ADP-stimulated Respiration Rate Research Articles

Treatment of cardiomyocytes isolated from left ventricular myocardium of dogs with chronic heart failure with ALDA-1, an activator of aldehyde dehydrogenase-2, improves mitochondrial function

Abstract Background Reactive oxygen species (ROS) generated by dysfunctional mitochondria (MITO) of the failing heart plays an important role in lipid peroxidation of polyunsaturated fatty acids in cell and MITO membranes resulting in the production of aldehydes, such as 4-hydroxy-2-nonenal (4-HNE). MITO aldehyde dehydrogenase-2 (mALDH2) is a key enzyme for the detoxification of endogenous aldehyde substrates through NAD dependent oxidation. ALDH2 catalyzes toxic 4HNE to nontoxic 4HNA in cardiac cells. We previously showed that mALDH2 expression and activity are markedly reduced in MITO of failing LV cardiomyocytes (CM), a maladaptation that can lead to considerable accumulation of 4-HNE in MITO and ultimately to progressive LV systolic and diastolic dysfunction. In this study, we tested the hypothesis that exposure of failing cardiomyocytes to ALDA-1, an activator of mALDH2, improves MITO function. Methods Freshly isolated CM from LV myocardium of dogs (n = 7) with heart failure (HF, LV ejection fraction ≤35%) and of normal (NL) dogs (n = 4) were incubated for 1 hour in absence (baseline) and presence of Alda-1 at concentration of 10 µM. After incubation, ADP-stimulated respiration (ADPresp) and maximal rate of ATP synthesis (ATPmax) were measured using Strathklein respirometer and bioluminescence ATP assay kit. Results Treatment of isolated CM from HF dogs with 10 µM Alda-1 significantly increased ADPresp (277 ± 18 vs. 189 ± 12 nAtom O/min/mg, p<0.05) and ATPmax (30 ± 3 vs. 16 ± 3 nmoles/min/mg, p<0.05) compared to untreated CM of HF dogs. Treatment of CM from NL dogs with 10 µM Alda-1 had no effect on ADPresp (488 ± 18 vs. 484 ± 19 nAtom O/min/mg) or ATPmax (54 ± 3 vs. 52 ± 2 nmoles/min/mg) compared to untreated NL CM. Conclusions Treatment of CM from HF dogs with Alda-1 improved overall MITO function. Activation of mALDH2 represents a potential viable therapeutic target for treating HF.

The Role of Swelling in the Regulation of OPA1-Mediated Mitochondrial Function in the Heart In Vitro.

Optic atrophy-1 (OPA1) plays a crucial role in the regulation of mitochondria fusion and participates in maintaining the structural integrity of mitochondrial cristae. Here we elucidate the role of OPA1 cleavage induced by calcium swelling in the presence of Myls22 (an OPA1 GTPase activity inhibitor) and TPEN (an OMA1 inhibitor). The rate of ADP-stimulated respiration was found diminished by both inhibitors, and they did not prevent Ca2+-induced mitochondrial respiratory dysfunction, membrane depolarization, or swelling. L-OPA1 cleavage was stimulated at state 3 respiration; therefore, our data suggest that L-OPA1 cleavage produces S-OPA1 to maintain mitochondrial bioenergetics in response to stress.

PCr/ATP ratios and mitochondrial function in the heart. A comparative study in humans

Cardiac energy status, measured as phosphocreatine (PCr)/adenosine triphosphate (ATP) ratio with 31P-Magnetic Resonance Spectroscopy (31P-MRS) in vivo, is a prognostic factor in heart failure and is lowered in cardiometabolic disease. It has been suggested that, as oxidative phosphorylation is the major contributor to ATP synthesis, PCr/ATP ratio might be a reflection of cardiac mitochondrial function. The objective of the study was to investigate whether PCr/ATP ratios can be used as in vivo marker for cardiac mitochondrial function. We enrolled thirty-eight patients scheduled for open-heart surgery in this study. Cardiac 31P-MRS was performed before surgery. Tissue from the right atrial appendage was obtained during surgery for high-resolution respirometry for the assessment of mitochondrial function. There was no correlation between the PCr/ATP ratio and ADP-stimulated respiration rates (octanoylcarnitine R2 < 0.005, p = 0.74; pyruvate R2 < 0.025, p = 0.41) nor with maximally uncoupled respiration (octanoylcarnitine R2 = 0.005, p = 0.71; pyruvate R2 = 0.040, p = 0.26). PCr/ATP ratio did correlate with indexed LV end systolic mass. As no direct correlation between cardiac energy status (PCr/ATP) and mitochondrial function in the heart was found, the study suggests that mitochondrial function might not the only determinant of cardiac energy status. Interpretation should be done in the right context in cardiac metabolic studies.

Calcium overload decreases net free radical emission in cardiac mitochondria

Elevated calcium and reactive oxygen species (ROS) are responsible for the bulk of cell death occurring in a variety of clinical settings that include acute coronary events, cerebrovascular accidents, and acute kidney injury. It is commonly believed that calcium and ROS participate in a viscous cycle during these events. However, the precise feedback mechanisms are unknown. We quantitatively demonstrate in this study that, on the contrary, calcium does not stimulate free radical production but suppresses it. Isolated mitochondria from guinea pig hearts were energized with a variety of substrates and exposed to calcium concentrations designed to induce moderate calcium overload conditions associated with ischemia/reperfusion injury but do not elicit the well-known mitochondrial permeability transition phenomenon. Metabolic function and free radical emission were simultaneously quantified using high-resolution respirometry and fluorimetry. Membrane potential, high amplitude swelling, and calcium dynamics were also quantified in parallel. Our results reveal that calcium overload does not lead to excessive ROS emission but does decrease ADP stimulated respiration rates for NADH-dependent pathways. Moreover, we developed an empirical model of mitochondrial free radical homeostasis to identify the processes that are different for each substrate and calcium condition. In summary, we show that in healthy guinea pig mitochondria, calcium uptake and free radical generation do not contribute to a viscous cycle and that the relationship between net free radical production and oxygen concentration is hyperbolic. Altogether, these results lay out an important foundation necessary to quantitatively determine the role of calcium in IR injury and ROS production.

PL-008 Adaptation of skeletal muscle to aerobic exercise: specific transcriptome response to acute exercise and training

Objective Variety of processes including circadian rhythm and systemic factors affect expression of many genes in skeletal muscle during a day. Therefore, post-exercise gene expression depends on many factors: contractile activity per seas well as circadian rhythm, nerve activity, concentration of different substances in blood, feeding and fasting. In our study, we investigated specific for contractile activity changes in the transcriptome in untrained and trained (after an aerobic training programme) human skeletal muscle. The second goal was to examine effect of aerobic training on gene expression in muscle in basal state.&#x0D; Methods Seven untrained males performed the one-legged knee extension exercise (for 60 min) with the same relative intensity before and after a 2 month aerobic training programme (1 h/day, 5/week). Biopsy samples were taken at rest (basal state, 48 h after the previous exercise), 1 and 4 h after one-legged exercise from m. vastus lateralisof either leg. This approach allowed us to evaluate specific changes in the transcriptome associated with contractile activity. RNA­sequencing (84 samples in total; ~42 million reads/sample) was performed by HiSeq 2500 (Illumina).&#x0D; Results Two months aerobic training increased the aerobic capacity of the knee-extensor muscles (power at anaerobic threshold in incremental one-legged and cycling tests), the maximum rate of ADP-stimulated mitochondrial respiration in permeabilized muscle fibres and amounts of oxidative phosphorylation proteins. After one-legged exercise, expression of many genes was changed in exercised muscle (~1500) as well as in non-exercised muscle (~400). Pronounced changes in gene expression in non-exercised muscle may be associated with many factors, including circadian rhythm (result of GO analysis).&#x0D; To examine transcriptome changes specific for contractile activity, the difference in gene expression between legs was examined. In untrained muscle, one-legged exercise changed expression of ~1200 genes specific for contractile activity at each time point. Despite the same relative intensity of one-legged exercise, transcriptomic response in trained muscle was markedly lower (~300 genes) compare to untrained.&#x0D; We observed a strong overlap between transcriptomic responses (~250 genes) and particularly between enriched transcription factor binding sites in promoters of these genes in untrained and trained muscles. These sets of genes and transcription factors play the key role in adaptation of muscle to contractile activity independently on the level of muscular fitness.&#x0D; Surprisingly, 2 months aerobic training changed the expression of more than 1500 genes in basal state. Noteworthy, these genes demonstrated a small overlap (~200 genes) with genes related to specific response to acute exercise. Moreover, these genes were associated with significantly different biological processes than genes related to specific response to acute exercise.&#x0D; Conclusions Specific for contractile activity changes in the transcriptome in untrained and trained human skeletal muscle were revealed for the first time. After 2 month aerobic training, the specific transcriptome response to acute exercise become much less pronounced. A computational approach reveals common transcription factors important for adaptation of both untrained and trained muscle. We found out that adaptation of muscle to aerobic training associates not only with the transitory changes in gene expression after each exercise, but also with the marked changes in transcriptome in basal state.&#x0D; This work was supported by the Russian Science Foundation (14­15­00768).

Modeling Calcium‐Induced Inhibition of Oxidative Phosphorylation

Ischemic myocardium is characterized by a chronic elevation in cellular calcium concentration. In this setting, mitochondria accumulate calcium to levels that compromise their ability to synthesize ATP. Although total mitochondrial calcium can reach up to 1 M, the vast majority is presumed to be present as insoluble calcium phosphate granules. We recently reported calcium levels below thresholds that trigger mitochondrial permeability transition significantly lowers the rate of ADP‐stimulated respiration. To explain these data, we tested the following hypotheses using a novel mathematical model of cardiac mitochondrial energetics and calcium handling: i) depressed membrane potential caused by sodium/calcium cycling lowering the driving force for ATP production; ii) direct inhibition of calcium on mitochondrial processes critical for ATP production; iii) calpain activation leading to a decrease in ATP synthesis capacity; iv) depletion/reduction of available ADP for phosphorylation caused by calcium biding, incorporation into calcium phosphate granules, or net loss of adenine nucleotide. The model includes mechanistic descriptions of mitochondrial metabolism and energetics in addition to calcium uptake, sequestration, and efflux pathways. Based on the model analysis, sodium/calcium cycling lowers mitochondrial membrane potential, but does not lead to lower rates of oxidative phosphorylation. However, direct inhibition of calcium on ATP production did agree with these data. We also found that calpain activation is a possible explanation, but we experimentally ruled this mechanism out. Finally, ADP complexation with calcium did not affect the rate of oxidative phosphorylation, but net loss of nucleotides via incorporation into calcium phosphate granules or efflux out of mitochondria can explain these data. The model will be used to design further experiments to determine the most likely mechanism capable of explaining the calcium‐induced inhibition of oxidative phosphorylation.Support or Funding InformationR00‐HL121160This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effects of Buffer Composition and Substrates on Calcium‐stimulated Mitochondrial Respiration and Free Radical Emission

Mitochondrial ATP synthesis is tightly coupled to oxygen consumption in a highly regulated process; however, unavoidable byproducts in the form of reactive oxygen species (ROS) are formed. An important regulator of this process is calcium, but the precise relationship between calcium and free radical production is obscure. Therefore, we measured ROS production under a variety of experimental conditions that mimic the pathological environmental setting. The Oroborous oxygraph system was used to simultaneously measure ROS emission and the oxygen consumption rate from isolated cardiac guinea pig mitochondria energized with different substrates and challenged with different calcium concentrations. The Amplex Red assay was used to quantify ROS emission rates. The substrates used utilized a mixture of the carbohydrate and fatty acid dependent metabolic pathways. Two respiration buffers, one lactobionate‐based and the other potassium chloride‐based, were used to quantify the differences in respiration and ROS emission rates. Relative to the potassium chloride‐based respiration buffer, the lactobionate‐based buffer resulted in higher ADP‐stimulated respiration rates and higher ROS emission rates for each substrate combination. On average, the presence of calcium depressed ROS emission rates during the leak respiration, sodium/calcium cycling, and ADP‐stimulated states. The lower respiration rates in the potassium chloride‐based buffer is likely due to the presence of inhibitory levels of chloride. The high chloride levels also appear to depress ROS emission. Overall, these data refute the hypothesis that calcium stimulates ROS emission from isolated mitochondria. Studies are ongoing to further characterize ROS emission from cardiac guinea pig mitochondria and elucidate the relationship between calcium and ROS emission.Support or Funding InformationR00‐HL121160This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Role of the mitochondrial ATP synthase central stalk subunits γ and δ in the activity and assembly of the mammalian enzyme

The central stalk of mitochondrial ATP synthase consists of subunits γ, δ, and ε, and along with the membraneous subunit c oligomer constitutes the rotor domain of the enzyme. Our previous studies showed that mutation or deficiency of ε subunit markedly decreased the content of ATP synthase, which was otherwise functionaly and structuraly normal. Interestingly, it led to accumulation of subunit c aggregates, suggesting the role of the ε subunit in assembly of individual enzyme domains. In the present study we focused on the role of subunits γ and δ. Using shRNA knockdown in human HEK293 cells, the protein levels of γ and δ were decreased to 30% and 10% of control levels, respectively. The content of the assembled ATP synthase decreased in accordance with the levels of the silenced subunits, which was also the case for most structural subunits. In contrast, the hydrophobic c subunit was increased to 130% or 180%, respectively and most of it was detected as aggregates of 150–400 kDa by 2D PAGE. In addition the IF1 protein was upregulated to 195% and 300% of control levels. Both γ and δ subunits silenced cells displayed decreased ATP synthase function - lowered rate of ADP-stimulated respiration, a two-fold increased sensitivity of respiration to inhibitor oligomycin, and impaired utilization of mitochondrial membrane potential for ADP phosphorylation. In summary, similar phenotype of γ, δ and ε subunit deficiencies suggest uniform requirement for assembled central stalk as driver of the c-oligomer attachment in the assembly process of mammalian ATP synthase.

The impact of a short-term high-fat diet on mitochondrial respiration, reactive oxygen species production, and dynamics in oxidative and glycolytic skeletal muscles of young rats.

Multiple aspects of mitochondrial function and dynamics remain poorly studied in the skeletal muscle of pediatric models in response to a short‐term high‐fat diet (HFD). This study investigated the impact of a short‐term HFD on mitochondrial function and dynamics in the oxidative soleus (SOL) and glycolytic extensor digitorum longus (EDL) muscles in young rats. Young male Wistar rats were submitted to either HFD or normal chow (NCD) diets for 14 days. Permeabilized myofibers from SOL and EDL were prepared to assess mitochondrial respiration and reactive oxygen species (ROS) production. The expression and content of protein involved in mitochondrial metabolism and dynamics (fusion/fission) were also quantified. While no effects of HFD was observed on mitochondrial respiration when classical complex I and II substrates were used, both SOL and EDL of rats submitted to a HFD displayed higher basal and ADP‐stimulated respiration rates when Malate + Palmitoyl‐L‐carnitine were used as substrates. HFD did not alter ROS production and markers of mitochondrial content. The expression of CPT1b was significantly increased in SOL and EDL of HFD rats. Although the expression of UCP3 was increased in SOL and EDL muscles from HFD rats, mitochondrial coupling efficiency was not altered. In SOL of HFD rats, the transcript levels of Mfn2 and Fis1 were significantly upregulated. The expression and content of proteins regulating mitochondrial dynamics was not modulated by HFD in the EDL. Finally, DRP1 protein content was increased by over fourfold in the SOL of HFD rats. Taken altogether, our findings show that exposing young animals to short‐term HFD results in an increased capacity of skeletal muscle mitochondria to oxidize fatty acids, without altering ROS production, coupling efficiency, and mitochondrial content. Our results also highlight that the impact of HFD on mitochondrial dynamics appears to be muscle specific.

In vivo study of hepatic oxidative stress and mitochondrial function in rabbits with severe hypotension after propofol prolonged infusion.

In humans, prolonged sedations with propofol or using high doses have been associated with propofol infusion syndrome. The main objective of this study was to evaluate the effects of prolonged high-dose administration of a specific propofol emulsion (Propofol Lipuro) and an improved lipid formulation (SMOFlipid) in liver mitochondrial bioenergetics and oxidative stress of rabbits, comparatively to a saline control. Twenty-one male New Zealand white rabbits were randomly allocated in three groups that were continuously treated for 20 h. Each group of seven animals received separately: NaCl 0.9 % (saline), SMOFlipid (lipid-based emulsion without propofol) and Lipuro 2 % (propofol lipid emulsion). An intravenous propofol bolus of 20 mg kg−1 was given to the propofol Lipuro group to allow blind orotracheal intubation and mechanical ventilation. Anesthesia was maintained using infusion rates of: 20, 30, 40, 50 and 60 mg kg−1 h−1, according to the clinical scale of anesthetic depth and the index of consciousness values. The SMOFlipid and saline groups received the same infusion rate as the propofol Lipuro group, which were infused during 20 consecutive hours. At the end, the animals were euthanized, livers collected and mitochondria isolated by standard differential centrifugation. Mitochondrial respiration, membrane potential, swelling and oxidative stress were evaluated. Data were processed using one-way ANOVA (p < 0.05). The animals revealed a significant decrease in cardiovascular parameters showing bradycardia and severe hypotension. No statistical differences were observed when using pyruvate as substrate, however, when using succinate as respiratory substrate, significant decrease in ADP-stimulated respiration rate was observed for SMOFlipid group (p = 0.002). Lipid peroxides (p < 0.01) and protein carbonyls (p = 0.01) showed a statistically significant difference between propofol Lipuro and the SMOFlipid groups. These results suggest that lipid-based emulsions can be involved in the regulation of different pathways that ultimately lead to a decrease of state 3 mitochondrial respiration rate. The infusion of propofol Lipuro during prolonged periods, in addition to marked hypotension and hypoperfusion, also showed to have higher anti-oxidant activity and lower impairment of the mitochondrial function comparatively to the improved lipid formulation, SMOFlipid, using the rabbit as animal model.Electronic supplementary materialThe online version of this article (doi:10.1186/s40064-016-2970-2) contains supplementary material, which is available to authorized users.

The Inhibitor Protein (IF1) of the F1F0-ATPase Modulates Human Osteosarcoma Cell Bioenergetics

The bioenergetics of IF1 transiently silenced cancer cells has been extensively investigated, but the role of IF1 (the natural inhibitor protein of F1F0-ATPase) in cancer cell metabolism is still uncertain. To shed light on this issue, we established a method to prepare stably IF1-silenced human osteosarcoma clones and explored the bioenergetics of IF1 null cancer cells. We showed that IF1-silenced cells proliferate normally, consume glucose, and release lactate as controls do, and contain a normal steady-state ATP level. However, IF1-silenced cells displayed an enhanced steady-state mitochondrial membrane potential and consistently showed a reduced ADP-stimulated respiration rate. In the parental cells (i.e. control cells containing IF1) the inhibitor protein was found to be associated with the dimeric form of the ATP synthase complex, therefore we propose that the interaction of IF1 with the complex either directly, by increasing the catalytic activity of the enzyme, or indirectly, by improving the structure of mitochondrial cristae, can increase the oxidative phosphorylation rate in osteosarcoma cells grown under normoxic conditions.

Submaximal ADP‐stimulated respiration is impaired in ZDF rats and recovered by resveratrol

Mitochondrial dysfunction and reactive oxygen species (ROS) have been implicated in the aetiology of skeletal muscle insulin resistance, although there is considerable controversy regarding these concepts. Mitochondrial function has been traditionally assessed in the presence of saturating ADP, but ATP turnover and the resultant ADP is thought to limit respiration in vivo. Therefore, we investigated the potential link between submaximal ADP-stimulated respiration rates, ROS generation and skeletal muscle insulin sensitivity in a model of type 2 diabetes mellitus, the ZDF rat. Utilizing permeabilized muscle fibres we observed that submaximal ADP-stimulated respiration rates (250-2000 μm ADP) were lower in ZDF rats than in lean controls, which coincided with decreased adenine nucleotide translocase 2 (ANT2) protein content. This decrease in submaximal ADP-stimulated respiration occurred in the absence of a decrease in electron transport chain function. Treating ZDF rats with resveratrol improved skeletal muscle insulin resistance and this was associated with elevated submaximal ADP-stimulated respiration rates as well as an increase in ANT2 protein content. These results coincided with a greater ability of ADP to attenuate mitochondrial ROS emission and an improvement in cellular redox balance. Together, these data suggest that mitochondrial dysfunction is present in skeletal muscle insulin resistance when assessed at submaximal ADP concentrations and that ADP dynamics may influence skeletal muscle insulin sensitivity through alterations in the propensity for mitochondrial ROS emission.

Corrections by melatonin of liver mitochondrial disorders under diabetes and acute intoxication in rats

The aim of the present work was to investigate the mechanisms of oxidative damage of the liver mitochondria under diabetes and intoxication in rats as well as to evaluate the possibility of corrections of mitochondrial disorders by pharmacological doses of melatonin. The experimental (30 days) streptozotocin-induced diabetes mellitus caused a significant damage of the respiratory activity in rat liver mitochondria. In the case of succinate as a respiratory substrate, the ADP-stimulated respiration rate V₃ considerably decreased (by 25%, p < 0·05) as well as the acceptor control ratio (ACR) V₃/V₂ markedly diminished (by 25%, p < 0·01). We observed a decrease of the ADP-stimulated respiration rate V₃ by 35% (p < 0·05), with glutamate as substrate. In this case, ACR also decreased (by 20%, p < 0·05). Surprisingly, the phosphorylation coefficient ADP/O did not change under diabetic liver damage. Acute rat carbon tetrachloride-induced intoxication resulted in considerable decrease of the phosphorylation coefficient because of uncoupling of the oxidation and phosphorylation processes in the liver mitochondria. The melatonin administration during diabetes (10 mg·kg⁻¹ body weight, 30 days, daily) showed a considerable protective effect on the liver mitochondrial function, reversing the decreased respiration rate V₃ and the diminished ACR to the control values both for succinate-dependent respiration and for glutamate-dependent respiration. The melatonin administration to intoxicated animals (10 mg·kg⁻¹ body weight, three times) partially increased the rate of succinate-dependent respiration coupled with phosphorylation. The impairment of mitochondrial respiratory plays a key role in the development of liver injury under diabetes and intoxication. Melatonin might be considered as an effector that regulates the mitochondrial function under diabetes.

Muscle Mitochondrial Oxidative Phosphorylation Activity, But Not Content, Is Altered with Abdominal Obesity in Sedentary Men: Synergism with Changes in Insulin Sensitivity

Abdominal obesity is a major risk factor for muscle insulin resistance. Mitochondria may play a key role in this etiology. Changes in muscle mitochondrial content and function were examined according to abdominal obesity and insulin sensitivity in men. The descriptive MitHyCal study was conducted on the general population of Clermont-Ferrand, France. Forty-two healthy sedentary men (41.7 +/- 4.3 yr) were divided into four groups according to waist circumference: 87 cm or less (group 1, n = 10); 88-93 cm (group 2, n = 12); 94-101 cm (group 3, n = 10); and 102 cm or greater (group 4, n = 10). Plasma metabolic check-up was performed, and insulin sensitivity index was calculated from glucose and insulin responses to a 3-h oral glucose tolerance test. Muscle biopsies were obtained to assess mitochondrial content, oxidative phosphorylation activity, and superoxide anion (reactive oxygen species) production. Assessment of muscle mitochondrial content and function was planned before data collection began. Abdominal obesity was negatively correlated to insulin sensitivity index (r = -0.39; P < 0.01), and only group 4 was insulin-resistant (P < 0.05). There were no between-group differences in muscle mitochondrial content and maximal activity of key oxidative enzymes. In contrast, muscle mitochondrial ADP-stimulated respiration rate was 24% higher in groups 2 and 3 compared to groups 1 and 4 (P < 0.05). Mitochondrial ATP and reactive oxygen species production rates were 27 and 48% lower in group 4 than in group 1 (P < 0.05). Abdominal obesity is associated with alterations in intrinsic muscle mitochondrial function but not content. These adaptations mainly result in reduced mitochondrial ATP production rate in response to insulin resistance.

CRYAB and HSPB2 deficiency alters cardiac metabolism and paradoxically confers protection against myocardial ischemia in aging mice

The abundantly expressed small molecular weight proteins, CRYAB and HSPB2, have been implicated in cardioprotection ex vivo. However, the biological roles of CRYAB/HSPB2 coexpression for either ischemic preconditioning and/or protection in situ remain poorly defined. Wild-type (WT) and age-matched ( approximately 5-9 mo) CRYAB/HSPB2 double knockout (DKO) mice were subjected either to 30 min of coronary occlusion and 24 h of reperfusion in situ or preconditioned with a 4-min coronary occlusion/4-min reperfusion x 6, before similar ischemic challenge (ischemic preconditioning). Additionally, WT and DKO mice were subjected to 30 min of global ischemia in isolated hearts ex vivo. All experimental groups were assessed for area at risk and infarct size. Mitochondrial respiration was analyzed in isolated permeabilized cardiac skinned fibers. As a result, DKO mice modestly altered heat shock protein expression. Surprisingly, infarct size in situ was reduced by 35% in hearts of DKO compared with WT mice (38.8 +/- 17.9 vs. 59.8 +/- 10.6% area at risk, P < 0.05). In DKO mice, ischemic preconditioning was additive to its infarct-sparing phenotype. Similarly, infarct size after ischemia and reperfusion ex vivo was decreased and the production of superoxide and creatine kinase release was decreased in DKO compared with WT mice (P < 0.05). In permeabilized fibers, ADP-stimulated respiration rates were modestly reduced and calcium-dependent ATP synthesis was abrogated in DKO compared with WT mice. In conclusion, contrary to expectation, our findings demonstrate that CRYAB and HSPB2 deficiency induces profound adaptations that are related to 1) a reduction in calcium-dependent metabolism/respiration, including ATP production, and 2) decreased superoxide production during reperfusion. We discuss the implications of these disparate results in the context of phenotypic responses reported for CRYAB/HSPB2-deficient mice to different ischemic challenges.

Impact of captopril on cardiac mitochondrial function in an in-vivo animal model of atherosclerosis

Recently, we have demonstrated that a twelve-week treatment with hypercholesterolemic atherogenic diet induced profound alterations in mitochondrial energy metabolism and oxidative capacity of isolated cardiac muscle’s mitochondria. The aim of the present study was to investigate the effects of treatment with an ACE inhibitor (captopril) on energy metabolism and oxidative capacity in cardiac muscle’s mitochondria using an in-vivo animal model of experimental atherosclerosis. Mitochondrial oxidative capacity was followed-up during the course of twelve weeks of atherogenic hypercholesterolic diet. Fifty five male Chinchilla rabbits, were randomized to one of four groups: a control group (A, n=17) received physiological saline orally; experimental group (B, n=18) received atherogenic 2% hypercholesterolemic diet; group C (n=10) received the atherogenic diet and two hours later on the aqueous solution of captopril (10 mg/kg/day); group D (n=10) received the aqueous solution of captopril, only. Isolation of mitochondrial fraction of the heart was performed by the method of Tyler. The oxygen consumption rate was studied at different respiration phases: as basal, unstimulated (V4) and as ADP-stimulated (V3), and expressed as indices of respiratory control ratio (RCR) and ADP/O. Hypercholesterolemic atherogenic diet induced profound alterations in mitochondrial energy metabolism and oxidative capacity. Basal oxygen consumption rate without ADP (V4) and the maximal ADP-stimulated respiration rate (V3) showed a marked reduction (quantitative changes i.e. dramatic decrease in oxidative capacities). The sensibility of mitochondria to ADP (ADP/O) was also reduced (qualitative change) in rabbits treated with the atherogenic diet (group B) compared to controls (group A). Respiratory control ratio was not significantly different among the studied groups. These results indicate that hypercholesterolemic atherogenic diet impairs mitochondrial oxidative capacity without affecting coupling of oxidation and phosphorilation. Captopril treatment had only limited effects on alterations of oxidative capacity in hypercholesterolemic rabbits. The main characteristic of oxidative capacity, i.e. the maximal respiration rate (V3), was not improved by captopril.

Cardiac muscle’s mitochondrial dysfunction in hypercholesterolemic rabbits

Cardiovascular diseases are often associated with energy deficit and in many cases this is also accompanied by lipid disorders such as hyperlipidemias and obesity. The aim of the study was to check mitochondrial oxidative capacity in the course of twelve weeks atherogenic hypercholesterolic diet. Thirty five Chinchilla rabbits, male, were randomized to one of two groups a control group (A, n=17) received (per os) physiological saline experimental group (B, n=18) received atherogenic 2% hypercholesterolemic diet. Isolation of the mitochondrial fraction of the heart was done by the method of Tyler. The oxygen consumption rate was studied in different respiration phases: as basal, unstimulated (V4) and as ADP-stimulated (V3) and expressed as indices: respiratory control ratio (RCR) and ADP/O. Hypercholesterolemic atherogenic diet induced profound perturbations in mitochondrial energy metabolism and oxidative capacity. Basal oxygen consumption rate without ADP (V4) and the maximal ADP-stimulated respiration rate (V3) showed a marked reduction (quantitative changes); sensibility of mitochondria to ADP (ADP/O) was also reduced (qualitative change) in rabbits treated by atherogenic diet (group B) compared to controls (group A). Respiratory control ratio was not significantly different among the groups. These results indicate that hypercholesterolemic atherogenic diet impairs mitochondrial oxidative capacity without affecting coupling of oxidative and phosphorilative processes.

Control of skeletal muscle mitochondria respiration by adenine nucleotides: differential effect of ADP and ATP according to muscle contractile type in pigs

Skeletal muscle exhibits considerable variation in mitochondrial content among fiber types, but it is less clear whether mitochondria from different fiber types also present specific functional and regulatory properties. The present experiment was undertaken on ten 170-day-old pigs to compare functional properties and control of respiration by adenine nucleotides in mitochondria isolated from predominantly slow-twitch (Rhomboideus (RM)) and fast-twitch (Longissimus (LM)) muscles. Mitochondrial ATP synthesis, respiratory control ratio (RCR) and ADP-stimulated respiration with either complex I or II substrates were significantly higher (25–30%, P<0.05) in RM than in LM mitochondria, whereas no difference was observed for basal respiration. Based on mitochondrial enzyme activities (cytochrome c oxidase [COX], F0F1-ATPase, mitochondrial creatine kinase [mi-CK]), the higher ADP-stimulated respiration rate of RM mitochondria appeared mainly related to a higher maximal oxidative capacity, without any difference in the maximal phosphorylation potential. Mitochondrial K m for ADP was similar in RM (4.4±0.9 μM) and LM (5.9±1.2 μM) muscles ( P>0.05) but the inhibitory effect of ATP was more marked in LM ( P<0.01). These findings demonstrate that the regulation of mitochondrial respiration by ATP differs according to muscle contractile type and that absolute muscle oxidative capacity not only relies on mitochondrial density but also on mitochondrial functioning per se.

Effects of osmoconditioning on mitochondrial respiration and phosphorylation in soybean seeds

Cotyledon mitochondrion respiration and oxidative phosphorylation activity were studied in two groups of soybean seeds. One group was primed with polyethylene glycol (PEG) for different periods of time, and the other was unprimed (control), and both were then exposed to imbibition at low temperatures before their germination. The results indicated that when L-Malate (L-Mal) and α-Ketoglutarate (α-Kg) were used as substrates, the ADP-stimulated mitochondria respiration rates of control seeds were markedly higher than state III respiration rates of primed seeds. However, the osmoconditioning pretreatment significantly enhanced the oxidative phosphorylation activity of cotyledon mitochondrion in 12 h. The oxidative phosphorylation activity of the mitochondrion of primed seeds was normal and the ADP/O value was consistent with the theoretical one. When reduced nicotinamide adenine dinucleotide (NADH) was used as the substrate, the mitochondria of control seeds still had oxidative phosphorylation activity, while ADP/O value was obviously lower than that of mitochondria of primed seeds. When Succinate (Succ) was used as the substrate, the oxidative phosphorylation activity of the primed seeds was normal after priming for 24 h. When different substrates were used, the emerging order of the oxidative phosphorylation activity of the primed seeds was NADH, α-Kg, Succ and in the last place L-Mal. The mechanism of soybean imbibitionl chilling injury and protective effect of PEG priming were discussed.

What controls the outer mitochondrial membrane permeability for ADP: facts for and against the role of oncotic pressure

In our study 10% of bovine serum albumin was added to the physiological incubation medium to mimic the oncotic pressure of the cellular cytoplasm and to test for its effect on the respiration of isolated rat heart mitochondria, saponin- or saponin plus crude collagenase (type IV)-treated heart muscle fibers and saponin-treated rat quadriceps muscle fibers. Pyruvate and malate were used as substrates. We found that albumin slightly decreased the maximal ADP-stimulated respiration rate only for saponin-treated heart muscle fibers. The apparent K m ADP of oxidative phosphorylation increased significantly, by 70–100%, for isolated heart mitochondria, saponin plus collagenase-treated heart muscle fibers and for saponin-treated quadriceps muscle fibers but remained unchanged for saponin-treated heart muscle fibers. The saponin-treated heart muscle fibers were characterized by a very high control apparent K m ADP value (234±24 μM ADP) compared with other preparations (14–28 μM ADP). The results suggest that in vivo the oncotic pressure is not the relevant factor causing the low outer mitochondrial membrane permeability for ADP in cardiomyocytes, in contrast to quadriceps muscle cells. It is likely that the outer mitochondrial membrane-bound protein(s) which is supposed to remain in saponin-treated heart muscle fibers is responsible for this property of the membrane.