Metabolomic and computational systems analysis of hypoxic metabolism

Metabolomic and computational systems analysis of hypoxic metabolism in Drosophila Jacob Feala 1, 2 Laurence Coquin, Ph. D 2 Andrew Mc. Culloch, Ph. D 1 Giovanni Paternostro, Ph. D 1, 2 1) UCSD Bioengineering 2) Burnham Institute for Medical Research

Cellular hypoxia response n n n 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 n Complex balances must be maintained to tolerate hypoxia n n n ATP supply and demand Redox potential Metabolic intermediates p. H Hypothesis: flexible metabolic regulation key to hypoxia tolerance Systems biology to understand model the complex control systems Hochachka, P. W. J Exp Biol 2003; 206: 2001 -2009

Drosophila as a model for hypoxia research n n 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 1 Haddad GG et. al. , Proc Natl Acad Sci U S A. 1997 Sep 30; 94(20): 10809 -12. 2 Chen 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) n Global (ATP production, biosynthesis, protein translation) n

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 (Nkx 2. 5) – ether-a-go-go (h. ERG) – 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 n Cardiac aging n n Cardiac hypoxia n n n [From Giovanni’s slides] Fruit flies are highly tolerant to oxygen fluctuations Genetic screen for hypoxia tolerance: Adar (Haddad, 1997) Cardiac systems biology n n n Several high-throughput datasets (protein interactions, genetic interactions, microarrays) Well annotated genome Ease of manipulation and short lifespan for phenotype screens

Response to low O 2 in flies and mammals n Standard physiological endpoints for resistance to ischemia n n n 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 n n Regulation of metabolism is key in hypoxia-tolerant organisms Systems biology to understand model the complex control systems

Hypoxia Genes and Human Disease n Apolipoprotein D Expressed in stroke, aging, alzheimers n Defense against hypoxia-reperfusion injury in flies n n CD 36 Fatty acid transporter n Mouse KO had reduced ischemia tolerance (41% drop in cardiac output during ischemia) n

Our systems approach to modeling ATPgenerating metabolism: Metabolomics to find all anaerobic pathways n Flux-balance analysis to simulate pathways under varying oxygen n Generate novel, specific, testable hypotheses for hypoxia tolerance n

The fly 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 N 2 gas anesthesia slide computer-controlled valve

Cardiac Mechanics Research Group

Heart rate measurement O 2 detected beat time microscope view M-mode image Heart rate is monitored at baseline, hypoxia (via N 2 gas), and during recovery

Preliminary Research: Cardiac phenotype Hypoxic cardiac phenotype n n n Decrease in rate and fractional shortening, fast recovery Recovery is affected by %O 2, duration Lots of data collected, needs better analysis 120 s hypoxia Aim 1

Environmental factors n n n Reflexive body contraction (loss of muscle tone? ) below 2%, hard to measure heart Myocardial stunning at < 5% Increased response at high temperature

1 H NMR spectroscopy of hypoxic fly muscle • 0. 5% O 2 • 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 1 H NMR spectroscopy of flight muscle at t=0, 1, 10, 60, 240 minutes

Reconstructing the Drosophila metabolic network n Database integration n n 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 n n n 162 genes, 143 proteins and 158 reactions Includes glycolysis, TCA cycle, oxidative phosphorylation, β-oxidation, amino acids Elementally- and charge. Stoichiometric matrix balanced 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 NH 4 NADH α-Oxoglutarate Glycolysis ATP Glutamate ATP NADH Pyruvate Alanine Lactate Acetyl-Co. A α-GPDH shuttle NADH Cytosol Mitochondria Pyruvate Acyl-carnitine shuttle FADH NADH CO 2 Acetyl-Co. A Oxaloacetate Citrate NADH/FADH 2 Known Drosophila pathways ATP O 2 TCA cycle NADH/FADH 2 CO 2 H 2 O Oxidative phosphorylation Hypothesized pathways Products seen in NMR ATP

Flux-balance analysis n n n Steady state assumption, flux constraints Optimize for objective function Mass and charge balance inherent n n n Null Space of S ATP supply and demand Redox potential p. H 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 O 2 uptake constraint - Objective: maximize ATP production

Hypoxia simulation: key fluxes (Pseudo-) Mammalian Drosophila Stable p. H Reduced glucose uptake Equivalent ATP Abbreviations: • atp: ATP production • co 2: CO 2 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 O 2 uptake constraint - Objective: maximize ATP production

Cardiac phenotypes and single deletion analysis WT vs mutant heart rate under hypoxia Colors represent effect of deletion on ATP production

Conclusions n n n 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 n Palsson lab, UCSD Bioengineering n n Adam Feist n Thuy Vo n Khoi Pham Questions