ATP Demand Research Articles

Modulation of OPC Mitochondrial Function by Inhibiting USP30 Promotes Their Differentiation.

Multiple lines of evidence indicate that mitochondrial dysfunction occurs in demyelinating diseases, such as multiple sclerosis (MS). Failure of remyelination is thought to be caused in part by a block of oligodendrocyte progenitor cell (OPC) differentiation into oligodendrocytes, which generate myelin sheaths around axons. The process of OPC differentiation requires a substantial amount of energy and high demand for ATP which is supplied through the mitochondria. In this study, we highlight mitochondrial gene expression changes during OPC differentiation in two murine models of remyelination and in human postmortem MS brains. Given these transcriptional alterations, we then investigate whether genetic alteration of USP30, a mitochondrial deubiquitinase, enhances OPC differentiation and myelination. By genetic knockout of USP30, we observe increased OPC differentiation and myelination without affecting OPC proliferation and survival in invitro and exvivo assays. We also find that OPC differentiation is accelerated invivo following focal demyelination in USP30 knockout mice. The promotion of OPC differentiation and myelination observed is associated with increased oxygen consumption rates in USP30 knockout OPCs. Together, these data indicate a role for mitochondrial function and USP30 in OPC differentiation and myelination.

ATP dynamics as a predictor of future podocyte structure and function after acute ischemic kidney injury in female mice

Acute kidney injury (AKI), typically caused by ischemia, is a common clinical complication with a poor prognosis. Although proteinuria is an important prognostic indicator of AKI, the underlying causal mechanism remains unclear. In vitro studies suggest that podocytes have high ATP demands to maintain their structure and function, however, analyzing their ATP dynamics in living kidneys has been technically challenging. Here, using intravital imaging to visualize a FRET-based ATP biosensor expressed systemically in female mice due to their suitability for glomerular imaging, we monitor the in vivo ATP dynamics in podocytes during ischemia reperfusion injury. ATP levels decrease during ischemia, but recover after reperfusion in podocytes, exhibiting better recovery than in glomerular endothelial cells. However, prolonged ischemia results in insufficient ATP recovery in podocytes, which is inversely correlated with mitochondrial fragmentation and foot process effacement during the chronic phase. Furthermore, preventing mitochondrial fission via pharmacological inhibition ameliorates podocyte injury in vitro, ex vivo, and in vivo. Thus, these findings provide several insights into how ATP depletion and mitochondrial fragmentation contribute to podocyte injury after ischemic AKI and could potentially be therapeutic targets.

Isolated Mouse Adult Cardiomyocytes Display Minimal Mitochondrial ATP Demand and Maximal Reliance on Glycolysis.

Mitochondrial maladaptation is a hallmark of heart failure, contributing to impaired energy production and contractile dysfunction. Understanding the bioenergetics of cardiomyocytes under healthy and pathological conditions is critical for characterizing mitochondrial maladaptation. While adult cardiomyocytes from rodents are a widely used model, recent studies have reported oligomycin insensitivity in these cells, a phenomenon often overlooked. This has led to incomplete assessments of key bioenergetic parameters, such as ATP-linked respiration and glycolytic capacity, focusing primarily on basal and maximal respiration. In this study, we performed a comprehensive characterization of bioenergetic and glycolytic profiles in three cardiomyocyte models: neonatal rat ventricular myocytes (NRVMs), human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), and mouse adult cardiomyocytes (mouse ACMs). Our findings demonstrate distinct metabolic adaptations in mouse ACMs, revealing critical insights into mitochondrial function, ATP demand, and glycolytic reliance. These results underscore the importance of selecting appropriate cellular models for studying mitochondrial bioenergetics in cardiac physiology and pathology.

Cellular ATP demand creates metabolically distinct subpopulations of mitochondria.

Mitochondria serve a crucial role in cell growth and proliferation by supporting both ATP synthesis and the production of macromolecular precursors. Whereas oxidative phosphorylation (OXPHOS) depends mainly on the oxidation of intermediates from the tricarboxylic acid cycle, the mitochondrial production of proline and ornithine relies on reductive synthesis1. How these competing metabolic pathways take place in the same organelle is not clear. Here we show that when cellular dependence on OXPHOS increases, pyrroline-5-carboxylate synthase (P5CS)-the rate-limiting enzyme in the reductive synthesis of proline and ornithine-becomes sequestered in a subset of mitochondria that lack cristae and ATP synthase. This sequestration is driven by both the intrinsic ability of P5CS to form filaments and the mitochondrial fusion and fission cycle. Disruption of mitochondrial dynamics, by impeding mitofusin-mediated fusion or dynamin-like-protein-1-mediated fission, impairs the separation of P5CS-containing mitochondria from mitochondria that are enriched in cristae and ATP synthase. Failure to segregate these metabolic pathways through mitochondrial fusion and fission results in cells either sacrificing the capacity for OXPHOS while sustaining the reductive synthesis of proline, or foregoing proline synthesis while preserving adaptive OXPHOS. These findings provide evidence of the key role of mitochondrial fission and fusion in maintaining both oxidative and reductive biosyntheses in response to changing nutrient availability and bioenergetic demand.

The mitochondrial physiology of torpor in ruby-throated hummingbirds, Archilochus colubris.

Hummingbirds save energy by facultatively entering torpor, but the physiological mechanisms underlying this metabolic suppression are largely unknown. We compared whole-animal and pectoralis mitochondrial metabolism between torpid and normothermic ruby-throated hummingbirds (Archilochus colubris). When fasting, hummingbirds were exposed to 10°C ambient temperature at night and they entered torpor; average body temperature decreased by nearly 25°C (from ∼37 to ∼13°C) and whole-animal metabolic rate (V̇O2) decreased by 95% compared with normothermia, a much greater metabolic suppression compared with that of mammalian daily heterotherms. We then measured pectoralis mitochondrial oxidative phosphorylation (OXPHOS) fueled by either carbohydrate or fatty acid substrates at both 39°C and 10°C in torpid and normothermic hummingbirds. Aside from a 20% decrease in electron transport system complex I-supported respiration with pyruvate, the capacity for OXPHOS at a common in vivo temperature did not differ in isolated mitochondria between torpor and normothermia. Similarly, the activities of pectoralis pyruvate dehydrogenase and 3-hydroxyacyl-CoA dehydrogenase did not differ between the states. Unlike heterothermic mammals, hummingbirds do not suppress muscle mitochondrial metabolism in torpor by active, temperature-independent mechanisms. Other mechanisms that may underly this impressive whole-animal metabolic suppression include decreasing ATP demand or relying on rapid passive cooling facilitated by the very small body size of A. colubris.

Metabolic Adaptation in Epilepsy: From Acute Response to Chronic Impairment.

Epilepsy is characterized by hypersynchronous neuronal discharges, which are associated with an increased cerebral metabolic rate of oxygen and ATP demand. Uncontrolled seizure activity (status epilepticus) results in mitochondrial exhaustion and ATP depletion, which potentially generate energy mismatch and neuronal loss. Many cells can adapt to increased energy demand by increasing metabolic capacities. However, acute metabolic adaptation during epileptic activity and its relationship to chronic epilepsy remains poorly understood. We elicited seizure-like events (SLEs) in an in vitro model of status epilepticus for eight hours. Electrophysiological recording and tissue oxygen partial pressure recordings were performed. After eight hours of ongoing SLEs, we used proteomics-based kinetic modeling to evaluate changes in metabolic capacities. We compared our findings regarding acute metabolic adaptation to published proteomic and transcriptomic data from chronic epilepsy patients. Epileptic tissue acutely responded to uninterrupted SLEs by upregulating ATP production capacity. This was achieved by a coordinated increase in the abundance of proteins from the respiratory chain and oxidative phosphorylation system. In contrast, chronic epileptic tissue shows a 25-40% decrease in ATP production capacity. In summary, our study reveals that epilepsy leads to dynamic metabolic changes. Acute epileptic activity boosts ATP production, while chronic epilepsy reduces it significantly.

Myosin ATPase inhibition fails to rescue the metabolically dysregulated proteome of nebulin-deficient muscle.

Nemaline myopathy (NM) is a genetic muscle disease, primarily caused by mutations in the NEB gene (NEB-NM) and with muscle myosin dysfunction as a major molecular pathogenic mechanism. Recently, we have observed that the myosin biochemical super-relaxed state was significantly impaired in NEB-NM, inducing an aberrant increase in ATP consumption and remodelling of the energy proteome in diseased muscle fibres. Because the small-molecule Mavacamten is known to promote the myosin super-relaxed state and reduce the ATP demand, we tested its potency in the context of NEB-NM. We first conducted in vitro experiments in isolated single myofibres from patients and found that Mavacamten successfully reversed the myosin ATP overconsumption. Following this, we assessed its short-term in vivo effects using the conditional nebulin knockout (cNeb KO) mouse model and subsequently performing global proteomics profiling in dissected soleus myofibres. After a 4week treatment period, we observed a remodelling of a large number of proteins in both cNeb KO mice and their wild-type siblings. Nevertheless, these changes were not related to the energy proteome, indicating that short-term Mavacamten treatment is not sufficient to properly counterbalance the metabolically dysregulated proteome of cNeb KO mice. Taken together, our findings emphasize Mavacamten potency in vitro but challenge its short-term efficacy in vivo. KEY POINTS: No cure exists for nemaline myopathy, a type of genetic skeletal muscle disease mainly derived from mutations in genes encoding myofilament proteins. Applying Mavacamten, a small molecule directly targeting the myofilaments, to isolated membrane-permeabilized muscle fibres from human patients restored myosin energetic disturbances. Treating a mouse model of nemaline myopathy in vivo with Mavacamten for 4weeks, remodelled the skeletal muscle fibre proteome without any noticeable effects on energetic proteins. Short-term Mavacamten treatment may not be sufficient to reverse the muscle phenotype in nemaline myopathy.

Defective mitochondrial COX1 translation due to loss of COX14 function triggers ROS-induced inflammation in mouse liver

Mitochondrial oxidative phosphorylation (OXPHOS) fuels cellular ATP demands. OXPHOS defects lead to severe human disorders with unexplained tissue specific pathologies. Mitochondrial gene expression is essential for OXPHOS biogenesis since core subunits of the complexes are mitochondrial-encoded. COX14 is required for translation of COX1, the central mitochondrial-encoded subunit of complex IV. Here we describe a COX14 mutant mouse corresponding to a patient with complex IV deficiency. COX14M19I mice display broad tissue-specific pathologies. A hallmark phenotype is severe liver inflammation linked to release of mitochondrial RNA into the cytosol sensed by RIG-1 pathway. We find that mitochondrial RNA release is triggered by increased reactive oxygen species production in the deficiency of complex IV. Additionally, we describe a COA3Y72C mouse, affected in an assembly factor that cooperates with COX14 in early COX1 biogenesis, which displays a similar yet milder inflammatory phenotype. Our study provides insight into a link between defective mitochondrial gene expression and tissue-specific inflammation.

Efficiency of acetate-based isopropanol synthesis in Escherichia coli W is controlled by ATP demand

BackgroundDue to increasing ecological concerns, microbial production of biochemicals from sustainable carbon sources like acetate is rapidly gaining importance. However, to successfully establish large-scale production scenarios, a solid understanding of metabolic driving forces is required to inform bioprocess design. To generate such knowledge, we constructed isopropanol-producing Escherichia coli W strains.ResultsBased on strain screening and metabolic considerations, a 2-stage process was designed, incorporating a growth phase followed by a nitrogen-starvation phase. This process design yielded the highest isopropanol titers on acetate to date (13.3 g L−1). Additionally, we performed shotgun and acetylated proteomics, and identified several stress conditions in the bioreactor scenarios, such as acid stress and impaired sulfur uptake. Metabolic modeling allowed for an in-depth characterization of intracellular flux distributions, uncovering cellular demand for ATP and acetyl-CoA as limiting factors for routing carbon toward the isopropanol pathway. Moreover, we asserted the importance of a balance between fluxes of the NADPH-providing isocitrate dehydrogenase (ICDH) and the product pathway.ConclusionsUsing the newly gained system-level understanding for isopropanol production from acetate, we assessed possible engineering approaches and propose process designs to maximize production. Collectively, our work contributes to the establishment and optimization of acetate-based bioproduction systems.Graphical

Abstract Tu107: Decreased Mitochondrial Protein Expression Accompanies Atrial Hypertrophy but Precedes Atrial Fibrillation Onset in a Mouse Model of Spontaneous Atrial Fibrillation

Introduction: Atrial fibrillation (AF), characterized by episodes of rapid and irregular heart rate, increases left atrial (LA) demand for ATP. While the heart is metabolically flexible, able to use fatty acids, lactate, glucose, ketones, and amino acids to generate ATP, fatty acids are the primary fuel source. AF is associated with altered mitochondrial structure and function, but the role of energetic pathways in early stages of AF progression is not clear. Hypothesis: Decreased expression of mitochondrial proteins contributes to AF progression. Aim: Evaluate expression of proteins that influence mitochondrial structure and energetic capacity in AF onset and progression. Methods: AF burden was assessed weekly by electrocardiography (ECG) in male wild-type (WT) and cAMP responsive element modulator IbΔC-X heterozygous mice (CREM) (n=3). LA (4 - 9 mg) was obtained at 7 weeks and 16 weeks. Global proteomics was conducted, and 139,316 peptides were identified that mapped to 6000 robustly quantified proteins. Results: LA hypertrophy was evident in 7-week and 16-week CREM mice (Figure 1). We detected 2408 proteins differentially expressed in CREM compared to WT. At 7 weeks, Hallmark pathways adipogenesis, fatty acid metabolism, oxidative phosphorylation, and myogenesis were decreased in CREM (Table 1). These changes persisted at 16 weeks. We identified 740 differentially expressed mitochondrial proteins using MitoCarta 3.0 (552 decreased, 188 increased). Mitochondrial pathways associated with energy production, including branched chain amino acid metabolism, fatty acid transport and oxidation, carbohydrate metabolism, and tricarboxylic acid cycle were decreased (Table 1). Mitochondrial cristae organizing system (MICOS) and mitophagy, pathways that influence mitochondrial structure and content, were decreased. Terminal ECGs confirmed AF in 16-week CREM mice only. The coefficient of variation of RR intervals (CVRR), a measure of heart rate variability, was increased in 16-week CREM (Figure 2). These data suggest that decreased expression of mitochondrial proteins accompanies LA hypertrophy but precedes onset of AF. Conclusions: Decreased expression of proteins that regulate mitochondrial structure and energy production prior to AF onset suggests that these proteins play an important role in early LA changes that promote AF onset and progression. Future studies will examine how these changes in protein expression relate to alterations in atrial energy homeostasis.

Abstract Tu110: CD36-Mediated Transendothelial Fatty Acid Transport Determines Cardiomyocyte Uptake and is Critical to Limiting a Lipotoxic Ceramide Profile and Supporting Cardiac Function during Pathological Stress.

The heart primarily relies on long chain fatty acid oxidation (LCFA) to meet ATP demand. CD36, is a transmembrane glycoprotein transferase for LCFA uptake widely expressed in several cell types including endothelial cells (ECs), cardiomyocytes (CMs), skeletal myocytes, adipocytes, and monocyte/macrophages. Notably, genetic deficiency of CD36 in humans is associated with hypertrophic cardiomyopathy. The role of CD36 in transendothelial LCFA delivery to CMs under physiologic vs pathologic conditions is unknown. We explored consequences of EC-specific CD36 deletion (EC-CD36 KO mice) on the cardiac response to pressure overload. Lack of EC-CD36 increased severity of left ventricular hypertrophy (LVH), and systolic and diastolic dysfunction at 8 weeks of pressure overload via transverse aortic constriction (TAC), with increases in HW:TL ratio, LV mass by echo, reduced EF (p<0.05), and elevated E/E’ in EC-CD36-KO vs. flox/flox littermate controls (f/f) (p<0.5) (Fig 1). Dynamic mode 13 C NMR revealed delayed 13 C LCFA uptake kinetics (time constant, τ) in CMs of isolated, sham and TAC EC-CD36 KO hearts provided 13 C palmitate + 13 C oleate (0.02 mM each) + 10 mM glucose + 1 mM lactate (Fig 2). 13 C LCFA esterification into triglyceride (TG) was also reduced in EC-CD36-KO vs f/f controls, but without evidence for an additional effect of TAC. The contribution of 13 C LCFA to acetyl CoA entry into oxidation via the TCA cycle, was reduced by lack of EC-CD36 in sham and TAC hearts (p<0.0001) (Fig 2). Consistent with diminished LCFA oxidation, phosphocreatine (PCr) to ATP ratio was also compromised in EC-CD36-KO-TAC hearts (p<0.05). Lack of EC-CD36 during TAC increased 13 C LCFA trafficking to lipotoxic C16 ceramide in EC-CD36 KO hearts (Fig 3). CD36 in ECs is critical to LCFA delivery to CMs during pathological stress on the heart. The imbalance in intramyocellular LCFA dynamics due to restricted transendothelial delivery of LCFA to CMs increased lipotoxic ceramide formation. Thus, EC CD36 is critical to the adaptive cardiac response to pathological stress, and the absence of EC CD36-dependent delivery of LCFA to CMs exacerbates reduced energy potential, adverse cardiac remodeling, and dysfunction during afterload stress.

A novel Nav1.5-dependent feedback mechanism driving glycolytic acidification in breast cancer metastasis

Solid tumours have abnormally high intracellular [Na+]. The activity of various Na+ channels may underlie this Na+ accumulation. Voltage-gated Na+ channels (VGSCs) have been shown to be functionally active in cancer cell lines, where they promote invasion. However, the mechanisms involved, and clinical relevance, are incompletely understood. Here, we show that protein expression of the Nav1.5 VGSC subtype strongly correlates with increased metastasis and shortened cancer-specific survival in breast cancer patients. In addition, VGSCs are functionally active in patient-derived breast tumour cells, cell lines, and cancer-associated fibroblasts. Knockdown of Nav1.5 in a mouse model of breast cancer suppresses expression of invasion-regulating genes. Nav1.5 activity increases ATP demand and glycolysis in breast cancer cells, likely by upregulating activity of the Na+/K+ ATPase, thus promoting H+ production and extracellular acidification. The pH of murine xenograft tumours is lower at the periphery than in the core, in regions of higher proliferation and lower apoptosis. In turn, acidic extracellular pH elevates persistent Na+ influx through Nav1.5 into breast cancer cells. Together, these findings show positive feedback between extracellular acidification and the movement of Na+ into cancer cells which can facilitate invasion. These results highlight the clinical significance of Nav1.5 activity as a potentiator of breast cancer metastasis and provide further evidence supporting the use of VGSC inhibitors in cancer treatment.

MITOCHONDRIAL DYSFUNCTION IN THE PATHOMORPHOGENESIS OF HYPERTROPHIC CARDIOMYOPATHY

The pathomorphogenesis of hypertrophic cardiomyopathy is a disruption of the arrangement of bundles of muscle cells in the myocardium and is associated with mutations in genes encoding the synthesis of myocardial contractile proteins. Metabolic changes in this pathology are caused by hypertrophy of the interventricular septum due to disruption of the myocardial contractile apparatus associated with these mutations, as well as mitochondrial dysfunction. Mutations in myofiber proteins can negatively affect mitochondria through increased oxidative stress due to increased ATP demand. Mitochondria are complex organelles with their own circular DNA, as well as the presence of enzyme complexes involved in redox reactions, which causes frequent damage to mitochondrial protein structures and membranes by reactive oxygen species. In this regard, mitochondrial dysfunction can also be caused by mutations in genes encoding mitochondrial proteins, which leads to disruption of mitophagy and mitochondrial dynamics. The functioning of defective mitochondria is associated with insufficient ATP synthesis and ineffective muscle contraction, which leads to the same consequences at the tissue level as mutations in contractile protein genes. In this review, we tried to summarize the role of mitochondrial dysfunction in the pathomorphogenesis of hypertrophic cardiomyopathy.

A Mitochondrial Basis for Heart Failure Progression.

In health, the human heart is able to match ATP supply and demand perfectly. It requires 6kg of ATP per day to satisfy demands of external work (mechanical force generation) and internal work (ion movements and basal metabolism). The heart is able to link supply with demand via direct responses to ADP and AMP concentrations but calcium concentrations within myocytes play a key role, signalling both inotropy, chronotropy and matched increases in ATP production. Calcium/calmodulin-dependent protein kinase (CaMKII) is a key adapter to increased workload, facilitating a greater and more rapid calcium concentration change. In the failing heart, this is dysfunctional and ATP supply is impaired. This review aims to examine the mechanisms and pathologies that link increased energy demand to this disrupted situation. We examine the roles of calcium loading, oxidative stress, mitochondrial structural abnormalities and damage-associated molecular patterns.

Synthesis, Biological, and Computational Evaluations of Conformationally Restricted NAD-Mimics as Discriminant Inhibitors of Human NMN-Adenylyltransferase Isozymes.

Nicotinamide adenine dinucleotide (NAD) cofactor metabolism plays a significant role in cancer development. Tumor cells have an increased demand for NAD and ATP to support rapid growth and proliferation. Limiting the amount of available NAD by targeting critical NAD biosynthesis enzymes has emerged as a promising anticancer therapeutic approach. In mammals, the enzyme nicotinamide/nicotinic acid adenylyltransferase (NMNAT) catalyzes a crucial downstream reaction for all known NAD synthesis routes. Novel nicotinamide/nicotinic acid adenine dinucleotide (NAD/NaAD) analogues 1-4, containing a methyl group at the ribose 2'-C and 3'-C-position of the adenosine moiety, were synthesized as inhibitors of the three isoforms of human NMN-adenylyltransferase, named hNMNAT-1, hNMNAT-2, and hNMNAT-3. An NMR-based conformational analysis suggests that individual NAD-analogues (1-4) have distinct conformational preferences. Biological evaluation of dinucleotides 1-4 as inhibitors of hNMNAT isoforms revealed structural relationships between different conformations (North-anti and South-syn) and enzyme-inhibitory activity. Among the new series of NAD analogues synthesized and tested, the 2'-C-methyl-NAD analogue 1 (Ki = 15 and 21 µM towards NMN and ATP, respectively) emerged as the most potent and selective inhibitor of hNMNAT-2 reported so far. Finally, we rationalized the in vitro bioactivity and selectivity of methylated NAD analogues with in silico studies, helping to lay the groundwork for rational scaffold optimization.

Hyperbaric oxygen treatment reveals spatiotemporal OXPHOS plasticity in the porcine heart.

Cardiomyocytes meet their high ATP demand almost exclusively by oxidative phosphorylation (OXPHOS). Adequate oxygen supply is an essential prerequisite to keep OXPHOS operational. At least two spatially distinct mitochondrial subpopulations facilitate OXPHOS in cardiomyocytes, i.e. subsarcolemmal (SSM) and interfibrillar mitochondria (IFM). Their intracellular localization below the sarcolemma or buried deep between the sarcomeres suggests different oxygen availability. Here, we studied SSM and IFM isolated from piglet hearts and found significantly lower activities of electron transport chain enzymes and F1FO-ATP synthase in IFM, indicative for compromised energy metabolism. To test the contribution of oxygen availability to this outcome, we ventilated piglets under hyperbaric hyperoxic (HBO) conditions for 240 min. HBO treatment raised OXPHOS enzyme activities in IFM to the level of SSM. Complexome profiling analysis revealed that a high proportion of the F1FO-ATP synthase in the IFM was in a disassembled state prior to the HBO treatment. Upon increased oxygen availability, the enzyme was found to be largely assembled, which may account for the observed increase in OXPHOS complex activities. Although HBO also induced transcription of genes involved in mitochondrial biogenesis, a full proteome analysis revealed only minimal alterations, meaning that HBO-mediated tissue remodeling is an unlikely cause for the observed differences in OXPHOS. We conclude that a previously unrecognized oxygen-regulated mechanism endows cardiac OXPHOS with spatiotemporal plasticity that may underlie the enormous metabolic and contractile adaptability of the heart.

Re-evaluating the energy balance of the many routes of carbon flow through and from photorespiration.

Photorespiration is an essential process related to photosynthesis that is initiated following the oxygenation reaction catalyzed by rubisco, the initial enzyme of the Calvin-Benson-Bassham cycle. This reaction produces an inhibitory intermediate that is recycled back into the Calvin-Benson-Bassham cycle by photorespiration which requires the use of energy and the release of a portion of the carbon as CO2. The energy use and CO2 release of canonical photorespiration form a foundation for biochemical models used to describe and predict leaf carbon exchange and energy use (ATP and NAPDH). The ATP and NADPH demand of canonical photorespiration is thought to be different than that of the Calvin-Benson-Bassham cycle, requiring increased flexibility in the ratio of ATP and NADPH from the light reactions. Photorespiration requires many reactions across the chloroplasts, mitochondria and peroxisomes and involves many intermediates. Growing evidence indicates that these intermediates do not all stay in photorespiration as typically assumed and instead feed into other aspects of metabolism and leave as glycine, serine, and methylene-THF. Here we discuss how alternative flux through and from canonical photorespiration alters the ATP and NADPH requirements of metabolism following rubisco oxygenation using additional derivations of biochemical models of leaf photosynthesis and energetics. Using these new derivations, we determine that the ATP and NADPH demands of photorespiration are highly sensitive to alternative flux in ways that fundamentally changes how photorespiration contributes to the ratio of total ATP and NADPH demand. Specifically, alternative flows of carbon through photorespiration could reduce ATP and NADPH demand ratio to values below what is produced from linear electron transport.

Remodeling of skeletal muscle myosin metabolic states in hibernating mammals.

Hibernation is a period of metabolic suppression utilized by many small and large mammal species to survive during winter periods. As the underlying cellular and molecular mechanisms remain incompletely understood, our study aimed to determine whether skeletal muscle myosin and its metabolic efficiency undergo alterations during hibernation to optimize energy utilization. We isolated muscle fibers from small hibernators, Ictidomys tridecemlineatus and Eliomys quercinus and larger hibernators, Ursus arctos and Ursus americanus. We then conducted loaded Mant-ATP chase experiments alongside X-ray diffraction to measure resting myosin dynamics and its ATP demand. In parallel, we performed multiple proteomics analyses. Our results showed a preservation of myosin structure in U. arctos and U. americanus during hibernation, whilst in I. tridecemlineatus and E. quercinus, changes in myosin metabolic states during torpor unexpectedly led to higher levels in energy expenditure of type II, fast-twitch muscle fibers at ambient lab temperatures (20 °C). Upon repeating loaded Mant-ATP chase experiments at 8 °C (near the body temperature of torpid animals), we found that myosin ATP consumption in type II muscle fibers was reduced by 77-107% during torpor compared to active periods. Additionally, we observed Myh2 hyper-phosphorylation during torpor in I. tridecemilineatus, which was predicted to stabilize the myosin molecule. This may act as a potential molecular mechanism mitigating myosin-associated increases in skeletal muscle energy expenditure during periods of torpor in response to cold exposure. Altogether, we demonstrate that resting myosin is altered in hibernating mammals, contributing to significant changes to the ATP consumption of skeletal muscle. Additionally, we observe that it is further altered in response to cold exposure and highlight myosin as a potentially contributor to skeletal muscle non-shivering thermogenesis.

Remodeling of skeletal muscle myosin metabolic states in hibernating mammals

Hibernation is a period of metabolic suppression utilized by many small and large mammal species to survive during winter periods. As the underlying cellular and molecular mechanisms remain incompletely understood, our study aimed to determine whether skeletal muscle myosin and its metabolic efficiency undergo alterations during hibernation to optimize energy utilization. We isolated muscle fibers from small hibernators, Ictidomys tridecemlineatus and Eliomys quercinus and larger hibernators, Ursus arctos and Ursus americanus. We then conducted loaded Mant-ATP chase experiments alongside X-ray diffraction to measure resting myosin dynamics and its ATP demand. In parallel, we performed multiple proteomics analyses. Our results showed a preservation of myosin structure in U. arctos and U. americanus during hibernation, whilst in I. tridecemlineatus and E. quercinus, changes in myosin metabolic states during torpor unexpectedly led to higher levels in energy expenditure of type II, fast-twitch muscle fibers at ambient lab temperatures (20 °C). Upon repeating loaded Mant-ATP chase experiments at 8 °C (near the body temperature of torpid animals), we found that myosin ATP consumption in type II muscle fibers was reduced by 77–107% during torpor compared to active periods. Additionally, we observed Myh2 hyper-phosphorylation during torpor in I. tridecemilineatus, which was predicted to stabilize the myosin molecule. This may act as a potential molecular mechanism mitigating myosin-associated increases in skeletal muscle energy expenditure during periods of torpor in response to cold exposure. Altogether, we demonstrate that resting myosin is altered in hibernating mammals, contributing to significant changes to the ATP consumption of skeletal muscle. Additionally, we observe that it is further altered in response to cold exposure and highlight myosin as a potentially contributor to skeletal muscle non-shivering thermogenesis.

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.