Mitochondrial OXPHOS Functions in R1H Rhabdomyosarcoma and Skeletal Muscles of the Rat

The aim of the study was to determinate mitochondrial oxidative phosphorylation (OXPHOS) functions in rat rhabdomyosarcoma R1H (R1H) and rat skeletal muscles. For that purpose skinned fiber technique and multiple substrate inhibitor titration were adapted to tumor samples. In our animal tumor model (R1H) functional abnormalities of OXPHOS were found compared to skeletal muscles. In R1H the state 3 respiration of pyruvate + malate was decreased: 0.56 +/- 0.28 nmol O(2)/mg/min versus 2.32 +/- 1.19 nmol O(2)/mg/min, P < 0.001, whereas the state 3 respiration of succinate + rotenone was increased: 36 +/- 14% versus 19 +/- 11%, P < 0.001. In R1H the rotenone-insensitive respiration reached higher levels than the antimycin A-insensitive respiration, whereas in normal muscles the converse was observed. Additionally, the obvious difference between the CAT- and the antimycin A-independent respiration indicates an increased part of leak respiration in R1H. By now, the high feasibility of these techniques is appreciated for the investigation of muscles and prospectively for tumors, too.     

Life-history Constraints on the Mechanisms that Control the Rate of ROS Production

The quest to understand why and how we age has led to numerous lines of investigation that have gradually converged to consider mitochondrial metabolism as a major player. During mitochondrial respiration a small and variable amount of the consumed oxygen is converted to reactive species of oxygen (ROS). For many years, these ROS have been perceived as harmful by-products of respiration. However, evidence from recent years indicates that ROS fulfill important roles as cellular messengers. Results obtained using model organisms suggest that ROS-dependent signalling may even activate beneficial cellular stress responses, which eventually may lead to increased lifespan. Nevertheless, when an overload of ROS cannot be properly disposed of, its accumulation generates oxidative stress, which plays a major part in the ageing process. Comparative studies about the rates of ROS production and oxidative damage accumulation, have led to the idea that the lower rate of mitochondrial oxygen radical generation of long-lived animals with respect to that of their short-lived counterpart, could be a primary cause of their slow ageing rate. A hitherto largely under-appreciated alternative view is that such lower rate of ROS production, rather than a cause may be a consequence of the metabolic constraints imposed for the large body sizes that accompany high lifespans. To help understanding the logical underpinning of this rather heterodox view, herein I review the current literature regarding the mechanisms of ROS formation, with particular emphasis on evolutionary aspects.     

Analysis of mitochondrial subunit assembly into respiratory chain complexes using Blue Native polyacrylamide gel electrophoresis

The mitochondrial respiratory chain consists of multi-subunit protein complexes embedded in the inner membrane. Although the majority of subunits are encoded by nuclear genes and are imported into mitochondria, 13 subunits in humans are encoded by mitochondrial DNA. The coordinated assembly of subunits encoded from two genomes is a poorly understood process, with assembly pathway defects being a major determinant in mitochondrial disease. In this study, we monitored the assembly of human respiratory complexes using radiolabeled, mitochondrially encoded subunits in conjunction with Blue Native polyacrylamide gel electrophoresis. The efficiency of assembly was found to differ markedly between complexes, and intermediate complexes containing newly synthesized mitochondrial DNA-encoded subunits could be observed for complexes I, III, and IV. In particular, we detected human cytochrome b as a monomer and as a component of a novel approximately 120 kDa intermediate complex at early chase times before being totally assembled into mature complex III. Furthermore, we show that this approach is highly suited for the rapid detection of respiratory complex assembly defects in fibroblasts from patients with mitochondrial disease and, thus, has potential diagnostic applications.     

Preservation of mitochondrial functional integrity in mitochondria isolated from small cryopreserved mouse brain areas

Studies of mitochondrial bioenergetics in brain pathophysiology are often precluded by the need to isolate mitochondria immediately after tissue dissection from a large number of brain biopsies for comparative studies. Here we present a procedure of cryopreservation of small brain areas from which mitochondrial enriched fractions (crude mitochondria) with high oxidative phosphorylation efficiency can be isolated. Small mouse brain areas were frozen and stored in a solution containing glycerol as cryoprotectant. Crude mitochondria were isolated by differential centrifugation from both cryopreserved and freshly explanted brain samples and were compared with respect to their ability to generate membrane potential and produce ATP. Intactness of outer and inner mitochondrial membranes was verified by polarographic ascorbate and cytochrome c tests and spectrophotometric assay of citrate synthase activity. Preservation of structural integrity and oxidative phosphorylation efficiency was successfully obtained in crude mitochondria isolated from different areas of cryopreserved mouse brain samples. Long-term cryopreservation of small brain areas from which intact and phosphorylating mitochondria can be isolated for the study of mitochondrial bioenergetics will significantly expand the study of mitochondrial defects in neurological pathologies, allowing large comparative studies and favoring interlaboratory and interdisciplinary analyses.     

Metabolic remodeling in human colorectal cancer and surrounding tissues: alterations in regulation of mitochondrial respiration and metabolic fluxes

The aim of the work was to evaluate whether or not there is glycolytic reprogramming in the neighboring cells of colorectal cancer (CRC). Using postoperative material we have compared the functional capacity of oxidative phosphorylation (OXPHOS) in CRC cells, their glycolytic activity and their inclination to aerobic glycolysis, with those of the surrounding and healthy colon tissue cells. Experiments showed that human CRC cannot be considered a hypoxic tumor, since the malignancy itself and cells surrounding it exhibited even higher rates of OXPHOS than healthy large intestine. The absence of acute hypoxia in colorectal carcinomas was also confirmed by their practically equal glucose-phosphorylating capacity as compared with surrounding non-tumorous tissue and by upregulation of VEGF family and their ligands. Studies indicated that human CRC cells in vivo exert a strong distant effect on the energy metabolism of neighboring cells, so that they acquire the bioenergetic parameters specific to the tumor itself. The growth of colorectal carcinomas was associated with potent downregulation of the creatine kinase system. As compared with healthy colon tissue, the tumor surrounding cells display upregulation of OXPHOS and have high values of basal and ADP activated respiration rates. Strong differences between the normal and CRC cells in the affinity of their mitochondria for ADP were revealed; the corresponding K_m values were measured as 93.6±7.7 µM for CRC cells and 84.9±9.9 µM for nearby tissue; both these apparent K_m (ADP) values were considerably (by almost 3 times) lower in comparison with healthy colon tissue cells (256±34 µM).     

A novel oxyconforming response in the freshwater fish Galaxias maculatus

How fish oxygen consumption is modulated by external PO₂ has long been a matter of interest, yet is an experimentally complicated question to answer. In this study closed and semi-closed respirometry were used to evaluate the oxygen consumption rate of the scaleless galaxiid fish, inanga (Galaxias maculatus) as a function of decreasing external PO₂. Both respirometry techniques showed that as environmental oxygen levels declined, oxygen consumption rates also decreased. At no point did inanga regulate oxygen consumption. This is strong evidence that inanga is an oxyconformer. Partitioned respirometry experiments showed that skin plays an important role in oxygen uptake in this fish species, and cutaneous oxygen uptake may have an important role in shaping the oxygen consumption response to hypoxia.     

Circumventing the Crabtree Effect: A method to induce lactate consumption and increase oxidative phosphorylation in cell culture

Most cells grown in glucose-containing medium generate almost all their ATP via glycolysis despite abundant oxygen supply and functional mitochondria, a phenomenon known as the Crabtree effect. By contrast, most cells within the body rely on mitochondrial oxidative phosphorylation (OXPHOS) to generate the bulk of their energy supply. Thus, when utilising the accessibility of cell culture to elucidate fundamental elements of mitochondria in health and disease, it is advantageous to adopt culture conditions under which the cells have greater reliance upon OXPHOS for the supply of their energy needs. Substituting galactose for glucose in the culture medium can provide these conditions, but additional benefit can be gained from alternate in vitro models. Herein we describe culture conditions in which complete autonomous depletion of medium glucose induces a lactate-consuming phase marked by increased MitoTracker Deep Red staining intensity, increased expression of Kreb’s cycle proteins, increased expression of electron transport chain subunits, and increased sensitivity to the OXPHOS inhibitor rotenone. We propose these culture conditions represent an alternate accessible model for the in vitro study of cellular processes and diseases involving the mitochondrion without limitations incurred via the Crabtree effect.     

The pathomechanism of cytochrome c oxidase deficiency includes nuclear DNA damage

Mitochondrial cytochrome c oxidase (COX, respiratory chain complex IV), contributes to ATP production via oxidative phosphorylation (OXPHOS). Clinical presentation of COX deficiency is heterogeneous ranging from mild to severe neuromuscular diseases. Anemia is among the symptoms and we have previously reported Fanconi anemia like features in COX4-1 deficiency, suggesting genomic instability and our preliminary results detected nuclear double stranded DNA breaks (DSB). We now quantified the DSB by phospho histone H2AX Ser139 staining of COX4-1 and COX6B1 deficient fibroblasts (225% and 215% of normal, respectively) and confirmed their occurrence by neutral comet assay. We further explored the mechanism of DNA damage by studying normal fibroblasts treated with micromolar concentrations of cyanide (KCN). Present results demonstrate elevated nuclear DSB in cells treated with 50?μM KCN for 24?h (170% of normal) in high-glucose medium conditions where ROS and ATP remain normal, although Glutathione content was partially decreased. In glucose-free and serum-free medium, where growth is hampered, DSB were not elevated. Additionally we demonstrate the benefit of nicotinamide riboside (NR) which ameliorated DSB in COX4-1, COX6B1 and KCN treated cells (130%, 154% and 87% of normal cells, respectively). Conversely a negative effect of a poly[ADP-ribose] polymerase (PARP) inhibitor was found. Although additional investigation is needed, our findings raise the possibility that the pathomechanism of COX deficiency and possibly also in other OXPHOS defects, include nuclear DNA damage resulting from nicotinamide adenine dinucleotide (NAD⁺) deficit combined with a replicative state, rather than oxidative stress and energy depletion.     

Inhibition of the mitochondrial permeability transition pore reduces “apoptosis like” changes during cryopreservation of stallion spermatozoa

In order to evaluate to what extent the changes that occur during cryopreservation involve the mitochondrial permeability transition pore (PT-pore), a specific inhibitor was used during the cryopreservation process of stallion spermatozoa. Four ejaculates from each of 7 stallions were frozen using a standard protocol. Before freezing, each ejaculate was split into three subsamples. The first was supplemented with 2.5 μM bongkrekic acid (BA) and the second with 5 μM BA. The third subsample served as control. The BA significantly reduced the percentage of spermatozoa depicting active caspases after thawing, reduced the percentage of spermatozoa with increased membrane permeability, and increased the mitochondrial membrane potential of thawed sperm. Sperm motility was reduced as a result of the treatment. It is concluded that the mitochondrial pathway of apoptosis seems to be an important factor involved in the sublethal damage that equine spermatozoa experience after freezing and thawing, and that sperm motility in the equine species is largely dependent on mitochondrial ATP produced by oxidative phosphorylation.