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Impaired mitochondria cause an impressive decrease in ATP production becoming a common condition of cardiovascular diseases. Myxomatous mitral valve disease (MMVD) is characterized by mitochondrial dysfunction. By a non-invasive procedure of metabolism analysis on peripheral blood mononuclear cells, we exploit ex-vivo studies that directly constitute a translational approach to evaluate the cell bioenergetics. Cell ATP production decreased in the presence of MMVD, whereas glycolysis was unaffected. In MMVD, the mitochondrial activity underwent a significant reduction of basal respiration, maximal respiration, and ATP production. Our results depicted a pathological condition of MMVD characterized by cell metabolism deprived of mitochondrial energy support., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Salvatore Nesci reports financial support was provided by University of Bologna. Chiara Bernardini reports financial support was provided by University of Bologna. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Of the two main sectors of the F-type ATP synthase, the membrane-intrinsic F O domain is the one which, during evolution, has undergone the highest structural variations and changes in subunit composition. The F O 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 F O 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 F O subunits produce mitochondrial dysfunctions and lead to severe pathologies. The F O variability in different species, pointed out by cryo-EM analysis, mirrors the multiple enzyme functions and opens a new scenario in mitochondrial biology.

The properties of the mitochondrial F 1 F O -ATPase catalytic site, which can bind Mg 2+ , Mn 2+ , or Ca 2+ and hydrolyze ATP, were explored by inhibition kinetic analyses to cast light on the Ca 2+ -activated F 1 F O -ATPase connection with the permeability transition pore (PTP) that initiates cascade events leading to cell death. While the natural cofactor Mg 2+ activates the F 1 F O -ATPase in competition with Mn 2+ , Ca 2+ is a noncompetitive inhibitor in the presence of Mg 2+ . Selective F 1 inhibitors (Is-F 1 ), namely NBD-Cl, piceatannol, resveratrol, and quercetin, exerted different mechanisms (mixed and uncompetitive inhibition) on either Ca 2+ - or Mg 2+ -activated F 1 F O -ATPase, consistent with the conclusion that the catalytic mechanism changes when Mg 2+ is replaced by Ca 2+ . In a partially purified F 1 domain preparation, Ca 2+ -activated F 1 -ATPase maintained Is-F 1 sensitivity, and enzyme inhibition was accompanied by the maintenance of the mitochondrial calcium retention capacity and membrane potential. The data strengthen the structural relationship between Ca 2+ -activated F 1 F O -ATPase and the PTP, and, in turn, on consequences, such as physiopathological cellular changes., (© 2019 New York Academy of Sciences.)

Background: The mitochondrial F 1 F O -ATPase has the main role in synthesizing most of ATP, thus providing energy to living cells, but it also works in reverse and hydrolyzes ATP, depending on the transmembrane electrochemical gradient. Within the same complex the vital role of the enzyme of life coexists with that of molecular switch to trigger programmed cell death. The two-faced vital/lethal role makes the enzyme complex an intriguing biochemical target to fight pathogens resistant to traditional therapies and diseases linked to mitochondrial dysfunctions. A variety of post-translational modifications (PTMs) of selected F 1 F O -ATPase aminoacids have been reported to affect the enzyme function., Scope of Review: By reviewing the known PTMs of aminoacid side chains of both F 1 and F O sectors according to the most recent advances, the main aim is to highlight how local chemical changes may constitute the molecular key leading to pathological or physiological events., Major Conclusions: PTMs represent the chemical tool to modulate the F 1 F O -ATPase activity in response to different stimuli. Some PTMs are required to ensure the enzyme catalysis or, conversely, to inactivate the enzyme function. Each covalent modification of the F 1 F O -ATPase, which occur in response to local changes, is the result of a selective molecular mechanism which, by translating a chemical modification into a biochemical effect, guarantees the enzyme tuning under changing conditions., General Significance: Once highlighted how the molecular mechanism works, some PTMs may be exploited to modulate the effect of drugs targeting the enzyme complex or constitute promising tools for F 1 F O -ATPase-targeted therapeutic strategies., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The ATP synthase can be imagined as a reversible H(+)-translocating channel embedded in the membrane, FO portion, coupled to a protruding catalytic portion, F1. Under physiological conditions the F1FO complex synthesizes ATP by exploiting the transmembrane electrochemical gradient of protons and their downhill movement. Alternatively, under other patho-physiological conditions it exploits ATP hydrolysis to energize the membrane by uphill pumping protons. The reversibility of the mechanism is guaranteed by the structural coupling between the hydrophilic F1 and the hydrophobic FO. Which of the two opposite processes wins in the energy-transducing membrane complex depends on the thermodynamic balance between the protonmotive force (Δp) and the phosphorylation potential of ATP (ΔG P). Accordingly, while Δp prevalence drives ATP synthesis by translocating protons from the membrane P-side to the N-side and generating anticlockwise torque rotation (viewed from the matrix), ΔG P drives ATP hydrolysis by chemomechanical coupling of FO to F1 with clockwise torque. The direction of rotation is the same in all the ATP synthases, due to the conserved steric arrangement of the chiral a subunit of FO. The ability of this coupled bi-functional complex to produce opposite rotations in ATP synthesis and hydrolysis is explained on the basis of the a subunit asymmetry.

The main and best known role of the mitochondrial ATP synthase is to synthesize ATP by exploiting the transmembrane electrochemical gradient of protons and their downhill movement. However, under different conditions, the same enzyme can also switch to the opposite function of ATP hydrolysis and exploits its energy to pump protons against their gradient and energize the membrane. The change in functionality is linked to the change of direction of rotation of the two matched sectors of this unique complex, namely the hydrophilic F1, which performs the catalysis, and the hydrophobic membrane-embedded FO, which channels protons. Accordingly, viewed from the matrix side, ATP synthesis is driven by counterclockwise rotation and ATP hydrolysis by clockwise rotation of the FO rotor which is transmitted to F1. ATP dissipation through this mechanism features some diseases such as myocardial ischemia. Increasing evidence shoulders the hypothesis that the asymmetry of the a subunit of FO and particularly the steric arrangement of the two inner semi-channels for protons, play a key role in conferring to the coupled bi-functional complex the ability to reverse rotation by switching from ATP synthesis to ATP hydrolysis and vice versa. Accordingly, the conserved steric arrangement of the chiral a subunit of FO yields the same direction of rotation for all the ATP synthases. According to this hypothesis, the a subunit chirality imposes the direction of rotation of the rotor according to the proton gradient across the membrane. It seems likely that the direction of rotation of the membrane-embedded c-ring, which is adjacent to the a-subunit and acts as a rotor, may be under multiple control, being rotation essential to make the whole enzyme machinery work. However, the asymmetric features of the a subunit would make it the master regulator, thus directly determining which of the two functions, ATP production or ATP dissipation, will be performed. The handedness of a subunit should be considered in drug design to counteract tissue damage under all pathological conditions linked to functional impairment of ATP synthase., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The molecular mechanism by which the membrane-embedded FO sector of the mitochondrial ATP synthase translocates protons, thus dissipating the transmembrane protonmotive force and leading to ATP synthesis, involves the neutralization of the carboxylate residues of the c-ring. Carboxylates are thought to constitute the binding sites for ion translocation. In order to cast light on this mechanism, we exploited N,N'-dicyclohexylcarbodiimide, which covalently binds to FO c-ring carboxylates, and ionophores which selectively modulate the transmembrane electric (Δφ) and chemical (ΔpH) gradients such as valinomycin, nigericin and dinitrophenol. ATP hydrolysis was evaluated in mitochondrial preparations and/or inside-out submitochondrial particles from mussel and mammalian tissues under different experimental conditions. The experiments pointed out striking similarities between mussel and mammalian mitochondrial ATP synthase. Our results support the hypothesis that the ATP synthase of Mytilus galloprovincialis induces intersubunit torque generation and translocates H(+) by coordinating the hydronium ion (H3O(+)) in the ion binding site of FO. Our results are consistent with the hypothesis that in mussel mitochondria the main component of the electrochemical gradient driving proton flux and ATP synthesis is Δφ. Therefore, mussel FO probably contains a small c-ring, which implies a low bioenergetic cost of making ATP as in mammals. These features which make mussel mitochondria as efficient in ATP production as mammalian ones may be especially advantageous in facultative aerobic species which intermittently exploit mitochondrial respiration to generate ATP.