Abstract 338: Limited Reduction of Heart Rate Promotes Cardiac Regeneration by Switching the Energy Metabolic Mode in Cardiomyocytes

2020 ◽  
Vol 127 (Suppl_1) ◽  
Author(s):  
Jing Tan ◽  
Ming Yang ◽  
Haiping Wang ◽  
Yandi Wu ◽  
Yuanlong Li ◽  
...  
Keyword(s):  
Heart Rate ◽  
Myocardial Injury ◽  
Metabolic Flux ◽  
Cardiac Regeneration ◽  
Clinical Treatment ◽  
Glycolytic Enzymes ◽  
Flux Analysis ◽  
Atp Production

Aims: Lack of cardiac regeneration with robust fibrosis response to the acute myocardial injury is the main obstacle to clinical treatment of cardiovascular diseases in humans. Stimulating the proliferation of endogenous cardiomyocytes (CMs) and replacing the scarred tissue with new functional CMs is a potential therapeutic strategy to the patients with heart failure. Heart rate reduction (HRR) is regarded as an effective clinical treatment for myocardial infarction. However, the mechanism of HRR promoting the recovery of cardiac function after injury still remains controversial, and whether there is any endogenous CM proliferation induced by HRR is undefined. Methods and results: The beating of CMs was reduced in vitro and heart rate (HR) of adult mice and different animal models of myocardial injury were modulated by six antiarrhythmic drugs to determine the role of HR in CM proliferation and cardiac repair. RNA-seq, extracellular flux analysis, metabolic flux analysis, and metabonomics were used to study the CM metabolism after HR modulation. We verified that reducing the beating can induce CM proliferation both in vitro and in vivo physiologically, and HRR also promoted cardiac regenerative repair after myocardial injury as well, reversely, increasing HR showed the opposite effect. Mechanistically, HRR reduced energy metabolism requirements and total ATP production of CMs but switched energy metabolic mode that the proportion of ATP production from aerobic glycolysis was increased, while from fatty acid oxidation was decreased. The switching of energy metabolic mode in CMs occurred in synchrony with the changes of glycolytic enzymes activities, these enzymes, including PFKFB3, PKM2, GAPDH, induced G1/S transition for cell cycle re-entry of CMs by upregulating the expression of cyclin D and CDK4/6 and facilitate substrates into the biomass needed to produce a new cell by biosynthesis. This coordinating function of glycolytic enzymes contributed to CM proliferation. Conclusion: Together, these results demonstrate that reduction of heart rate promotes CM proliferation by switching the energy metabolic mode, and highlight the potential therapeutic role of HRR in regenerative medicine.

scholarly journals Bifunctional Malic/Malolactic Enzyme Provides a Novel Mechanism for NADPH-Balancing in Bacillus subtilis

mBio ◽  
2021 ◽  
Vol 12 (2) ◽  
Author(s):  
Manuel Hörl ◽  
Tobias Fuhrer ◽  
Nicola Zamboni
Keyword(s):  
Bacillus Subtilis ◽  
Metabolic Flux ◽  
Central Carbon Metabolism ◽  
Flux Analysis ◽  
Content Type ◽  
Malolactic Enzyme ◽  
First Time

ABSTRACT The redox cofactor NADPH is required as a reducing equivalent in about 100 anabolic reactions throughout metabolism. To ensure fitness under all conditions, the demand is fulfilled by a few dehydrogenases in central carbon metabolism that reduce NADP+ with electrons derived from the catabolism of nutrients. In the case of Bacillus subtilis growing on glucose, quantitative flux analyses indicate that NADPH production largely exceeds biosynthetic needs, suggesting a hitherto unknown mechanism for NADPH balancing. We investigated the role of the four malic enzymes present in B. subtilis that could bring about a metabolic cycle for transhydrogenation of NADPH into NADH. Using quantitative 13C metabolic flux analysis, we found that isoform YtsJ alone contributes to NADPH balancing in vivo and demonstrated relevant NADPH-oxidizing activity by YtsJ in vitro. To our surprise, we discovered that depending on NADPH, YtsJ switches activity from a pyruvate-producing malic enzyme to a lactate-generating malolactic enzyme. This switch in activity allows YtsJ to adaptively compensate for cellular NADPH over- and underproduction upon demand. Finally, NADPH-dependent bifunctional activity was also detected in the YtsJ homolog in Escherichia coli MaeB. Overall, our study extends the known redox cofactor balancing mechanisms by providing first-time evidence that the type of catalyzed reaction by an enzyme depends on metabolite abundance. IMPORTANCE A new mechanism for NADPH balancing was discovered in Bacillus subtilis. It pivots on the bifunctional enzyme YtsJ, which is known to catalyze NADP-dependent malate decarboxylation. We found that in the presence of excessive NADPH, the same enzyme switches to malolactic activity and creates a transhydrogenation cycle that ultimately converts NADPH to NADH. This provides a regulated mechanism to immediately adjust NADPH/NADP+ in response to instantaneous needs.

scholarly journals Phosphoketolase Pathway Dominates in Lactobacillus reuteri ATCC 55730 Containing Dual Pathways for Glycolysis

2007 ◽  
Vol 190 (1) ◽  
pp. 206-212 ◽  
Author(s):  
Emma Årsköld ◽  
Elke Lohmeier-Vogel ◽  
Rong Cao ◽  
Stefan Roos ◽  
Peter Rådström ◽  
...  
Keyword(s):  
Metabolic Pathways ◽  
Metabolic Flux ◽  
Lactobacillus Reuteri ◽  
Intermediate Stage ◽  
Exponential Growth Phase ◽  
Flux Analysis ◽  
Atp Production ◽  
Phosphoketolase Pathway ◽  
Fructose 1,6 Bisphosphate

ABSTRACT Metabolic flux analysis indicated that the heterofermentative Lactobacillus reuteri strain ATCC 55730 uses both the Embden-Meyerhof pathway (EMP) and phosphoketolase pathway (PKP) when glucose or sucrose is converted into the three-carbon intermediate stage of glycolysis. In all cases studied, the main flux is through the PKP, while the EMP is used as a shunt. In the exponential growth phase, 70%, 73%, and 84% of the flux goes through the PKP in cells metabolizing (i) glucose plus fructose, (ii) glucose alone, and (iii) sucrose alone, respectively. Analysis of the genome of L. reuteri ATCC 55730 confirmed the presence of the genes for both pathways. Further evidence for the simultaneous operation of two central carbon metabolic pathways was found through the detection of fructose-1,6-bisphosphate aldolase, phosphofructokinase, and phosphoglucoisomerase activities and the presence of phosphorylated EMP and PKP intermediates using in vitro 31P NMR. The maximum specific growth rate and biomass yield obtained on glucose were twice as low as on sucrose. This was the result of low ATP levels being present in glucose-metabolizing cells, although the ATP production flux was as high as in sucrose-metabolizing cells due to a twofold increase of enzyme activities in both glycolytic pathways. Growth performance on glucose could be improved by adding fructose as an external electron acceptor, suggesting that the observed behavior is due to a redox imbalance causing energy starvation.

scholarly journals The Role of Herbal Bioactive Components in Mitochondria Function and Cancer Therapy

2019 ◽  
Vol 2019 ◽  
pp. 1-12 ◽  
Author(s):  
Fangfang Tao ◽  
Yanrong Zhang ◽  
Zhiqian Zhang
Keyword(s):  
Cancer Therapy ◽  
Cancer Cell ◽  
Apoptotic Cell ◽  
Redox Status ◽  
Cancer Therapies ◽  
Bioactive Components ◽  
Atp Production

Mitochondria are highly dynamic double-membrane organelles which play a well-recognized role in ATP production, calcium homeostasis, oxidation-reduction (redox) status, apoptotic cell death, and inflammation. Dysfunction of mitochondria has long been observed in a number of human diseases, including cancer. Targeting mitochondria metabolism in tumors as a cancer therapeutic strategy has attracted much attention for researchers in recent years due to the essential role of mitochondria in cancer cell growth, apoptosis, and progression. On the other hand, a series of studies have indicated that traditional medicinal herbs, including traditional Chinese medicines (TCM), exert their potential anticancer effects as an effective adjunct treatment for alleviating the systemic side effects of conventional cancer therapies, for reducing the risk of recurrence and cancer mortality and for improving the quality of patients’ life. An amazing feature of these structurally diverse bioactive components is that majority of them target mitochondria to provoke cancer cell-specific death program. The aim of this review is to summarize the in vitro and in vivo studies about the role of these herbs, especially their bioactive compounds in the modulation of the disturbed mitochondrial function for cancer therapy.

scholarly journals Molecular Basis for Anaerobic Growth of Saccharomyces cerevisiae on Xylose, Investigated by Global Gene Expression and Metabolic Flux Analysis

2004 ◽  
Vol 70 (4) ◽  
pp. 2307-2317 ◽  
Author(s):  
Marco Sonderegger ◽  
Marie Jeppsson ◽  
Bärbel Hahn-Hägerdal ◽  
Uwe Sauer
Keyword(s):  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Metabolic Flux Analysis ◽  
Metabolic Flux ◽  
Global Gene Expression ◽  
Anaerobic Growth ◽  
Flux Analysis ◽  
Atp Production ◽  
Redox Imbalance ◽  
Chemostat Cultures

ABSTRACT Yeast xylose metabolism is generally considered to be restricted to respirative conditions because the two-step oxidoreductase reactions from xylose to xylulose impose an anaerobic redox imbalance. We have recently developed, however, a Saccharomyces cerevisiae strain that is at present the only known yeast capable of anaerobic growth on xylose alone. Using transcriptome analysis of aerobic chemostat cultures grown on xylose-glucose mixtures and xylose alone, as well as a combination of global gene expression and metabolic flux analysis of anaerobic chemostat cultures grown on xylose-glucose mixtures, we identified the distinguishing characteristics of this unique phenotype. First, the transcript levels and metabolic fluxes throughout central carbon metabolism were significantly higher than those in the parent strain, and they were most pronounced in the xylose-specific, pentose phosphate, and glycerol pathways. Second, differential expression of many genes involved in redox metabolism indicates that increased cytosolic NADPH formation and NADH consumption enable a higher flux through the two-step oxidoreductase reaction of xylose to xylulose in the mutant. Redox balancing is apparently still a problem in this strain, since anaerobic growth on xylose could be improved further by providing acetoin as an external NADH sink. This improved growth was accompanied by an increased ATP production rate and was not accompanied by higher rates of xylose uptake or cytosolic NADPH production. We concluded that anaerobic growth of the yeast on xylose is ultimately limited by the rate of ATP production and not by the redox balance per se, although the redox imbalance, in turn, limits ATP production.

Abstract MP247: Mitochondrial Complex V Is Regulated By GRK2 In The Heart

2021 ◽  
Vol 129 (Suppl_1) ◽  
Author(s):  
Kimberly Ferrero ◽  
Jessica M Pfleger ◽  
Kurt Chuprun ◽  
Eric Barr ◽  
Erhe Gao ◽  
...  
Keyword(s):  
Atp Synthase ◽  
Cardiac Injury ◽  
Atp Synthesis ◽  
Cardiac Metabolism ◽  
Neonatal Rat ◽  
Interacting Proteins ◽  
Atp Production

The GPCR kinase GRK2 is highly expressed the heart; importantly, during cardiac injury or heart failure (HF) both levels and activity of GRK2 increase. The role of GRK2 during HF is canonically studied upstream of β-adrenergic desensitization. However, GRK2 has a large interactome and noncanonical functions for this kinase are being uncovered. We have discovered that in the heart, GRK2 translocates to mitochondria ( mtGRK2 ) following injury and is associated with negative effects on cardiac metabolism. Thus, we have sought to identify the mechanism(s) by which GRK2 can regulate mitochondrial function. We hypothesize that mtGRK2 interacts with proteins which regulate bioenergetics and substrate utilization, and this never-before-described role may partially explain the altered mitochondrial phenotype seen following cardiac injury or HF. Stress-induced mitochondrial translocation of GRK2 was validated in neonatal rat ventricular myocytes, murine heart tissue and a cardiac-derived cell line. Consequently, the GRK2 interactome was mapped basally and under stress conditions in vitro, in vivo , and with tagged recombinant peptides. GRK2-interacting proteins were isolated via immunoprecipitation and analyzed via liquid chromatography-mass spectroscopy (LCMS). Proteomics analysis (IPA; Qiagen) identified mtGRK2 interacting proteins which were also involved in mitochondrial dysfunction. Excitingly, Complexes I, II, IV and V (ATP synthase) of the electron transport chain (ETC) were identified in the subset of mtGRK2-dysfunction partners. Several mtGRK2-ETC interactions were increased following stress, particularly those in Complex V. We further established that mtGRK2 phosphorylates some of the subunits of Complex V, particularly the ATP synthase barrel which is critical for ATP production in the heart. Specific amino acid residues on these subunits have been identified using PTM-LCMS and are currently being validated in a murine model of myocardial infarction. To support these data, we have also determined that alterations in either the levels or kinase activity of GRK2 appear to alter the enzymatic activity of Complex V in vitro , thus altering ATP production. In summary, the phosphorylation of the ATP synthesis machinery by mtGRK2 may be regulating some of the phenotypic effects of injured or failing hearts such as increased ROS production and reduced fatty acid metabolism. Research is ongoing in our lab to elucidate the novel role of GRK2 in regulating mitochondrial bioenergetics and cell death, thus uncovering an exciting, druggable novel target for rescuing cardiac function in patients with injured and/or failing hearts.

Measuring non-steady-state metabolic fluxes in starch-converting faecal microbiota in vitro

2010 ◽  
Vol 1 (4) ◽  
pp. 391-405 ◽  
Author(s):  
T. Binsl ◽  
A. De Graaf ◽  
K. Venema ◽  
J. Heringa ◽  
A. Maathuis ◽  
...  
Keyword(s):  
Steady State ◽  
Metabolic Flux ◽  
Flux Distribution ◽  
Computational Modelling ◽  
Total Carbon ◽  
Flux Analysis ◽  
Small Contribution ◽  
Faecal Microbiota ◽  
Isotope Labelling

This paper explores human gut bacterial metabolism of starch using a combined analytical and computational modelling approach for metabolite and flux analysis. Non-steady-state isotopic labelling experiments were performed with human faecal microbiota in a well-established in vitro model of the human colon. After culture stabilisation, [U-13C] starch was added and samples were taken at regular intervals. Metabolite concentrations and 13C isotopomeric distributions were measured amongst other things for acetate, propionate and butyrate by mass spectrometry and NMR. The vast majority of metabolic flux analysis methods based on isotopomer analysis published to date are not applicable to metabolic non-steady-state experiments. We therefore developed a new ordinary differential equation-based representation of a metabolic model of human faecal microbiota to determine eleven metabolic parameters that characterised the metabolic flux distribution in the isotope labelling experiment. The feasibility of the model parameter quantification was demonstrated on noisy in silico data using a downhill simplex optimisation, matching simulated labelling patterns of isotopically labelled metabolites with measured metabolite and isotope labelling data. Using the experimental data, we determined an increasing net label influx from starch during the experiment from 94±1 µmol/l/min to 133±3 µmol/l/min. Only about 12% of the total carbon flux from starch reached propionate. Propionate production mainly proceeded via succinate with a small contribution via acrylate. The remaining flux from starch yielded acetate (35%) and butyrate (53%). Interpretation of 13C NMR multiplet signals further revealed that butyrate, valerate and caproate were mainly synthesised via cross-feeding, using acetate as a co-substrate. This study demonstrates for the first time that the experimental design and the analysis of the results by computational modelling allows the determination of time-resolved effects of nutrition on the flux distribution within human faecal microbiota in metabolic non-steady-state.

scholarly journals YtsJ Has the Major Physiological Role of the Four Paralogous Malic Enzyme Isoforms in Bacillus subtilis

2006 ◽  
Vol 188 (13) ◽  
pp. 4727-4736 ◽  
Author(s):  
Guillaume Lerondel ◽  
Thierry Doan ◽  
Nicola Zamboni ◽  
Uwe Sauer ◽  
Stéphane Aymerich
Keyword(s):  
Enzyme Activity ◽  
Bacillus Subtilis ◽  
Malic Enzyme ◽  
Metabolic Flux ◽  
Physiological Role ◽  
Extreme Case ◽  
Growth Defect ◽  
Flux Analysis ◽  
Malic Enzyme Activity

ABSTRACT The Bacillus subtilis genome contains several sets of paralogs. An extreme case is the four putative malic enzyme genes maeA, malS, ytsJ, and mleA. maeA was demonstrated to encode malic enzyme activity, to be inducible by malate, but also to be dispensable for growth on malate. We report systematic experiments to test whether these four genes ensure backup or cover different functions. Analysis of single- and multiple-mutant strains demonstrated that ytsJ has a major physiological role in malate utilization for which none of the other three genes could compensate. In contrast, maeA, malS, and mleA had distinct roles in malate utilization for which they could compensate one another. The four proteins exhibited malic enzyme activity; MalS, MleA, and MaeA exhibited 4- to 90-fold higher activities with NAD+ than with NADP+. YtsJ activity, in contrast, was 70-fold higher with NADP+ than with NAD+, with Km values of 0.055 and 2.8 mM, respectively. lacZ fusions revealed strong transcription of ytsJ, twofold higher in malate than in glucose medium, but weak transcription of malS and mleA. In contrast, mleA was strongly transcribed in complex medium. Metabolic flux analysis confirmed the major role of YtsJ in malate-to-pyruvate interconversion. While overexpression of the NADP-dependent Escherichia coli malic enzyme MaeB did not suppress the growth defect of a ytsJ mutant on malate, overexpression of the transhydrogenase UdhA from E. coli partially suppressed it. These results suggest an additional physiological role of YtsJ beyond that of malate-to-pyruvate conversion.

scholarly journals The Role of Pool Size Measurements in Improving Flux Estimations in Non-Stationary Metabolic Flux Analysis

2019 ◽  
Vol 116 (3) ◽  
pp. 132a
Author(s):  
Anna Sher ◽  
Daniel Fridman ◽  
Jamey Young ◽  
Cynthia J. Musante
Keyword(s):  
Metabolic Flux Analysis ◽  
Metabolic Flux ◽  
Pool Size ◽  
Flux Analysis

scholarly journals Compartmentalized Glucose Metabolism in Pseudomonas putida Is Controlled by the PtxS Repressor

2010 ◽  
Vol 192 (17) ◽  
pp. 4357-4366 ◽  
Author(s):  
Abdelali Daddaoua ◽  
Tino Krell ◽  
Carlos Alfonso ◽  
Bertrand Morel ◽  
Juan-Luis Ramos
Keyword(s):  
Pseudomonas Putida ◽  
Metabolic Flux ◽  
Glucose Dehydrogenase ◽  
Consensus Sequence ◽  
Binding Domain ◽  
Titration Calorimetry ◽  
Flux Analysis ◽  
Scanning Calorimetry ◽  
Host Chromosome

ABSTRACT Metabolic flux analysis revealed that in Pseudomonas putida KT2440 about 50% of glucose taken up by the cells is channeled through the 2-ketogluconate peripheral pathway. This pathway is characterized by being compartmentalized in the cells. In fact, initial metabolism of glucose to 2-ketogluconate takes place in the periplasm through a set of reactions catalyzed by glucose dehydrogenase and gluconate dehydrogenase to yield 2-ketogluconate. This metabolite is subsequently transported to the cytoplasm, where two reactions are carried out, giving rise to 6-phosphogluconate, which enters the Entner-Doudoroff pathway. The genes for the periplasmic and cytoplasmic set of reactions are clustered in the host chromosome and grouped within two independent operons that are under the control of the PtxS regulator, which also modulates its own synthesis. Here, we show that although the two catabolic operons are induced in vivo by glucose, ketogluconate, and 2-ketogluconate, in vitro we found that only 2-ketogluconate binds to the regulator with an apparent KD (equilibrium dissociation constant) of 15 μM, as determined using isothermal titration calorimetry assays. PtxS is made of two domains, a helix-turn-helix DNA-binding domain located at the N terminus and a C-terminal domain that binds the effector. Differential scanning calorimetry assays revealed that PtxS unfolds via two events characterized by melting points of 48.1°C and 57.6°C and that, in the presence of 2-ketogluconate, the unfolding of the effector binding domain occurs at a higher temperature, providing further evidence for 2-ketogluconate-PtxS interactions. Purified PtxS is a dimer that binds to the target promoters with affinities in the range of 1 to 3 μM. Footprint analysis revealed that PtxS binds to an almost perfect palindrome that is present within the three promoters and whose consensus sequence is 5′-TGAAACCGGTTTCA-3′. This palindrome overlaps with the RNA polymerase binding site.

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