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 Km 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.

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.

PURPOSE: 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. METHODS: Thirty-two patients (mean age 39 years; mean body mass index [BMI] 36 kg∙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. RESULTS: 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). CONCLUSIONS: 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.

GSK3ß is a key regulator of the ROS-dependent necrotic death induced by the quinone DMNQ.

Signaling pathways controlling necrosis are still mysterious and debated. We applied a shRNA-based viability screen to identify critical elements of the necrotic response. We took advantage from a small molecule (G5) that makes covalent adducts with free thiols by Michael addition and elicits multiple stresses. In cells resistant to apoptosis, G5 triggers necrosis through the induction of protein unfolding, glutathione depletion, ER stress, proteasomal impairments, and cytoskeletal stress. The kinase GSK3ß was isolated among the top hits of the screening. Using the quinone DMNQ, a ROS generator, we demonstrate that GSK3ß is involved in the regulation of ROS-dependent necrosis. Our results have been validated using siRNA and by knocking-out GSK3ß with the CRISPR/Cas9 technology. In response to DMNQ GSK3ß is activated by serine 9 dephosphorylation, concomitantly to Akt inactivation. During the quinone-induced pro-necrotic stress, GSK3ß gradually accumulates into the nucleus, before the collapse of the mitochondrial membrane potential. Accumulation of ROS in response to DMNQ is impaired by the absence of GSK3ß. We provide evidence that the activities of the obligatory two-electrons reducing flavoenzymes, NQO1 (NAD(P)H quinone dehydrogenase 1) and NQO2 are required to suppress DMNQ-induced necrosis. In the absence of GSK3ß the expression of NQO1 and NQO2 is dramatically increased, possibly because of an increased transcriptional activity of NRF2. In summary, GSK3ß by blunting the anti-oxidant response and particularly NQO1 and NQO2 expression, favors the appearance of necrosis in response to ROS, as generated by the quinone DMNQ.

Mitochondrial Adaptations in Elderly and Young Men Skeletal Muscle Following 2 Weeks of Bed Rest and Rehabilitation.

The aim of the study was to evaluate the expression levels of proteins related to mitochondrial biogenesis regulation and bioenergetics in vastus lateralis muscle biopsies from 16 elderly and 7 young people subjected to 14 days of bed-rest, causing atrophy, and subsequent 14 days of exercise training. Based on quantitative immunoblot analyses, in both groups a reduction of two key regulators of mitochondrial biogenesis/remodeling and activity, namely PGC-1α and Sirt3, was revealed during bed-rest, with a subsequent up-regulation after rehabilitation, indicating an involvement of PGC-1α-Sirt3 axis in response to the treatments. A difference was observed comparing the young and elderly subjects as, for both proteins, the abundance in the elderly was more affected by immobility and less responsive to exercise. The expression levels of TOM20 and Citrate Synthase, assayed as markers of outer mitochondrial membrane and mitochondrial mass, showed a noticeable sensitivity in the elderly group, where they were affected by bed-rest and rehabilitation recalling the pattern of PGC-1α. TOM20 and CS remained unchanged in young subjects. Single OXPHOS complexes showed peculiar patterns, which were in some cases dissimilar from PGC-1α, and suggest different influences on protein biogenesis and degradation. Overall, exercise was capable to counteract the effect of immobility, when present, except for complex V, which was markedly downregulated by bed-rest, but remained unaffected after rehabilitation, maybe as result of greater extent of degradation processes over biogenesis. Phosphorylation extent of AMPK, and its upstream activator LKB1, did not change after bed-rest and rehabilitation in either young or elderly subjects, suggesting that the activation of energy-sensing LKB1-AMPK signaling pathway was "missed" due to its transient nature, or was not triggered under our conditions. Our study demonstrates that, as far as the expression of various proteins related to mitochondrial biogenesis/remodeling, adaptations to bed-rest and rehabilitation in the two populations were different. The impact of bed-rest was greater in the elderly subjects, where the pattern (decrease after bed rest and recovery following rehabilitation) was accompanied by changes of mitochondrial mass. Modifications of protein abundance were matched with data obtained from gene expression analyses of four public human datasets focusing on related genes.

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

KEY POINTS: Superposition of hypoxia on 21 day 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. ABSTRACT: Skeletal muscle oxidative function was evaluated in 11 healthy males (mean ± SD age 27 ± 5 years) prior to (baseline data collection, BDC) and following a 21 day horizontal bed rest (BR), carried out in normoxia ( PIO2  = 133 mmHg; N-BR) and hypoxia ( PIO2  = 90 mmHg; H-BR). H-BR was aimed at simulating reduced gravity habitats. The effects of a 21 day hypoxic ambulatory confinement ( PIO2  = 90 mmHg; H-AMB) were also assessed. Pulmonary O2 uptake ( V̇O2 ), 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. V̇O2peak decreased (P < 0.05) after N-BR (0.98 ± 0.13 L min-1 ) and H-BR (0.96 ± 0.17 L min-1 ) vs. BDC (1.05 ± 0.14 L min-1 ). In the presence of a decreased (by ∼6-8%) thigh muscle volume, V̇O2peak 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 V̇O2peak 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 21 day 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.

Mitochondrial energy metabolism and signalling in human glioblastoma cell lines with different PTEN gene status.

Glioblastomas epidemiology and aggressiveness demand for a well characterization of biochemical mechanisms of the cells. The discovery of oxidative tumours related to chemoresistance is changing the prevalent view of dysfunctional mitochondria in cancer cells. Thus, glioblastomas metabolism is now an area of intense research, wherein was documented a high heterogeneity in energy metabolism and in particular in mitochondrial OxPhos. We report results gained by investigating mitochondrial OxPhos and bioenergetics, in a model of three human glioblastoma cell lines characterized by a different PTEN gene status. Functional data are analysed in relation to the expression levels of some main transcription factors and signalling proteins, which can be involved in the regulation of mitochondrial biogenesis and activity. Collectively, our observations indicate for the three cell lines a similar bioenergetic phenotype maintaining a certain degree of mitochondrial oxidative activity, with some difference for PTEN-wild type SF767 cells respect to PTEN-deleted A172 and U87MG characterized by a loss-of-function point mutation of PTEN. SF767 has lower ATP content and higher ADP/ATP ratio, higher AMPK activating-phosphorylation evoking energy impairment, higher OxPhos complexes and PGC1α-Sirt3-p53 protein abundance, in line with a higher respiration. Finally, SF767 shows a similar mitochondrial energy supply, but higher non-phosphorylating respiration linked to dissipation of protonmotive force. Intriguingly, it is now widely accepted that a regulated mitochondrial proton leak attenuate ROS generation and in tumours may be at the base of pro-survival advantage and chemoresistance.

Exercise training in Tgαq*44 mice during the progression of chronic heart failure: cardiac vs. peripheral (soleus muscle) impairments to oxidative metabolism.

Cardiac function, skeletal (soleus) muscle oxidative metabolism, and the effects of exercise training were evaluated in a transgenic murine model (Tgαq*44) of chronic heart failure during the critical period between the occurrence of an impairment of cardiac function and the stage at which overt cardiac failure ensues (i.e., from 10 to 12 mo of age). Forty-eight Tgαq*44 mice and 43 wild-type FVB controls were randomly assigned to control groups and to groups undergoing 2 mo of intense exercise training (spontaneous running on an instrumented wheel). In mice evaluated at the beginning and at the end of training we determined: exercise performance (mean distance covered daily on the wheel); cardiac function in vivo (by magnetic resonance imaging); soleus mitochondrial respiration ex vivo (by high-resolution respirometry); muscle phenotype [myosin heavy chain (MHC) isoform content; citrate synthase (CS) activity]; and variables related to the energy status of muscle fibers [ratio of phosphorylated 5'-AMP-activated protein kinase (AMPK) to unphosphorylated AMPK] and mitochondrial biogenesis and function [peroxisome proliferative-activated receptor-γ coactivator-α (PGC-1α)]. In the untrained Tgαq*44 mice functional impairments of exercise performance, cardiac function, and soleus muscle mitochondrial respiration were observed. The impairment of mitochondrial respiration was related to the function of complex I of the respiratory chain, and it was not associated with differences in CS activity, MHC isoforms, p-AMPK/AMPK, and PGC-1α levels. Exercise training improved exercise performance and cardiac function, but it did not affect mitochondrial respiration, even in the presence of an increased percentage of type 1 MHC isoforms. Factors "upstream" of mitochondria were likely mainly responsible for the improved exercise performance.NEW & NOTEWORTHY Functional impairments in exercise performance, cardiac function, and soleus muscle mitochondrial respiration were observed in transgenic chronic heart failure mice, evaluated in the critical period between the occurrence of an impairment of cardiac function and the terminal stage of the disease. Exercise training improved exercise performance and cardiac function, but it did not affect the impaired mitochondrial respiration. Factors "upstream" of mitochondria, including an enhanced cardiovascular O2 delivery, were mainly responsible for the functional improvement.

Transformation by different oncogenes relies on specific metabolic adaptations.

Metabolic adaptations are emerging as common traits of cancer cells and tumor progression. In vitro transformation of NIH 3T3 cells allows the analysis of the metabolic changes triggered by a single oncogene. In this work, we have compared the metabolic changes induced by H-RAS and by the nuclear resident mutant of histone deacetylase 4 (HDAC4). RAS-transformed cells exhibit a dominant aerobic glycolytic phenotype characterized by up-regulation of glycolytic enzymes, reduced oxygen consumption and a defect in complex I activity. In this model of transformation, glycolysis is strictly required for sustaining the ATP levels and the robust cellular proliferation. By contrast, in HDAC4/TM transformed cells, glycolysis is only modestly up-regulated, lactate secretion is not augmented and, instead, mitochondrial oxygen consumption is increased. Our results demonstrate that cellular transformation can be accomplished through different metabolic adaptations and HDAC4/TM cells can represent a useful model to investigate oncogene-driven metabolic changes besides the Warburg effect.

Separate and combined effects of a 10-d exposure to hypoxia and inactivity on oxidative function in vivo and mitochondrial respiration ex vivo in humans.

An integrative evaluation of oxidative metabolism was carried out in 9 healthy young men (age, 24.1 ± 1.7 yr mean ± SD) before (CTRL) and after a 10-day horizontal bed rest carried out in normoxia (N-BR) or hypoxia (H-BR, FiO2 = 0.147). H-BR was designed to simulate planetary habitats. Pulmonary O2 uptake (V̇o2) and vastus lateralis fractional O2 extraction (changes in deoxygenated hemoglobin+myoglobin concentration, Δ[deoxy(Hb+Mb)] evaluated using near-infrared spectroscopy) were evaluated in normoxia and during an incremental cycle ergometer (CE) and one-leg knee extension (KE) exercise (aimed at reducing cardiovascular constraints to oxidative function). Mitochondrial respiration was evaluated ex vivo by high-resolution respirometry in permeabilized vastus lateralis fibers. During CE V̇o2peak and Δ[deoxy(Hb+Mb)]peak were lower (P < 0.05) after both N-BR and H-BR than during CTRL; during KE the variables were lower after N-BR but not after H-BR. During CE the overshoot of Δ[deoxy(Hb+Mb)] during constant work rate exercise was greater in N-BR and H-BR than CTRL, whereas during KE a significant difference vs. CTRL was observed only after N-BR. Maximal mitochondrial respiration determined ex vivo was not affected by either intervention. In N-BR, a significant impairment of oxidative metabolism occurred downstream of central cardiovascular O2 delivery and upstream of mitochondrial function, possibly at the level of the intramuscular matching between O2 supply and utilization and peripheral O2 diffusion. Superposition of hypoxia on bed rest did not aggravate, and partially reversed, the impairment of muscle oxidative function in vivo induced by bed rest. The effects of longer exposures will have to be determined.

F1FO ATP Synthase Is Expressed at the Surface of Embryonic Rat Heart-Derived H9c2 Cells and Is Affected by Cardiac-Like Differentiation.

Taking advantage from the peculiar features of the embryonic rat heart-derived myoblast cell line H9c2, the present study is the first to provide evidence for the expression of F1FO ATP synthase and of ATPase Inhibitory Factor 1 (IF1) on the surface of cells of cardiac origin, together documenting that they were affected through cardiac-like differentiation. Subunits of both the catalytic F1 sector of the complex (ATP synthase-ß) and of the peripheral stalk, responsible for the correct F1-FO assembly/coupling, (OSCP, b, F6) were detected by immunofluorescence, together with IF1. The expression of ATP synthase-ß, ATP synthase-b and F6 were similar for parental and differentiated H9c2, while the levels of OSCP increased noticeably in differentiated cells, where the results of in situ Proximity Ligation Assay were consistent with OSCP interaction within ecto-F1FO complexes. An opposite trend was shown by IF1 whose ectopic expression appeared greater in the parental H9c2. Here, evidence for the IF1 interaction with ecto-F1FO complexes was provided. Functional analyses corroborate both sets of data. i) An F1FO ATP synthase contribution to the exATP production by differentiated cells suggests an augmented expression of holo-F1FO ATP synthase on plasma membrane, in line with the increase of OSCP expression and interaction considered as a requirement for favoring the F1-FO coupling. ii) The absence of exATP generation by the enzyme, and the finding that exATP hydrolysis was largely oligomycin-insensitive, are in line in parental cells with the deficit of OSCP and suggest the occurrence of sub-assemblies together evoking more regulation by IF1.

Proteomic analysis of F1F0-ATP synthase super-assembly in mitochondria of cardiomyoblasts undergoing differentiation to the cardiac lineage.

Mitochondria are essential organelles with multiple functions, especially in energy metabolism. An increasing number of data highlighted their role for cellular differentiation processes. We investigated differences in ATP synthase supra-molecular organization occurring in H9c2 cardiomyoblasts in the course of cardiac-like differentiation, along with ATP synthase biogenesis and maturation of mitochondrial cristae morphology. Using BN-PAGE analysis combined with one-step mild detergent extraction from mitochondria, a significant increase in dimer/monomer ratio was observed, indicating a distinct rise in the stability of the enzyme super-assembly. Remarkably, sub-stoichiometric mean values for ATP synthase subunit e were determined in both parental and cardiac-like H9c2 by an MS-based quantitative proteomics approach. This indicates a similar high proportion of complex molecules lacking subunit e in both cell types, and suggests a minor contribution of this component in the observed changes. 2D BN-PAGE/immunoblotting analysis and MS/MS analysis on single BN-PAGE band showed that the amount of inhibitor protein IF1 bound within the ATP synthase complexes increased in cardiac-like H9c2 and appeared greater in the dimer. In concomitance, a consistent improvement of enzyme activity, measured as both ATP synthesis and ATP hydrolysis rate, was observed, despite the increase of bound IF1 evocative of a greater inhibitory effect on the enzyme ATPase activity. The results suggest i) a role for IF1 in promoting dimer stabilization and super-assembly in H9c2 with physiological IF1 expression levels, likely unveiled by the fact that the contacts through accessory subunit e appear to be partially destabilized, ii) a link between dimer stabilization and enzyme activation.

Skeletal muscle oxidative function in vivo and ex vivo in athletes with marked hypertrophy from resistance training.

Oxidative function during exercise was evaluated in 11 young athletes with marked skeletal muscle hypertrophy induced by long-term resistance training (RTA; body mass 102.6 ± 7.3 kg, mean ± SD) and 11 controls (CTRL; body mass 77.8 ± 6.0 kg). Pulmonary O2 uptake (Vo2) and vastus lateralis muscle fractional O2 extraction (by near-infrared spectroscopy) were determined during an incremental cycle ergometer (CE) and one-leg knee-extension (KE) exercise. Mitochondrial respiration was evaluated ex vivo by high-resolution respirometry in permeabilized vastus lateralis fibers obtained by biopsy. Quadriceps femoris muscle cross-sectional area, volume (determined by magnetic resonance imaging), and strength were greater in RTA vs. CTRL (by ∼40%, ∼33%, and ∼20%, respectively). Vo2peak during CE was higher in RTA vs. CTRL (4.05 ± 0.64 vs. 3.56 ± 0.30 l/min); no difference between groups was observed during KE. The O2 cost of CE exercise was not different between groups. When divided per muscle mass (for CE) or quadriceps muscle mass (for KE), Vo2 peak was lower (by 15-20%) in RTA vs. CTRL. Vastus lateralis fractional O2 extraction was lower in RTA vs. CTRL at all work rates, during both CE and KE. RTA had higher ADP-stimulated mitochondrial respiration (56.7 ± 23.7 pmol O2·s(-1)·mg(-1) ww) vs. CTRL (35.7 ± 10.2 pmol O2·s(-1)·mg(-1) ww) and a tighter coupling of oxidative phosphorylation. In RTA, the greater muscle mass and maximal force and the enhanced mitochondrial respiration seem to compensate for the hypertrophy-induced impaired peripheral O2 diffusion. The net results are an enhanced whole body oxidative function at peak exercise and unchanged efficiency and O2 cost at submaximal exercise, despite a much greater body mass.

Glucose-modulated mitochondria adaptation in tumor cells: a focus on ATP synthase and inhibitor Factor 1.

Warburg's hypothesis has been challenged by a number of studies showing that oxidative phosphorylation is repressed in some tumors, rather than being inactive per se. Thus, treatments able to shift energy metabolism by activating mitochondrial pathways have been suggested as an intriguing basis for the optimization of antitumor strategies. In this study, HepG2 hepatocarcinoma cells were cultivated with different metabolic substrates under conditions mimicking "positive" (activation/biogenesis) or "negative" (silencing) mitochondrial adaptation. In addition to the expected up-regulation of mitochondrial biogenesis, glucose deprivation caused an increase in phosphorylating respiration and a rise in the expression levels of the ATP synthase ß subunit and Inhibitor Factor 1 (IF1). Hyperglycemia, on the other hand, led to a markedly decreased level of the transcriptional coactivator PGC-α suggesting down-regulation of mitochondrial biogenesis, although no change in mitochondrial mass and no impairment of phosphorylating respiration were observed. Moreover, a reduction in mitochondrial networking and in ATP synthase dimer stability was produced. No effect on ß-ATP synthase expression was elicited. Notably, hyperglycemia caused an increase in IF1 expression levels, but it did not alter the amount of IF1 associated with ATP synthase. These results point to a new role of IF1 in relation to high glucose utilization by tumor cells, in addition to its well known effect upon mitochondrial ATP synthase regulation.

Mitochondrial bioenergetic profile and responses to metabolic inhibition in human hepatocarcinoma cell lines with distinct differentiation characteristics.

The classical view of tumour cell bioenergetics has been recently revised. Then, the definition of the mitochondrial profile is considered of fundamental importance for the development of anti-cancer therapies, but it still needs to be clarified. We investigated two human hepatocellular carcinoma cell lines: the partially differentiated HepG2 and the undifferentiated JHH-6. High resolution respirometry revealed a marked impairment/uncoupling of OXPHOS in JHH-6 compared with HepG2, with the phosphorylation system limiting the capacity for electron transport much more in JHH-6. Blocking glycolysis or mitochondrial ATP synthase we demonstrated that in JHH-6 ATP synthase functions in reverse and consumes glycolytic ATP, thereby sustaining ΔΨm. A higher expression level of ATP synthase Inhibitor Factor 1 (IF1), a higher extent of IF1 bound to ATP synthase and a lower ATPase/synthase capacity were documented in JHH-6. Thus, here IF1 appears to down-regulate the reverse mode of ATPsynthase activity, thereby playing a crucial role in controlling energy waste and ΔΨm. These results, while confirming the over-expression of IF1 in cancer cells, are the first to indicate an inverse link between cell differentiation status and IF1 (expression level and regulatory function).

Knock-in reconstitution studies reveal an unexpected role of Cys-65 in regulating APE1/Ref-1 subcellular trafficking and function.

Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1) protects cells from oxidative stress via the base excision repair pathway and as a redox transcriptional coactivator. It is required for tumor progression/metastasis, and its up-regulation is associated with cancer resistance. Loss of APE1 expression causes cell growth arrest, mitochondrial impairment, apoptosis, and alterations of the intracellular redox state and cytoskeletal structure. A detailed knowledge of the molecular mechanisms regulating its different activities is required to understand the APE1 function associated with cancer development and for targeting this protein in cancer therapy. To dissect these activities, we performed reconstitution experiments by using wild-type and various APE1 mutants. Our results suggest that the redox function is responsible for cell proliferation through the involvement of Cys-65 in mediating APE1 localization within mitochondria. C65S behaves as a loss-of-function mutation by affecting the in vivo folding of the protein and by causing a reduced accumulation in the intermembrane space of mitochondria, where the import protein Mia40 specifically interacts with APE1. Treatment of cells with (E)-3-(2-[5,6-dimethoxy-3-methyl-1,4-benzoquinonyl])-2-nonyl propenoic acid, a specific inhibitor of APE1 redox function through increased Cys-65 oxidation, confirm that Cys-65 controls APE1 subcellular trafficking and provides the basis for a new role for this residue.

Cardiac differentiation promotes mitochondria development and ameliorates oxidative capacity in H9c2 cardiomyoblasts.

H9c2 undergoing cardiac differentiation induced by all-trans-retinoic acid were investigated for mitochondria structural features together with the implied functional changes, as a model for the study of mitochondrial development in cardiogenic progenitor cells. As the expression of cardiac markers became detectable, mitochondrial mass increased and mitochondrial morphology and ultrastructure changed. Reticular network organization developed and more bulky mitochondria with greater numbers of closely packed cristae and more electron-dense matrix were detected. Increased expression of PGC-1α proved the occurrence of mitochondrial biogenesis. Improvements in mitochondrial energetic competence were also documented, linked to better assembly between F(0) and F(1) sectors of the F(0)F(1)ATPsynthase enzyme complex.

Stoichiometry and topology of the complex of the endogenous ATP synthase inhibitor protein IF(1) with calmodulin.

IF(1), the natural inhibitor protein of F(O)F(1)ATP synthase able to regulate the ATP hydrolytic activity of both mitochondrial and cell surface enzyme, exists in two oligomeric states depending on pH: an inactive, highly helical, tetrameric form above pH 6.7 and an active, inhibitory, dimeric form below pH 6.7 [ Cabezon , E. , Butler , P. J. , Runswick , M. J. , and Walker , J. E. ( 2000 ) J. Biol. Chem. 275 , 25460 -25464 ]. IF(1) is known to interact in vitro with the archetypal EF-hand calcium sensor calmodulin (CaM), as well to colocalize with CaM on the plasma membrane of cultured cells. Low resolution structural data were herein obtained in order to get insights into the molecular interaction between IF(1) and CaM. A combined structural proteomic strategy was used which integrates limited proteolysis and chemical cross-linking with mass spectrometric analysis. Specifically, chemical cross-linking data clearly indicate that the C-terminal lobe of CaM molecule contacts IF(1) within the inhibitory, flexible N-terminal region that is not involved in the dimeric interface in IF(1). Nevertheless, native mass spectrometry analysis demonstrated that in the micromolar range the stoichiometry of the IF(1)-CaM complex is 1:1, thereby indicating that binding to CaM promotes IF(1) dimer dissociation without directly interfering with the intersubunit contacts of the IF(1) dimer. The relevance of the finding that only the C-terminal lobe of CaM is involved in the interaction is two fold: (i) the IF(1)-CaM complex can be included in the category of noncanonical structures of CaM complexes; (ii) it can be inferred that the N-terminal region of CaM might have the opportunity to bind to a second target.

Mitochondrial and cell-surface F0F1ATPsynthase in innate and acquired cardioprotection.

Mitochondria are central to heart function and dysfunction, and the pathways activated by different cardioprotective interventions mostly converge on mitochondria. In a context of perspectives in innate and acquired cardioprotection, we review some recent advances in F(0)F(1)ATPsynthase structure/function and regulation in cardiac cells. We focus on three topics regarding the mitochondrial F(0)F(1)ATPsynthase and the plasma membrane enzyme, i.e.: i) the crucial role of cardiac mitochondrial F(0)F(1)ATPsynthase regulation by the inhibitory protein IF(1) in heart preconditioning strategies; ii) the structure and function of mitochondrial F(0)F(1)ATPsynthase oligomers in mammalian myocardium as possible endogenous factors of mitochondria resistance to ischemic insult; iii) the external location and characterization of plasma membrane F(0)F(1) ATP synthase in search for possible actors of its regulation, such as IF(1) and calmodulin, at cell surface.

Caspase-independent apoptosis in Friend's erythroleukemia cells: role of mitochondrial ATP synthesis impairment in relocation of apoptosis-inducing factor and endonuclease G.

Mitochondria have emerged as the central components of both caspase-dependent and independent apoptosis signalling pathways through release of different apoptogenic proteins. We previously documented that parental and differentiated Friend's erythroleukemia cells were induced to apoptosis by oligomycin and H(2)O(2) exposure, showing that the energy impairment occurring in both cases as a consequence of a severe mitochondrial F(0)F(1)ATPsynthase inactivation was a common early feature. Here we provide evidence for AIF and Endo G mitochondrio-nuclear relocation in both cases, as a component of caspase-independent apoptosis pathways. No detectable change in mitochondrial transmembrane potential and no variation in mitochondrial levels of Bcl-2 and Bax are observed. These results point to the osmotic rupture of the mitochondrial outer membrane as occurring in response to cell exposure to the two energy-impairing treatments under conditions preserving the mitochondrial inner membrane. A critical role of the mitochondrial F(0)F(1)ATP synthase inhibition in this process is also suggested.

Functional and stoichiometric analysis of subunit e in bovine heart mitochondrial F(0)F(1)ATP synthase.

The role of the integral inner membrane subunit e in self-association of F(0)F(1)ATP synthase from bovine heart mitochondria was analyzed by in situ limited proteolysis, blue native PAGE/iterative SDS-PAGE, and LC-MS/MS. Selective degradation of subunit e, without disrupting membrane integrity or ATPase capacity, altered the oligomeric distribution of F(0)F(1)ATP synthase, by eliminating oligomers and reducing dimers in favor of monomers. The stoichiometry of subunit e was determined by a quantitative MS-based proteomics approach, using synthetic isotope-labelled reference peptides IAQL*EEVK, VYGVGSL*ALYEK, and ELAEAQEDTIL*K to quantify the b, gamma and e subunits, respectively. Accuracy of the method was demonstrated by confirming the 1:1 stoichiometry of subunits gamma and b. Altogether, the results indicate that the integrity of a unique copy of subunit e is essential for self-association of mammalian F(0)F(1)ATP synthase.