Mitochondrial Energy Transduction Research Articles

DIPG-14. METABOLIC REPROGRAMMING AS A RESISTANCE MECHANISM IN DIFFUSE MIDLINE GLIOMA

Abstract BACKGROUND Diffuse midline gliomas (DMG) are uniformly fatal pediatric brain cancers, refractory to current standards of care. Based on genomic and transcriptomic profiling data, we previously proposed that platelet-derived growth factor receptor alpha (PDGFRA) is crucial to the tumorigenic potential and maintenance of stem-like DMG cells and that targeting PDGFRA is an important therapeutic avenue in DMG. In comprehensive pre-clinical studies, we identified avapritinib, an FDA-approved PDGFRA inhibitor, to be highly effective in patient-derived xenograft (PDX) DMG models. We further reported the first clinical experience using avapritinib in recurrent DMG and demonstrated preliminary safety, tolerability, and efficacy of the drug in this population. While a subset of patients experienced a strong clinical response from avapritinib, tumor progression was eventually observed in all patients. METHODS Here, we investigated the molecular mechanisms that DMG cells upregulate following avapritinib treatment, with the aim to exploit them therapeutically. To uncover these mechanisms, we performed transcriptomic, metabolic and functional assays along with a combinatorial drug screening. RESULTS Transcriptional analysis of patient-derived DMG cell lines revealed an upregulation of genes associated with fatty acid metabolism and oxidative phosphorylation (OXPHOS) following treatment with avapritinib. Functional assays confirmed elevated OXPHOS in avapritinib-treated cells with significant (i) increase in mitochondrial energy transduction, (ii) increase in palmitate- (a product of fatty acid metabolism) driven oxygen consumption rate and (iii) greater incorporation of palmitate-derived carbons into the tricarboxylic acid cycle. To determine which therapies could target this dependency on OXPHOS and fatty acid metabolism, we performed a metabolic drug screening in patient-derived DMG cell lines and identified two metabolic drugs to have synergistic cytotoxic effects with avapritinib. CONCLUSION We revealed distinct metabolic reprogramming in avapritinib-treated DMG cells and showed that targeting these metabolic vulnerabilities might further increase the clinical benefit of PDGFRA inhibitor avapritinib.

Revealing and targeting metabolic drivers contributing to treatment escape in diffuse midline glioma.

10038 Background: Diffuse midline glioma (DMG) is an aggressive pediatric brain tumor that predominately affects children, with a median survival of 9-11 months. DMGs are driven by self-renewing, stem-like glioma cells that have been stalled in an oligodendrocyte precursor cell (OPC)-like state and that highly express PDGFRA. This overexpression of PDGFRA has been shown to be pivotal in DMG development. Thus, targeting PDGFRA may serve as a viable treatment approach for DMG. In our previous studies, we revealed that avapritinib, a next generation tyrosine kinase inhibitor of PDGFRA, is highly effective in DMG cells. While a subset of patients receiving treatment with avapritinib experienced a promising clinical response, all patients eventually experienced tumor progression due to treatment escape. In this study, we investigated which mechanisms DMG cells use to escape avapritinib treatment, and how we can therapeutically exploit these resistance mechanisms. Methods: Transcriptomic profiling and functional assays were performed on patient-derived DMG cell lines. A combinatorial drug screening identified drug candidates evaluated in this study. Results: Bulk RNA sequencing analysis of two patient derived DMG cell lines revealed an upregulation of genes associated with both fatty acid metabolism and oxidative phosphorylation (OXPHOS) following avapritinib treatment. Functional assays confirmed elevated OXPHOS in avapritinib-treated cells with significant increases in mitochondrial energy transduction, palmitate- (a product of fatty acid metabolism) driven oxygen consumption rates, and incorporation of palmitate-derived carbons into the tricarboxylic acid (TCA) cycle. CRISPR-Cas9-mediated knockout of PDGFRAin one DMG cell line confirmed the metabolic changes observed following avapritinib treatment. Analysis of bulk RNA sequencing data revealed an upregulation of key fatty acid- related transcription factors in avapritinib-treated DMG cells. Specifically, Peroxisome Proliferator-Activated Receptor alpha (PPAR-alpha), Sterol Regulatory Binding Element Binding Transcription Factors 1 and 2, and Fatty Acid Synthase (FASN) were most prominently upregulated. To determine which therapies could target the dependency of avapritinib-treated cells on fatty acid metabolism, we performed a combinatorial drug screening and found three lipid pathway inhibitors that have synergistic cytotoxic effects with avapritinib. Specifically, a FASN inhibitor, cholesterol pathway inhibitor, and PPAR-alpha inhibitor. Conclusions: In this study, we revealed metabolic drivers that may contribute to avapritinib resistance in DMG cells, and identified compounds capable of inhibiting these drivers that demonstrate synergy with avapritinib. We now intend on testing these combination therapies in vivo as we aim to provide a long-term clinical benefit to DMG patients receiving avapritinib.

The allometry of mitochondrial efficiency is tissue dependent: a comparison between skeletal and cardiac muscles of birds.

Body mass is known to be a fundamental driver of many biological traits, including metabolism. However, the effect of body mass on mitochondrial energy transduction is still poorly understood and has mainly been described in mammals. Using 13 species of birds ranging from 15 g (finches) to 160 kg (ostrich), we report here that the mitochondrial production of ATP, and the corresponding oxygen consumption, are negatively dependent on body mass in skeletal muscles but not in the heart. Results also showed that mitochondrial efficiency was positively correlated with body mass at sub-maximal phosphorylating states in the skeletal muscle, but not in the heart. This difference between muscle tissues is potentially linked to the difference in energetic demand expandability and the heavy involvement of skeletal muscle in thermoregulation.

Raman Spectroscopy Imaging As a Tool to Classify Diffused Large B-Cell Lymphoma Subtypes

INTRODUCTION: Diffuse large B-cell lymphoma (DLBCL) is a clinically heterogenous, aggressive disease. The ‘cell-of-origin’ classification (COO) distinguishes two major DLBCL subtypes, GCB (germinal center B cell-like) and ABC (activated B cell-like) that are derived from distinct putative cell of origin, differ in prognosis and transcriptional pattern. Comprehensive cluster classification (CCC) based on unbiased “consensus clustering” of transcriptomic microarray data has led to the identification of subtypes that differ in energy source utilization and bioenergetic profiles The oxPhos DLBCL subtype, unlike non-oxPhos, is characterized by increased mitochondrial oxidative phosphorylation and energy transduction. Although COO class assignment can be achieved with multiple clinical diagnostic tools (including transcript-based or immunohistochemical approaches), there is no clinical diagnostic tool appropriate for COO classification. In Raman effect, laser light, as a result of internal oscillations of molecules, is scattered with a change in wavelength, giving a spectrum characteristic for a given substance. This allows the chemical composition of mixtures and even cells to be determined on the basis of the obtained spectra. Thus, it also allows to determine differences in metabolism, including energy metabolism of lymphoma cells. The aim of the study was to use the RS images to recognize and classify DLBCL cells. MATERIALS AND METHODS: DLBCL cell lines (OCI-Ly4, Pfeiffer, Toledo, DHL4, DHL6 , OCI-Ly1 and OCI-Ly7) with known COO and CCC subtype assignments, and B cells isolated from the blood of healthy donors were used. For Raman analysis cells were fixed in 0.5% glutaraldehyde and imaged at 533 and 633 nm emission wavelengths under a WITec Raman microscope. The collected spectral images were subjected to chemometric analyses: characteristic bands analysis, k-means cluster analysis (KMCA), principal component analysis (PCA) and least squares discriminant analysis (PLS-DA) using dedicated software. RESULTS: The analysis of Raman spectra revealed clear changes in the structure and composition of cells after neoplastic transformation. Chemometric analysis of spectral bands showed greater proportion of proteins (1008, 1041 and 1660 cm-1) and lipids (1440, 1240 and 2850 cm-1) in DLBCL cells then B cells from healthy donors. More nucleic acids were observed (733, 790 , 1583 cm-1) in normal B cells that at least partially resulted from the greater ratio of the nucleus to the cytoplasm. The differences resulted in clear separation of malignant and healthy cells in PCA and PLS-DA. Similar approach allowed to characterize differences between ABC vs GCB subtype cell lines and oxPhos vs non-oxPhos subtype. Based on the specific differences between healthy B cells and DLBCL cells a predictive model was built using PLS-DA that accurately determines the chance of the analyzed cell, first, being a lymphoma cell, and second, belonging to the ABC vs GCB or oxPhos vs non-oxPhos DLBCL subtype. The good performance of the constructed predictive model was evaluated by prediction on unknown samples with sensitivity and specificity of 98%. CONCLUSIONS: We present a model of a classification of B cell lymphomas based on label-free Raman spectral imagines, capable of automatic and efficient identification of DLBCL cells spectra. In addition, we describe each lymphoma subtype by its unique spectral profile, linking it to biochemical features. Acknowledgements. The studies were performed as a part of the “ Label-free and rapid optical imaging, detection and sorting of leukemia cells” project carried out within the Team-Net programme of the Foundation for Polish Science co-financed by the European Union under the European Regional Development Fund.

Use of Transcriptional Signatures to Differentiate Pathogen-Specific and Treatment-Specific Host Responses in Patients With Bacterial Bloodstream Infections.

Clinical outcomes in bacterial bloodstream infections (BSIs) are influenced by bacterial species, host immunity, and antibiotic therapy. The mechanisms by which such factors influence outcomes are poorly understood. We aimed to identify bacterial- and antibiotic-specific host transcriptional signatures in patients with bacterial BSI. RNA sequencing was performed on blood samples from patients with BSI due to gram-negative (GN) versus gram-positive (GP) pathogens: Escherichia coli (n = 30) or Klebsiella pneumoniae (n = 28) versus methicillin-susceptible Staphylococcus aureus (MSSA) (n = 24) or methicillin-resistant S. aureus (MRSA) (n = 58). Patients were matched by age, sex, and race. No significant host transcriptome differences were detected in patients with E. coli versus K. pneumoniae BSI, so these were considered together as GN BSI. Relative to S. aureus BSI, patients with GN BSI had increased activation of the classic complement system. However, the most significant signal was a reduction in host transcriptional signatures involving mitochondrial energy transduction and oxidative burst in MRSA versus MSSA. This attenuated host transcriptional signature remained after controlling for antibiotic therapy. Given the importance of immune cellular energetics and reactive oxygen species in eliminating hematogenous or intracellular MRSA, these findings may offer insights into its persistence relative to other bacterial BSIs.

Abstract 13959: A Role for Striated Muscle Nuclear Receptor Co-Regulator RIP140 ( Nrip1 ) in the Control of Endurance Fitness

During the development of heart failure (HF), the capacity for myocardial fatty acid oxidation (FAO) and oxidative phosphorylation is reduced, leading to an energy-starved state. Similar metabolic derangements occur in skeletal muscle during HF and with physical inactivity associated with chronic disease states. Receptor Interacting Protein 140 (RIP140) is a nuclear receptor co-regulator that has been shown to repress the transcription of genes involved in mitochondrial energy transduction in highly metabolic tissues. We recently demonstrated that striated muscle-specific RIP140 KO mice (str-RIP140 -/- ) were protected against the development of HF and had an adaptive form of cardiac hypertrophy (PMID: 36927960). To assess the impact of RIP140 deficiency on skeletal muscle function, we conducted physiological and genomic interrogation of str-RIP140 -/- mice. We found that the str-RIP140 -/- mice display an endurance performance phenotype. Specifically, str-RIP140 -/- mice run significantly longer on an endurance treadmill regimen (str-RIP140 -/- = 118.0 min, WT= 73.38 min; p= <0.0001) with increased VO 2max compared to controls (str-RIP140 -/- = 9779 ml/kg/hr, WT= 7748 ml/kg/hr; p= <0.0001). Respiratory Exchange Ratio (RER) was maintained at a decreased level during exercise in str-RIP140 -/- mice vs controls, indicative of increased utilization and capacity for FAO. The response of str-RIP140 -/- mice to endurance exercise training was also assessed. str-RIP140 -/- mice achieved a greater VO 2max following 8 weeks of training compared to control trained mice (str-RIP140 -/- = 9589 ml/kg/hr, WT= 8366 ml/kg/hr; p= 0.002). RNA-sequence analysis comparing RIP140 -/- and control fast-twitch muscle (EDL) prior to and following exercise training identified differentially upregulated genes involved in an array of pathways activated with endurance training including mitochondrial FAO, triglyceride turnover, the slow-twitch fiber program, angiogenesis, and components of the neuromuscular junction. We conclude that RIP140 serves as a co-repressor of a network of genes involved in skeletal muscle endurance. These results suggest that RIP140 could prove to be a target for novel metabolic therapies aimed at cardiac and skeletal muscle diseases.

DIPG-28. IDENTIFICATION AND TARGETING OF METABOLIC VULNERABILITIES IN COMBINATION WITH PDGFRA-INHIBITION IN DIFFUSE MIDLINE GLIOMA

Abstract Diffuse midline gliomas (DMG) are driven by self-renewing, stem-like glioma cells stalled in an oligodendrocyte precursor cell (OPC)-like state that are characterized by high expression of PDGFRA. This lineage overexpression of PDGFRA, together with commonly found genomic amplifications and mutations in PDGFRA, has been shown to cooperate with H3-K27M mutations in tumor development. Therefore, targeting PDGFRA has long been proposed as a potential treatment strategy for DMG. Our previous work revealed that Avapritinib, a small molecule inhibitor of PDGFRA, is highly effective in DMG cells in vitro and in vivo. Following these preclinical findings, nine DMG patients were treated with Avapritinib for the first time. While 50% of all treated patients experienced a strong clinical response from Avapritinib, tumor progression and treatment escape were eventually observed in most patients. Here, we investigated which mechanisms DMG cells use to escape Avapritinib treatment and explored how these resistance mechanisms can be therapeutically exploited. Transcriptional analysis of a DMG cell line previously identified as Avapritinib-resistant revealed an upregulation of genes associated with fatty acid metabolism and oxidative phosphorylation (OXPHOS) following treatment with Avapritinib. Functional assays confirmed elevated OXPHOS in Avapritinib-resistant cells with significant (i) increase in mitochondrial energy transduction, (ii) increase in palmitate- (a product of fatty acid metabolism) driven oxygen consumption rate and (iii) greater incorporation of palmitate-derived carbons into the tricarboxylic acid cycle. These metabolic changes could be genetically reproduced in the same cell line after CRISPR-Cas9-mediated knockout of PDGFRA. To determine which therapies could target the dependency of Avapritinib-resistant cells on OXPHOS and fatty acid metabolism, we performed a metabolic drug screening and identified two metabolic drugs to have synergistic cytotoxic effects with Avapritinib. Together, we revealed distinct metabolic changes in Avapritinib-resistant DMG cells and showed that targeting these metabolic vulnerabilities might further increase the clinical benefit of Avapritinib.

Mechanisms of Energy Transduction in Mitochondria

Mitochondria play a key role in providing energy for cellular functions, and on the other hand, they are a target, decoder and switch of intracellular signals, generator of secondary messengers and pro-apoptotic factors. Modern ideas about the mechanisms of energy transduction in the mitochondrial membrane were formed after Peter Mitchell created the chemiosmotic theory of ATP synthesis. The data presented in the article can serve as a fundamental basis for further study of the brain in normal and pathological conditions with further extrapolation of the obtained data to humans. Mitochondria play a key role in providing energy for cellular functions, and on the other hand, they are a target, decoder and switch of intracellular signals, generator of secondary messengers and pro-apoptotic factors [1]. Modern ideas about the mechanisms of energy transduction in the mitochondrial membrane were formed after Peter Mitchell created the chemiosmotic theory of ATP synthesis. The energy resources used by the inner mitochondrial membrane are initially directed at the transport of the conjugating proton through the membrane to an area with a higher concentration (energization of the membrane), then the proton gradient is used by the mitochondria as a driving force capable of leading to the synthesis of ATP. Isolation of mitochondrial proteins by D.E. Green and co-workers led to the formation of the idea of “complexes” of the respiratory chain [2].

Ischemic damage to every segment of the oxidative phosphorylation cascade elevates ETC driving force and ROS production in cardiac mitochondria.

Myocardial ischemia has long-lasting negative impacts on cardiomyocyte mitochondrial ATP production. However, the location(s) of damage to the oxidative phosphorylation pathway responsible for altered mitochondrial function is unclear. Mitochondrial reactive oxygen species (ROS) production increases following ischemia, but the specific factors controlling this increase are unknown. To determine how ischemia affects the mitochondrial energy conversion cascade and ROS production, mitochondrial driving forces [redox potential and membrane potential (ΔΨ)] were measured at resting, intermediate, and maximal respiration rates in mitochondria isolated from rat hearts after 60 min of control flow (control) or no-flow ischemia (ischemia). The effective activities of the dehydrogenase enzymes, the electron transport chain (ETC), and ATP synthesis and transport were computed using the driving forces and flux. Ischemia lowered maximal mitochondrial respiration rates and diminished the responsiveness of respiration to both redox potential and ΔΨ. Ischemia decreased the activities of every component of the oxidative phosphorylation pathway: the dehydrogenase enzymes, the ETC, and ATP synthesis and transport. ROS production was linearly related to driving force down the ETC; however, ischemia mitochondria demonstrated a greater driving force down the ETC and higher ROS production. Overall, results indicate that ischemia ubiquitously damages the oxidative phosphorylation pathway, reduces mitochondrial sensitivity to driving forces, and augments the propensity for electrons to leak from the ETC. These findings underscore that strategies to improve mitochondrial function following ischemia must target the entire mitochondrial energy conversion cascade.NEW & NOTEWORTHY This integrative analysis is the first to assess how myocardial ischemia alters the mitochondrial driving forces and the degree to which individual segments of the mitochondrial energy transduction pathway contribute to diminished function following ischemia. This investigation demonstrates that increased reactive oxygen species production following ischemia is related to a lower effective activity of the electron transport chain and a greater driving force down the electron transport chain.

Prescription drugs and mitochondrial metabolism.

Mitochondria are central to the physiology and survival of nearly all eukaryotic cells and house diverse metabolic processes including oxidative phosphorylation, reactive oxygen species buffering, metabolite synthesis/exchange, and Ca2+ sequestration. Mitochondria are phenotypically heterogeneous and this variation is essential to the complexity of physiological function among cells, tissues, and organ systems. As a consequence of mitochondrial integration with so many physiological processes, small molecules that modulate mitochondrial metabolism induce complex systemic effects. In the case of many commonly prescribed drugs, these interactions may contribute to drug therapeutic mechanisms, induce adverse drug reactions, or both. The purpose of this article is to review historical and recent advances in the understanding of the effects of prescription drugs on mitochondrial metabolism. Specific ‘modes’ of xenobiotic–mitochondria interactions are discussed to provide a set of qualitative models that aid in conceptualizing how the mitochondrial energy transduction system may be affected. Findings of recent in vitro high-throughput screening studies are reviewed, and a few candidate drug classes are chosen for additional brief discussion (i.e. antihyperglycemics, antidepressants, antibiotics, and antihyperlipidemics). Finally, recent improvements in pharmacokinetics models that aid in quantifying systemic effects of drug–mitochondria interactions are briefly considered.

Mitochondrial Bioenergetic and Proteomic Phenotyping Reveals Organ-Specific Consequences of Chronic Kidney Disease in Mice.

Chronic kidney disease (CKD) results in reduced kidney function, uremia, and accumulation of uremic metabolites. Mitochondrial alterations have been suggested to play a role in the disease pathology within various tissues. The purpose of this study was to perform a comprehensive bioenergetic and proteomic phenotyping of mitochondria from skeletal muscle (SkM), cardiac muscle (CM), and renal tissue from mice with CKD. The 5-month-old C57BL/6J male mice were fed a casein control or adenine-supplemented diet for 6 months. CKD was confirmed by blood urea nitrogen. A mitochondrial diagnostic workflow was employed to examine respiratory function, membrane and redox potential, reactive oxygen species production, and maximal activities of matrix dehydrogenases and electron transport system (ETS) protein complexes. Additionally, tandem-mass-tag-assisted proteomic analyses were performed to uncover possible differences in mitochondrial protein abundance. CKD negatively impacted mitochondrial energy transduction (all p < 0.05) in SkM, CM, and renal mitochondria, when assessed at physiologically relevant cellular energy demands (ΔGATP) and revealed the tissue-specific impact of CKD on mitochondrial health. Proteomic analyses indicated significant abundance changes in CM and renal mitochondria (115 and 164 proteins, p < 0.05), but no differences in SkM. Taken together, these findings reveal the tissue-specific impact of chronic renal insufficiency on mitochondrial health.

Plate-Based Respirometry to Assess Thermal Sensitivity of Zebrafish Embryo Bioenergetics in situ.

Oxygen consumption allows measuring the metabolic activity of organisms. Here, we adopted the multi-well plate-based respirometry of the extracellular flux analyzer (Seahorse XF96) to investigate the effect of temperature on the bioenergetics of zebrafish embryos (Danio rerio) in situ. We show that the removal of the embryonic chorion is beneficial for oxygen consumption rates (OCR) and penetration of various mitochondrial inhibitors, and confirm that sedation reduces the variability of OCR. At 48h post-fertilization, embryos (maintained at a routine temperature of 28°C) were exposed to different medium temperatures ranging from 18°C to 37°C for 20h prior OCR measurement. Measurement temperatures from 18°C to 45°C in the XF96 were achieved by lowering the room temperature and active in-built heating. At 18°C assay temperature, basal OCR was low due to decreased ATP-linked respiration, which was not limited by mitochondrial power, as seen in substantial spare respiratory capacity. Basal OCR of the embryos increased with assay temperature and were stable up to 37°C assay temperature, with pre-exposure of 37°C resulting in more thermo-resistant basal OCR measured at 41°C. Adverse effects of the mitochondrial inhibitor oligomycin were seen at 37°C and chemical uncouplers disrupted substrate oxidation gradually with increasing assay temperature. Proton leak respiration increased at assay temperatures above 28°C and compromised the efficiency of ATP production, calculated as coupling efficiency. Thus, temperature impacts mitochondrial respiration by reduced cellular ATP turnover at lower temperatures and by increased proton leak at higher temperatures. This conclusion is coherent with the assessment of heart rate, an independent indicator of systemic metabolic rate, which increased with exposure temperature, peaking at 28°C, and decreased at higher temperatures. Collectively, plate-based respirometry allows assessing distinct parts of mitochondrial energy transduction in zebrafish embryos and investigating the effect of temperature and temperature acclimation on mitochondrial bioenergetics in situ.

The photosensitiser azure A disrupts mitochondrial bioenergetics through intrinsic and photodynamic effects

Azure A (AA) is a cationic molecule of the class of phenothiazines that has been applied in vitro as a photosensitising agent in photodynamic antimicrobial chemotherapy. It is a di-demethylated analogue of methylene blue (MB), which has been demonstrated to be intrinsically and photodynamically highly active on mitochondrial bioenergetics. However, as far as we know, there are no studies about the photodynamic effects of AA on mammalian mitochondria. Therefore, this investigation aimed to characterise the intrinsic and photodynamic acute effects of AA (0.540 μM) on isolated rat liver mitochondria, isolated hepatocytes, and isolated perfused rat liver. The effects of AA were assessed by evaluating several parameters of mitochondrial bioenergetics, oxidative stress, cell viability, and hepatic energy metabolism. The photodynamic effects of AA were assessed under simulated hypoxic conditions, a suitable way for mimicking the microenvironment of hypoxic solid tumour cells. AA interacted with the mitochondria and, upon photostimulation (10 min of light exposure), produced toxic amounts of reactive oxygen species (ROS), which damaged the organelle, as demonstrated by the high levels of lipid peroxidation and protein carbonylation. The photostimulated AA also depleted the GSH pool, which could compromise the mitochondrial antioxidant defence. Bioenergetically, AA photoinactivated the complexes I, II, and IV of the mitochondrial respiratory chain and the F1FO–ATP synthase complex, sharply inhibiting the oxidative phosphorylation. Upon photostimulation (10 min of light exposure), AA reduced the efficiency of mitochondrial energy transduction and oxidatively damaged lipids in isolated hepatocytes but did not decrease the viability of cells. Despite the useful photobiological properties, AA presented noticeable dark toxicity on mitochondrial bioenergetics, functioning predominantly as an uncoupler of oxidative phosphorylation. This harmful effect of AA was evidenced in isolated hepatocytes, in which AA diminished the cellular ATP content. In this case, the cells exhibited signs of cell viability reduction in the presence of high AA concentrations, but only after a long time of incubation (at least 90 min). The impairments on mitochondrial bioenergetics were also clearly manifested in intact perfused rat liver, in which AA diminished the cellular ATP content and stimulated the oxygen uptake. Consequently, gluconeogenesis and ureogenesis were strongly inhibited, whereas glycogenolysis and glycolysis were stimulated. AA also promoted the release of cytosolic and mitochondrial enzymes into the perfusate concomitantly with inhibition of oxygen consumption. In general, the intrinsic and photodynamic effects of AA were similar to those of MB, but AA caused some distinct effects such as the photoinactivation of the complex IV of the mitochondrial respiratory chain and a diminution of the ATP levels in the liver. It is evident that AA has the potential to be used in mitochondria-targeted photodynamic therapy, even under low oxygen concentrations. However, the fact that AA directly disrupts mitochondrial bioenergetics and affects several hepatic pathways that are linked to ATP metabolism, along with its ability to perturb cellular membranes and its little potential to reduce cell viability, could result in significant adverse effects especially in long-term treatments.

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

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

Extreme Acetylation of the Cardiac Mitochondrial Proteome Does Not Promote Heart Failure.

Circumstantial evidence links the development of heart failure to posttranslational modifications of mitochondrial proteins, including lysine acetylation (Kac). Nonetheless, direct evidence that Kac compromises mitochondrial performance remains sparse. This study sought to explore the premise that mitochondrial Kac contributes to heart failure by disrupting oxidative metabolism. A DKO (dual knockout) mouse line with deficiencies in CrAT (carnitine acetyltransferase) and Sirt3 (sirtuin 3)-enzymes that oppose Kac by buffering the acetyl group pool and catalyzing lysine deacetylation, respectively-was developed to model extreme mitochondrial Kac in cardiac muscle, as confirmed by quantitative acetyl-proteomics. The resulting impact on mitochondrial bioenergetics was evaluated using a respiratory diagnostics platform that permits comprehensive assessment of mitochondrial function and energy transduction. Susceptibility of DKO mice to heart failure was investigated using transaortic constriction as a model of cardiac pressure overload. The mitochondrial acetyl-lysine landscape of DKO hearts was elevated well beyond that observed in response to pressure overload or Sirt3 deficiency alone. Relative changes in the abundance of specific acetylated lysine peptides measured in DKO versus Sirt3 KO hearts were strongly correlated. A proteomics comparison across multiple settings of hyperacetylation revealed ≈86% overlap between the populations of Kac peptides affected by the DKO manipulation as compared with experimental heart failure. Despite the severity of cardiac Kac in DKO mice relative to other conditions, deep phenotyping of mitochondrial function revealed a surprisingly normal bioenergetics profile. Thus, of the >120 mitochondrial energy fluxes evaluated, including substrate-specific dehydrogenase activities, respiratory responses, redox charge, mitochondrial membrane potential, and electron leak, we found minimal evidence of oxidative insufficiencies. Similarly, DKO hearts were not more vulnerable to dysfunction caused by transaortic constriction-induced pressure overload. The findings challenge the premise that hyperacetylation per se threatens metabolic resilience in the myocardium by causing broad-ranging disruption to mitochondrial oxidative machinery.

MCL-1Matrix maintains neuronal survival by enhancing mitochondrial integrity and bioenergetic capacity under stress conditions

Mitochondria play a crucial role in neuronal survival through efficient energy metabolism. In pathological conditions, mitochondrial stress leads to neuronal death, which is regulated by the anti-apoptotic BCL-2 family of proteins. MCL-1 is an anti-apoptotic BCL-2 protein localized to mitochondria either in the outer membrane (OM) or inner membrane (Matrix), which have distinct roles in inhibiting apoptosis and promoting bioenergetics, respectively. While the anti-apoptotic role for Mcl1 is well characterized, the protective function of MCL-1 Matrix remains poorly understood. Here, we show MCL-1OM and MCL-1Matrix prevent neuronal death through distinct mechanisms. We report that MCL-1Matrix functions to preserve mitochondrial energy transduction and improves respiratory chain capacity by modulating mitochondrial oxygen consumption in response to mitochondrial stress. We show that MCL-1Matrix protects neurons from stress by enhancing respiratory function, and by inhibiting mitochondrial permeability transition pore opening. Taken together, our results provide novel insight into how MCL-1Matrix may confer neuroprotection under stress conditions involving loss of mitochondrial function.

Chronic Kidney Disease Causes Skeletal Mitochondrial Myopathy Through Disruption of the Electron Transport System

IntroductionChronic kidney disease (CKD) is associated with debilitating myopathic symptoms including muscle wasting, weakness, and fatigue. Mitochondria have been implicated as a mediator of skeletal muscle dysfunction in CKD, however, the underlying mechanisms of mitochondrial dysfunction in CKD are unknown.PurposeThe purpose of this study was to define the impact of CKD on muscle mitochondrial energy transduction, metabolomic profiles, and muscle contractile function.Methods8‐week old C57BL/6J mice (N=6–10/group/sex) were fed a casein control or adenine supplemented‐diet for 10‐weeks. CKD was confirmed through measurement of glomerular filtration rate (GFR), blood urea nitrogen, and kidney histology. A comprehensive mitochondrial phenotyping platform included respiratory function, H2O2 emission, matrix dehydrogenase and OXPHOS enzyme activity, and ATP synthase activity was employed. Muscle function, size, metabolomic profiles were also assessed.ResultsCKD resulted in a 30–40% decrease in OXPHOS sensitivity supported by pyruvate/malate, glutamate/malate, succinate/rotenone, and octanoylcarnitine/malate (all P&lt;0.05) using a creatine kinase clamp to control the extracellular energy demand for mitochondria. CKD decreased maximal ADP‐stimulated respiratory capacity (4139.1 ± 564.4 vs 3448.7 ± 782.2 pmols/s/mg, P&lt;0.05), despite no change in mitochondrial content (citrate synthase activity and western blotting). Significant correlations between GFR and skeletal muscle mitochondrial respiratory function were observed in all substrate conditions (all P&lt;0.03). Enzymatic assays demonstrated significant decreases (~35%) in the activity of pyruvate and alpha‐ketoglutarate dehydrogenases (P&lt;0.05), and non‐significant decreases in glutamate dehydrogenase, malic enzyme, and isocitrate dehydrogenase (12–36%). Mitochondrial H2O2 production was modestly increased with pyruvate/malate (P=0.043) but not different with octanoylcarnitine/malate or glutamate/malate (P=0.18 and 0.23 respectively). With both sexes combined, muscle force production was not different, however, when separated male CKD mice had lower force production (293.1 ± 20.8 vs 236.8 ± 25.5, P&lt;0.05). Muscle weights and myofiber areas confirmed atrophy in male CKD mice. Metabolomics analyses indicated alterations in amino acid catabolism including arginine, tryptophan, phenylalanine/tyrosine, increased allantoin, and accumulation of uremic metabolites previously found to negatively impact mitochondrial energetics.ConclusionTo our knowledge, this is the first study to define the exact mechanisms by which CKD alters skeletal muscle mitochondrial function. Using mitochondrial phenotyping, CKD was found to disrupt OXPHOS enzymes and matrix dehydrogenases resulting in impaired oxidative phosphorylation and elevated H2O2 production. These discoveries provide the foundation for targeted mitochondrial therapies aimed to treat skeletal myopathy in CKD patients.Support or Funding InformationSupported by R01 HL148597 (STS and TER).

A Critical Role for Estrogen-Related Receptor Signaling in Cardiac Maturation.

The heart undergoes dramatic developmental changes during the prenatal to postnatal transition, including maturation of cardiac myocyte energy metabolic and contractile machinery. Delineation of the mechanisms involved in cardiac postnatal development could provide new insight into the fetal shifts that occur in the diseased heart and unveil strategies for driving maturation of stem cell-derived cardiac myocytes. To delineate transcriptional drivers of cardiac maturation. We hypothesized that ERR (estrogen-related receptor) α and γ, known transcriptional regulators of postnatal mitochondrial biogenesis and function, serve a role in the broader cardiac maturation program. We devised a strategy to knockdown the expression of ERRα and γ in heart after birth (pn-csERRα/γ [postnatal cardiac-specific ERRα/γ]) in mice. With high levels of knockdown, pn-csERRα/γ knockdown mice exhibited cardiomyopathy with an arrest in mitochondrial maturation. RNA sequence analysis of pn-csERRα/γ knockdown hearts at 5 weeks of age combined with chromatin immunoprecipitation with deep sequencing and functional characterization conducted in human induced pluripotent stem cell-derived cardiac myocytes (hiPSC-CM) demonstrated that ERRγ activates transcription of genes involved in virtually all aspects of postnatal developmental maturation, including mitochondrial energy transduction, contractile function, and ion transport. In addition, ERRγ was found to suppress genes involved in fibroblast activation in hearts of pn-csERRα/γ knockdown mice. Disruption of Esrra and Esrrg in mice during fetal development resulted in perinatal lethality associated with structural and genomic evidence of an arrest in cardiac maturation, including persistent expression of early developmental and noncardiac lineage gene markers including cardiac fibroblast signatures. Lastly, targeted deletion of ESRRA and ESRRG in hiPSC-CM derepressed expression of early (transcription factor 21 or TCF21) and mature (periostin, collagen type III) fibroblast gene signatures. ERRα and γ are critical regulators of cardiac myocyte maturation, serving as transcriptional activators of adult cardiac metabolic and structural genes, an.d suppressors of noncardiac lineages including fibroblast determination.

Deregulating mitochondrial metabolite and ion transport has beneficial effects in yeast and human cellular models for NARP syndrome.

The m.8993T>G mutation of the mitochondrial MT-ATP6 gene has been associated with numerous cases of neuropathy, ataxia and retinitis pigmentosa and maternally inherited Leigh syndrome, which are diseases known to result from abnormalities affecting mitochondrial energy transduction. We previously reported that an equivalent point mutation severely compromised proton transport through the ATP synthase membrane domain (FO) in Saccharomyces cerevisiae and reduced the content of cytochrome c oxidase (Complex IV or COX) by 80%. Herein, we report that overexpression of the mitochondrial oxodicarboxylate carrier (Odc1p) considerably increases Complex IV abundance and tricarboxylic acid-mediated substrate-level phosphorylation of ADP coupled to conversion of α-ketoglutarate into succinate in m.8993T>G yeast. Consistently in m.8993T>G yeast cells, the retrograde signaling pathway was found to be strongly induced in order to preserve α-ketoglutarate production; when Odc1p was overexpressed, this stress pathway returned to an almost basal activity. Similar beneficial effects were induced by a partial uncoupling of the mitochondrial membrane with the proton ionophore, cyanide m-chlorophenyl hydrazone. This chemical considerably improved the glutamine-based, respiration-dependent growth of human cytoplasmic hybrid cells that are homoplasmic for the m.8993T>G mutation. These findings shed light on the interdependence between ATP synthase and Complex IV biogenesis, which could lay the groundwork for the creation of nutritional or metabolic interventions for attenuating the effects of mtDNA mutations.

Energetic Modelling of Mitochondrial Redox Reactions

Mitochondria make use of redox reactions to provide the energy driving many living systems. Energetic modelling of the key components of mitochondria is thus an important challenge to systems biology. Like engineering systems, living systems are subject to the laws of physics in general and the laws of thermodynamics in particular. This fact gives the opportunity of applying engineering approaches to the modelling, analysis and understanding of living systems. The bond graph method of Paynter is one such well-established engineering approach. The mitochondrial electron transport chain transduces chemical energy to the trans-membrane protonic potential; the energy stored in this trans-membrane protonic potential is then transduced back to chemical energy by driving ATP hydrolysis in the reverse direction clatalysed by ATPase. Redox reactions are a fundamental part of the electron transport chain. Redox reactions are conveniently split into two half-reactions coupled by the transfer of electrons and thus themselves transduce chemical and electrical energy. As well as being the driver of protonic energy transduction, the electrons coupling the redox half reactions are also the source of the reactive oxygen species (ROS) superoxide and its derivatives. Thus explicit modelling of electron transfer also provides insight into ROS generation. The bond graph approach focuses on energy transduction across physical domains and is thus an ideal modelling tool in this context. Modular bond graphs provide a way of decomposing complex biomolecular systems into manageable parts. We show that such a modular bond graph approach, provides a flexible and powerful energy-inspired modelling method which brings engineering expertise to the analysis of biomolecular systems in general and redox reactions, ROS generation and mitochondrial chemiosmotic energy transduction in particular.