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We investigated simultaneously the correlations between dobutamine-induced contractile reserve (CC), thallium-201 reverse redistribution (RR) and a mismatch between perfusion and metabolism (MM) to the magnitude of functional recovery. In 32 patients with coronary angioplasty early after infarction, echocardiography was performed at low-dose dobutamine stress within 1 week and at resting state at 1 month. Thallium-201/iodine-123 beta-methyl-iodophenyl pentadecanoic acid (BMIPP) dual-isotope single photon emission tomography was performed at 1 month. Wall motion and the uptake of each tracer were scored as 0 to 2 in the infarct-related segments, and CC, RR, and MM were evaluated in the infarct-related segments. In 71 akinetic or dyskinetic segments before reperfusion, the initial thallium-201 uptake and initial BMIPP uptake scores and the 4 hour redistribution thallium-201 uptake scores were less severe in the group with complete functional recovery (group A), followed by the group with incomplete recovery (group B) and then the group with no recovery (group C) (each P < 0.0001). CC was the greatest in group A, followed by group B, and then group C (76.2% in 16/21, 60% in 15/25, 36% in 9/25, P = 0.0212). RR and MM were greater in group B (52% in 13 and 64% in 16) than in groups A and C (19% in 4 and 8% in 2, 33.3% in 7 and 24% in 6, P = 0.0013 and P = 0.0113). The intensity of functional damage reflects perfusion and metabolism, but the delayed and incomplete functional recovery after reperfusion may be closely related to RR, MM, and CC.

Objective: ST-segment depression is believed as a common electrocardiographic sign of myocardial ischemia during exercise testing. Ischemia is generally defined as oxygen deprivation due to reduced perfusion. However, the exact relationship of the ischemic definition to ST-segment depression remains unclear. This study was conducted to evaluate the correlation between myocardial oxygen demand and myocardial blood flow (MBF) when ischemic (horizontal or downsloping) ST-segment depression of > or = 0.1 mV 80 ms after the J point developed during low-level exercise., Methods and Results: Seventy-two patients with angiographically proven coronary artery disease (CAD) and 9 healthy volunteers underwent exercise positron emission tomography (PET). Myocardial oxygen demand was defined as a rate-pressure product (RPP, heart rate x systolic blood pressure) during exercise and MBF was quantified by nitrogen-13 ammonia perfusion PET. The myocardial demand-supply balance (MDSB) index was calculated from the MBF ratio (values during exercise/values at rest) against the RPP ratio (values during exercise/values at rest). The MDSB index was significantly lower in patients with ischemic ST-segment depression than in patients with non-ischemic ST depression and healthy volunteers (0.82 +/- 0.16 vs. 1.02 +/- 0.17, p < 0.0001 and vs. 0.99 +/- 0.14, p = 0.0109). Further, the presence of inadequate increase in MBF of < or = 10% (2 SD below the mean % value of healthy volunteers) during exercise in regional myocardium perfused by stenotic CAD significantly correlated with exercise-induced ischemic ST-segment depression (p = 0.0105)., Conclusions: Our study could demonstrate that exercise-induced ischemic ST-segment depression is associated with myocardial ischemia due to exercise-induced imbalance between myocardial oxygen demand and global and regional MBF supply in patients with proven CAD.

Ischemic myocardium avidly incorporates fluorine-18 fluorodeoxyglucose (F-18 FDG) in the fasting state, in contrast to the relative absence of F-18 FDG uptake in normal myocardium with sufficient blood flow in the fasting state. Although many studies have attempted to use F-18 FDG uptake to discriminate ischemic but viable myocardium from scarred myocardium, little is known clinically about the correlation between blood flow and F-18 FDG uptake in ischemic myocardium. We studied the critical level of blood flow that causes avid F-18 FDG uptake in myocardium in 9 patients. All patients had angiographically proven ischemic heart disease but no diabetes. Regional myocardial blood flow (RMBF) was measured quantitatively by positron emission tomography (PET) using nitrogen-13 ammonia in the resting state, in which the normal value was 80.2 +/- 13.0 ml/min/100 cm3. The F-18 FDG uptake in myocardium was assessed with the differential uptake ratio (DUR) scale. We constructed circumferential profiles of radioactivity uptake in myocardium for each study, and chose 780 sections of myocardium in which the relation between the two factors could be analyzed. In moderately ischemic to normal myocardium with RMBF of 50 to 90 ml/min/100 cm3, RMBF and F-18 FDG uptake were negatively correlated (r = -0.44, p < 0.01). When RMBF was 50 to 60 ml/min/100 cm3 (n = 121), the peak DUR value of F-18 FDG uptake was 4.0 +/- 2.0. The two factors were not correlated when RMBF was less than 50 ml/min/100 cm3 or 90 ml/min/100 cm3 or higher. Our results suggest that RMBF and F-18 FDG uptake values as measured with PET may provide valuable information on the possible benefit of intervention in ischemic heart disease.

Diabetes mellitus is associated with alterations in lipid metabolism and cardiac dysfunction despite an absence of coronary arteriosclerotic changes. To investigate mechanisms of cardiac dysfunction in diabetic cardiomyopathy, we studied the relation between activities of membrane-bound enzymes and surrounding phospholipids in rats with diabetes induced with a single intravenous injection of streptozotocin (65 mg/kg). We found that total phospholipid content of sarcoplasmic reticulum membrane increased significantly 8 weeks after treatment with streptozotocin owing to increases in phosphatidylcholine and phosphatidylethanolamine, a decrease in arachidonic acid, and an increase in docosahexaenoic acid in the early stage of diabetes. Sarcolemmal Na+/K(+)-ATPase activity and the number of receptors decreased in isolated cardiomyocytes of diabetic rats 8 weeks after streptozotocin administration. The Ca2+ uptake of both sarcoplasmic reticulum and mitochondria decreased simultaneously in permeabilized, isolated cardiomyocytes from diabetic rats. The depression of membrane-bound enzyme activities was correlated with alterations in phospholipids, which are closely related to the microenvironment of membrane-bound enzymes and influence intracellular Ca2+ metabolism. Because these changes in phospholipids and fatty acids were reversible with insulin therapy, they are diabetes-specific and might be a cause of cardiac dysfunction in diabetes.

We evaluated the effects of angiotensin-converting enzyme (ACE) inhibition on metabolic changes in myocardial organelles, myocardial hypertrophy, and interstitial fibrosis in the early stage of hypertension. An ACE inhibitor, imidapril (2.5 mg/kg per day), a calcium-channel blocker, diltiazem (30 mg/kg per day), or vehicle was given to spontaneously hypertensive rats (SHRs) from 10 to 18 weeks of age. Single myocytes were isolated enzymatically from the left ventricles of these SHRs and normotensive Wistar-Kyoto (WKY) controls at 18 weeks of age. In single ventricular myocytes, enzyme activities in the sarcoplasmic reticulum (SR) and the sarcolemma (SL) and the mitochondrial respiratory control ratio (RCR) were determined. In 18-week-old SHRs receiving vehicle, myocardial hypertrophy and interstitial fibrosis developed, and SR Ca2+ AT-Pase activity and the mitochondrial RCR were significantly lower and SL Na+, K(+)-ATPase activity was significantly higher than in age-matched WKYs. However, compared with diltiazem, imidapril was better able to prevent the development of myocardial hypertrophy and interstitial fibrosis, to improve SR Ca(2+)-ATPase activity and the mitochondrial RCR, and to increase SL Na+, K(+)-ATPase activity. These results suggest that ACE inhibition can prevent the development of morphologic changes associated with hypertension-induced left ventricular remodeling, such as myocardial hypertrophy and interstitial fibrosis, and can counteract ongoing dysfunction of organelle metabolism early in the development of hypertension.

Changes in myocardial lactate metabolism during ramp exercise were investigated through great cardiac vein catheterization in 15 patients with effort angina (EA) and 7 patients with microvascular angina (MVA). The exercise test was performed using a supine bicycle ergometer. Blood samples were obtained from the great cardiac vein (GCV) and the radial artery each minute during exercise. Patients in the EA group showed a point at which the lactate extraction ratio (LER) and the ST level decreased rapidly during exercise. This point was clearly recognized in 12 of 13 patients, and may represent the ischemic threshold. Both the LER and ST level showed similar changes during exercise, and these values were significantly different between the ischemic threshold and peak exercise (p < 0.01). Both the LER and ST level were strongly correlated with the duration of exercise until the threshold (r = 0.703). In the MVA group, both the LER and ST level during exercise continuously decreased throughout exercise without an ischemic threshold in all of the subjects. Endomyocardial biopsy revealed sclerosis of small arteries in the myocardium in all of the patients. In both effort angina and microvascular angina, a close correlation was noted between the change in ST and that in the myocardial lactate extraction ratio.

Myocellular injury induced by acute ischemia and subsequent reperfusion was studied in 38 dogs, with special reference to sarcolemmal permeability as determined by the vital ionic lanthanum (La3+) probe technique and electron microscopy. The left anterior descending coronary artery (LAD) was occluded in 14 dogs for 10 to 60 min, and the ischemic zone was perfused slowly for 7 min with a La(3+)-containing solution. In 21 dogs, the LAD was released for 10 min after occlusion and was then reperfused for 7 min with arterial blood plus the La(3+)-containing solution. Subsequently, in both groups of animals, the ischemic myocardium was subjected to perfusion fixation in preparation for electron microscopy. In normal cardiac myocytes, La3+ was localized exclusively in the extracellular space. After 10 to 20 min of ischemia, more than 80% of myocytes appeared normal or were damaged only slightly, and the majority continued to exclude La3+. After 10 min of ischemia, deposits of lanthanum were detected in 1 and 6% of myocytes in the absence or presence of reperfusion, respectively. The number of cells with such deposits was markedly increased after 30 min of ischemia (19%), as well as after 20 min of ischemia followed by reperfusion (17%), prior to the development of irreversible myocardial damage. After 60 min of ischemia with or without reperfusion, about 30% of myocytes showed severe injury with particulate deposits of lanthanum throughout the entire cell. These results indicate that sarcolemmal permeability increases during the early stage of myocardial injury due to ischemia or ischemia-reperfusion and contributes to the development of myocardial damage.

We studied the ultrastructure of cardiac myocytes and small blood vessels obtained by endomyocardial biopsy from 21 patients with microvascular angina. Ischemic ST segment depression during atrial pacing was recognised in all the patients who had normal coronary arteriograms and biopsy tissues were examined by light and electron microscopy. In patients with microvascular angina, insufficient increases in coronary sinus blood flow and in myocardial oxygen consumption measured with a Webster's catheter were apparent during atrial pacing. Biopsy samples under the light microscope showed evidence of myocardial hypertrophy and sclerosis of small arteries and arterioles with perivascular fibrosis in 18 of 19 (95%) patients. Electron microscopy revealed that many endothelial nuclei in capillaries were swollen and that lumina of small arteries and arterioles were irregularly narrowed with proliferated and deformed medial smooth muscle cells. These findings suggest that disturbances in the coronary microcirculation in these patients is responsible for the ischemic changes in electrocardiograms.

To investigate the pathologic changes of the myocardium and the adverse effects of radiofrequency (RF) catheter ablation, we observed canine hearts that had been ablated by RF energy. Catheter ablation was administered to three sites of the hearts of 46 dogs: endomyocardium, coronary sinus, and atrioventricular junction. The dogs were sacrificed 0-12 weeks after ablation and the lesions were stained with hematoxylin-eosin and Azan stain. Each ablated lesion was well demarcated from the adjacent normal tissue. The margin between the ablated lesion and normal tissue became clearer with time. The lesions were hemispherical and measured about 2 to 10 mm in diameter and 1 to 6 mm in depth. Histologic examination in the acute phase demonstrated the presence of coagulation necrosis of the myocardium, interstitial hemorrhage and edema, followed by infiltration of inflammatory cells. Granulation tissue replaced the normal myocardium. In the chronic phase, the lesions became fibrotic and fatty. A mural thrombus was found in 30% of the cases of endomyocardial ablation and 50% of the right ventricular lesions were transmural. Ablated lesions in the coronary sinus extended to the left atrium and left ventricle. Obstruction of the coronary sinus was observed in 8% of the ablated dogs. Most of the lesions with complete atrioventricular block were ablated at the His penetrating region of the conduction system. Thus, since the ablated lesions were clearly delineated, catheter ablation using RF energy appears to eliminate only the target area.

We studied the relative synthetic phase (S-phase) ratio of cardiac myocytes of spontaneously hypertensive rats (SHR) utilizing monoclonal antibody to 5-bromo 2'-deoxyuridine (BrdU), a halogen derivative of thymidine. BrdU was administered intraperitoneally to SHR and Wistar Kyoto rats at 66.7 mg/kg/day for 3 consecutive days. The rats were sacrificed 1 week after the first injection of BrdU. Using anti-BrdU antibody, myocardial tissue was stained by the avidin biotinylated peroxidase complex method. We assessed the ability of the nuclei to synthesize DNA. The percentage of BrdU-labeled myocytes was higher in SHR than in control rats at 9 weeks. The hyperplastic phase of heart growth in rats was presumed to occur about 3 weeks after birth. From these results, the ability of the myocardial cell to synthesize DNA is probably enhanced under the load of high blood pressure in the early hypertrophic phase following hyperplasia.

Impairment of sarcolemmal permeability after the initiation of acute ischemic myocardial injury was studied using an ionic lanthanum (La3+) probe in electron microscopy. Acute ischemic myocardium was induced by ligation of the left anterior descending coronary artery in dogs. In normal cardiac myocytes La is localized exclusively in the extracellular space. i.e. on the glycocalyx, in the T-system and in intercalated discs, with normal fine structures. In ischemic myocardial cells, La deposits were found in the cytosolic space in 22% of subendocardial cells with mild to moderate, but non-necrotic, fine structural changes as early as 30 min. The number of myocytes with La deposition increased with advancing ischemic injury, and necrotic fine structural changes were recognized following ischemia for 60 min. These results indicate that deposition of La occurs before the appearance of irreversible morphologic alterations in ischemic myocardial cells, and suggest an increased permeability of the sarcolemma for Ca2+ and the development of degradation of plasma membrane integrity.

Reperfusion injury in early myocardial ischemia was studied in the dog with special reference to sarcoplasmic reticulum (SR) and contraction bands. Acute myocardial ischemia (I) was induced by occlusion of the left anterior descending coronary artery (LAD) for 10, 20 and 30 min followed by reperfusion for 15 min (R). Ca(++)-ATPase activity of SR in 10-min-R-Group was significantly reduced to 60% of control activity, but activity of 10-min-I-Group remained near the control level in subendomyocardium (Endo). ATPase activity in 30-min-I-Group diminished to 60% of control activity in Endo and it was similar for 30-min-R-Group. In ischemic myocardium, composition of major ATPase protein decreased significantly in 30-min-I-Group and similar reduction was observed in 20-min-R-Group in Endo. In morphology proportion of appearance of contraction bands in Endo was significantly increased in 20-min or longer-R-Groups. These results suggest that reperfusion injury is likely to occur when coronary artery is reflowed after 10 min of ischemia. This may be caused by increased intracellular Ca++ at a very early stage of reperfusion period, and reperfusion injury may be induced due to acceleration in the necrotic process of the membrane system in the myocytes during ischemia.

We studied alterations in respiratory activity of mitochondria (Mt) in non-infarcted myocardium (NIZ) under severe pump failure complicated by acute myocardial infarction (AMI). Dogs in which AMI was induced were divided into two groups; one in which left ventricular systolic pressure (LVPs) was maintained higher than 70% of preligation level (ND group); and one in which LVPs was diminished to less than 70% (D group). Regional myocardial blood flow (MBF) in NIZ reduced significantly in proportion to decreases in LVPs and cardiac output (CO). State -III activity and RCR decreased in proportion to reductions in MBF, LVPs, and CO in Mt from NIZ of D group. Complex-I and DNP-stimulated ATPase activities were also reduced in NIZ of D group. Morphologic studies revealed slight swelling and fusion of mitochondria in NIZ cells of D group, but no changes such as the appearance of a dense deposit indicating ischemic damage were seen. Pump failure in AMI is likely to be caused partly by impaired function of Mt in NIZ induced by hypoperfusion. Improvement of metabolic impairment in NIZ is important in the treatment of pump failure.

Ultrastructural localization and intensity of ATPase activity were studied in myocardial cells from biopsies with reference to fine-structural alterations and cardiac functions in patients with various heart diseases. ATPase activity was found to be intense in the sarcoplasmic reticulum (SR), the matrices of the mitochondria (Mt), on the myofilaments (Mf) and along the gap-junctions of intercalated discs in the control myocardial cells. ATPase activity was more intense in cardiac myocytes from well-functioning or ultrastructurally well preserved hearts. In failing and degenerating hearts, ATPase activity was decreased. ATPase activity was more intense in clinically-improving than in clinically-worsening patients. However, the localized pattern of ATPase activity was similar in each heart disease. These results suggest that cytochemical observation of ATPase activity can reflect not only fine structural changes in cardiac myocytes, but also the metabolic state in the diseased heart, and is valuable therefore from the standpoint of clinical medicine.

Comparative studies of the fine structural changes and histochemical examination of adenosine triphosphatase (ATPase) activity were performed in canine ischemic heart muscle cells following left coronary artery ligation. In the intact myocardial cells, the ATPase activities were observed most intensely in the sarcoplasmic reticulum (SR), particularly in the terminal cisternae (TC), and moderately around gap junctions of the intercalated discs and on myofilaments. Ischemic cellular changes occurred three to 24 hours after coronary ligation and became severe after three to seven days. ATPase activities decreased at around 48 hours and also became weak at three to seven days. Two to three weeks after ligation, improvement of the fine structure and increase in ATPase activities were observed in the ischemic cells, suggesting recovery. ATPase activities in the ischemic cardiac cells appeared to be decreased in parallel with the process of the fine structural changes.

1. Quantitative analysis of structural proteins from a minute amount of myocardial tissue was performed from 10 mu thick cardiac tissue slices weighing 2 to 5 mg by the extraction in glycerol solution and by sodium dodecylsulfate (SDS) gel electrophoresis, and these changes were compared with the histologic alterations in the striated structure of the adjacent cardiac slices in the experimental myocardial infarction in the dog. 2. Approximately 69 micrograms of structural proteins were obtained from 1 mg of the normal heart muscle. In the central portions of the myocardial infarction, reductions in myosin heavy chain (HC), light chain (LC) 1 and alpha-actinin were observed at 12 to 24 hours after the coronary occlusion followed by the decrease in myosin LC 2 at 48 hours. Those changes became intense at 72 hours to 7 days, but restored gradually at 14 to 28 days. 3. Alterations in the striated structure of cardiac muscle fibers of the adjacent tissues slices were found simultaneously with the changes in structural proteins. At 12 to 24 hours after the coronary ligation increase in eosinophilia and overstretch of cross-striation were observed. The findings of coagulation necrosis, loss of striation, fragmentation, swelling of A-band, etc. of the infarcted fibers were markedly observed at 48 hours to 7 days, but the histologic restoration of cardiac fibers was found with the recovery of the infarcted tissue at 14 to 28 days after the coronary ligation. 4. Changes in the compositions of structural proteins corresponded well to the alterations in the striated structure in chronology and in quantity.

1. Morphologic as well as biochemical alterations in chronic ischemic myocardial tissue without infarction were studied in dogs utilizing the Ameroid constrictor. 2. Serum creatine kinase activity elevated at around three weeks after placing the Ameroid constrictor around the circumflex branch of the left coronary artery suggestive of myocardial tissue injury followed by the initial activation caused by surgery. 3. Subendocardial proliferation of connective tissue was observed in about 60% of the experiments, but the middle and the subepicardial muscles were morphologically intact. 4. The marked increase in glycogen particles was observed in the subendocardial muscle cells in most of the experiments, and mild features of myocardial cellular necrosis were found in approximately 60% of the experiments. 5. ATPase activities of the structural proteins as well as sarcoplasmic reticulum in the ischemic myocardium shoed relatively higher values than those in the non-ischemic myocardium. However, no substructural changes were observed in SDS gel electrophoresis in both the fractions. 6. The alterations in the chronic myocardial ischemia are supposed to be essentially the same as those in myocardial necrosis followed by acute coronary occlusion.

The healing process of myocardial infarction was studied with special reference to the production of collagen by the determinations of prolylhydroxylase (PHase) activity and contents of hydroxyproline (Hyp) of the collagen subfractions, i.e., neutral salt soluble (NSC), acid soluble (ASC) and insoluble collagens (ISC) in comparison with histologic observation of the proliferation of connective tissue in the canine heart. The contents of Hyp in NSC and ASC increased in the infarcted tissue 5 days after coronary ligation prior to its increase in ISC. The content of Hyp in NSC, which is a precursor of ASC and ISC, reached the maximal value at 7 days and that reached the maximal value in ASC 7 to 14 days after coronary ligation. ISC increased markedly after 14 days. PHase activity increased on the second day and attained to the highest at 5 to 7 days. Proliferation of fibroblasts paralleled with the increases in PHase activity and NSC, and an increase in collagen fiber did with the contents of ISC. From these results it is concluded that in the healing stage of myocardial infarction, PHase was activated earlier than the increase in immature collagen in the infarcted area which was observed 5 to 7 days after coronary ligation and was followed by the production of mature collagen after 14 days.

1. Alterations in troponin subunits in myocardial infarction were studied in the dog heart by the analysis of troponin-tropomyosin complex, i.e, native tropomyoin, of total structural proteins in SDS gel electrophoresis, and by the measurement of degree of activation of actomyosin-ATPase by Ca++. 2. At 12 to 24 hours after coronary ligation, reductions in TN-C and tropomyosin were observed followed by a decrease in TN-I at 48 hours. The relative contents of these subunits were the lowest at 72 hours to 7 days falling to less than 10% of those in the non-ischemic myocardium. On the contrary, TN-T was preserved through the course of myocardial infarction. 3. Actomyosin-ATPase activity was increased at 12 to 24 hours after coronary ligation and then reduced rapidly at 24 to 48 hours together with the degradation of myosin. However, the activation of actomyosin-ATPase by Ca++-troponin-tropomyosin was reduced already at 12 hours simultaneously with the reduction in TN-C, and almost completely lost at 48 hours. 4. Troponin subunits and actomyosin-ATPase activity returned to those of the control myocardium at 28 days after coronary ligation indicating the recovery of infarcted tissue.

1. Changes of structural proteins in experimental and human myocardial infarction were studied by the determination of myosin- and actomyosin-ATPase activities and gel electrophoretic analysis in the presence of sodium dodecyl sulfate (SDS). 2. In animal experiments using dogs, the relative amounts of myosin and alpha-actinin decreased at 24 to 48 hours after coronary ligation, became lowest at 72 hours, and remained at this level for 2 weeks and returned to almost normal value at 28 days. 3. Myosin- and actomyosin-ATPase activities decreased rapidly during 24 to 48 hours after ligation with temporary increase in their activities in the initial stage of ischemia and followed the similar time course as that of the amounts of myosin and alpha-actinin. 4. SDS gel electrophoretic analysis of structural proteins of infarcted tissues of the human hearts obtained from 5 cadavers showed also marked decrease of the contents of myosin and alpha-actinin with relative preservation of actin, tropomyosin and troponin-T.

Ultracytochemical changes in ATPase activity of ischemic myocardial cells were studied in the dog heart by electron microscopy in the early stage of myocardial infarction and compared to the fine structural alterations in the ischemic myocardium. 1) In the normal myocardial cell, ATPase activity was observed intensely in the terminal cisternae (TC) of the sarcoplasmic reticulum (SR), and moderately in the myofilaments in the longitudinal SR and around the gap junctions of the intercalated discs. 2) The most striking change in the ischemic myocardial cells was the reduction in the ATPase reaction product in the TC of the SR and along the gap junctions 60 min after coronary ligation, simultaneously with swelling of the TC and the appearance of mitochondrial dense deposits. The reaction product began to decrease at 30 min on the myofilaments, and for 3 to 12 hours no reaction product was observed except irreversible morphologic changes in 60 to 70% of the longitudinal SR in the ischemic subendocardial cells. 3) A decrease in ATPase activity was recognized in the early stage of myocardial ischemia simultaneously with the fine structural changes of myocardial cells and it is considered to be one of the signs of ischemic irreversibility.

1. Alterations in subcellular distribution of electrolytes were studied with particular emphasis on Ca++ in normal and ischemic myocardium produced by ligating the coronary artery in the dog, by means of the histochemical method using potassium pyroantimonate. 2. In the normal myocardium, the antimonate precipitates were observed most abundantly in TC of SR and on N-line within sarcomeric I-band, but found scarcely in mitochondria, M-line, Z-line, intercalated disc or T-system. 3. In the infarcted myocardium, the precipitates were strikingly increased in both number and size in disrupted mitochondria, while they were markedly decreased in swollen TC with the progress of ischemia. 4. Most of the precipitates in TC and on N-line were removed by chelation with EGTA but a few larger precipitates were consistently found mainly on N-line. And the presence of Ca++ in the antimonate precipitates was confirmed by energy dispersive X-ray microanalysis.

Alterations in the localization and the intensity of acid phosphatase activity were studied electron microscopically in acute ischemic myocardial cells in the dog, in relation to processes of cellular degradation. In normal myocardial cells, the acid phosphatase activity was concentrated in the terminal cisternae, the longitudinal elements and the subsarcolemmal cisternae of the sarcoplasmic reticulum and the primary lysosomes. Activity was moderate in the secondary lysosomes, residual bodies and Golgi apparatus. As early as 15 min after coronary ligation the intensity of acid phosphatase activity increased in the enlarged lysosomes and the sarcoplasmic reticulum of the ischemic myocardial cells. Fine deposits of the reaction product were distributed in the sarcoplasm around lysosomes and the sarcoplasmic reticulum after 30 min, and the activity began to decrease in lysosomes and in the sarcoplasmic reticulum. One to 3 hours after ligation, intramitochondrial dense deposits appeared, and the reaction product decreased both in lysosomes and the sarcoplasmic reticulum. The fine reaction product, which leaked from lysosomes and the sarcoplasmic reticulum, was scattered in the sarcoplasm and was accompanied by fine structural changes indicating cellular necrosis. From these findings it is strongly suggested that acid hydrolases in lysosomes and the sarcoplasmic reticulum are closely related to the necrotic process in ischemic myocardial cells.

We investigated the morphologic changes in the sarcoplasmic reticulum (SR) in acute myocardial ischemia, induced by occlusion of the coronary artery in the canine heart, by freeze-fracture of SR in situ and in vitro, and they were compared to the alterations in Ca++-stimulated ATPase activity and protein composition of the isolated SR. Both SR in situ and the isolated SR exhibited typical intramembranous particles with diameters of 70 to 90 A in freeze-fracture replicas. The intramembranous particle density of the P face in SR in situ was 3,319 +/- 75 (mean +/- SE) per micrometer2 and that in the isolated SR was 2,667 +/- 60; particles were more numerous on the concave (P) face than on the convex (E) face. In ischemia for 30 to 60 min a significant decrease in the number of particles was found in SR in situ, and the corresponding change was noted in the isolated SR. Simultaneous decreases in Ca++-stimulated ATPase activity and the major ATPase protein of the SR were recognized. The close correlation of the changing pattern between the reduction in Ca++-ATPase and that in intramembranous particle density during ischemia supports the suggestion that a large part of the intramembranous particles represent ATPase protein itself. Decrease in the SR membrane particles strongly suggests the degradation of ATPase protein in the process of ischemic myocardial injury.

An analytical procedure for the quantitative determination of the structural proteins from a biopsied human cardiac muscle weighing approximately 1 mg was described to be applicable in clinical studies in 20 patients with various heart diseases. The principle of the method is glycerinization of heart muscle and analysis by SDS gel electrophoresis. In 6 control heart muscles obtained from patients having almost normal hearts, the pattern of the structural proteins was similar to that of the normal canine heart. Myosin heavy chain-actin ratio ranged 1.26 +/- 0.44. In 5 cases with secondary cardiac hypertrophy, the pattern of the structural proteins was the same as that of the control heart. In 4 cases with hypertrophic cardiomyopathy, an increase in myosin heavy chain was observed in 2 cases, while myosin heavy chain and alpha-actinin decreased in another 2 cases. Hypertrophy and severe disarray of myofibrils were noted in the former, and atrophy and degradation were done in the latter in electron microscopy. In 5 cases with dilated cardiomyopathies, the relative contents of myosin heavy chain and alpha-actinin was reduced in all cases together with atrophy and degradation of myofibrils.

1. Gel electrophoretic analysis of structural proteins extracted from myocardial tissues in the presence of sodium dodecylsulfate (SDS) is applicable to study the relative amount of structural proteins from less than 1 Gm of heart tissue. 2. The compositions of structural proteins of the right and left atria and ventricles of the normal canine heart were identical and similar to those of skeletal muscle. 3. The compositions of structural proteins of the atrium and ventricle of the normal human heart were also identical.

Activities and subcellular distributions of acid hydrolases, cathepsin D, acid phosphatase and beta-glucuronidase in myocardial subfractions were determined serially with reference to the initiation of myocardial necrosis in dog hearts with acute ischemia. The following results were obtained: 1) In the normal myocardium, respectable activities of three enzymes were obtained either in the sarcoplasmic reticulum or in the lysosome-containing fraction. 2) Thirty min after coronary ligation, an increase in the activities was observed in both lysosome and sarcoplasmic reticulum fractions of the ischemic heart muscle. After 60 to 90 min these activities were decreased rapidly in both fractions to about 70% of those of the normal myocardium with an increase in the cytosolic activity. Two to 3 hours after ligation, the reduction in the cytosolic activity was noted, indicating an escape of the enzymes from the necrotic myocardium. The subcellular distribution of these enzymes was further altered in the ischemic heart muscle for 12 to 14 hours reflecting an infiltration of the interstitial cells. These findings suggest that activation and release of acid hydrolases not only in lysosomes but also in the sarcoplasmic reticulum are one of the primary and the earliest factors for the evolution of ischemic myocardial injury which leads to necrosis.

Ultrastructural and biochemical alterations in myofibrils (Mf) were studied in biopsied myocardial tissues in 11 patients with dilated cardiomyopathy (DCM) and compared with those in non-hypertrophic control and secondary hypertrophic (SHT) heart muscles. Transverse diameters of biopsied cardiac myocytes increased significantly in both of SHT and DCM, and ultrastructural changes were similar in quality in both of them. Volume densities of Mf were 61.1 and 59.9% on average in control and SHT myocardial cells, respectively, and they were not significantly different. But in DCM volume density was significantly less (49.8% in left ventricular myocytes), and inverse relation between that and diameter of cardiac myocytes was observed (p less than 0.01). Electrophoretic pattern and relative composition of major structural proteins from control and SHT heart muscles were similar and statistically insignificant. In DCM, relative contents of myosin heavy chain and alpha-actinin decreased significantly and distinctively in all of cases suggesting primary degradation of myofibrils.

The degradation of the sarcoplasmic reticulum (SR) in acute myocardial ischemia was studied with references to the regional irreversibility and to the mechanism of ischemic degradation by the measurements of Ca++-stimulated ATPase activity and composition of the major ATPase protein of the SR and activity of cathepsin B of the SR and lysosome (Ly) fractions. Ca++-stimulated ATPase activity decreased to 66% of that of the nonischemic portion at 20 min after coronary ligation in the subendocardium (Endo) and to 44% at 30 min in the subepicardium (Epi). Composition of the major ATPase protein decreased to 55% and 73% at 30 min in Endo and Epi, respectively. In both SR and Ly fractions cathepsin B exhibited the maximal activity at 6.0-6.5, and pH dependent. And incubation of the SR at pH 6.0 induced the degradation of the ATPase protein quite similarly to that in vivo ischemia. These results suggest that the degradation of the SR membrane of ischemic myocardial cells begins earlier in Endo 20 to 30 min after the cease of the coronary blood flow, and extends to Epi later. Cathepsin B is strongly conceivable to play an initial role of necrotic process of the ischemic myocardial cells by activation inside of the SR in ischemic acidic state.

Impairment of mitochondrial respiration in early myocardial ischemia was studied with special reference to myocellular irreversible injury. The technique used was total ligation of the left anterior descending coronary artery, followed by reconstruction of coronary blood flow, in the dog. State 3 respiratory activity reduced significantly to 76% of that of the non-ischemic myocardium in subendocardial muscle (Endo) as early as 30 min after occlusion, and at 60 min to 84% in the subepicardium (Epi). The activity was not recovered by reperfusion. The activity of complex I of sonicated submitochondrial particles decreased at 30 min to 67% in Endo and at 60 min to 71% in Epi, and was not recovered by reperfusion. Complex II and IV activities were kept in the control level until 60 min of ischemia. DNP-stimulated ATPase activity reduced to 79% in Endo at 15 min and to 70% in Epi at 30 min, but recovered significantly by reperfusion until 30 min of ischemia. Mitochondrial respiratory activity was impaired irreversibly in ischemia for 30 min in Endo and this spread to Epi later. Degradation of complex I is considered to be one of the causes of myocardial irreversibility in early ischemia.