ADP-stimulated Respiration Research Articles

Hepatic mitochondrial adaptations to physical activity: impact of sexual dimorphism, PGC1α and BNIP3-mediated mitophagy.

Hepatic mitochondrial adaptations to physical activity may be regulated by mitochondrial biogenesis (PGC1α) and mitophagy (BNIP3). Additionally, these adaptations may be sex-dependent. Chronic increase in physical activity lowers basal mitochondrial respiratory capacity in mice. Female mice have higher hepatic electron transport system protein content, elevated respiratory capacity, lowered mitophagic flux, and emit less mitochondrial H2 O2 independent of physical activity. Males require chronic daily physical activity to attain a similar mitochondrial phenotype compared to females. In contrast, females have limited hepatic adaptations to chronic physical activity. Livers deficient in PGC1α and BNIP3 display similar mitochondrial adaptations to physical activity to those found in wild-type mice. Hepatic mitochondrial adaptations to physical activity may be regulated by biogenesis- and mitophagy-associated pathways in a sex-dependent manner. Here, we tested if mice with targeted deficiencies in liver-specific peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α; LPGC1α+/- ) and BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3)-mediated mitophagy (BNIP3-/- ) would have reduced physical activity-induced adaptations in respiratory capacity, H2 O2 emission and mitophagy compared to wild-type (WT) controls and if these effects were impacted by sex. Male and female WT, LPGC1α+/- and BNIP3-/- C57BL6/J mice were divided into groups that remained sedentary or had access to daily physical activity via voluntary wheel running (VWR) (n= 6-10/group) for 4weeks. Mice had ad libitum access to low-fat diet and water. VWR reduced basal mitochondrial respiration, increased mitochondrial coupling and altered ubiquitin-mediated mitophagy in a sex-specific manner in WT mice. Female mice of all genotypes displayed higher electron transport system content, displayed increased ADP-stimulated respiration, produced less mitochondrially derived reactive oxygen species, exhibited reduced mitophagic flux, and were less responsive to VWR compared to males. Males responded more robustly to VWR-induced changes in hepatic mitochondrial function resulting in a match to adaptations found in females. Deficiencies in PGC1α and BNIP3 alone did not largely alter mitochondrial adaptations to VWR. However, VWR restored sex-dependent abnormalities in mitophagic flux in LPGC1α+/- . Finally, BNIP3-/- mice had elevated mitochondrial content and increased mitochondrial respiration putatively through repressed mitophagic flux. In conclusion, hepatic mitochondrial adaptations to physical activity are more dependent on sex than PGC1α and BNIP3.

Abstract 417: Targeting the NAD/NADH Ratio for Heart Failure Therapy

Energy deficiency and mitochondrial dysfunction are strongly implicated in the development of heart failure (HF). Yet, the exact mechanisms remain poorly understood. Recently, a causal relationship between loss of cardiac NAD(H) homeostasis and mitochondrial dysfunction have been proposed in HF. A number of studies have demonstrated the benefit of raising NAD levels prior to or at the time of inducing heart failure, but it is unknown whether this strategy benefits hearts with established or pre-existing dysfunction. Therefore, in the present study we increased NAD level in wild-type (WT) mice after pathological hypertrophy and dysfunction were induced by transverse aortic constriction (TAC) surgery. WT mice were treated daily for six-weeks with nicotinamide riboside (NR) starting four-weeks after TAC. The NR treatments significantly elevated NAD level in the TAC hearts (3.1 fold, n=4) compared to vehicle treated mice, accompanied by an improvement in fractional shortening (FS) (sham vehicle 51%±2% vs TAC Vehicle 31%±3% vs TAC NR 43%±5%, n=4 each group). Most evidence thus far suggest the benefit of raising NAD level in the failing heart involves activation of sirtuin 3 (Sirt3), a NAD dependent deacylase. We hypothesize there are Sirt3 independent mechanisms that are activated by increasing NAD levels to benefit the failing heart. To test this hypothesis, we treated Sirt3 deficient (Sirt3-/-) mice with NR starting four-weeks after TAC surgery and extended the study to ten-weeks post TAC. NR treatments elevated cardiac NAD level (2.9-fold, n=4) compared to vehicle treated controls. This correlated with lowered global protein acetylation in the hearts and an improvement in FS (sham vehicle 50%±5% vs TAC vehicle 24%±6% vs TAC NR 41%±8%). We also looked at mitochondrial function in Sirt3-/- mice ten-weeks after TAC ± NR treatments and observed an increase in state 3 ADP-stimulated respiration in the NR treated Sirt3-/- mice (sham vehicle 183±17 vs TAC vehicle 103±23 vs TAC NR 127±21 nmol/min/mg). These findings suggest increasing NAD can improve cardiac function through Sirt3 independent mechanisms including one that enhances mitochondrial respiration. Further elucidation of these mechanisms could identify novel NAD sensitive therapeutic targets.

Muscle mass and inspired oxygen influence oxygen extraction at maximal exercise: Role of mitochondrial oxygen affinity.

We examined the Fick components together with mitochondrial O2 affinity (p50mito ) in defining O2 extraction and O2 uptake during exercise with large and small muscle mass during normoxia (NORM) and hyperoxia (HYPER). Seven individuals performed 2 incremental exercise tests to exhaustion on a bicycle ergometer (BIKE) and 2 on a 1-legged knee extension ergometer (KE) in NORM or HYPER. Leg blood flow and VO2 were determined by thermodilution and the Fick method. Maximal ADP-stimulated mitochondrial respiration (OXPHOS) and p50mito were measured exvivo in isolated mitochondria. Mitochondrial excess capacity in the leg was determined from OXPHOS in permeabilized fibres and muscle mass measured with magnetic resonance imaging in relation to peak leg O2 delivery. The exvivo p50mito increased from 0.06±0.02 to 0.17±0.04kPa with varying substrate supply and O2 flux rates from 9.84±2.91 to 16.34±4.07pmolO2 ·s-1 ·μg-1 respectively. O2 extraction decreased from 83% in BIKE to 67% in KE as a function of a higher O2 delivery and lower mitochondrial excess capacity. There was a significant relationship between O2 extraction and mitochondrial excess capacity and p50mito that was unrelated to blood flow and mean transit time. O2 extraction varies with mitochondrial respiration rate, p50mito and O2 delivery. Mitochondrial excess capacity maintains a low p50mito which enhances O2 diffusion from microvessels to mitochondria during exercise.

PlanHab* : hypoxia does not worsen the impairment of skeletal muscle oxidative function induced by bed rest alone.

Superposition of hypoxia on 21day bed rest did not worsen the impairment of skeletal muscle oxidative function induced by bed rest alone. A significant impairment of maximal oxidative performance was identified downstream of cardiovascular O2 delivery, involving both the intramuscular matching between O2 supply and utilization and mitochondrial respiration. These chronic adaptations appear to be relevant in terms of exposure to spaceflights and reduced gravity habitats (Moon or Mars), as characterized by low gravity and hypoxia, in patients with chronic diseases characterized by hypomobility/immobility and hypoxia, as well as in ageing. Skeletal muscle oxidative function was evaluated in 11 healthy males (mean±SD age 27±5years) prior to (baseline data collection, BDC) and following a 21day horizontal bed rest (BR), carried out in normoxia ( =133mmHg; N-BR) and hypoxia ( =90mmHg; H-BR). H-BR was aimed at simulating reduced gravity habitats. The effects of a 21day hypoxic ambulatory confinement ( =90mmHg; H-AMB) were also assessed. Pulmonary O2 uptake ( ), vastus lateralis fractional O2 extraction (changes in deoxygenated haemoglobin+myoglobin concentration, Δ[deoxy(Hb+Mb)]; near-infrared spectroscopy) and femoral artery blood flow (ultrasound Doppler) were evaluated during incremental one-leg knee-extension exercise (reduced constraints to cardiovascular O2 delivery) carried out to voluntary exhaustion in a normoxic environment. Mitochondrial respiration was evaluated ex vivo by high-resolution respirometry in permeabilized vastus lateralis fibres. decreased (P<0.05) after N-BR (0.98±0.13Lmin-1 ) and H-BR (0.96±0.17Lmin-1 ) vs. BDC (1.05±0.14L min-1 ). In the presence of a decreased (by ∼6-8%) thigh muscle volume, normalized per unit of muscle mass was not affected by both interventions. Δ[deoxy(Hb+Mb)]peak decreased (P<0.05) after N-BR (65±13% of limb ischaemia) and H-BR (62±12%) vs. BDC (73±13%). H-AMB did not alter or Δ[deoxy(Hb+Mb)]peak . An overshoot of Δ[deoxy(Hb+Mb)] was evident during the first minute of unloaded exercise after N-BR and H-BR. Arterial blood flow to the lower limb during both unloaded and peak knee extension was not affected by any intervention. Maximal ADP-stimulated mitochondrial respiration decreased (P<0.05) after all interventions vs. control. In 21day N-BR, a significant impairment of oxidative metabolism occurred downstream of cardiovascular O2 delivery, affecting both mitochondrial respiration and presumably the intramuscular matching between O2 supply and utilization. Superposition of H on BR did not worsen the impairment induced by BR alone.

Patterns of Suppressed Mitochondrial Respiration in Isolated Muscle Fibers from Type 2 Diabetics

The quality of skeletal muscle is markedly influenced by mitochondrial function. Aberrant mitochondrial function has been associated with reduced contractile and metabolic dysfunction in skeletal muscle, potentially leading to insulin resistance and type 2 diabetes. However, the direct relationship between mitochondrial dysfunction and type 2 diabetes remains to be fully understood. In the present study, vastus lateralis muscle biopsies were obtained from individuals with type 2 diabetes (N = 7) and non‐diabetic, age, gender and weight‐matched controls (N = 7). Maximal mitochondrial oxygen consumption was assessed in isolated muscle fibers and normalized to wet tissue weight. Preliminary findings illustrate that reductions in carbohydratedependent ADP‐stimulated respiration (~30%) were sustained in muscle fibers from type 2 diabetics, even with the addition of the Krebs Cycle anaplerotic substrates, glutamate (~30%) and succinate (~32%). Interestingly, trends of similar magnitudes persisted for lipid substrate metabolism, as fatty acid‐dependent ADP‐stimulated respiration also tended to be lower (~35%) compared to controls. Furthermore, rotenone‐treated respiration in diabetic skeletal muscle tended to be reduced (~37%). To conclude, these initial findings provide support for a systematic impairment in the oxidative metabolism of both carbohydrates and lipids in skeletal muscle of type 2 diabetic individuals. In addition, findings from rotenone‐treated conditions indicate that investigating functional deficits of Complex I of the Electron Transport Chain (ETC) may provide a more insightful understanding of the relationship between mitochondrial respiratory control and type 2 diabetes.Support or Funding Information*This study is supported by funds from the American Diabetic Association, ADA‐1‐16‐ICIS‐015, (D.P.C.) and start‐up funds from the Department of Health and Human Physiology at the University of Iowa (V.A.L.).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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.

The effect of respiration buffer composition on mitochondrial metabolism and function.

Functional studies on isolated mitochondria critically rely on the right choice of respiration buffer. Differences in buffer composition can lead to dramatically different respiration rates leading to difficulties in comparing prior studies. The ideal buffer facilities high ADP-stimulated respiratory rates and minimizes substrate transport effects so that the ability to distinguish between various treatments and conditions is maximal. In this study, we analyzed a variety of respiration buffers and substrate combinations to determine the optimal conditions to support mitochondrial function through ADP-stimulated respiration and uncoupled respiration using FCCP. The buffers consisted of a standard KCl based buffer (B1) and three modified buffers with chloride replaced by the K-lactobionate, sucrose, and the antioxidant taurine (B2) or K-gluconate (B3). The fourth buffer (B4) was identical to B2 except that K-lactobionate was replaced with K-gluconate. The substrate combinations consisted of metabolites that utilize different pathways of mitochondrial metabolism. To test mitochondrial function, we used isolated cardiac guinea pig mitochondria and measured oxygen consumption for three respiratory states using an Oroboros Oxygraph-2k. These states were the leak state (energized mitochondria in the absence of adenylates), ADP-stimulated state (energized mitochondria in the presence of saturating ADP concentrations), and uncoupled state (energized mitochondria in the presence of FCCP). On average across all substrate combinations, buffers B2, B3, and B4 had an increase of 16%, 26%, and 35% for the leak state, ADP-simulated state, and uncoupled state, respectively, relative to rates using B1. The common feature distinguishing these buffers from B1 is the notable lack of high chloride concentrations. Based on the respiratory rate metrics obtained with the substrate combinations, we conclude that the adenine nucleotide translocase, the dicarboxylate carrier, and the alpha-ketoglutarate exchanger are partially inhibited by chloride. Therefore, when the goal is to maximize ADP-stimulated respiration, buffers containing K-lactobionate or K-gluconate are superior choices compared to the standard KCl-based buffers.

Ablating the protein TBC1D1 impairs contraction-induced sarcolemmal glucose transporter 4 redistribution but not insulin-mediated responses in rats

TBC1 domain family member 1 (TBC1D1), a Rab GTPase-activating protein and paralogue of Akt substrate of 160 kDa (AS160), has been implicated in both insulin- and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase-mediated glucose transporter type 4 (GLUT4) translocation. However, the role of TBC1D1 in contracting muscle remains ambiguous. We therefore explored the metabolic consequence of ablating TBC1D1 in both resting and contracting skeletal muscles, utilizing a rat TBC1D1 KO model. Although insulin administration rapidly increased (p < 0.05) plasma membrane GLUT4 content in both red and white gastrocnemius muscles, the TBC1D1 ablation did not alter this response nor did it affect whole-body insulin tolerance, suggesting that TBC1D1 is not required for insulin-induced GLUT4 trafficking events. Consistent with findings in other models of altered TBC1D1 protein levels, whole-animal and ex vivo skeletal muscle fat oxidation was increased in the TBC1D1 KO rats. Although there was no change in mitochondrial content in the KO rats, maximal ADP-stimulated respiration was higher in permeabilized muscle fibers, which may contribute to the increased reliance on fatty acids in resting KO animals. Despite this increase in mitochondrial oxidative capacity, run time to exhaustion at various intensities was impaired in the KO rats. Moreover, contraction-induced increases in sarcolemmal GLUT4 content and glucose uptake were lower in the white gastrocnemius of the KO animals. Altogether, our results highlight a critical role for TBC1D1 in exercise tolerance and contraction-mediated translocation of GLUT4 to the plasma membrane in skeletal muscle.

Abstract 453: FADS2 Overexpression Exacerbates Myocardial Ischemia-reperfusion Injury in Mice: Role of Mitochondria

Common FADS2 haplotypes associated with hyperactivity of its gene product, delta-6-desaturase (D6D), predict cardiovascular morbidity and mortality in humans. D6D is the rate-limiting enzyme in essential polyunsaturated fatty acid (PUFA) metabolism, but its role in the pathogenesis of cardiovascular disease is unclear. To investigate this, we generated mice with global (CMV promoter) transgenic overexpression of FADS2 and evaluated their cardiometabolic phenotype. FADS2 -tg mice exhibit mild glucose intolerance and aortic stiffening with advancing age, but no overt cardiac pathology. However, when challenged with ischemia-reperfusion (I/R) ex vivo , FADS2 -tg mouse hearts have greater infarct sizes compared to their wild-type (WT; FVB) counterparts. Mitochondrial phospholipid analyses revealed a 65% increase in the membrane arachidonic/linoleic acid (AA/LA) ratio of FADS2- tg versus WT hearts, consistent with previous work in our lab linking D6D hyperactivity with mitochondrial phospholipid remodeling and disease progression in heart failure. Thus, we hypothesized that FADS2 might exacerbate myocardial injury by altering mitochondrial responses to ischemia-reperfusion. To examine this further, mitochondria were isolated from FADS2 -tg or WT mouse hearts for functional studies following 45/90 minutes of global I/R ex vivo . FADS2- tg - I/R mitochondria exhibited lower ADP-stimulated respiration and higher H 2 O 2 emission per O 2 consumed compared to WT-I/R and non-ischemic controls , suggesting impaired cardiac energetics and greater oxidant burden following ischemia. I/R augmented Ca 2+ -induced swelling in both WT and FADS2 -tg mitochondria. This was attenuated in the presence of K + in WT, but not FADS2 -tg, implicating FADS2 -dependent alterations post-ischemic in mitochondrial K + handling. Interestingly, ischemia led to a 38% depletion of mitochondrial membrane AA in FADS2 -tg, but not WT hearts, suggesting an interaction of FADS2 and mitochondrial membrane AA liberation following ischemia. Ongoing studies in our laboratory are investigating the potentially novel links between FADS2 expression with mitochondrial phospholipases, AA signaling, and mitoK ATP channel activity in the pathogenesis of myocardial I/R injury.

Long-term rates of mitochondrial protein synthesis are increased in mouse skeletal muscle with high-fat feeding regardless of insulin-sensitizing treatment.

Skeletal muscle mitochondrial protein synthesis is regulated in part by insulin. The development of insulin resistance with diet-induced obesity may therefore contribute to impairments to protein synthesis and decreased mitochondrial respiration. Yet the impact of diet-induced obesity and insulin resistance on mitochondrial energetics is controversial, with reports varying from decreases to increases in mitochondrial respiration. We investigated the impact of changes in insulin sensitivity on long-term rates of mitochondrial protein synthesis as a mechanism for changes to mitochondrial respiration in skeletal muscle. Insulin resistance was induced in C57BL/6J mice using 4 wk of a high-fat compared with a low-fat diet. For 8 additional weeks, diets were enriched with pioglitazone to restore insulin sensitivity compared with nonenriched control low-fat or high-fat diets. Skeletal muscle mitochondrial protein synthesis was measured using deuterium oxide labeling during weeks 10-12 High-resolution respirometry was performed using palmitoyl-l-carnitine, glutamate+malate, and glutamate+malate+succinate as substrates for mitochondria isolated from quadriceps. Mitochondrial protein synthesis and palmitoyl- l-carnitine oxidation were increased in mice consuming a high-fat diet, regardless of differences in insulin sensitivity with pioglitazone treatment. There was no effect of diet or pioglitazone treatment on ADP-stimulated respiration or H2O2 emission using glutamate+malate or glutamate+malate+succinate. The results demonstrate no impairments to mitochondrial protein synthesis or respiration following induction of insulin resistance. Instead, mitochondrial protein synthesis was increased with a high-fat diet and may contribute to remodeling of the mitochondria to increase lipid oxidation capacity. Mitochondrial adaptations with a high-fat diet appear driven by nutrient availability, not intrinsic defects that contribute to insulin resistance.

The gap junction modifier ZP1609 decreases cardiomyocyte hypercontracture following ischaemia/reperfusion independent from mitochondrial connexin 43.

Dysregulation of gap junction-mediated cell coupling contributes to development of arrhythmias and myocardial damage after ischaemia/reperfusion (I/R). Connexin 43 (Cx43) is present at ventricular gap junctions and also in the mitochondria of cardiomyocytes. The dipeptide (2S, 4R)-1-(2-aminoacetyl)-4-benzamidopyrrolidine-2-carboxylic acid (ZP1609) has antiarrhythmic properties and reduces infarct size when given at reperfusion. However, it is unclear, whether ZP1609 targets Cx43-containing mitochondria and affects cardiomyocyte hypercontracture following I/R. We studied the effects of ZP1609 on the function of murine sub-sarcolemmal mitochondria (SSM, containing Cx43) and interfibrillar mitochondria (IFM, lacking Cx43). Murine isolated cardiomyocytes were subjected to simulated I/R without and with ZP1609 (applied during I/R or at the onset of reperfusion only), and the number of cardiomyocytes undergoing hypercontracture was quantified. Biochemical pathways targeted by ZP1609 in cardiomyocytes were analysed. ZP1609 inhibited ADP-stimulated respiration and ATP production in SSM and IFM. ROS formation and calcium retention capacities in SSM and IFM were not affected by ZP1609, whereas potassium uptake was enhanced in IFM. The number of rod-shaped cardiomyocytes was increased by ZP1609 (10μM) when administered either during I/R or reperfusion. ZP1609 altered the phosphorylation of proteins contributing to the protection against I/R injury. ZP1609 reduced mitochondrial respiration and ATP production, but enhanced potassium uptake of IFM. Additionally, ZP1609 reduced the extent of cardiomyocytes undergoing hypercontracture following I/R. The protective effect was independent of mitochondrial Cx43, as ZP1609 exerts its effects in Cx43-containing SSM and Cx43-lacking IFM.

Mechanism of triclosan toxicity: Mitochondrial dysfunction including complex II inhibition, superoxide release and uncoupling of oxidative phosphorylation

Triclosan (5-chloro-2′-(2,4-dichlorophenoxy)phenol), a widely used antibacterial agent, exerts adverse effects on the organism of mammals. Recent research reviled that triclosan at low micromolar concentrations causes mitochondrial dysfunction in many cell types, but the mechanisms of its effect are not fully understood. Here we show that exposure to triclosan disrupted membrane potential, prevented the calcium uptake-driven high-amplitude mitochondrial swelling, stimulated the respiration in the presence of complex I substrates, and suppressed the ADP-stimulated respiration in the presence of complex II substrate, succinate. Triclosan directly inhibited complex II activity. Similar to the complex II inhibitor thenoyltrifluoroacetone, triclosan induced the oxidation of the cytochromes b566 and b562 and caused the release of mitochondrial superoxide. Opposite to thenoyltrifluoroacetone, triclosan increased superoxide release synergistically with myxothiazol but not with antimycin A, indicating different topology of superoxide-producing sites. We concluded that triclosan is unique by its capability of acting as both a protonophore and an unusual complex II inhibitor, which interferes with the mitochondrial respiration by blocking the electron transfer between ubiquinone at the Qd-binding site and heme b. Our data can provide an insight into the mechanisms of the carcinogenic effect of triclosan in the liver and other tissues.

Associations of Mitochondrial Fatty Acid Oxidation with Body Fat in Premenopausal Women.

Higher in vivo fatty acid (FA) oxidation rates have been reported in obese individuals compared to lean counterparts; however whether this reflects a shift in substrate-specific oxidative capacity at the level of the skeletal muscle mitochondria has not been examined. The purpose of this study was to test the hypothesis that in situ measures of skeletal muscle mitochondria FA oxidation would be positively associated with total body fat. Participants were 38 premenopausal women (BMI = 26.5 ± 4.3 kg/m2). Total and regional fat were assessed by dual-energy X-ray absorptiometry (DXA). Mitochondrial FA oxidation was assessed in permeabilized myofibers using high-resolution respirometry and a palmitoyl carnitine substrate. We found positive associations of total fat mass with State 3 (ADP-stimulated respiration) (r = 0.379, p < 0.05) and the respiratory control ratio (RCR, measure of mitochondrial coupling) (r = 0.348, p < 0.05). When participants were dichotomized by high or low body fat percent, participants with high total body fat displayed a higher RCR compared to those with low body fat (p < 0.05). There were no associations between any measure of regional fat and mitochondrial FA oxidation independent of total fat mass. In conclusion, greater FA oxidation in obesity may reflect molecular processes that enhance FA oxidation capacity at the mitochondrial level.

Abstract 12977: Neladenoson, a Partial Adenosine A1-receptor Agonist, Improves Mitochondrial Function in Left Ventricular Cardiomyocytes Isolated From Dogs With Chronic Heart Failure

Background: Mitochondria (MITO) of failing cardiomyocytes (CMs) manifest structural and functional abnormalities characterized by hyperplasia, reduced organelle size and reduced respiration. These abnormalities lead to reduced rate of ATP synthesis and excess production of reactive oxygen species. We previously showed that chronic therapy with a partial adenosine A1-receptor agonist (pA1RA) improves LV function and normalizes tissue levels of MITO uncoupling proteins 2 and 3 in dogs with heart failure (HF). Hypothesis: Neladenoson, a new pA1RA, improves MITO function in freshly isolated CMs from dogs with chronic HF. Methods: CMs were isolated from LV of 7 microembolization-induced HF dogs (LV ejection fraction &lt;30%) and from 6 normal (NL) dogs using a collagenase-based enzymatic process that yielded ~70% rod-shaped CMs that excluded trypan blue. Equal aliquotes of CMs were incubated in 0 (vehicle), 3, 10, and 30 nM concentration of Neladenoson for one hour at 37°C. MITO ADP-stimulated state-3 respiration, maximum rate of ATP synthesis and complex-IV activity (COX-IV) were measured at end of incubation. Respiration was measured with a Strathkelvin respirometer and COX-IV activity was measured polarographically and both expressed as nAtom Oxygen/min/mg protein. Rate of ATP synthesis was measured using the bioluminescent ApoSENSOR assay and quantified in RFU/μg protein. Results: Data are shown in the table. Increasing concentration of Neladenoson had no effect on measures of MITO function in CMs from NL dogs. In CMs from HF dogs, depressed levels of MITO respiration, ATP synthesis and COX-IV increased significantly and in a dose-dependent manner after exposure to Neladenoson. Conclusions: Neladenoson has beneficial effects on MITO function in CMs from HF dogs. Furthermore, the improvement in MITO function after treatment with Neladenoson can occur early after initiation of therapy (within one hour) and is dose-dependent up to concentrations of 30 nM.

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.

Abstract 106: G Protein-coupled Receptor Kinase 2 Associates With Mitochondrial Proteins and Negatively Alters Mitochondrial Respiration After Myocardial Infarction

During cardiac injury or stress, G protein-coupled receptor (GPCR) kinase 2 (GRK2) expression levels and activity are increased, leading to a desensitization of myocardial β-adrenergic receptors (βARs) and contributing to the loss of contractile reserve. Up-regulated GRK2 has been shown to be pathogenic in the post-injured heart and is involved in the promotion of heart failure (HF). Consequently, inhibition of GRK2 has been shown to rescue several animal models of HF, as demonstrated by cardiac expression of the βARKct, a peptide inhibitor of GRK2. There is new evidence that GRK2 has other, non-GPCR dependent pathological functions within cardiomyocytes. For example, during conditions that increase oxidative stress, GRK2 has been shown to localize to the mitochondria, where it is a pro-death kinase. βARKct has also been shown to inhibit the mitochondrial translocation of GRK2 due to inhibition of Hsp90 binding, a chaperone found to bind and localize GRK2 to mitochondria. Our objective is to identify the functions and targets of GRK2 in the mitochondria and its role in the development of HF. We hypothesize that GRK2 has mitochondrial targets that impair mitochondrial function in vivo, contributing to the pathogenesis of HF. Our results show that ADP-stimulated and maximal respiration are significantly decreased in mitochondria isolated from mouse hearts 2 weeks post-myocardial infarction (MI). Cardiac-specific expression of the βARKct rescued the reduction in mitochondrial respiration post-MI, in the presence of pyruvate or palmitoylcarnitine. To identify the mitochondrial targets of GRK2, we performed interactome and phospho-interactome analyses on GRK2 immunoprecipitates from neonatal rat cardiomyocytes subjected to oxidative stress. These results revealed that GRK2 interacts with several mitochondrial proteins, including those involved in metabolism, oxidative stress, calcium handling and cell death. Thus we propose that during ischemia, an increase in mitochondrial GRK2 results in its association with proteins that compromise respiration and contribute to myocardial damage and HF. This demonstrates that understanding both the canonical and non-canonical roles of GRK2 in HF is essential for more targeted intervention for therapy.

Demonstration of a day-night rhythm in human skeletal muscle oxidative capacity

ObjectiveA disturbed day-night rhythm is associated with metabolic perturbations that can lead to obesity and type 2 diabetes mellitus (T2DM). In skeletal muscle, a reduced oxidative capacity is also associated with the development of T2DM. However, whether oxidative capacity in skeletal muscle displays a day-night rhythm in humans has so far not been investigated. MethodsLean, healthy subjects were enrolled in a standardized living protocol with regular meals, physical activity and sleep to reflect our everyday lifestyle. Mitochondrial oxidative capacity was examined in skeletal muscle biopsies taken at five time points within a 24-hour period. ResultsCore-body temperature was lower during the early night, confirming a normal day-night rhythm. Skeletal muscle oxidative capacity demonstrated a robust day-night rhythm, with a significant time effect in ADP-stimulated respiration (state 3 MO, state 3 MOG and state 3 MOGS, p < 0.05). Respiration was lowest at 1 PM and highest at 11 PM (state 3 MOGS: 80.6 ± 4.0 vs. 95.8 ± 4.7 pmol/mg/s). Interestingly, the fluctuation in mitochondrial function was also observed in whole-body energy expenditure, with peak energy expenditure at 11 PM and lowest energy expenditure at 4 AM (p < 0.001). In addition, we demonstrate rhythmicity in mRNA expression of molecular clock genes in human skeletal muscle. ConclusionsOur results suggest that the biological clock drives robust rhythms in human skeletal muscle oxidative metabolism. It is tempting to speculate that disruption of these rhythms contribute to the deterioration of metabolic health associated with circadian misalignment.