Mitochondrial bioenergetics: An integrated platform to study interactions of multiple stressors

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