Toshikuni Yanagishita

Changes in microsomal membrane phospholipids and fatty acids and in activities of membrane-bound enzyme in diabetic rat heart.

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 cardiomyoctes of diabetic rats 8 weeks after streptozotocin administration. The Ca2+ uptake of both sarcoplasmic reticulum and mitochondria decreased simultancously 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.

Biochemical and Morphologic Alterations in Cardiac Myocytes in Streptozotocin-Induced Diabetic Rats

Complication of congestive heart failure in patients with diabetes mellitus has well been known in clinical practice. Impairment of cardiac function was recognized in the absence of arteriosclerotic changes in diabetics, and the concept of “diabetic cardiomyopathy” has been proposed as the diabetes specific myocardial disorder (1,2). In the clinical practice, diabetes mellitus occurs in the mature to elder age and complication of coronary arterio- or athero-sclerosis is usual. Therefore, influence of ischemia could not be excluded in clinical cases. To elucidate effect of hyperglycemic state on cardiac myocytes without vascular changes, experimental diabetes was induced in relatively young rats by single intravenous injection of streptozotocin (STZ), and the effects of abrupt and long-standing severe hyperglycemia on the cardiac micro organs such as the sarcoplasmic reticulum (SR) and the structural proteins were investigated in comparison with the fine structures of cardiac myocytes.

Production of Hydrogen Peroxide During Hypoxia-Reoxygenation in Isolated Myocytes

Active oxygen species, including hydrogen peroxide (H2O2), have been implicated in myocardial reperfusion injury. Recently, spin-trap agents and biochemical techniques applied to intact hearts have shown that H2O2 is generated by leukocytes, by endothelial cells, and by mitochondria in myocytes. In this study, we used electron microscopy and the cerium (Ce) method to histologically investigate H2O2 formation during hypoxia—reoxygenation and its toxic effects on myocardium. This Ce method involves the formation of an electron-dense precipitate when H2O2 reacts with cerium chloride (CeCl3). Single myocytes were obtained from rat hearts by the collagenase method. Isolated myocytes were reoxygenated for 15 minutes after 30 minutes of hypoxia. Digitonin and CeCl3, were added to make cell membranes permeable and to detect intracellular H2O2 by electron microscopy. In the control group, the ultrastructure was well preserved and no dense deposits were found in myocytes. However, in the hypoxia—reoxygenation group, precipitates, which were cerium—H2O2 reaction products, were found along swollen mitochondria. Moreover, in the hypoxia-reoxygenation group, cell viability was reduced to 72% of control. These results indicate that H2O2 is generated by mitochondria and that its relese into cytosol may lead to myocyte death during hypoxia—reperfusion.

Effects of Catecholamine and Amrinone on the Metabolism of Noninfarcted Myocardium in Cardiogenic Shock

We investigated the effects of catecholamine and amrinone (AMR) on the metabolism of noninfarcted myocardium (NIM) during heart failure in acute myocardial infarction. Acute myocardial ischemia was induced by left circumflex coronary artery ligation on dogs divided into two groups: in the C group, left ventricular pressive (LVP) remained at >70% and in the S group, LVP decreased to <70% of preligation values. In part of the S group, 10 μg/kg/min of dopamine (DOA) or dobutamine (DOB), or 60μg/kg/min of AMR, were given intravenously beginning 90 min after ligation. At the end of 120 min of ischemia, mitochondria were extracted from NIM, and respiratory and electron transport system enzyme activities were measured. In the DOA and DOB groups, LVP, myocardial blood flow, cardiac output, and max LV dp/dt recovered significantly. In the AMR group, in spite of LVP reduction, other hemodynamic parameters increased. In the S group, state III respiration, complex I, and DNP-ATPase activities in NIM decreased to 62%, 65%, and 68% of preligaton levels, respectively. These values improved markedly with DOA, DOB, and AMR treatments. Electron microscopy showed swelling and fusion of mitochondria in the S group. These results indicate that catecholamine and AMR improve energy production in NIM and ultimately improve cardiac function.

Metabolic Changes in Nonischemic Myocardium During Pump Failure

Metabolic changes in the nonischemic myocardium after acute myocardial infarction in canine hearts were studied. Ca2+-ATPase activity and the major ATPase protein of the sarcoplasmic reticulum, tissue levels of ATP, mitochondrial respiratory, and complex I activities were decreased in the noninfarcted zone in proportion to heart function. It is suggested that recovery of these functions may be important in any treatment of pump failure.

Ultrastructure of sarcoplasmic reticulum in the ischemic myocardium

Fine structural changes in the sarcoplasmic reticulum (SR) in myocardial ischemia, induced by occlusion of the anterior descending branch of the left coronary artery in the canine heart, were studied by the freeze-fracture technique in situ and in vitro and compared to the alterations in Ca2+-stimulated ATPase activity and sodium dodecyl sulfate gel electrophoresis of the isolated SR. Both SR in situ and the isolated SR exhibited the typical intramembranous particles of 70-90 A in freeze-fracture replicas, and the numbers of particles were more numerous in the concave face (PF) than in the convex face (EF). The numbers of particles in the PF were 2748/micron 2 on the average. In ischemia for 1-2 hr, a significant decrease in the numbers of the particles was found in SR in situ, and corresponding changes were noted in the isolated SR. Decreases in Ca2+-stimulated ATPase activity and in the major protein band of ATPase were recognized simultaneously. The close correlation of the changing patterns between the reduction in Ca2+-ATPase and that in the intramembranous particle density during ischemia supports the suggestion that a large part of the intramembranous particles represents ATPase protein itself. The decrease in the particles of SR membrane indicates the degradation of ATPase in the process of ischemic myocardial necrosis.