ATP-synthetase activity, respiration and cytochromes of rat heart mitochondria in aging and hyperthyroidism

The ATP-synthetase activity, the rate of oxygen uptake under different metabolic conditions, the tightness of coupling of respiration to oxidative phosphorylation and the cytochrome contents in heart mitochondria of rats from different age groups were studied under normal conditions and in hyperthyroidism. It was found that heart mitochondria of aged animals did not practically differ in terms of their functional activity from those of the young animals. Administration of thyroxin to the animals from all age groups produced no significant effects on the state of mitochondria, increasing the rate of ATP synthesis on alpha-glycerophosphate, which was especially well-pronounced in aged animals, and the cytochrome content in 1-month-old rats.     

Quantitative protein profiling in heart mitochondria from diabetic rats

Quantitative protein profiling based on in vitro stable isotope labeling, two-dimensional polyacrylamide gel electrophoresis, and mass spectrometry is an accurate and reliable approach to measure simultaneously the relative abundance of many individual proteins within two different samples. In the present study, it was used to define a set of alterations caused by diabetes in heart mitochondria from streptozotocin-treated rats. We demonstrated that the expression of proteins from the myocardial tricarboxylic acid cycle was not altered in diabetes. However, up-regulation of the fatty acid beta-oxidation favored fatty acids over glucose as a source of acetyl CoA for the tricarboxylic acid cycle. Protein levels for several proteins involved in electron transport were modestly decreased. Whether this may depress overall ATP production remains to be established, since the protein level of ATP synthase seems to be unchanged. Other changes include down-regulation of protein levels for creatine kinase, voltage-dependent anion channel 1 (VDAC-1), HSP60, and Grp75. The mitochondria-associated level of albumin was decreased, while the level of catalase was substantially increased. All of the changes were evident as early as 1 week after streptozotocin administration. Taken together, these data point to a rapid and highly coordinated regulation of mitochondrial protein expression that occurs during the heart adaptation to diabetes.     

The features of mitochondria of cardiomyocytes from rats with chronic heart failure

Electron-microscopy study of rat myocardium 2 weeks after a heart attack revealed significant alterations in the ultrastructure of cardiomyocytes than for the control. The location of myofibrils was less regular than for normal cells. The population of interfibrillar mitochondria decreased. Mitochondrial cristae were located less densely and formed cellated structures. Swollen mitochondria were observed in the periinfarction and intact areas, indicating the development of ischemia in the myocardium as a whole. Six months after the occlusion of coronary vessels alterations in the location of myofibrils and mitochondria were mainly observed in the peri-infarction area. Mitochondria also formed cellated structures. A 30% decrease in the density of the arrangement of the inner membranes of mitochondria on an area unit was found in the periinfarction zone. The ratio between the relative volumes of mitochondria and myofibrils in the cardiomyocytes of the peri-infarction area was increased by 20%. The area of mitochondria in the intact zone of the left ventricle was 30% greater than for the control. A study of isolated living cardiomyocytes revealed that the mitochondrial- membrane potential in the rats subjected to myocardial infarction half a year ago previously was significantly lower than for the mitochondrial-membrane potential in the control rats. Thus, cardiomyocytes that were similar to healthy cardiomyocytes in their morphology exhibited lower total mitochondrial-membrane potential, indicating their decreased energy state.     

Effect of quercetin on daunorubicin-induced heart mitochondria changes in rats

Cancer therapy with daunorubicin is limited by its cardiotoxicity. It has been suggested that daunorubicin-induced free radical generation can be involved. The precise molecular mechanism of daunorubicin-induced cardiotoxicity is still not well understood but it is believed that mitochondria play an important role in this process. It has been reported that flavonoids with antioxidant properties may prevent anthracycline-induced cardiotoxicity. In this work, we investigated the effects of daunorubicin and quercetin on mitochondrial enzyme activities such as ATPase, glutathione peroxidase (GPx) and glutathione reductase (GR). Moreover, we also studied the changes of outer mitochondrial membrane using synchronous fluorescence spectra. The activity of ATPase and GR were significantly increased after daunorubicin application. Pretreatment with quercetin significantly alleviated this increase. On the other hand, GPx activity was significantly decreased and quercetin prevented this decrease. Treatment with quercetin alone had no significant effect on the enzyme activity studied. Quercetin also completely prevented daunorubicin-induced changes in fluorescence of the outer mitochondrial membrane. In conclusion, our data indicate that quercetin may be useful in mitigating daunorubicin-induced cardiotoxicity.     

Creatine kinase of heart mitochondria. Control of oxidative phosphorylation by the extramitochondrial concentrations of creatine and phosphocreatine

Defining how extramitochondrial high-energy phosphate acceptors influence the rates of heart oxidative phosphorylation is essential for understanding the control of myocardial respiration. When the production of phosphocreatine is coupled to electron transport via mitochondrial creatine kinase, the net reaction can be expressed by the balanced equation: creatine + Pi----phosphocreatine + H2O. This suggests that rates of oxygen consumption could be regulated by changes in [creatine], [Pi], or [phosphocreatine], alone or in combination. The effects of altering these metabolites upon mitochondrial rates of respiration were examined in vitro. Rat heart mitochondria were incubated in succinate-containing oxygraph medium (pH 7.2, 37 degrees C) supplemented with five combinations of creatine (1.0-20 mM), phosphocreatine (0-25 mM), and Pi (0.25-5.0 mM). In all cases, the mitochondrial creatine kinase reaction was initiated by additions of 0.5 mM ATP. To emphasize the duality of control, the results are presented as three-dimensional stereoscopic projections. Under physiological conditions, with 5.0 mM creatine, increases in Pi or decreases in phosphocreatine had little influence upon mitochondrial respiration. When phosphocreatine was held constant (15 mM), changes in [creatine] modestly stimulated respiratory rates, whereas Pi again showed little effect. With 1.0 mM Pi, respiration clearly became dependent upon changes in [creatine] and [phosphocreatine]. Initially, respiratory rates increased as a function of [creatine]. However, at [phosphocreatine] values below 10 mM, product "deinhibition" was observed, and respiratory rates rapidly increased to 80% State 3. With 2.0 mM Pi or higher, respiration could be regulated from State 4 to 100% State 3. Overall, the data show how increasing [creatine] and decreasing [phosphocreatine] influence the rates of oxidative phosphorylation when mediated by mitochondrial creatine kinase. Thus, these changes may become secondary cytoplasmic signals regulating heart oxygen consumption.     

Effects of hypothyroidism and hyperthyroidism on GDP binding to brown-adipocyte mitochondria from rats.

1. Rats were made hypothyroid by giving them a low-iodine diet with propylthiouracil for 4 weeks, or were made hyperthyroid by injection with tri-iodothyronine (T3) over a 3-day period. 2. Brown adipocytes were isolated from the interscapular depots of these animals or from their euthyroid controls, followed by isolation of mitochondria from the cells. 3. Relative to cell DNA content, hypothyroidism decreased the maximum binding (Bmax.) of [3H]GDP to mitochondria by 50%. T3 treatment increased binding by 37%. 4. These findings, which are discussed in relation to previously observed changes in brown adipose tissue after alteration of thyroid status, suggest that mitochondrial uncoupling for thermogenesis is less or more effective in hypothyroidism or hyperthyroidism respectively.     

Deficit of coenzyme Q in heart and liver mitochondria of rats with streptozotocin-induced diabetes

Mitochondrial dysfunction and oxidative stress participate in the development of diabetic complications, however, the mechanisms of their origin are not entirely clear. Coenzyme Q has an important function in mitochondrial bioenergetics and is also a powerful antioxidant. Coenzyme Q (CoQ) regenerates alpha-tocopherol to its active form and prevents atherogenesis by protecting low-density lipoproteins against oxidation. The aim of this study was to ascertain whether the experimentally induced diabetes mellitus is associated with changes in the content of endogenous antioxidants (alpha-tocopherol, coenzymes Q9 and Q10) and in the intensity of lipoperoxidation. These biochemical parameters were investigated in the blood and in the isolated heart and liver mitochondria. Diabetes was induced in male Wistar rats by a single intravenous injection of streptozotocin (45 mg x kg(-1)), insulin was administered once a day for 8 weeks (6 U x kg(-1)). The concentrations of glucose, cholesterol, alpha-tocopherol and CoQ homologues in the blood of the diabetic rats were increased. The CoQ9/cholesterol ratio was reduced. In heart and liver mitochondria of the diabetic rats we found an increased concentration of alpha-tocopherol, however, the concentrations of CoQ9 and CoQ10 were decreased. The formation of malondialdehyde was enhanced in the plasma and heart mitochondria. The results have demonstrated that experimental diabetes is associated with increased lipoperoxidation, in spite of the increased blood concentrations of antioxidants alpha-tocopherol and CoQ. These changes may be associated with disturbances of lipid metabolism in diabetic rats. An important finding is that heart and liver mitochondria from the diabetic rats contain less CoQ9 and CoQ10 in comparison with the controls. We suppose that the deficit of coenzyme Q can participate in disturbances of mitochondrial energy metabolism of diabetic animals.     

Changes in the cytochrone C and A (A3) content in the liver mitochondria of rats with hyperthyroidism

Thyroxin was administered daily to male Wistar rats aged 1, 3, 12 and 24 months in a dose of 250 g per 100 g body weight. After 9 days there was a significant increase in the content of cytochromes c and a (a(3)). An appreciable rise in the cytochrome c content was recorded as long as after one day, whereas the content of cytochromes a (a(3)) did not exceed normal even after 2 days. Following one day the content of cytochromes a (a(3)) in 3-, 12-, and 24-month-old rats was sightly lower than in normal rats. A significant temporary increase in the c/a (a(3)) ratio was observed after 1-2 days only in 12- and 24-month-old rats. The c/a (a(3)) ratio increased with age. It is suggested that application of thyroid hormones may be promising in studies on the regulation of mitochondrial biogenesis.     

Structural and functional changes in heart mitochondria from sucrose-fed hypertriglyceridemic rats

In the heart of sugar-induced hypertriglyceridemic (HTG) rats, cardiac performance is impaired with glucose as fuel, but not with fatty acids. Accordingly, the glycolytic flux and the transfer of energy diminish in the HTG heart, in comparison to control heart. To further explore the biochemical nature of such alteration in the HTG heart, the components of the non-glycolytic energy systems involved were evaluated. Total creatine kinase (CK) activity in the myocardial tissue was depressed by 30% in the HTG heart whereas the activity of the mitochondrial CK (mitCK) isoenzyme fraction that is functionally associated with oxidative phosphorylation decreased in isolated HTG heart mitochondria by 45%. Adenylate kinase (AK) was 20% lower in the HTG heart. In contrast, respiratory rates with 2-oxoglutarate (2-OG) and pyruvate/malate (pyr) were significantly higher in HTG heart mitochondria than in control mitochondria. 2-OG dehydrogenase activity was also higher in HTG mitochondria. Respiration with succinate was similar in both groups. Content of cytochromes b, c + c₁ and a + a₃, and cytochrome c oxidase activity, were also similar in the two kinds of mitochondria. A larger content of saturated and monounsaturated fatty acids was found in the HTG mitochondrial membranes with no changes in phospholipids composition or cholesterol content. Mitochondrial membranes from HTG hearts were more rigid, which correlated with the generation of higher membrane potentials. As the mitochondrial function was preserved or even enhanced in the HTG heart, these results indicated that deficiency in energy transfer was associated with impairment in mitCK and AK. This situation brought about uncoupling between the site of ATP production and the site of ATP consumption (contractile machinery), in spite of compensatory increase in mitochondrial oxidative capacity and membrane potential generation.     

Structural and functional changes in heart mitochondria from sucrose-fed hypertriglyceridemic rats

In the heart of sugar-induced hypertriglyceridemic (HTG) rats, cardiac performance is impaired with glucose as fuel, but not with fatty acids. Accordingly, the glycolytic flux and the transfer of energy diminish in the HTG heart, in comparison to control heart. To further explore the biochemical nature of such alteration in the HTG heart, the components of the non-glycolytic energy systems involved were evaluated. Total creatine kinase (CK) activity in the myocardial tissue was depressed by 30% in the HTG heart whereas the activity of the mitochondrial CK (mitCK) isoenzyme fraction that is functionally associated with oxidative phosphorylation decreased in isolated HTG heart mitochondria by 45%. Adenylate kinase (AK) was 20% lower in the HTG heart. In contrast, respiratory rates with 2-oxoglutarate (2-OG) and pyruvate/malate (pyr) were significantly higher in HTG heart mitochondria than in control mitochondria. 2-OG dehydrogenase activity was also higher in HTG mitochondria. Respiration with succinate was similar in both groups. Content of cytochromes b, c + c1 and a + a3, and cytochrome c oxidase activity, were also similar in the two kinds of mitochondria. A larger content of saturated and monounsaturated fatty acids was found in the HTG mitochondrial membranes with no changes in phospholipids composition or cholesterol content. Mitochondrial membranes from HTG hearts were more rigid, which correlated with the generation of higher membrane potentials. As the mitochondrial function was preserved or even enhanced in the HTG heart, these results indicated that deficiency in energy transfer was associated with impairment in mitCK and AK. This situation brought about uncoupling between the site of ATP production and the site of ATP consumption (contractile machinery), in spite of compensatory increase in mitochondrial oxidative capacity and membrane potential generation.     

Formation of lamellar bodies in rat liver mitochondria in hyperthyroidism

In the present work, ultrastructural changes of rat liver mitochondria in hyperthyroidism were studied. Hyperthyroidism was induced in male Wistar rats by daily administration of 100 μg thyroxin per 100 g body weight for 5 days. The level of triiodothyronine and thyroxine increased 3- and 4-fold, respectively, in comparison with the same parameters in the control group, indicating the development of hyperthyroidism in experimental animals. It was found that under this experimental pathology 58% of the mitochondria are swollen, with their matrix enlightened, as compared to the control. In 40% of the profiles, the swollen mitochondria in the liver under hyperthyroidism exhibited rounded mono- or multilayer membrane structures, called lamellar bodies (LBs), presumably at different stages of their development: from the formation to the release from the organelles. Most LBs were located in the mitochondria near the nuclear zone (27%), while their number was reduced in the part of the cell adjacent to the plasma membrane. In a number of swollen mitochondria the cristae were shown to change their orientation, being directed radially toward the center of the mitochondria. We suggested that it is the first stage of formation of LBs. The second stage can be attributed to the formation of monomembrane structures in the center of the organelles. The third stage is characterized by the fact that the membrane of the lamellar bodies consists of several layers, and in this case the bodies were located closer to the outer mitochondrial membrane. The evagination of the outer mitochondrial membrane and its connection with lamellar structure can be recognized as the fourth stage of formation of LBs. At the fifth stage the developed lamellar formations exited the mitochondria. At the same time, following the exit of LBs from the mitochondria, no damage to the mitochondrial membrane was registered, and the structure of the remaining part of the mitochondria was similar to the control. The nucleus of the hepatocyte also underwent structural changes in hyperthyroidism, exhibiting changes in the membrane configuration, and chromatin condensation. The nature and structure of the LBs, as well as their functional role in the liver mitochondria in hyperthyroidism, require further investigation.     

Decrease of cytochrome c oxidase protein in heart mitochondria of copper-deficient rats

Copper deficiency has been reported to be associated withdecreased cytochrome c oxidase activity, whichin turn may be responsible for theobserved mitochondrial impairment and cardiac failure. We isolatedmito-chondriafrom hearts of copper-deficient rats: cytochrome c oxidase activity was found to be lowerthan incopper-adequate mitochondria. The residual activity paralleled coppercontent of mitochondria and also corresponded with the heme amount associated with cytochromeaa3. In fact, lower absorption in thea-band region of cytochrome aa3 was foundfor copper-deficient rat heart mitochondria. Gel electrophoresisof protein extractedfrom mitochondrial membranes allowed measurements of protein content of thecomplexes ofoxidative phosphorylation, revealing a lower content of complex IV protein incopper-deficientrat heart mitochondria. The alterations caused by copper deficiency appear to bespecific forcytochrome c oxidase. Changes were not observed for F 0 F 1 ATP synthase activity,for heme contents ofcytochrome c and b, and for protein contents of complexes I, III and V.The present study demonstrates that the alteration of cytochrome c oxidase activityobserved in copper deficiency is due to a diminishedcontent of assembled protein and that shortnessof copper impairs heme insertion into cytochrome c oxidase.     

Synthesis of ordinary and -hydroxy fatty acids by heart mitochondria

The fatty acids (FA) synthesized from acetate by intact rabbit heart mitochondria were identified. These FA were mainly 12 to 16 carbons long. One-half were β-hydroxy FA, and mass spectrometric analysis after [1-¹³C)acetate incorporation showed them to be synthesized de novo. The latter were oxidized by the mitochondria with an ADP pulse, which means that they were L(+) isomers. β-Hydroxymyristate was the predominant endogenous saturated β-hydroxy FA detected in heart mitochondria.     

Synthesis of proteins in heart mitochondria during postnatal development of the rat

Proteins of inner mitochondrial membranes of the albino rat myocardium during postnatal development of 1, 3 and 6 months old animals were electrophoretically separated in 10% polyacrylamide gel. The rate of 14C-amino acids incorporation into examined proteins was determined in vitro. Specific radioactivity of the total mitochondrial fraction decreased in the course of the postnatal development. That of outer membranes remained unchanged, though it sharply increased in inner membranes of mature animals as compared with animals aged one month. Levels of radioactive precursor incorporation in separate protein fractions of inner membranes of the myocardium mitochondria were estimated.