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Mitochondrial bioenergetics: An integrated platform to study interactions of multiple stressors
Authors
Publication date
1 January 2015
Publisher
'Robertson Library, University of Prince Edward Island'
Abstract
Animals must expend energy to deal with a wide array of stressors associated with
environmental change. Because mitochondria produce >90% of the energy requirement of the
cell, they are likely the fundamental drivers of responses to environmental change. The primary
goal of my thesis was to explore the interactions of three common stressors in aquatic systems –
temperature, metals (copper: Cu) and hypoxia– on mitochondrial bioenergetics. To achieve this,
I first performed a series of in vitro experiments using rainbow trout, Oncorhynchus mykiss liver
mitochondria energized with complex I and II substrates to characterize the acute interactive
responses of temperature and Cu on mitochondrial function. These studies revealed that Cu
altered the basal and maximal mitochondrial oxidation rates differently depending on the metal
dose and temperature. Mechanistically, I showed that Cu impairs oxidative phosphorylation in
part by inhibiting the electron transport system (ETS) enzymes, stimulating proton leak, inducing
mitochondrial permeability transition pore and dissipating inner membrane potential.
Importantly, temperature exacerbated the effects of Cu suggesting that environmental warming,
e.g., due to climate change, may sensitize fish to Cu toxicity.
The next study combined in vitro and in vivo approaches to shed light on how persistent elevated
temperature (warm acclimation) modulates the effects of acute temperature increase, hypoxiareoxygenation
(HRO) and/or Cu on mitochondrial function. Sequential inhibition and activation
of mitochondrial ETS enzyme complexes permitted the measurement of respiratory activities
supported by ETS complexes I-IV in one run and allowed me to identify segments/components
of the ETS that are resilient or susceptible to single and combined effects of temperature, Cu and
HRO. This study also revealed that warm acclimation blunted the sensitivity of the ETS to acute
temperature rise and, together with HRO, sensitized the ETS to Cu. My fourth study examined how warm acclimation influences the ability of fish to handle
individual and joint effects of subsequent acute temperature shifts, hypoxia and Cu stress by
exposing fish in vivo to the three stressors. Here I measured mitochondrial oxidation and apical
endpoints indicative of stress and organismal energy status to assess the relevance of energy
metabolism endpoints in vivo. I showed that warm acclimation reduced fish condition, promoted
anaerobic metabolism, decelerated the ETS and altered the responses of fish to acute temperature
shifts, hypoxia and Cu. Moreover, Cu and hypoxia showed reciprocal antagonistic interaction on
the ETS and plasma metabolites, with modest additive actions limited to proton leak.
The final study highlighted the functional-biochemical and transcriptional responses of fish to
warm acclimation and short-term exposures to Cu and hypoxia. In this in vivo study, activities of
ETS enzyme complexes and targeted analyses of transcripts encoding for proteins involved in
mitochondrial oxidation, metals detoxification/stress response and energy sensing were done in
isolated liver mitochondria and in whole liver and gill tissues by RT-qPCR. Warm acclimation
inhibited activities of ETS enzymes while effects of Cu and hypoxia depended on the enzyme
and thermal acclimation status. The genes encoding for proteins involved in mitochondrial
oxidation, metals detoxification/stress response and energy sensing were all strongly regulated
by warm acclimation while Cu and hypoxia clearly increased transcript levels of genes encoding
for proteins involved in metals detoxification/stress response. Overall, the studies I carried out
not only provided the mechanistic underpinnings of responses of fish to thermal stress, hypoxia
and Cu, but unveiled novel interactive effects of multiple stressors. Importantly, I showed that
mitochondria are a viable platform for integrating effects of multiple stressors
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