Mitochondrial DNA synthesis studied autoradiographically in various cell types in vivo

It is generally accepted that mitochondria are able to proliferate even in postmitotic cells due to their natural turnover and also to satisfy increased cell energy requirements. However, no detailed studies are available, particularly with respect to specific cell types. Since [3H]-thymidine is incorporated not only into nuclear (n) DNA but also into the DNA of cytoplasmic mitochondria, an autoradiographic approach was developed at the light microscopy level in order to study basic questions of mitochondrial (mt) proliferation in organs of rodents in situ via the cytoplasmic incorporation of [3H]-thymidine injected into the animals 1 h before sacrifice. Experiments carried out on mice after X-irradiation showed that cytoplasmic labeling was not due to a process such as unscheduled nuclear DNA synthesis (nUDS). Furthermore, half-lives of mitochondria between 8-23 days were deduced specifically in relation to cell types. The phase of mtDNA synthesis was about 75 min. Finally, mt proliferation was measured in brain cells of mice as a function of age. While all neurons showed a decreasing extent of mtDNA synthesis during old age, nUDS decreased only in distinct cell types of the cortex and hippocampus. We conclude that the leading theories explaining the phenomenon of aging are closely related, i.e., aging is due to a decreasing capacity of nDNA repair, which leads to unrepaired nDNA damage, or to an accumulation of mitochondria with damaged mtDNA, which leads to a deficit of cellular energy production

Alternative oxidase in the branched mitochondrial respiratory network: an overview on structure, function, regulation, and role

Plants and some other organisms including protists possess a complex branched respiratory network in their mitochondria. Some pathways of this network are not energy-conserving and allow sites of energy conservation to be bypassed, leading to a decrease of the energy yield in the cells. It is a challenge to understand the regulation of the partitioning of electrons between the various energy-dissipating and -conserving pathways. This review is focused on the oxidase side of the respiratory chain that presents a cyanide-resistant energy-dissipating alternative oxidase (AOX) besides the cytochrome pathway. The known structural properties of AOX are described including transmembrane topology, dimerization, and active sites. Regulation of the alternative oxidase activity is presented in detail because of its complexity. The alternative oxidase activity is dependent on substrate availability: total ubiquinone concentration and its redox state in the membrane and O2 concentration in the cell. The alternative oxidase activity can be long-term regulated (gene expression) or short-term (post-translational modification, allosteric activation) regulated. Electron distribution (partitioning) between the alternative and cytochrome pathways during steady-state respiration is a crucial measurement to quantitatively analyze the effects of the various levels of regulation of the alternative oxidase. Three approaches are described with their specific domain of application and limitations: kinetic approach, oxygen isotope differential discrimination, and ADP/O method (thermokinetic approach). Lastly, the role of the alternative oxidase in non-thermogenic tissues is discussed in relation to the energy metabolism balance of the cell (supply in reducing equivalents/demand in energy and carbon) and with harmful reactive oxygen species formation.

Characterization of six rat strains (Rattus norvegicus) by mitochondrial DNA restriction fragment length polymorphism

Restriction fragment length polymorphism (RFLP) was used to examine the extent of mtDNA polymorphism among six strains of rats (Rattus norvegicus) - Wistar, Wistar Munich, Brown Norway, Wistar Kyoto, SHR and SHR-SP. A survey of 26 restriction enzymes has revealed a low level of genetic divergence among strains. The sites of cleavage by EcoRI, NcoI and XmnI were shown to be polymorphic. The use of these three enzymes allows the 6 strains to be classified into 4 haplotypes and identifies specific markers for each one. The percentage of sequence divergence among all pairs of haplotypes ranged from 0.035 to 0.33%, which is the result of a severe population constriction undergone by the strains. These haplotypes are easily demonstrable and therefore RFLP analysis can be employed for genetic monitoring of rats within animal facilities or among different laboratories.

Alternative mitochondrial functions in cell physiopathology: beyond ATP production

It is well known that mitochondria are the main site for ATP generation within most tissues. However, mitochondria also participate in a surprising number of alternative activities, including intracellular Ca2+ regulation, thermogenesis and the control of apoptosis. In addition, mitochondria are the main cellular generators of reactive oxygen species, and may trigger necrotic cell death under conditions of oxidative stress. This review concentrates on these alternative mitochondrial functions, and their role in cell physiopathology.

Restriction site heteroplasmy in the mitochondrial DNA of Brycon opalinus (Cuvier, 1819) (Characiformes, Characidae, Bryconiae)

Homoplasmy is a feature usually found in the mtDNA of higher animal taxa. On the other hand, the presence of two classes of mtDNA in the same cell or organism is rare and may appear in length or site variation. Data from mtDNA RFLP analysis of Brycon opalinus populations (Cuvier, 1819; Characiformes, Characidae, Bryconinae) revealed site heteroplasmy from endonuclease NheI digestion. Southern blotting hybridization was used to survey a total of 257 specimens with 24 restriction enzymes. Three different restriction fragment patterns of mtDNA were obtained from NheI digestion. Two individuals from hatchery broodstock were found to have two of them. NheI digests of heteroplasmic individuals yielded two fragments of approximately 1180 and 1260 bp. Despite the low frequency of this type of heteroplasmy in the whole B. opalinus population, the presence of site heteroplasmy in this species supports the evidence of this phenomenon in lower vertebrate groups.

Evaluation by blue native polyacrylamide electrophoresis colorimetric staining of the effects of physical exercise on the activities of mitochondrial complexes in rat muscle

Blue native polyacrylamide electrophoresis (BN-PAGE) is a technique developed for the analysis of membrane complexes. Combined with histochemical staining, it permits the analysis and quantification of the activities of mitochondrial oxidative phosphorylation enzymes using whole muscle homogenates, without the need to isolate muscle mitochondria. Mitochondrial complex activities were measured by emerging gels in a solution containing all specific substrates for NADH dehydrogenase and cytochrome c oxidase enzymes (complexes I and IV, respectively) and the colored bands obtained were measured by optique densitometry. The objective of the present study was the application of BN-PAGE colorimetric staining for enzymatic characterization of mitochondrial complexes I and IV in rat muscles with different morphological and biochemical properties. We also investigated these activities at different times after acute exercise of rat soleus muscle. Although having fewer mitochondria than oxidative muscles, white gastrocnemius muscle presented a significantly higher activity (26.7 ± 9.5) in terms of complex I/V ratio compared to the red gastrocnemius (3.8 ± 0.65, P < 0.05) and soleus (9.8 ± 0.9, P < 0.001) muscles. Furthermore, the complex IV/V ratio of white gastrocnemius muscle was always significantly higher when compared to the other muscles. Ninety-five minutes of exhaustive physical exercise induced a decrease in complex I/V and complex IV/V ratios after all resting times (0, 3 and 6 h) compared to control (P < 0.05), probably reflecting the oxidative damage due to increasing free radical production in mitochondria. These results demonstrate the possible and useful application of BN-PAGE-histochemical staining to physical exercise studies.

Mitochondrial K+ transport and cardiac protection during ischemia/reperfusion

Mitochondrial ion transport, oxidative phosphorylation, redox balance, and physical integrity are key factors in tissue survival following potentially damaging conditions such as ischemia/reperfusion. Recent research has demonstrated that pharmacologically activated inner mitochondrial membrane ATP-sensitive K+ channels (mitoK ATP) are strongly cardioprotective under these conditions. Furthermore, mitoK ATP are physiologically activated during ischemic preconditioning, a procedure which protects against ischemic damage. In this review, we discuss mechanisms by which mitoK ATP may be activated during preconditioning and the mitochondrial and cellular consequences of this activation, focusing on end-effects which may promote ischemic protection. These effects include decreased loss of tissue ATP through reverse activity of ATP synthase due to increased mitochondrial matrix volumes and lower transport of adenine nucleotides into the matrix. MitoK ATP also decreases the release of mitochondrial reactive oxygen species by promoting mild uncoupling in concert with K+/H+ exchange. Finally, mitoK ATP activity may inhibit mitochondrial Ca2+ uptake during ischemia, which, together with decreased reactive oxygen release, can prevent mitochondrial permeability transition, loss of organelle function, and loss of physical integrity. We discuss how mitochondrial redox status, K+ transport, Ca2+ transport, and permeability transitions are interrelated during ischemia/reperfusion and are determinant factors regarding the extent of tissue damage.

Liver mitochondrial dysfunction and oxidative stress in the pathogenesis of experimental nonalcoholic fatty liver disease

Oxidative stress and hepatic mitochondria play a role in the pathogenesis of nonalcoholic fatty liver disease. The aim of the present study was to evaluate the role of hepatic mitochondrial dysfunction and oxidative stress in the pathogenesis of the disease. Fatty liver was induced in Wistar rats with a choline-deficient diet (CD; N = 7) or a high-fat diet enriched with PUFAs-omega-3 (H; N = 7) for 4 weeks. The control group (N = 7) was fed a standard diet. Liver mitochondrial oxidation and phosphorylation were measured polarographically and oxidative stress was estimated on the basis of malondialdehyde and glutathione concentrations. Moderate macrovacuolar liver steatosis was observed in the CD group and mild liver steatosis was observed in the periportal area in the H group. There was an increase in the oxygen consumption rate by liver mitochondria in respiratory state 4 (S4) and a decrease in respiratory control rate (RCR) in the CD group (S4: 32.70 ± 3.35; RCR: 2.55 ± 0.15 ng atoms of O2 min-1 mg protein-1) when compared to the H and control groups (S4: 23.09 ± 1.53, 17.04 ± 2.03, RCR: 3.15 ± 0.15, 3.68 ± 0.15 ng atoms of O2 min-1 mg protein-1, respectively), P < 0.05. Hepatic lipoperoxide concentrations were significantly increased and the concentration of reduced glutathione was significantly reduced in the CD group. A choline-deficient diet causes moderate steatosis with disruption of liver mitochondrial function and increased oxidative stress. These data suggest that lipid peroxidation products can impair the flow of electrons along the respiratory chain, causing overreduction of respiratory chain components and enhanced mitochondrial reactive oxygen species. These findings are important in the pathogenesis of nonalcoholic fatty liver disease.

Prevalence of the A1555G (12S rRNA) and tRNA Ser(UCN) mitochondrial mutations in hearing-impaired Brazilian patients

Mitochondrial mutations are responsible for at least 1% of the cases of hereditary deafness, but the contribution of each mutation has not yet been defined in African-derived or native American genetic backgrounds. A total of 203 unselected hearing-impaired patients were screened for the presence of the mitochondrial mutation A1555G in the 12S rRNA gene and mutations in the tRNA Ser(UCN) gene in order to assess their frequency in the ethnically admixed Brazilian population. We found four individuals with A1555G mutation (2%), which is a frequency similar to those reported for European-derived populations in unselected samples. On the other hand, complete sequencing of the tRNA Ser(UCN) did not reveal reported pathogenic substitutions, namely A7445G, 7472insC, T7510C, or T7511C. Instead, other rare substitutions were found such as T1291C, A7569G, and G7444A. To evaluate the significance of these findings, 110 "European-Brazilians" and 190 "African-Brazilians" unrelated hearing controls were screened. The T1291C, A7569G and G7444A substitutions were each found in about 1% (2/190) of individuals of African ancestry, suggesting that they are probably polymorphic. Our results indicate that screening for the A1555G mutation is recommended among all Brazilian deaf patients, while testing for mutations in the tRNA Ser(UCN) gene should be considered only when other frequent deafness-causing mutations have been excluded or in the presence of a maternal transmission pattern.

Partial purification of tightly bound mitochondrial hexokinase from maize (Zea mays L.) root membranes

In mammals, hexokinase (HK) is strategically located at the outer membrane of mitochondria bound to the porin protein. The mitochondrial HK is a crucial modulator of apoptosis and reactive oxygen species generation. In plants, these properties related to HK are unknown. In order to better understand the physiological role of non-cytosolic hexokinase (NC-HK) in plants, we developed a purification strategy here described. Crude extract of 400 g of maize roots (230 mg protein) contained a specific activity of 0.042 µmol G6P min-1 mg PTN-1. After solubilization with detergent two fractions were obtained by DEAE column chromatography, NC-HK 1 (specific activity = 3.6 µmol G6P min-1 mg PTN-1 and protein recovered = 0.7 mg) and NC-HK 2. A major purification (yield = 500-fold) was obtained after passage of NC-HK 1 through the hydrophobic phenyl-Sepharose column. The total amount of protein and activity recovered were 0.04 and 18%, respectively. The NC-HK 1 binds to the hydrophobic phenyl-Sepharose matrix, as observed for rat brain HK. Mild chymotrypsin digestion did not affect adsorption of NC-HK 1 to the hydrophobic column as it does for rat HK I. In contrast to mammal mitochondrial HK, glucose-6-phosphate, clotrimazole or thiopental did not dissociate NC-HK from maize (Zea mays) or rice (Oryza sativa) mitochondrial membranes. These data show that the interaction between maize or rice NC-HK to mitochondria differs from that reported in mammals, where the mitochondrial enzyme can be displaced by modulators or pharmacological agents known to interfere with the enzyme binding properties with the mitochondrial porin protein.

A mechanistic view of mitochondrial death decision pores

Mitochondria increase their outer and inner membrane permeability to solutes, protons and metabolites in response to a variety of extrinsic and intrinsic signaling events. The maintenance of cellular and intraorganelle ionic homeostasis, particularly for Ca2+, can determine cell survival or death. Mitochondrial death decision is centered on two processes: inner membrane permeabilization, such as that promoted by the mitochondrial permeability transition pore, formed across inner membranes when Ca2+ reaches a critical threshold, and mitochondrial outer membrane permeabilization, in which the pro-apoptotic proteins BID, BAX, and BAK play active roles. Membrane permeabilization leads to the release of apoptogenic proteins: cytochrome c, apoptosis-inducing factor, Smac/Diablo, HtrA2/Omi, and endonuclease G. Cytochrome c initiates the proteolytic activation of caspases, which in turn cleave hundreds of proteins to produce the morphological and biochemical changes of apoptosis. Voltage-dependent anion channel, cyclophilin D, adenine nucleotide translocase, and the pro-apoptotic proteins BID, BAX, and BAK may be part of the molecular composition of membrane pores leading to mitochondrial permeabilization, but this remains a central question to be resolved. Other transporting pores and channels, including the ceramide channel, the mitochondrial apoptosis-induced channel, as well as a non-specific outer membrane rupture may also be potential release pathways for these apoptogenic factors. In this review, we discuss the mechanistic models by which reactive oxygen species and caspases, via structural and conformational changes of membrane lipids and proteins, promote conditions for inner/outer membrane permeabilization, which may be followed by either opening of pores or a rupture of the outer mitochondrial membrane.

The use of light-emitting diodes to stimulate mitochondrial function and liver regeneration of partially hepatectomized rats

The biostimulating effect of laser radiation has been observed in many areas of Medicine. However, there are still several questions to be answered, among them the importance of light coherence in the stimulatory process. In the present study, we used light-emitting diodes (LED) to promote the stimulation of liver regeneration after partial hepatectomy in rats. Fourteen male Wistar rats weighing 200-250 g were submitted to partial hepatectomy (70%) followed by LED light irradiation (630 nm) of the remaining part of the liver at two doses, i.e., 10 (N = 7) and 140 (N = 7) J/cm². A group irradiated with laser, 590 nm (N = 7, 15 J/cm²) was performed for the study of proliferating cell nuclear antigen-labeling index. Data are reported as mean ± SEM. Statistical comparisons of the groups were performed by analysis of variance for parametric measurements followed by the Bonferroni post-test, with the level of significance set at P < 0.05. Respiratory mitochondrial activity was increased in the irradiated groups (states 3 and 4; P < 0.05), with better results for the group exposed to the lower LED dose (10 J/cm²). The proliferating cell nuclear antigen-labeling index, by immunohistochemical staining, was similar for both LED-exposed groups (P > 0.05) and higher than for the control group (P < 0.05). The cell proliferation index obtained with LED and laser were similar (P > 0.05). In conclusion, the present results suggest that LED irradiation promotes biological stimulatory effects during the early stage of liver regeneration and that LED is as effective as laser light, independent of the coherence, divergence and cromaticity.

Mitochondrial control region haplotypes of the South American sea lion Otaria flavescens (Shaw, 1800)

The South American sea lion, Otaria flavescens, is widely distributed along the Pacific and Atlantic coasts of South America. However, along the Brazilian coast, there are only two nonbreeding sites for the species (Refúgio de Vida Silvestre da Ilha dos Lobos and Refúgio de Vida Silvestre do Molhe Leste da Barra do Rio Grande), both in Southern Brazil. In this region, the species is continuously under the effect of anthropic activities, mainly those related to environmental contamination with organic and inorganic chemicals and fishery interactions. This paper reports, for the first time, the genetic diversity of O. flavescens found along the Southern Brazilian coast. A 287-bp fragment of the mitochondrial DNA control region (D-loop) was analyzed. Seven novel haplotypes were found in 56 individuals (OFA1-OFA7), with OFA1 being the most frequent (47.54%). Nucleotide diversity was moderate (π = 0.62%) and haplotype diversity was relatively low (67%). Furthermore, the median joining network analysis indicated that Brazilian haplotypes formed a reciprocal monophyletic clade when compared to the haplotypes from the Peruvian population on the Pacific coast. These two populations do not share haplotypes and may have become isolated some time back. Further genetic studies covering the entire species distribution are necessary to better understand the biological implications of the results reported here for the management and conservation of South American sea lions.

Relationship between work rate and oxygen uptake in mitochondrial myopathy during ramp-incremental exercise

We determined the response characteristics and functional correlates of the dynamic relationship between the rate (Δ) of oxygen consumption ( O2) and the applied power output (work rate = WR) during ramp-incremental exercise in patients with mitochondrial myopathy (MM). Fourteen patients (7 males, age 35.4 ± 10.8 years) with biopsy-proven MM and 10 sedentary controls (6 males, age 29.0 ± 7.8 years) took a ramp-incremental cycle ergometer test for the determination of the O2 on-exercise mean response time (MRT) and the gas exchange threshold (GET). The ΔO2/ΔWR slope was calculated up to GET (S1), above GET (S2) and over the entire linear portion of the response (S T). Knee muscle endurance was measured by isokinetic dynamometry. As expected, peak O2 and muscle performance were lower in patients than controls (P < 0.05). Patients had significantly lower ΔO2/ΔWR than controls, especially the S2 component (6.8 ± 1.5 vs 10.3 ± 0.6 mL·min-1·W-1, respectively; P < 0.001). There were significant relationships between ΔO2/ΔWR (S T) and muscle endurance, MRT-O2, GET and peak O2 in MM patients (P < 0.05). In fact, all patients with ΔO2/ΔWR below 8 mL·min-1·W-1 had severely reduced peak O2 values (<60% predicted). Moreover, patients with higher cardiopulmonary stresses during exercise (e.g., higher Δ ventilation/carbon dioxide output and Δ heart rate/ΔO2) had lower ΔO2/ΔWR (P < 0.05). In conclusion, a readily available, effort-independent index of aerobic dysfunction during dynamic exercise (ΔO2/ΔWR) is typically reduced in patients with MM, being related to increased functional impairment and higher cardiopulmonary stress.

Normal skin and hypertrophic scar fibroblasts differentially regulate collagen and fibronectin expression as well as mitochondrial membrane potential in response to basic fibroblast growth factor

Basic fibroblast growth factor (bFGF) regulates skin wound healing; however, the underlying mechanism remains to be defined. In the present study, we determined the effects of bFGF on the regulation of cell growth as well as collagen and fibronectin expression in fibroblasts from normal human skin and from hypertrophic scars. We then explored the involvement of mitochondria in mediating bFGF-inducedeffects on the fibroblasts. We isolated and cultivated normal and hypertrophic scar fibroblasts from tissue biopsies of patients who underwent plastic surgery for repairing hypertrophic scars. The fibroblasts were then treated with different concentrations of bFGF (ranging from 0.1 to 1000 ng/mL). The growth of hypertrophic scar fibroblasts became slower with selective inhibition of type I collagen production after exposure to bFGF. However, type III collagen expression was affected in both normal and hypertrophic scar fibroblasts. Moreover, fibronectin expression in the normal fibroblasts was up-regulated after bFGF treatment. bFGF (1000 ng/mL) also induced mitochondrial depolarization in hypertrophic scar fibroblasts (P < 0.01). The cellular ATP level decreased in hypertrophic scar fibroblasts (P < 0.05), while it increased in the normal fibroblasts following treatment with bFGF (P < 0.01). These data suggest that bFGF has differential effects and mechanisms on fibroblasts of the normal skin and hypertrophic scars, indicating that bFGF may play a role in the early phase of skin wound healing and post-burn scar formation.