Original articleEffects of copper and temperature on heart mitochondrial hydrogen peroxide production
Graphical abstract
Introduction
The heart is the central organ of the cardiovascular system and its continuous contraction-relaxation cycles require constant energy (ATP) supply which is sustained by mitochondrial metabolism within cardiomyocytes. Mitochondrial energy conversion involves finely coordinated transfer of electrons from reducing equivalents (NADH and FADH2) generated in the Krebs cycle through the electron transport system (ETS) to reduce the terminal acceptor, molecular oxygen (O2) [1,2]. This electron transfer is coupled with pumping of protons creating a protonmotive force across the inner mitochondrial membrane which is used by ATP synthase to make ATP. During this process, electron leakage along the ETS results in incomplete reduction of O2 with formation of reactive oxygen species (ROS). At physiological concentrations, ROS take part in cellular signaling networks and can protect the failing heart [3]. However, excessive levels of ROS, often as a consequence of cellular stress, may overwhelm the capacity of the antioxidant defense system precipitating oxidative stress [4,5]. Notably, the high energy demand together with the large number of mitochondria in cardiomyocytes (20–40% of cell volume in vertebrates) may predispose the heart to excessive ROS production which has been implicated in mitochondrial impairment [6] and cardiomyopathy [[6], [7], [8]].
Superoxide anion radical (O2•−) and hydrogen peroxide (H2O2) are the primary ROS produced by the mitochondria [[9], [10], [11]]. When produced as O2•−, subsequent dismutation by mitochondrial matrix (Mn) and cytosolic (Cu–Zn) superoxide dismutases (SODs) converts it to H2O2 [12]. At present, our understanding of mitochondrial ROS production is based primarily on mammalian mitochondria [9,11,13,14] where 12 sites have been identified as sources of ROS [11,15]. While ETS complexes I and III are often regarded as the major sites of ROS production in mammalian heart mitochondria [[16], [17], [18]], complex II was recently shown to generate large amounts of ROS [19,20]. However, there are only a few reports on ROS production by fish heart mitochondria [[21], [22], [23]] and the sites of ROS production are yet to be identified and characterized in this species. Importantly, it is necessary to accurately measure ROS before ascribing causality of pathological states to oxidative stress.
Effects of environmental stressors on cardiac function have been widely investigated [[24], [25], [26]] and it is known that temperature and copper (Cu) influence mitochondrial function [[27], [28], [29], [30]]. The mitochondria are a primary target for thermal stress-induced adverse effects [[29], [30], [31]] wherein acute temperature rise impairs heart mitochondrial function [32] in part because of denaturation of enzymes and disruption of membrane structure at high temperature [33,34]. Notably, impairment of ETS enzymes as a result of thermal stress has been implicated in heart failure [32,35,36]. Furthermore, elevated temperature increases mitochondrial respiration [29,30,37] and ROS production [22,38]. Similarly, cold stress has been shown to alter mitochondrial membrane composition [39] resulting in increased ROS production [40]. To-date, the majority of the studies on heart mitochondria focus on respiratory function and enzyme activity and only a few have examined the influence of thermal stress on ROS production [21,22]. In this regard, ROS production mechanisms in mitochondria of aquatic ectotherms may be highly sensitive to temperature fluctuations because internal temperature in ectotherms varies directly with environmental temperature [41,42].
Cu is an essential transition metal required in trace amounts for normal physiology but becomes toxic when accumulated in cells in excess amounts [43,44]. Among its many functions, Cu is a component of cuproproteins such as complex IV and Cu-ZnSOD that take part in aerobic metabolism and cellular antioxidant defense mechanisms, respectively [44,45]. The toxicity and biochemical activity of Cu are based on its ability to gain or lose electrons, and its effect on mitochondrial function is concentration-dependent [[28], [29], [30]]. Indeed, ROS induction has been suggested as the basic mechanism of Cu toxicity in biological systems [46]. Specifically for mitochondria, excessive Cu concentration disrupts ROS homeostasis (i), by directly impairing ETS enzyme complexes [[28], [29], [30]] which induces ROS formation [28], and catalyzing Fenton/Haber-Weiss reactions resulting in the production of hydroxyl radicals [47,48] and (ii), indirectly by impairing the antioxidant defense system. Importantly, the effects of combined exposures to Cu and temperature on heart mitochondrial ROS metabolism remain to be investigated.
The aim of our study was to investigate the effects of Cu and temperature on ROS emission by fish heart mitochondria. First, we optimized the AUR-HRP detection system for measuring ROS emission by fish heart mitochondria and then tested the hypothesis that Cu would alter the efficiency of the system. Second, we probed how thermal stress and Cu exposure influence ROS emission in heart mitochondria oxidizing different substrates. Third, we tested the idea that Cu exposure modulates individual mitochondrial sites of ROS production in fish heart mitochondria differently. We find that the effects of Cu and temperature on mitochondrial ROS production depend on respiratory substrate type, and that Cu imposes concentration-dependent stimulatory and/or inhibitory effects on heart mitochondrial ROS production sites.
Section snippets
Animals
Rainbow trout (Oncorhynchus mykiss) were obtained from Ocean Trout Farm, Brookvale, PE, Canada. The fish were kept in a 250-l tank supplied with aerated flow-through well-water maintained at 11 °C at the aquatic facility of the Atlantic Veterinary College. Fish were fed 1% of their body weight daily with commercial trout chow pellets (Corey Feed Mills, Fredericton, NB). The study and all of the experimental procedures were approved by the University of Prince Edward Island Animal Care Committee
Optimizing mitochondrial protein, substrate and HRP concentrations for measurement of H2O2 emission and respiration
To optimize the conditions for measurement of fluorescence generated by the HRP-catalyzed reaction of AUR and H2O2, we first titrated mitochondrial protein at different malate-glutamate and succinate concentrations and, surprisingly, obtained biphasic concentration-response curves (Fig. 1A&B). Increasing the concentration of malate-glutamate imposed a concentration-dependent increase in fluorescence intensity across all protein concentrations (F2,48 = 28.0, p < 0.0001), with the highest
Optimized assay conditions are essential for measurement of mitochondrial H2O2 emission and respiration
Quantitation of ROS emission by isolated mitochondria is characterized by variable results even in tissues of the same organism [61]. Among the factors that may affect ROS emission are the amounts of mitochondrial protein, substrates and HRP in the assay. We tested mitochondrial protein concentrations encompassing the range commonly used for measuring ROS emission by isolated heart mitochondria [17,18,62]. The results indicated that heart mitochondrial H2O2 emission increased with protein
Funding
This work was supported by a discovery grant award to CK (RGPIN-2017-05386) from the Natural Sciences and Engineering Research Council of Canada.
Declaration of competing interest
The authors have no conflict of interest to declare.
Acknowledgments
We are grateful to Ms. Nicole McDonald for technical support.
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