1) The document describes a study aiming to characterize mitochondrial adaptations that allow some species to survive periods of hypoxia and reoxygenation by comparing hypoxia-tolerant and hypoxia-sensitive species. 2) The study will examine representatives of ancient hypoxia-tolerant elasmobranch species and modern hypoxia-tolerant fish and tilapia species, comparing them to hypoxia-sensitive ray and fish species. 3) In vivo and in vitro experiments will measure mitochondrial oxygen affinity, calcium buffering capacity, reactive oxygen species production, and redox state when exposed to normoxia and induced hypoxia, in order to understand the adaptations that enhance mitochondrial efficiency and function during oxygen deprivation.

1. !1
Tolerance to hypoxia and re-oxygenation: analysis of mitochondrial adaptations.
The central problem and its significance:
Adequate oxygen (O2) is crucial for vertebrate survival. Metabolically active organs such as
the brain, heart and liver, require a continuous ATP supply derived almost exclusively, in the
presence of available substrate producing reduced equivalents (NADH2 and FADH2) and O2, from
OXPHOS within mitochondria (Mito).
Hypoxic conditions followed by re-oxygenation leads to ATP depletion, an elevated redox
state (NADH) with elevated ROS production and Ca2+ overload, leading ultimately to cell death and
organismal death. During evolution, hypoxia-tolerant (HT) species developed Mito adaptations to
withstand hours of prolonged hypoxia at less than 3% of our atmospheric O2, or even at zero
oxygen (anoxia), while most hypoxia-sensitive (HS) vertebrates would die within minutes.
Previous studies reveal that HT species have more stable Mito with greater OXPHOS
efficiency, produce less ROS and are able to better buffer [ATP]. No study has characterised the
precise mitochondrial adaptations that HT species have to routinely survive under hypoxia/anoxia.
As the best way to reveal adaptational changes is through the comparison of different
species, this project is aimed at characterising Mito responses/adaptations in HT vs. HS with a
particular emphasis on O2 affinity (P50), Ca2+ buffering, ROS production and NADH/NAD ratio.
Knowledge of the adaptive mechanisms used to maintain mitochondrial function following
hypoxia and re-oxygenation could contribute to developing novel interventions that can be used in a
clinical setting, for example to protect the heart and brain during ischemia.
Experimental design:
Representatives of both ancient HT species (elasmobranchs) and modern HT species will be
examined to investigate the repertoire of mitochondrial adaptations used to survive hypoxia
followed by re-oxygenation.
Jules Devaux

2. !2
The hypoxia tolerant species to be examined are: the Epaulette shark (Hemiscyllium
ocellatum); the Grey Carpet shark (Chiloscyllium puncatum); the Mozambique tilapia (Oreochromis
mosambicus) and a Damselfish (Chromis atripectoralis).
The HS species to be examined are: the Whiptail ray (Hymantura Fai); the Shovel nose ray
(Rhinobatus typus); the Coral trout (Plectropomus leopardus) and a Damsel fish (Pomacentrus
moluccensis). They (n=20/species) will be collected under permit or purchased from licenced
collectors.
In vivo baseline measurements.
In vivo P50 will be measured for each vertebrate under progressive hypoxia, induced with N2
titration, until loss of equilibrium.
In vitro measurements on untreated animals.
Mito function will be assessed in saponin-permeabilised fibres and isolated Mito from
freshly collected brain, heart, skeletal muscle and liver. Experiments will use high resolution
respirometers O2k-Oroboros™ coupled with TIP-2k microPumps.
Experiment 1: Samples will be exposed to normoxia, induced hypoxia (40% of P50) to determine
Mito efficiency, complexes (CI and CII) capacity and leak state.
Experiment 2: Mito electron transport system (ETS) composition will be compared in repsonse to
hypoia and re-oxygenation, using a multiple substrate-inhibitor titration (protocol derived from
previous studies in A/Prof Renshaw’s lab).
Experiment 3: Ca2+ efflux and total uptake capacity will be monitored using CalciumGreen™,
tracked using purpose built LED array (506/531nm), in the presence of mKATP channel inhibitors 5-
desoxydecanoate or glibenclamide, and Ca2+ efflux agonist succinate and lactate.
Experiment 4: ROS production will be evaluated by measuring [H2O2] using Amplex®UltraRed
reagent, monitored by the use of fluorimeters (568/581nm).
Jules Devaux

3. !3
Experiment 5: Redox state through NADH/NAD ratio will be followed using purpose built UV
array, to reveal the NADH absorbance switch at 340nm.
Timeline:
J a n t o
May 2015
April to
July 2015
August to
Nov 2015
Dec to Jan
2016
Feb to June
2016
July to Dec
2016
Jan to May
2017
June - Dec
2017
Literature
review
Expt 1.
Literature
review
Expt 2.
Literature
Review
Conference
Preparation
(Poster)
Data analysis,
RHD
confirmation
Expt 3
Data
analysis,
Conference
Preparation
(Oral)
Expt 4
Data
analysis.
Thesis
preparation
Expt 5,
Data
analysis.
Thesis
preparation
FinalisingT
hesis and
submission
Jules Devaux