Coupling Of Oxidative Phosphorylation Research Articles

A protet-based model that can account for energy coupling in oxidative and photosynthetic phosphorylation

Two-stage (e.g. light-dark) phosphorylation experiments showed that there is a stored ‘high-energy’ intermediate linking electron transport and phosphorylation. Large, artificial electrochemical proton gradients (protonmotive forces or pmfs) can also drive phosphorylation, a fact seen as strongly supportive of the chemiosmotic coupling hypothesis that a pmf is the ‘high-energy’ intermediate. However, in such experiments there is an experimental threshold (pmf >170 mV, equivalent to ΔpH ∼2.8) below which no phosphorylation is in fact observed, and 220 mV are required to recreate in vivo rates. This leads to the correct question, which is then whether those values of the pmf generated by electron transport are large enough. Even the lower ones as required for any phosphorylation (leave alone those required to explain in vivo rates) are below the threshold [1, 2], whether measured directly with microelectrodes or via the use of membrane-permeant ions and/or acids/bases (which are always transporter substrates [3], so all such measurements are in fact artefactual). The single case that seemed large enough (220 mV) is now admitted to be a diffusion potential artefact [4]. Many other observables (inadequate bulk H+ in ‘O2-pulse’-type experiments, alkaliphilic bacteria, dual-inhibitor titrations, uncoupler-binding proteins, etc.) are consistent with the view that values of the pmf, and especially of Δψ, are actually very low. A protet-based charge separation model [2], a protonic version analogous to how energy may be stored in devices called electrets, provides a high-energy intermediate that can explain the entire literature, including the very striking demonstration [5] that close proximity is required between electron transport and ATP synthase complexes for energy coupling between them to allow phosphorylation to occur. A chief purpose of this article is thus to summarise the extensive and self-consistent literature, much of which is of some antiquity and rarely considered by modern researchers, despite its clear message of the inadequacy of chemiosmotic coupling to explain these phenomena.

Activation of innate immunity selectively compromises mitochondrial complex I, proline oxidation, and flight activity in the major arbovirus vector Aedes aegypti.

Aedes aegypti females are natural vectors of important arboviruses such as dengue, zika, and yellow fever. Mosquitoes activate innate immune response signaling pathways upon infection, as a resistance mechanism to fight pathogens and limit their propagation. Despite the beneficial effects of immune activation for insect vectors, phenotypic costs ultimately affect their fitness. However, the underlying mechanisms that mediate these fitness costs remain poorly understood. Given the high energy required to mount a proper immune response, we hypothesized that systemic activation of innate immunity would impair flight muscle mitochondrial function, compromising tissue energy demand and flight activity. Here, we investigated the dynamic effects of activation of innate immunity by intra-thoracic zymosan injection on A. aegypti flight muscle mitochondrial metabolism. Zymosan injection significantly increased defensin A expression in fat bodies in a time-dependent manner that compromised flight activity. Although oxidant levels in flight muscle were hardly altered, ATP-linked respiratory rates driven by mitochondrial pyruvate+proline oxidation were significantly reduced at 24 h upon zymosan injection. Oxidative phosphorylation coupling was preserved regardless of innate immune response activation along 24 h. Importantly, rotenone-sensitive respiration and complex I-III activity were specifically reduced 24 h upon zymosan injection. Also, loss of complex I activity compromised ATP-linked and maximal respiratory rates mediated by mitochondrial proline oxidation. Finally, the magnitude of innate immune response activation negatively correlated with respiratory rates, regardless of the metabolic states. Collectively, we demonstrate that activation of innate immunity is strongly associated with reduced flight muscle complex I activity with direct consequences to mitochondrial proline oxidation and flight activity. Remarkably, our results indicate a trade-off between dispersal and immunity exists in an insect vector, underscoring the potential consequences of disrupted flight muscle mitochondrial energy metabolism to arbovirus transmission.

Abnormal mitochondrial function and morphology in heart transplanted patients with cardiac allograft vasculopathy.

Cardiac allograft vasculopathy (CAV) remains the Achilles' heel of long-term survival of HTx patients. Mitochondrial dysfunction has been reported in both arteriosclerotic coronary disease and heart failure. However, myocardial mitochondrial function has not been examined in HTx patients with CAV. 43 HTx patients (21 patients with CAV and 22 patients without CAV) ≥12 months after HTx were enrolled. Endomyocardial biopsies were analyzed using high-resolution respirometry for glucose-coupled mitochondrial respiration. Number and area of mitochondria profiles as well as cristae morphology were assessed by transmission electron microscopy. Echocardiography and coronary angiography were used to measure global longitudinal strain (GLS) and grade CAV. Complex I+II-linked respiration was reduced in patients with CAV compared with patients without CAV (82.7 ± 31.9 pmol O2/(s•mg) vs 116 ± 35.9 pmol O2/(s•mg), p=0.003). Mitochondrial respiratory function measured as oxidative phosphorylation coupling efficiency was positively associated with left ventricular GLS (r=0.49, p=0.002) and negatively associated with elevated biomarkers (Troponin T: r=-0.33, p=0.04 and NT-proBNP: r=-0.41, p=0.009). Mitochondrial profile number and area did not differ. However, patients with CAV had a larger proportion of mitochondria with abnormal cristae morphology (p < 0.001). Myocardial mitochondrial respiration is impaired in patients with CAV and is associated with an abnormal cristae morphology. The mitochondrial dysfunction appears to be associated with reduced myocardial contractile function and elevated biomarkers. These results highlight that mitochondrial targeted treatment in patients with CAV should be assessed in future clinical studies.

Heat Stress More Negatively Impacts Cardiac Muscle Mitochondria in Female Versus Male Pigs

With continued trends toward more frequent and more severe heat events there is a greater risk of environment‐induced heat stress (HS). How persistently elevated core temperatures impacts body tissues remains largely unknown. Our preliminary evidence indicates HS may negatively impact cardiac tissue and that biological sex may play a role in HS‐mediated pathology. To further test the hypothesis that cardiac muscle mitochondria are more negatively impacted by HS in females than in males, samples were isolated from the left ventricle (LV) of the heart from 3‐month‐old castrated male and prepubertal female pigs that were exposed to thermoneutral (TN) conditions (n = 4 per sex) or HS conditions for 24 h at 37°C (HS; n = 6 per sex). Samples were analyzed for citrate synthase (CS) and cytochrome c oxidase (COX) activities, and for mitochondrial oxidative phosphorylation (P) and electron transfer (E) capacities via high resolution respirometry. Data were analyzed by mixed linear models in SAS v9.4 with treatment, sex, and treatment × sex as fixed effects. A traditional marker of mitochondrial volume density, CS activity, tended to be greater in TN males compared to HS males (P = 0.08) but was unaffected by HS in female pigs. Conversely, intrinsic (relative to CS activity) mitochondrial function, as measured by COX activity, was greater in TN females than HS females (P = 0.04) and within HS, males maintained greater intrinsic mitochondrial function than females (P = 0.02). Both integrative (relative to tissue wet weight) and intrinsic mitochondrial leak respiration were greater in HS than TN pigs (P = 0.03) but the contribution of leak to total E (flux control ratio; FCRLEAK) tended to be greater in males than females (P = 0.07). Conversely, both integrative and intrinsic maximal E (ECI+II) tended to be greater in female than male pigs (P = 0.08) indicating a greater degree of excess electron transfer capacity in females. The contribution of complex I‐supported P (PCI) to total E (FCRPCI) tended to be greater in TN compared to HS pigs regardless of sex (P = 0.09) and the contribution of maximal P to E (FCRPCI+II) was greater in TN females compared to HS females (P = 0.05). This resulted in the FCRPCI+IIbeing greater in males compared to females in the HS condition (P = 0.01), suggesting greater coupling of oxidative phosphorylation and electron transfer in cardiac mitochondria in males during 24 h of HS. These results suggest that HS may negatively impact cardiac muscle mitochondria in female pigs compared to males. Moreover, these data indicate that persistent HS has deleterious consequences for the myocardium and may represent an additional health concern caused by global climate trends. This project was supported by USDA NIFA Award #2020‐68014‐31954.

Mitochondria Bioenergetic Functions and Cell Metabolism Are Modulated by the Bergamot Polyphenolic Fraction.

The bergamot polyphenolic fraction (BPF) was evaluated in the F1FO-ATPase activity of swine heart mitochondria. In the presence of a concentration higher than 50 µg/mL BPF, the ATPase activity of F1FO-ATPase, dependent on the natural cofactor Mg2+, increased by 15%, whereas the enzyme activity in the presence of Ca2+ was inhibited by 10%. By considering this opposite BPF effect, the F1FO-ATPase activity involved in providing ATP synthesis in oxidative phosphorylation and triggering mitochondrial permeability transition pore (mPTP) formation has been evaluated. The BPF improved the catalytic coupling of oxidative phosphorylation in the presence of a substrate at the first phosphorylation site, boosting the respiratory control ratios (state 3/state 4) by 25% and 85% with 50 µg/mL and 100 µg/mL BPF, respectively. Conversely, the substrate at the second phosphorylation site led to the improvement of the state 3/state 4 ratios by 15% only with 100 µg/mL BPF. Moreover, the BPF carried out its beneficial effect on the mPTP phenomenon by desensitizing the pore opening. The acute effect of the BPF on the metabolism of porcine aortica endothelial cells (pAECs) showed an ATP rate index greater than one, which points out a prevailing mitochondrial oxidative metabolism with respect to the glycolytic pathway, and this ratio rose by about three times with 100 µg/mL BPF. Consistently, the mitochondrial ATP turnover, in addition to the basal and maximal respiration, were higher in the presence of the BPF than in the controls, and the MTT test revealed an increase in cell viability with a BPF concentration above 200 µg/mL. Therefore, the molecule mixture of the BPF aims to ensure good performance of the mitochondrial bioenergetic parameters.

Pioglitazone Is a Mild Carrier-Dependent Uncoupler of Oxidative Phosphorylation and a Modulator of Mitochondrial Permeability Transition.

Pioglitazone (PIO) is an insulin-sensitizing antidiabetic drug, which normalizes glucose and lipid metabolism but may provoke heart and liver failure and chronic kidney diseases. Both therapeutic and adverse effects of PIO can be accomplished through mitochondrial targets. Here, we explored the capability of PIO to modulate the mitochondrial membrane potential (ΔΨm) and the permeability transition pore (mPTP) opening in different models in vitro. ΔΨm was measured using tetraphenylphosphonium and the fluorescent dye rhodamine 123. The coupling of oxidative phosphorylation was estimated polarographically. The transport of ions and solutes across membranes was registered by potentiometric and spectral techniques. We found that PIO decreased ΔΨm in isolated mitochondria and intact thymocytes and the efficiency of ADP phosphorylation, particularly after the addition of Ca2+. The presence of the cytosolic fraction mitigated mitochondrial depolarization but made it sustained. Carboxyatractyloside diminished the PIO-dependent depolarization. PIO activated proton transport in deenergized mitochondria but not in artificial phospholipid vesicles. PIO had no effect on K+ and Ca2+ inward transport but drastically decreased the mitochondrial Ca2+-retention capacity and protective effects of adenine nucleotides against mPTP opening. Thus, PIO is a mild, partly ATP/ADP-translocase-dependent, uncoupler and a modulator of ATP production and mPTP sensitivity to Ca2+ and adenine nucleotides. These properties contribute to both therapeutic and adverse effects of PIO.

Respiration responses of wheat seedlings to treatment with trehalose under heat stress

Heat stress limits wheat production and trehalose can improve stress tolerance. How trehalose affects wheat respiration is unclear. In this study, we investigated the effects of exogenous trehalose on the respiration of wheat seedlings during heat stress and the subsequent recovery period. Trehalose pretreatment significantly increased the expression of the alternative oxidase genes AOX1a and AOX1c under heat stress, indicating that trehalose pretreatment increased the capacity of the alternative respiration pathway (AP) in response to heat stress. Trehalose pretreatment also enhanced the activity of the malate-oxaloacetate (Mal-OAA) shuttle and ameliorated the decrease in photosynthetic activity caused by heat stress. However, when the AP was inhibited by salicylhydroxamic acid under heat stress, both Mal-OAA shuttle activity and photosynthetic efficiency were substantially reduced in the control and trehalose pretreatment groups. In addition, trehalose pretreatment helped to maintain inner mitochondrial respiratory activity and the activity of Complex II during heat stress, particularly the coupling of oxidative phosphorylation with the Complex II electron transport chain, thereby mitigating heat-related damage to the cytochrome pathway (CP). Taken together, these results suggest that exogenous trehalose enhanced the AP and reduced damage to the CP under heat stress in wheat seedlings, thus maintaining cellular energy metabolism. Up-regulation of the AP by trehalose pretreatment may improve the heat tolerance of wheat seedlings by dissipating excess reducing equivalents transported through the Mal-OAA shuttle, thereby protecting photosynthetic performance.

Combined Therapy With Polyethylene Glycol-20k and MCC950 Preserves Post-Resuscitated Myocardial Function in a Rat Model of Cardiac Arrest and Cardiopulmonary Resuscitation.

BackgroundTo investigate the therapeutic potential of combined therapy with polyethylene glycol‐20k (PEG‐20k) and MCC950 on post‐resuscitation myocardial function in a rat model of cardiac arrest.Methods and ResultsThirty rats were randomized into 5 groups: Sham, Control, PEG‐20k, MCC950, PEG‐20k+ MCC950. Except for sham, animals were subjected to 6 minutes of ventricular fibrillation followed by 8 minutes cardiopulmonary resuscitation. Two milliliters PEG‐20k was administered by intravenous injection coincident with the start of cardiopulmonary resuscitation; MCC950 (10 mg/kg), a highly selective NLRP3 inflammasome inhibitor, was delivered immediately after restoration of spontaneous circulation. Myocardial function, sublingual microcirculation, mitochondrial function, plasma cardiac troponin I, and interleukin‐1β, expression of proteins in SIRT1 (sirtuin 1)/PGC‐1α (peroxisome proliferator‐activated receptor gamma coactivator 1‐alpha) and NLRP3 (the NOD‐like receptor family protein 3) inflammasome pathways were evaluated. Following cardiopulmonary resuscitation, myocardial function was compromised with a significantly decreased cardiac output, ejection fraction, and increased myocardial performance index, cardiac troponin I. Sublingual microcirculation was disturbed with impaired perfused vessel density and microvascular flow index. Cardiac arrest reduced mitochondrial routine respiration, Complex I‐linked respiration, respiratory control rates and oxidative phosphorylation coupling efficiency. PEG‐20k or MCC950 alone restored mitochondrial respiratory function, restituted sublingual microcirculation, and preserved myocardial function, whereas a combination of PEG‐20k and MCC950 further improved these aspects. PEG‐20k restored the expression of SIRT1 and PGC‐1α, and blunted activation of NLRP3 inflammasomes. MCC950 suppressed expression of cleaved‐caspase‐1/pro‐caspase‐1, ASC (apoptosis‐associated speck‐like protein), GSDMD [gasdermin d], and interleukin‐1β.ConclusionsCombined therapy with PEG‐20k and MCC950 is superior to either therapy alone for preserving post‐resuscitated myocardial function, restituting sublingual microcirculation at restoration of spontaneous circulation at 6 hours. The responsible mechanisms involve upregulated expression of SIRT1/PGC1‐α in tandem with inhibition of NLRP3 inflammasomes.

A protet-based, protonic charge transfer model of energy coupling in oxidative and photosynthetic phosphorylation.

Textbooks of biochemistry will explain that the otherwise endergonic reactions of ATP synthesis can be driven by the exergonic reactions of respiratory electron transport, and that these two half-reactions are catalyzed by protein complexes embedded in the same, closed membrane. These views are correct. The textbooks also state that, according to the chemiosmotic coupling hypothesis, a (or the) kinetically and thermodynamically competent intermediate linking the two half-reactions is the electrochemical difference of protons that is in equilibrium with that between the two bulk phases that the coupling membrane serves to separate. This gradient consists of a membrane potential term Δψ and a pH gradient term ΔpH, and is known colloquially as the protonmotive force or pmf. Artificial imposition of a pmf can drive phosphorylation, but only if the pmf exceeds some 150-170mV; to achieve in vivo rates the imposed pmf must reach 200mV. The key question then is 'does the pmf generated by electron transport exceed 200mV, or even 170mV?' The possibly surprising answer, from a great many kinds of experiment and sources of evidence, including direct measurements with microelectrodes, indicates it that it does not. Observable pH changes driven by electron transport are real, and they control various processes; however, compensating ion movements restrict the Δψ component to low values. A protet-based model, that I outline here, can account for all the necessary observations, including all of those inconsistent with chemiosmotic coupling, and provides for a variety of testable hypotheses by which it might be refined.

Tafazzin deficiency impairs CoA-dependent oxidative metabolism in cardiac mitochondria

Barth syndrome is a mitochondrial myopathy resulting from mutations in the tafazzin (TAZ) gene encoding a phospholipid transacylase required for cardiolipin remodeling. Cardiolipin is a phospholipid of the inner mitochondrial membrane essential for the function of numerous mitochondrial proteins and processes. However, it is unclear how tafazzin deficiency impacts cardiac mitochondrial metabolism. To address this question while avoiding confounding effects of cardiomyopathy on mitochondrial phenotype, we utilized Taz-shRNA knockdown (TazKD ) mice, which exhibit defective cardiolipin remodeling and respiratory supercomplex instability characteristic of human Barth syndrome but normal cardiac function into adulthood. Consistent with previous reports from other models, mitochondrial H2O2 emission and oxidative damage were greater in TazKD than in wild-type (WT) hearts, but there were no differences in oxidative phosphorylation coupling efficiency or membrane potential. Fatty acid and pyruvate oxidation capacities were 40-60% lower in TazKD mitochondria, but an up-regulation of glutamate oxidation supported respiration rates approximating those with pyruvate and palmitoylcarnitine in WT. Deficiencies in mitochondrial CoA and shifts in the cardiac acyl-CoA profile paralleled changes in fatty acid oxidation enzymes and acyl-CoA thioesterases, suggesting limitations of CoA availability or "trapping" in TazKD mitochondrial metabolism. Incubation of TazKD mitochondria with exogenous CoA partially rescued pyruvate and palmitoylcarnitine oxidation capacities, implicating dysregulation of CoA-dependent intermediary metabolism rather than respiratory chain defects in the bioenergetic impacts of tafazzin deficiency. These findings support links among cardiolipin abnormalities, respiratory supercomplex instability, and mitochondrial oxidant production and shed new light on the distinct metabolic consequences of tafazzin deficiency in the mammalian heart.

The Impairment Of Oxidative Metabolism After 10-day Of Bed Rest Is Upstream Of Skeletal-Muscle Mitochondria

PURPOSE: Exposure to microgravity, as simulated by bed rest (BR), leads to an impairment of oxidative metabolism. The sites of this impairment are still debated. Aim of this study was to identify markers of impaired oxidative metabolism along the O2 pathway, from ambient air to skeletal muscle mitochondria, following 10 days of BR. METHODS: Measurements were carried out on 10 recreationally active young males (age 23 ± 5 years [mean±SD]) before (PRE) and after (POST) 10 days of horizontal BR. Pulmonary O2 uptake (V̇O2) and other respiratory, cardiovascular and skeletal muscle variables were determined during an incremental exercise on a cycle ergometer. Peripheral vascular and endothelial functions were evaluated by the blood flow response (Doppler ultrasound) in the femoral artery during 1-min passive leg movement (PLM). Mitochondrial respiration was evaluated by high-resolution respirometry on permeabilized vastus lateralis fibers obtained by biopsy. RESULTS: Peak V̇O2 was lower (P=0.001) in POST (41.5 ± 6.5 ml.kg-1.min-1) vs. PRE (44.5 ± 7.4). The area under the blood flow vs. time curve during PLM was lower (P=0.038) in POST (274 ± 233 mL) vs. PRE (427 ± 291). Skeletal muscle citrate synthase activity, an estimate of mitochondrial mass, was not different (P=0.115) in POST (131.2 ± 15.9 UI.mg-1.protein) vs. PRE (137.9 ± 18.8). Maximal ADP-stimulated mitochondrial respiration (66.4 ± 17.5 pmol.s-1.mg-1 wet weight [POST] vs. 72.3 ± 14.0 [PRE], P=0.127) and oxidative phosphorylation coupling efficiency (respiratory control ratio, 4.10 ± 1.19 [PRE] vs. 3.59 ± 1.11 [POST], P=0.443) were not affected by BR. CONCLUSIONS: These preliminary data suggest that the whole-body impairment of oxidative metabolism during exercise, following 10 days of horizontal BR, is associated with an impairment of peripheral vascular and endothelial functions, whereas mitochondrial volume and respiratory function are unaffected. Data related to several other markers (pulmonary function, cardiac output, submaximal exercise tolerance, skeletal muscle fractional O2 extraction, skeletal muscle V̇O2 recovery kinetics, mitochondrial respiration sensitivity to submaximal [ADP]) are yet to be analyzed. Funding: ASI, MARS-PRE Project, n. DC-VUM-2017-006

Bioactivity of the Novel Fungicide SYP-14288 Against Plant Pathogens and the Study of its Mode of Action Based on Untargeted Metabolomics.

Plant disease is a major threat to crop production, and fungicide application is one of the most effective methods to control plant disease. With emerging issues related to toxic residues and pathogen resistance, new fungicides with novel modes of action are urgently needed. SYP-14288 is a novel fungicide that could efficiently promote respiration and inhibit ATP biosynthesis in target organisms, but its bioactivity against various plant pathogens and exact mode of action are still unknown. In this study, we found that SYP-14288 is highly effective against 31 important plant pathogens belonging to a range of taxonomic groups. In addition, SYP-14288 has demonstrated excellent activity against all life stages of the important fungal plant pathogen Magnaporthe oryzae and is especially effective during the pathogen's high energy consumption stages. SYP-14288 showed good preventative control efficacy against pepper blight and rice blast in the greenhouse and field, respectively. In an untargeted metabolomics assay designed to determine the exact mode of action of SYP-14288, significant changes occurred in 25 metabolites, with the accumulation of seven fatty acid metabolites and a decrease in 18 starch and sugar metabolites (e.g., from the tricarboxylic acid cycle). This suggests that SYP-14288 is an uncoupling agent similar to 2,4-dinitrophenol, which can allow for accumulation of various fatty acids after destroying oxidative phosphorylation coupling, thereby inhibiting the growth of the phytopathogen. These results indicate that the novel uncoupler SYP-14288 is a promising agrochemical in plant disease management.

Effect of acute nitrite infusion on contractile economy and metabolism in isolated skeletal muscle in situ during hypoxia.

Increased plasma nitrite concentrations may have beneficial effects on skeletal muscle function. The physiological basis explaining these observations has not been clearly defined and it may involve positive effects on muscle contraction force, microvascular O2 delivery and skeletal muscle oxidative metabolism. In the isolated canine gastrocnemius model, we evaluated the effects of acute nitrite infusion on muscle force and skeletal muscle oxidative metabolism. Under hypoxic conditions, but in the presence of normal convective O2 delivery, an elevated plasma nitrite concentration affects neither muscle force, nor muscle contractile economy. In accordance with previous results suggesting limited or no effects of nitrate/nitrite administrations in highly oxidative and highly perfused muscle, our data suggest that neither mitochondrial respiration, nor muscle force generation are affected by acute increased concentrations of NO precursors in hypoxia. Contrasting findings have been reported concerning the effects of augmented nitric oxide (NO) on skeletal muscle force production and oxygen consumption ( ). The present study examined skeletal muscle mitochondrial respiration and contractile economy in an isolated muscle preparation during hypoxia (but normal convective O2 delivery) with nitrite infusion. Isolated canine gastrocnemius muscles in situ (n=8) were studied during 3min of electrically stimulated isometric tetanic contractions corresponding to ∼35% of . During contractions, sodium nitrite (NITRITE) or sodium chloride (SALINE) was infused into the popliteal artery. was calculated from the Fick principle. Experiments were carried out in hypoxia ( =0.12), whereas convective O2 delivery was maintained at normal levels under both conditions by pump-driven blood flow ( ). Muscle biopsies were taken and mitochondrial respiration was evaluated by respirometry. Nitrite infusion significantly increased both nitrite and nitrate concentrations in plasma. No differences in force were observed between conditions. was not significantly different between NITRITE (6.1±1.8mL 100g-1 min-1 ) and SALINE (6.2±1.8mL100g-1 min-1 ), even after being 'normalized' per unit of developed force (muscle contractile economy). No differences between conditions were found for maximal ADP-stimulated mitochondrial respiration (both for complex I and complex II), leak respiration and oxidative phosphorylation coupling. In conclusion, in the absence of changes in convective O2 delivery, muscle force, muscle contractile economy and mitochondrial respiration were not affected by acute infusion of nitrite. The previously reported positive effects of elevated plasma nitrite concentrations are presumably mediated by the increased microvascular O2 availability.

Mitochondrial Function is Impaired in Heart Transplant Patients with Cardiac Allograft Vasculopathy

Cardiac allograft vasculopathy (CAV) remains the Achilles' heel of long-term survival of heart transplanted (HTx) patients. CAV affects approximately 50 % of HTx patients 10 years after transplant. We evaluated whether cardiac allograft vasculopathy is associated with mitochondrial dysfunction. A total of 36 HTx patients (18 patients with CAV and 18 patients without CAV) ≥ 12 months after HTx were enrolled. Endomyocardial biopsies were analyzed using high-resolution respirometry for glucose-coupled mitochondrial respiration. Echocardiography and coronary angiography were used to grade CAV according to ISHLT nomenclature. Maximally coupled respiration of mitochondrial complex I and II was significantly reduced in patients with CAV compared with patients without CAV (97.2 pmol O2/(s*mg) [65.5; 133.6] vs. 59.1 pmol O2/(s*mg) [53.9; 83.0], p = 0.009) (Fig. 1). Patients with positive donor specific antibodies (MFI > 3000) had a lower maximally coupled respiration compared with patients without (56.5 pmol O2/(s*mg) [53.3; 69.0] vs. 90.6 pmol O2/(s*mg) [59.8; 113.9], p = 0.009). Mitochondrial respiratory function measured as oxidative phosphorylation coupling efficiency was positively associated with both left ventricular function (global longitudinal strain, r2=0.239, p = 0.0045) and right ventricular function (tricuspid annular plane systolic excursion, r2=0.133, p=0.03). Mitochondrial respiration is impaired in patients with CAV compared with patients without CAV. Decreased mitochondrial respiratory function is associated with impaired myocardial contractile function.

Powerhouses in the cold: mitochondrial function during thermal acclimation in montane mayflies.

Mitochondria provide the vast majority of cellular energy available to eukaryotes. Therefore, adjustments in mitochondrial function through genetic changes in mitochondrial or nuclear-encoded genes might underlie environmental adaptation. Environmentally induced plasticity in mitochondrial function is also common, especially in response to thermal acclimation in aquatic systems. Here, we examined mitochondrial function in mayfly larvae (Baetis and Drunella spp.) from high and low elevation mountain streams during thermal acclimation to ecologically relevant temperatures. A multi-substrate titration protocol was used to evaluate different respiratory states in isolated mitochondria, along with cytochrome oxidase and citrate synthase activities. In general, maximal mitochondrial respiratory capacity and oxidative phosphorylation coupling efficiency decreased during acclimation to higher temperatures, suggesting montane insects may be especially vulnerable to rapid climate change. Consistent with predictions of the climate variability hypothesis, mitochondria from Baetis collected at a low elevation site with highly variable daily and seasonal temperatures exhibited greater thermal tolerance than Baetis from a high elevation site with comparatively stable temperatures. However, mitochondrial phenotypes were more resilient than whole-organism phenotypes in the face of thermal stress. These results highlight the complex relationships between mitochondrial and organismal genotypes, phenotypes and environmental adaptation. This article is part of the theme issue 'Linking the mitochondrial genotype to phenotype: a complex endeavour'.

Tissue-specific dysregulation of mitochondrial respiratory capacity and coupling control in colon-26 tumor-induced cachexia.

In addition to skeletal muscle dysfunction, cancer cachexia is a systemic disease involving remodeling of nonmuscle organs such as adipose and liver. Impairment of mitochondrial function is associated with multiple chronic diseases. The tissue-specific control of mitochondrial function in cancer cachexia is not well defined. This study determined mitochondrial respiratory capacity and coupling control of skeletal muscle, white adipose tissue (WAT), and liver in colon-26 (C26) tumor-induced cachexia. Tissues were collected from PBS-injected weight-stable mice, C26 weight-stable mice and C26 mice with moderate (10% weight loss) and severe cachexia (20% weight loss). The respiratory control ratio [(RCR) an index of oxidative phosphorylation (OXPHOS) coupling efficiency] was low in WAT during the induction of cachexia because of high nonphosphorylating LEAK respiration. Liver RCR was low in C26 weight-stable and moderately cachexic mice because of reduced OXPHOS. Liver RCR was further reduced with severe cachexia, where Ant2 but not Ucp2 expression was increased. Ant2 was inversely correlated with RCR in the liver (r = -0.547, P < 0.01). Liver cardiolipin increased in moderate and severe cachexia, suggesting this early event may also contribute to mitochondrial uncoupling. Impaired skeletal muscle mitochondrial respiration occurred predominantly in severe cachexia, at complex I. These findings suggest that mitochondrial function is subject to tissue-specific control during cancer cachexia, whereby remodeling in WAT and liver arise early and may contribute to altered energy balance, followed by impaired skeletal muscle respiration. We highlight an under-recognized role of liver and WAT mitochondrial function in cancer cachexia and suggest mitochondrial function of multiple tissues to be therapeutic targets.

Effect of hypothermia on the functional activity of liver mitochondria of grass snake (Natrix natrix): inhibition of succinate-fueled respiration and K+ transport, ROS-induced activation of mitochondrial permeability transition.

The article considers the comparative analysis of the functional activity of mitochondria isolated from the liver of grass snakes, Natrix natrix (Linnaeus, 1758) that were kept at different temperatures (23-26°C and 4-5°C). It was found that liver mitochondria of hypothermia-exposed grass snakes are characterized by weak coupling of oxidative phosphorylation as compared to mitochondria of active animals which is caused by inhibition of succinate-fuelled respiration in ADP-stimulated state, as well as by activation of basal non-phosphorylating rate. Inhibition of mitochondrial respiration in hibernating animals is associated with a decrease in the activity of the respiratory chain complexes of organelles. A significant decrease in the rate of K+ transport in the liver mitochondria of hibernating animals has been established. Under these conditions, a decrease in the calcium capacity of the organelles was also revealed, which indicates a decrease in the resistance of the mitochondria of hibernating animals to the induction of the Ca2+-dependent mitochondrial pore. All these changes in the functional activity of mitochondria are observed on the background of increasing H2O2 production as well as increasing the proportion of polyunsaturated fatty acids in phospholipid composition of mitochondrial membranes, which are the targets of reactive oxygen species. It can lead to increased formation of lipid peroxides and activation of destructive processes associated with the induction of Ca2+-dependent mitochondrial pore.

Pancreatic β-cells detoxify H2O2 through the peroxiredoxin/thioredoxin antioxidant system

Oxidative stress is thought to promote pancreatic β-cell dysfunction and contribute to both type 1 and type 2 diabetes. Reactive oxygen species (ROS), such as superoxide and hydrogen peroxide, are mediators of oxidative stress that arise largely from electron leakage during oxidative phosphorylation. Reports that β-cells express low levels of antioxidant enzymes, including catalase and GSH peroxidases, have supported a model in which β-cells are ill-equipped to detoxify ROS. This hypothesis seems at odds with the essential role of β-cells in the control of metabolic homeostasis and organismal survival through exquisite coupling of oxidative phosphorylation, a prominent ROS-producing pathway, to insulin secretion. Using glucose oxidase to deliver H2O2 continuously over time and Amplex Red to measure extracellular H2O2 concentration, we found here that β-cells can remove micromolar levels of this oxidant. This detoxification pathway utilizes the peroxiredoxin/thioredoxin antioxidant system, as selective chemical inhibition or siRNA-mediated depletion of thioredoxin reductase sensitized β-cells to continuously generated H2O2 In contrast, when delivered as a bolus, H2O2 induced the DNA damage response, depleted cellular energy stores, and decreased β-cell viability independently of thioredoxin reductase inhibition. These findings show that β-cells have the capacity to detoxify micromolar levels of H2O2 through a thioredoxin reductase-dependent mechanism and are not as sensitive to oxidative damage as previously thought.

Improving mitochondrial function with SS-31 reverses age-related redox stress and improves exercise tolerance in aged mice

Sarcopenia and exercise intolerance are major contributors to reduced quality of life in the elderly for which there are few effective treatments. We tested whether enhancing mitochondrial function and reducing mitochondrial oxidant production with SS-31 (elamipretide) could restore redox balance and improve skeletal muscle function in aged mice. Young (5 mo) and aged (26 mo) female C57BL/6Nia mice were treated for 8-weeks with 3 mg/kg/day SS-31. Mitochondrial function was assessed in vivo using 31P and optical spectroscopy. SS-31 reversed age-related decline in maximum mitochondrial ATP production (ATPmax) and coupling of oxidative phosphorylation (P/O). Despite the increased in vivo mitochondrial capacity, mitochondrial protein expression was either unchanged or reduced in the treated aged mice and respiration in permeabilized gastrocnemius (GAS) fibers was not different between the aged and aged+SS-31 mice. Treatment with SS-31 also restored redox homeostasis in the aged skeletal muscle. The glutathione redox status was more reduced and thiol redox proteomics indicated a robust reversal of cysteine S-glutathionylation post-translational modifications across the skeletal muscle proteome. The gastrocnemius in the age+SS-31 mice was more fatigue resistant with significantly greater mass compared to aged controls. This contributed to a significant increase in treadmill endurance compared to both pretreatment and untreated control values. These results demonstrate that the shift of redox homeostasis due to mitochondrial oxidant production in aged muscle is a key factor in energetic defects and exercise intolerance. Treatment with SS-31 restores redox homeostasis, improves mitochondrial quality, and increases exercise tolerance without an increase in mitochondrial content. Since elamipretide is currently in clinical trials these results indicate it may have direct translational value for improving exercise tolerance and quality of life in the elderly.

214 - The “not-so-vulnerable” β-cell

Oxidative stress, mediated by reactive oxygen species (ROS) like superoxide and H2O2, is thought to promote pancreatic β-cell dysfunction and contribute to type 1 and type 2 diabetes. When blood glucose is chronically elevated, it is thought that increased oxidative phosphorylation results in accumulation of ROS in β-cells due to electron leak from the mitochondrial electron transport chain. However, early reports that β-cells express low levels of detoxifying enzymes, including catalase and glutathione peroxidases, suggested that β-cells are ill-equipped to detoxify ROS. This notion has been propagated by the common use of bolus H2O2 delivery to cells. However, the logic supporting the “vulnerable β-cell” model is unclear when considering the importance of β-cells to survival of the organism as well as the tight coupling of oxidative phosphorylation to insulin secretion. Here, using glucose oxidase to deliver H2O2 continuously over time, rather than the more traditional bolus delivery, we found that β-cells are capable of detoxifying micromolar levels of this oxidant. Treatment with H2O2 bolus, but not glucose oxidase, results in DNA damage, activation of the DNA damage response, and depletion of cellular energy stores. We find that β-cells readily express peroxiredoxins, thioredoxins, and thioredoxin reductase, essential components of the peroxiredoxin/thioredoxin antioxidant system. Either inhibition or specific knockdown of thioredoxin reductase or peroxiredoxins 1 or 3 sensitizes β-cells to continuously-generated H2O2, suggesting that this mechanism is essential for β-cells to detoxify this ROS. Finally, inhibition of thioredoxin reductase sensitizes β-cells to peroxynitrite, a potent RNS generated by the reaction of superoxide and nitric oxide. Together, these studies, which directly contradict the current dogma, suggest that β-cells are able to detoxify H2O2 and peroxynitrite through a peroxiredoxin-dependent mechanism, and may not be so vulnerable after all.