ADP-stimulated Respiration Research Articles

Mitochondrial Sensitivity To Submaximal ADP Concentrations Following Bed Rest: A Double Exponential Model Analysis

We recently demonstrated that a 10-day exposure to inactivity/microgravity (bed rest, BR) determined impairments of peak oxygen uptake (V̇O2), peak cardiac output, peripheral and microvascular function, whereas maximal ADP-stimulated mitochondrial respiration ex vivo in isolated permeabilized fibers and muscle V̇O2 off-kinetics in vivo were unaffected. PURPOSE: To evaluate mitochondrial sensitivity to submaximal [ADP]. METHODS: Isolated permeabilized vastus lateralis fibers were analyzed by high-resolution respirometry, before (PRE) and after (POST) a 10-day BR in 10 young males. 11 submaximal titrations of ADP (from 12.5 to 10,000 μM) were utilized to assess complex I + II-linked ADP sensitivity which was analyzed by both a Michaelis-Menten (MM) kinetics equation, with the calculation of the apparent Km by standard methods, and by applying a double-exponential function, with the calculation of [ADP] corresponding to 50% of the maximal mitochondrial respiration ([ADP] at 50% JO2max). RESULTS: The double-exponential function provided a better fitting of the data according to AIC model selection. The sum of squared residuals was substantially lower with the double-exponential vs. MM fitting (411 ± 419 and 1209 ± 495 [pmol/s/mg]2, respectively). R2 was higher with the double-exponential vs. the MM fitting (0.97 ± 0.04 and 0.90 ± 0.05, respectively). Analysis of residuals showed that the fitting by MM markedly overestimated JO2 from 1000 to 2000 μM, and underestimated JO2 from 6000 to 10,000 μM. The apparent Km for MM was lower than the [ADP] at 50% JO2max, both in PRE (843 ± 706 and 1270 ± 1015 μM, respectively) and in POST (375 ± 173 and 409 ± 398 μM, respectively). Only for the [ADP] at 50% JO2max the difference between POST vs. PRE was statistically significant (P = 0.046). CONCLUSIONS: The double-exponential function provided a better fit of experimental data. It might also give insights into cellular mechanisms regulating mitochondrial respiration, possibly related to the transport of nucleotides and phosphate across external and internal mitochondrial membranes, ATP synthase activity and respiratory chain activity. The enhanced mitochondrial sensitivity to submaximal [ADP] after BR could represent an attempt to maintain metabolic homeostasis in the presence of impairments upstream of mitochondria.

Reverse and Forward Electron Flow-Induced H2O2 Formation Is Decreased in α-Ketoglutarate Dehydrogenase (α-KGDH) Subunit (E2 or E3) Heterozygote Knock Out Animals.

α-ketoglutarate dehydrogenase complex (KGDHc), or 2-oxoglutarate dehydrogenase complex (OGDHc) is a rate-limiting enzyme in the tricarboxylic acid cycle, that has been identified in neurodegenerative diseases such as in Alzheimer’s disease. The aim of the present study was to establish the role of the KGDHc and its subunits in the bioenergetics and reactive oxygen species (ROS) homeostasis of brain mitochondria. To study the bioenergetic profile of KGDHc, genetically modified mouse strains were used having a heterozygous knock out (KO) either in the dihydrolipoyl succinyltransferase (DLST+/−) or in the dihydrolipoyl dehydrogenase (DLD+/−) subunit. Mitochondrial oxygen consumption, hydrogen peroxide (H2O2) production, and expression of antioxidant enzymes were measured in isolated mouse brain mitochondria. Here, we demonstrate that the ADP-stimulated respiration of mitochondria was partially arrested in the transgenic animals when utilizing α-ketoglutarate (α-KG or 2-OG) as a fuel substrate. Succinate and α-glycerophosphate (α-GP), however, did not show this effect. The H2O2 production in mitochondria energized with α-KG was decreased after inhibiting the adenine nucleotide translocase and Complex I (CI) in the transgenic strains compared to the controls. Similarly, the reverse electron transfer (RET)-evoked H2O2 formation supported by succinate or α-GP were inhibited in mitochondria isolated from the transgenic animals. The decrease of RET-evoked ROS production by DLST+/− or DLD+/− KO-s puts the emphasis of the KGDHc in the pathomechanism of ischemia-reperfusion evoked oxidative stress. Supporting this notion, expression of the antioxidant enzyme glutathione peroxidase was also decreased in the KGDHc transgenic animals suggesting the attenuation of ROS-producing characteristics of KGDHc. These findings confirm the contribution of the KGDHc to the mitochondrial ROS production and in the pathomechanism of ischemia-reperfusion injury.

Dysregulated Mitochondrial Respiration Occurs With Aging

Numerous metabolic changes occur in skeletal muscle with aging. At the crossroads of these changes lies the cellular energy highway, the mitochondrial reticulum. Aging is associated with altered patterns of mitochondrial dynamics and morphology, which are thought to be primarily driven by mitochondrial fragmentation. This fragmentation phenomenon may also lead to altered mitochondrial respiration with aging. We used a mouse model and measured parameters of respiration in muscle mitochondrial preparations to evaluate the hypothesis of aging‐related effects on metabolism and energy substrate partitioning. Mixed skeletal muscle of C57/Bl6 mice (n=32) was excised from single hind‐limbs of young males (YM) (n=8), old males (OM) (n=8), young females (YF) (n=8) and old females (OF) (n=8). Tissues were homogenized in an isotonic solution supplemented with 0.15% protease inhibitor. Mitochondria were isolated by differential centrifugation and their concentrations were determined by BCA analysis. Isolated mitochondria (0.1mg of mitochondria/mL of respiration medium) were then respired using a Clarke‐Type Electrode at 25°C with 100 nM of ADP and either 10 mM Pyruvate+ 2.5 mM Malate (P+M) or 40 uM Palmitoyl‐L‐Carnitine+ 1 mM Malate (PC+M) as substrates. Respiratory Control Ratios (RCR) were calculated as the quotient of State 3 (ADP‐stimulated respiration) and State 4 (recovery rate post ATP synthesis) respiratory rates (State 3/State 4) and compared between substrate and age. ADP to oxygen ratios (P:O) were calculated by dividing the amount of ADP added to the respiratory chamber by the oxygen consumption rate (nmol of O2/mg of mitochondria/minute) of state 3 respiration. RCR in both P+M and PC+M significantly decreased with aging [P+M (p<0.05): YM (4.1±0.9) vs. OM (2.7±1.1); YF (5.1±1.4) vs. OF (3.2±2)] [PC+M (p<0.05): YM (4.2±0.9) vs. OM (2.4±0.7); YF (3.4.±0.5) vs. OF (2.2±0.9)]. P:O in both P+M and PC+M did not significantly change with aging [P+M: (p=0.69): YM (2.9±0.3) vs. OM (2.9±0.2); YF: (p=0.07): (3±0.1) vs. OF (2.9±0.1)] [PC+M:(p=0.65); YM (2.9±0.4) vs. OM (2.9±0.3); (p=0.22): YF (2.9±0.2) vs. OF (2.7±0.3)]. While there were no significant changes in the P:O the age related decreases in the RCR were characterized by a lower capacity to respire derivatives of both CHO and FA. Decreased State 3 respiration and RCR typically corresponds to loss of respiratory capacity and loosening of coupling efficiency. Because exercise increases mitochondrial function and biogenesis it may be prudent to study older animals after exercise training. We plan to further investigate the structural changes in the mitochondrial reticulum and proteome that may be altering mitochondrial function in aging.

NMR Spectroscopy Identifies Chemicals in Cigarette Smoke Condensate That Impair Skeletal Muscle Mitochondrial Function.

Tobacco smoke-related diseases such as chronic obstructive pulmonary disease (COPD) are associated with high healthcare burden and mortality rates. Many COPD patients were reported to have muscle atrophy and weakness, with several studies suggesting intrinsic muscle mitochondrial impairment as a possible driver of this phenotype. Whereas much information has been learned about muscle pathology once a patient has COPD, little is known about how active tobacco smoking might impact skeletal muscle physiology or mitochondrial health. In this study, we examined the acute effects of cigarette smoke condensate (CSC) on muscle mitochondrial function and hypothesized that toxic chemicals present in CSC would impair mitochondrial respiratory function. Consistent with this hypothesis, we found that acute exposure of muscle mitochondria to CSC caused a dose-dependent decrease in skeletal muscle mitochondrial respiratory capacity. Next, we applied an analytical nuclear magnetic resonance (NMR)-based approach to identify 49 water-soluble and 12 lipid-soluble chemicals with high abundance in CSC. By using a chemical screening approach in the Seahorse XF96 analyzer, several CSC-chemicals, including nicotine, o-Cresol, phenylacetate, and decanoic acid, were found to impair ADP-stimulated respiration in murine muscle mitochondrial isolates significantly. Further to this, several chemicals, including nicotine, o-Cresol, quinoline, propylene glycol, myo-inositol, nitrosodimethylamine, niacinamide, decanoic acid, acrylonitrile, 2-naphthylamine, and arsenic acid, were found to significantly decrease the acceptor control ratio, an index of mitochondrial coupling efficiency.

S-15176 Difumarate Salt Can Impair Mitochondrial Function through Inhibition of the Respiratory Complex III and Permeabilization of the Inner Mitochondrial Membrane.

Simple SummaryThe metabolic agent S-15176 difumarate salt belongs to a potent class of drugs called partial fatty acid oxidation inhibitors. The possible therapeutic effects of this agent have been widely investigated in models of oxidative damage to different tissues, but the underlying mechanisms are currently unclear and require further research. In the present study, we aimed to investigate in more detail the effect of S-15176 on the mitochondria, power stations inside the cell, and pivotal regulators of many cellular processes. We found that S-15176 affected the key indicators of the function of rat liver and thymocyte mitochondria at the concentrations reached in target tissues. S-15176 disturbed the catalytic function of enzyme complex III of the mitochondrial electron transfer system and induced nonspecific membrane permeability, which was related to the dissipation of the mitochondrial membrane potential and a decline in ATP production. These findings lead us to predict that S-15176 can induce mitochondrial dysfunction in mammal tissues and organs that are most vulnerable to chemical toxicity or exposed to higher concentrations of the drug.S-15176 difumarate salt, a derivative of the anti-ischemic metabolic drug trimetazidine, has been intensively studied for its impact on cellular metabolism in animal models of ischemia-reperfusion injury of the liver, heart, spinal cord, and other organs. Despite evidence of some reduction in oxidative damage to cells, the results of therapy with S-15176 have been mostly disappointing, possibly because of the lack of data on its underlying mechanisms. Here, we aimed to investigate in more detail the role of complexes I-IV of the electron transport chain and membrane permeability transition in mitochondrial toxicity associated with S-15176. Using rat thymocyte and liver mitochondria, we demonstrated that: (1) acute exposure to S-15176 (10 to 50 μM) dose-dependently decreased the mitochondrial membrane potential; (2) S-15176 suppressed the ADP-stimulated (State 3) and uncoupled (State 3UDNP) respiration of mitochondria energized with succinate or malate/glutamate, but not ascorbate/TMPD, and increased the resting respiration (State 4) when using all the substrate combinations; (3) S-15176 directly inhibited the activity of the respiratory complex III; (4) low doses of S-15176 diminished the rate of H2O2 production by mitochondria; (5) at concentrations of above 30 μM, S-15176 reduced calcium retention capacity and contributed to mitochondrial membrane permeabilization. Taken together, these findings suggest that S-15176 at tissue concentrations reached in animals can impair mitochondrial function through suppression of the cytochrome bc1 complex and an increase in the nonspecific membrane permeability.

Oxyhaemoglobin Level Measured Using Near-Infrared Spectrometer Is Associated with Brain Mitochondrial Dysfunction After Cardiac Arrest in Rats.

Cerebral blood oxygenation (CBO), measured using near-infrared spectroscopy (NIRS), can play an important role in post-cardiac arrest (CA) care as this emerging technology allows for noninvasive real-time monitoring of the dynamic changes of tissue oxygenation. We recently reported that oxyhaemoglobin (oxy-Hb), measured using NIRS, may be used to evaluate the quality of chest compressions by monitoring the brain tissue oxygenation, which is a critical component for successful resuscitation. Mitochondria are the key to understanding the pathophysiology of post-CA oxygen metabolism. In this study, we focused on mitochondrial dysfunction, aiming to explore its association with CBO parameters such as oxy-Hb and deoxyhaemoglobin (deoxy-Hb) or tissue oxygenation index (TOI). Male Sprague-Dawley rats were used in the study. We applied NIRS between the nasion and the upper cervical spine. Following 10 min of CA, the rats underwent cardiopulmonary resuscitation (CPR) with a bolus injection of 20μg/kg epinephrine. At 10 and 20 min after CPR, brain, and kidney tissues were collected. We isolated mitochondria from these tissues and evaluated the association between CBO and mitochondrial oxygen consumption ratios. There were no significant differences in the mitochondrial yields (10 vs. 20 min after resuscitation: brain, 1.33±0.68 vs. 1.30±0.75mg/g; kidney, 19.5±3.2 vs. 16.9±5.3mg/g, respectively). State 3 mitochondrial oxygen consumption rates, known as ADP-stimulated respiration, demonstrated a significant difference at 10 vs. 20 min after CPR (brain, 170±26 vs. 115±17nmol/min/mg protein; kidney, 170±20 vs. 130±16nmol/min/mg protein, respectively), whereas there was no significant difference in ADP non-dependent state 4 oxygen consumption rates (brain, 34.0±6.7 vs. 31.8±10nmol/min/mg protein; kidney, 29.8±4.8 vs. 21.0±2.6nmol/min/mg protein, respectively). Consequently, the respiratory control ratio (RCR=state 3/state 4) showed a significant difference over time, but this was only noted in the brain (brain, 5.0±0.29 vs. 3.8±0.64; kidney, 5.8±0.53 vs. 6.2±0.25nmol/min/mg protein, respectively). The oxy-Hb levels had a dynamic change after resuscitation, and they had a significant association with the RCR of the brain mitochondria (r=0.8311, p=0.0102), whereas deoxy-Hb and TOI did not (r=-0.1252, p=0.7677; r=0.4186, p=0.302, respectively). The RCRs of the kidney mitochondria did not have a significant association with CBO (oxy-Hb, r=-0.1087, p=0.7977; deoxy-Hb, r=0.1565, p=0.7113; TOI, r=-0.1687, p=0.6896, respectively). The brain mitochondrial respiratory dysfunction occurred over time, and it was seen at the time points between 10 and 20 min after CPR. The oxy-Hb level was associated with brain mitochondrial dysfunction during the early post-resuscitation period.

NME6 is a phosphotransfer-inactive, monomeric NME/NDPK family member and functions in complexes at the interface of mitochondrial inner membrane and matrix

BackgroundNME6 is a member of the nucleoside diphosphate kinase (NDPK/NME/Nm23) family which has key roles in nucleotide homeostasis, signal transduction, membrane remodeling and metastasis suppression. The well-studied NME1-NME4 proteins are hexameric and catalyze, via a phospho-histidine intermediate, the transfer of the terminal phosphate from (d)NTPs to (d)NDPs (NDP kinase) or proteins (protein histidine kinase). For the NME6, a gene/protein that emerged early in eukaryotic evolution, only scarce and partially inconsistent data are available. Here we aim to clarify and extend our knowledge on the human NME6.ResultsWe show that NME6 is mostly expressed as a 186 amino acid protein, but that a second albeit much less abundant isoform exists. The recombinant NME6 remains monomeric, and does not assemble into homo-oligomers or hetero-oligomers with NME1-NME4. Consequently, NME6 is unable to catalyze phosphotransfer: it does not generate the phospho-histidine intermediate, and no NDPK activity can be detected. In cells, we could resolve and extend existing contradictory reports by localizing NME6 within mitochondria, largely associated with the mitochondrial inner membrane and matrix space. Overexpressing NME6 reduces ADP-stimulated mitochondrial respiration and complex III abundance, thus linking NME6 to dysfunctional oxidative phosphorylation. However, it did not alter mitochondrial membrane potential, mass, or network characteristics. Our screen for NME6 protein partners revealed its association with NME4 and OPA1, but a direct interaction was observed only with RCC1L, a protein involved in mitochondrial ribosome assembly and mitochondrial translation, and identified as essential for oxidative phosphorylation.ConclusionsNME6, RCC1L and mitoribosomes localize together at the inner membrane/matrix space where NME6, in concert with RCC1L, may be involved in regulation of the mitochondrial translation of essential oxidative phosphorylation subunits. Our findings suggest new functions for NME6, independent of the classical phosphotransfer activity associated with NME proteins.

Fission accomplished: Uncovering the role of Drp1 in regulating mitochondrial dysfunction and age-related muscle atrophy.

Fission accomplished: Uncovering the role of Drp1 in regulating mitochondrial dysfunction and age-related muscle atrophy.

Peripheral impairments of oxidative metabolism after a 10-day bed rest are upstream of mitochondrial respiration.

In order to identify peripheral biomarkers of impaired oxidative metabolism during exercise following a 10‐day bed rest, 10 males performed an incremental exercise (to determine peak pulmonary V̇O2 (V̇O2 p)) and moderate‐intensity exercises, before (PRE) and after (POST) bed rest. Blood flow response was evaluated in the common femoral artery by Eco‐Doppler during 1 min of passive leg movements (PLM). The intramuscular matching between O2 delivery and O2 utilization was evaluated by near‐infrared spectroscopy (NIRS). Mitochondrial respiration was evaluated ex vivo by high‐resolution respirometry in isolated muscle fibres, and in vivo by NIRS by the evaluation of skeletal muscle V̇O2 (V̇O2 m) recovery kinetics. Resting V̇O2 m was estimated by NIRS. Peak V̇O2 p was lower in POST vs. PRE. The area under the blood flow vs. time curve during PLM was smaller (P = 0.03) in POST (274 ± 233 mL) vs. PRE (427 ± 291). An increased (P = 0.03) overshoot of muscle deoxygenation during a metabolic transition was identified in POST. Skeletal muscle citrate synthase activity was not different (P = 0.11) in POST (131 ± 16 nmol min–1 mg–1) vs. PRE (138 ± 19). Maximal ADP‐stimulated mitochondrial respiration (66 ± 18 pmol s–1 mg–1 (POST) vs. 72 ± 14 (PRE), P = 0.41) was not affected by bed rest. Apparent K m for ADP sensitivity of mitochondrial respiration was reduced in POST vs. PRE (P = 0.04). The V̇O2 m recovery time constant was not different (P = 0.79) in POST (22 ± 6 s) vs. PRE (22 ± 6). Resting V̇O2 m was reduced by 25% in POST vs. PRE (P = 0.006). Microvascular‐endothelial function was impaired following a 10‐day bed rest, whereas mitochondrial mass and function (both in vivo and ex vivo) were unaffected or slightly enhanced.Key points Ten days of horizontal bed rest impaired in vivo oxidative function during exercise.Microvascular impairments were identified by different methods.Mitochondrial mass and mitochondrial function (evaluated both in vivo and ex vivo) were unchanged or even improved (i.e. enhanced mitochondrial sensitivity to submaximal [ADP]).Resting muscle oxygen uptake was significantly lower following bed rest, suggesting that muscle catabolic processes induced by bed rest/inactivity are less energy‐consuming than anabolic ones.

Alisporivir Treatment Alleviates Mitochondrial Dysfunction in the Skeletal Muscles of C57BL/6NCrl Mice with High-Fat Diet/Streptozotocin-Induced Diabetes Mellitus.

Diabetes mellitus is a systemic metabolic disorder associated with mitochondrial dysfunction, with mitochondrial permeability transition (MPT) pore opening being recognized as one of its pathogenic mechanisms. Alisporivir has been recently identified as a non-immunosuppressive analogue of the MPT pore blocker cyclosporin A and has broad therapeutic potential. The purpose of the present work was to study the effect of alisporivir (2.5 mg/kg/day i.p.) on the ultrastructure and functions of the skeletal muscle mitochondria of mice with diabetes mellitus induced by a high-fat diet combined with streptozotocin injections. The glucose tolerance tests indicated that alisporivir increased the rate of glucose utilization in diabetic mice. An electron microscopy analysis showed that alisporivir prevented diabetes-induced changes in the ultrastructure and content of the mitochondria in myocytes. In diabetes, the ADP-stimulated respiration, respiratory control, and ADP/O ratios and the level of ATP synthase in the mitochondria decreased, whereas alisporivir treatment restored these indicators. Alisporivir eliminated diabetes-induced increases in mitochondrial lipid peroxidation products. Diabetic mice showed decreased mRNA levels of Atp5f1a, Ant1, and Ppif and increased levels of Ant2 in the skeletal muscles. The skeletal muscle mitochondria of diabetic animals were sensitized to the MPT pore opening. Alisporivir normalized the expression level of Ant2 and mitochondrial susceptibility to the MPT pore opening. In parallel, the levels of Mfn2 and Drp1 also returned to control values, suggesting a normalization of mitochondrial dynamics. These findings suggest that the targeting of the MPT pore opening by alisporivir is a therapeutic approach to prevent the development of mitochondrial dysfunction and associated oxidative stress in the skeletal muscles in diabetes.

Short-term Bed Rest Exposure Impairs Peripheral Vascular And Endothelial Functions Whereas Mitochondrial Respiration Is Unaffected

PURPOSE: Impairments of oxidative metabolism after exposure to microgravity are documented, but the sites of impairment are still debated. Aim of this study was to identify markers of impaired oxidative metabolism along the O2 pathway, following 10-day of horizontal bed rest (BR). METHODS: fore (PRE) and after (POST) BR, 10 young males performed on a cycle ergometer an incremental exercise (to determine peak pulmonary V̇O2 [V̇O2p] and peak cardiac output [CO]), and moderate-intensity exercise (MOD). Vastus lateralis muscle fractional O2 extraction was measured by near-infrared spectroscopy (NIRS). Blood flow response was evaluated in the common femoral artery by Eco-Doppler during 1-min passive leg movements (PLM). Mitochondrial respiration was evaluated ex vivo by high-resolution respirometry in isolated muscle fibers obtained by biopsy, and non-invasively in vivo by NIRS, by calculating skeletal muscle V̇O2 (V̇O2m) recovery kinetics (repeated occlusions method) following MOD. Resting V̇O2m was estimated by NIRS by the rate of muscle oxygenation during limb blood flow occlusions at rest. RESULTS: Peak V̇O2p and peak cardiac output were lower in POST vs. PRE (41.5 ± 6.5 ml/kg/min vs. 44.5 ± 7.4, P = 0.001; 19.0 ± 3.2 L/min vs. 25.2 ± 5.5, P = 0.004). 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, estimate of mitochondrial mass, was not different (P = 0.115) in POST (131.2 ± 15.9 UI/mg) vs. PRE (137.9 ± 18.8). Maximal ADP-stimulated mitochondrial respiration (66.4 ± 17.5 pmol/s/mg [POST] vs. 72.3 ± 14.0 [PRE], P = 0.127) was not affected by BR. Apparent Km for ADP sensitivity was reduced in POST vs. PRE (P = 0.04). The time-constant of V̇O2m recovery was not different (P = 0.079) in POST (22.2 ± 5.9 s) vs. PRE (21.7 ± 5.7). V̇O2m at rest was reduced by about 25% in POST vs. PRE (P < 0.01). CONCLUSIONS: Cardiovascular and peripheral vascular-endothelial functions were impaired following a 10-day BR, whereas mitochondrial mass and function (both in vivo and ex vivo) were unaffected, or even improved (enhanced mitochondrial sensitivity to submaximal [ADP]). Resting V̇O2m was lower after BR; muscle catabolic processes induced by BR are less energy-consuming than anabolic ones. Funding: ASI, MARS-PRE, n. DC-VUM-2017-006.

Effects of plant alkaloids on mitochondrial bioenergetic parameters

Mitochondria are among the first responders to various stress factors that challenge cell and tissue homeostasis. Various plant alkaloids have been investigated for their capacity to modulate mitochondrial activities. In this study, we used isolated mitochondria from mouse brain and liver tissues to assess nicotine, anatabine and anabasine, three alkaloids found in tobacco plant, for potential modulatory activity on mitochondrial bioenergetics parameters. All alkaloids decreased basal oxygen consumption of mouse brain mitochondria in a dose-dependent manner without any effect on the ADP-stimulated respiration. None of the alkaloids, at 1 nM or 1.25 μM concentrations, influenced the maximal rate of swelling of brain mitochondria. In contrast to brain mitochondria, 1.25 μM anatabine, anabasine and nicotine increased maximal rate of swelling of liver mitochondria suggesting a toxic effect. Only at 1 mM concentration, anatabine slowed down the maximal rate of Ca2+-induced swelling and increased the time needed to reach the maximal rate of swelling.The observed mitochondrial bioenergetic effects are probably mediated through a pathway independent of nicotinic acetylcholine receptors, as quantitative proteomic analysis could not confirm their expression in pure mitochondrial fractions isolated from mouse brain tissue.

Impact of presymptomatic COVID-19 on vascular and skeletal muscle function: a case study.

The impact of COVID-19 has been largely described after symptom development. Although the SARS-CoV-2 virus elevates heart rate (HR) prior to symptom onset, whether this virus evokes other presymptomatic alterations is unknown. This case study details the presymptomatic impact of COVID-19 on vascular and skeletal muscle function in a young woman [24 yr, 173.5 cm, 89 kg, body mass index (BMI): 29.6 kg·m–2]. Vascular and skeletal muscle function were assessed as part of a separate study with the first and second visits separated by 2 wk. On the evening following the second visit, the participant developed a fever and a rapid antigen test confirmed a positive COVID-19 diagnosis. Compared with the first visit, the participant presented with a markedly elevated HR (∼30 beats/min) and a lower mean blood pressure (∼8 mmHg) at the second visit. Vascular function measured by brachial artery flow-mediated dilation, reactive hyperemia, and passive leg movement were all noticeably attenuated (25%–65%) as was leg blood flow during knee extension exercise. Muscle strength was diminished as was ADP-stimulated respiration (30%), assessed in vitro, whereas there was a 25% increase in the apparent Km. Lastly, an elevation in IL-10 was observed prior to symptom onset. Notably, 2.5 mo after diagnosis symptoms of fatigue and cough were still present. Together, these findings provide unique insight into the physiological responses immediately prior to onset of COVID-19 symptoms; they suggest that SARS-CoV-2 negatively impacts vascular and skeletal muscle function prior to the onset of common symptoms and may set the stage for the widespread sequelae observed following COVID-19 diagnosis.NEW & NOTEWORTHY This unique case study details the impact of SARS-CoV-2 infection on vascular and skeletal muscle function in a young predominantly presymptomatic woman. Prior to COVID-19 diagnosis, substantial reductions in vascular, skeletal muscle, and mitochondrial function were observed along with an elevation in IL-10. This integrative case study indicates that the presymptomatic impact of COVID-19 is widespread and may help elucidate the acute and long-term sequelae of this disease.

The effect of DS16570511, a new inhibitor of mitochondrial calcium uniporter, on calcium homeostasis, metabolism, and functional state of cultured cortical neurons and isolated brain mitochondria

BackgroundDisorders of mitochondrial Ca2+ homeostasis play a key role in the glutamate excitotoxicity of brain neurons. DS16570511 (DS) is a new penetrating inhibitor of mitochondrial Ca2+ uniporter complex (MCUC). The paper examines the effects of DS on the cultivated cortical neurons and isolated mitochondria of the rat brain. MethodsThe functions of neurons and mitochondria were examined using fluorescence microscopy, XF24 microplate-based сell respirometry, ion-selective microelectrodes, spectrophotometry, and polarographic technique. ResultsAt the doses of 30 and 45 μM, DS reliably slowed down the onset of glutamate-induced delayed calcium deregulation of neurons and suppressed their death. 30 μM DS caused hyperpolarization of mitochondria of resting neurons, and 45 μM DS temporarily depolarized neuronal mitochondria. It was also demonstrated that 30–60 μM DS stimulated cellular respiration. DS was shown to suppress Ca2+ uptake by isolated brain mitochondria. In addition, DS inhibited ADP-stimulated mitochondrial respiration and ADP-induced decrease in the mitochondrial membrane potential. It was found that DS inhibited the activity of complex II of the respiratory chain. In the presence of Ca2+, high DS concentrations caused a collapse of the mitochondrial membrane potential. ConclusionsThe data obtained indicate that, in addition to the inhibition of MCUC, DS affects the main energy-transducing functions of mitochondria. General significanceThe using DS as a tool for studying MCUC and its functional role in neuronal cells should be done with care, bearing in mind multiple effects of DS, a proper evaluation of which would require multivariate analysis.

High-fat diet activates liver iPLA2γ generating eicosanoids that mediate metabolic stress

High-fat (HF) diet–induced obesity precipitates multiple metabolic disorders including insulin resistance, glucose intolerance, oxidative stress, and inflammation, resulting in the initiation of cell death programs. Previously, we demonstrated murine germline knockout of calcium-independent phospholipase A2γ (iPLA2γ) prevented HF diet–induced weight gain, attenuated insulin resistance, and decreased mitochondrial permeability transition pore (mPTP) opening leading to alterations in bioenergetics. To gain insight into the specific roles of hepatic iPLA2γ in mitochondrial function and cell death under metabolic stress, we generated a hepatocyte-specific iPLA2γ-knockout (HEPiPLA2γKO). Using this model, we compared the effects of an HF diet on wild-type versus HEPiPLA2γKO mice in eicosanoid production and mitochondrial bioenergetics. HEPiPLA2γKO mice exhibited higher glucose clearance rates than WT controls. Importantly, HF-diet induced the accumulation of 12-hydroxyeicosatetraenoic acid (12-HETE) in WT liver which was decreased in HEPiPLA2γKO. Furthermore, HF-feeding markedly increased Ca2+ sensitivity and resistance to ADP-mediated inhibition of mPTP opening in WT mice. In contrast, ablation of iPLA2γ prevented the HF-induced hypersensitivity of mPTP opening to calcium and maintained ADP-mediated resistance to mPTP opening. Respirometry revealed that ADP-stimulated mitochondrial respiration was significantly reduced by exogenous 12-HETE. Finally, HEPiPLA2γKO hepatocytes were resistant to calcium ionophore-induced lipoxygenase-mediated lactate dehydrogenase release. Collectively, these results demonstrate that an HF diet increases iPLA2γ-mediated hepatic 12-HETE production leading to mitochondrial dysfunction and hepatic cell death.

Skeletal muscle mitochondrial fragmentation and impaired bioenergetics from nutrient overload are prevented by carbon monoxide.

Nutrient excess increases skeletal muscle oxidant production and mitochondrial fragmentation that may result in impaired mitochondrial function, a hallmark of skeletal muscle insulin resistance. This led us to explore whether an endogenous gas molecule, carbon monoxide (CO), which is thought to prevent weight gain and metabolic dysfunction in mice consuming high-fat diets, alters mitochondrial morphology and respiration in C2C12 myoblasts exposed to high glucose (15.6 mM) and high fat (250 µM BSA-palmitate) (HGHF). Also, skeletal muscle mitochondrial morphology, distribution, respiration, and energy expenditure were examined in obese resistant (OR) and obese prone (OP) rats that consumed a high-fat and high-sucrose diet for 10 wk with or without intermittent low-dose inhaled CO and/or exercise training. In cells exposed to HGHF, superoxide production, mitochondrial membrane potential (ΔΨm), mitochondrial fission regulatory protein dynamin-related protein 1 (Drp1) and mitochondrial fragmentation increased, while mitochondrial respiratory capacity was reduced. CO decreased HGHF-induced superoxide production, Drp1 protein levels and mitochondrial fragmentation, maintained ΔΨm, and increased mitochondrial respiratory capacity. In comparison with lean OR rats, OP rats had smaller skeletal muscle mitochondria that contained disorganized cristae, a normal mitochondrial distribution, but reduced citrate synthase protein expression, normal respiratory responses, and a lower energy expenditure. The combination of inhaled CO and exercise produced the greatest effect on mitochondrial morphology, increasing ADP-stimulated respiration in the presence of pyruvate, and preventing a decline in resting energy expenditure. These data support a therapeutic role for CO and exercise in preserving mitochondrial morphology and respiration during metabolic overload.

Undesirable effects of chemical inhibitors of NAD(P)+ transhydrogenase on mitochondrial respiratory function

NAD(P)+ transhydrogenase (NNT) is located in the inner mitochondrial membrane and catalyzes a reversible hydride transfer between NAD(H) and NADP(H) that is coupled to proton translocation between the intermembrane space and mitochondrial matrix. NNT activity has an essential role in maintaining the NADPH supply for antioxidant defense and biosynthetic pathways. In the present report, we evaluated the effects of chemical compounds used as inhibitors of NNT over the last five decades, namely, 4-chloro-7-nitrobenzofurazan (NBD-Cl), N,N′-dicyclohexylcarbodiimide (DCC), palmitoyl-CoA, palmitoyl-l-carnitine, and rhein, on NNT activity and mitochondrial respiratory function. Concentrations of these compounds that partially inhibited the forward and reverse NNT reactions in detergent-solubilized mouse liver mitochondria significantly impaired mitochondrial respiratory function, as estimated by ADP-stimulated and nonphosphorylating respiration. Among the tested compounds, NBD-Cl showed the best relationship between NNT inhibition and low impact on respiratory function. Despite this, NBD-Cl concentrations that partially inhibited NNT activity impaired mitochondrial respiratory function and significantly decreased the viability of cultured Nnt−/− mouse astrocytes. We conclude that even though the tested compounds indeed presented inhibitory effects on NNT activity, at effective concentrations, they cause important undesirable effects on mitochondrial respiratory function and cell viability.

Drp1 knockdown induces severe muscle atrophy and remodelling, mitochondrial dysfunction, autophagy impairment and denervation.

The maintenance of optimal mitochondrial content and function is critical for muscle health. Mitochondrial dynamics play key roles in mitochondrial quality control; however, the exact role that mitochondrial fission plays in skeletal muscle health remains unclear. Here we report knocking down Drp1 (a protein regulating mitochondrial fission) for 4months in adult mouse skeletal muscle resulted in severe muscle atrophy (40-50%). Drp1 knockdown also led to a reduction in ADP-stimulated respiration, an increase in markers of impaired autophagy and increased muscle regeneration, denervation, fibrosis and oxidative stress. Our data indicate that Drp1 is crucial for the maintenance of normal mitochondrial function and that Drp1 depletion severely impairs muscle health. Mitochondria play central roles in skeletal muscle physiology, including energy supply, regulation of energy-sensitive signalling pathways, reactive oxygen species production/signalling, calcium homeostasis and the regulation of apoptosis. The maintenance of optimal mitochondrial content and function is therefore critical for muscle cells. Mitochondria are now well known as highly dynamic organelles, able to change their morphology through fusion and fission processes. Solid experimental evidence indicates that mitochondrial dynamics play key roles in mitochondrial quality control, and alteration in the expression of proteins regulating mitochondrial dynamics have been reported in many conditions associated with muscle atrophy and wasting. However, the exact role that mitochondrial fission plays in skeletal muscle health remains unclear. To address this issue, we investigated the impact of Drp1 (a protein regulating mitochondrial fission) knockdown, introduced via intramuscular injection of adeno-associated virus (AAV) on adult mouse skeletal muscle. Knocking down Drp1 for 4months resulted in very severe muscle atrophy (40-50%). Drp1 knockdown also led to a reduction in ADP-stimulated respiration and increases in markers of muscle regeneration, denervation, fibrosis, oxidative stress and impaired autophagy. Our findings indicate that Drp1 is essential for the maintenance of normal mitochondrial function and that Drp1 suppression severely impairs muscle health.

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

Effects of 3-month high-intensity interval training vs. moderate endurance training and 4-month follow-up on fat metabolism, cardiorespiratory function and mitochondrial respiration in obese adults.

The purpose of this study was to investigate, in obese adults, changes in body composition, physical capacities, fat oxidation and ex vivo mitochondrial respiration induced by a 3-month either moderate-intensity continuous training (MICT) or high-intensity interval training (HIIT); afterwards, the patients were followed for four months. Thirty-two patients (mean age 39years; mean body mass index [BMI] 36kg∙m-2) participated in this study attending ~ 34 sessions of training. At baseline (PRE), at the end of the program (POST) and after follow-up, body composition, peak O2 uptake (V'O2peak) and fat oxidation rate were measured. Vastus lateralis biopsies for the evaluation of mitochondrial respiration were performed only at PRE and POST. At POST, body mass (BM) and fat mass (FM) decreased (- 6 and - 14%, respectively, P < 0.05) in MICT and HIIT; V'O2peak increased in both groups (+ 6 and + 16%, respectively, P < 0.05). Maximal fat oxidation rate increased only after HIIT (P < 0.001). Maximal ADP-stimulated mitochondrial respiration normalized by citrate synthase increased (P < 0.05) by 67% and 36% in MICT and HIIT, respectively, without significant difference. After follow-up, BM and FM were still lower (- 4 and - 20%, respectively, P < 0.050) compared with baseline in both groups. Only after HIIT, V'O2peak (+ 8%) and maximal fat oxidation rate were still higher (P < 0.05). HIIT was more effective in improving and maintaining V'O2peak and fat oxidation. These results may be relevant for an appropriate prescription of training programs designed to optimize aerobic fitness in obese subjects.