Proteolysis of isolated mitochondria by myocardial lysosomal enzymes.

1. Solubilized mitochondria and lysosomal fractions were obtained from guinea-pig heart by differential centrifugation and selective membrane disruption. 2. Mitochondria incubated at 37 degrees C in the presence of lysosomal enzymes underwent proteolysis. The rate of protein degradation was inversely dependent on pH. 3. The use of proteinase inhibitors showed that at low pH the major enzyme involved in mitochondrial digestion was cathepsin D. 4. At neutral pH carboxyl proteinases were still active, but thiol proteinases accounted for most of the protein breakdown. 5. The role of lysosomal enzymes as mediators of mitochondrial damage in ischaemic myocardium is discussed.

Myocardial infarction in the dog: effects of intravenous propranolol.

The effects of propranolol on myocardial perfusion and metabolism during acute myocardial infarction were studied in 18 mongrel dogs. A reversible snare was placed on the left anterior descending coronary artery; regional myocardial perfusion was continuously measured using the short-lived isotope krypton-81m, and myocardial metabolism was assessed using the epicardial electrocardiogram and measurement of release of creatine kinase activity from the affected segment of myocardium. Six dogs with no arterial occlusion acted as "sham operated" dogs; six others in which the snare was occluded acted as a control group and a third group of six were given propranolol, 0.5 mg/kg, 30 minutes after coronary occlusion. All variables were recorded before and for 5 hours after coronary occlusion. Dogs treated with propranolol showed a significant improvement in regional myocardial perfusion to the affected segment, decreased loss of electrically active myocardium at the end of each experiment for any given degree of early S-T segment elevation and a delay in the local release of creatine kinase activity compared with that in the control dogs. These results suggest that propranolol exerts a beneficial effect on the progress of ischemic myocardial damage when given shortly after the onset of infarction.

Plasma lysosomal enzyme activity in acute myocardial infarction and the effects of drugs.

The activity of N-acetyl-beta-glucosaminidase (NAG) was found to be increased in serial plasma samples from patients with acute myocardial infarction (AMI). Maximum activity occurred 18 hr after the onset of chest pain, and a further peak of activity was found at 72 hr. Four isozymes of NAG were resolved from samples of human myocardium. All four isozymes were present in plasma from patients with AMI but not in normal plasma. beta-Glucuronidase, which is also present in myocardium, had increased activity in plasma at 18 hr but not at 72 hr in patients with AMI. Lysozyme, a lysosomal enzyme in white blood cells, had increased activity in plasma at 72 hr. There was a linear relationship (r = 0.98) between peak levels of NAG at 18 hr and the peak activity of the MB-isozyme of creatine kinase (CK-MB). Three groups of 10 patients were treated with drugs known to stabilize lysosomes during experimental myocardial anoxia. The first group received 25 mg/kg methylprednisolone sodium succinate i.v. within 4 hr of the onset of chest pain. The second group received propranolol, 5-mg, i.v. within 4 hr of the onset of chest pain. The second group received propranolol, 5-mg, i.v. within 4 hr of the onset of chest pain, and the third group comprised patients who developed AMI while on propranolol therapy and were maintained on this drug after admission to the hospital. All three groups showed an alteration in the pattern of lysosomal and cytosolic enzyme activity and a relative reduction in NAG activity compared to CK-MB.

The effects of propranolol on myocardial perfusion and metabolism during acute regional ischaemia.

The unique physical properties of the short-lived inert and freely diffusing isotope 81mkrypton allow a continuous observation to be made of regional myocardial perfusion. Eighteen dogs were anaesthetised and a reversible snare placed on the left anterior descending coronary artery (LAD). 81mKrypton was used to study regional myocardial perfusion, and myocardial metabolism was assessed using the epicardial ECG and release of creatine kinase activity (CK). Six dogs did not undergo LAD occlusion ("sham operated"); in six other dogs the LAD was occluded (controls), and another six dogs were given propranolol, 0.5 mg/kg, 20 min after LAD occlusion. All the parameters were measured before and for 5 h after LAD occlusion. When compared to controls, dogs treated with propranolol showed significant improvement (p less than 0.01) in regional myocardial perfusion; smaller loss of electrically active myocardium for any given degree of early ST-segment elevation; and a delay in the release of CK activity from a local coronary vein. These results suggest that propranolol exerts a beneficial effect following the development of acute myocardial infarction.

An experimental model for angina pectoris using krypton-81m. The dynamic balance between myocardial perfusion and metabolism.

16 anesthetized and open chest dogs were studied. Regional myocardial perfusion was assessed using a constant infusion of krypton-81m (half-life 13 sec) into the aortic sinuses and a gamma camera linked to a digital computer. The epicardial electrocardiogram was recorded and the plasma activity of creatine kinase was measured in serial blood samples from the aorta and a local coronary vein draining the area of myocardium supplied by the left anterior descending coronary artery (LAD). These parameters were observed throughout the whole period of a 5-h experiment. Two reversible snares were positioned on the middle portion of this artery. A critical narrowing of this vessel was produced and a peripheral venous infusion of isoproterenol (causing a 5--10% increase in heart rate and a 10--15% fall in blood pressure) was used to increase myocardial oxygen demand. During infusion there was both a relative and absolute fall in regional myocardial perfusion together with evidence of myocardial ischemia in the epicardial electrocardiogram. Provided the infusion was discontinued within 30 min (8 dogs) myocardial perfusion and the epicardial electrocardiogram returned to normal during a 5-h recovery period. In addition there was no efflux of creatine kinase activity from the ischemic area. When infusion was continued for 1 h (4 dogs) permanent alterations in myocardial perfusion and the epicardial electrocardiogram occurred and there was increased creatine kinase activity released from the area of myocardium by the narrowed vessel. Infusion for 40 min in 4 dogs produced permanent alterations in the parameters measured in 2 and complete recovery in the remaining 2. A further 4 dogs were studied in the same way but without a snare on the coronary artery. Isoproterenol given for 1 h produced no effects on any of the parameters either during or after infusion.

Precordial mapping of ST-segment elevation and Q waves following anterior myocardial infarction. The effects of established beta-blocking drugs.

The electrocardiographic (ECG) signs of ST-segment elevation and the development of Q was using 72-lead precordial surface mapping, and the release of creatine kinase (CK) activity has been studied in 47 patients with uncomplicated anterior myocardial infarction. These findings were compared with a further nine patients who had acute myocardial infarction but were receiving long-term beta-blocking drugs. It was found that ST-segment elevation and Q waves had rapidly changing and different natural histories and that beta-blocking drugs altered the natural history of ST-segment changes but had no effect on the pattern and time course for the loss of electrically active myocardium. There was a close relationship between the precordial area of ST-segment elevation at 2--3 h and the final development of Q waves in the patients with uncomplicated anterior myocardial infarction. No similar relationship could be found in those on beta-blocking drugs. The pattern of changes in plasma CK and its MB isoenzymes activity were similar for both groups. The relationship between early ST-segment elevation and the final area of Q waves may prove useful in clinical practice. This may not apply where beta-blocking drugs are commenced before the initial recording of ST-segment elevation.

Stabilization of lysosomes in anoxic myocardium by propranolol.

1. Isolated hearts of guinea-pigs were perfused aerobically and anoxically for 60 min. (+/-)-Propranolol was added to the perfusion medium in concentrations ranging from 10 ng to 10 microgram/ml. 2. Lysosome stability was assessed by measurements of latent acid hydrolase activity in homogenates of left ventricular tissue. 3. In the absence of propranolol, the integrity of the myocardial lysosomes was considerably reduced after 60 min anoxia. Lysosome stability was enhanced by the presence of (+/-)-propranolol. The optimal concentration was found to be 0.1 microgram/ml. This concentration of the (+)-isomer alone was less effective. 4. It was concluded that beta-adrenoceptor blockade was the major mechanism by which lysosome disruption was prevented but that some direct membrane effect of propranolol may also be involved.

The effects of nifedipine on acute experimental myocardial ischemia and infarction in dogs.

We studied 25 anesthetized and thoracotamized dogs before and during 5 hours of acute regional myocardial ischemia. Krypton-81m (81mKr) was infused constantly into the aortic sinuses. The myocardial equilibrium of this tracer was used to image and assess the distribution of regional myocardial perfusion using a gamma camera and digital computer. The epicardial ECG was recorded, S-T segment elevation and the loss of R and appearance of Q waves were measured, and the plasma activity of creatine kinase (CK) was determined in aortic and coronary venous blood throughout these experiments. Ten dogs underwent left anterior descending coronary artery (LAD) narrowing for 5 hours and received no drugs. Five dogs received nifedipine 13 microgram/kg, and another five received 1.0 microgram/kg intravenously 30 minutes after LAD narrowing. Those dogs receiving nifedipine, 13 microgram/kg, showed a 30% fall in aortic pressure, a 12% rise in heart rate, and an extension of regional ischemia. The ECG showed an extension of infarct size, and CK release into the coronary vein appeared earlier than in the controls. Dogs receiving nifedipine, 1 microgram/kg, showed a 12% fall in blood pressure, no rise in heart rate, an improvement in regional perfusion, and ECG signs that suggested limitation of infarct size. There also was delayed release of coronary venous CK. The effects of nifedipine on the natural history of regional myocardial perfusion, the electrocardiogram, and enzyme release from the heart were dose related and cannot be generalized. These observations warrant further clinical investigation to improve the use of this agent in man.

The effect of established beta-adrenoreceptor-blocking therapy on the release of cytosolic and lysosomal enzymes after acute myocardial infarction in man.

1. Serial venous blood samples were obtained from 45 patients with acute myocardial infarction. Ten of these patients were receiving beta-adreno-receptor-blocking drugs at the time of onset of chest pain and continued on these drugs during their stay in the coronary care unit. The activities of creatine kinase and its MB-isoenzyme (CK-MB) were assayed in the plasma. A lysosomal enzyme, beta-N-acetylglucosaminidase, was also assayed. 2. In the 35 untreated patients it was found that creatine kinase activity was maximal at a mean time of 21.3 +/- 1.3 h after the onset of chest pain, whereas in the patients receiving beta-adrenoreceptor-blocking drugs peak activity of the enzyme occurred at 24.4 +/- 0.7 h. 3. Peak CK-MB acitivity was also delayed from 18.1 +/- 1.6 h in the control group to 22.4 +/- 1.2 h in the treated patients. 4. The lysosomal enzyme showed a similar pattern of changes to that of CK-MB. Maximum activity in plasma occurred at 18.0 +/- 1.0 h after the onset of chest pain in the control group of patients. In the treated patients peak lysosomal enzyme activity was not found until 24.2 +/- 1.2 h. 5. These alterations in the time-course of plasma enzyme changes after acute myocardial infarction are consistent with the suggestion that beta-receptor antagonists may delay tissue damage during myocardial ischaemia.

Electrocardiographic precordial mapping in anterior myocardial infarction. The critical period for interventions as exemplified by methylprednisolone.

Serial 72-point precordial mapping of ECG has been recorded to describe the natural history of changes in the precordial areas of ST segment elevation and the development of Q waves in 51 patients with acute uncomplicated anterior myocardial infarction. Eight patients have been studied in the same way but received 25 mg/kg of methylprednisolone sodium succinate as a single intravenous injection within 6 hours from the onset of chest pain. There was a linear relationship between the stable precordial area of Q waves at 24 hours and the rapidly changing precordial areas of ST segment elevation at 2--3 hours, 5--6 hours and 12 hours after the onset of pain in the untreated patients. When methylprednisolone was given, the treated patients developed a smaller precordial area of Q waves at 24 hours than was predicted from the precordial area of ST elevation recorded before the drug was given. This study has introduced a technique that can provide a qualitative assessment of the relationship between ECG evidence of ischemia and infarction in each patient.

Electrocardiographic signs in experimental myocardial ischemia and infarction.

Epicardial ECG signs have been studied in 26 anesthetized and thoracotomized dogs in an attempt to follow the progress of tissue damage during regional myocardial ischemia. Epicardial ECG's were recorded before and during 15 min, 1 and 5 h of severe left anterior descending coronary artery narrowing. Epicardial ST segment elevation followed a complicated natural history. An analysis of variance showed the significant effects of respiration, heart rate and changes in time during myocardial ischemia. Regional epicardial R waves showed a transient increase in amplitude following coronary narrowing. There was no loss of electrically active myocardium following 15 min of ischemia. Irreversible loss of R waves were noted at between 30 and 45 min and progressed to full development within 5 h following coronary artery narrowing. The loss of electrically active myocardium (R loss plus Q waves) at 5 h was closely related to the myocardial depletion of creatine kinase activity (mu/mg DNA-1) at 24 h in each dog. The early manifestation of myocardial ischemia (ST segment elevation at 17 min) was closely related in the later evidence of cell death (R loss plus Q waves) in each dog. These relationships were less precise when the results were combined and this showed the variability between dogs in heart size and infarct size. The study suggested that the individual complete natural history of these ECG signs must be studied before they can be used to assess the extent and progress of myocardial ischemia and cell death.

Effects of nifedipine on creatine kinase release during myocardial ischemia in dogs.

25 anesthetized mongrel dogs underwent a left thoracotomy. Creatine kinase (CK) activity was measured in serial blood samples drawn simultaneously from the aorta and a coronary vein. The distribution of myocardial perfusion was determined by a continuous infusion of krypton-81m (half-life 13 sec) into the aortic sinuses. Heart rate and arterial blood pressure were also measured throughout the procedure. In 20 dogs regional myocardial ischemia was produced by ligation of a major branch of the left anterior descending coronary artery. Five of these dogs received 1 microgram.kg-1 nifedipine i.v. and a further 5 received 13 microgram.kg-1. Thoracotomy alone produced a slight rise in plasma CK activity but the arteriovenous difference (AV) across the segment of the heart remained positive over 5 h. Myocardial ischemia in the untreated dogs caused a considerable increase in CK activity and the AV difference became negative at 90 min. Treatment with the lower dose of nifedipine considerably reduced the plasma CK activity and the AV difference did not become negative until 3 h. Regional myocardial perfusion showed a significant improvement. Conversely, the higher dose of nifedipine produced a marked increase in the area of ischemia and an acceleration of CK release from the heart. This was associated with a decrease in arterial pressure and an increase in heart rate. These results show that nifedipine can be beneficial in experimental myocardial infarction but care must be taken to avoid hypotension and increases in heart rate.