H+-slip correlated to rotor free-wheeling as cause of F1FO-ATPase dysfunction in primary mitochondrial disorders.

Inborn errors of metabolism are related to mitochondrial disorders caused by dysfunction of the oxidative phosphorylation (OXPHOS) system. Congenital hypermetabolism in the infant is a rare disease belonging to Luft syndrome, nonthyroidal hypermetabolism, arising from a singular example of a defect in OXPHOS. The mitochondria lose coupling of mitochondrial substrates oxidation from the ADP phosphorylation. Since Luft syndrome is due to uncoupled cell respiration responsible for deficient in ATP production that originates in the respiratory complexes, a de novo heterozygous variant in the catalytic subunit of mitochondrial F1FO-ATPase arises as the main cause of an autosomal dominant syndrome of hypermetabolism associated with dysfunction in ATP production, which does not involve the respiratory complexes. The F1FO-ATPase works as an embedded molecular machine with a rotary action using two different motor engines. The FO, which is an integral domain in the membrane, dissipates the chemical potential difference for H+, a proton motive force (Δp), across the inner membrane to generate a torsion. The F1 domain-the hydrophilic portion responsible for ATP turnover-is powered by the molecular rotary action to synthesize ATP. The structural and functional coupling of F1 and FO domains support the energy transduction for ATP synthesis. The dissipation of Δp by means of an H+ slip correlated to rotor free-wheeling of the F1FO-ATPase has been discovered to cause enzyme dysfunction in primary mitochondrial disorders. In this insight, we try to offer commentary and analysis of the molecular mechanism in these impaired mitochondria.

Emerging Roles for the Mitochondrial ATP Synthase Supercomplexes.

As pointed out by Gu et al. (Science 2019) in mammalian mitochondria, the H-shaped tetrameric structure of the ATP synthase, the cell powerhouse, consists of two V-shaped dimers linked by two IF1 in antiparallel arrangement. This supramolecular structure reveals new functional/structural roles of the enzyme complex in mitochondria.

Inflammatory signaling in NASH driven by hepatocyte mitochondrial dysfunctions.

Liver steatosis, inflammation, and variable degrees of fibrosis are the pathological manifestations of nonalcoholic steatohepatitis (NASH), an aggressive presentation of the most prevalent chronic liver disease in the Western world known as nonalcoholic fatty liver (NAFL). Mitochondrial hepatocyte dysfunction is a primary event that triggers inflammation, affecting Kupffer and hepatic stellate cell behaviour. Here, we consider the role of impaired mitochondrial function caused by lipotoxicity during oxidative stress in hepatocytes. Dysfunction in oxidative phosphorylation and mitochondrial ROS production cause the release of damage-associated molecular patterns from dying hepatocytes, leading to activation of innate immunity and trans-differentiation of hepatic stellate cells, thereby driving fibrosis in NASH.

1,5-disubstituted-1,2,3-triazoles counteract mitochondrial dysfunction acting on F1FO-ATPase in models of cardiovascular diseases.

The compromised viability and function of cardiovascular cells are rescued by small molecules of triazole derivatives (Tzs), identified as 3a and 3b, by preventing mitochondrial dysfunction. The oxidative phosphorylation improves the respiratory control rate in the presence of Tzs independently of the substrates that energize the mitochondria. The F1FO-ATPase, the main candidate in mitochondrial permeability transition pore (mPTP) formation, is the biological target of Tzs and hydrophilic F1 domain of the enzyme is depicted as the binding region of Tzs. The protective effect of Tz molecules on isolated mitochondria was corroborated by immortalized cardiomyocytes results. Indeed, mPTP opening was attenuated in response to ionomycin. Consequently, increased mitochondrial roundness and reduction of both length and interconnections between mitochondria. In in-vitro and ex-vivo models of cardiovascular pathologies (i.e., hypoxia-reoxygenation and hypertension) were used to evaluate the Tzs cardioprotective action. Key parameters of porcine aortic endothelial cells (pAECs) oxidative metabolism and cell viability were not affected by Tzs. However, in the presence of either 1 µM 3a or 0.5 µM 3b the impaired cell metabolism of pAECs injured by hypoxia-reoxygenation was restored to control respiratory profile. Moreover, endothelial cells isolated from SHRSP exposed to high-salt treatment rescued the Complex I activity and the endothelial capability to form vessel-like tubes and vascular function in presence of Tzs. As a result, the specific biochemical mechanism of Tzs to block Ca2+-activated F1FO-ATPase protected cell viability and preserved the pAECs bioenergetic metabolism upon hypoxia-reoxygenation injury. Moreover, SHRSP improved vascular dysfunction in response to a high-salt treatment.

Effects of cryopreservation on the mitochondrial bioenergetics of bovine sperm.

This study evaluated the bioenergetic map of mitochondria metabolism in cryopreserved bovine sperm. The detected oligomycin-sensitive basal respiration supported ATP production; frozen-thawed spermatozoa were found to have a coupling efficiency higher than 0.80. Cell respiration, however, was not stimulated by the protonophoric action of FCCP, as its titration with 1, 2, 4 and 6 µM did not stimulate the uncoupling activity on oxidative phosphorylation as highlighted by unresponsive oxygen consumption. The unusual effect on the stimulation of maximal respiration was not related to fibronectin- or PDL-coated plates used for cellular metabolism analysis. Conversely, irradiation of frozen-thawed bovine sperm with the red light improved mitochondrial parameters. In effect, the maximal respiration of red-light-stimulated sperm in PDL-coated plates was higher than the non-irradiated. In spite of this, red-light irradiation had no impact on membrane integrity and mitochondrial activity evaluated by epifluorescence microscopy.

Cell Metabolism Therapy by Small Natural Compounds.

Cellular metabolism therapy counteracting metabolic dysfunction performs a preeminent role in the pathophysiology of different diseases, such as cancer, diabetes, metabolic syndrome, and cardiovascular and neurodegenerative diseases [...].

Inflammation, Mitochondria and Natural Compounds Together in the Circle of Trust.

Human diseases are characterized by the perpetuation of an inflammatory condition in which the levels of Reactive Oxygen Species (ROS) are quite high. Excessive ROS production leads to DNA damage, protein carbonylation and lipid peroxidation, conditions that lead to a worsening of inflammatory disorders. In particular, compromised mitochondria sustain a stressful condition in the cell, such that mitochondrial dysfunctions become pathogenic, causing human disorders related to inflammatory reactions. Indeed, the triggered inflammation loses its beneficial properties and turns harmful if dysregulation and dysfunctions are not addressed. Thus, reducing oxidative stress with ROS scavenger compounds has proven to be a successful approach to reducing inflammation. Among these, natural compounds, in particular, polyphenols, alkaloids and coenzyme Q10, thanks to their antioxidant properties, are capable of inhibiting the activation of NF-κB and the expression of target genes, including those involved in inflammation. Even more, clinical trials, and in vivo and in vitro studies have demonstrated the antioxidant and anti-inflammatory effects of phytosomes, which are capable of increasing the bioavailability and effectiveness of natural compounds, and have long been considered an effective non-pharmacological therapy. Therefore, in this review, we wanted to highlight the relationship between inflammation, altered mitochondrial oxidative activity in pathological conditions, and the beneficial effects of phytosomes. To this end, a PubMed literature search was conducted with a focus on various in vitro and in vivo studies and clinical trials from 2014 to 2022.

A Lethal Channel between the ATP Synthase Monomers.

The molecular structure of the transmembrane domain of ATP synthases is responsible for the inner mitochondrial membrane bending. According to the hypothesized mechanism, ATP synthase dissociation from dimers to monomers, triggered by Ca2+ binding to F1, allows the mitochondrial permeability transition pore formation at the interface between the detached monomers.

Mitochondrial F-type ATP synthase: multiple enzyme functions revealed by the membrane-embedded FO structure.

Of the two main sectors of the F-type ATP synthase, the membrane-intrinsic FO domain is the one which, during evolution, has undergone the highest structural variations and changes in subunit composition. The FO complexity in mitochondria is apparently related to additional enzyme functions that lack in bacterial and thylakoid complexes. Indeed, the F-type ATP synthase has the main bioenergetic role to synthesize ATP by exploiting the electrochemical gradient built by respiratory complexes. The FO membrane domain, essential in the enzyme machinery, also participates in the bioenergetic cost of synthesizing ATP and in the formation of the cristae, thus contributing to mitochondrial morphology. The recent enzyme involvement in a high-conductance channel, which forms in the inner mitochondrial membrane and promotes the mitochondrial permeability transition, highlights a new F-type ATP synthase role. Point mutations which cause amino acid substitutions in FO subunits produce mitochondrial dysfunctions and lead to severe pathologies. The FO variability in different species, pointed out by cryo-EM analysis, mirrors the multiple enzyme functions and opens a new scenario in mitochondrial biology.

Protein folding and unfolding: proline cis-trans isomerization at the c subunits of F1 FO -ATPase might open a high conductance ion channel.

The c subunits, which constitute the c-ring apparatus of the F1 FO -ATPase, could be the main components of the mitochondrial permeability transition pore (mPTP). The well-known modulator of the mPTP formation and opening is the cyclophilin D (CyPD), a peptidyl-prolyl cis-trans isomerase. On the loop, which connects the two hairpin α-helix of c subunit, is present the unique proline residue (Pro40 ) that could be a biological target of CyPD. Indeed, the proline cis-trans isomerization might provide the switch that interconverts the open/closed states of the pore by pulling out the c-ring lipid plug.

Cellular Metabolism and Bioenergetic Function in Human Fibroblasts and Preadipocytes of Type 2 Familial Partial Lipodystrophy.

LMNA mutation is associated with type-2 familial partial lipodystrophy (FPLD2). The disease causes a disorder characterized by anomalous accumulation of body fat in humans. The dysfunction at the molecular level is triggered by a lamin A/C mutation, impairing the cell metabolism. In human fibroblasts and preadipocytes, a trend for ATP production, mainly supported by mitochondrial oxidative metabolism, is detected. Moreover, primary cell lines with FPLD2 mutation decrease the mitochondrial ATP production if compared with the control, even if no differences are observed in the oxygen consumption rate of bioenergetic parameters (i.e., basal and maximal respiration, spare respiratory capacity, and ATP turnover). Conversely, glycolysis is only inhibited in FPLD2 fibroblast cell lines. We notice that the amount of ATP produced in the fibroblasts is higher than in the preadipocytes, and likewise in the control, with respect to FPLD2, due to a more active oxidative phosphorylation (OXPHOS) and glycolysis. Moreover, the proton leak parameter, which characterizes the transformation of white adipose tissue to brown/beige adipose tissue, is unaffected by FPLD2 mutation. The metabolic profile of fibroblasts and preadipocytes is confirmed by the ability of these cell lines to increase the metabolic potential of both OXPHOS and glycolysis under energy required independently by the FPLD2 mutation.

The Impairment of Cell Metabolism by Cardiovascular Toxicity of Doxorubicin Is Reversed by Bergamot Polyphenolic Fraction Treatment in Endothelial Cells.

The beneficial effects of bergamot polyphenolic fraction (BPF) on the mitochondrial bioenergetics of porcine aortic endothelial cells (pAECs) were verified under the cardiotoxic action of doxorubicin (DOX). The cell viability of pAECs treated for 24 h with different concentrations of DOX was reduced by 50%, but the negative effect of DOX was reversed in the presence of increasing doses of BPF (100 µg/mL and 200 µg/mL BPF). An analysis of the protective effect of BPF on the toxic action of DOX was also carried out on cell respiration. We observed the inhibition of the mitochondrial activity at 10 µM DOX, which was not restored by 200 µg/mL BPF. Conversely, the decrease in basal respiration and ATP production caused by 0.5 or 1.0 µM DOX were improved in the presence of 100 or 200 µg/mL BPF, respectively. After 24 h of cell recovery with 100 µg/mL or 200 µg/mL BPF on pAECs treated with 0.5 µM or 1.0 µM DOX, respectively, the mitochondrial parameters of oxidative metabolism impaired by DOX were re-boosted.

Ca2+ as cofactor of the mitochondrial H+ -translocating F1 FO -ATP(hydrol)ase.

The mitochondrial F1 FO -ATPase in the presence of the natural cofactor Mg2+ acts as the enzyme of life by synthesizing ATP, but it can also hydrolyze ATP to pump H+ . Interestingly, Mg2+ can be replaced by Ca2+ , but only to sustain ATP hydrolysis and not ATP synthesis. When Ca2+ inserts in F1 , the torque generation built by the chemomechanical coupling between F1 and the rotating central stalk was reported as unable to drive the transmembrane H+ flux within FO . However, the failed H+ translocation is not consistent with the oligomycin-sensitivity of the Ca2+ -dependent F1 FO -ATP(hydrol)ase. New enzyme roles in mitochondrial energy transduction are suggested by recent advances. Accordingly, the structural F1 FO -ATPase distortion driven by ATP hydrolysis sustained by Ca2+ is consistent with the permeability transition pore signal propagation pathway. The Ca2+ -activated F1 FO -ATPase, by forming the pore, may contribute to dissipate the transmembrane H+ gradient created by the same enzyme complex.

Relationship between serum concentration, functional parameters and cell bioenergetics in IPEC-J2 cell line.

The foetal bovine serum (FBS) concentration could influence functional parameters of IPEC-J2 cells. IPEC-J2 is a non-transformed continuous epithelial cell line that represents an established in vitro model to study porcine gut inflammation and alterations of intestinal integrity. This cell line also represents a good translational model thanks to the high similitudes between pig and human gastrointestinal tract. With the aim to assess if the FBS-dependent functional variations are linked to the bioenergetic aspects, the addition of 5% and 10% FBS in the IPEC-J2 culture medium were tested. Doubling time and TEER measurement indicated that cells cultured at higher FBS dose grow faster and as a more compact monolayer. 10% FBS increases ATP production and mitochondrial oxidative phosphorylation (OxPhos) and does not affect glycolysis. Both at 5% and 10% FBS ATP production mainly comes from OxPhos and FBS concentration does not affect the cell respiration bioenergetic parameters. Noteworthy, IPEC-J2 treated with 5% and 10% FBS have a metabolic potential since both OxPhos and glycolysis increase by > 100% and < 50%, respectively in comparison with baseline metabolism. Moreover, glucose, fatty acids and glutamine constitute the preferred metabolic fuel for mitochondrial respiration at both FBS conditions tested. Accordingly, the cells flexibility to oxidize these substrates shows that IPEC-J2 mitochondria cannot maintain the basal ATP production without oxidizing all the substrates available irrespective of FBS concentration. To sum up, in IPEC-J2 cells OxPhos increases with the FBS-stimulated functional physiological parameters to fulfil ATP requirements.

The mitochondrial F1FO-ATPase exploits the dithiol redox state to modulate the permeability transition pore.

The dithiol reagents phenylarsine oxide (PAO) and dibromobimane (DBrB) have opposite effects on the F1FO-ATPase activity. PAO 20% increases ATP hydrolysis at 50 µM when the enzyme activity is activated by the natural cofactor Mg2+ and at 150 µM when it is activated by Ca2+. The PAO-driven F1FO-ATPase activation is reverted to the basal activity by 50 µM dithiothreitol (DTE). Conversely, 300 µM DBrB decreases the F1FO-ATPase activity by 25% when activated by Mg2+ and by 50% when activated by Ca2+. In both cases, the F1FO-ATPase inhibition by DBrB is insensitive to DTE. The mitochondrial permeability transition pore (mPTP) formation, related to the Ca2+-dependent F1FO-ATPase activity, is stimulated by PAO and desensitized by DBrB. Since PAO and DBrB apparently form adducts with different cysteine couples, the results highlight the crucial role of cross-linking of vicinal dithiols on the F1FO-ATPase, with (ir)reversible redox states, in the mPTP modulation.

Sulfide affects the mitochondrial respiration, the Ca2+-activated F1FO-ATPase activity and the permeability transition pore but does not change the Mg2+-activated F1FO-ATPase activity in swine heart mitochondria.

In mammalian cells enzymatic and non-enzymatic pathways produce H2S, a gaseous transmitter which recently emerged as promising therapeutic agent and modulator of mitochondrial bioenergetics. To explore this topic, the H2S donor NaHS, at micromolar concentrations, was tested on swine heart mitochondria. NaHS did not affect the F1FO-ATPase activated by the natural cofactor Mg2, but, when Mg2+ was replaced by Ca2+, a slight 15% enzyme inhibition at 100 µM NaHS was shown. Conversely, both the NADH-O2 and succinate-O2 oxidoreductase activities were totally inhibited by 200 µM NaHS with IC50 values of 61.6 ± 4.1 and 16.5 ± 4.6 µM NaHS, respectively. Since the mitochondrial respiration was equally inhibited by NaHS at both first or second respiratory substrates sites, the H2S generation may prevent the electron transfer from complexes I and II to downhill respiratory chain complexes, probably because H2S competes with O2 in complex IV, thus reducing membrane potential as a consequence of the cytochrome c oxidase activity inhibition. The Complex IV blockage by H2S was consistent with the linear concentration-dependent NADH-O2 oxidoreductase inhibition and exponential succinate-O2 oxidoreductase inhibition by NaHS, whereas the coupling between substrate oxidation and phosphorylation was unaffected by NaHS. Even if H2S is known to cause sulfhydration of cysteine residues, thiol oxidizing (GSSG) or reducing (DTE) agents, did not affect the F1FO-ATPase activities and mitochondrial respiration, thus ruling out any involvement of post-translational modifications of thiols. The permeability transition pore, the lethal channel which forms when the F1FO-ATPase is stimulated by Ca2+, did not open in the presence of NaHS, which showed a similar effect to ruthenium red, thus suggesting a putative Ca2+ transport cycle inhibition.

The mitochondrial permeability transition pore in cell death: A promising drug binding bioarchitecture.

Bioenergetic failure often features programmed cell death involved in some severe pathologies. When the cell is fated to die, the inner mitochondrial membrane becomes permeable to ions and solutes, due to the formation and opening of a channel known as mitochondrial permeability transition pore (mPTP). Up to now, the still-elusive mPTP structure and mechanism prevented any attempt to identify/design drugs to rule its formation and limit cell death. Latest advances, which strongly suggest that the F1 FO -ATPase can coincide with the mPTP, open new perspectives in therapy. Compounds targeting and inhibiting cyclophilin D, a known mPTP promoter, could be exploited to block mPTP formation. Moreover, if the mPTP-F1 FO -ATPase connection will be consolidated, selected F1 FO -ATPase inhibitors could represent novel therapeutic options to attenuate mPTP-related diseases by directly acting on mPTP molecular mechanism. This intriguing perspective, which raises new hopes to counteract mPTP-related diseases, stimulates further studies to clarify the mPTP architecture and mechanism.

Phenylglyoxal inhibition of the mitochondrial F1FO-ATPase activated by Mg2+ or by Ca2+ provides clues on the mitochondrial permeability transition pore.

Phenylglyoxal (PGO), known to cause post-translational modifications of Arg residues, was used to highlight the role of arginine residues of the F1FO-ATPase, which may be crucial to yield the mitochondrial permeability transition pore (mPTP). In swine heart mitochondria PGO inhibits ATP hydrolysis by the F1FO-ATPase either sustained by the natural cofactor Mg2+ or by Ca2+ by a similar uncompetitive inhibition mechanism, namely the tertiary complex (ESI) only forms when the ATP substrate is already bound to the enzyme, and with similar strength, as shown by the similar K'i values (0.82 ± 0.07 mM in presence of Mg2+ and 0.64 ± 0.05 mM in the presence of Ca2+). Multiple inhibitor analysis indicates that features of the F1 catalytic sites and/or the FO proton binding sites are apparently unaffected by PGO. However, PGO and F1 or FO inhibitors can bind the enzyme combine simultaneously. However they mutually hinder to bind the Mg2+-activated F1FO-ATPase, whereas they do not mutually exclude to bind the Ca2+-activated F1FO-ATPase. The putative formation of PGO-arginine adducts, and the consequent spatial rearrangement in the enzyme structure, inhibits the F1FO-ATPase activity but, as shown by the calcium retention capacity evaluation in intact mitochondria, apparently favours the mPTP formation.

Effects of Hydrogen Sulfide Donor NaHS on Porcine Vascular Wall-Mesenchymal Stem Cells.

Hydrogen sulfide (H2S) is now considered not only for its toxicity, but also as an endogenously produced gas transmitter with multiple physiological roles, also in maintaining and regulating stem cell physiology. In the present work, we evaluated the effect of a common H2S donor, NaHS, on porcine vascular wall-mesenchymal stem cells (pVW-MSCs). pVW-MSCs were treated for 24 h with increasing doses of NaHS, and the cell viability, cell cycle, and reactive oxygen species (ROS) production were evaluated. Moreover, the long-term effects of NaHS administration on the noteworthy characteristics of pVW-MSCs were analyzed. The MTT test revealed no alteration in cell viability, however, the cell cycle analysis demonstrated that the highest NaHS dose tested (300 µM) determined a block in S phase, which did not depend on the ROS production. Moreover, NaHS (10 µM), continuously administered in culture for 21 days, was able to significantly reduce NG2, Nestin and PDGFR-ß expression. The pro-angiogenic attitude of pVW-MSCs was partially reduced by NaHS: the cells maintained the ability to grow in spheroid and sprouting from that, but endothelial markers (Factor VIII and CD31) were reduced. In conclusion, NaHS can be toxic for pVW-MSCs in high doses, while in low doses, it influences cellular physiology, by affecting the gene expression with a slowing down of the endothelial lineage.

Characterization of metabolic profiles and lipopolysaccharide effects on porcine vascular wall mesenchymal stem cells.

The link between metabolic remodeling and stem cell fate is still unclear. To explore this topic, the metabolic profile of porcine vascular wall mesenchymal stem cells (pVW-MSCs) was investigated. At the first and second cell passages, pVW-MSCs exploit both glycolysis and cellular respiration to synthesize adenosine triphosphate (ATP), but in the subsequent (third to eighth) passages they do not show any mitochondrial ATP turnover. Interestingly, when the first passage pVW-MSCs are exposed to 0.1 or 10 µg/ml lipopolysaccharides (LPSs) for 4 hr, even if ATP synthesis is prevented, the spare respiratory capacity is retained and the glycolytic capacity is unaffected. In contrast, the exposure of pVW-MSCs at the fifth passage to 10 µg/ml LPS stimulates mitochondrial ATP synthesis. Flow cytometry rules out any reactive oxygen species (ROS) involvement in the LPS effects, thus suggesting that the pVW-MSC metabolic pattern is modulated by culture conditions via ROS-independent mechanisms.