Conference Agenda

Introduction: The chronic kidney disease (CKD) patients are prone to develop cardio-renal syndrome (CRS) type IV, where CKD favors heart failure. Recent evidence suggests that after CKD, kidney cells released to blood stream extracellular vesicles (EVs) capable of reprogramming recipient cells and induce mitochondrial damage, key processes in CRS development. This work was focused in study the effect of N-acetylcysteine (NAC) pre administration, on mitochondrial damage and oxidative stress induced by plasma EVs from animals with CKD addition in H9c2 cardiac recipient cells, to determine the role of EV-induced metabolic reprogramming in the CRS development. Method: CKD was induced in male Wistar rats (250-300 g) by 5/6 nephrectomy. After 2 weeks, renal damage was confirmed by clinic markers and histology and EVs was isolated from the plasma. EVs was marked with BODIPY® -FA and added to the cell lines for 24 h. In the NAC groups the cells were treated with 10 mM before EVs addition. Results: EVs addition induced in H9c2 cardiac the cell viability reduction, mitochondrial complex activity and OXPHOS derived ATP production decrease, increase in ROS and mitochondrial oxidative stress markers. This was associated with GSH and NADPH levels and the enzymatic antioxidant system activity decrease, which induce inflammatory and apoptotic markers enhance. In contrast, NAC partially prevented these alterations, which was asocieted with mitochondrial preservation by GSH regulation. Conclusion: Our results suggested that EVs plays a key role as cardio-renal connectors in mitochondrial impairment in CRS type 4. Therefore, the mitochondrial protection of EVs induced alteration, could be a promising strategy to prevent the CRS progression.

[1] O.E. Aparicio-Trejo, ..., E. Tapia, Extracellular Vesicles in Redox Signaling and Metabolic Regulation in Chronic Kidney Disease, Antioxidants. 11 (2022) 356.

[2] I. Amador-Martínez, O…, E. Tapia, Mitochondrial Impairment: A Link for Inflammatory Responses Activation in the Cardiorenal Syndrome Type 4, Int. J. Mol. Sci. 24 (2023) 15875.

[3] B. Cuevas-López, …, O.E. Aparicio-Trejo, NAC Pre-Administration Prevents Cardiac Mitochondrial Bioenergetics, Dynamics, Biogenesis, and Redox Alteration in Folic Acid-AKI-Induced Cardio-Renal Syndrome Type 3, Antioxidants. 12 (2023) 1592.

Quality control (QC) in mitochondrial respiration is essential to ensure accuracy and reproducibility on three levels: (1) sample preparation, (2) instrumental and technical reproducibility, and (3) chemicals and respiration medium. In the present study, we focus on the quality of mitochondrial respiration medium, which plays a crucial role in maintaining and supporting mitochondrial function.

We evaluated the results obtained with mitochondrial respiration medium MiR05, prepared from 6 lots of MiR05-Kit (Oroboros Instruments) with storage times from 1 to 51 months at room temperature. Substrate-uncoupler-inhibitor titration (SUIT) reference protocols RP1 and RP2 were used with cryopreserved HEK 293T cells in the Oroboros O2k. The two protocols interrogate 20 different respiratory pathway and coupling control states, of which four are comparable with the following rates: ROUTINE respiration of living cells; residual endogenous respiration after permeabilization by digitonin; electron transfer capacity after uncoupler titration in the presence of succinate, rotenone and glycerophosphate; and CIV activity stimulated by ascorbate and TMPD.

Respiration in all 20 respiratory states was reproducible between different lots of MiR05-Kit and independent of storage time up to 51 months.

CIV activity was corrected for the chemical O₂ background caused by autoxidation of cytochrome c, ascorbate and TMPD, which is a linear function of O₂ concentration down to 50 µM. The chemical O₂ background flux was comparable for each lot of MiR05-Kit evaluated at different O₂ concentrations and independent of storage time. This provides another evidence of the stability of the MiR05-Kit.

Our 4-years study demonstrates a constant quality of lots of MiR05-Kit up to several years of storage. The long-term stability of MiR05-Kit and consistency between different lots represent essential elements to obtain reliable and reproducible results with high-resolution respirometry in research and clinical investigations.

Breast carcinoma is the most prevalent type of cancer among women. The primary therapeutic approach often involves hormone therapy targeting the estrogen receptor (ER), which is, however, frequently impeded by primary or secondary resistance. The molecular mechanisms underlying hormone therapy resistance remain largely unknown, and predictive molecular biomarkers suitable for diagnosis are lacking.

In this study, we employed metabolomic and lipidomic profiling of a novel cell model resistant to tamoxifen (Tam5R cells) and identified pathways significantly correlated with tamoxifen resistance in two model cell lines, MCF7 and T47D. The two most significantly deregulated lipid classes in Tam5R cells were lysophosphatidylinositol (LPI) and cardiolipins (CL). LPI is a potent activator of the GPR55 G-coupled receptor, which acts as an upstream activator of extracellular activated kinases 1 and 2 (ERK1 and 2). We aimed to identify enzymes contributing to excessive LPI production in Tam5R cells. Additionally, the levels of cardiolipins were significantly reduced compared to controls. We demonstrate that the mitochondrial network in Tam5R cells is severely fragmented, and we further investigated cristae morphology in relation to cardiolipin synthesis and remodeling defects in Tam5R cells.

Our research suggests a role for glycerolipid remodeling enzymes in the development of hormone therapy resistance in ER-positive breast carcinoma and provides a valuable foundation for further investigations into anabolic metabolism in cancer.

Supported by Czech Science Foundation grant 23-06208S to KS.

Although glucose and ketone bodies are the main energy sources of the brain, fatty acid oxidation (FAO) plays a relevant role in the pathogenesis of central nervous system disorders. Given the low contribution of FAO to brain metabolism, protocols for its assessment must be carefully designed.

High-resolution respirometry protocols were developed to avoid FAO overestimation by malate-linked anaplerotic activity in brain mitochondria. We compared octanoylcarnitine (medium-chain) and palmitoylcarnitine (long-chain acylcarnitine) as substrates of β-oxidation in the brain. The capacity of FA oxidative phosphorylation (F-OXPHOS) with palmitoylcarnitine was up to 4 times higher than respiration with octanoylcarnitine. The optimal concentration of palmitoylcarnitine was 10 µM which corresponds to the total concentration of long-chain acylcarnitines in the brain. Higher concentrations of palmitoylcarnitine inhibited respiration, showing that optimization is essential to avoid inhibitory concentrations of this substrate. Maximal respiration with octanoylcarnitine was reached at 20 µM, however, this concentration exceeds medium-chain acylcarnitine concentrations in the brain 15 times.

Brain mitochondrial F-OXPHOS capacity was higher in mice and rats compared to Drosophila. However, compared to other respiratory rates, FAO-linked respiration had a higher contribution in Drosophila brains. F-OXPHOS capacity was highest in mouse cerebellum, intermediate in the cortex, prefrontal cortex, and hypothalamus, and hardly detectable in the hippocampus.

In a rat model of endothelin-1-induced stroke, OXPHOS capacities showed a trend to decline which was not limited to F-OXPHOS, whereas medium- and long-chain acylcarnitine levels increased. In aged rats, F-OXPHOS capacity declined more in comparison to other OXPHOS capacities, being 2-fold lower than in the younger control group, while concentrations of long-chain acylcarnitines were 2-fold higher.

These data indicate that, although FAO is not a dominant pathway in brain energy metabolism, it plays important roles, such as avoiding the accumulation of long-chain acylcarnitines. Further studies of fatty acid oxidation deserve attention in brain bioenergetics and its relations to physiology and pathology.

Mitochondrial Cyclophilin D (CyPD), endowed with peptidyl-prolyl cis-trans isomerase activity, is a master regulator of the mitochondrial permeability transition pore (PTP). PTP is a Ca²⁺-dependent, unselective channel involved in mediating fast Ca²⁺release from mitochondria (short open times) and in generating a bioenergetic failure eventually leading to cell death (long open times). Previously, we demonstrated that CyPD interacts with the OSCP subunit of ATP synthase, one of the most promising candidate as the PTP.

In this study we identified two forms of CyPD in various cell lines and tissues. Mass spectrometry analyses showed that one form is ~1 kDa shorter than the other at the N-terminus, suggesting that CyPD could be cleaved by a mitochondrial protease, whose molecular identity is under investigation. Nuclear Magnetic Resonance (NMR) spectroscopy revealed that the human full-length CyPD (FL-CyPD) and a N-terminal truncated form (ΔN-CyPD) are structurally identical, except for the N-terminal tail (absent in the truncated form), which is highly flexible, in sharp contrast with the remaining globular rigid part. In vitro studies using FL-CyPD and ΔN-CyPD highlighted that the latter interacts with OSCP more avidly than FL-CyPD in a KCl buffer, which best resembles the physiological environment. Consistently, electrophysiological studies on highly purified bovine heart ATP synthase showed that in the absence of the phosphate only ΔN-CyPD rapidly triggers PTP opening upon Ca²⁺ stimulation. We could explain these differences in terms of structure. Indeed, NMR analysis showed that KCl affected the dynamics of FL-CyPD at the putative OSCP-binding site, possibly making it incompatible with OSCP binding. Overall, our data suggest that: i) the CyPD N-terminus plays a key role in regulating CyPD interaction with OSCP and ATP synthase transformation into PTP; ii) a N-terminally cleaved CyPD forms in cell, thus offering new insights into PTP regulation.

Equine skeletal muscle provides a rich source of mitochondria, suitable for both basic and applied studies of cellular energetics. Inclusion of fluorophores and the associated endpoints provides valuable information to complement measurements of mitochondrial oxygen flux, but investigators must be aware of the possible artifacts created by those fluorophores. Our lab compiled data from two separate studies to facilitate a better understanding of potential artifact created by three common flurophores used in respiromentry: Amplex UltraRed, Magnesium Green, and tetramethylrhodamine methlyester. Study #1 compared respirometry variables obtained in the presence of either Magnesium Green or Amplex UltraRed. Study #2 utilized cross-wise comparison of all three dyes and fluorophore-free conditions. Our studies demonstrate that Magnesium Green has no significant effect on respiration of isolated mitochondria, whereas tetramethylrhodamine inhibits phosphorylating respiration and increases LEAK respiration. Amplex UltraRed increases phosphorylating and non-phosphorylating respiration through Complex 1. The effect of Amplex UltraRed is a novel finding, and further study is necessary to determine the mechanism underlying this artifact.

The link between endoplasmic reticulum (ER) stress and mitochondrial dysfunction, focusing on the IRE1α arm of the unfolded protein response (UPR), is discussed. In addition to mitochondrial respiration, we have determined the impact of both thapsigargin- and tunicamycin-induced ER stress on the generation of ΔΨm in neuroblastoma SH-SY5Y cells. We have shown that treatment of cells with either thapsigargin or tunicamycin is associated with a significant decrease in ROUTINE and ATP-coupled mitochondrial respiration as well as significant changes in mitochondrial membrane potential (ΔΨm) generation, which is mainly associated with the reversal of the succinyl-CoA ligase reaction and a decreased activity of complex II. Despite the induction of the ER UPR, as documented by the increased expression of HRD1, ER stress did not induce the mitochondrial UPR, since the expression of both the mitochondrial protease LONP1 and the mitochondrial chaperone HSP60 were not significantly altered.

Finally, we evaluated the impact of an inhibitor of IRE1α endonuclease activity STF-083010 (STF) on ER stress-induced mitochondrial dysfunction, as the majority of the recent studies have suggested a central involvement of IRE1α in the process of transmission of ER stress to mitochondria. Inhibition of IRE1α ribonuclease with STF did not protect the SH-SY5Y cells from ER stress-induced mitochondrial dysfunction. STF itself had a significant impact on both mitochondrial respiration and the generation of ΔΨm. We also showed that STF has an important impact on mitochondrial functions independent of its ability to inhibit the endonuclease activity of IRE1α, which is involved in the activation of the IRE1α-XBP1 arm of the unfolded protein response following ER stress. The lowered respiratory reserve induced by STF may be associated with increased sensitivity of the cells to another cytotoxic agent in combination with STF. The impact of STF on mitochondrial functions could be associated with its possible off-target effect.

The experiments were funded by grants VEGA 1/0085/24 and 1/0183/23 of the Scientific Grant Agency of the Ministry of Education, Science and Sports of the Slovak Republic.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) constitute a class of natural compounds exhibiting both great chemical diversity and broad spectrum of biological activities, stemming from different maturation processes the genetically encoded precursor peptide can undergo [1]. This class includes thioalbamide, a thioviridamide-like molecule biosynthesized by Amycolatopsis alba and belonging to the thioamitide family, a rare group of specialized microbial metabolites known for their distinctive post-translational modifications and potential biological activities.

Thioalbamide is highly cytotoxic to cancer cells, while exhibiting significantly less activity toward a noncancerous epithelial cell line; in breast cancer cell lines, it triggered apoptotic cell death by inducing mitochondrial dysfunction and oxidative stress. Furthermore, it was able to inhibit cancer stem-like cells growth, a cell population strongly dependent on mitochondrial function for survival and propagation, responsible for chemotherapy resistance, metastasis, and tumor recurrence [2]. The antitumor potential of thioalbamide, as well as its ability to reduce cell migration and tumor invasiveness in breast cancer, has also been successfully confirmed by in vivo models.

Different experimental procedures detected that thioalbamide activity could be due to a highly selective inhibition of the FoF1-ATPase complex [2]. However, the mechanisms through which this microbial peptide exerts its inhibitory activity have not yet been established. Recently, we have found that thioalbamide induces cytotoxicity in glioblastoma, an extremely aggressive brain tumor, demonstrating greater efficacy against cell lines that heavily rely on mitochondrial energy metabolism for their proliferation. These new findings are consistent with those obtained in breast cancer and appear to confirm the FoF1-ATPase complex as a target of thioalbamide.

[1] L. Frattaruolo, R. Lacret, A.R. Cappello, A.W. Truman, A Genomics-Based Approach Identifies a Thioviridamide-Like Compound with Selective Anticancer Activity, ACS Chem. Biol., 12(11) (2017) 2815-2822.

[2] L. Frattaruolo, R. Malivindi, M. Brindisi, V. Rago, R. Curcio, G. Lauria, M. Fiorillo, V. Dolce, A.W. Truman, A.R. Cappello, Thioalbamide inhibits F_oF₁-ATPase in breast cancer cells and reduces tumor proliferation and invasiveness in breast cancer in vivo model, Mol. Metab., 68 (2023) 101674.

The mitochondrial transacylase tafazzin is responsible for cardiolipin remodeling, iteratively optimizing phospholipids side chain configuration to a mature form. Mutations in the TAFAZZIN gene lead to Barth syndrome, an X-linked inherited disorder presenting with heart and skeletal muscle myopathy, and neutropenia. Tafazzin-deficiency is characterized by monolyso-cardiolipin accumulation, cardiolipin depletion, and loss of cardiolipin unsaturation. These effects can be partially countered by alterations in the lipid environment.

We investigated the impact of fatty acids supplementation of HEK 293T cells mitochondrial respiration, respiratory complexes content and activity. Control and tafazzin-deficient cells were treated with increasing concentrations of oleic (OA) and linoleic acid (LA) for 5 days. Cardiolipin profile was monitored by mass spectrometric analysis of lipid extracts. Western blotting and CI-, and CII-activity measurements were performed in mitochondria-enriched fractions. Trypsin-harvested cells were used for high-resolution respirometry analysis with sequential titrations of digitonin, pyruvate+malate, ADP, cytochrome c, glutamate, succinate, CCCP, rotenone and antimycin A.

We found that OA supplementation had minimal impact on the cardiolipin profile. However, LA supplementation led to a substantial increase in unsaturated cardiolipins in both cell lines. As the LA concentration increased, the discrepancy in the lipid phenotype between control and tafazzin-deficient cells was consistently reduced.

Despite the strong reconfiguration of mitochondrial membrane lipids, CI expression presented a non-significant trend towards higher levels upon LA supplementation, while CII expression was not impacted. Likewise, enzymatic activities of CI and CII were not altered significantly with both supplementations. Neither LA nor OA supplementation affected ROUTINE respiration of living cells nor NADH-linked LEAK respiration or OXPHOS capacity, NADH- and succinate-linked OXPHOS and electron transfer capacities, or succinate-linked electron transfer capacity.

These results show that modification of cardiolipin remodeling by OA and LA in tafazzin-deficient and control cells does not necessarily result in functional changes in mitochondrial respiration. In standard culturing conditions, cells demonstrate the ability to adapt to their genetic deficiency in cardiolipin remodeling capabilities and alterations in lipid composition. This could be partly due to cardiolipin metabolism being already evolutionarily well-adapted to be compatible with diverse lipid environments in tissues and different cell types.

Opening of the mitochondrial permeability transition (MPT) pore often underlies heart dysfunction. This pore is activated in response to calcium ions in a process dependent of Cyclophilin D (CypD) isomerase activity. CypD can bind to several proteins and enzymes inside the mitochondrial matrix including subunits of the ATP synthase and mitochondrial Slc25a carriers [1]. We have previously found Complement 1q-binding protein (C1qbp) as an in vitro CypD-binding target [2]. Here we further tested the interactions between C1qbp, CypD, and the MPT pore using molecular dynamics simulations and by generating mice with either C1qbp overexpression or gene silencing. Our results show that C1qbp binds to CypD and alters MPT. Moreover, mice with C1qbp downregulation show abnormal oxidative phosphorylation, cardiac dysfunction and cell death in a model of myocardial infarction. Overall, the results highlight the relevance of C1qbp as a regulator of the calcium-induced MPT pore, mitochondrial homeostasis and sensitivity to injury in the heart.

Statins, cholesterol-lowering drugs, and bisphosphonates, drugs that slow bone loss, inhibit the activity of the mevalonate pathway, one of whose products is coenzyme Q, an important cellular antioxidant and a key electron carrier in the mitochondrial respiratory chain. The aim of this study was to investigate the effects of chronic exposure to statins (atorvastatin and simvastatin) or bisphosphonates (alendronate and zolendronate) on cell function and oxidative metabolism of cultured astrocytes or endothelial cells, respectively. Although both types of drugs significantly reduce the level of coenzyme Q in cells and mitochondria in a similar way, they induce a different reaction of aerobic metabolism (oxidation of respiratory substrates) and the resulting oxidative stress (level of formation of reactive oxygen species and level of antioxidant defense). Our studies indicate that statins and bisphosphonates, which reduce coenzyme Q levels, modulate cellular energy metabolism, leading to changes in cellular energy status, coenzyme Q redox homeostasis, mitochondrial turnover, and mitochondrial respiratory function.

This research was funded by National Science Centre, Poland, OPUS 2020/37/B/NZ1/01188

Insulin secretion in pancreatic β-cells is stimulated by so-called secretagogues, among which glucose is the most notable one. Insulin secretion requires also a redox signal, besides elevated ATP/ADP ratio in the cytosol, upon the glucose stimulated insulin secretion (GSIS) [1]. Other type of secretagogues is represented by branched-chain (BC) keto acids (BCKAs), i.e., α-ketoisocaproate (KIC), α-ketoisovalerate (KIV), and α-ketomethylvalerate (KMV); derivatives of BC amino acids (BCAA). BCKAs are metabolized by so-called β-like oxidation, in which each of BCKAs yield FADH₂ as well as one or several NADH molecules, with their increasing number in order of KIC<KMV<KIV. Finally, acetyl-CoA (for KIC and KMV) or succinyl-CoA (for KIV and KMV) are yielded. Asking which of these products contribute to redox (H₂O₂) signaling determining insulin secretion (IS), i.e., BCKA-stimulated IS (BCKA-SIS) in pancreatic β-cells, we studied Amplex UltraRed-monitored H₂O₂ release to the exterior of cells and pancreatic islets (PIs) upon BCKA-SIS. Such redox signal determined a closure of the ATP-sensitive K⁺ channels and Ca²⁺oscillations and was partly inhibited by mitochondrial antioxidant SkQ1, and nearly completely by silencing of electron-transfer flavoprotein (ETF) : ubiquinone (Q) oxidoreductase (ETFQOR). The ETFQOR QH₂ input retards respiratory chain electron transport, giving superoxide and the concomitant redox (H₂O₂) signal. In conclusion, BCKA-SIS requires both peri-plasma membrane ATP and H₂O₂ elevation, while both originate from BCKA β-like oxidation.

Supported by the GACR grant 24-10132S to P.J.

The mitochondrial Dynamin-like protein Optic Atrophy 1 (OPA1) is a small GTPase involved in mitochondrial fusion, cristae remodeling, cytochrome c release and apoptosis. OPA1 upregulation has been increasingly identified as an exploitable vulnerability in cancer cells. From a previous screening of >10,000 drug-like molecules for inhibition of OPA1 GTPase activity, we identified MYLS22 as a promising hit. MYLS22 is not mitochondriotoxic but causes mitochondrial fragmentation and cristae remodeling, enhancing cytochrome c release following proapoptotic stimuli, recapitulating the effect of OPA1 downregulation in cells. Following a SAR analysis of MYLS22, multiple possible derivatives have been identified with increased water solubility. Three of these derivatives showed enhanced inhibitory effects on OPA1 GTPase activity in vitro and caused mitochondrial fragmentation in cells. To identify hits with further enhanced potency, solubility and overall availability of the leads, we generated a virtual, combinatorial library of 81,000 compounds. We docked these compounds to the GTPase domain of the currently available structure of OPA1 (PDB: 6JTG) and performed a virtual screening to discover second generation OPA1 inhibitors. We synthesized the best hits and analyzed their potency as OPA1 inhibitors in vitro and in cells. Our work identifies specific and potent OPA1 inhibitors with the potential to treat cancers where OPA1 is upregulated.

Heart failure is the leading cause of death in the elderly population and the heart is largely powered by oxidative phosphorylation which takes place in the mitochondria. Indeed, the heart is packed with these organelles and it is conceivable that the failure to uphold energy requirements during old age is related to alterations in mitochondrial function. To investigate specifically the role of ATP Synthase during aging, we established a cell model of human induced pluripotent stem cell derived cardiomyocytes and induced senescence. This allowed us to perform experiments that specifically focused on ATP production as well as ultrastructural changes of the inner mitochondrial membrane and the spatio-temporal organisation of the ATP Synthase in control and senescent cardiomyocytes. We found that ATP production is decreased during senescence, while the membrane potential is increased. This appears to be caused by increased activity of Complex I and we hypothesise that the built-up membrane potential is not depleted due to malfunctioning of the ATP Synthase. We see reduced cristae density and altered morphology, however, this is can not be explained by differences in ATP Synthase dimerization patterns or expression levels. Interestingly, we observed a reduced mobility of the enzyme in senescent cells, using single molecule localisation microscopy. However, differences are only minor, albeit statistically significant, leaving room for further studies regarding the functionality of the complex.

Mitochondria have been well studied as the primary producers of ATP in the cell, but more recent research has shown that mitochondria have a myriad of roles in the diagnosis and progression of multiple different diseases. At the forefront of monitoring mitochondrial status is respirometry, an assay which uses oxygen consumption as a surrogate readout for mitochondrial activity. While the current standards for mitochondrial respirometry assess the primary tissue for mitochondrial monitoring, these techniques require invasive sampling methods and intensive processing requirements, making them largely prohibitive to utilize. Recent research has identified a possible solution to this challenge though; the respirometry profiles of systemic markers like PBMCs may be used indirectly measure the mitochondrial status of the tissue of interest. While this breakthrough has made repeated patient sampling for mitochondrial monitoring more feasible, the intensive processing requirements still prevent large-scale adoption of respirometry methods for clinical and research use. Our lab has identified that salivary leukocytes from salivary mouthwash may be used as a systemic marker of mitochondrial health. These cells may be repeatedly sampled in a non-invasive manner and require very little processing and storage requirements, overcoming the hurdles preventing large-scale assessments of mitochondrial respirometry. Our poster will detail the isolation of this novel sample type and the optimizations required to run respirometry on these cells using a Seahorse respirometer.

The sequential conversion of choline to betaine aldehyde and subsequently to betaine in mitochondria expressing choline dehydrogenase (CDH) and betaine aldehyde dehydrogenase (BADH) results in the reduction of ubiquinone, supporting downstream Complex III (CIII) and Complex IV (CIV) operation. This process is sustained by NAD⁺ provided by Complex I (CI), which is necessary for the activity of BADH. We established a model to assess the rate of ATP export, which was produced by substrate-level phosphorylation catalyzed by succinate-CoA ligase (mtSLP), in isolated mitochondria with inhibited OXPHOS at the CI level. There, we show that addition of choline to substrate cocktails metabolized through ketoglutarate dehydrogenase complex (KGDHC) enhanced the rate of ATP exported from mitochondria by the adenine nucleotide translocator (ANT). In CI-inhibited mitochondria when KGDHC was additionally blocked by arsenite, choline boosted ATP export in the presence of exogenous quinones. This was partially abolished by dicoumarol, or -for some quinones- in mitochondria obtained from NQO1^-/- mice. None of the effects of choline were observed when mitochondria were inhibited at the CIII or CIV levels instead of CI. This implies that choline sustained ATP export by maintaining the membrane potential at values more negative than the reversal potential of the ANT (Erev_ANT), as verified by safranin O fluorescence measurements. Mechanistically, our work describes a metabolic network through which a suitable CI bypasser (choline) sustains the export of ATP made by mtSLP, the latter supported by NAD⁺ provided by diaphorases (at least one being NQO1), by maintaining the mitochondrial membrane potential more negative than Erev_ANT by proton pumping through CIII and CIV.

A known metabolic consequence of obesity and insulin resistance is decreased mitochondrial activity (i.e., complex activity, membrane potential, oxidative phosphorylation) in skeletal muscle. This is, in part, due to the incomplete breakdown of lipid droplets which occurs with metabolic disease. It remains unclear if mitochondria next to lipid droplets observe changes in mitochondrial activity to a greater extent due to the limitations of fat breakdown with metabolic disease. Therefore, the purpose of this investigation was to perform mitochondrial functional imaging in isolated skeletal muscle fibers of obese and wild-type animals, and determine if metabolic changes occur in proximity to lipid droplets. Cells from the flexor digitorum brevis were isolated from seven wild-type mice and six ob/ob mice (Leptin knockout). Functional live cell imaging was performed to measure mitochondrial membrane potential, mitochondrial content, lipid droplet content, NADH redox and NADH flux using an upright Leica SP8 microscope. To measure mitochondrial function in proximity to lipid droplets, we defined lipid droplet associated mitochondria were defined as mitochondria within 0.5 micron of a lipid droplet. Statistical analysis for whole-cell measurements was performed using unpaired t-tests in Prism. Statistical analysis for lipid bound versus non-lipid bound mitochondria was performed using a paired t-test in Prism. Ob/ob animals were significantly heavier than wild-type animals (48.64±6.86 g vs 26.28±2.36 g, p<0.0001), confirming the obese phenotype of the ob/ob animals. NADH redox was no different between ob/ob and wild-type animals (p=0.099). NADH flux was significantly lower in the ob/ob animals (1.31±0.53 %NADH reduced/sec) compared to wild-type animals (2.31±0.89 %NADH reduced/sec, p=0.011). Despite no change in the redox state, mitochondrial membrane potential was decreased in ob/ob animals compared to wild-type animals (p=0.023). Lipid droplet associated mitochondria had higher mitochondrial membrane potential compared to non-lipid droplet associated mitochondria (p<0.0001) of wild-type animals. However, this energetic enhancement of lipid droplet associated mitochondria was lost in ob/ob animals (p=0.085). Overall, these data demonstrate a deficiency in the electron transport chain in the skeletal muscle of obese mice. Moreover, the energetic advantage provided by direct proximity to lipid droplets is lost in skeletal muscle mitochondria from obese mice, which could add evidence to limited ability of these cells to participate in fat oxidation.

Phospholipases play a crucial role in various cellular functions, including membrane properties, lipid second messenger production, and cellular bioenergetics. Redox-activated mitochondrial calcium-independent phospholipase A2γ (iPLA2γ) was implicated in cleaving both saturated and unsaturated fatty acids from the phospholipids of mitochondrial membranes and participating in post-synthetic remodeling of cardiolipin [1]. It has been shown that redox-activated iPLA2γ is involved in regulating GPR40-dependent insulin secretion in β-cell model insulinoma INS1-E cells [2]. Here, we investigated the impact of iPLA2γ ablation on insulin secretion in vivo. While iPLA2γ knockout mice (iPLA2γ-KO) did not show alterations in glucose-stimulated insulin secretion, they exhibited a suppressed and delayed insulin release in response to oral administration of liposomal triglycerides (Intralipid) as observed over a period of up to 6 hours. Treatment with the specific iPLA2γ inhibitor prior to Intralipid intake resulted in a similar effect as observed in iPLA2γ-KO mice, highlighting the role of iPLA2γ in amplifying the fatty acid-stimulated insulin secretion (FASIS) in vivo. The impaired insulin response in iPLA2γ-KO mice is consistent with the role of iPLA2γ-cleaved fatty acids in GPR40-mediated insulin secretion. These findings further emphasize the contribution of iPLA2γ to whole-animal bioenergetics by modulating insulin release and sensitivity.

Supported by the Czech Science Foundation grant 22-17173S.

[1] P. Průchová, K. Gotvaldová, K. Smolková, L. Alán, B. Holendová, J. Tauber, A. Galkin, P. Ježek, M. Jabůrek, Antioxidant role and cardiolipin remodeling by redox-activated mitochondrial Ca²⁺-independent phospholipase A2γ in the brain, Antioxidants (Basel), 11 (2022) 198.

[2] J. Ježek, A. Dlasková, J. Zelenka, M. Jabůrek, P. Ježek, H₂O₂-activated mitochondrial phospholipase iPLA₂γ prevents lipotoxic oxidative stress in synergy with UCP2, amplifies signaling via G-protein-coupled receptor GPR40, and regulates insulin secretion in pancreatic β-cells. Antioxid Redox Signal, 23 (2015), 958-972.

Mitochondria play an essential role in mitochondrial and cellular homeostasis; in ATP synthesis and in death. Oxidative phosphorylation (OxPhos) is vital for ATP production. In the mitochondrial inner membrane (MIM) the permeability transition pore (mPTP) has large effects on the efficiency to produce ATP. It is considered a physiological uncoupling mechanism [1]. When mPTP opens, ion and proton gradients across MIM are depleted and ATP synthesis decreases. If PTP remains open for long, it causes cell death [2]. In cardiomyocytes and myocytes mPTP undergoes frequent transitory opening/closing events and is associated with Ca²⁺ homeostasis and protection against stress [3]. However, in tissues such as the liver PTP dynamics do not seem to occur. To explore a possible tissue-specific difference in mPTP physiology, its reversible opening and closing was compared in heart and liver mitochondria using different effectors while alternating Ca²⁺/EGTA additions. We monitored the rate of O₂ consumption, mitochondrial swelling, transmembrane potential and reactive oxygen species (ROS) production. Our results suggest that liver mPTP loses opening reversibility earlier that heart mPTP. Differences in structure of the putative protein components in each of these organs were found suggesting an explanation for our results.

[1] Vianello A, Casolo V, Petrussa E, Peresson C, Patui S, Bertolini A, Passamonti S, Braidot E, Zancani M. The mitochondrial permeability transition pore (PTP) - an example of multiple molecular exaptation? Biochim Biophys Acta. 1817(2012):2072-86.

2. Manon S, Roucou X, Guérin M, Rigoulet M, Guérin B. Characterization of the yeast mitochondria unselective channel: a counterpart to the mammalian permeability transition pore? J Bioenerg Biomembr. 5 (1998): 419-29.

3. Hurst, S., Hoek, J., & Sheu, S. S. Mitochondrial Ca 2+ and regulation of the permeability transition pore. Journal of bioenergetics and biomembranes, 49(2017), 27-47.

AMP-activated protein kinase (AMPK) evolved as the central cellular energy sensor and regulator. It can detect a critical increase in cellular AMP/ATP and ADP/ATP concentration ratios as a signal for limiting energy availability, and trigger compensatory mechanisms to maintain energy homeostasis. We have engineered AMPfret, a genetically encoded energy nanosensor, which makes the AMPK-based sensing process detectable by an increased fluorescence resonance energy transfer (FRET) between donor and acceptor fluorophores [1]. In this study, we generated HEK293T clonal cell lines stably expressing AMPfret and utilized them to assess the energy status of cell populations over time and space [2]. Our findings reveal that the early time course of the AMPfret signal effectively distinguishes between physiological and toxic stress. While physiological stress induces only mild or transient increases in the AMPfret signal, toxic stress leads to a possibly slow, but persistent and robust elevation of the signal. Further, we observed significant heterogeneity in the AMPfret response of clonal cell populations to external stressors, highlighting the importance of single-cell approaches in studying cell energetics.

[1] M. Pelosse, C. Cottet-Rousselle, C. Bidan, A. Dupont, K. Gupta, I. Berger, U. Schlattner, Synthetic energy sensor AMPfret deciphers adenylate-dependent AMPK activation mechanism, Nat. Commun. 10 (2019) 1038.

[2] R. Abi Nahed, R., M. Pelosse, F. Aulicino, F. Cottaz, I. Berger, U. Schattner, FRET-based sensor for measuring adenine nucleotide binding to AMPK, Meth. Mol. Biol. (2014); preprint on BioRxiv doi: https://doi.org/10.1101/2023.09.05.553069

Citrin Deficiency (CD) is an inborn error of metabolism and secondary urea cycle disorder that is pan-ethnic with three distinct age-related pathological stages: neonatal intrahepatic cholestasis (NICCD), failure to thrive and dyslipidemia (FTTDCD) and adult-onset citrullinemia type-2 (CTLN2). CD results from mutations in the slc25a13 gene, encoding the mitochondrial aspartate-glutamate carrier isoform 2, also called Citrin, which imports glutamate alongside a proton into the mitochondria, and exports aspartate. Citrin lies at a nexus of metabolic pathways in the cell, and mutations in the protein lead to disruption of the urea cycle, gluconeogenesis and the malate-aspartate shuttle alongside mitochondrial ATP production. Citrin is the dominant aspartate-glutamate carrier isoform in the liver, and as such, CD mutations primarily affect the liver in patients. In order to understand the changes in metabolism and the associated bioenergetic profile, here we utilised siRNA-mediated knockdown and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 gene editing to experimentally disrupt Citrin expression in the established hepatoblastoma cell line, HepG2, in both a transient and stable manner. Using high-resolution respirometry, we were able to probe the bioenergetic changes accompanying partial or complete ablation of Citrin expression in HepG2 cells. We show significant derailment of bioenergetic capacity even following transient knockdown, and retention of 70% of wild type expression. Deletion of slc25a13 from HepG2 cells results in a severe growth defect accompanying these bioenergetic deficits, which remains evident even in heterozygous cells. Future work will investigate whether nutritional supplements proposed to be of use to patients may rescue either the bioenergetic or growth phenotypes observed in these cell models. Further work will also focus on identifying the specific metabolic pathways disrupted in these knock-out cells and determine how the level of citrin expression influences the specific phenotype observed. This work provides greater insight into the bioenergetic phenotype of citrin deficiency and thus may lead to the discovery of new therapeutic avenues.

Complex III (ubiquinol:cytochrome c oxidoreductase) is a multisubunit membrane bound enzyme and in its native form is a symmetrical homodimer (CIII₂). CIII₂ is central for mitochondrial respiratory chain and is associated with different stoichiometry with Complex I and Complex IV to form supramolecular assemblies, called supercomplexes (SCs). Defects in CIII₂ are rare and mostly associated with mutations in MT-CYB gene that encodes for one of the catalytic subunits, cytochrome b (cyt b). It has been suggested that pathogenic mutations in MT-CYB are mitigated when CIII₂ is assembled in SCs [1]. Therefore, we applied biochemical approaches in human cellular models carrying pathogenic point mutations in cyt b to analyse the structural stability and enzymatic activity of CIII₂. Our preliminary results showed that pathogenic mutation differently affected the kinetics of the assembly of CIII₂ and its SCs after the treatment with a reversible mitochondrial translation inhibitor, suggesting a role of these mutations not only in CIII₂ activity but also in its biogenesis. Interestingly, the rescue of the oxygen consumption profile was further delayed compared to the formation of enzymatic complexes. In addition, we applied the Protein Stability Prediction with a Gaussian Network Model (PSP-GNM) approach [2] to evaluate global changes in the unfolding Gibbs free energy change and study the effects of single amino acid mutations on cyt b stability on the available isolated and SC-bound CIII₂ structures. Preliminary results indicate that some pathogenic mutations may affect the unfolding free energy of CIII₂, stiffening the structure of the enzyme, in agreement with the reduction of CIII₂ activity. This dual experimental and biocomputational approach may be very useful to dissect assembly processes and function of the respiratory chain to better understand the effect of these rare pathogenic mutations and to design new strategies for possible therapeutic options.

[1] Rugolo M, Zanna C, Ghelli AM. Organization of the Respiratory Supercomplexes in Cells with Defective Complex III: Structural Features and Metabolic Consequences. Life (Basel). 2021 Apr 17;11(4):351

[2] Mishra SK. PSP-GNM: Predicting Protein Stability Changes upon Point Mutations with a Gaussian Network Model. Int J Mol Sci. 2022 Sep 14;23(18):10711

The aim of the study was to understand the connections between neuro-inflammation, glutamate toxicity and mitochondrial dysfunction, three pathologic events, which accompany brain injury and majority of neurodegenerative diseases. We examined neuronal culture, experimental brain injury in rats as well as patients with subarachnoid hemorrhage (SAH). In rats we showed that during the acute post-traumatic phase, NO levels in the brain reached several micromoles. These data are consistent with the amount of NO determined in brain micro-dialysates from patients with SAH. The experiments with cortex homogenates and neuronal cell cultures showed that these NO levels first significantly reduce the activity of oxoglutarate dehydrogenase complex (OGDHC); further increase in NO levels impairs mitochondrial respiration. The loss of OGDHC activity inhibits uptake of glutamate by mitochondria, thus facilitating its extracellular accumulation and stimulating toxic glutamate pathway. High levels of extracellular glutamate lead to dysregulation of intracellular redox homeostasis and cause severe mitochondrial dysfunction leading to cell death. In vivo and in vitro experiments show that thiamine, a precursor of OGDHC cofactor, recovers mitochondrial function and improves the viability of neurons. Similarly, elevated levels of NO metabolites and glutamate in brain micro-dialysates were associated with poor outcome in SAH patients. Therefore, during brain injury, mitochondria influence neuron function in a biphasic manner. First, they inhibit glutamate absorption and induce glutamate toxicity, and second, they are themselves affected by the glutamate toxicity that they have induced.

Skin cutaneous melanoma (SKCM) is the deadliest form of skin cancer due to its high heterogeneity that drives tumor aggressiveness. Melanoma plasticity consists of two distinct phenotypic states that co-exist in the tumor niche, the proliferative and the invasive, respectively associated with a high and low expression of MITF (Microphthalmia-associated transcription factor), the master regulator of melanocyte lineage. However, despite efforts, melanoma research is still far from exhaustively dissecting this phenomenon.

We discovered a key function of Transglutaminase Type-2 (TG2) in regulating melanogenesis by modulating MITF transcription factor expression and its transcriptional activity. Importantly, we demonstrated that TG2 expression affects melanoma invasiveness, highlighting its positive value in SKCM. These results suggest that TG2 may have implications in the regulation of the phenotype switching by promoting melanoma differentiation and impairing its metastatic potential. Our findings offer potential perspectives to unravel melanoma vulnerabilities via tuning intra-tumor heterogeneity.

To investigate the possible interplay between TG2 expression and melanoma cell metabolism, we assayed mitochondrial fitness as well as other metabolic cellular pathways in wild-type and TG2 KO melanoma cells. We demonstrated that TG2 KO cells show an impairment in mitochondrial respiratory chain complexes expression and activity, thus impacting on mitochondrial respiration, hence the TG2 key role in metabolic homeostasis. In TG2 KO melanoma cells, some complexes of the respiratory chain, in particular the complex I, seem to be downregulated. Therefore, this down-modulation results in an impairment of mitochondrial respiration. These data let us to hypothesis that TG2 expression may impact on the modulation of tumor metabolism in melanoma cells.

TG2 capability to modulate oxidative phosphorylation, in addition to its ability to drive the phenotypic switch, indicates interesting perspectives for its use as a therapeutic target in melanoma cells. Its modulation can potentially reverse resistance to currently available drugs or enhance their effects.

Wayne Willis¹, Benjamin Willis², Paul Langlais¹, and Lawrence Mandarino¹

¹Department of Medicine, University of Arizona, Tucson, Arizona, United States of America

²Department of Electrical, Computer, and Energy Engineering, Arizona State University, Tempe, Arizona, United States of America

E-mail: waynewillis@arizona.edu

Interpretation of ³¹P-MRS Assessment of ΔG_ATP:J_ATP Elasticity in Human Skeletal Muscle Requires Estimation of the Oxidative Fuel Mixture

Magnetic resonance spectroscopy (³¹P-MRS) can be used to non-invasively evaluate the linear relationship between the cytosolic free energy of ATP hydrolysis (ΔG_ATP) and the rate of mitochondrial oxidative phosphorylation (J_ATP). Based on the assumptions of nonequilibrium thermodynamic (NET) metabolic control and that only one “fuel battery” drives the matrix redox potential [1], the slope of this relationship (the elasticity of J_ATP with respect to ΔG_ATP) reflects the conductance of the oxidative pathway. This important question, whether ΔG_ATP:J_ATP elasticity does in fact provide fundamental insight into the structural and functional capacity of mitochondria to deliver ATP power to ATP utilization sites, was studied in 16 healthy human volunteers. Proteomic abundances of over 500 mitochondrial proteins, along with the muscle total creatine concentration (TCr), were measured in biopsies of vastus lateralis muscle. Fuel selection during mild intensity cycle ergometer exercise was measured using indirect calorimetry. Finally, during a four-minute recovery from mild exercise, ³¹P-MRS measurements of muscle energy phosphates were used to evaluate tau (τ), the time constant of ΔG_ATP recovery. Proteomics revealed a 3-fold span of oxidative phosphorylation (OxPhos) protein abundance ([Mito]) in the subjects and nearly constant ratios of OxPhos pathway segments to each other in all subjects. [Mito] predicted fuel selection during mild exercise (r=0.71, p=0.002); the fractional contribution of carbohydrate rose as [Mito] fell. However, there was no relationship between [Mito] and either ΔG_ATP:J_ATP elasticity or the TCr/τ ratio (in the “one-fuel model”, TCr/τ equals the conductance of the OxPhos pathway [1]). A two-fuel NET-based computational model was developed, which accounts for the dramatically different K_eq values of β-oxidation (K_eq < 10) vs Pyruvate Dehydrogenase (K_eq > 500,000). Model simulations of mild exercise revealed that low [Mito] muscle compensates for lower OxPhos conductance by preferentially oxidizing carbohydrate, which provides stronger defense of matrix redox energy thus higher than expected ΔG_ATP:J_ATP elasticity. We conclude that ΔG_ATP:J_ATP elasticity must be interpreted in the context of the fuel selected by the exercising muscle.

[1] R Meyer, A linear model of muscle respiration explains monoexponential phosphocreatine changes. Am J Physiol 254 (1988) C548-C553.

Heart attack (cardiac ischemia) is a significant cause of mortality and morbidity worldwide. During reperfusion (the restoration of organ blood flow), reactive oxygen species (ROS) are released from mitochondrial complex I by reverse electron transport (RET) or from complex III, leading to cellular damage. Importantly, drugs that protect mitochondria provide direct protection against cardiac infarction. Another effective method to protect the organ from ischemic damage involves inducing short periods of ischemia intercalated with brief periods of reperfusion, also known as ischemic preconditioning (IP). The IP leads to the activation of the mitochondrial ATP-sensitive potassium channels (mitoKATP). This channel is located in the inner mitochondrial membrane and possesses protective cellular properties. Indeed, glibenclamide (a mitoKATP antagonist) prevents the cardiac protection induced by IP or by the diazoxide-induced mitoKATP opening. However, the mechanisms by which these drugs function and their effects under control and IP conditions remain poorly understood. Here we show that a truncated glibenclamide which lacks the sulphonylurea portion (called IMPA) is incapable of blocking the diazoxide or IP-induced cardiac protection in a Langendorff rat model of cardiac ischemia/reperfusion. Indeed, glibenclamide blocked IP/diazoxide-induced cardiac protection. Our data also indicate that isolated mitochondria from hearts subjected to ischemia/reperfusion produced higher levels of ROS when fed with succinate in a manner sensitive to rotenone (characterizing RET-induced ROS) or when fed with malate/glutamate, compared to mitochondria from hearts subjected to IP. Interestingly, glibenclamide blocked IP protection by increasing ROS in both situations, but IMPA was not active in doing so. Mitochondria isolated from IP-treated hearts had preserved levels of protein sulfhydryls, which were negatively impacted by glibenclamide but not by IMPA treatment. Using a clark-type electrode we also found that IP and IP plus IMPA had preserved malate/glutamate or succinate-driven mitochondrial respiration in state 3 (high O₂ consumption coupled to ATP synthesis). Interestingly, blocking complex I with rotenone restored succinate-driven respiration in mitochondria isolated from hearts treated with IP plus glibenclamide. This effect was absent in ischemic mitochondria. These results demonstrate that the cyclohexylurea group is essential for glibenclamide's actions toward blocking mitoKATP and IP protection. These findings could pave the way for the development of new therapeutic approaches to address ischemia-reperfusion injury.

Individual complexes of the mitochondrial oxidative phosphorylation system (OXPHOS) are not linked solely by their function; they also share dependencies at the maintenance/assembly level, where one complex depends on the presence of a different individual complex. Despite the relevance of this ‘interdependence’ behavior for mitochondrial diseases, its true nature remains elusive. To understand the mechanism that can explain this phenomenon, we examined the consequences of the aberration of different OXPHOS complexes in human cells. We demonstrate here that complete disruption of each of the OXPHOS complexes resulted in a perturbation in energy deficiency sensing pathways, including the integrated stress response (ISR) pathway. The secondary decrease of complex I (CI) level was triggered by both complex IV and complex V deficiency, and it was independent of ISR signaling. On the other hand, we identified the unifying mechanism behind cI downregulation in the downregulation of mitochondrial ribosomal proteins and, thus, mitochondrial translation. We conclude that the secondary cI defect is due to mitochondrial protein synthesis attenuation, while the responsible signaling pathways could differ based on the origin of the OXPHOS defect.

Funded by the Czech Science Foundation (22-21082S, 21-18993S), Czech Health Research Council (NU22-01-00499), and Next Generation EU project National Institute for Research of Metabolic and Cardiovascular Diseases (Programme EXCELES, ID LX22NPO5104).

RATIONALE

Alveolar Macrophages (AMs) are the sentinel cells in the lung, which clear bacteria and initiate inflammation. Gram-negative bacteria release outer membrane vesicles (OMVs) into the extracellular environment and antibiotics increase OMV production. OMVs of bacteria contain different types of cargo such as proteins, lipids, and nucleic acids. As colonization with Klebsiella pneumoniae (K. pneumoniae) constitutes a risk factor for infection, we hypothesized that OMVs of K. pneumoniae might alter bactericidal properties of AMs to facilitate pneumonia.

METHODS

K. pneumoniae were cultured in vitro, treated with different subinhibitory concentrations of antibiotics, and the secreted OMVs were isolated. Murine AMs were harvested by bronchoalveolar lavage and treated with OMVs. Bactericidal properties were assessed in AMs infected with viable K. pneumoniae ex vivo. Reactive oxygen species (ROS) were quantified by flow cytometry. Cytokines were measured using a multiplex bead-based assay. Oxygen consumption rate (OCR) and glycolysis were measured using an extracellular flux analyzer.

RESULTS

Preincubation with OMVs significantly decreased the killing capacity of AMs. In line, intratracheal instillation of OMVs facilitated bacterial outgrowth in a subsequent infection with K. pneumoniae. Whereas, OMVs did not alter cytosolic ROS production, they abrogated mitochondrial (mt) ROS release and decreased cellular respiration in AMs in response to K. pneumoniae. Specifically, OMVs isolated from K. pneumoniae treated with meropenem or piperacillin/tazobactam had the strongest effect. Human AMs were functionally similarly altered by OMVs. Inactivation of proteins and not DNA or RNA in permeabilized OMVs (perOMVs) abrogated inhibition of mtROS release upon bacterial encounter. Subsequently, the inactivation of proteins in perOMVs reverse the killing capacity of AMs. The proteomics data showed that OMVs of K. pneumoniae treated with meropenem revealed different protein composition compared to OMVs of non-treated bacteria. By using a BamA inhibitor to stop producing outer membrane proteins, we showed the effects of OMVs are reversed.

CONCLUSIONS

In summary, we found that OMVs of K. pneumoniae dampen the killing capacity of AMs. Therefore, we suggest that OMVs might facilitate the transition from bacterial colonization to infection in the lung by decreasing bactericidal properties of AMs.

Muscle mitochondrial function is altered in insulin resistant states but its assessment requires invasive muscle biopsies. While blood cell-based bioenergetics hold potential for revealing systemic mitochondrial changes, its association with muscle mitochondrial function remains unexplored. We evaluated respiratory capacity of skeletal muscle mitochondria and peripheral blood mononuclear cells (PBMCs) from patients with type 2 diabetes (T2D) and healthy participants (CON) and assessed, whether the latter reflect muscle mitochondrial respirometric measures.

This study involved 17 patients with T2D (35% female, 57±7 years, BMI 28±4 kg/m²) and 17 age- and sex-matched CON (35% female, 57±7 years, BMI 26±4 kg/m²). We took muscle biopsies from the M. vastus lateralis and venous blood samples to isolate PBMCs. High-resolution respirometry was performed in duplicate to assess mitochondrial respiration from permeabilized muscle fibers and PBMCs using an established SUIT-protocol.

Skeletal muscle NADH-linked OXPHOS capacity, NADH- and succinate-linked OXPHOS capacity as well as maximal respiratory capacity were lower in T2D vs CON (all p<0.01). No such between-group differences were found for PBMC mitochondrial function. In CON only, there was a significant correlation between the PBMC and muscle mitochondrial P/E ratio (r=0.63; p<0.01) while no other associations were found for PBMCs and muscle bioenergetics across several respiratory states in both groups (all p>0.05). In T2D, muscle NADH- and succinate-linked OXPHOS capacity correlated negatively with diabetes disease duration (r=-0.50, p=0.02).

These results suggest an impairment of muscle, but not PBMC mitochondrial function in individuals with type 2 diabetes. In this way, the duration of diabetes negatively correlates with muscle mitochondrial function. In CON only, certain bioenergetic features of PBMCs associate with muscle mitochondrial function.

Endothelial cells (ECs) form the inner layer of blood vessels and play a key role in various physiological and pathological processes. In response to various stimuli, ECs can switch between states of quiescence and activation. It is a generally accepted view that, despite having functional mitochondria, ECs primarily rely on glycolysis for ATP production. However, ECs are highly heterogeneous and their phenotypic features strongly depend on the vascular bed that may influence their energy metabolism pattern. In particular, liver sinusoidal ECs (LSECs) maintain the main functional aspects typical for ECs in other locations; however, they also have unique morphological and functional features. Structurally, LSECs have dynamically regulated fenestrae. Functionally, LSECs show high endocytic capacity, scavenge blood-born waste macromolecules, and present antigens, playing an important role in the immunological response. In addition, LSECs inhabit the environment of the liver, which is rich in metabolic substrates and can also influence their energy metabolism.

In fact, our results clearly demonstrate that in primary murine LSECs, mitochondrial ATP production prevails over glycolysis. To further explore this peculiarity of LSECs, we comprehensively characterised the energy metabolism of LSECs using the Seahorse XF technique, mass spectrometry–based proteomics and metabolomics, and Raman spectroscopy imaging. The oxidative phosphorylation in LSECs was efficiently fuelled by glucose-derived pyruvate, short- and medium-chain fatty acids, and glutamine. In turn, despite its high availability, palmitic acid was not directly oxidised in mitochondria, as evidenced by the acylcarnitine profile and the lack of the effects of etomoxir on oxygen consumption. Surprisingly, together with L-carnitine, palmitic acid supported mitochondrial respiration, suggesting the chain-shortening role of peroxisomal β-oxidation of long-chain fatty acids before further degradation and ATP production in mitochondria. This phenomenon might be instrumental in the harmless use of fatty acids in LSECs in the liver microenvironment. In conclusion, LSECs show a unique bioenergetic profile of highly metabolically plastic ECs adapted to the liver environment, the functional consequences of which will be discussed.

Coenzyme Q (CoQ) is a lipophilic electron carrier in the oxidative phosphorylation system (OXPHOS), whose biosynthesis is regulated by the multiprotein complex Q localised on the mitochondrial inner membrane. ARCA2 is a primary CoQ deficiency with heterogeneous symptoms, among which cerebellar ataxia is the main clinical symptom in patients with mutations in the COQ8A gene. Less than 100 patients have been described worldwide. We present a model HEK293 cell line (COQ8A-KD) showing only a mild OXPHOS deficiency, like the findings in a 38-year-old ARCA2 patient with slowly progressive cerebellar ataxia and cerebellar atrophy diagnosed at the age of 30.

HEK293 cells were modified by CRISPR/Cas9 plasmid-based technique. Total CoQ was measured by HPLC, OXPHOS activities by spectrophotometry. Gene expression was analysed by qPCR and Western blot, respiration by polarography.

In COQ8A-KD cells, where less than 7% of WT COQ8A mRNA was present, the CoQ content decreased to 17%. COQ8B expression was unchanged. The mild OXPHOS deficiency was detected in respiration with complex I substrates. The ARCA2 patient carries two mutations (trans) in the COQ8A gene resulting in c.1012G>A (p.A338T) and c.1018T>G (p.S340A), COQ8B expression in fibroblasts was unchanged. CoQ levels in her lymphocytes were significantly reduced. OXPHOS activity and content in her fibroblasts remained unaffected. The patient noticed an improvement since the beginning of the administration of bioactive ubiquinol (19 mg/kg/day) for 8 months.

Here we present biochemical properties of COQ8A mutant HEK293 and patient cells showing only a mild defect in OXPHOS, although CoQ content was severely reduced. Moreover, there is apparently no association and compensatory effect in COQ8B expression in COQ8A-KD and ARCA2 patient fibroblasts.

This work was supported by VFN: NU22-01-00499, RVO VZ 64165; and 1.LF UK SVV 260631.

Severe impairment of respiratory complex I (CI) has been demonstrated to slow down tumor progression, thus prompting preclinical studies on the use of its inhibitors in different types of cancers. However, we demonstrated that cancer cells lacking CI are able to activate adaptive responses that support survival despite the energetic deficit, thus allowing the subsequent tumor repopulation. Since such mechanisms are far from being understood, we generated and exploited multiple cancer cells of different origin lacking CI (CI^null) to elucidate their response to energetic stress. Upon glucose restriction, CI^null cells showed significant mitochondrial network fragmentation and depolarization due to activation of OMA1, as shown by OPA1 and PGAM5 cleavage and DRP1 dephosphorylation. These mitochondrial alterations were accompanied by a consistent activation of Integrated Stress Response (ISR), which is orchestrated by OMA1 and suppressed when the protease expression is downregulated. Similar results were obtained by specific CI pharmacological inhibition with EVP-4593. Moreover, we found that glucose starvation triggered the accumulation of lipid droplets (LDs) in contact with deranged mitochondria in vitro and in vivo, accompanied by a profound remodelling of lipid content, in particular of triacylglycerols and in cholesteryl esters. However, inhibition of LDs biogenesis did not affect cell viability, suggesting that they are not implicated in cell survival upon glucose restriction. On the other hand, OMA1 silencing and the consequent block of ISR in CI^null cells trigger cell death, indicating that mechanism is necessary to support cell survival. Overall, this study revealed that CI dysfunction, glucose restriction and ISR are in synthetic lethality, thus pointing to a combined metabolic approach to eradicate tumors.

Luisa Iommarini is supported by the National Recovery and Resilience Plan “Italia Domani”, Mission 4 - Component 2 - Investment 1.4 - National Center for Gene Therapy and Drugs based on RNA Technology (Spoke: 1 – Genetic diseases)

Changes in mitochondrial morphology are associated with nutrient utilization, but the precise causalities and the underlying mechanisms remain unknown. Using cellular models representing a wide spectrum of mitochondrial shapes, we show a strong linear correlation between mitochondrial fragmentation and increased fatty acid oxidation (FAO) rates. Forced mitochondrial elongation following MFN2 over-expression or DRP1 depletion diminishes FAO, while forced elongation upon knockdown/knockout of MFN2 augments FAO as evident from respirometry and metabolic tracing studies. Importantly, genetic induction of fragmentation phenocopies the distinct cell type-specific biologic functions of FAO. These includes stimulation of gluconeogenesis in hepatocytes, induction of insulin secretion in islet β-cells exposed to fatty acids, and survival of FAO-dependent lymphoma subtypes. We find that fragmentation stimulates long-chain but not short-chain FAO, identifying CPT1 as the downstream effector of mitochondrial morphology in regulation of FAO. Mechanistically, we determined that fragmentation reduces malonyl-CoA inhibition of CPT1, while elongation increases CPT1 sensitivity to malonyl-CoA inhibition. Overall, these findings underscore a physiologic role for fragmentation as a mechanism whereby cellular fuel preference and FAO capacity are determined.

Adverse childhood experiences (ACEs) encompass a variety of stressors encountered by children and adolescents, including household dysfunctions and experiences of violence, abuse, and neglect. ACEs affect more than 20% of all children worldwide. ACEs often occur repeatedly and have profound effects not only on mental health, like Major Depression Disorder (MDD), but also on various physical illnesses. Individuals with a history of ACEs show an increased risk for severe health complications, while the biological mechanisms and the underlying pathophysiology remain hardly understood.

A growing body of evidence suggests that mitochondrial physiology could present a biological function underlying this risk. Mitochondria are sensitive to endogenous and exogenous stressors, exhibiting susceptibility to oxidative stress and resulting inflammatory states. Studies have shown increased mitochondrial activity in individuals with ACEs, suggesting a dose-response relationship with the severity and timing of childhood trauma. Therefore, it is important to better understand the impact of ACEs on the transition from (mental) health to disease, with mitochondrial bioenergetics in PBMC from individuals with and without depression.

In a collaboration with the University Hospital Zurich (project CCO-NIRS), participants with and without MDD completed the Childhood Trauma Questionnaire (CTQ). In addition, the severity of depressive symptoms was determined using the Beck Depression Inventory (BDI), together with the Hamilton Depression Rating Scale(HDRS). PBMC were isolated from whole blood, and cryopreserved cells were analyzed with O2K oxygraph technology. High-resolution respirometry was performed using a SUIT protocol for the assessment of oxygen consumption states and flux control ratios.

Data analysis investigated the relationships between ACEs, mitochondrial respiration in PBMC, and the clinical severity.

The 140 participants were divided into four groups (ACE^- MDD^-with N=61, ACE^- MDD⁺ with N=24, ACE⁺MDD^- with N= 19 and ACE⁺MDD⁺ with N= 37) and the group differences were examined. The statistical examination is currently being processed, and results will be presented at EBEC 2024.

Coenzyme A (CoA) is a central cofactor in cellular metabolism. The final two steps of CoA de-novo synthesis are catalyzed by the bifunctional CoA synthase (COASY). Pathogenic biallelic variants in COASY gene result in COASY protein-associated neurodegeneration (CoPAN).

In our study, we present a complex clinical, tissue, biochemical, and molecular genetic case study of a male patient affected by a fatal fulminant neonatal-onset hepathopathy with hypoglycaemia, hyperammonaemia and lactic acidosis. The patient died at 6 days of age due to multi-organ dysfunction dominated by liver failure.

Two novel pathogenic COASY variants were identified in the patient by whole exome sequencing. While c.612C>G (p.His204Gln) variant compromised the predicted active of the COASY 4’-phosphopantetheine adenylyltransferase domain, c.1485+1G>T (p.Lys464Cysfs*12) resulted in skipping of exon 7 and non-sense mediated decay of the COASY transcript. No enzymatic COASY activity was detected in the patient’s fibroblasts which corresponded to minimal amounts of residual mutated protein by Western blotting. Parallel negative impacts of COASY deficiency on the mitochondrial function were hallmarked by overall reduction of the amount and activity of cytochrome c oxidase (complex IV) and decreased mitochondrial respiration and ATP production.

Supported by: AZV NU22-07-00614; AZV NU22-01-00499; RVO VFN 64165; Cooperacio Programme, Pediatrics 207040-1; UNCE/24/MED/022, LX22NPO5107.

Mitochondrial dysfunction is implicated in a vast array of rare and common disease, but the exact mechanisms by which this dysfunction is involved in the aetiology and progression of many diseases remain to be elucidated. Additionally, the tissue specific effects of mitochondrial dysfunction are poorly understood [1]. Thus, investigations focused on the role of mitochondrial function in health and disease are very complex. Here we utilised a comprehensive multi-omics approach to generate hypotheses [2] on the mechanistic processes responsible for perturbations related to mitochondrial disease, using the NDUFS4 KO (complex I) mitochondrial disease mouse model. We produced large proteomics and transcriptomics datasets for six tissues (heart, liver, kidney, olfactory bulb, cerebellum and brainstem) of these mice and show significantly differentially expressed proteins and transcripts in all tissues. We focused on tissue specificity by first identifying proteins and transcripts that were uniquely affected in each tissue, before conducting pathway enrichment analysis. We show that distinct processes of the same pathways were affected in different tissues, suggesting compensatory mechanisms between different tissues/organs. We go on to discuss selected pathways of interest that were identified for follow-up hypothesis-testing studies. In this way, we hope to better understand the exact mechanisms that underline disease instead of only understanding the overall perturbations, to further identify novel therapy targets [3].

[1] D. Pacheu-Grau, R. Rucktäschel, M. Deckers, Mitochondrial dysfunction and its role in tissue-specific cellular stress, Cell Stress, 2 (2018) 184-199.

[2] A.C. Schrimpe-Rutledge, S.G. Codreanu, S.D. Sherrod, J.A. McLean, Untargeted Metabolomics Strategies-Challenges and Emerging Directions, J Am Soc Mass Spectrom, 27 (2016) 1897-1905.

[3] M. Di Nottia, D. Verrigni, A. Torraco, T. Rizza, E. Bertini, R. Carrozzo, Mitochondrial Dynamics: Molecular Mechanisms, Related Primary Mitochondrial Disorders and Therapeutic Approaches, Genes (Basel), 12 (2021).

To measure membrane potential, a crucial parameter in studying mitochondrial bioenergetics, safranin serves as a convenient and economical fluorescent probe, but also possesses electroactive properties. Using a voltametric electrode coupled with an Oroboros Oxygraph system, we detected a previously undescribed redox interaction between safranin and isolated mitochondria. This interaction became evident as the oxygen tension in the suspension decreased to levels undetectable by an oxygen electrode, following exhaustive respiration in the presence of excess substrates and ADP. The appearance of a reduced electroactive substance derived from safranin slightly preceded a decrease in safranin fluorescence with similar kinetics.

Triphenyl tetrazolium chloride, but not tetramethylrhodamine methyl ester (TMRM) or rhodamine 123, exhibited similar reducibility to safranin. Reduction of safranin under anoxic conditions occurred in isolated mitochondria in the presence of NADH-linked substrates, or in permeabilized mitochondria (albeit at lower levels) in the presence of exogenously added NADH, but not NAD+.

Succinate dehydrogenase operating in reverse during anoxia, fueled by fumarate, prevented the accumulation of reduced safranin species in an atpenin- or malonate-sensitive manner. However, no ETC inhibitor of any respiratory complex influenced the reduction of safranin under anoxic conditions. Although the specific mitochondrial entity responsible for the 'forward' redox interaction with safranin remains unidentified, a practical application of observing the reduced safranin species fluorimetrically could be to check for anoxic conditions in spaces where an oxygen electrode is not present.

Rapid environmental alterations, particularly hypobaric hypoxia encountered at high altitudes, impose various physical challenges that can lead to severe health consequences even during short exposures. Such conditions will have an acute impact on the cardiovascular, pulmonary, and neurological systems, triggering significant physiological and psychological stress responses. Understanding these responses during a short-term high-altitude exposure is critical for disclosing the pathophysiology of acute altitude illnesses and potential chronic health implications.

This study aims to comprehensively assess whether there are any physiological and psychological maladaptive reactions caused by acute hypoxic stress through monitoring various physiological values ​​of healthy adults exposed to an altitude of 2,500 meters for 3 days.

We compared multiple indexes such as blood oxygenase-1 (HO-1) metabolite (bilirubin), psychological stress marker (urinary biopyrrin), mitochondrial oxygen consumption, hematological assessments (red/white blood cell count, hemoglobin concentration), and arterial oxygen tension (PaO2) to improve our understanding of human tolerance to sudden environmental stress. These detailed analyses help develop strategies for emergency medicine, aviation and aerospace medicine, and could guide the development of clinical protocols to prevent and treat acute high-altitude illness.

S. cerevisiae can generate energy through fermentation and aerobic respiration, either separately or simultaneously, depending on the availability of carbon sources [1]. Acetic acid (AA) inhibits fermentation by inducing the accumulation of reactive oxygen species, which can damage proteins and DNA [2]. This study aims to investigate the action mechanisms of AA on S. cerevisiae ATCC 9804 (wine) and ATCC 13007 (beer) strains at pH 3.0, under aerobic and oxygen-limited conditions. The specific growth rates (SGR) were determined by monitoring the yeast culture growth and sodium flux across cell membranes was measured using selective electrodes. In yeast batch culture, the dissolved oxygen level was 5 times lower in oxygen-limited growth conditions compared to aerobic conditions (17.5%). Increasing the AA concentration during growth reduces the SGR in a concentration-dependent manner. 10 mM AA did not have a significant effect on the SGR of both strains under aerobic and oxygen-limited conditions. 20 mM AA inhibits the growth of both yeast strains by 1.3-fold, regardless of the availability of oxygen in the growth medium. Furthermore, 50 mM AA reduced the SGR of ATCC 9804 and ATCC 13007 strains by 4.4 and 15.5-fold under aerobic conditions, and by 5.8 and 27-fold under oxygen-limited conditions, respectively. The sodium flux in the ATCC 9804 strain is N′N'-dicyclohexylcarbodiimide (DCCD)-dependent under aerobic and physiological conditions, unlike the ATCC 13007 strain. During oxygen-limited growth, sodium flux is DCCD-dependent in both strains and is twice as high in the ATCC 9804 strain compared to aerobic conditions. The total and DCCD-dependent sodium efflux decreases by 1.8-fold (ATCC 9804) and 1.6-fold (ATCC 13007) when exposed to 10 mM AA compared to physiological conditions. Under stress conditions caused by higher AA concentrations, there are no significant changes in the total sodium flux rate, but the DCCD-dependent sodium flux increases significantly, especially under oxygen-limited conditions. Specifically, under the influence of 50 mM AA, the DCCD-sensitive sodium flux is ~25% and ~60% in the ATCC 13007 strain under aerobic and oxygen-limited conditions, respectively. In the ATCC 9804 strain, regardless of the presence of oxygen during growth, the DCCD-sensitive sodium flux is 32%. These findings demonstrate that plasma membrane sodium ion channels and ATPases, as well as their interactions, play a crucial role in the response and adaptation to AA, especially under low pH and oxygen conditions and will be important for optimizing yeast metabolism to produce alcoholic beverages, biofuels, and other biotechnological applications.

Alternative quinol oxidase (AOX) is an enzyme that transfers electrons from reduced quinone directly to oxygen without proton translocation. When AOX from C.intestinalis is expressed in transgenic mice, it can substitute the combined activity of mitochondrial respiratory complexes III/IV [1]. Here, we characterized bioenergetics of such a synthetic respiratory chain using brain mitochondria from AOX-expressing mice. The content and activity of respiratory chain complexes were not affected by AOX expression. The respiratory control ratio (RCR) and ADP:O were similar in mitochondria from wild-type and transgenic mice, indicating that the efficiency of oxidative phosphorylation was not compromised by AOX expression. If measured at temperatures below 37°C, RCR was decreased in AOX-containing mitochondria oxidizing succinate due to the accelerated respiration in non-phosphorylating State 2. When oxidizing malate/pyruvate, RCR was not affected.

The complex IV inhibitor cyanide only partially blocked respiration by AOX-containing mitochondria. The fraction of cyanide-insensitive respiration increased with temperature decrease due to the attenuation of the activity of mammalian respiratory enzymes at lower temperatures and therefore, lesser contribution to the overall respiration.

Mitochondrial transmembrane potential in AOX-containing mitochondria was more sensitive to cyanide during succinate oxidation than during malate/pyruvate-supported respiration. This indicates a difference in the proton-translocating capacity upstream of the quinone pool for these substrates. High concentrations of cyanide fully collapsed membrane potential during succinate, but not malate/pyruvate-supported respiration, confirming electroneutral redox activity of AOX.

Succinate oxidation promotes reverse electron transfer (RET) towards complex I, supporting the highest rate of H₂O₂ production in intact mitochondria. AOX expression significantly decreased RET-induced H₂O₂ formation with the effect more pronounced at low temperatures. Inhibitor-sensitivity analysis showed that the AOX-induced decrease in H₂O₂ production rate was due to the lower contribution of complex I to the net ROS production.

Overall our results suggest AOX as a useful tool to study electron transfer in the synthetic mammalian respiratory chain when both AOX and complexes III/IV are present.

[1]. M. Szibor, P. Dhandapani, E. Dufour, K. Holmström, Y. Zhuang, et al. Broad AOX expression in a genetically tractable mouse model does not disturb normal physiology. Dis.Model Mech. 10 (2017) 163-171.

The ATP synthase inhibitor Bedaquiline (BDQ) is the cornerstone of new regimens that have accelerated the treatment multidrug-resistant tuberculosis. Despite its clear importance, its precise bactericidal mechanism continues to debated: specifically whether BDQ binding to ATP synthase disrupts the proton motive force by inducing a leaky ‘uncoupled’ state, or simply blocks catalysis. To unravel its mechanism of action we applied non-invasive methods to study the effects of BDQ on the oxidative phosphorylation system in live mycobacteria. Via the use of a specialised bioenergetic chamber we are able to monitor changes in cytochrome oxidation states through multi-wavelength remission spectroscopy while measuring the oxygen consumption rate of cells in real time. We find that BDQ’s effects do not match those of established uncouplers/ionophores that disrupt the proton motive force. Our results demonstrate BDQ acts as a direct inhibitor of ATP synthase and its effects on oxygen consumption are the result of electrons being routed through cytochrome bd oxidase. This strategy by the bacteria limits their susceptibility to back pressure and highlights bd oxidase as a key target for future combination therapies to increase the effectiveness of BDQ.

Cytochrome bd from Mycobacterium tuberculosis (Mtbd) is a menaquinol oxidase that has gained considerable interest as an antibiotic target due to its importance in survival under infectious conditions. Mtbd contains a characteristic disulfide bond that has been hypothesized to confer a redox regulatory role during infection by constraining the movement of the menaquinone-binding Q-loop. Here, the role of the disulfide bond and quinone specificity of Mtbd has been determined by the reconstitution of a minimal respiratory chain consisting of a NADH dehydrogenase and Mtbd, both in detergent and native-like lipid environments. Comparison to cytochrome bd from Escherichia coli (Ecbd) confirms that Mtbd is under tight redox regulation and is selective for menaquinol, unable to oxidize either ubiquinol or demethylmenaquinol. Reduction of the Mtbd disulfide bond resulted in a decrease in oxidase activity up to 90%. The cryo-EM structure of Mtbd at 2.9 Å resolution confirms that the disulfide bond is broken in this inactive form, but no major conformational changes are observed outside the Q-loop. We propose a new menaquinol binding site in the Q-loop of Mtbd and signify Mtbd as the first redox-sensing terminal oxidase, enabling Mtbd to adapt its activity in defence against reactive oxygen species encountered during infection by M. tuberculosis.

Macrophages can be roughly divided in vitro into 2 subtypes: M1 proinflammatory cells, which display a glycolytic metabolism, and M2 or reparative cells, which largely rely on oxidative phosphorylation (OxPhos) for ATP supply. Previous research has indicated that induction of the M1 phenotype leads to an increase in ROS-induced DNA damage, resulting in the consumption of nicotinamide adenine dinucleotide (NAD⁺) by poly (ADP-ribose) polymerase 1 (PARP1). Consequently, cells heavily rely on the NAD⁺ salvage pathway to sustain glycolysis and other cellular functions. Blockade of this pathway thus interferes with M1 function. Conversely, inhibition of the de novo NAD⁺ synthesis pathway disrupts OxPhos and promotes glycolysis, thereby enhancing M1 differentiation. These seemingly contradictory findings suggest a model in which the compartmentalization of NAD⁺ between the cytosol and the mitochondria emerges as a critical modulator of macrophage differentiation. Recently, SLC25A51, also known as MCART1, was identified as the mitochondrial NAD⁺ carrier.

Utilizing genetically encoded fluorescent mitochondrial biosensors to monitor free NAD⁺, we observed a reduction in mitochondrial NAD⁺ levels during M1 differentiation, contrasting with increased levels of the cofactor during M2 differentiation. These findings underscore the pivotal role of NAD⁺ in driving M2 polarity. To specifically investigate the role of mitochondrial NAD⁺ in macrophage differentiation, we conducted experiments involving the silencing and overexpression of MCART1. Our findings revealed that knockdown of MCART1 led to increased phosphorylation of the transcription factor NF-κB. Moreover, the secretion of proinflammatory cytokines such as TNFα also increases in M1 cells lacking the transporter, suggesting that a decrease in mitochondrial NAD⁺ levels favors M1 polarity. Conversely, overexpression of MCART1 resulted in an upregulation of M2 macrophage markers, such as arginase, accompanied by a reduction in the release of pro-inflammatory cytokines, indicating that an increase in mitochondrial NAD⁺ levels promotes M2 polarity.

Given the pivotal role of macrophages in orchestrating inflammation and tissue repair, understanding the delicate balance between M1 pro-inflammatory and M2 reparative phenotypes is crucial for elucidating the pathogenesis of various diseases, including autoimmune disorders and cardiovascular conditions. Therefore, this research could unveil potential therapeutic targets in disease settings.

Background:

Organ acceptance is based on donor parameters, surgeons' experience and logistic factors. Viability assessment is often performed in already accepted livers during ex-situ machine perfusion and often includes downstream parameters of liver injury with limited sensitivity. New tests to identify suitable donors earlier are key to increasing organ utilization, reducing the number of dry runs and tailoring preservation. This is the first systematic analysis of markers of mitochondrial metabolism in organ donors.

Methodology:

Donor risk factors, blood and liver tissues were analyzed. Donor plasma was used to quantify mitochondrial injury through spectroscopy for Flavin-mononucleotide (FMN) and to measure danger-associated molecular patterns (Damps) and cytokines. Such results were correlated with parameters commonly used to decline donors and were linked to viability tests during hypothermic oxygenated (HOPE) and normothermic machine perfusion (NMP) and posttransplant outcomes.

Results:

Between 5/2023 and 12/2023, blood samples from 100 human donors were analyzed. Overall, 20 livers were transplanted, 12 at our center with end ischemic NMP (n=10, OrganOx-MetraÒ) or static cold storage (n=2) (Figure 1). Both donor types were present among discarded livers. Twelve livers underwent HOPE-treatment and assessment (VitasmartÒ). Donor factors (i.e., age, body-mass-index, cause of death, hospital stay, liver enzymes, inotrope support) did not correlate with mitochondrial markers or damps/cytokines in donors or perfusates. Median donor plasma FMN was 1.46mg/mL (IQR: 0.86; 2.04), with a good correlation to perfusate FMN during machine perfusion (r=0.7951, p=0.01). FMN values correlated further with damps-/cytokine perfusate levels.

Conclusion:

This is the first analysis of mitochondrial biomarkers in organ donors linking the metabolic donor risk with viability criteria during machine perfusion and outcome after transplantation. Mitochondrial biomarkers allow a more objective interpretation of donor and liver quality on a subcellular level, when compared to donor-derived data and other viability parameters. Donor plasma FMN correlated well with viability markers during both types of machine perfusion.

Obesity and its associated comorbidities, particularly metabolic syndrome, are a growing problem in developed societies. Due to its polygenic nature, the genetic component of metabolic syndrome is only slowly being elucidated. Common mitochondrial DNA (mtDNA) sequence variants have been associated with late-onset human diseases, including cardiovascular disease or type 2 diabetes mellitus, and may therefore be relevant players in the genetics of metabolic syndrome.

In the current project, we investigated the effect of physiological variation in the mitochondrial DNA sequence on symptoms of metabolic syndrome using our unique models of conplastic rats carrying mtDNA from the spontaneously hypertensive rat strain (mtSHR), Brown Norway strain (mtBN) or Fischer strain (mtF344) on the identical nuclear background (SHR). Sequence differences in mtDNA between these strains include structural genes for oxidative phosphorylation proteins, tRNAs, and rRNAs. We found that mtF344 developed insulin resistance on a high-fat diet. This could not be explained by the changes in inflammatory markers or mitochondrial ROS production. On the contrary, it was associated with reduced oxidative capacity of the heart but not liver mitochondria. Reduced fatty acid oxidation led to the accumulation of bioactive diacylglycerols and subsequent inhibition of insulin signalling. In the case of the mtF344 strain, we propose that these metabolic perturbations result from variation in the 12S rRNA sequence, which affects mitochondrial ribosome assembly and, subsequently, mitochondrial protein translation.

Our work has demonstrated that physiological sequence variation in mitochondrial rRNA may be a relevant underlying factor in the progression of metabolic syndrome.

Funded by the Next Generation EU project National Institute for Research of Metabolic and Cardiovascular Diseases (Programme EXCELES, ID LX22NPO5104).

Stargardt disease (STGD1) is the most prevalent juvenile inherited macular dystrophy, caused by mutations in the gene encoding the ATP-binding cassette transporter 4 (ABCA4). Loss of ABCA4 in STGD1 patients accelerates the build-up of lipofuscin pigments in the retinal pigment epithelium (RPE), ultimately leading to RPE and photoreceptors (PRs) degeneration. To date, no effective treatments are available for this disorder.

Abca4KO mice have been widely used as a model of STGD1. They are characterized by abnormal accumulation of lipofuscin, signs of chronic inflammation, and elevated levels of lipids peroxidation. Interestingly, we uncovered substantial mitochondrial alterations that have not been previously reported in these mice. Indeed, Abca4KO mice display a significant reduction of mitochondria in both RPE and PRs.Moreover, PRs mitochondria exhibit altered mitochondria morphology i.e. round-shaped cristae and partially swollen electron-lucent matrix associated with reduced level of Opa1 protein and transcript. Notable STGD1 patients-derived retinal organoids exhibit similar mitochondrial phenotype.

MicroRNAs (miRNAs) are small noncoding RNAs that finely regulate gene expression by simultaneous modulation of multiple biological pathways. The eye-expressed miR-181a/b regulates genes involved in mitochondrial biogenesis and function and ROS detoxification. Their inhibition preserves retinal cells from death and ameliorates visual function in different models of retinal disorders associated with mitochondrial dysfunction, indicating these miRNAs as attractive therapeutic targets for these pathologies. We tested the effect of miR-181a/b genetic inactivation in Abca4KO mice that results in a significant improvement of mitochondrial biogenesis, and in the preservation of PRs mitochondrial morphology recovering Opa1 levels. Moreover, Abca4KO;miR-181a/bKO mice show a strong reduction of lipofuscin content and lipids peroxides, and an amelioration of PRs and RPE phenotypes that results in increased cell survival. To test the translational potential of our approach, we generated AAV vectors encoding a miR-181a/b sponge, which allows long-term inhibition of miR-181a/b in vivo. Notably, the therapeutic agent is safe and effective in Abca4KO mice and STGD1 organoids ameliorating retinal phenotypes. Interestingly our results indicate the potential application of miR-181a/b modulation as a novel therapeutic strategy in STGD1 and in other common disorders affecting the macula such as the Age-Related Macular Degeneration.

LHON is the most common mitochondrial DNA disorder and causes optic nerve atrophy and blindness. Idebenone, the only EMA-approved therapy for LHON, is activated after reduction by the NAD(P)H oxidoreductase I (NQO1) enzyme and can shuttle the electrons directly to Complex III (CIII) bypassing the defective Complex I (CI). The use of idebenone for LHON treatment has shown efficacy, although there is a consistent subset of patients (about 50%) that fail to benefit visual improvement.

We investigated the role of NQO1 expression and activity on the efficacy of idebenone in control and LHON patient fibroblasts showing that only cells expressing NQO1 protein were able to increase or maintain respiration in presence of idebenone, when CI was inhibited. To explain the different levels of NQO1 expression in fibroblasts, we sequenced NQO1 gene identifying the presence of the two common polymorphic variants that are known to induce instability and degradation of the protein. The two variants were present in homozygous or in compound heterozygote combination in cells lacking NQO1 expression. Evaluation of the effect of the NQO1 variants on neuronal precursors respiration in absence/presence of idebenone confirmed the same results observed in fibroblasts. In addition, the retrospective analysis of a large cohort of idebenone-treated LHON patients showed that patients with homozygous or compound heterozygous NQO1 variants had the poorest therapy response. Hence, fibroblasts carrying NQO1 polymorphic variants were treated with drugs that increase NQO1 protein amount and stability and respirometry analysis revealed that, after treatments, cells mainly maintained respiration after CI inhibition.

Overall, we showed that NQO1 protein level is genetically defined, it is tightly associated with idebenone capability to restore mitochondrial respiration and it may be pharmacologically increased to improve the therapeutic efficacy of idebenone.

Mitochondrial diseases are a group of clinically heterogeneous hereditary disorders caused by faulty oxidative phosphorylation (OXPHOS). COX15 is an enzyme involved in the biogenesis of heme A, the prosthetic group of cytochrome c oxidase (COX). In humans, COX15 mutations are associated to Leigh syndrome and cardioencephalomyopathy. A skeletal-muscle specific knock-out mouse for Cox15 (Cox15^skm/skm), characterized by a severe myopathy with profound COX deficiency and impaired mitophagy, was crossed with a strain expressing the mitophagy reporter Mito‑QC (Cox15^skm/skm-MQC). MQC encodes a tandem GFP-mCherry fluorescent protein targeted to mitochondria. Once a tagged mitochondrion is delivered to the lysosome, the GFP signal is quenched in the acidic environment, while the mCherry remains visible as distinct, red puncta, allowing for the quantification of mitolysosomes number.

The aim of this work is to identify new mitophagy-inducing compounds whose mechanism of action is mediated by the activation of Transcription Factor EB (TFEB), the main transcription factor regulating autophagy and lysosomal biogenesis. We carried out a two-step in vitro screening of an FDA-approved drug library to identify compounds able to induce the translocation to the nucleus of TFEB and the activation of mitophagy.

TFEB-GFP overexpressing Mouse Adult Fibroblasts (MAFs) from wild-type animals were treated with 1971 drugs, followed by quantification of the GFP-positive nuclei in which nuclear translocation of TFEB occurred. This screening identified 294 putative TFEB activators, among which 147 drugs were selected for the secondary screening to assess mitophagy induction and mitochondrial fragmentation on MQC-expressing MAFs. MQC quantification identified 9 drugs causing a significant increase in mitolysosomes without fragmentation of the mitochondrial network.

We then evaluated these 9 compounds in patients-derived fibroblasts with COX defects as relevant cellular model of mitochondrial diseases. Among these, 5 drugs increased mitophagy flux by >20% without significantly altering the mitochondrial membrane potential, thus suggesting that they did not induce mitophagy by promoting depolarization. We also investigated their capacity to improve OXPHOS defects through mitophagy activation by measuring cellular respiration.

Positive hits will undergo validation on Cox15^‑/‑ cells before being tested in vivo on Cox15^skm/skm‑MQC mice.

Parkinson's disease (PD) is an incurable neurodegenerative disease and is associated with changes in the concentration of neurotransmitters, the accumulation of α-synuclein, the loss of dopamine production, and the death of dopaminergic neurons (DA) in the substantia nigra. The idiopathic form of PD (iPD) is the most common form of this disease, which affects more than 10 million people worldwide. There are currently no markers available in the clinic that would have a predictive character, and therefore the determination of the speed of disease progression is usually empirical and largely influenced by the neurologist's experience with this disease. One of the important factors in the development of PD is the impairment of mitochondrial functions and the occurrence of oxidative stress due to the aggregation of α-synuclein, which leads to the inhibition of complex I of the respiratory chain. Since the degree of the disease's progression and degradation of mitochondrial homeostasis (MiH) in neurons may be correlated with its stage and complexity, we suggested a set of techniques that would enable a multiparametric assessment of mitochondrial fitness (MiF). This relates especially to the integrity of the electron transport chain's constituent parts and mitochondrial respiration. We created a combination of procedures that are able to determine MiF as a parameter describing the state of MiH in the peripheral blood leukocytes of a patient with iPD. Our aim was to observe and compare the MiF parameters and to compare the iPD cohort, which consists of 26 newly diagnosed patients with disease stage 1 to 2 (according to the Hoehn-Yahr scale) with an average age of 66.8 ± 9.76 years, with a control cohort with a mean age of 65.64 ± 8.74 years, corresponding by gender and age to the iPD cohort with disease stage 1 to 2. The division of iPD patients into the cohort of patients with disease stages 1–2 is based on empirical observations of the different dynamics of disease progression in patients. We used high-resolution respiration (HRR) (Oroboros, AT) to analyze mitochondrial respiration using SUIT protocol 003 ce-cpe O2 and DatLab software. As part of the results, more than 200 parameters were evaluated, which we sorted according to importance in the importance plot. Furthermore, we worked with 68 parameters that were significant according to mathematical analyses. We found that up to 19 parameters specifically related to mitochondrial respiration are significant and thus represent key factors and markers influencing mitochondrial respiration.

The oxidation of nutrients generates reduced coenzymes (NADH and FADH₂) which drives oxidative phosphorylation (OXPHOS). Complex I of the OXPHOS system oxidizes NADH back to NAD⁺, allowing recurring transport of electrons from nutrients to the electron transport chain. When complex I is deficient, it not only impairs ATP production but also the recycling of NAD⁺, which leads to a redox (NADH: NAD⁺) imbalance and reductive stress (especially in the brain and heart). Several pathways (like fatty acid oxidation and ketogenesis) are activated with complex I deficiency in an attempt to restore energy levels; but because of the redox imbalance, fail to do so and accumulate toxic metabolic intermediates. Reduced intermediates (like lactate and 3-hydroxybutyrate) effectively transport electrons through the body and therefor play a role in each organ’s redox balance – which this study aimed to elucidate. Liver, brain and heart samples from Ndufs4 knockout (complex I deficient) and wild-type mice were collected. Redox metabolites were extracted and analysed with GC-MS/MS. Metabolite concentrations and ratios were determined and compared between the mice. Metabolites involved in redox metabolism were markedly perturbed in the brain and heart of Ndufs4 knockout (KO) mice, while the liver coped surprisingly well. 3-hydroxybutyrate levels were markedly higher in the brain and heart of the KO mice. Elevated 3-hydroxybutyrate: acetoacetate and lactate: pyruvate confirmed a high NADH: NAD⁺ ratio in these organs while a markedly lower ratio was found in the KO mice liver. The results indicate that the liver is able to alleviate reductive stress by exporting electrons to extrahepatic tissue via 3-hydroxybutyrate. Hepatic ketogenesis in the KO mice favours the reduction of acetoacetate to 3-hydroxybutyrate to recycle NAD⁺. 3-hydroxybutyate shuttled to extrahepatic tissue effectively transport electrons and therefor augment reductive stress in these organs. This result highlight potential targets for therapy aimed at restoring redox balance.

Background:

Mitochondrial bioenergetic dysfunction is a key factor driving the decline in cardiac contractile function in type 2 diabetic (T2D) patients and relevant animal models. Exogenous administration of mitochondria-derived peptides, notably MOTS-c, has emerged as a promising approach to enhance mitochondrial function in metabolic diseases. Clinical research and my data reveal decreased plasma MOTS-c levels in individuals with diabetes and coronary endothelial dysfunction as well as lower MOTS-c protein expression in the heart tissues of diabetic male rats respectively. This study investigates therapeutic effects of MOTS-c treatment on mitochondrial oxygen flux linking to ATP synthesis and reactive oxygen species (ROS) production rates in diabetic heart tissues.

Methods:

Male Wistar rats (150-200 g) were divided into three groups: control, untreated T2D, and treated T2D. The T2D groups were fed a high-fat diet (HFD) for 15 weeks and received streptozotocin injection (25 mg/kg) at week 8 post-HFD. The control group received an equivalent volume of saline injection and was fed with normal chow. At week 12, the treated T2D group received daily intraperitoneal MOTS-c injections (15 mg/kg/day) for 3 weeks, while untreated groups received saline injections. Blood glucose and body weight were monitored weekly. Glucose tolerance tests were conducted at week 15 post-HFD and heart tissues were collected for multi-scale experiments. All procedures adhered to the University of Auckland Animal Ethics requirements (AEC22653).

High-resolution fluo-respirometers were used to simultaneously measure mitochondrial oxygen flux alongside ATP synthesis rate (Magnesium Green) and ROS production rate (Amplex UltraRed). Specific titration assays employing various substrates and inhibitors were employed to selectively target respiratory complexes, offering insight into in vivo changes within the electron transport system complexes.

Results:

Compared to control rats, untreated T2D rats displayed elevated fasting blood glucose levels, impaired glucose tolerance handling, and lower mass-specific oxygen flux during oxidative phosphorylation with unchanged steady-state ATP synthesis and ROS rates. MOTS-c treatment improved glucose regulation with decreased blood glucose levels, and increased mitochondrial respiration and ATP synthesis rates compared to the untreated T2D group.

Conclusion:

The findings from this study offer compelling evidence of mitochondrial dysfunction in T2D hearts and suggest that MOTS-c treatment can effectively improve the mitochondrial bioenergetic function to supply energy.

Acknowledgement:

The participation at the 22^nd European Bioenergetics Conference was financially supported by Oroboros Instruments.

Glutaric aciduria type 1 (GA1) is an inherited neurometabolic disease caused by glutaryl-CoA dehydrogenase (GCDH) deficiency. GCDH catalyzes both the α,β-dehydrogenation and decarboxylation of glutaryl-CoA, which is largely supplied by dicarboxylic odd-chain fatty acids and lysine catabolism. GA1 patients commonly present with acute encephalopathy associated with severe striatum degeneration and progressive cortical and striatal injury whose pathogenesis is still poorly known. Neurological manifestations like dyskinesia, dystonia, hypotonia, muscle stiffness, and spasticity are also observed. Current guidelines for treatment include dietary protein/lysine restriction and L-carnitine supplementation. In this study, we evaluated the potential benefit of five mitochondria-targeted antioxidants and a selective peroxisome proliferator-activated receptor δ (PPARδ) agonist on bioenergetics function and gene expression in fibroblasts from GA1 patients. Cells were treated with the compounds for 48 hours in the absence of glucose, to assess their ability to accommodate an energy source shift from glucose to fatty and amino acids and to mimic a metabolic stressor. O₂-consumption rate and mRNA expression were assessed using the Cell Mito Stress assay (Seahorse oximetry) and RT-qPCR, respectively. Untreated GCDH-deficient fibroblasts showed decreased mitochondrial respiration and ATP levels when compared to control cells. In contrast, cells treated with the mitochondria-targeted antioxidants and the PPARδ agonist showed improved cellular bioenergetics and upregulated genes involved in mitochondrial function. These results are consistent with data from previous studies showing that bezafibrate, a pan-PPAR agonist, has neuroprotective effects in a knockout GA1 mice model. These findings provide evidence that mitochondrial dysfunction observed in GCDH-deficient fibroblasts can be improved by treatment with mitochondrial antioxidants as well as a PPARδ agonist.

Barth syndrome (BTHS) is an inherited cardioskeletal myopathy that results from non-functional mutations in the Tafazzin gene encoding a phospholipid transacylase required for the remodeling of cardiolipin. Cardiolipin is an inner mitochondrial membrane phospholipid essential for the function of numerous mitochondrial proteins and processes, but exactly how tafazzin deficiency impacts cardiac mitochondrial metabolism is unclear. While several forms of mitochondrial dysfunction have been linked to tafazzin deficiency, accumulating evidence suggests that oxidation of long-chain fatty acids (LCFAs) is selectively impaired in cardiac mitochondria other fuel sources. Recent analyses of cardiac tissue from BTHS patients identified specific alterations in the mitochondrial LCFA oxidation machinery that distinguished BTHS from other forms of pediatric cardiomyopathy [1] and paralleled evidence from a tafazzin-deficient mouse model [2]. This led to the hypothesis that providing alternative fuels that bypass the LCFA oxidation system could improve functional capacity of BTHS patients. Using the TAFAZZIN shRNA (TazKD) mouse model, we found that 4 weeks of dietary sodium beta-hydroxybutyrate (BHB) supplementation (1% w/w in chow) significantly improved aerobic exercise capacity on a graded treadmill running test. This was associated with improvements in cardiac mitochondrial oxidative capacity supported by pyruvate or palmitoylcarnitine, and an augmentation of multi-substrate oxidation capacity (malate, pyruvate, glutamate, succinate and fatty acid) in the presence of BHB in vitro. Taken together, these findings shed new light on the distinct bioenergetic defects that result from TAFAZZIN deficiency, and suggest that dietary BHB supplementation may be an effective strategy for circumventing these defects to improve functional capacity in BTHS patients.

[1] Chatfield KC, Sparagna GC, Specht KS, Whitcomb L, Omar A, Wolfe LA, Chicco AJ. Long-chain fatty acid oxidation and respiratory complex I deficiencies distinguish Barth Syndrome from idiopathic pediatric cardiomyopathy. J Inher Metab Dis 45 (2022)111-124.

[2] Le CH, Benage LG, Specht KS, Li Puma LA, Mulligan CM, Heuberger AL, Prenni JE, Claypool SM, Chatfield KC, Sparagna GC, Chicco AJ. Tafazzin deficiency impairs CoA-dependent oxidative metabolism in cardiac mitochondria. J Biol Chem, 295 (2020) 12485-12497.

According to World Health Organization, 60% of adults are overweight or obese. As an adaptation to excessive food intake, hepatocytes accumulate triacylglycerols in a safe lipid droplet form and enhance fatty acid oxidation. When cells' capacity to safely store and remove excessive lipids is exceeded, metabolic dysfunction-associated steatotic liver disease (MASLD) can be developed. MASLD has been estimated to affect roughly 30% of the global population. In around 20% of these patients, the disease progresses to a more severe condition – metabolic dysfunction-associated steatohepatitis (MASH).

The aim of this study was to investigate the impact of progression from steatosis to steatosis with inflammation on bioenergetic parameters in HepG2/C3A cells. A cellular model mimicking the progressive steatotic condition in hepatocytes of MASLD patients is based on HepG2/C3A cells incubated with a mixture of free fatty acids (FFAs), reflecting the lipid composition in hepatocytes of patients with fatty liver, and FFAs in combination with tumor necrosis factor alpha (TNFα), mimicking steatosis with inflammation in MASH patients’ liver. The investigated parameters included ATP level, general metabolic activity (dehydrogenases activity), glycolysis pathway activity (along with lactate levels and lactate dehydrogenase level), as well as mitochondrial oxygen consumption rate (along with the level of mitochondrial respiratory chain complexes). The obtained results provide valuable information on the impact of progression from simple steatosis to steatosis with inflammation on cellular bioenergetic parameters. This, in turn, represents important insight into the mechanism of MASLD progression, which remains poorly understood.

The research was funded by the National Science Centre, Poland (grant OPUS-22 + LAP; UMO-2021/43/I/NZ3/00510) and Czech Science Foundation (22-04100L).

Pathogenic variants affecting genes of respiratory complex I (CI) subunits show a variable phenotypic expression, that ranges from severe and lethal infantile encephalopathy (Leigh syndrome) to adult-onset milder phenotypes, as the Leber hereditary optic neuropathy (LHON). Nowadays, it is difficult to establish a priori whether a single or a combination of variants may impact on CI mechanism. In this frame, computational approaches can be used to support the experimental studies. Here, a computational approach based on coarse-grained molecular dynamics simulations was applied to investigate mutations on CI variants. Specifically, one of the primary CI variants involved in LHON onset (m.14484T>C/MT-ND6) [1] was analysed alone and in combination with two rare CI variants, whose role remains uncertain [2]. The primary variant, positioned in a fundamental region for CI function called E-channel, stiffens the enzyme dynamics. Moreover, one of the rare variants, located next to the primary one, seems to further worsens the stiffening, while the other probably does not affect CI function. This approach may be extended for future studies to other variants to analyse the pathogenic impact on CI dynamics, or to investigate multiple variants.

I would like to thank University of Bologna (Department of Pharmacy and Biotechnology) and CINECA consortium for providing the resources for our project.

[1] M.D. Brown, I.A. Trounce, A.S. Jun, J.C. Allen, D.C. Wallace, Functional analysis of lymphoblast and cybrid mitochondria containing the 3460, 11778, or 14484 Leber's hereditary optic neuropathy mitochondrial DNA mutation, J Biol Chem, 275(51) (2000) 39831-6.

[2] J. Yang, Y. Zhu, Y. Tong, Z. Zhang, L. Chen, S. Chen, Z. Cao, C. Liu, J. Xu, X. Ma, The novel G10680A mutation is associated with complete penetrance of the LHON/T14484C family, Mitochondrion 9(4) (2009) 273-8.

The molecule adenosine triphosphate (ATP) serves as the central energy currency of life. The majority of ATP is synthesized by oxidative phosphorylation, catalyzed by ATP synthase and dependent on generation of the proton motive force across the membranes (pmf) by respiratory complexes in animal cells and bacterial cells.There are two types of ATP synthases: the F type (F_oF₁) and the V type ATP synthases. Similar to F-ATPase, the V/A-ATPase from T. thermophilus (Tth) synthesizes ATP utilizing pmf in contrast to eukaryotic V-ATPases which function as proton pumps powered by ATP hydrolysis.

In this study, we determined the structures of the V_o domain of the full V/A-ATPase and the isolated V_o domain using cryo-electron microscopy. The structure of the V_o domain of V/A-ATPase was almost identical to that of the isolated V_o. By analyzing the structure of the 2.8 Å resolution V_o, we indicated precise identification of glutamate residue (Glu) side chain orientations within the c₁₂-ring.MD simulations based on this structure revealed an asymmetric protonated state of the Glu residues in the two half-channels by the pmf. We propose a model in which the asymmetric protonation states of the Glu residues of the c-subunits on either side of the two channels provide the unidirectional bias for the Brownian motion of the c₁₂-ring.

The physiological adaptability strategy of complex IV (cytochrome c oxidase) is handling several
isoforms encoded by paralog genes that may vary according to different tissues, oxygen tension.
Among these, subunit COX7A presents three different isoforms that can be alternatively selected
for CIV assembly, and each one has a specific impact on the functionality of the OXPHOS system
by affecting the ability of CIV of forming monomers, dimers or supercomplexes. However, the
mechanism of selection of the different isoforms and how they modulate metabolism remains
unclear.
On the other hand, one of these tissues where the organization of the electron transport chain
(ETC) complexes presents high sensitivity to changes in isoforms is brown adipose tissue (BAT). In
BAT, the supercomplexes formation is notably affected by the presence or absence of the different
isoforms of COX7A. Furthermore, it has been described that cold exposure can affect
supercomplex formation in BAT by rotating CIII in the interaction between CIII and CI.
Therefore, to gain a better understanding of the underlying mechanisms for isoform selection and
functionality we are performing computational analysis that combine genetic and structural
information of the ETC complexes. In this project, we use high-precision cryo-electron mycroscopy
(CryoEM) evidence to study isoform selection in CIV and its effects on the organization of the ETC
complexes and supercomplexes formation. Thus, we analyzed supercomplex I+III2+IV (also called
N-respirasome) from BAT by Cryo-EM.
We aim to obtain a structural model of the N-respirasome complex in which we explore the
presence of each of the COX7A isoforms. At the same time, we are evaluating the effect of cold
exposure that has been observed in CIV and CIII binding in our models of N-respirasome .
Therefore, in our images, we expect to observe four populations of respirasomes. We contemplate
to find respirasomes that present SCAF1, in which all the complexes are bound between them, and
respirasomes where CIII and CIV are both bound to CI but not to each other. Also, we expect to
see the effect of cold exposure affecting these respirasomes in different ways, as the movement of
CIII respect to CI will affect CIV only when they are covalently bound by SCAF1.

The four mitochondrial respiratory complexes (II, III, IV and V) of Toxoplasma gondii are essential for energy metabolism and survival. Of particular interest, is T. gondii complex II (succinate dehydrogenase, TgCII) due to it being one of the entry points to the electron transport chain (with the absence of complex I from T. gondii) and the only link to the TCA cycle. Our previous studies have proposed seven putative new subunits in TgCII for a total of nine, more than the usual four subunits found in mammals and yeast [1]. The contribution of these subunits to TgCII have been experimentally validated through microscopy, cell-biology, and biochemical methods [2], raising the question of what the functional implications of the new complex composition are. Importantly, two of the canonical four subunits, that make critical contribution to the catalytic sites, don’t seem to have clear homologs in the T. gondii complex. Furthermore, structural predictions do not produce a structure that includes the required active sites for TgCII known activity. Sequence alignments provided some progress by revealing that one of the subunits, S10, contains a highly conserved DY motif involved in activity, and this was validated via genetics. To complete the picture and elucidate the molecular mechanisms of TgCII function, we plan to solve the structure using single particle analysis via cryoEM. Here I show my progress in optimising purification of TgCII, and of the data analysis pipelines via single particle analysis.

[1] A. E. Maclean, H. Bridges, M. F. Silva, S. Ding, J. Ovciarikova, J. Hirst, & L. Sheiner, Complexome profile of Toxoplasma gondii mitochondria identifies divergent subunits of respiratory chain complexes including new subunits of cytochrome bc1 complex. PLoS pathogens, 17(3) (2021).

[2] M. F. Silva, K. Douglas, S. Sandalli, A. E. Maclean, & L. Sheiner, Functional and biochemical characterization of the Toxoplasma gondii succinate dehydrogenase complex. PLoS pathogens, 19(12) (2023).

The electron transport chain complexes in the inner mitochondrial membrane (IMM) associate to form supra-molecular assemblies known as supercomplexes (SCs). SCs have major biomedical implications as defects in respiratory complexes contribute to a broad spectrum of diseases. While the existence of SCs is now widely accepted, the molecular mechanisms regulating its assembly and stability remain elusive. We report the finding of SCARI, a small-ORF encoded peptide localized in the IMM with functions in regulating the SC formation. In C2C12 myoblasts, SCARI forms a supra-molecular complex, and its deletion increases the steady state levels of Complex I and associated SCs. Deficiency of SCARI enhances mitochondrial respiration and confers growth advantage under oxidative stress conditions. We have defined the interactome of SCARI and identified Prohibitin-AFG3L2 (m-AAA protease) complex and Complex I subunits to be the major binding partners. Here, we propose that SCARI acts as an adaptor within prohibitin complex to regulate the turnover of Complex I subunits via AFG3L2 protease. Clarifying the regulatory mechanisms of SCARI in SC assemblies will aid in understanding the roles of OXPHOS defects in disease pathogenesis and to develop SCARI based therapeutics for mitochondrial diseases characterized by destabilized respiratory complex assembly.

Mitochondrial disorders are multisystemic diseases mainly associated with neurological features, but cardiac manifestations are also common traits and can be the major cause of significant comorbidity and mortality [1]. Kearns-Sayre syndrome (KSS), a heterogeneous neurodegenerative disorder in which many patients require pacemaker implantation, is caused by the clonal expansion of mutant mitochondrial DNA (mtDNA) with a single heteroplasmic large-scale deletion (common deletion). Here, we used patient-specific induced pluripotent stem cells (hiPSCs) derived from skin fibroblasts of an 8-year-old male patient carrying the common deletion, to generate cardiomyocytes (CMs). We obtained two clones with different levels of heteroplasmy, 60%, and 0% respectively, with the latter serving as an isogenic control to assess whether nuclear DNA can influence the pathological phenotype. As an additional control, CMs from an unrelated healthy subject were differentiated. When compared to isogenic and unrelated controls, the clone carrying the deletion had a lower oxygen consumption rate, a higher spontaneous beating rate with a shorter repolarization duration measured with MultiElectrode Arrays (MEAs) and a higher propensity for arrhythmias such as delayed afterdepolarizations (DADs) measured with patch clamp. Preliminary data from calcium transient measurements (Fluo-4AM) linked the deletion with a shorter calcium transient duration and with higher propensity for abnormal calcium transients. RNAseq analysis was performed to clarify which pathways might be involved in the pathogenic mechanisms. The main dysregulated pathways were: OXPHOS, calcium homeostasis, solute carrier transporters, vesicular trafficking and mitophagy. To investigate the presence of adaptive mechanisms, such as mitochondrial biogenesis, mtDNA copy number was measured by qPCR, which indicated the presence of increased level of mtDNA in deleted CMs as compared to isogenic and unrelated controls The ultrastructural morphology of mitochondria showed increased mitochondrial size.

[1] P. Kabunga, et al., Systematic review of cardiac electrical disease in Kearns–Sayre syndrome and mitochondrial cytopathy, International Journal of Cardiology 181 (2015) 303–310. https://doi.org/10.1016/j.ijcard.2014.12.038.

Sulfite oxidase (SO) deficiency may arise from mutations in the SUOX gene, which encodes SO (isolated SO deficiency, ISOD), or mutations in the genes encoding enzymes involved in the biosynthesis of the molybdenum cofactor (molybdenum cofactor deficiency, MoCD). Mutations in MOCS1, MOCS2 and GPHN result in MoCD type A, B and C, respectively. Both ISOD and MoCD are biochemically characterized by tissue accumulation of sulfite. Patients present with severe neurological symptoms and brain abnormalities, the pathophysiology of which is not fully established. Considering that mitochondrial dysfunction has been shown in different animal models for SO deficiency and that metformin induces mitochondrial biogenesis, we evaluated the effects of this molecule in fibroblasts from a patient with MoCD.

Fibroblasts derived from a patient with MOCS1 deficiency (MoCD type A) were incubated with metformin (2.5 or 5 µM) for 12, 24, and 48 hours in a medium devoid of glucose. After incubation, mitochondrial respiration was determined using the Seahorse Extracellular Flux Analyzer. Expression levels of genes and content of proteins involved in mitochondrial biogenesis and dynamics were determined by RT-qPCR and western blotting respectively.

A reduction in basal, maximal, and ATP-linked respiration and reserve respiratory capacity was verified in MOCS1-deficient fibroblasts. The protein content of MFN1/2, OPA1, and DRP1 were reduced in these cells. In addition, metformin treatment increased mitochondrial respiration. Furthermore, expression levels of PRKAA1, PPARGC1A, and SIRT1 were upregulated after 24 hours of metformin incubation. Moreover, mRNA expression levels of mitofusin 1 and DNM1L were enhanced in deficient cells following 48 hours of metformin treatment. Additionally, metformin increases MFN1/2, OPA1, and DRP1 protein levels in MOCS1-deficient fibroblasts.

Our data show mitochondrial respiration disruption along with mitochondrial biogenesis and dynamics disturbances in MOCS1-deficient fibroblasts. Moreover, metformin increased mitochondrial respiration and levels of mitochondrial biogenesis and dynamics proteins, suggesting that this molecule activates AMPK/PGC-1α pathway in these cells. Therefore, metformin is a potential adjuvant therapy for the treatment of patients with ISOD and MoCD.

We present a method to analyze fast rotation trajectories in F1-ATPase using the distribution of angular velocity. The analysis involves the transitions during the stepping between pauses. A theoretical-computational approach is used to model the fluctuation of the imaging probe as the molecular motor undergoes stepping rotation. This method relies on the concept of an angle-dependent velocity and torque which are extracted from experimental data and calculated from theory. When applying the method on Thermophilic Bacillus F1-ATPase rotation data, we detected the presence of a short-lived substep previously not detectable in the histograms. The comparison between the experimental and theory reveals that an 80^O substep of the “concerted” ATP binding and ADP release involves an intermediate state reminiscent of a 3-occupancy structure. Its lifetime (~10 µs) is about six orders of magnitude smaller than the lifetime for spontaneous ADP release. By detecting this short-lived state the method provides “temporal super-resolution”. Most recently, this method was applied to single-molecule imaging data from Paracoccus Denitrificans F1-ATPase and it yielded a similar hidden state in the transitions between dwells. Our recent findings indicate a common mechanism for the acceleration of ADP release in the F1-ATPase motor of the two species.

Mildly elevated concentrations of total bilirubin (BR), the end product of heme catabolism, are considered protective (e.g. up to 60 µM in individuals with Gilbert syndrome). However, high BR concentrations are toxic and pose risks, as evidenced by the adverse effects of newborn jaundice. BR can reduce mitochondrial respiration, initiate apoptosis, or induce changes in cell membranes [1]. Despite this, the specific effects of BR on mitochondrial respiration or respiratory complexes remain poorly understood.

This study aimed to elucidate the direct effects of toxic BR concentrations on mitochondrial respiration in Human Embryonic Kidney cells.

Mitochondrial respiration was measured using Oroboros-O2k Oxygraphs (Oroboros Instruments, Innsbruck, Austria) with cryopreserved HEK293T cells and MiR05 respiration medium containing bovine serum albumin (1 g·mL^-1). The direct effect of BR was assessed during succinate-stimulated respiration in both the LEAK state (low adenylates, high mitochondrial membrane potential, MMP) and the OXPHOS state (high ADP, low MMP). After introducing HEK293T cells into the chamber (0.5–0.75·10⁶ cells·mL^-1), cells were permeabilized using digitonin (5 µg·mL^-1). Succinate (10 mM), rotenone (0.5 µM), and cytochrome c (10 µM) were titrated to initiate LEAK respiration, followed by the addition of ADP + Mg²⁺ (1 mM) to induce the OXPHOS state. BR (up to 35 µM dissolved in DMSO) was subsequently titrated to observe its direct effects on mitochondrial respiration in both the LEAK and OXPHOS states.

Our results indicated that BR at 35 µM had no effect on the mitochondrial respiration of HEK293T cells in the OXPHOS state. However, BR at concentrations from 15 µM significantly increased LEAK respiration in a concentration-dependent manner up to 30 µM suggesting dyscoupling the mitochondrial inner membrane (mtIM). The addition of cytochrome c significantly improved LEAK respiration at toxic bilirubin levels, suggesting defects also in the mitochondrial outer membrane (mtOM). However, this effect on mtOM was not observed in the OXPHOS state.

In conclusion, our data suggest that BR may affect mitochondrial membranes in the LEAK state. Further experiments are needed to clarify the mechanisms and to elucidate why BR did not have this effect in the OXPHOS state.

Supported by Oroboros Science Scholarship, Foundation of the Czech Society of Hepatology and Mobility Fund of Charles University (application FM/c/2024-1-032).

[1] Vidimce J et al.; (2021) Mitochondrial Function, Fatty Acid Metabolism, and Body Composition in the Hyperbilirubinemic Gunn Rat. Front. Pharmacol. 12:586715. doi: 10.3389/fphar.2021.586715

Cytochrome b₆f, a multi-subunit enzyme, plays a crucial role in the photosynthetic electron transport chain, linking Photosystems I and II and facilitating the transfer of electrons between plastoquinone (PQ) and plastocyanin. This process is vital for the conversion of light energy into chemical energy during photosynthesis. Although the atomic structure of cytochrome b₆f has been elucidated, the intricate details of its catalytic mechanism have remained elusive and a subject of intense scientific investigation. In this study, we present new high-resolution cryo-EM structures of spinach cytochrome b₆f, which provide unprecedented insights into the molecular interactions and orientation of the substrate PQ within the enzyme's active quinone reduction site. Notably, our findings indicate that PQ binds to the active site in a manner distinct from that of known inhibitors. Instead, PQ adopts a unique orientation, suggesting a different interaction mode. We also captured cytochrome b₆f in different conformations, including various positions of the iron-sulfur protein (ISP). By elucidating these structural nuances, our research offers a deeper understanding of the dynamic mechanisms involved in quinone catalysis within cytochrome b₆f. This work not only enhances our comprehension of the fundamental processes underlying photosynthesis but also sheds light on the broader molecular mechanisms that sustain life on our planet, highlighting the intricate interplay between structure and function in biological systems.

Coenzyme Q (CoQ, ubiquinone) plays a pivotal role in the mitochondrial respiratory system, acting as an electron carrier within the electron transport chain (ETC). CoQ serves as a substrate for various mitochondrial enzymes, facilitating electron transfer from NADH and FADH2 to complex III (cytochrome bc1 complex) [1]. This electron transfer is crucial for the production of ATP through oxidative phosphorylation. CoQ is utilized by several dehydrogenases, including NADH dehydrogenase (complex I), succinate dehydrogenase (complex II), dihydroorotate dehydrogenase, choline dehydrogenase, and mitochondrial glycerol-3-phosphate dehydrogenase, among others [1]. These enzymes are involved in diverse metabolic pathways such as amino acid and fatty acid oxidation, nucleotide biosynthesis, and hydrogen sulfide detoxification [1]. The interaction of CoQ with these enzymes is not merely diffusion-controlled but involves specific binding and electron transfer mechanisms. For instance, the reduction of CoQ by NADH:ubiquinone oxidoreductase (complex I) follows a ping-pong kinetic mechanism, indicating a complex interaction between the enzyme and CoQ [2]. Additionally, the CoQH2/CoQ ratio serves as a sensor for the efficiency of the respiratory chain, modulating the configuration of the ETC to match the metabolic demands and substrate availability [3]. This dynamic regulation ensures optimal electron flux and minimizes the generation of reactive oxygen species, thereby maintaining mitochondrial function and cellular health [3].

In summary, CoQ is integral to the mitochondrial respiratory system, interfacing with multiple enzymes to drive essential metabolic processes and regulate the efficiency of the electron transport chain [1-4].

[1] R. Banerjee, J. Purhonen, J. Kallijärvi, The mitochondrial coenzyme Q junction and complex III: biochemistry and pathophysiology, FEBS J., 289 (2022) 6936-6958.

[2] R. Fato, E Estornell, S Di Bernardo, …, and G. Lenaz, Steady-state kinetics of the reduction of coenzyme Q analogs by complex I (NADH:ubiquinone oxidoreductase) in bovine heart mitochondria and submitochondrial particles, Biochemistry, 35 (1996) 2705-2716.

[3] A.M. Guaras, E. Perales-Clemente, R. Acin-Perez, …, and J.A. Enriquez, The CoQH2/CoQ ratio serves as a sensor of respiratory chain efficiency, Cell reports, 15 (2016) 197-209.

[4] The ConsensusNLP Copilot (2024) provided primary research articles and created this document when prompted by the author with search terms on Coenzyme Q (ubiquinone) at the crossroads of metabolic pathways in the mitochondrial respiratory system.

Sarcopenic obesity, or the loss of skeletal muscle function mediated by age-related adiposity, is an increasingly prevalent disease with ineffective medical treatments and poor survival. Notably, the mechanisms underlying the onset and progression of sarcopenic obesity remain largely unclear. We established a mouse model of sarcopenic obesity by placing aged mice on a high fat diet (60% kcal) for 4 weeks which increased body fat while decreasing muscle function and lean mass relative to age-matched low fat diet controls. We employed 10 weeks of exercise training (3-4 day/week; 1 hr/day; 70% of max speed) as a benchmark to differentiate putative cellular mediators associated with gain and loss of muscle function in the context of sarcopenic obesity. Mice with sarcopenic obesity exhibited defective NADH- and succinate-linked oxidative phosphorylation (OXPHOS) capacity, which were entirely normalized by exercise training. Defects in oxidative capacity were restored experimentally by bypassing the Q pool with durohydroquinone, a coenzyme Q (CoQ) analogue and electron donor for Complex III. To test whether these observations occurred due to CoQ deficiency, oxidation of the Q pool, or inadequate electron flow to Complex III, mice with sarcopenic obesity were chronically administered mitoquinone mesylate (MitoQ), a mitochondrially targeted CoQ10 derivative that selectively reduces the Q pool without donating electrons to Complex III. 8 weeks of MitoQ treatment enhanced muscle function in mice with sarcopenic obesity to the level of an aged low fat diet control while restoring CoQ redox poise and NADH- and succinate-linked OXPHOS capacity. Importantly, mice with sarcopenic obesity displayed a deficiency in the absolute CoQ abundance, which was not rescued by MitoQ treatment. Taken together, the skeletal muscle CoQ pool becomes overly oxidized in mice with sarcopenic obesity, limiting OXPHOS capacity and muscular fitness. Restoring CoQ redox poise by exercise training or mitochondrially targeted CoQ therapy reverses the defects in OXPHOS capacity and locomotor function independent of CoQ abundance. Thus, maintaining CoQ redox poise represents a promising therapeutic strategy for sarcopenic obesity.

Soumitra Chowdhury¹, Kiron Viswanath¹, Francisco Peixoto², S. Mariza Monteiro^3,4, Luis Felix^3,4
¹Faculty of Biosciences and Aquaculture, Nord University, Bodø, Norway

²Chemistry Center of Vila Real, University of Trás-os-Montes and Alto Douro (UTAD), Vila Real, Portugal

³Centre for the Research and Technology of Agro-Environmental and Biological Sciences (CITAB), UTAD, Vila Real, Portugal

⁴Instituto para a Inovação, Capacitação e Sustentabilidade da Produção Agroalimentar (Inov4Agro), UTAD, Vila Real, Portugal

lfelix@utad.pt

Bioenergetic Cost on Zebrafish Intestine Caused by a Ubiquitous Pollutant, Glyphosate

Glyphosate is a widely known herbicide that has become a pervasive contaminant in aquatic ecosystems due to its extensive use in agriculture. Despite its widespread presence, glyphosate is often considered safe based on earlier toxicological assessments [1]. However, recent studies have increasingly highlighted its potential hazardous effects on aquatic organisms, which has raised significant concerns about its ecological footprint [2, 3]. To understand more of the physiological impact on aquatic organisms, we assessed the bioenergetic cost in the intestine of zebrafish (Danio rerio), following exposure to environmentally relevant concentrations of glyphosate.

We designed an experiment involving three groups: a control group and two treatment groups (exposed to low and high concentrations of glyphosate added to the rearing water). Each group had 5 replicates, with 15 adult fish per tank. After 21 days of exposure, intestinal tissues were collected to assess oxidative stress using standard plate-reader assays, mitochondrial membrane potential through fluorescence-based techniques, and mitochondrial respiratory functions via Oroboros™ O2k instrument. Preliminary results did not reveal any statistically significant differences for most parameters, except for a significant reduction in respiratory capacity of Complex I (CI), and Complex II (CII) in the glyphosate-exposed groups, pointing to potential mitochondrial dysfunction. These findings warrant further investigations to better understand the specific impacts on mitochondrial functions. Our ongoing research aims to further explore the mechanisms underlying mitochondrial dysfunction in zebrafish intestine when exposed to glyphosate.

References:

[1] O. Matthias, Glyphosate: useful, dangerous? To license or to phase out?, Medycyna Środowiskowa-Environmental Medicine, 4 (2016) 7-11.

[2] F.D. dos Santos, J.C. Cruz, A. Swarowsky, R.A. Fantinel, Use of the glyphosate herbicide: an integrated review, Acta Scientiarum. Technology, 46 (2024) e65057.

[3] B.B. Gonçalves, P.C. Giaquinto, D. dos Santos Silva, A.A. de Lima, A.A.B. Darosci, J.L. Portinho, T.L. Rocha, Ecotoxicology of glyphosate-based herbicides on aquatic environment, Biochemical toxicology-heavy metals and nanomaterials, 1 (2019) 1-24.