Tuan H. Kuo

Defective Oxidative Metabolism of Heart Mitochondria from Genetically Diabetic Mice

Long chain saturated β-hydroxy fatty acid content and oxidative metabolism were studied in hearts of diabetic mice (C57BL/KsJ db/db) with a progressive cardiomy-opathy at intervals of 7, 10, 16, and 26 wk of age. Total β-hydroxy fatty acid (BHFA) content increases progressively with age in diabetic hearts with a mean value of 143.5 nmoi/g dry wt as compared with a mean of 59.6 nmol/g dry wt in control hearts. There was also a redistribution of BHFA in myocardium of diabetic mice when compared with controls, with a relative decrease in β-hydroxymyristate and an increase of β-hy-droxypalmitate. Oxidative phosphorylation studies using isolated mitochondria from diabetic mice demonstrated depressed state 3 oxidation rates with both palmityl carnitine and pyruvate as substrates. While mitochondrial NADH-oxidase activity was not statistically different from that of controls, there was a significant decrease in mitochondrial total NAD + NADH content in diabetic hearts. In addition, treatment of my-ocardfal tissue with lanthanum demonstrated an abnormal permeability of sarcolemmal, intercalated disc as well as mitochondrial membranes in myocytes of diabetic mice. The data indicate that deficiencies in total NAD + NADH content can account for the depressed state 3 oxidation of palmitylcarnitine and pyruvate in diabetic mice that in turn may explain the abnormal accumulation of BFHA. The latter could play a role in altering the permeability of cardiac cell membranes.

Defective Ca2+-pumping ATPase of heart sarcolemma from cardiomyopathic hamster.

The Syrian cardiomyopathic hamster has a hereditary disease characterized by a progressive myocyte necrosis and intracellular calcium overload. Several systems in the heart sarcolemma that regulate the rate of Ca2+ entry or efflux were examined. There is a selective decrease of Ca2+-pumping ATPase activity in the heart sarcolemma of 40-day-old myopathic hamsters, while the Na+-Ca2+ exchange system and the ouabain-sensitive (Na+ + K+)-ATPase activity remain intact. This age-dependent decrease in Ca2+-ATPase activity closely parallels the time course of lesion development. Both the affinity for Ca2+ (Km) and the maximal velocity (Vmax) of the Ca2+-dependent ATP hydrolysis are altered. In addition, there is also an increased number of calcium channel receptor binding sites. Thus the data suggest that the imbalance in Ca2+ fluxes across the cardiac plasma membrane may be involved in the pathogenesis of this cardiomyopathy.

Altered calcium regulation in the cardiac plasma membrane in experimental renal hypertension

The factors regulating calcium homeostasis in the cardiac plasma membrane of renal hypertension in the rat (two kidney-one clip, Goldblatt model) have been studied. Comparison of the cardiac sarcolemma from control (C) and hypertensive (H) rats indicates similar protein yield and purity. Study of longer term hypertension (4 to 12 weeks) shows a decrease in the number of calcium channel receptor binding sites (Bmax C: 549 +/- 122 fmol/mg; H: 334 +/- 74 fmol/mg) as well as a depressed calcium pumping ATPase activity (C: 7.6 +/- 2.5 nmol/mg/min; H: 3.8 +/- 1.5 nmol/mg/min). Furthermore, there is a decreased rate of Na+-Ca2+ exchange (C: 5.4 +/- 1.9 nmol/mg/5 s; H: 2.3 +/- 0.9 nmol/mg/5 s). Study of short-term hypertension (1 to 4 weeks) indicates that the earliest change occurs at 1 week with decreased calcium pumping ATPase due to a change of the Vmax of Ca2+ transport (C: 9.7 +/- 1.6 nmol/mg/min; H: 5.4 +/- 1.4 nmol/mg/min). This is then followed by the decreased calcium channel receptor binding. However, the rate and the extent of depression in Ca2+-ATPase activity are much greater than that of Ca2+ channel receptor binding. Since alteration of Ca2+-ATPase is accompanied by an increase in intracellular Ca2+ concentration and there is a temporal association with the onset of myocardial lesions in the hypertensive rats, it is suggested that elevated intracellular calcium concentration as a result of altered Ca2+-ATPase activity may play a significant role in the development of hypertensive cardiomyopathy.

Some effects of Ca2+, Mg2+ , and Mn2+ on the ultrastructure, light-scattering properties, and malic enzyme activity of adrenal cortex mitochondria.

Abstract The ultrastructure and 90 ° light-scattering capacity of adrenal cortex mitochondria have been examined under conditions which lead to an activation of malic enzyme activity in these mitochondria. After isolation, the mitochondria display an aggregate ultrastructure which does not resemble the vesicular (orthodox) form normally seen in vivo . Under conditions of malic enzyme activation (presence of malate, NADP + , Mg 2+ and 1 m m Ca 2+ ), the ultrastructure reverts to a vesicular form as seen in vivo . Of these required components, only Ca 2+ affects the ultrastructure. The ultrastructural transformation from the aggregate to the orthodox form is always accompanied by a decrease in the 90 ° light-scattering capacity. When produced by Ca 2+ , transformation requires energy-dependent Ca 2+ uptake if an oxidizable substrate is present. In the absence of substrate, the transformation occurs as an apparent energy-independent effect. Mn 2+ can substitute for Ca 2+ only in the presence of substrate. In de-energized mitochondria, Mn 2+ prevents the effects of Ca 2+ . The activation of malic enzyme is always preceded by a decrease in light scattering and transformation to the orthodox ultrastructure; however, the presence of the orthodox form is not a sufficient condition since subsequent chelation of free Ca 2+ fails to reverse either the decrease in light scattering or ultrastructural transformation but does reverse the enzyme activation. In addition, levels of Mn 2+ which effectively depress light-scattering capacity and produce the orthodox form, fail to activate malic enzyme significantly. The data are discussed as they relate to Ca 2+ -induced damage in mitochondria.

Biochemical characterization and cellular localization of serine protease in myopathic hamster

Abstract Purified serine protease was obtained from skeletal and heart muscle of 150-day-old myopathic hamsters. The skeletal muscle enzyme showed identical molecular weight and characteristics toward various reagents and inhibitors when compared with the cardiac muscle enzyme. In addition, no antigenic differences were detected between the two enzymes using double-immunodiffusion test. Further characterization of the properties of the two enzymes was carried out by using individual myofibrillar proteins as substrates. Both enzymes were capable of degrading myosin, tropomyosin and troponin but not actin. The divalent cations Ca 2+ or Mg 2+ were able to protect the myosin light chain 2 against proteolytic cleavage. The cellular localization of the serine protease of skeletal and cardiac muscle was studied by immunohistochemical techniques. The observations indicate that serine protease is contained within the cytoplasmic granules of cardiac and skeletal muscle mast cells of myopathic and control hamsters. The number of labeled mast cells is about three-fold greater in myopathic than in control tissues. This increase correlates with the increase in enzyme activity as detected by biochemical assay in tissue homogenates.

Changes in some biochemical parameters including cytochrome P-450 after hypophysectomy and their restoration by acth administration in rats four months post hypophysectomy☆

Abstract Following withdrawal of ACTH by hypophysectomy, rat adrenal gland P450 content falls to a low level with a half life time of 3.5 days. The content can be restored by subsequent ACTH treatment in animals as long as four months post hypophysectomy with parallel regeneration of in vitro steroid synthetic capacity. The recovery pattern of a number of other gland parameters suggests that the regeneration of P450 and steroidogenic capacity in these severely atrophied glands occurs by a mechanism which is largely dependent on cell replication.

Photoaffinity labeling of the calcium channel antagonist receptor in the heart of the cardiomyopathic hamster.

The high affinity 1,4-dihydropyridine receptors of the cardiac membrane calcium channel from Syrian Cardiomyopathic hamsters were studied using [3H] PN200-110 and [3H]azidopine as ligands. [3H]Azidopine was photoincorporated covalently into bands of 180, 100, 79, 45 and 31 kDa, as determined by SDS/polyacrylamide gel electrophoresis. Photolabeling of the 180 kDa band is protected by 2 microM [1H]PN200-110 whereas the lower Mr bands are not. Thus, only the 180 kDa band is the calcium channel linked 1,4 dihydropyridine receptor. The photoincorporation into this 180 kDa band is doubled with samples of myopathic hamsters vs. control hamsters. It is suggested that the increase in calcium channel receptors may be involved in the pathogenesis of this cardiomyopathy.

Oxidative metabolism of Polytron versus Nagarse mitochondria in hearts of genetically diabetic mice.

We have shown previously that heart mitochondria obtained by the Nagarse method from genetically diabetic mice (C57BL/KsJ db/db) exhibit a defect in oxidizing NAD+-linked substrates (Kuo, T.H., Moore, K.H., Giacomelli, F. and Wiener, J. (1983) Diabetes 32, 781-787). In this study, the oxidative phosphorylation characteristics of cardiac mitochondria isolated by the Polytron method were compared with that of Nagarse mitochondria. Evidence is presented here that in the diabetic heart both Nagarse and Polytron mitochondria have defective pyruvate oxidation, whereas only the former exhibit impaired fatty acid oxidation. Assay of two rate-limiting beta-oxidation enzymes, namely beta-hydroxyacyl-CoA dehydrogenase and beta-ketothiolase, indicates no alteration in specific activities from diabetic mice vs. controls. The data suggest that two populations of mitochondria are present in myocardium and that the defective oxidative metabolism in the cardiac mitochondria of db/db mice may be linked to deficiencies in total NAD + NADH content.

Pyruvate dehydrogenase activity in cardiac mitochondria from genetically diabetic mice

Pyruvate dehydrogenase (PDH) was studied in isolated heart mitochondria from genetically diabetic mice (C57BL/KsJ db/db) with progressive cardiomyopathy. Both the basal activity (active enzyme) and the total activity (the sum of active and inactive enzyme) were determined. In mitochondrjal extracts from 8–18-wk-old db/db mice, there was a 73% decrease of basal activity accompanied by a 38% decrease of total activity as compared with controls. The lower basal activity at 8 wk of age suggests an increased conversion of the enzyme into the phosphorylated(inactive) form. Evidence is also given that the conversion of inactive (phosphorylated) enzyme into active (dephosphorylated) enzyme is inhibited in cardiac mitochondria prepared from 8-wk and older db/db mice. These changes coincide with the onset of defective oxidative metabolism andcan explain the depressed pyruvate oxidation reported previously.

Purification and characterizaton of two distinct Ca2+-activated proteinases from hearts of hypertensive rats

Two distinct Ca2+-activated proteinases were purified and characterized from hearts of hypertensive rats. Ca2+-activated proteinases I and II, having low and high Ca2+ requirements, respectively, were first separated by DEAE-cellulose chromatography. The enzymes were then purified individually by different column procedures: chromatography on phenyl-Sepharose, then Sephadex G-200 for proteinase I and reactive-red agarose for proteinase II. The apparent molecular weight of purified proteinase I was 125 000 and that for purified proteinase II was 110 000. Both enzymes are heterodimers made up of a larger catalytic subunit and a smaller subunit devoid of proteinase activity. Ca2+ concentrations for half-maximal activation were 5 microM for proteinase I and 200 microM for proteinase II. Both enzymes were inhibited by sulfhydryl-modifying agents, but exhibited different characteristics in the auto-digestion reaction in the presence of Ca2+. Proteinases I and II were also purified from hearts of normotensive rats and shown to be identical to their respective counterparts from hearts of hypertensive rats. However, proteinase II activity in hypertensive rat hearts was significantly elevated as compared to controls.