Bioenergetic cluster analysis – mitochondrial respiratory control in human fibroblasts

In this article the spontaneous chemiluminescence and the steady-state concentration of hydrogen peroxide were determined in rat liver as indicators of oxidative stress in the tissue. Hydroperoxide-initiated chemiluminescence and the activity of antioxidant enzymes (catalase, superoxide dismutase and glutathione peroxidase) were also measured to evaluate antioxidant defenses and serum activity of lactate dehydrogenase and aspartate aminotransferase. Mitochondrial morphology and mitochondrial respiratory control ratio were measured as indicators of cell and mitochondrial damage. Xanthine dehydrogenase and xanthine oxidase activities were determined as a possible source of oxyradicals. No significant changes were observed after 10 or 30 min of vena cava occlusion in any of the measured parameters. In contrast, 10 min of occlusion followed by 10 min of reperfusion increased chemiluminescence (from 18 +/- 3 to 32 +/- 5 cps/cm2), hydrogen peroxide (from 0.10 +/- 0.01 to 0.17 +/- 0.01 mumol/L), lactate dehydrogenase (from 80 +/- 2 to 330 +/- 30 U/L), and aspartate aminotransferase (from 42 +/- 2 to 100 +/- 10 U/L). Liver reperfusion was also associated with mitochondrial swelling and decreased mitochondrial respiratory control (from 5.6 +/- 0.3 to 2.6 +/- 0.1). The activity of the antioxidant enzymes and xanthine oxidase was instead without change. After 30 min of vena cava occlusion and 10 min of reperfusion a more marked increase in chemiluminescence (37 +/- 5 cps/cm2), hydrogen peroxide (0.30 +/- 0.01 mumol/L), lactate dehydrogenase (730 +/- 10 U/L) and aspartate aminotransferase (140 +/- 10 U/L) was observed. No further changes were found in either mitochondrial morphology or respiratory control (2.4 +/- 0.1) in isolated mitochondria.(ABSTRACT TRUNCATED AT 250 WORDS)

The ability of cells to maintain a bioenergetically favorable ATP/ADP ratio confers a tight balance between cellular events that consume ATP and the rate of ATP production. However, after growth factor withdrawal, the cellular ATP/ADP ratio declines. To investigate these changes, mitochondria from growth factor-deprived cells isolated before the onset of apoptosis were characterized in vitro. Mitochondria from growth factor-deprived cells have lost their ability to undergo matrix condensation in response to ADP, which is accompanied by a failure to perform ADP-coupled respiration. At the time of analysis, mitochondria from growth factor-deprived cells were not depleted of cytochrome c and cytochrome c-dependent respiration was unaffected, demonstrating that the inhibition of the respiratory rate is not due to loss of cytochrome c. Agents that disrupt the mitochondrial outer membrane, such as digitonin, or maintain outer membrane exchange of adenine nucleotide, such as Bcl-x(L), restored ADP-dependent control of mitochondrial respiration. Together, these data suggest that the regulation of mitochondrial outer membrane permeability contributes to respiratory control.

During the last decade many reports on the isolation from plant tissues of particulate fractions containing many respiratory enzymes, have been published (5). These fractions proved to be very suitable for a detailed study of respiratory metabolism, the more so because plant mitochondria which after addition of substrate and cofactors gave ?2 uptake coupled with phosphorylating processes, could be isolated. The effect of 2,4-dinitrophenol upon the respiration rate of potato tuber tissue (15) strongly suggests that under normal conditions respiratory activity and oxidative phosphorylation are coupled, and regulated by the availability of phosphate acceptor (ADP). Therefore, also after isolation, the mitochondria should have a substrate oxidation which is coupled with phosphorylation, and the rate of which is controlled by ADP. Hackett et al. (8) were the first who described the ability of potato tuber mitochondria to carry out oxidative phosphorylation. In their experiments using the Warburg respirometer and using succinate as the substrate they found P/O ratios of around 1.3, but they did not obtain evidence for a respiratory control by ADP. The best method available for investigating respiratory control in mitochondria is the oxygen electrode method (9) which allows short-term experiments. Bonner and Voss (2) using this highly sensitive method were the first who succeeded in demonstrating a respiratory control by ADP in isolated plant mitochondria. Later Wiskich and Bonner (17) were able to prepare mitochondrial fractions from potato tuber tissue, which not only showed accelerated 02 uptake upon addition of ADP, but also a decreased rate of substrate oxidation when the ADP had been consumed. They foundl respiratory control ratios (R.C. ratios) of around 2.0. Although Hackett et al. (8) and Wiskich and Bonner (17) made great progress in isolating mitochondria from potato tuber tissue, their preparations had some properties which suggested that the condition of the particles might not have been very good. Wiskich and Bonner could already eliminate the marked increase of the respiration upon addition of cytochrome c, as described by Hackett et al. (8), but their preparations exhibited a considerable induction period for the ADP stimulation of the succinate oxidation. Such an induction period could be avoided by preincubation of the mitochondria with ATP. Now it is known that ATP has an effect on the maintenance of contraction in mitochondria (7, 13). So the ATP induction phenomenon may be an indication that the mitochondria require a recovery, induced by ATP, from changes occurring during the isolation procedure, before they can react to addition of ADP. Therefore, the conditions for the isolation of mitochondria from potato tuber tissue have been reinvestigated in order to obtain mitochondria in a better condition than described up until now.

We studied respiratory activity and microviscosity of mitochondrial membranes from the left ventricular myocardium of 2-month-old (n=8) and 15-month-old (n=8) rats. The respiratory control (RC) during substrate phosphorylation was calculated as the ratio of oxygen absorption rates in the presence of ADP and after its utilization. In 2-month-old rats, RC was 4.66 (4.56; 4.71); in 15-month-old animals, it was significantly (р<0.05) lower: 3.57 (3.50; 3.62). Pyrene probe eximerization indices for regions of protein-lipid and lipid-lipid interactions in mitochondria of 2-month-old animals were significantly lower than in the group of 15-month-old rats, which indicated reduced microviscosity of the lipid environment of proteins. Thus, the decrease in RC of mitochondria from the left ventricle of 15-month-old animals and the increase in the microviscosity coefficients of their membranes indicate age-related changes in the structural and functional activity of mitochondria.

The lack of effective disease-modifying therapeutics to tackle Alzheimer’s disease (AD) is unsettling considering the actual prevalence of this devastating neurodegenerative disorder worldwide. Intermittent hypoxic conditioning (IHC) is a powerful non-pharmacological procedure known to enhance brain resilience. In this context, the aim of the present study was to investigate the potential long-term protective impact of IHC against AD-related phenotype, putting a special focus on cognition and mitochondrial bioenergetics and dynamics. For this purpose, six-month-old male triple transgenic AD mice (3×Tg-AD) were submitted to an IHC protocol for two weeks and the behavioral assessment was performed at 8.5 months of age, while the sacrifice of mice occurred at nine months of age and their brains were removed for the remaining analyses. Interestingly, IHC was able to prevent anxiety-like behavior and memory and learning deficits and significantly reduced brain cortical levels of amyloid-β (Aβ) in 3×Tg-AD mice. Concerning brain energy metabolism, IHC caused a significant increase in brain cortical levels of glucose and a robust improvement of the mitochondrial bioenergetic profile in 3×Tg-AD mice, as mirrored by the significant increase in mitochondrial membrane potential (ΔΨm) and respiratory control ratio (RCR). Notably, the improvement of mitochondrial bioenergetics seems to result from an adaptative coordination of the distinct but intertwined aspects of the mitochondrial quality control axis. Particularly, our results indicate that IHC favors mitochondrial fusion and promotes mitochondrial biogenesis and transport and mitophagy in the brain cortex of 3×Tg-AD mice. Lastly, IHC also induced a marked reduction in synaptosomal-associated protein 25 kDa (SNAP-25) levels and a significant increase in both glutamate and GABA levels in the brain cortex of 3×Tg-AD mice, suggesting a remodeling of the synaptic microenvironment. Overall, these results demonstrate the effectiveness of the IHC paradigm in forestalling the AD-related phenotype in the 3×Tg-AD mouse model, offering new insights to AD therapy and forcing a rethink concerning the potential value of non-pharmacological interventions in clinical practice.

Mitochondrial resistance to massive irradiation in vivo: V. Repair and the repair overshoot

A model system for mitochondrial ion transport and respiratory control

Acute administration of magnesium orotate (Mg-Or) at reperfusion has been previously shown to elicit significant protection in isolated rat hearts. Since recovery of mitochondrial function is mandatory for cardioprotection, the...

Assessment of the toxicity of silver nanoparticles in vitro: A mitochondrial perspective

The effect of prolonged administration of alcohol on mitochondrial function and high-energy phosphate (ATP) of heart muscle was investigated in dogs. Animals were divided into two groups, a control group and a group that received alcohol. In the experimental series, dogs received 400 ml of a 25% solution of alcohol added to the food and drinking water. Measurements were carried out after ethanol had been withheld for 2 days. Total myocardial blood flow, cardiac output, and myocardial O(2) consumption remained at control levels. Measurement of cardiac contractility using the maximal rate of left ventricular pressure rise (dP/dt(max)) showed no change in animals exposed to alcohol. When the afterload of the heart was increased with angiotensin, a slight but not significant decline in cardiac contractility was observed. Activities of various intramitochondrial and extramitochondrial enzymes were measured in both groups. After alcohol administration, the primarily intramitochondrial isocitrate dehydrogenase diminished. ATP in heart muscle of dogs exposed to alcohol declined, and mitochondrial oxygen consumption and respiratory control indices diminished. These observations suggest that the primary lesion leading to alteration of myocardial performance is a biochemical malfunction of the mitochondria, which at this early stage is not reflected in changes in myocardial contractility.

The lack of effective treatments for mitochondrial disease has seen the development of new approaches, including those that aim to stimulate mitochondrial biogenesis to boost ATP generation above a critical disease threshold. Here, we examine the effects of the peroxisome proliferator-activated receptor γ (PPARγ) activator pioglitazone (PioG), in combination with deoxyribonucleosides (dNs), on mitochondrial biogenesis in cybrid cells containing >90% of the m.3243A>G mutation associated with mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS). PioG + dNs combination treatment increased mtDNA copy number and mitochondrial mass in both control (CON) and m.3243A>G (MUT) cybrids, with no adverse effects on cell proliferation. PioG + dNs also increased mtDNA-encoded transcripts in CON cybrids, but had the opposite effect in MUT cybrids, reducing the already elevated transcript levels. Steady-state levels of mature oxidative phosphorylation (OXPHOS) protein complexes were increased by PioG + dNs treatment in CON cybrids, but were unchanged in MUT cybrids. However, treatment was able to significantly increase maximal mitochondrial oxygen consumption rates and cell respiratory control ratios in both CON and MUT cybrids. Overall, these findings highlight the ability of PioG + dNs to improve mitochondrial respiratory function in cybrid cells containing the m.3243A>G MELAS mutation, as well as their potential for development into novel therapies to treat mitochondrial disease.

Malonaldehyde acts as a mitochondrial toxin: Inhibitory effects on respiratory function and enzyme activities in isolated rat liver mitochondria

Re-evaluation of mitochondrial permeability transition as a primary neuroprotective target of minocycline

The electrostatic interactions of cytochrome c with its redox partners and membrane lipids, as well as other protein interactions and biochemical reactions, may be modulated by the ionic strength of the intermembrane space of the mitochondrion. FITC-BSA was used to determine the relative value of the mitochondrial intermembrane ionic strength with respect to bulk medium external to the mitochondrial outer membrane. FITC-BSA exhibited an ionic strength-dependent fluorescence change with an affinity in the mM range as opposed to its pH sensitivity in the microM range. A controlled, low pH-induced membrane fusion procedure was developed to transfer FITC-BSA encapsulated in asolectin liposomes, to the intermembrane space of intact mitochondria. The fusion procedure did not significantly affect mitochondrial ultrastructure, electron transport, or respiratory control ratios. The extent of fusion of liposomes with the mitochondrial outer membrane was monitored by fluorescence dequenching assays using a membrane fluorescent probe (octadecylrhodamine B) and the soluble FITC-BSA fluorescent probe, which report membrane and contents mixing, respectively. Assays were consistent with a rapid, low pH-induced vesicle-outer membrane fusion and delivery of FITC-BSA into the intermembrane space. Similar affinities for the ionic strength- dependent change in fluorescence were found for bulk medium, soluble (9.8 +/- 0.8 mM) and intermembrane space-entrapped FITC-BSA (10.2 +/- 0.6 mM). FITC-BSA consistently reported an ionic strength in the intermembrane space of the functionally and structurally intact mitochondria within +/- 20% of the external bulk solution. These findings reveal that the intermembrane ionic strength changes as does the external ionic strength and suggest that cytochrome c interactions, as well as other protein interactions and biochemical reactions, proceed in the intermembrane space of mitochondria in the intact cell at physiological ionic strength, i.e., 100-150 mM.

The renin‐angiotensin system promotes oxidative stress, apoptosis, necrosis, fibrosis, and thus heart failure. Secretory renin plays a central role in these processes, initiating the generation of angiotensins. Nevertheless, alternative renin transcripts exist, which code for a cytosolically localized renin isoform (cyto‐renin) that is cardioprotective. We tested the hypothesis that the protective effects are associated with a beneficial switch of metabolic and mitochondrial functions. To assess H9c2 cell mitochondrial parameters, we used the Seahorse XF analyser. Cardiac H9c2 cells overexpressing cyto‐renin exhibited enhanced nonmitochondrial oxygen consumption, lactate accumulation, and LDH activity, reflecting a switch to more aerobic glycolysis known as Warburg effect. Additionally, mitochondrial spare capacity and cell respiratory control ratio were enhanced, indicating an increased potential to tolerate stress conditions. Renin knockdown induced opposite effects on mitochondrial functions without influencing metabolic parameters. Thus, the protective effects of cyto‐renin are associated with an altered bioenergetic profile and an enhanced stress tolerance, which are favourable under ischaemic conditions. Therefore, cyto‐renin is a promising new target for the prevention of ischaemia‐induced myocardial damage.