Download metabolomic and computational systems analysis

Metabolomic and computational
systems analysis of hypoxic
metabolism in Drosophila
Jacob Feala1,2
Laurence Coquin, PhD2
Andrew McCulloch, PhD1
Giovanni Paternostro, PhD1,2
1) UCSD Bioengineering
2) Burnham Institute for Medical Research
Cellular hypoxia response
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Hypoxia is the cause of cell death in many
pathologies, mechanism not known
All cells have intrinsic defenses
Hypoxia tolerant organisms have highly
orchestrated metabolic regulation
Metabolic response is immediate and global
Drosophila is hypoxia tolerant model
Systems analysis of hypoxia response
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Complex balances must be
maintained to tolerate hypoxia
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ATP supply and demand
Redox potential
Metabolic intermediates
pH
Hypothesis: flexible metabolic
regulation key to hypoxia tolerance
Systems biology to understand and
model the complex control systems
Hochachka, P. W. J Exp Biol 2003; 206:2001-2009
Drosophila as a model for hypoxia research
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Flies are hypoxia tolerant
Simple system, genetic tools and libraries
Genetic screen found gene required for tolerance 1
Hypoxia tolerance gene was successfully transferred to
mammalian cells 2
human
fly
1Haddad
GG et. al., Proc Natl Acad Sci
U S A. 1997 Sep 30;94(20):10809-12.
2Chen Q et. al., J Biol Chem. 2003 Dec
5;278(49):49113-8. Epub 2003 Sep 16.
Phylogenetic tree
General hypothesis for hypoxia
tolerance
Flexible metabolic regulation is the major source
of hypoxia tolerance
Immediate (minutes)
 Global (ATP production, biosynthesis, protein
translation)
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Drosophila as a genetic model
yeast
Drosophila is the most
genetically tractable
model organism with a
circulatory system.
human
No heart
fly
worm
Phylogenetic tree
•
Humans and flies share many cardiac genes:
– tinman (Nkx2.5)
– ether-a-go-go (hERG)
– troponin
•
Sequence similarity: Flies vs. humans
714 out of 929 (77%) of human disease
genes had a Drosophila homolog (26
cardiovascular) (Reiter, 2001)
Drosophila as a model organism
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Cardiac aging
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Cardiac hypoxia
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[From Giovanni’s slides]
Fruit flies are highly tolerant to oxygen fluctuations
Genetic screen for hypoxia tolerance: Adar (Haddad, 1997)
Cardiac systems biology
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Several high-throughput datasets (protein interactions, genetic
interactions, microarrays)
Well annotated genome
Ease of manipulation and short lifespan for phenotype screens
Response to low O2 in flies and mammals
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Standard physiological endpoints for resistance to ischemia
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Infarct size: gold standard for mammals
Myocardial stunning
Recovery of mechanical function
Post-ischemic arrhythmias
Measurable in flies
Sensitivity of fly heart to oxygen fluctuations not yet characterized
Systems analysis of hypoxia response
in Drosophila heart
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Regulation of metabolism is key in
hypoxia-tolerant organisms
Systems biology to understand and
model the complex control systems
Hypoxia Genes and Human Disease
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Apolipoprotein D
Expressed in stroke, aging, alzheimers
 Defense against hypoxia-reperfusion injury in flies
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CD36
Fatty acid transporter
 Mouse KO had reduced ischemia tolerance (41%
drop in cardiac output during ischemia)
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Our systems approach to modeling ATPgenerating metabolism:
Metabolomics to find all anaerobic pathways
 Flux-balance analysis to simulate pathways under
varying oxygen
 Generate novel, specific, testable hypotheses for
hypoxia tolerance
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The fly heart
heart organ
Microscope view
Automated measurement of heart
function in Drosophila
Gene Disruption Project
Mail-order mutants
(Bloomington, Indiana)
Computer automated
Detect and measure
heart
Anesthetize and
mount on slide
Breed and cross
Automated cardiac phenotyping
Automated anesthesia and mounting
pressurized
air
anesthesia
chamber
fly vial
vacuum
N2 gas
anesthesia
slide
computer-controlled valve
Cardiac Mechanics
Research Group
Heart rate measurement
O2
detected
beat
time
microscope view
M-mode image
Heart rate is monitored at baseline, hypoxia (via
N2 gas), and during recovery
Preliminary Research:
Cardiac phenotype
Aim 1
Hypoxic cardiac phenotype
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Decrease in rate and fractional shortening, fast recovery
Recovery is affected by %O2, duration
Lots of data collected, needs better analysis
Heart rate under 2% oxygen (one fly)
10
1/RR
120s hypoxia
5
0
0
20
40
60
80
time (s)
100
120
140
160
Environmental factors
Measurement errors due to body movement
100
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90
Percent completing measurement
80
70
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60
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Reflexive body contraction (loss of muscle tone?)
below 2%, hard to measure heart
Myocardial stunning at < 5%
Increased response at high temperature
50
40
30
20
10
0
0
1
2
3
4
5
6
7
8
9
10
11
Percent oxygen
Hypoxia and recovery normalized to baseline
Cycle length in fly heart under 1 min of 3% O2
7
1.4
6
Baseline (20s)
1.2
Hypoxia (2 min)
5
Baseline (20s)
Recovery (20s)
1
Hypoxia (1 min)
Recovery (20s)
3
RR interval
Normalized RR interval
4
2
0.8
0.6
1
0.4
0
0.2
-1
-2
1
2
3
4
5
6
7
Percent oxygen
8
9
10
11
0
24
26
28
30
Temperature (deg C)
32
34
36
1H
NMR
spectroscopy
of hypoxic fly
muscle
• 0.5% O2
• 240 minutes
• supervised by Laurence Coquin
MAMMALIAN TISSUE:
Troy H et. al. Metabolomics 2005;
1: 293-303
Global metabolic profile
• Concentrations measured by targeted profiling (Chenomx): peak identification, alignment, subtraction
• Lower confidence group due to spectra overlap
Significant metabolites
1H
NMR spectroscopy of flight muscle at
t=0,1,10,60,240 minutes
Reconstructing the Drosophila
metabolic network
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Database integration
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KEGG: metabolic genes, enzymes, reactions, EC numbers,
pathways
Flybase: complete genome, proteins, function,
compartment, mutant stocks, references
Filtered gene index
Pathways
109
EC numbers
437
Genes
1322
Genes (mitochondrial)
125
Genes (stocks available)
507
Reconstructing the network
Network model of central metabolism
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162 genes, 143 proteins and 158 reactions
Includes glycolysis, TCA cycle, oxidative
phosphorylation, β-oxidation, amino acids
Elementally- and chargeStoichiometric
balanced
matrix
Metabolic network reconstruction
Literature and Databases
Gene-protein-reaction
associations
Annotated Genome
Reed JL et. al., Nat Rev Genet. 2006 Feb;7(2):130-41.
Drosophila central
metabolism
Main energetic pathways in model
Glucose
NADH
Acetate
NH4
NADH
α-Oxoglutarate
Glycolysis
ATP
Glutamate
ATP
NADH
Pyruvate
Alanine
Lactate
Acetyl-CoA
α-GPDH shuttle
NADH
Cytosol
Mitochondria
Pyruvate
Acyl-carnitine
shuttle
FADH
NADH
CO2
Acetyl-CoA
Oxaloacetate
Known Drosophila pathways
ATP
Citrate
TCA cycle
NADH/FADH2
CO2
ATP
O2
H2O
Oxidative phosphorylation
Hypothesized pathways
Products seen in NMR
NADH/FADH2
Flux-balance analysis
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Steady state assumption, flux constraints
Optimize for objective function
Mass and charge balance inherent
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Null Space of S
ATP supply and demand
Redox potential
pH
S matrix
Metabolic network
reconstruction
Solution space
Particular solution
(optimal)
Price, et. al. (2004)
Nat Rev Microbiol
2, 886-897
Flux-balance analysis of
hypoxia
glc
Simulation conditions
ac
lac
ala
- Glucose (and equivalents) only carbon substrate
- Lactate, alanine, acetate constrained to NMR
fluxes
- Varied O2 uptake constraint
- Objective: maximize ATP production
Hypoxia simulation: key fluxes
(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
Flux-balance analysis of
hypoxia
glc
Simulation conditions
ac
lac
ala
- Glucose (and equivalents) only carbon substrate
- Lactate, alanine, acetate constrained to NMR
fluxes
- Varied O2 uptake constraint
- Objective: maximize ATP production
Cardiac phenotypes and single
deletion analysis
16775 (red)
1.4
1.2
1
20862 (red)
1.4
0.8
1.2
0.6
1
0.4
0.8
0.2
0.6
0
0
20
40
60
80
100
0.4
0.2
0
0
20
40
60
80
100
WT vs mutant heart rate
under hypoxia
Colors represent effect
of deletion on ATP
production
11055 (red)
1.4
1.2
1
0.8
0.6
0.4
0.2
0
0
20
40
60
80
100
Conclusions
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Multiple 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
Acknowledgements and many thanks
Polly Huang
 Palsson lab, UCSD Bioengineering
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Adam Feist
 Thuy Vo
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Khoi Pham
Questions