Phosphocreatine Concentrations Research Articles

Modelling Skeletal Muscle Motor Unit Recruitment Contributions to Contractile Function: Part 3 - Substrate Oxidation of Phosphagen, Lipid, and Carbohydrate Metabolism

This study aimed to apply and expand a prior model of motor unit recruitment of the vastus lateralis muscle (VL) to explore theoretical motor unit-specific substrate oxidation. The model utilized repeated contractions of varied frequency and fraction of motor unit recruitment, and four different genetic expressions of motor unit proportions. The study applied prior modelled data of the vastus lateralis (VL) motor unit contractile power and turnover of adenosine triphosphate (ATPto) based on non-linear functions of estimated percentage contributions of different energy systems across various muscle fibre types and contraction frequencies. Using LabVIEW™ programming, the model then used the prior data of ATPto and known ATPto coefficients for substrate oxidation for the energy systems of phosphagen, glycolytic, and mitochondrial respiration from fatty acid and carbohydrate to calculate total and fibre type (motor unit) specific creatine phosphate catabolism, glycogenolysis, glycolytic glucose oxidation, fatty acid oxidation in mitochondrial respiration, glucose oxidation in mitochondrial respiration, and lactate production. Results revealed that for the phosphagen system, substrate turnover was far larger than research-based expressions of decreasing concentrations of creatine phosphate and ATP. This is to be expected for modelled research involving temporal summation of metabolism. Creatine phosphate is continually broken down and partially replenished during low intensity exercise, with such partial replenishment sustained during more intense exercise thanks to the creatine kinase shuttle. For carbohydrate oxidation, mitochondrial respiration accounted for greatest substrate oxidation in type I, and I-IIa motor units, where glycolysis accounted for most substrate oxidation in type IIa, IIab and IIb motor units. Fatty acid oxidation was larger for lower motor unit recruitment conditions, and highest for type I-IIa and IIa motor units. This result is logical based on the larger net muscle fibre recruitment for contraction conditions necessitating progression to fast twitch motor units causing higher substrate oxidation. Such findings reinforce the need for more research on fibre type specific substrate oxidation during different exercise intensities and durations.

Blunted Cardiac Mitophagy in Response to Metabolic Stress Contributes to HFpEF.

Metabolic remodeling and mitochondrial dysfunction are hallmarks of heart failure with reduced ejection fraction. However, their role in the pathogenesis of HF with preserved ejection fraction (HFpEF) is poorly understood. In a mouse model of HFpEF, induced by high-fat diet and Nω-nitrol-arginine methyl ester, cardiac energetics was measured by 31P NMR spectroscopy and substrate oxidation profile was assessed by 13C-isotopmer analysis. Mitochondrial functions were assessed in the heart tissue and human induced pluripotent stem cell-derived cardiomyocytes. HFpEF hearts presented a lower phosphocreatine content and a reduced phosphocreatine/ATP ratio, similar to that in heart failure with reduced ejection fraction. Decreased respiratory function and increased reactive oxygen species production were observed in mitochondria isolated from HFpEF hearts suggesting mitochondrial dysfunction. Cardiac substrate oxidation profile showed a high dependency on fatty acid oxidation in HFpEF hearts, which is the opposite of heart failure with reduced ejection fraction but similar to that in high-fat diet hearts. However, phosphocreatine/ATP ratio and mitochondrial function were sustained in the high-fat diet hearts. We found that mitophagy was activated in the high-fat diet heart but not in HFpEF hearts despite similar extent of obesity suggesting that mitochondrial quality control response was impaired in HFpEF hearts. Using a human induced pluripotent stem cell-derived cardiomyocyte mitophagy reporter, we found that fatty acid loading stimulated mitophagy, which was obliterated by inhibiting fatty acid oxidation. Enhancing fatty acid oxidation by deleting ACC2 (acetyl-CoA carboxylase 2) in the heart stimulated mitophagy and improved HFpEF phenotypes. Maladaptation to metabolic stress in HFpEF hearts impairs mitochondrial quality control and contributed to the pathogenesis, which can be improved by stimulating fatty acid oxidation.

Repeated-sprint training in hypoxia: A review with 10 years of perspective.

Over the past decade, numerous studies have investigated an innovative "live low-train high" approach based on the repetition of short (<30 s) "all-out" sprints with incomplete recoveries in hypoxia; the so-called Repeated-Sprint training in Hypoxia (RSH). The aims of the present review are therefore threefold. First, this study summarizes the available evidence on putative additional performance enhancement after RSH comparing to the same training in normoxia (RSN). Second, a critical analysis of underpinning mechanisms discusses how advantages can be obtained through RSH for sea-level performance enhancement. An enhanced microcirculatory vasodilation leading to improved muscle perfusion and/or oxygenation and an increase in muscular phosphocreatine content may help explain the superiority of RSH vs. RSN. Third, the present review aims to provide guidelines for coaches, athletes and scientists to apply RSH interventions with regard to the interval duration, exercise-to-rest ratio and training volume. In conclusion, this review supports repeated-sprint training in hypoxia as an efficient (but not magic) training intervention with 77% of the controlled studies reporting an additional benefit with added hypoxia, mainly for team-, combat- and racket-sports athletes but also for all other sports (e.g. endurance) that require repeated accelerations with lesser fatigue.

Decreased mitochondrial creatine kinase 2 impairs skeletal muscle mitochondrial function independently of insulin in type 2 diabetes.

Increased plasma creatine concentrations are associated with the risk of type 2 diabetes, but whether this alteration is associated with or causal for impairments in metabolism remains unexplored. Because skeletal muscle is the main disposal site of both creatine and glucose, we investigated the role of intramuscular creatine metabolism in the pathophysiology of insulin resistance in type 2 diabetes. In men with type 2 diabetes, plasma creatine concentrations were increased, and intramuscular phosphocreatine content was reduced. These alterations were coupled to reduced expression of sarcomeric mitochondrial creatine kinase 2 (CKMT2). In C57BL/6 mice fed a high-fat diet, neither supplementation with creatine for 2 weeks nor treatment with the creatine analog β-GPA for 1 week induced changes in glucose tolerance, suggesting that increased circulating creatine was associated with insulin resistance rather than causing it. In C2C12 myotubes, silencing Ckmt2 using small interfering RNA reduced mitochondrial respiration, membrane potential, and glucose oxidation. Electroporation-mediated overexpression of Ckmt2 in skeletal muscle of high-fat diet-fed male mice increased mitochondrial respiration, independent of creatine availability. Given that overexpression of Ckmt2 improved mitochondrial function, we explored whether exercise regulates CKMT2 expression. Analysis of public data revealed that CKMT2 content was up-regulated by exercise training in both humans and mice. We reveal a previously underappreciated role of CKMT2 in mitochondrial homeostasis beyond its function for creatine phosphorylation, independent of insulin action. Collectively, our data provide functional evidence for how CKMT2 mediates mitochondrial dysfunction associated with type 2 diabetes.

The Effects of Water Flow Speed on Swimming Capacity and Energy Metabolism in Adult Amur Grayling (Thymallus grubii)

The present study aimed to explore whether water flow velocity could affect the swimming ability and overall energy metabolism of wild Amur grayling (Thymallus grubii). Swimming performance was assessed by measuring critical swimming speed (Ucrit), burst speed (Uburst), and oxygen consumption rate (MO2) based on the stepped velocity test method. Our results showed that the absolute values of Ucrit and Uburst tended to increase with body length. In contrast, the relative values of Ucrit and Uburst tended to decrease and increase, respectively. MO2 in Amur grayling was elevated with increasing velocity, suggesting relatively high swimming efficiency. We also measured the biochemical indices related to energy metabolism. Lactate dehydrogenase, hexokinase, and pyruvate kinase activities significantly increased (p &lt; 0.05). Hepatic glycogen, glucose, and muscle glycogen contents decreased with the increasing trend of velocity (p &lt; 0.05), the lactic acid contents of the blood and muscles increased significantly with the increase in velocities (p &lt; 0.05), and changes in creatine phosphate content showed no significant difference (p &gt; 0.05). The results not only denote the relationship between body size and swimming speed but also show the effects of water flow velocity on energy metabolism in Amur grayling. The results provide basic data for the construction of fish passage.

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 (&amp;gt;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.

Estimating the synaptic density deficit in Alzheimer's disease using multi-contrast CEST imaging.

In vivo noninvasive imaging of neurometabolites is crucial to improve our understanding of the underlying pathophysiological mechanism in neurodegenerative diseases. Abnormal changes in synaptic organization leading to synaptic degradation and neuronal loss is considered as one of the primary factors driving Alzheimer's disease pathology. Magnetic resonance based molecular imaging techniques such as chemical exchange saturation transfer (CEST) and magnetic resonance spectroscopy (MRS) can provide neurometabolite specific information which may relate to underlying pathological and compensatory mechanisms. In this study, CEST and short echo time single voxel MRS was performed to evaluate the sensitivity of cerebral metabolites to beta-amyloid (Aβ) induced synaptic deficit in the hippocampus of a mouse model of Alzheimer's disease. The CEST based spectra (Z-spectra) were acquired on a 9.4 Tesla small animal MR imaging system with two radiofrequency (RF) saturation amplitudes (1.47 μT and 5.9 μT) to obtain creatine-weighted and glutamate-weighted CEST contrasts, respectively. Multi-pool Lorentzian fitting and quantitative T1 longitudinal relaxation maps were used to obtain metabolic specific apparent exchange-dependent relaxation (AREX) maps. Short echo time (TE = 12 ms) single voxel MRS was acquired to quantify multiple neurometabolites from the right hippocampus region. AREX contrasts and MRS based metabolite concentration levels were examined in the ARTE10 animal model for Alzheimer's disease and their wild type (WT) littermate counterparts (age = 10 months). Using MRS voxel as a region of interest, group-wise analysis showed significant reduction in Glu-AREX and Cr-AREX in ARTE10, compared to WT animals. The MRS based results in the ARTE10 mice showed significant decrease in glutamate (Glu) and glutamate-total creatine (Glu/tCr) ratio, compared to WT animals. The MRS results also showed significant increase in total creatine (tCr), phosphocreatine (PCr) and glutathione (GSH) concentration levels in ARTE10, compared to WT animals. In the same ROI, Glu-AREX and Cr-AREX demonstrated positive associations with Glu/tCr ratio. These results indicate the involvement of neurotransmitter metabolites and energy metabolism in Aβ-mediated synaptic degradation in the hippocampus region. The study also highlights the feasibility of CEST and MRS to identify and track multiple competing and compensatory mechanisms involved in heterogeneous pathophysiology of Alzheimer's disease in vivo.

From periphery immunity to central domain through clinical interview as a new insight on schizophrenia

Identifying disease predictors through advanced statistical models enables the discovery of treatment targets for schizophrenia. In this study, a multifaceted clinical and laboratory analysis was conducted, incorporating magnetic resonance spectroscopy with immunology markers, psychiatric scores, and biochemical data, on a cohort of 45 patients diagnosed with schizophrenia and 51 healthy controls. The aim was to delineate predictive markers for diagnosing schizophrenia. A logistic regression model was used, as utilized to analyze the impact of multivariate variables on the prevalence of schizophrenia. Utilization of a stepwise algorithm yielded a final model, optimized using Akaike’s information criterion and a logit link function, which incorporated eight predictors (White Blood Cells, Reactive Lymphocytes, Red Blood Cells, Glucose, Insulin, Beck Depression score, Brain Taurine, Creatine and Phosphocreatine concentration). No single factor can reliably differentiate between healthy patients and those with schizophrenia. Therefore, it is valuable to simultaneously consider the values of multiple factors and classify patients using a multivariate model.

Energetics of sinusoidal exercise below and across critical power and the effects of fatigue

PurposePrevious studies investigating sinusoidal exercise were not devoted to an analysis of its energetics and of the effects of fatigue. We aimed to determine the contribution of aerobic and anaerobic lactic metabolism to the energy balance and investigate the fatigue effects on the cardiorespiratory and metabolic responses to sinusoidal protocols, across and below critical power (CP).MethodsEight males (26.6 ± 6.2 years; 75.6 ± 8.7 kg; maximum oxygen uptake 52.8 ± 7.9 ml·min−1·kg−1; CP 218 ± 13 W) underwent exhausting sinusoidal cycloergometric exercises, with sinusoid midpoint (MP) at CP (CPex) and 50 W below CP (CP-50ex). Sinusoid amplitude (AMP) and period were 50 W and 4 min, respectively. MP, AMP, and time-delay (tD) between mechanical and metabolic signals of expiratory ventilation (V˙E\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$${\\dot{V}}_{E}$$\\end{document}), oxygen uptake (V˙O2\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$${\\dot{V}}_{{{\ ext{O}}}_{2}}$$\\end{document}), and heart rate (fH\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$${f}_{H}$$\\end{document}) were assessed sinusoid-by-sinusoid. Blood lactate ([La−]) and rate of perceived exertion (RPE) were determined at each sinusoid.ResultsV˙O2\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$${\\dot{V}}_{{{\ ext{O}}}_{2}}$$\\end{document} AMP was 304 ± 11 and 488 ± 36 ml·min−1 in CPex and CP-50ex, respectively. Asymmetries between rising and declining sinusoid phases occurred in CPex (36.1 ± 7.7 vs. 41.4 ± 9.7 s for V˙O2\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$${\\dot{V}}_{{{\ ext{O}}}_{2}}$$\\end{document}tD up and tD down, respectively; P < 0.01), with unchanged tDs. V˙O2\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$${\\dot{V}}_{{{\ ext{O}}}_{2}}$$\\end{document} MP and RPE increased progressively during CPex. [La−] increased by 2.1 mM in CPex but remained stable during CP-50ex. Anaerobic contribution was larger in CPex than CP-50ex.ConclusionThe lower aerobic component during CPex than CP-50ex associated with lactate accumulation explained lower V˙O2\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$${\\dot{V}}_{{{\ ext{O}}}_{2}}$$\\end{document} AMP in CPex. The asymmetries in CPex suggest progressive decline of muscle phosphocreatine concentration, leading to fatigue, as witnessed by RPE.

Dietary creatine promotes creatine reserves, protein deposition, and myofiber hyperplasia in muscle of juvenile largemouth bass (Micropterus salmoides)

Creatine can increase muscle mass, enhance performance, prevent disease-induced muscle atrophy, and improve overall cellular energy. This study investigated the effects of creatine as a feed additive on muscle growth and intrinsic molecular regulation mechanism in largemouth bass (Micropterus salmoides). Seven diets supplemented with different concentrations of creatine (0%, 0.1%, 0.25%, 0.5%, 1%, 2%, and 4%) were fed to juvenile largemouth bass (4.5–5.1 g) for eight weeks. Dietary creatine significantly increased body weight and body length, and reduced feed conversion ratio. High-performance liquid chromatography analysis revealed that dietary creatine addition notably increased creatine and phosphocreatine levels by 19.94% and 16.54% in the muscle of largemouth bass, respectively. Histological observation showed that dietary creatine significantly increased the total muscle fiber number and decreased the mean muscle fiber diameter. The Western blot results indicated that creatine ingestion significantly increased the protein level of MyoD1 in the muscle and promoted muscle fiber hyperplasia. In addition, dietary creatine also contributed to mTOR-S6K/4EBP1-mediated protein synthesis by maintaining AMPKα Thr172 dephosphorylation and AKT Thr308 phosphorylation. Moreover, the protein levels of the muscle-specific E3 ligases Atrogin1 and Murf1 responsible for ubiquitination degradation exhibited no significant change after feeding with creatine in largemouth bass. Overall, our data showed that creatine as a feed additive increased creatine and phosphocreatine contents, thus promoting muscle protein deposition and myofiber hyperplasia, eventually elevating growth performance and feed utilization efficiency in juvenile largemouth bass. The regression analysis of weight gain rate, specific growth rate, and feed conversion ratio determined 0.46%–0.54% creatine concentration range as optimal supplementation concentration for the culture of juvenile largemouth bass.

A generalized QUCESOP method with evaluating CEST peak overlap.

The overlapping peaks of the target chemical exchange saturation transfer (CEST) solutes and other unknown CEST solutes affect the quantification results and accuracy of the chemical exchange parameters-the fractional concentration, , exchange rate, , and transverse relaxation rate, -for the target solutes. However, to date, no method has been established for assessing the overlapping peaks. This study aimed to develop a method for quantifying the , , and values of a specific CEST solute, as well as assessing the overlap between the CEST peaks of the specific solute(s) and other unknown solutes. A simplified model was proposed, assuming linear approximation of the other solutes' contributions to . A CEST data acquisition scheme was applied with various saturation offsets and saturation powers. In addition to fitting the , , and values of the specific solute, the overlapping condition was evaluated based on the root mean square error (RMSE) between the trajectories of the acquired and synthesized data. Single-solute and multi-solute phantoms with various phosphocreatine (PCr) concentrations and pH values were used to calculate the and of PCr and the corresponding RMSE. The feasibility of RMSE for evaluating the overlapping condition, and the accurate fitting of and in weak overlapping conditions, were verified. Furthermore, the method was employed to quantify the nuclear Overhauser effect signal in rat brains and the PCr signal in rat skeletal muscles, providing results that were consistent with those reported in previous studies. In summary, the proposed approach can be applied to evaluate the overlapping condition of CEST peaks and quantify the , , and values of specific solutes, if the weak overlapping condition is satisfied.

Utilization of Near Infrared Spectroscopy Imaging at Nanoscale for the Identification of Biomarkers in Sports-Induced Muscle Fatigue

This study integrates Near Infrared Spectroscopy (NIRS) and nanoscale imaging technologies to discern alterations in muscle tissue biomarkers, thereby enhancing the precision of non-invasive monitoring of muscle fatigue. Experimental investigations were carried out on the biceps brachii muscle of 12 subjects, categorized into mild, moderate, and severe fatigue groups. Concurrently, a specific wavelength of Near Infrared Laser Diode (NIR-LD) was employed to acquire spectral data. The application of Atomic Force Microscopy (AFM) in conjunction with NIRS imaging facilitated the attainment of high-resolution images of the biceps brachii tissue. The absorption characteristics of distinct biomarkers in muscle tissue, responsive to near-infrared light, were captured to calculate concentration variations and evaluate muscle fatigue levels. The findings revealed substantial variations in the concentrations of Oxy-hemoglobin (HbO), Deoxy-hemoglobin (HbR), Lactic Acid (LA), Phosphocreatine (PCr), Troponin (Tn), Creatine Kinase (CK), and Glutamine (Gln) across different fatigue groups. Muscle fatigue assessment exhibited an average sensitivity, accuracy, specificity, and F1 score of 0.96, 0.96, 0.95, and 0.96, respectively, for the 12 subjects. The average Area Under the Curve (AUC) values for detecting mild, moderate, and severe fatigue were 0.96, 0.96, and 0.98, respectively. This method demonstrates notable accuracy in the identification of muscle fatigue biomarkers, rendering it suitable for sports-related muscle fatigue assessment.

Changes in sperm metabolome during carp sperm short-term storage in different media: In search of sperm quality and storage capability markers

Although short-term hypothermic storage of sperm is already widely used in artificial fish reproduction and is considered very useful for resolving multiple problems related to logistics and broodstock maintenance, this approach continues to be explored and developed. In the present study, targeted and global metabolome LC/MS analyses of common carp sperm during refrigerated storage in undiluted and diluted (in a buffer: NaCl 55 mmol/L, KCl 40 mmol/L, CaCl2 1 mmol/L, MgSO4 0.5 mmol/L, sucrose 87.5 mmol/L, Tris 10 mmol/L, pH 8, with or without 5% methanol) conditions were used to find potential metabolic markers of fish sperm quality and storage capability evaluated by sperm motility parameters. Sperm motility percentage started to decline from the 6th day of the storage, followed by a significant drop in ATP content in all studied groups, ADP content in undiluted sperm, and creatine phosphate content in sperm diluted by the methanol-containing buffer. Correlation analysis of whole metabolome and sperm motility data allowed us to suggest several potential markers belonging to different chemical classes (mainly nucleotides and their components). Considering motility preservation and changes in the content of proposed markers of sperm quality/storage ability, sperm storage in the methanol-containing buffer is more promising for further application. At the same time, further searches for reliable markers and their validation remain necessary.

Modulation of Cellular Levels of Adenosine Phosphates and Creatine Phosphate in Cultured Primary Astrocytes

Adenosine triphosphate (ATP) is the main energy currency of all cells, while creatine phosphate (CrP) is considered as a buffer of high energy-bond phosphate that facilitates rapid regeneration of ATP from adenosine diphosphate (ADP). Astrocyte-rich primary cultures contain ATP, ADP and adenosine monophosphate (AMP) in average specific contents of 36.0 ± 6.4 nmol/mg, 2.9 ± 2.1 nmol/mg and 1.7 ± 2.1 nmol/mg, respectively, which establish an adenylate energy charge of 0.92 ± 0.04. The average specific cellular CrP level was found to be 25.9 ± 10.8 nmol/mg and the CrP/ATP ratio was 0.74 ± 0.28. The specific cellular CrP content, but not the ATP content, declined with the age of the culture. Absence of fetal calf serum for 24 h caused a partial loss in the cellular contents of both CrP and ATP, while application of creatine for 24 h doubled the cellular CrP content and the CrP/ATP ratio, but did not affect ATP levels. In glucose-deprived astrocytes, the high cellular ATP and CrP contents were rapidly depleted within minutes after application of the glycolysis inhibitor 2-deoxyglucose and the respiratory chain inhibitor antimycin A. For those conditions, the decline in CrP levels always preceded that of ATP contents. In contrast, incubation of glucose-fed astrocytes for up to 30 min with antimycin A had little effect on the high cellular ATP content, while the CrP level was significantly lowered. These data demonstrate the importance of cellular CrP for maintaining a high cellular ATP content in astrocytes during episodes of impaired ATP regeneration.

Metabolic profile of blood serum in experimental arterial hypertension.

The etiology of essential hypertension is intricate, since it employs simultaneously various body systems related to the regulation of blood pressure in one way or another: the sympathetic nervous system, renin-angiotensin-aldosterone and hypothalamic-pituitary-adrenal systems, renal and endothelial mechanisms. The pathogenesis of hypertension is influenced by a variety of both genetic and environmental factors, which determines the heterogeneity of the disease in human population. Hence, there is a need to perform research on experimental models - inbred animal strains, one of them being ISIAH rat strain, which is designed to simulate inherited stress-induced arterial hypertension as close as possible to primary (or essential) hypertension in humans. To determine specific markers of diseases, various omics technologies are applied, including metabolomics, which makes it possible to evaluate the content of low-molecular compounds - amino acids, lipids, carbohydrates, nucleic acids fragments - in biological samples available for clinical analysis (blood and urine). We analyzed the metabolic profile of the blood serum of male ISIAH rats with a genetic stress-dependent form of arterial hypertension in comparison with the normotensive WAG rats. Using the method of nuclear magnetic resonance spectroscopy (NMR spectroscopy), 56 metabolites in blood serum samples were identified, 18 of which were shown to have significant interstrain differences in serum concentrations. Statistical analysis of the data obtained showed that the hypertensive status of ISIAH rats is characterized by increased concentrations of leucine, isoleucine, valine, myo-inositol, isobutyrate, glutamate, glutamine, ornithine and creatine phosphate, and reduced concentrations of 2-hydroxyisobutyrate, betaine, tyrosine and tryptophan. Such a ratio of the metabolite concentrations is associated with changes in the regulation of glucose metabolism (metabolic markers - leucine, isoleucine, valine, myo-inositol), of nitric oxide synthesis (ornithine) and catecholamine pathway (tyrosine), and with inflammatory processes (metabolic markers - betaine, tryptophan), all of these changes being typical for hypertensive status. Thus, metabolic profiling of the stress-dependent form of arterial hypertension seems to be an important result for a personalized approach to the prevention and treatment of hypertensive disease.

Abstract P3182: Promoting Mitophagy By Enhanced Fatty Acid Oxidation Improves Phenotypes Of Heart Failure With Preserved Ejection Fraction

Although metabolic remodeling is known to contribute to the development of heart failure with reduced ejection fraction (HFrEF), the role of energy metabolism in HF with preserved ejection fraction (HFpEF) is poorly understood. Here we assessed cardiac energy metabolism in both HFpEF and HFrEF mice by multi-nuclear NMR spectroscopy. HFpEF was induced by a 2-hit of high fat diet (HFD) and Nω-nitrol-arginine-methyl-ester (L-NAME, in drinking water) for 10 weeks. Mice fed with chow diet, HFD or L-NAME administration alone were used as controls. HFpEF phenotypes were assessed as the exercise intolerance, pulmonary congestion, high E/e’ by echocardiography and left shift of pressure-volume relationship obtained from isolated hearts. High energy phosphate content, assessed by 31P NMR spectroscopy of isolated perfused hearts, showed a lower phosphocreatine (PCr) content in HFpEF hearts while ATP level was unaltered compared to all the controls, resulting a reduced PCr/ATP ratio similar to that in HFrEF induced by TAC. Decreased respiratory function and increased ROS production were observed in mitochondria isolated from HFpEF hearts suggesting mitochondrial dysfunction. Cardiac substrate oxidation profile, assessed by 13C-isotopmer analysis, showed a high dependency on fatty acid oxidation (FAO) in HFpEF hearts, which is opposite of HFrEF but similar to that in HFD hearts. However, PCr/ATP ratio and mitochondrial function were sustained in the HFD hearts with the same duration of treatment. We found that mitophagy was activated in HFD heart but did not change in HFpEF hearts despite similar extent of obesity suggesting that HFpEF heart is maladaptive to the metabolic stress. Promoting mitophagy by TAT-Beclin1 treatment improved mitochondrial function in HFpEF hearts and partially rescued cardiac diastolic dysfunction and lung congestion. Furthermore, enhancing FAO in HFpEF hearts by deleting acetyl-CoA carboxylase 2 stimulated mitophagy and improved HFpEF phenotypes. In summary, our results show that mitochondrial dysfunction is an important pathogenic mechanism for cardiac dysfunction in HFpEF. Maladaptation to metabolic stress in HFpEF hearts impairs mitochondrial quality control which can be improved by stimulating FAO.

Real-time influence of intracellular acidification and Na+ /H+ exchanger inhibition on in-cell pyruvate metabolism in the perfused mouse heart: A 31 P-NMR and hyperpolarized 13 C-NMR study.

Disruption of acid-base balance is linked to various diseases and conditions. In the heart, intracellular acidification is associated with heart failure, maladaptive cardiac hypertrophy, and myocardial ischemia. Previously, we have reported that the ratio of the in-cell lactate dehydrogenase (LDH) to pyruvate dehydrogenase (PDH) activities is correlated with cardiac pH. To further characterize the basis for this correlation, these in-cell activities were investigated under induced intracellular acidification without and with Na+ /H+ exchanger (NHE1) inhibition by zoniporide. Male mouse hearts (n= 30) were isolated and perfused retrogradely. Intracellular acidification was performed in two ways: (1) with the NH4 Cl prepulse methodology; and (2) by combining the NH4 Cl prepulse with zoniporide. 31 P NMR spectroscopy was used to determine the intracellular cardiac pH and to quantify the adenosine triphosphate and phosphocreatine content. Hyperpolarized [1-13 C]pyruvate was obtained using dissolution dynamic nuclear polarization. 13 C NMR spectroscopy was used to monitor hyperpolarized [1-13 C]pyruvate metabolism and determine enzyme activities in real time at a temporal resolution of a few seconds using the product-selective saturating excitation approach. The intracellular acidification induced by the NH4 Cl prepulse led to reduced LDH and PDH activities (-16% and -39%, respectively). This finding is in line with previous evidence of reduced myocardial contraction and therefore reduced metabolic activity upon intracellular acidification. Concomitantly, the LDH/PDH activity ratio increased with the reduction in pH, as previously reported. Combining the NH4 Cl prepulse with zoniporide led to a greater reduction in LDH activity (-29%) and to increased PDH activity (+40%). These changes resulted in a surprising decrease in the LDH/PDH ratio, as opposed to previous predictions. Zoniporide alone (without intracellular acidification) did not change these enzyme activities. A possible explanation for the enzymatic changes observed during the combination of the NH4 Cl prepulse and NHE1 inhibition may be related to mitochondrial NHE1 inhibition, which likely negates the mitochondrial matrix acidification. This effect, combined with the increased acidity in the cytosol, would result in an enhanced H+ gradient across the mitochondrial membrane and a temporarily higher pyruvate transport into the mitochondria, thereby increasing the PDH activity at the expense of the cytosolic LDH activity. These findings demonstrate the complexity of in-cell cardiac metabolism and its dependence on intracellular acidification. This study demonstrates the capabilities and limitations of hyperpolarized [1-13 C]pyruvate in the characterization of intracellular acidification as regards cardiac pathologies.

The exchange rate of creatine CEST in mouse brain.

To estimate the exchange rate of creatine (Cr) CEST and to evaluate the pH sensitivity of guanidinium (Guan) CEST in the mouse brain. Polynomial and Lorentzian line-shape fitting (PLOF) were implemented to extract the amine, amide, and Guan CEST signals from the brain Z-spectrum at 11.7T. Wild-type (WT) and knockout mice with the guanidinoacetate N-methyltransferase deficiency (GAMT-/- ) that have low Cr and phosphocreatine (PCr) concentrations in the brain were used to extract the CrCEST signal. To quantify the CrCEST exchange rate, a two-step Bloch-McConnell (BM) fitting was used to fit the CrCEST line-shape, B1 -dependent CrCEST, and the pH response with different B1 values. The pH in the brain cells was altered by hypercapnia to measure the pH sensitivity of GuanCEST. Comparison between the Z-spectra of WT and GAMT-/- mice suggest that the CrCEST is between 20% and 25% of the GuanCEST in the Z-spectrum at 1.95 ppm between B1 = 0.8 and 2 μT. The CrCEST exchange rate was found to be around 240-480 s-1 in the mouse brain, which is significantly lower than that in solutions (∼1000 s-1 ). The hypercapnia study on the mouse brain revealed that CrCEST at B1 = 2 μT and amineCEST at B1 = 0.8 μT are highly sensitive to pH change in the WT mouse brain. The in vivo CrCEST exchange rate is slow, and the acquisition parameters for the CrCEST should be adjusted accordingly. CrCEST is the major contribution to the opposite pH-dependence of GuanCEST signal under different conditions of B1 in the brain.

On the interdependence of ketone body oxidation, glycogen content, glycolysis and energy metabolism in the heart.

In heart, glucose and glycolysis are important for anaplerosis and potentially therefore for d-β-hydroxybutyrate (βHB) oxidation. As a glucose store, glycogen may also furnish anaplerosis. We determined the effects of glycogen content on βHB oxidation and glycolytic rates, and their downstream effects on energetics, in the isolated rat heart. High glycogen (HG) and low glycogen (LG) containing hearts were perfused with 11mM [5-3 H]glucose and/or 4mM [14 C]βHB to measure glycolytic rates or βHB oxidation, respectively, then freeze-clamped for glycogen and metabolomic analyses. Free cytosolic [NAD+ ]/[NADH] and mitochondrial [Q+ ]/[QH2 ] ratios were estimated using the lactate dehydrogenase and succinate dehydrogenase reaction, respectively. Phosphocreatine (PCr) and inorganic phosphate (Pi ) concentrations were measured using 31 P-nuclear magnetic resonance spectroscopy. Rates of βHB oxidation in LG hearts were half that in HG hearts, with βHB oxidation directly proportional to glycogen content. βHB oxidation decreased glycolysis in all hearts. Glycogenolysis in glycogen-replete hearts perfused with βHB alone was twice that of hearts perfused with βHB and glucose, which had significantly higher levels of the glycolytic intermediates fructose 1,6-bisphosphate and 3-phosphoglycerate, and higher free cytosolic [NAD+ ]/[NADH]. βHB oxidation increased the Krebs cycle intermediates citrate, 2-oxoglutarate and succinate, the total NADP/H pool, reduced mitochondrial [Q+ ]/[QH2 ], and increased the calculated free energy of ATP hydrolysis (∆GATP ). Although βHB oxidation inhibited glycolysis, glycolytic intermediates were not depleted, and cytosolic free NAD remained oxidised. βHB oxidation alone increased Krebs cycle intermediates, reduced mitochondrial Q and increased ∆GATP . We conclude that glycogen facilitates cardiac βHB oxidation by anaplerosis. KEY POINTS: Ketone bodies (d-β-hydroxybutyrate, acetoacetate) are increasingly recognised as important cardiac energetic substrates, in both healthy and diseased hearts. As 2-carbon equivalents they are cataplerotic, causing depletion of Krebs cycle intermediates; therefore their utilisation requires anaplerotic supplementation, and intra-myocardial glycogen has been suggested as a potential anaplerotic source during ketone oxidation. It is demonstrated here that cardiac glycogen does indeed provide anaplerotic substrate to facilitate β-hydroxybutyrate oxidation in isolated perfused rat heart, and this contribution was quantified using a novel pulse-chase metabolic approach. Further, using metabolomics and 31 P-MR, it was shown that glycolytic flux from myocardial glycogen increased the heart's ability to oxidise βHB, and βHB oxidation increased the mitochondrial redox potential, ultimately increasing the free energy of ATP hydrolysis.

Responses of the Macroalga Ulva prolifera Müller to Ocean Acidification Revealed by Complementary NMR- and MS-Based Omics Approaches.

Ocean acidification (OA) is a dramatic perturbation of seawater environments due to increasing anthropogenic emissions of CO2. Several studies indicated that OA frequently induces marine biota stress and a reduction of biodiversity. Here, we adopted the macroalga Ulva prolifera as a model and applied a complementary multi-omics approach to investigate the metabolic profiles under normal and acidified conditions. Our results show that U. prolifera grows at higher rates in acidified environments. Consistently, we observed lower sucrose and phosphocreatine concentrations in response to a higher demand of energy for growth and a higher availability of essential amino acids, likely related to increased protein biosynthesis. In addition, pathways leading to signaling and deterrent compounds appeared perturbed. Finally, a remarkable shift was observed here for the first time in the fatty acid composition of triglycerides, with a decrease in the relative abundance of PUFAs towards an appreciable increase of palmitic acid, thus suggesting a remodeling in lipid biosynthesis. Overall, our studies revealed modulation of several biosynthetic pathways under OA conditions in which, besides the possible effects on the marine ecosystem, the metabolic changes of the alga should be taken into account considering its potential nutraceutical applications.