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 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 Cellular hypoxia is root of necrosis and apoptosis in myocardial infarction and stroke. Want to better understand cellular hypoxia because, although much work has been done, actual mechanisms of cell death and the cellular defense are not well known. All cells have intrinsic defenses to oxygen fluctuations, but some organisms better than others. These hypoxia-tolerant organisms generally have highly orchestrated regulation of metabolism to prevent cellular injury due to lowered ATP production in the absence of oxygen. We want to better understand this regulation in order to try to improve hypoxia sensitivity in human tissues.

Systems analysis of hypoxia response Complex balances must be maintained to tolerate hypoxia 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 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) One general hypothesis for the essence of hypoxia tolerance of certain organisms is that these cells have very flexible, highly orchestrated regulation of metabolism, and that this regulation occurs quickly (within minutes) and globally (affecting almost all metabolic processes in the cell)

Drosophila as a genetic model No heart yeast human fly worm Drosophila is the most genetically tractable model organism with a circulatory system. Phylogenetic tree Humans and flies share many cardiac genes: tinman (Nkx2.5) ether-a-go-go (hERG) troponin 714 out of 929 (77%) of human disease genes had a Drosophila homolog (26 cardiovascular) (Reiter, 2001) Metazoa  …  Arthropoda  …  Insecta  …  Diptera  …  Drosophilidae  …  Drosophila  melanogaster Disease gene study: PMID 11381037 (Reiter, 2001) RNA and protein sequence match Homologous disease alleles (cardiovascular?) Sequence similarity: Flies vs. humans

Drosophila as a model organism Cardiac aging [From Giovanni’s slides] Cardiac hypoxia Fruit flies are highly tolerant to oxygen fluctuations Genetic screen for hypoxia tolerance: Adar (Haddad, 1997) Cardiac systems biology Several high-throughput datasets (protein interactions, genetic interactions, microarrays) Well annotated genome Ease of manipulation and short lifespan for phenotype screens Original haddad paper (genetic screen): PMID: 9380715 Several reviews and follow-ups in recent years Adar = pre-mRNA adenosine deaminase. Edits a sodium channel mRNA (gene name: paralytic or para)

Response to low O2 in flies and mammals Standard physiological endpoints for resistance to ischemia Infarct size: gold standard for mammals Myocardial stunning Recovery of mechanical function Post-ischemic arrhythmias Sensitivity of fly heart to oxygen fluctuations not yet characterized Measurable in flies

Systems analysis of hypoxia response in Drosophila heart Regulation of metabolism is key in hypoxia-tolerant organisms Systems biology to understand and model the complex control systems Hochachka 1996 - PMID: 8790358 Hochachka, 2003 - PMID: 12756282

Hypoxia Genes and Human Disease Apolipoprotein D Expressed in stroke, aging, alzheimers Defense against hypoxia-reperfusion injury in flies CD36 Fatty acid transporter Mouse KO had reduced ischemia tolerance (41% drop in cardiac output during ischemia) ApoD: Walker, et. al., 2006 CD36: Null mouse had same baseline CO as wild-type, but after 6 min of ischemia CO dropped 41%

Our systems approach to modeling ATP-generating metabolism: Metabolomics to find all anaerobic pathways Flux-balance analysis to simulate pathways under varying oxygen Generate novel, specific, testable hypotheses for hypoxia tolerance We chose to apply our systems biology approach to first understand regulation of ATP-generating central metabolism in Drosophila flight muscle. To find all anaerobic pathways used in flies, we gathered global metabolomic profiles across several hypoxic timepoints using NMR spectroscopy. We reconstructed the pathways of central metabolism to simulate these pathways using flux-balance analysis, and in the end came up with novel, specific, and testable hypotheses for hypoxia tolerance mechanisms within the cell.

The fly heart heart organ Microscope view Normal beating

Automated measurement of heart function in Drosophila Gene Disruption Project Mail-order mutants Breed and cross (Bloomington, Indiana) Computer automated Mutants generated by individual investigators and BDGP (P-element insertion) >40% of genome (Bellen, et. al., 2004 - PMID: 15238527) Explain back-crossings. Balancer to prevent homologous recombination introduces genetic noise and is removed by crossing. Trait selection based on curly wing marker on balancer. Heterozygous mutations against a uniform background (WT is Oregan S). Ideally would do more back-crossings against WT strain Detect and measure heart Anesthetize and mount on slide

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 Decrease in rate and fractional shortening, fast recovery Recovery is affected by %O2, duration Lots of data collected, needs better analysis 120s hypoxia

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

1H NMR spectroscopy of hypoxic fly muscle 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 Database integration 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 162 genes, 143 proteins and 158 reactions Includes glycolysis, TCA cycle, oxidative phosphorylation, β-oxidation, amino acids Elementally- and charge- balanced Metabolic network reconstruction Stoichiometric matrix Drosophila central metabolism Literature and Databases Gene-protein-reaction associations Annotated Genome Reed JL et. al., Nat Rev Genet. 2006 Feb;7(2):130-41.

Acyl-carnitine shuttle Main energetic pathways in model Glucose NADH Acetate NH4 Glycolysis NADH ATP α-Oxoglutarate Glutamate ATP NADH Acetyl-CoA Alanine Pyruvate Lactate α-GPDH shuttle NADH Cytosol Mitochondria Acyl-carnitine shuttle Pyruvate FADH CO2 NADH Acetyl-CoA Oxaloacetate Citrate NADH/FADH2 ATP O2 H2O Known Drosophila pathways TCA cycle ATP Oxidative phosphorylation Hypothesized pathways NADH/FADH2 Products seen in NMR CO2

Flux-balance analysis Steady state assumption, flux constraints Optimize for objective function Mass and charge balance inherent ATP supply and demand Redox potential pH Null Space of S We then used flux balance analysis to generate network simulations. FBA relies on steady state assumption to create a solution space of all possibilities for flux distributions created by the network from a set of constraints A particular solution can be chosen within this solution space by optimizing for some objective function (can be one reaction or linear combination of rxns) Nice thing about FBA is that many of the complexities of hypoxic metabolism discussed before are inherently taken care of by the mass-balance requirement of FBA. Depending on boundary conditions, these conditions can be met: S matrix Solution space Particular solution (optimal) Metabolic network reconstruction

Price, et. al. (2004) Nat Rev Microbiol 2, 886-897

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

Hypoxia simulation: key fluxes Drosophila (Pseudo-) Mammalian 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 - Glucose (and equivalents) only carbon substrate - Lactate, alanine, acetate constrained to NMR fluxes - Varied O2 uptake constraint - Objective: maximize ATP production ac lac ala

Cardiac phenotypes and single deletion analysis WT vs mutant heart rate under hypoxia Colors represent effect of deletion on ATP production Here are a few example cardiac hypoxia traces overlaid on the single deletion analysis. Warm colors are enzymes whose deletions have a greater effect on ATP production in hypoxia

Conclusions 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

Questions Acknowledgements and many thanks Polly Huang Palsson lab, UCSD Bioengineering Adam Feist Thuy Vo Khoi Pham Questions