dysrhythmias induced by coronary occlusion alone (without reperfusion) were attenuated markedly by alpha-receptor blockade under conditions in which perfusion (measured with radiolabeled microspheres) within ischemic zones was not affected. Alternative sympatholytic interventions including pretreatment with 6-hydroxydopamine to deplete myocardial norepinephrine from 8.8 +/- 1.4 to 0.83 +/- 0.2 ng/mg protein and render the heart unresponsive to tyramine (120 microgram/kg) attenuated dysrhythmias induced by both coronary occlusion and reperfusion in a fashion identical to that seen with alpha-receptor blockade. Although efferent sympathetic activation induced by left stellate nerve stimulation increased idioventricular rate from 66 +/- 6 to 144+/- 7 beats/min (P less than 0.01) before coronary occlusion, this response was blocked by propranolol but not by phentolamine. In contrast, during reperfusion the increase in idioventricular rate induced by left stellate nerve stimulation (to 203 +/- 14) was not inhibited by propranolol but was abolished by phentolamine (79 +/- 10). Intracoronary methoxamine (0.1 microM) in animals depleted of myocardial catecholamines by 6-hydroxydopamine pretreatment did not affect idioventricular rate before coronary occlusion. However, early after coronary reperfusion, methoxamine increased idioventricular rate from 33 +/- 7 to 123 +/- 21 beats/min (P less than 0.01). Thus, enhanced alpha-adrenergic responsiveness occurs during myocardial ischemia and appears to be primary mediator of the electrophysiological derangements and resulting malignant dysrhythmias induced by catecholamines during myocardial ischemia and reperfusion.

Early estimation of myocardial damage in conscious dogs and patients with evolving acute myocardial infarction.

To estimate the ultimate extent of myocardial damage during evolving myocardial infarction in conscious dogs and patients, we analyzed early serum creatine phosphokinase (CPK) changes with nonlinear curve-fitting techniques. In experiments with dogs, serial serum CPK changes were fit to a log-normal function by the least squares method; the extent of the completed infarct was calculated by analysis of observed serum CPK changes and verified by measurement of myocardial CPK depletion 24 h after coronary occlusion. Early prediction of myocardial damage was based on projected serum CPK values from best fit curves based on data obtained during the first 5 h after initial elevation of enzyme activity. The correlation between predicted and observed values was close (r > 0.96, n = 11). In 11 additional conscious animals subjected to coronary occlusion, isoproterenol was administered continuously as soon as damage had been estimated from projected serum CPK values. The extent of the completed infarct was assessed by analysis of all serial serum CPK values and verified by analysis of myocardial CPK depletion 24 h after coronary occlusion. In each experiment the calculated completed infarct size exceeded infarct size projected before administration of isoproterenol (average increase = 44+/-10 [SE]%). When similar calculations were applied in experiments with eight dogs treated with propranolol, myocardial salvage was detected in 50% of the animals. In 30 patients with uncomplicated acute myocardial infarction the extent of the completed infarct, measured by analysis of CPK activity in serum samples obtained every 2 h, was compared with damage estimated from CPK values projected by the best fit log-normal curve derived from data obtained during the first 7 h after the initial serum CPK elevation. The estimate of damage based on early data correlated closely with the extent of infarction calculated from all available serial serum CPK values (r = 0.93, n = 30). Thus, the extent of the completed infarct could be estimated accurately during the early evolution of infarction. In patients with spontaneous extension of infarction manifested by chest pain and electrocardiographic changes, the calculated extent of the completed infarct exceeded that predicted. Conversely, salvage of myocardium, after reduction of myocardial oxygen requirements by administration of trimethaphan, was reflected by reduction of the extent of the calculated completed infarct with respect to that predicted from early serum CPK changes.

Reduction of experimental myocardial infarct size by corticosteroid administration.

The influence of the administration of pharmacologic doses of hydrocortisone on the extent and severity of acute myocardial ischemic injury and on subsequent necrosis after acute coronary occlusion was investigated in 28 dogs. In order to study acute myocardial injury, repeated epicardial electrocardiograms were recorded from 10 to 15 sites on the anterior surface of the left ventricle. Average ST segment elevation (ST) and the number of sites in which ST segment elevation exceeded 2 mV (NST), indices of the magnitude and extent of myocardial injury, respectively, were analyzed at 30 and 60 min after coronary occlusion. In the control group ST and NST did not change significantly in this time interval while in the treated group, which received 50 mg/kg hydrocortisone just after the 30 min recording, ST fell from 3.5+/-0.8 to 1.1+/-0.4 mV (P<0.01) and NST was reduced from 6.7+/-1.1 to 1.4+/-0.8 (P<0.01). In order to study the influence of hydrocortisone on necrosis, epicardial ST segment elevation 15 min after coronary occlusion was compared to myocardial creatine phosphokinase activity (CPK) and histologic appearance 24 h later in each site. In a control group (14 dogs) a relationship was established between ST segment elevation at 15 min (in millivolts) and CPK activity (in international units per milligram of protein) 24 h later: log CPK = -0.0611ST + 1.26 (N = 102 specimens, r = -0.79). In the treated groups, hydrocortisone (50 mg/kg i.v.) was given either at 30 min after occlusion (seven dogs) or at 6 h after occlusion (six dogs). Both groups received supplementary doses of hydrocortisone (25 mg/kg) 12 h after occlusion. The two treated groups exhibited less CPK depression than that expected from ST segment elevation at each site, with slopes of the regression lines which were significantly less steep: log CPK = -0.0288ST + 1.26 (N = 48, r = -0.71) and log CPK = -0.0321ST + 1.31 (N = 48, r = -0.76) in the (1/2) h and 6 h groups, respectively. Histologically, sites with ST segment elevations of less than 2 mV at 15 min after occlusion exhibited normal appearance 24 h later. Sites with ST segment elevations (> 2 mV) in the control group showed histologic changes compatible with early myocardial infarction in 96% of specimens, while this occurred only in 61% and 63% of specimens, respectively, in the treated groups, showing that over one third of the sites were protected from undergoing necrosis due to the intervening hydrocortisone treatment. Thus pharmacological doses of hydrocortisone prevent myocardial cells from progressing to ischemic necrosis even when administration is initiated 6 h after coronary occlusion.

Accumulation of lysophosphoglycerides with arrhythmogenic properties in ischemic myocardium.

Lysophosphoglycerides, products of membrane phospholipid catabolism known to influence membrane function in several systems, appeared in the effluents of anoxic isolated rabbit hearts perfused at low flow and accumulated in perfused hearts and myocardium rendered ischemic in situ. Comparable concentrations of lysophosphoglycerides bound to albumin markedly and reversibly altered action potentials of isolated canine Purkinje fibers in vitro. Changes induced included diminution of the maximum diastolic potential, peak dV/dt of phase zero, amplitude, and action potential duration--alterations resembling those seen in ischemic myocardium in vivo. These electrophysiological alterations are compatible with changes implicated in predisposing to dysrhythmia dependent on reentry, a phenomenon potentiated by the presence of zones of decreased conduction. Thus, accumulation of lysophosphoglycerides induced by ischemia may contribute to the genesis of malignant dysrhythmia early after its onset.

Alpha adrenergic-mediated accumulation of calcium in reperfused myocardium.

Reperfusion of ischemic myocardium is associated with increases in total myocardial calcium (Ca+2), which may influence the ultimate extent of ischemic damage as well as the development of arrhythmias. Since reperfusion is also associated with enhanced alpha-adrenergic responsivity, this study was performed to determine the potential interactions between alpha-adrenergic receptors and myocardial calcium during reperfusion. Cats were subjected to 35 min of left anterior descending coronary artery occlusion and 10 min of reperfusion. Total myocardial calcium was measured by atomic absorption spectrometry. Intracellular calcium was calculated from measurements of extracellular space [( 3H]inulin). In control animals with reperfusion, total calcium increased from 0.32 +/- 0.03 to 0.65 +/- 0.05 mmol/100 g dry tissue (P less than 0.0001), while intracellular calcium increased from 0.15 +/- 0.03 to 0.40 +/- 0.05 mmol/100 g dry tissue (P less than 0.001). Pretreatment with the alpha-adrenergic blocking agents phentolamine or prazosin prevented the increase in total and intracellular calcium. Phentolamine and the aqueous soluble alpha 1-adrenergic antagonist BE-2254 administered as late as 2 min before reperfusion similarly attenuated the increase in tissue calcium. Although administration of BE-2254 2 min before reperfusion failed to block the reperfusion-induced increase in extracellular space, the increase in calculated intracellular calcium was prevented. beta-Adrenergic blockade with propranolol partially attenuated but did not prevent an increase in total tissue calcium. Labetalol, a combined alpha- and beta-adrenergic blocking agent completely blocked the increase in tissue calcium during reperfusion. Additional experiments performed after 70 min of ischemia with reperfusion demonstrated a 49% attenuation of the increase in tissue calcium with alpha-adrenergic blockade. Electron microscopy with pyroantimonate and x-ray microprobe analysis demonstrated a large increase in calcium precipitate in mitochondria after reperfusion in untreated animals. Though alpha-adrenergic blockade prevented the calcium deposition in mitochondria, other criteria of ischemia persisted. Thus, alpha-adrenergic blockade specifically prevents the increase in intracellular calcium during reperfusion in reversibly injured tissue, independent of alterations in extracellular space and tissue water.

Electrophysiologic effects of intracellular lysophosphoglycerides and their accumulation in cardiac lymph with myocardial ischemia in dogs.

Lysophosphatidylcholine (LPC) accumulates in ischemic tissue, and exogenous LPC (20-100 microM) induces electrophysiologic alterations in vitro. However, to determine whether compartmentalization is critical, intracellular pressure microinjection of LPC was performed with simultaneous recording of the transmembrane action potential. Intracellular LPC in concentrations as high as 500 microM (n = 18), calculated based on calibration of injectate volume and cellular volume, did not induce electrophysiologic alterations. The concentrations and efflux of phospholipids and lysophospholipids were assessed in lymph obtained from the supracardiac lymph vessel in anesthetized dogs to assess the extent of extracellular accumulation. Prior to ischemia, phosphatidylcholine (PC) was the major phospholipid in lymph (79 +/- 2%) with substantial quantities of sphingomyelin (11 +/- 2%) and LPC (6 +/- 1%). With ischemia, the concentration of LPC increased by 18%, and net efflux of LPC increased by 24% (P less than 0.01) with no net efflux of PC or other assayed phospholipids. The calculated concentration of LPC increased from 84 to 197 microM in lymph within the ischemic region, a concentration sufficient to induce electrophysiologic derangements.

A relationship between ultrasonic integrated backscatter and myocardial contractile function.

We have shown previously that the physiologic, mechanical cardiac cycle is associated with a parallel, cardiac cycle-dependent variation of integrated backscatter (IB). However, the mechanisms responsible are not known. The mathematical and physiological considerations explored in the present study suggest that the relationship between backscatter and myocardial contractile function reflects cyclic alterations in myofibrillar elastic parameters, with the juxtaposition of intracellular and extracellular elastic elements that have different intrinsic acoustic impedances providing an appropriately sized scattering interface at the cellular level. Cardiac cycle-dependent changes in the degree of local acoustic impedance mismatch therefore may elicit concomitant changes in backscatter. Because acoustic impedance is determined partly by elastic modulus, changes in local elastic moduli resulting from the non-Hookian behavior of myocardial elastic elements exposed to stretch may alter the extent of impedance mismatch. When cardiac cell mechanical behavior is represented by a three-component Maxwell-type model of muscle mechanics, the systolic decrease in IB that we have observed experimentally is predicted. Our prior observations of regional intramural differences in IB and the dependence of IB on global contractile function are accounted for as well. When the model is tested experimentally by assessing its ability to predict the regional and global behavior of backscatter in response to passive left ventricular distention, good concordance is observed.

External quantification of myocardial perfusion by exponential infusion of positron-emitting radionuclides.

A technique was developed and evaluated using the exponential infusion of positron-emitting diffusible tracers to quantitate myocardial perfusion. The approach employs a parameter that rapidly reaches a constant value as a function of tracer delivery rate, isotope decay constant, and the monotonically increasing tissue radioactivity. Isolated rabbit hearts with controlled flow were used to evaluate the approach, because tracer kinetics in such preparations mimic those in vivo. Accordingly, exponential infusions of H2 15O and [11C]butanol were administered to 25 isolated rabbit hearts perfused with Krebs-Henseleit solution (KH) alone or KH enriched with erythrocytes (KH-RBC, hematocrit = 40). With flow varied from 1.2 to 5 ml/g per min in eight KH hearts infused with H2 15O, actual and estimated flow correlated closely (r = 0.95, n = 52 determinations). For the KH-RBC hearts, flow was varied from 0.3 to 1.5 ml/g per min. Actual and estimated flow correlated significantly for both the 14 KH-RBC hearts infused with H2 15O (r = 0.90, n = 89 determinations) and the 3 KH-RBC hearts infused with [11C]butanol (r = 0.93, n = 13 determinations). In addition, the required exponentially increasing arterial tracer concentrations were shown to be attainable in vivo in dogs and rhesus monkeys after intravenous exponential administrations of tracer. The results suggest that the approach developed employing exponential tracer infusion permits accurate measurement of myocardial perfusion and that it should prove useful in the noninvasive measurement of regional myocardial perfusion in vivo by positron emission tomography.

Coronary artery reperfusion. I. Early effects on local myocardial function and the extent of myocardial necrosis.

The effects of coronary artery reperfusion 3 hr after coronary occlusion on contractile function and the development of myocardial damage at 24 hr was studied experimentally. In 14 control and 6 reperfused dogs, relationships between epicardial ST segment elevation 15 min after coronary occlusion and myocardial creatine phosphokinase activity (CPK) and histologic appearance 24 hr later were examined. The electrocardiograms were recorded from 10 to 15 sites on the left ventricular epicardium and transmural samples for CPK and histology were obtained from the same sites where epicardial electrocardiograms had been recorded. An inverse relation existed between ST segment elevation (mv) 15 min after occlusion and log CPK activity (IU/ mg of protein) 24 hr later, log CPK = - 0.06ST + 1.26. In dogs subjected to coronary artery reperfusion, there was significantly less CPK depression (log CPK = - 0.01ST + 1.31, [P < 0.01]) than that expected from the control group. In the control group 97% of specimens showing ST segment elevations over 2 mv at 15 min showed abnormal histology 24 hr later. In contrast, in the reperfused group 43% of sites exhibiting elevated ST segment at 15 min showed abnormal histology 24 hr later. In six additional dogs it was shown that the paradoxical movement of the left ventricular wall could be reversed within 1 hr of perfusion. Therefore, by enzymatic and histologic criteria, as well as by functional assessment, coronary artery reperfusion 3 hr after occlusion resulted in salvage of myocardial tissue.

Coronary artery reperfusion. II. Reduction of myocardial infarct size at 1 week after the coronary occlusion.

The question of whether or not the size of an area of myocardial infarction, measured at 1 wk after coronary occlusion, can be influenced by coronary artery reperfusion was examined in dogs. In seven control experiments the anterior descending coronary artery was ligated, while in seven other studies the occlusion was released after 3 hr. In all animals calibrated photographs were used to assess the zone of hypoperfusion and the acutely injured area of epicardial ST segment elevation, as well as the extent of damage at postmortem 1 wk later. In control dogs, the gross infarct size at postmortem averaged 63.8+/-7.3% of that predicted from the acutely injured zone. However, in reperfused hearts the average gross infarct size at 1 wk was only 10.2+/-4.4% of that predicted. Transmural specimens were obtained at autopsy for histology and measurement of myocardial creatine phosphokinase (CPK) activity from sites initially used for epicardial electrocardiography. In control animals, there was a direct relationship between the degree of ST segment elevation and the degree of cell necrosis in transmural histologic sections. ST segment elevation also predicted myocardial CPK (international units per milligram protein): log CPK = - 0.0613 ST + 1.17 (r = 0.66, n = 56 sites). In the reperfused animals, log CPK = - 0.166 ST + 1.36 (r = 0.69, n = 46 sites) showing almost complete preservation of CPK activity at 1 wk, sparing being most prominent in the epicardial zone. Similarly, there was a good correlation between myocardial CPK activity and the histological assessment of cell destruction, the degree of cell damage = - 0.152 CPK + 3.86 (r = 0.86; n = 102 sites). Thus, control dogs showed severe myocardial CPK depletion and histologic evidence of extensive cell destruction, whereas animals subjected to coronary artery reperfusion had little CPK depletion and much less evidence of myocardial cell necrosis 1 wk later.

Prophylaxis of early ventricular fibrillation by inhibition of acylcarnitine accumulation.

Hypoxia in isolated myocytes results in accumulation of long-chain acylcarnitines (LCA) in sarcolemma. Inhibition of carnitine acyltransferase I (CAT-I) with sodium 2-[5-(4-chlorophenyl)-pentyl]-oxirane-2-carboxylate (POCA) prevents both the accumulation of LCA in the sarcolemma and the initial electrophysiologic derangements associated with hypoxia. Another amphiphilic metabolite, lysophosphatidylcholine (LPC), accumulates in the ischemic heart in vivo, in part because of inhibition of its catabolism by accumulating LCA. It induces electrophysiologic alterations in vitro analogous to early changes induced by ischemia in vivo. The present study was performed to determine whether POCA could prevent accumulation of both LCA and LPC induced by ischemia in vivo and if so, whether attenuation of early arrhythmogenesis would result. LAD coronary artery occlusions were induced for 5 min in chloralose-anesthetized cats. Coronary occlusion in untreated control animals elicited prompt, threefold increases of LCA (73 +/- 8 to 286 +/- 60 pmol/mg protein) and twofold increase of LPC (3.3 +/- 0.4 to 7.5 +/- 0.9 nmol/mg protein) selectively in the ischemic zone, associated with ventricular tachycardia (VT) or ventricular fibrillation (VF) occurring within the 5-min interval before acquisition of myocardial samples in 64% of the animals. POCA prevented the increase of both LCA and LPC. It also prevented the early occurrence of VT or VF (within 5 min of occlusion) in all animals studied. The antiarrhythmic effect of POCA was not attributable to favorable hemodynamic changes or to changes in myocardial perfusion measured with radiolabeled microspheres. Thus, inhibition of CAT-I effectively reduced the incidence of lethal arrhythmias induced early after the onset of ischemia. Accordingly, pharmacologic inhibition of this enzyme provides a promising approach for prophylaxis of sudden cardiac death, that typically occurs very soon after the onset of acute ischemia, in man.

Ethanolamine plasmalogen methylation by rabbit myocardial membranes.

Enzymatic methylation of alkenylacylglycerophosphoethanolamine to form alkenylacylglycerophosphocholine was observed in rabbit myocardial membranes, and was compared to the corresponding methylation sequence for diacyl substrates. Membranes were incubated with S-adenosyl-L-[methyl-3H]methionine and assayed for incorporation of radioactivity into selected lipids. The rate of incorporation of methyl groups into diacylglycerophosphocholine exceeded that for alkenylacylglycerophosphocholine, 12.0 +/- 3.6 vs. 3.9 +/- 0.7 pmol product formed/mg per h (mean +/- S.D.), even when normalized for ethanolamine substrate concentration (5.7 +/- 1.6 vs. 1.8 +/- 0.4 pmol CH3 incorporated/mumol diradylglycerophosphoethanolamine). Rabbit myocardial phospholipid methyltransferase activity is optimal at basic pH for each substrate, is moderately stimulated by added Ca2+ or Mg2+, and is completely inhibited by S-adenosylhomocysteine. An apparent Km of 0.2 mM for S-adenosylmethionine applies to diacyl- and alkenylacylglycerophosphocholine formation; at low concentrations of methyl donor (0.003 mM), the monomethylated products accumulate.