Age-related difference in bioenergetics of lung and heart mitochondria from rats exposed to ozone

Bioenergetics of isolated lung and heart mitochondria from adult and aged rats were examined in the presence of glutamate (NAD-linked substrate) or succinate + rotenone (FAD-linked substrate) following ozone exposure (3.0 ppm, 8 hr). In controls, several differences were observed between adults and aged in both organ preparations. Following exposure, all bioenergetic parameters were decreased significantly in lung preparations from both adult and aged rats. In heart mitochondria, the respiration rates in state 3 and in uncoupled state, and the ADP/O ratio were decreased significantly in both exposed age groups. The respiratory control ratio (RCR) was decreased significantly only in the aged exposed rats. These results suggest that acute exposure to high levels of ozone alters energy production in both lung and heart mitochondria of adult and aged rats.     

Age-related difference in pulmonary response to ozone

Acute exposure to 1.5 ppm O3 produced different responses in adult and aged rat lungs. Total triphosphonucleotides were only slightly decreased in adult animals, but were markedly decreased in aged animals. Also, adult animals maintained a greater proportion of their available triphosphonucleotides in the reduced form (NADPH) compared to aged animals. These results suggest that aged animals may not be able to maintain pulmonary reducing equivalents as efficiently as adult animals in the face of an oxidant insult.     

Alteration of lung lipids in ozone exposed rats.

Exposure of adult male rats to 1.1 ± 0.3 ppm of ozone gave a 10-fold elevation of arachidonic acid in the lipid of the endobronchial washings. Arachidonate and linoleate increased in the cholesteryl esters from 6.5% to 55.5% and 4.7% to 20.4%, respectively. Similar changes also occurred in the composition of phosphatidyl choline. Serum lecithin:cholesterol acyl transferase (LCAT) activity was increased by exposure to ozone and returned to normal levels upon reexposure to an atmosphere of uncontaminated air. The results suggest that the lipid enzyme systems are strongly influenced by ozone exposure.     

Age-related sensitivity to lung oxidative stress during ozone exposure

As immature and aged rats could be more sensitive to ozone (O(3))-linked lung oxidative stress we have attempted to shed more light on age-related susceptibility to O(3) with focusing our interest on lung mitochondrial respiration, reactive oxygen species (ROS) production and lung pro/antioxidant status. For this purpose, we exposed to fresh air or O(3) (500 ppb 12 h per day, for 7 days) 3 week- (immature), 6 month- (adult) and 20 month-old rats (aged). We determined, in lung, H(2)O(2) release by mitochondria, activities of major antioxidant enzymes [superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT)], heat shock protein (HSP(72)) content and 8-oxodG and dG-HNE nDNA contents, as DNA oxidative damage markers. In adult rats we did not observe alteration of pro/antioxidant status. In contrast to adults, immature rats exposed to O(3) higher nDNA 8-oxodG content and HSP(72) and without antioxidant enzymes modification. Aged rats displayed mild uncoupled lung mitochondria, increased SOD and GPx activities, and higher 8-oxodG content after O(3) exposure. Thus, in contrast to adults, immature and aged rats displayed lung oxidative stress after O(3) exposure. Higher sensitivity of immature to O(3) was partly related to ventilatory parameters and to the absence of antioxidant enzyme response. In aged rats, the increase in cytosolic SOD and GPx activities during O(3) exposure was not sufficient to prevent the impairment in mitochondrial function and accumulation in lung 8- oxodG. Finally, we showed that mitochondria seem not to be a major source of ROS under O(3) exposure.     

Essential roles of mitochondrial and heme function in lung cancer bioenergetics and tumorigenesis

Contrary to Warburg’s hypothesis, mitochondrial oxidative phosphorylation (OXPHOS) contributes significantly to fueling cancer cells. Several recent studies have demonstrated that radiotherapy-resistant and chemotherapy-resistant cancer cells depend on OXPHOS for survival and progression. Several cancers exhibit an increased risk in association with heme intake. Mitochondria are widely known to carry out oxidative phosphorylation. In addition, mitochondria are also involved in heme synthesis. Heme serves as a prosthetic group for several proteins that constitute the complexes of mitochondrial electron transport chain. Therefore, heme plays a pivotal role in OXPHOS and oxygen consumption. Further, lung cancer cells exhibit heme accumulation and require heme for proliferation and invasion in vitro. Abnormalities in mitochondrial biogenesis and mutations are implicated in cancer. This review delves into mitochondrial OXPHOS and lesser explored area of heme metabolism in lung cancer.     

Regulation of mitochondrial bioenergetics by the non-canonical roles of mitochondrial dynamics proteins in the heart

Recent advancement in mitochondrial research has significantly extended our knowledge on the role and regulation of mitochondria in health and disease. One important breakthrough is the delineation of how mitochondrial morphological changes, termed mitochondrial dynamics, are coupled to the bioenergetics and signaling functions of mitochondria. In general, it is believed that fusion leads to an increased mitochondrial respiration efficiency and resistance to stress-induced dysfunction while fission does the contrary. This concept seems not applicable to adult cardiomyocytes. The mitochondria in adult cardiomyocytes exhibit fragmented morphology (tilted towards fission) and show less networking and movement as compared to other cell types. However, being the most energy-demanding cells, cardiomyocytes in the adult heart possess vast number of mitochondria, high level of energy flow, and abundant mitochondrial dynamics proteins. This apparent discrepancy could be explained by recently identified new functions of the mitochondrial dynamics proteins. These “non-canonical” roles of mitochondrial dynamics proteins range from controlling inter-organelle communication to regulating cell viability and survival under metabolic stresses. Here, we summarize the newly identified non-canonical roles of mitochondrial dynamics proteins. We focus on how these fission and fusion independent roles of dynamics proteins regulate mitochondrial bioenergetics. We also discuss potential molecular mechanisms, unique intracellular location, and the cardiovascular disease relevance of these non-canonical roles of the dynamics proteins. We propose that future studies are warranted to differentiate the canonical and non-canonical roles of dynamics proteins and to identify new approaches for the treatment of heart diseases. This article is part of a Special issue entitled Cardiac adaptations to obesity, diabetes and insulin resistance, edited by Professors Jan F.C. Glatz, Jason R.B. Dyck and Christine Des Rosiers.     

Abnormal mitochondrial bioenergetics and heart rate dysfunction in mice lacking very-long-chain acyl-CoA dehydrogenase

Mitochondrial very-long-chain acyl-CoA dehydrogenase (VLCAD) deficiency is associated with severe hypoglycemia, cardiac dysfunction, and sudden death in neonates and children. Sudden death is common, but the underlying mechanisms are not fully understood. We report on a mouse model of VLCAD deficiency with a phenotype induced by the stresses of fasting and cold, which includes hypoglycemia, hypothermia, and severe bradycardia. The administration of glucose did not rescue the mice under stress conditions, but rewarming alone consistently led to heart rate recovery. Brown adipose tissue (BAT) from the VLCAD-/- mice showed elevated levels of the uncoupling protein isoforms and peroxisome proliferator-activated receptor-alpha. Biochemical assessment of the VLCAD(/- mice BAT showed increased oxygen consumption, attributed to uncoupled respiration in the absence of stress. ADP-stimulated respiration was 23.05 (SD 4.17) and 68.24 (SD 6.3) nmol O2.min(-1).mg mitochondrial protein(-1) for VLCAD+/+ and VLCAD-/- mice, respectively (P < 0.001), and carbonyl cyanide p-trifluoromethoxyphenylhydrazone-stimulated respiration was 35.9 (SD 3.6) and 49.3 (SD 9) nmol O2.min(-1).mg mitochondrial protein(-1) for VLCAD+/+ and VLCAD-/- mice, respectively (P < 0.20), but these rates were insufficient to protect them in the cold. We conclude that disturbed mitochondrial bioenergetics in BAT is a critical contributing factor for the cold sensitivity in VLCAD deficiency. Our observations provide insights into the possible mechanisms of stress-induced death in human newborns with abnormal fat metabolism and elucidate targeting of specific substrates for particular metabolic needs.     

Substrate-dependent differential regulation of mitochondrial bioenergetics in the heart and kidney cortex and outer medulla

The kinetics and efficiency of mitochondrial oxidative phosphorylation (OxPhos) can depend on the choice of respiratory substrates. Furthermore, potential differences in this substrate dependency among different tissues are not well-understood. Here, we determined the effects of different substrates on the kinetics and efficiency of OxPhos in isolated mitochondria from the heart and kidney cortex and outer medulla (OM) of Sprague-Dawley rats. The substrates were pyruvate+malate, glutamate+malate, palmitoyl-carnitine+malate, alpha-ketoglutarate+malate, and succinate±rotenone at saturating concentrations. The kinetics of OxPhos were interrogated by measuring mitochondrial bioenergetics under different ADP perturbations. Results show that the kinetics and efficiency of OxPhos are highly dependent on the substrates used, and this dependency is distinctly different between heart and kidney. Heart mitochondria showed higher respiratory rates and OxPhos efficiencies for all substrates in comparison to kidney mitochondria. Cortex mitochondria respiratory rates were higher than OM mitochondria, but OM mitochondria OxPhos efficiencies were higher than cortex mitochondria. State 3 respiration was low in heart mitochondria with succinate but increased significantly in the presence of rotenone, unlike kidney mitochondria. Similar differences were observed in mitochondrial membrane potential. Differences in H₂O₂ emission in the presence of succinate±rotenone were observed in heart mitochondria and to a lesser extent in OM mitochondria, but not in cortex mitochondria. Bioenergetics and H₂O₂ emission data with succinate±rotenone indicate that oxaloacetate accumulation and reverse electron transfer may play a more prominent regulatory role in heart mitochondria than kidney mitochondria. These studies provide novel quantitative data demonstrating that the choice of respiratory substrates affects mitochondrial responses in a tissue-specific manner.     

Structure-functional states of liver mitochondrial membranes of rats exposed to irradiation

The effect of the low dose gamma-irradiation (270 cGy--one-fold; 90 cGy per day during 3 days) on oxidative phosphorylation, lipid peroxidation, microviscosity of the annular and free lipids membrane, and membrane protein structural state was studied. The post-radiation influence on membrane functional activity and structural state in accordance with the irradiation regimes was established.     

Age-related difference in cardiac adaptation to chronic hypertension in rats,with and without nifedipine treatement

Three myosin isozymes, V1 ( MHC = Myosin Heavy Chain gene), V2 ( MHC) and V3 ( MHC) that are identified in the cardiac ventricles of most mammals have been shown to shift to a V3 predominance pattern during cardiac growth and in response to left ventricular pressure overload, and to V1 predominance following anti hypertensive treatment. This study examined whether long-term hypertension impairs the ability of the adult heart to restructure myosin isozyme proportions. Using pyrophosphate gel electrophoresis, we studied proportions of cardiac myosin isozymes (V1 and V3) in young (16 weeks) and adult (36 weeks) spontaneously hypertensive rats (SHR), and following 12 weeks of nifedipine (N) treatment in age-matched SHR rats (SHR-N). The values of V1 and V3 myosin isozymes were derived by adding half of the value of V2 to each isozyme proportion. The V3 proportion in the young SHR control (SHR-C) group (49%) was 34% higher (p < 0.05) than in the young Wistar Kyoto control (WKY-C) group (37%). However, the proportion was similarly high, though not statistically significant, in both the adult SHRC (73%) and WKY-C (71%) groups. The proportion in the young SHR-N group (29%) was 41% lower (p < 0.05) than in the young SHR-C group (49%), and the proportion in the adult SHR-N group (47%) was 34% lower (p < 0.05) than in the adult SHR-C group (73%). The ratio of left ventricular weight to body weight (LVW/BW), which determines left ventricular hypertrophy (LVH), was higher in both young and adult SHR-C (26%, p < 0.05, and 42%, p < 0.05, respectively) than in WKY-C groups. The mean LVW/BW was 27% (p lt; 0.05) greater in adult than in young SHR-C rats. The LVW/BW in both age groups of treated SHR-N was similar to that in age matched WKY-C rats. Conclusion: Our study showed that a rise in the V3 level occurs in young hypertensive rats, but no rise occurs in the V3 level in adult hypertensive rats. High blood pressure seems to contribute to the high V3 level in young hypertensive rats, but in adult hypertensive rats, high blood pressure does not accentuate the V3 rise already acquired due to the aging process. Nifedipine treatment in both young and adult hypertensive rats prevented the V3 rise due to hypertension and to the aging process. This effect of nifedipine seems to be through its antihypertensive action.     

Functional role of cardiolipin in mitochondrial bioenergetics

Cardiolipin is a unique phospholipid which is almost exclusively located in the inner mitochondrial membrane where it is biosynthesized. Considerable progress has recently been made in understanding the role of cardiolipin in mitochondrial function and bioenergetics. This phospholipid is associated with membranes designed to generate an electrochemical gradient that is used to produce ATP, such as bacterial plasma membranes and inner mitochondrial membrane. This ubiquitous and intimate association between cardiolipin and energy transducing membranes indicates an important role for cardiolipin in mitochondrial bioenergetic processes. Cardiolipin has been shown to interact with a number of proteins, including the respiratory chain complexes and substrate carrier proteins. Over the past decade, the significance of cardiolipin in the organization of components of the electron transport chain into higher order assemblies, termed respiratory supercomplexes, has been established. Moreover, cardiolipin is involved in different stages of the mitochondrial apoptotic process, as well as in mitochondrial membrane stability and dynamics. This review discusses the current understanding of the functional role that cardiolipin plays in several reactions and processes involved in mitochondrial bioenergetics. This article is part of a Special Issue entitled: Dynamic and ultrastructure of bioenergetic membranes and their components.     

Liver and brain mitochondrial ATPase activities in rats exposed to high ambient temperature

Liver and brain mitochondrial ATPase activities in rats exposed to high ambient temperature. Acta physiol. pol., 1985, 36 (3): 185-192. Rat liver and brain mitochondrial ATPase activities were investigated after a single exposure (6 h) of the animals to temperatures of 21 degrees, 28 degrees and 37 degrees C. An increase of ATPase activity stimulated by Ca++ ions was noted in the mitochondrial fractions of the liver at 28 degrees C and of the brain at 28 degrees and 37 degrees C. Only in liver mitochondria of rats exposed to 28 degrees C a depression of Mg++-ATPase activity was found.     

Age-related induction and disappearance of carcinogen-DNA-adducts in livers of rats exposed to low levels of 2-acetylaminofluorene

It was investigated whether in vivo aging of rat liver is associated with changes in the induction and rate of disappearance of DNA damage. For this purpose 6- and 36-month-old rats were intraperitoneally injected with a single, low dose (5 mg/kg body wt.) of the model liver carcinogen 2-acetylaminofluorene (AAF). Using the 32P-postlabeling assay we found that N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF) was the major DNA-adduct formed. The minor adduct N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-C8-AAF) could only be detected after doses of 20 mg/kg or more. Quantitation of adduct levels at various time points after treatment indicated a rapid induction of AF-adducts, which were already present at 6 h after treatment. The subsequent loss of AF-adducts was relatively slow, as was indicated by the presence of a substantial amount of AF-adducts as late as 21 days after treatment. Slight age-related differences in the pattern of induction and disappearance of AF-adducts and a somewhat higher level of persisting lesions in old than in young rats were observed.     

Exposure of rats to ozone: evidence of damage to heart and brain.

Ozone is a strong oxidizing agent, and in many locations it is a major atmospheric pollutant. It is phytotoxic and an important cause of lung dysfunction in humans. Recently, a significant association has been established between total atmospheric oxidants, of which ozone is one, and daily cardiovascular mortality rates. In this article, we show that exposure of rats to ozone for 5 days, in a concentration found in major urban centers, results in an increased concentration of thiobarbituric acid-reactive material (an indicator of lipid peroxidation) in heart and brain tissue as well as elevated activity of catalase and glutathione peroxidase (enzymic scavengers of peroxides) in these tissues. We examined the heart anatomically and found evidence of extracellular and intracellular edema. These findings indicate that the heart and brain are damaged by a concentration of ozone present in major urban centers; they may have important implications for chronic illness and degenerative processes in humans.     

Augmentation of mitochondrial oxidative metabolism in lung tissue during recovery of animals from acute ozone exposure

After O₃-mediated lung injury in rats (3 ppm O₃ exposure for 4 hr) recovery was studied in terms of alteration in lung mitochondrial oxidative metabolism. As judged from O₂ consumption, succinate oxidation in lung homogenate exhibited a 20% (P < 0.05) decrease at 0 hr but attained the control rate (0.6 μmole O₂/min/lung) within 12 hr and the peak rate (55% over control, P < 0.001) within 48 hr of recovery. Thereafter, the rate plateaued and at about the fifth day began to decline, exhibiting only a 15% (P < 0.05) increase over control after 21 days. The half-life for duration of this augmentation appeared to be 10 days. During recovery, the yield of isolated mitochondria was increasingly greater for exposed lungs relative to control (viz., 25–30% increase after 96 hr) as viewed from mitochondrial packed volume and protein content. Mitochondria from exposed lungs exhibited a 17–24% (P < 0.05) increase in activity (per mg of protein) for oxidation of 2-oxoglutarate, succinate, glycerol-1-phosphate, and ascorbate-Wurster's blue. The over-all augmentation of O₂ consumption observed in exposed rat lungs, therefore, would be attributable primarily to increase in population of mitochondria. Enhanced mitochondrial metabolism might serve as an index for assessing the repair process of injured lung.     

Elevated hepatic mitochondrial oxidative capacities in cold exposed rats

1. Hepatic mitochondrial oxidative capacities were studied in rats exposed to cold for periods ranging from 5 to 15 days. The mitochondria obtained in this study were well coupled as shown by RCR and ADP/O ratios. 2. The liver mitochondria of cold exposed rats showed significantly increased respiratory rates (ng atoms of oxygen consumed min-1 mg prot-1), starting from day 10 of cold exposure, using lipid and non-lipid substrates. 3. For non-lipid substrates, the elevated respiratory rates found in the mitochondria could indicate an increased capacity to oxidize these substrates. For the lipid substrate, on the other hand, an enhanced oxidation through Krebs-cycle of a part of acetyl-CoA otherwise utilized to form ketone bodies, could also occur. 4. Taken together the results suggest that, during cold exposure, liver mitochondria could participate in cold adaptation mechanisms, by improving ATP production.