Flexibility in energy metabolism supports hypoxia tolerance in Drosophila flight muscle: metabolomic

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Flexibility in energy metabolism supports hypoxia
tolerance in Drosophila flight muscle
metabolomic and computational systems analysis

• Jacob Feala
• Laurence Coquin, PhD
• Andrew McCulloch, PhD
• Giovanni Paternostro, PhD
• Cardiac Mechanics Research Group, UCSD
  Bioengineering Degenerative Diseases, Burnham
  Institute for Medical Research

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Systems analysis of hypoxia response

• Hypoxia is a cause of cell death in many diseases
• All cells have intrinsic defenses
• Hypoxia tolerant organisms have highly
  orchestrated regulation
• Complex balances
• ATP charge
• Redox potential
• Metabolic intermediates
• pH
• Systems biology to understand and model the
  complex control systems

Hochachka, 1996
Hochachka, 2003
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Drosophila as a model for hypoxia research

• Flies are hypoxia tolerant
• Simple system, genetic tools and libraries
• A previous screen found genes required for
  tolerance Ref
• One gene for hypoxia tolerance was successfully
  transferred to mammals Ref

Adams, et. al., 2000
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• General hypothesis flexible metabolic
  regulation major source of hypoxia tolerance
• Immediate (minutes)
• Global (ATP production, biosynthesis, protein
  translation)
• Systems approach (ATP supply)
• Metabolomics to find all anaerobic pathways
• Flux-balance analysis to simulate pathways under
  restricted oxygen
• Generate specific hypotheses for hypoxia
  tolerance

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1H NMR spectroscopy of hypoxic fly muscle

• 0.5 O2
• 240 minutes
• supervised by Laurence Coquin
• MAMMALIAN TISSUE

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Global metabolic profile

• Concentrations measured by targeted profiling
  (Chenomx) peak identification, alignment,
  subtraction
• Lower confidence group due to spectra overlap

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Significant metabolites

• 1H NMR spectroscopy of flight muscle at
  t0,1,10,60,240 minutes

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Reconstructing the Drosophila metabolic network

• Database integration
• KEGG metabolic genes, enzymes, reactions, EC
  numbers, pathways
• Flybase complete genome, proteins, function,
  compartment, mutant stocks, references

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Reconstructing the network

• Network model of central metabolism
• 162 genes, 143 proteins and 158 reactions
• Includes glycolysis, TCA cycle, oxidative
  phosphorylation, ß oxidation, amino acids
• Elementally- and charge-balanced

Metabolic network reconstruction
Stoichiometric matrix
Drosophila central metabolism
Literature and Databases
Gene-protein-reactionassociations
Annotated Genome
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(No Transcript)
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Flux-balance analysis

• Steady state assumption
• Optimize for objective function
• Mass and charge balance inherent
• ATP supply and demand
• Redox potential
• pH

Null Space of S
S matrix
Solution space
Particular solution (optimal)
Metabolic network reconstruction
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Flux-balance analysis of hypoxia
glc

• Simulation conditions
• - Glucose (and equivalents) only carbon substrate
• - Lactate, alanine, acetate constrained to NMR
  fluxes
• - Varied O2 uptake constraint
• - Objective maximize ATP production

ac
lac
ala
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Hypoxia simulation 3 pyruvate pathways vs 1
(Pseudo-) Mammalian
Drosophila
Stable pH
Reduced glucose uptake
Equivalent ATP

• Abbreviations
• atp ATP production
• co2 CO2 production
• glc glucose uptake
• h proton production

• ac acetate accumulation
• lac lactate accumulation
• ala alanine accumulation

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Conclusions

• Exotic anaerobic pyruvate pathways in fly may
  contribute to hypoxia tolerance
• New hypotheses to test alanine and
  acetate production essential under hypoxia
• Systems modeling revealed emergent
  behavior

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Perturbation Analysis of Energy Metabolism in
Hypoxic Myocardium
Model
Experiment
Genetic perturbation
Validate
Refine
NMR metabonomics
Candidate genes
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Questions

• Acknowledgements
• Polly Huang
• Palsson lab

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Future work Metabolic reconstruction
Aim 2

• Expand reconstruction to whole-cell myocyte
  (explore automated tools)
• Integrate fluxes from isotopomer study
• Further refine for cardiomyocyte
• Cardiac phenotypes of enzyme mutations
• Existing heart models (human, mouse)
• No biochemical data! In-vitro study?

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Research PlanIterative model building
Aim 3

• Hypoxic cardiac phenotype of unmeasured genes
  from modules
• Metabolomic analysis of control point mutations
• Detailed follow-up for novel genes of high
  interest
• Overexpression with UAS-GAL4 system,
  cardiospecific promoters
• Gene deletion with assay to confirm loss of
  function
• Transfection to mammals for cardioprotective
  effects
• Use the refined model to study cardiac aging
• Metabolomics of aging flies
• Test hypotheses with the model
• Loss of metabolic flexibility (flux variability
  analysis)
• Loss of regulation at control points
• Degradation of highly connected enzymes

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Constraint-based modeling
Metabolic network reconstruction
Null Space of S
Reaction 2 Flux (v2)
S matrix
Reaction 3 Flux (v3)
Flux balance analysis Sv dx/dt S
stoichiometric matrix v reaction flux vector x
metabolite concentration vector Steady state
assumption Sv 0
Particular solution (optimal)
Reaction 1 Flux (v1)
Solution space
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Flux variability