Rate Of ATP Synthesis Research Articles

Metabolic regulation of mitochondrial morphologies in pancreatic beta cells: coupling of bioenergetics and mitochondrial dynamics

Cellular bioenergetics and mitochondrial dynamics are crucial for the secretion of insulin by pancreatic beta cells in response to elevated levels of blood glucose. To elucidate the interactions between energy production and mitochondrial fission/fusion dynamics, we combine live-cell mitochondria imaging with biophysical-based modeling and graph-based network analysis. The aim is to determine the mechanism that regulates mitochondrial morphology and balances metabolic demands in pancreatic beta cells. A minimalistic differential equation-based model for beta cells is constructed that includes glycolysis, oxidative phosphorylation, calcium dynamics, and fission/fusion dynamics, with ATP synthase flux and proton leak flux as main regulators of mitochondrial dynamics. The model shows that mitochondrial fission occurs in response to hyperglycemia, starvation, ATP synthase inhibition, uncoupling, and diabetic conditions, in which the rate of proton leakage exceeds the rate of mitochondrial ATP synthesis. Under these metabolic challenges, the propensities of tip-to-tip fusion events simulated from the microscopy images of the mitochondrial networks are lower than those in the control group and prevent the formation of mitochondrial networks. The study provides a quantitative framework that couples bioenergetic regulation with mitochondrial dynamics, offering insights into how mitochondria adapt to metabolic challenges.

EARLY NODULIN93 acts via cytochrome c oxidase to alter respiratory ATP production and root growth in plants.

EARLY NODULIN 93 (ENOD93) has been genetically associated with biological nitrogen fixation in legumes and nitrogen use efficiency in cereals, but its precise function is unknown. We show that hidden Markov models define ENOD93 as a homolog of the N-terminal domain of RESPIRATORY SUPERCOMPLEX FACTOR 2 (RCF2). RCF2 regulates cytochrome oxidase (CIV), influencing the generation of a mitochondrial proton motive force in yeast (Saccharomyces cerevisiae). Knockout of ENOD93 in Arabidopsis (Arabidopsis thaliana) causes a short root phenotype and early flowering. ENOD93 is associated with a protein complex the size of CIV in mitochondria, but neither CIV abundance nor its activity changed in ruptured organelles of enod93. However, a progressive loss of ADP-dependent respiration rate was observed in intact enod93 mitochondria, which could be recovered in complemented lines. Mitochondrial membrane potential was higher in enod93 in a CIV-dependent manner, but ATP synthesis and ADP depletion rates progressively decreased. The respiration rate of whole enod93 seedlings was elevated, and root ADP content was nearly double that in wild type without a change in ATP content. We propose that ENOD93 and HYPOXIA-INDUCED GENE DOMAIN 2 (HIGD2) are the functional equivalent of yeast RCF2 but have remained undiscovered in many eukaryotic lineages because they are encoded by two distinct genes.

Effects of microplastics and combined pollution of polystyrene and di-n-octyl phthalate on photosynthesis of cucumber (Cucumis sativus L.)

Photosynthesis provides carbon sources and energy for crop growth and development, and the widespread presence of microplastics and plastic plasticisers in agricultural soils affects crop photosynthesis, but the mechanism of the effect is not clear. This study aims to investigate the effects of different microplastics and plasticizers on cucumber photosynthesis. Using polyvinyl chloride (PVC), polyethylene (PE), polystyrene (PS), and di-n-octyl phthalate (DOP) as representative microplastics and plasticizers, we assessed their impact on cucumber photosynthesis. Our results reveal significant alterations in key parameters: intercellular CO2 concentration (Ci) and transpiration rate (Tr) increased across all treatments, whereas stomatal limit value (Ls) and water use efficiency (WUE) decreased. Notably, PS + DOP treatment led to a significant reduction in the maximum efficiency of photosystem II (Fv/Fm) and ATP accumulation. Furthermore, PE and PS + DOP treatments decreased lycopene and ɛ-carotene synthesis rates, as well as abscisic acid (ABA) accumulation. All treatments inhibited the conversion of β-carotene into strigolactone (SL) and decreased chlorophyll synthesis rates, with PS + DOP exhibiting the most severe impact. Regarding chlorophyll degradation pathways, PVC and PE treatments reduced chlorophyll decomposition rates, whereas DOP with PS promoted degradation. PE and PS treatments also impaired light energy capture, electron transport, and the structural stability of photosystems I and II, as well as photosynthetic capacity and NADPH and ATP synthesis rates. Our findings underscore the differential impacts of microplastics and plasticizers on cucumber photosynthesis, with PS + DOP having the most detrimental effect. These results shed light on the complex interactions between microplastics and plant physiology, highlighting the urgent need for mitigation strategies in agricultural practices to safeguard crop productivity and environmental sustainability.

Cobra Venom Cytotoxins as a Tool for Probing Mechanisms of Mitochondrial Energetics and Understanding Mitochondrial Membrane Structure.

In this paper, we provide an overview of mitochondrial bioenergetics and specific conditions that lead to the formation of non-bilayer structures in mitochondria. Secondly, we provide a brief overview on the structure/function of cytotoxins and how snake venom cytotoxins have contributed to increasing our understanding of ATP synthesis via oxidative phosphorylation in mitochondria, to reconcile some controversial aspects of the chemiosmotic theory. Specifically, we provide an emphasis on the biochemical contribution of delocalized and localized proton movement, involving direct transport of protons though the Fo unit of ATP synthase or via the hydrophobic environment at the center of the inner mitochondrial membrane (proton circuit) on oxidative phosphorylation, and how this influences the rate of ATP synthesis. Importantly, we provide new insights on the molecular mechanisms through which cobra venom cytotoxins affect mitochondrial ATP synthesis, mitochondrial structure, and dynamics. Finally, we provide a perspective for the use of cytotoxins as novel pharmacological tools to study membrane bioenergetics and mitochondrial biology, how they can be used in translational research, and their potential therapeutic applications.

Neuronal activity-driven O-GlcNAcylation promotes mitochondrial plasticity

Neuronal activity is an energy-intensive process that is largely sustained by instantaneous fuel utilization and ATP synthesis. However, how neurons couple ATP synthesis rate to fuel availability is largely unknown. Here, we demonstrate that the metabolic sensor enzyme O-linked N-acetyl glucosamine (O-GlcNAc) transferase regulates neuronal activity-driven mitochondrial bioenergetics in hippocampal and cortical neurons. We show that neuronal activity upregulates O-GlcNAcylation in mitochondria. Mitochondrial O-GlcNAcylation is promoted by activity-driven glucose consumption, which allows neurons to compensate for high energy expenditure based on fuel availability. To determine the proteins that are responsible for these adjustments, we mapped the mitochondrial O-GlcNAcome of neurons. Finally, we determine that neurons fail to meet activity-driven metabolic demand when O-GlcNAcylation dynamics are prevented. Our findings suggest that O-GlcNAcylation provides a fuel-dependent feedforward control mechanism in neurons to optimize mitochondrial performance based on neuronal activity. This mechanism thereby couples neuronal metabolism to mitochondrial bioenergetics and plays a key role in sustaining energy homeostasis.

Bioenergetics and mitochondrial deregulation underlie self-sustained persistency of atrial fibrillation in an ovine model

Abstract Background Atrial fibrillation (AF) often transitions from paroxysmal to more sustained forms in a 4-15/100 person-year range(1). It is well-recognized from both clinical and animal studies that some individuals undergo this transition much more readily than others, but the underlying mechanisms remain unclear. Purpose To identify potential differences in atrial bioenergetics between animals developing stable AF (AF-S) versus those resistant to this transition (AF-R) in a sheep AF-model. Methods AF was induced through repeated bursts of atrial tachystimulation (2). Stability of AF was monitored with telemetry. In-vivo electrophysiological remodeling was assessed via contact mapping. Metabolic and bioenergetic remodeling were evaluated using frozen left atrial appendage (LAA) tissues and isolated LAA mitochondria. Results AF-S sheep developed stable (>24-hour self-sustained) AF after 13 (range 5-22) days on average, whereas AF-R sheep failed to develop stable AF despite 120-day tachystimulation. Contact mapping and histological analysis revealed similar electro-anatomical remodeling in AF-S and AF-R groups, with the only significant difference observed in the decrease of activation time and conduction velocity in the AF-S atria. Both AF-S and AF-R animals displayed a notable increase in mitochondrial mass. Proteomic analysis showed significant differences in metabolism-related proteins and pathways between AF-S and AF-R, in particular concerning the anaplerotic supplementation of the tricarboxylic acid cycle at the level of α-ketoglutarate. Furthermore, compared to AF-R, AF-S LAA exhibited a 45% increase in succinate content, and a 23% decrease in ATP and Creatine Phosphate (PCr) content, suggesting a pathological metabolic alteration linked to a bioenergetic default. AF-S mitochondria presented abnormal succinate-oxidation, associated with a significant 20% decrease in ATP-synthesis rate, 22% increase in ROS-production, mitochondrial-membrane hyperpolarization, and decreased Complex I/II specific activity ratio. AF-S mitochondria alterations were prevented by complex I inhibitor S1QEL1.1, hinting to a participation of mitochondrial ROS into the mechanism of AF stabilization. In vivo acute intravenous perfusion of succinate in rats made them more susceptible to more frequent (OR 35 CI=[1.63-449.80]) and longer AF episodes upon treansesophageal stimulation; this vulnerability to AF was prevented by intraperitoneal pretreatment with SQEL1.1. Conclusions Resistance to AF-stabilization is associated with specific bioenergetic remodeling. These findings provide new mechanistic insights into AF-resistance and might open avenues for novel therapeutic strategies for AF-management.

Thermodynamic analysis of energy coupling by determination of the Onsager phenomenological coefficients for a 3×3 system of coupled chemical reactions and transport in ATP synthesis and its mechanistic implications

The nonequilibrium coupled processes of oxidation and ATP synthesis in the fundamental process of oxidative phosphorylation (OXPHOS) are of vital importance in biosystems. These coupled chemical reaction and transport bioenergetic processes using the OXPHOS pathway meet >90% of the ATP demand in aerobic systems. On the basis of experimentally determined thermodynamic OXPHOS flux-force relationships and biochemical data for the ternary system of oxidation, ion transport, and ATP synthesis, the Onsager phenomenological coefficients have been computed, including an estimate of error. A new biothermokinetic theory of energy coupling has been formulated and on its basis the thermodynamic parameters, such as the overall degree of coupling, q and the phenomenological stoichiometry, Z of the coupled system have been evaluated. The amount of ATP produced per oxygen consumed, i.e. the actual, operating P/O ratio in the biosystem, the thermodynamic efficiency of the coupled reactions, η, and the Gibbs free energy dissipation, Φ have been calculated and shown to be in agreement with experimental data. At the concentration gradients of ADP and ATP prevailing under state 3 physiological conditions of OXPHOS that yield Vmax rates of ATP synthesis, a maximum in Φ of ∼0.5J(hmgprotein)−1, corresponding to a thermodynamic efficiency of ∼60% for oxidation on succinate, has been obtained. Novel mechanistic insights arising from the above have been discussed. This is the first report of a 3 × 3 system of coupled chemical reactions with transport in a biological context in which the phenomenological coefficients have been evaluated from experimental data.

Quantifying in vivo Mitochondrial Effciency at Rest and during Exercise in Human Skeletal Muscle

Introduction: Mitochondrial effciency provides an additional mechanism to fine-tune oxidative phosphorylation rate to match ATP demand in skeletal muscle with far-reaching consequences on exercise tolerance. However, the direct measurement of both oxygen consumption (VO2) and ATP production from the same region of the contracting muscle in vivo remains a technical challenge. Purpose: Using an innovative approach combining noninvasive phosphorus and proton magnetic resonance spectroscopy (31P/1H-MRS), the present study aimed to characterize skeletal muscle ATP synthesis rate and muscle VO2 at rest and during a graded dynamic plantar flexion exercise to determine mitochondrial effciency in the gastrocnemius muscle. Method: We assessed mitochondrial effciency in the gastrocnemius muscles of 12 healthy adults (21± 1 yrs.). 31P and 1H magnetic resonance spectroscopy (MRS) was performed using a whole-body 3T MR system (Skyra, Siemens) at rest and during a series of constant workloads coupled to brief transient ischemia to quantify both oxidative ATP synthesis (ATPox) rate and myoglobin-derived oxygen consumption (Mb-derived VO2). The MRS data were acquired with a dual-tuned 31P/1H surface coil with linear polarization positioned over the gastrocnemius muscle. Results: As expected, the end-exercise PCr concentration decreased from 29 ± 2 mM of WRmax to 18 ± 1 mM with increasing workload. Likewise, the end-exercise Mb oxygenation level declined linearly with increasing workload. As the exercise workload increased, the ATPox synthesis rate increased linearly (r2 = 0.45), whereas there was no significant change in Mb-derived VO2. At rest, the estimated P/O ratio in the gastrocnemius muscle was 1.95 ± 0.68, whereas non-physiological values were obtained during exercise. Conclusion: During exercise, Mb-derived VO2 was likely limited by O2 availability with the methodology employed (30-sec occlusion, spectrum time resolution of 10 sec), which precluded the accurate quantification of mitochondrial effciency. However, mitochondrial effciency calculated at rest is in agreement with previously documented values ex vivo and thus provides an additional parameter to evaluate mitochondrial function in vivo. This work was funded in part by National Heart, Lung, and Blood Institute Grant R00HL125756. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

EFFECT OF MEMANTINE AS N-METHYL D-ASPARTATE RECEPTOR ANTAGONIST ON CASPASE-9 LEVEL OF RETINAL GANGLION CELL IN METHANOL TOXIC OPTIC NEUROPATHY RAT MODEL

Abstract
 Introduction & Objectives : Methanol toxic optic neuropathy (MTON) remains as a health issue in developing countries and there are no current definitive treatments. Accumulation of formic acid as metabolite of methanol causes mitochondrial dysfunction that can reduce ATP synthesis rate, disrupt axoplasmic transport, and elevate the reactive oxygen species (ROS). All of these mechanisms lead to the death of retinal ganglion cell (RGC) and optic nerve. RGC destruction in MTON has similar patterns with those that have been observed in glaucoma, ischemia, and trauma cases, that is through excessive activation of glutamate neurotransmitter via N-Methyl D-Aspartate receptors (NMDAR). Memantine is a NMDAR antagonist that inhibit the ion channel of NMDAR. This inhibitory effect of NMDAR by memantine is expected to reduce intracellular calcium level, prevent apoptosis of RGC, and prevent further mitochondrial dysfunction. Caspase-9 is essential to eliminate cells by executing apoptotic Objective: To observe the effect of Memantine on Caspase-9 level of RGC in Methanol-Intoxicated Rats.
 Methods : This experimental study was performed on 28 Methanol-Intoxicated Rats that are assigned into four treatment groups: K1 (MTON;enucleated day-3), K2 (MTON+Memantine; enucleated day-3), K3 (MTON; enucleated day-7), and K4 (MTON+Memantine; enucleated day-7). During the experiment, all groups were exposed to N2O:O2 gas and were given methanol orally. Caspase-9 level of RGC were measured immediately after enucleation.
 Results : The average of Caspase-9 level (ng/ml): K1=197,88±33,98; K2=205,64±63,66; K3=243,15±40,13; K4=150,08±119,60. The Caspase-9 level was significantly lower in K4 compared to K3 (p value =0.038).
 Conclusion : Memantine decrease the level of Caspase-9 of RGC in Methanol Toxic Optic Neuropathy rat model.

Mitochondrial atp1 mRNA knockdown by a custom-designed pentatricopeptide repeat protein alters ATP synthase.

Spontaneous mutations are rare in mitochondria and the lack of mitochondrial transformation methods has hindered genetic analyses. We show that a custom-designed RNA-binding pentatricopeptide repeat (PPR) protein binds and specifically induces cleavage of ATP synthase subunit1 (atp1) mRNA in mitochondria, significantly decreasing the abundance of the Atp1 protein and the assembled F1Fo ATP synthase in Arabidopsis (Arabidopsis thaliana). The transformed plants are characterized by delayed vegetative growth and reduced fertility. Five-fold depletion of Atp1 level was accompanied by a decrease in abundance of other ATP synthase subunits and lowered ATP synthesis rate of isolated mitochondria, but no change to mitochondrial electron transport chain complexes, adenylates, or energy charge in planta. Transcripts for amino acid transport and a variety of stress response processes were differentially expressed in lines containing the PPR protein, indicating changes to achieve cellular homeostasis when ATP synthase was highly depleted. Leaves of ATP synthase-depleted lines showed higher respiratory rates and elevated steady-state levels of numerous amino acids, most notably of the serine family. The results show the value of using custom-designed PPR proteins to influence the expression of specific mitochondrial transcripts to carry out reverse genetic studies on mitochondrial gene functions and the consequences of ATP synthase depletion on cellular functions in Arabidopsis.

Protein-Rich Rafts in Hybrid Polymer/Lipid Giant Unilamellar Vesicles.

Considerable attention has been dedicated to lipid rafts due to their importance in numerous cell functions such as membrane trafficking, polarization, and signaling. Next to studies in living cells, artificial micrometer-sized vesicles with a minimal set of components are established as a major tool to understand the phase separation dynamics and their intimate interplay with membrane proteins. In parallel, mixtures of phospholipids and certain amphiphilic polymers simultaneously offer an interface for proteins and mimic this segregation behavior, presenting a tangible synthetic alternative for fundamental studies and bottom-up design of cellular mimics. However, the simultaneous insertion of complex and sensitive membrane proteins is experimentally challenging and thus far has been largely limited to natural lipids. Here, we present the co-reconstitution of the proton pump bo3 oxidase and the proton consumer ATP synthase in hybrid polymer/lipid giant unilamellar vesicles (GUVs) via fusion/electroformation. Variations of the current method allow for tailored reconstitution protocols and control of the vesicle size. In particular, mixing of protein-free and protein-functionalized nanosized vesicles in the electroformation film results in larger GUVs, while separate reconstitution of the respiratory enzymes enables higher ATP synthesis rates. Furthermore, protein labeling provides a synthetic mechanism for phase separation and protein sequestration, mimicking lipid- and protein-mediated domain formation in nature. The latter means opens further possibilities for re-enacting phenomena like supercomplex assembly or symmetry breaking and enriches the toolbox of bottom-up synthetic biology.

Protective Effect of Uridine on Structural and Functional Rearrangements in Heart Mitochondria after a High-Dose Isoprenaline Exposure Modelling Stress-Induced Cardiomyopathy in Rats.

The pyrimidine nucleoside uridine and its phosphorylated derivates have been shown to be involved in the systemic regulation of energy and redox balance and promote the regeneration of many tissues, including the myocardium, although the underlying mechanisms are not fully understood. Moreover, rearrangements in mitochondrial structure and function within cardiomyocytes are the predominant signs of myocardial injury. Accordingly, this study aimed to investigate whether uridine could alleviate acute myocardial injury induced by isoprenaline (ISO) exposure, a rat model of stress-induced cardiomyopathy, and to elucidate the mechanisms of its action related to mitochondrial dysfunction. For this purpose, a biochemical analysis of the relevant serum biomarkers and ECG monitoring were performed in combination with transmission electron microscopy and a comprehensive study of cardiac mitochondrial functions. The administration of ISO (150 mg/kg, twice with an interval of 24 h, s.c.) to rats caused myocardial degenerative changes, a sharp increase in the serum cardiospecific markers troponin I and the AST/ALT ratio, and a decline in the ATP level in the left ventricular myocardium. In parallel, alterations in the organization of sarcomeres with focal disorganization of myofibrils, and ultrastructural and morphological defects in mitochondria, including disturbances in the orientation and packing density of crista membranes, were detected. These malfunctions were improved by pretreatment with uridine (30 mg/kg, twice with an interval of 24 h, i.p.). Uridine also led to the normalization of the QT interval. Moreover, uridine effectively inhibited ISO-induced ROS overproduction and lipid peroxidation in rat heart mitochondria. The administration of uridine partially recovered the protein level of the respiratory chain complex V, along with the rates of ATP synthesis and mitochondrial potassium transport, suggesting the activation of the potassium cycle through the mitoKATP channel. Taken together, these results indicate that uridine ameliorates acute ISO-induced myocardial injury and mitochondrial malfunction, which may be due to the activation of mitochondrial potassium recycling and a mild uncoupling leading to decreased ROS generation and oxidative damage.

MRNA Translation Inhibition Targets Bioenergetic Homeostasis in AML Cells in Vitro and In Vivo and Synergizes with Cytarabine and Venetoclax

Acute myeloid leukemia (AML) is an aggressive hematological cancer resulting from uncontrolled proliferation of differentiation-blocked myeloid cells. Seventy percent of AML patients are currently not cured with available treatments, highlighting the need of novel therapeutic strategies. Recently, inhibition of BCL-2 with venetoclax in combination with hypomethylating agents has emerged as an attractive strategy for high-risk AML cases. Another promising target in AML is the mammalian target of rapamycin complex 1 (mTORC1) (Oki et al, Nature Comm, 2021). However, clinical inhibition of mTORC1 is limited by its reactivation through compensatory and regulatory feedback loops. To curtail these drawbacks, we adopted a strategy of inhibiting an important effector of the mTORC1 signaling pathway controlling mRNA translation - the eukaryotic initiation factor 4A (eIF4A), subunit of the translation initiation complex eIF4F. Recent evidence suggests that translational programs mediated by the mTORC1/4E-BP/eIF4F axis can support resistance to therapy in various cancer models driven by oncogenic kinases, in part by allowing cellular metabolic plasticity (Hulea L et al, Cell Metab. 2018). In fact, metabolism, and specifically mitochondrial oxidative phosphorylation, has emerged as a central dependency of AML cells, sustaining resistance to therapy and recurrence. Using the MOLM-14 human AML cell line to model therapy-resistant disease, we previously demonstrated the anti-leukemic effect of a potent and specific eIF4A inhibitor (eIF4Ai), CR-1-31-B, both in vitro and in vivo (Fooks et al, J Exp Clin Cancer Res 2022). eIF4Ai affected cellular metabolism, by reducing mitochondrial membrane potential (MMP) and the rate of ATP synthesis from mitochondrial respiration and glycolysis. Concomitantly, eIF4Ai decreased intracellular levels of specific metabolic intermediates of the tricarboxylic acid cycle (TCA cycle) and glucose metabolism, while enhancing mitochondrial ROS. Furthermore, eIF4i enhanced apoptotic priming while reducing the expression levels of the antiapoptotic factors BCL2, BCL-XL and MCL1. Importantly, CR-1-31-B acted synergistically in vitro in combination with cytarabine or venetoclax. Recently, we have expanded our characterization of the eIF4Ai/venetoclax combination in MOML-14 cells, and showed that venetoclax potentiates the CR-1-31-B-induced inhibition of cellular respiration, glycolysis and ATP production (Seahorse). The combination provokes a robust apoptotic response with different temporal dynamics to single treatment, as measured using the Incucyte platform. In vivo, we have validated the strong anti-leukemic response induced by CR-1-31-B in a MOLM-14 transplantation model, after only 7 days of treatment, and showed that venetoclax slightly potentiates this effect. In addition, eIF4A inhibition reduces the levels of several metabolic proteins (GLS1, IDH1), which supports our previous observations of decreased levels of TCA cycle metabolites. We have confirmed the metabolic and pro-apoptotic effect of eIF4Ai in a second cellular model of AML, U937. Consistent with previous results, in U937 cells CR-1-31-B induces apoptosis at low nM concentrations, reduces cellular bioenergetics, ATP production, as well as levels of pro-proliferative (cyclin D3, CDK4) and anti-apoptotic (BCL-2, MCL-1) proteins. Our collective studies highlight (i) the importance of the crosstalk between mRNA translation and metabolic regulation and (ii) that direct inhibition of translation represents an appealing therapeutic strategy for clinical cases of therapy resistance that are dependent on the mTORC1/eIF4F axis. This is of interest as several translation inhibitors are currently tested in phase 1/2 clinical trials in solid malignancies. # co-corresponding authors

Treatment of cardiomyocytes isolated from left ventricular myocardium of dogs with chronic heart failure with ALDA-1, an activator of aldehyde dehydrogenase-2, improves mitochondrial function

Abstract Background Reactive oxygen species (ROS) generated by dysfunctional mitochondria (MITO) of the failing heart plays an important role in lipid peroxidation of polyunsaturated fatty acids in cell and MITO membranes resulting in the production of aldehydes, such as 4-hydroxy-2-nonenal (4-HNE). MITO aldehyde dehydrogenase-2 (mALDH2) is a key enzyme for the detoxification of endogenous aldehyde substrates through NAD dependent oxidation. ALDH2 catalyzes toxic 4HNE to nontoxic 4HNA in cardiac cells. We previously showed that mALDH2 expression and activity are markedly reduced in MITO of failing LV cardiomyocytes (CM), a maladaptation that can lead to considerable accumulation of 4-HNE in MITO and ultimately to progressive LV systolic and diastolic dysfunction. In this study, we tested the hypothesis that exposure of failing cardiomyocytes to ALDA-1, an activator of mALDH2, improves MITO function. Methods Freshly isolated CM from LV myocardium of dogs (n = 7) with heart failure (HF, LV ejection fraction ≤35%) and of normal (NL) dogs (n = 4) were incubated for 1 hour in absence (baseline) and presence of Alda-1 at concentration of 10 µM. After incubation, ADP-stimulated respiration (ADPresp) and maximal rate of ATP synthesis (ATPmax) were measured using Strathklein respirometer and bioluminescence ATP assay kit. Results Treatment of isolated CM from HF dogs with 10 µM Alda-1 significantly increased ADPresp (277 ± 18 vs. 189 ± 12 nAtom O/min/mg, p<0.05) and ATPmax (30 ± 3 vs. 16 ± 3 nmoles/min/mg, p<0.05) compared to untreated CM of HF dogs. Treatment of CM from NL dogs with 10 µM Alda-1 had no effect on ADPresp (488 ± 18 vs. 484 ± 19 nAtom O/min/mg) or ATPmax (54 ± 3 vs. 52 ± 2 nmoles/min/mg) compared to untreated NL CM. Conclusions Treatment of CM from HF dogs with Alda-1 improved overall MITO function. Activation of mALDH2 represents a potential viable therapeutic target for treating HF.

Abstract 13305: Energetic Basis of Recovered Ejection Fraction in Human Heart Failure

Introduction: Left ventricular ejection fraction (LVEF) improvement in HFrEF patients is classified as heart failure with improved ejection fraction (HFimpEF) and is associated with better long-term outcomes than persistent HFrEF. HFimpEF is difficult to predict and underlying mechanisms remain poorly understood. Hypothesis: Better myocardial high-energy phosphate metabolism at baseline predicts future LVEF recovery and outcomes in a general HFrEF population Methods: We measured cardiac energetics, the ratio of the two main high-energy phosphates, phosphocreatine and ATP (PCr/ATP), and the rate of ATP synthesis through the primary cardiac energy reserve reaction creatine kinase (“CK flux”), in 37 nonischemic HFrEF patients (LVEF ≤40%) with 31 P cardiac magnetic resonance spectroscopy (MRS) at 3T. Baseline transthoracic echocardiogram (TTE) was obtained prior to 31 P MRS and follow-up consecutive TTEs with the two highest LVEFs meeting HFimpEF criteria (LVEF >40% and ≥10% increase over baseline LVEF) over a median follow-up of 6.9 years were averaged and used to classify HFimpEF. Results: There were no significant differences in demographics, HF etiology, baseline TTE metrics, or medications between persistent HFrEF (n=22) and eventual HFimpEF (n=15). Cardiac CK flux was significantly higher at baseline in patients who later recovered to HFimpEF than in those who remained HFrEF (p=0.031). Better baseline cardiac CK flux was associated with increases in LVEF (p=0.025) and decreases in LV end-diastolic diameter (p=0.016) over time. By both dichotomous and continuous metrics, better preserved CK flux was associated with future HFimpEF. Fourteen subjects (38%), all persistent HFrEF, experienced cardiac death, transplant, or LVAD and had significantly lower baseline CK flux than outcome-free patients (p=0.017). Cardiac PCr/ATP was not associated with eventual HFimpEF, outcomes, or changes in TTE indices. Conclusion: Better preserved cardiac CK flux is associated with future LVEF recovery and LV reverse remodeling while lower CK flux is associated with worse cardiac outcomes in HFrEF patients. Interventions restoring CK flux, and possibly other energy-generating metabolic reactions, may elicit myocardial recovery in HFrEF patients.

Effect of Burosumab on Muscle Function and Strength, and Rates of ATP Synthesis in Skeletal Muscle in Adults With XLH.

In clinical trials, burosumab ameliorates symptoms of pain, fatigue, and stiffness and improves performance on certain muscle function studies in patients with X-linked hypophosphatemia (XLH). This work aimed to determine if burosumab increases adenosine triphosphate (ATP) synthesis in skeletal muscle of treatment-naive adults with XLH, and if so, whether that correlates with improved muscle function. Ten untreated, symptomatic adults with XLH had ATP synthesis rates measured in the right calf using the 31P magnetic resonance spectroscopy saturation transfer technique. Baseline muscle function tests and symptoms of pain, fatigue, stiffness, and lower-extremity joint pain were quantified. All participants were treated with burosumab, 1 mg/kg every 4 weeks for 12 weeks. ATP synthesis rates and muscle function tests were repeated 2 weeks ("peak") and 4 weeks ("trough") after the third dose of burosumab. All symptoms improved with treatment. Performance on the 6-Minute Walk Test (6MWT) and Sit to Stand (STS) tests also improved. Muscle strength and ATP synthesis rates did not change over the 3 months of the study. When individuals whose performances on the 6MWT and STS test were at or better than the median outcome for those tests were compared to those whose outcomes were below the median, no difference was observed in the rate of change in ATP synthesis. Intracellular muscle concentrations of phosphate were normal. The improvement in the 6MWT and STS test without changes in muscle strength or ATP synthesis rates suggests that reductions in pain, fatigue, and stiffness may partly explain the improved performance. Intracellular phosphate in skeletal muscle is insulated from hypophosphatemia in XLH.

Molecular Bulkiness ofa Single Amino Acid inthe F1 α-Subunit Determines the Robustness ofCyanobacterial ATP Synthase.

Cyanobacteria are promising photosynthetic organisms owing to their ease of genetic manipulation. Among them, Synechococcus elongatus UTEX 2973 exhibits faster growth, higher biomass production efficiency and more robust stress tolerance compared with S. elongatus PCC 7942. This is due to specific genetic differences, including four single-nucleotide polymorphisms (SNPs) in three genes. One of these SNPs alters an amino acid at position 252 of the FoF1 ATP synthase α-subunit from Tyr to Cys (αY252C) in S. elongatus 7942. This change has been shown to significantly affect growth rate and stress tolerance, specifically in S. elongatus. Furthermore, experimental substitutions with several other amino acids have been shown to alter the ATP synthesis rate in the cell. In the present study, we introduced identical amino acid substitutions into Synechocystis sp. PCC 6803 at position 252 to elucidate the amino acid's significance and generality across cyanobacteria. We investigated the resulting impact on growth, intracellular enzyme complex levels, intracellular ATP levels and enzyme activity. The results showed that the αY252C substitution decreased growth rate and high-light tolerance. This indicates that a specific bulkiness of this amino acid's side chain is important for maintaining cell growth. Additionally, a remarkable decrease in the membrane-bound enzyme complex level was observed. However, the αY252C substitution did not affect enzyme activity or intracellular ATP levels. Although the mechanism of growth suppression remains unknown, the amino acid at position 252 is expected to play an important role in enzyme complex formation.

The pro-oncogenic protein IF1 does not contribute to the Warburg effect and is not regulated by PKA in cancer cells

The endogenous inhibitor of mitochondrial F1Fo-ATPase (ATP synthase), IF1, has been shown to exert pro-oncogenic actions, including reprogramming of cellular energy metabolism (Warburg effect). The latter action of IF1 has been reported to be hampered by its PKA-dependent phosphorylation, but both reprogramming of metabolism and PKA-dependent phosphorylation are intensely debated. To clarify these critical issues, we prepared stably IF1-silenced clones and compared their bioenergetics with that of the three parental IF1-expressing cancer cell lines. All functional parameters: respiration rate, ATP synthesis rate (OXPHOS), and mitochondrial membrane potential were similar in IF1-silenced and control cells, clearly indicating that IF1 cannot inhibit the ATP synthase in cancer cells when the enzyme works physiologically. Furthermore, all cell types exposed to PKA modulators and energized with NAD+-dependent substrates or succinate showed similar OXPHOS rate regardless of the presence or absence of IF1. Therefore, our results rule out that IF1 action is modulated by its PKA-dependent phosphorylated/dephosphorylated state. Notably, cells exposed to a negative PKA modulator and energized with NAD+-dependent substrates showed a significant decrease of the OXPHOS rate matching previously reported inactivation of complex I. Overall, this study definitively demonstrates that IF1 inhibits neither mitochondrial ATP synthase nor OXPHOS in normoxic cancer cells and does not contribute to the Warburg effect. Thus, currently the protection of cancer cells from severe hypoxia/anoxia and apoptosis remain the only unquestionable actions of IF1 as pro-oncogenic factor that may be exploited to develop therapeutic approaches.

Loss of mitochondrial performance at high temperatures is correlated with upper thermal tolerance among populations of an intertidal copepod.

Environmental temperatures have pervasive effects on the performance and tolerance of ectothermic organisms, and thermal tolerance limits likely play key roles underlying biogeographic ranges and responses to environmental change. Mitochondria are central to metabolic processes in eukaryotic cells, and these metabolic functions are thermally sensitive; however, potential relationships between mitochondrial function, thermal tolerance limits and local thermal adaptation in general remain unresolved. Loss of ATP synthesis capacity at high temperatures has recently been suggested as a mechanistic link between mitochondrial function and upper thermal tolerance limits. Here we use a common-garden experiment with seven locally adapted populations of intertidal copepods (Tigriopus californicus), spanning approximately 21.5° latitude, to assess genetically based variation in the thermal performance curves of maximal ATP synthesis rates in isolated mitochondria. These thermal performance curves displayed substantial variation among populations with higher ATP synthesis rates at lower temperatures (20-25°C) in northern populations than in southern populations. In contrast, mitochondria from southern populations maintained ATP synthesis rates at higher temperatures than the temperatures that caused loss of ATP synthesis capacity in mitochondria from northern populations. Additionally, there was a tight correlation between the thermal limits of ATP synthesis and previously determined variation in upper thermal tolerance limits among populations. This suggests that mitochondria may play an important role in latitudinal thermal adaptation in T. californicus, and supports the hypothesis that loss of mitochondrial performance at high temperatures is linked to whole-organism thermal tolerance limits in this ectotherm.

Developmental rate displays effects of inheritance but not of sex in interpopulation hybrids of Tigriopus californicus.

Coevolved genetic interactions within populations can be disrupted by hybridization resulting in loss of fitness in hybrid individuals (i.e., hybrid breakdown). However, the extent to which variation in fitness-related traits among hybrids is inherited across generations remains unclear, and variation in these traits may be sex-specific in hybrids due to differential effects of genetic incompatibilities in females and males. Here we present two experiments investigating variation in developmental rate among reciprocal interpopulation hybrids of the intertidal copepod Tigriopus californicus. Developmental rate is a fitness-related trait in this species that is affected by interactions between mitochondrial-encoded and nuclear-encoded genes in hybrids that result in variation in mitochondrial ATP synthesis capacities. First, we show that F2 -hybrid developmental rate is equivalent in two reciprocal crosses and is unaffected by sex, suggesting that breakdown of developmental rate is likely experienced equally by females and males. Second, we demonstrate that variation in developmental rate among F3 hybrids is heritable; times to copepodid metamorphosis of F4 offspring of fast-developing F3 parents (12.25 ± 0.05 days, μ ± SEM)were significantly faster than those of F4 offspring of slow-developing parents (14.58 ± 0.05 days). Third, we find that ATP synthesis rates in these F4 hybrids are unaffected by the developmental rates of their parents, but that mitochondria from females synthesize ATP at faster rates than mitochondria from males. Taken together, these results suggest that sex-specific effects vary among fitness-related traits in these hybrids, and that effects likely associated with hybrid breakdown display substantial inheritance across hybrid generations.