Journal of Thermal Biology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

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Temperature and sex dependent effects on cardiac mitochondrial metabolism in Atlantic cod (Gadus morhua L.) Kenneth J. Rodnick a,n, A. Kurt Gamperl b, Gordon W. Nash b, Douglas A. Syme c a

Department of Biological Sciences, Idaho State University, 921 South 8th Avenue, Mail Stop 8007, Pocatello, ID 83209-8007, USA Department of Ocean Sciences, Memorial University of Newfoundland, St. John's, NL, Canada A1C 5S7 c Department of Biological Sciences, University of Calgary, Calgary, AB, Canada T2N 1N4 b

art ic l e i nf o

Keywords: Temperature Mitochondria Oxidative phosphorylation Sex differences Thermal tolerance

a b s t r a c t To test the hypothesis that impaired mitochondrial respiration limits cardiac performance at warm temperatures, and examine if any effect(s) are sex-related, the consequences of high temperature on cardiac mitochondrial oxidative function were examined in 10 1C acclimated, sexually immature, male and female Atlantic cod. Active (State 3) and uncoupled (States 2 and 4) respiration were measured in isolated ventricular mitochondria at 10, 16, 20, and 24 1C using saturating concentrations of malate and pyruvate, but at a submaximal (physiological) level of ADP (200 mM). In addition, citrate synthase (CS) activity was measured at these temperatures, and mitochondrial respiration and the efficiency of oxidative phosphorylation (P:O ratio) were determined at [ADP] ranging from 25–200 mM at 10 and 20 1C. Cardiac morphometrics and mitochondrial respiration at 10 1C, and the thermal sensitivity of CS activity (Q10 ¼ 1.51), were all similar between the sexes. State 3 respiration at 200 mM ADP increased gradually in mitochondria from females between 10 and 24 1C (Q10 ¼ 1.48), but plateaued in males above 16 1C, and this resulted in lower values in males vs. females at 20 and 24 1C. At 10 1C, State 4 was  10% of State 3 values in both sexes [i.e. a respiratory control ratio (RCR) of  10] and P:O ratios were approximately 1.5. Between 20 and 24 1C, State 4 increased more than State 3 (by  70 vs. 14%, respectively), and this decreased RCR to  7.5. The P:O ratio was not affected by temperature at 200 μM ADP. However, (1) the sensitivity of State 3 respiration to increasing [ADP] (from 25 to 200 μM) was reduced at 20 vs. 10 1C in both sexes (Km values 10577 vs. 68710 μM, respectively); and (2) mitochondria from females had lower P:O values at 25 vs. 100 μM ADP at 20 1C, whereas males showed a similar effect at 10 1C but a much more pronounced effect at 20 1C (P:O 1.05 at 25 μM ADP vs. 1.78 at 100 μM ADP). In summary, our results demonstrate several sex-related differences in ventricular mitochondrial function in Atlantic cod, and suggest that myocardial oxidative function and possibly phosphorylation efficiency may be limited at temperatures of 20 1C or above, particularly in males. These observations could partially explain why cardiac function in Atlantic cod plateaus just below this species' critical thermal maximum ( 22 1C) and may contribute to yet unidentified sex differences in thermal tolerance and swimming performance. & 2014 Elsevier Ltd. All rights reserved.

1. Introduction Aquatic ectotherms are in thermal equilibrium with their environment. This enables water temperature to act as an ‘ecological master factor’, and in fish, affect behavior, distribution, energy expenditure, growth, stress, and immunology (Brett, 1971; Jobling, 1981; Pörtner and Knust, 2007; Pérez-Casanova et al., 2008 a, b; Hori et al., 2013). Oxygen delivery to the tissues via the cardiovascular system and efficient mitochondrial ATP production are vital regardless of body temperature, especially for organs with high metabolic

n

Corresponding author. Tel.: þ 1 208 282 3790. E-mail address: [email protected] (K.J. Rodnick).

rates and limited tolerance of extracellular hypoxia. During an acute temperature increase there may be a leveling off, or decline, of whole animal oxygen consumption as the upper thermal limit is approached. This limitation has been correlated with the uncoupling of mitochondria, decreased efficiency of oxidative phosphorylation (OXPHOS) due to the leak of protons, cytochrome c and NADH across the mitochondrial membrane, an inability to increase the activity of mitochondrial respiratory complexes, and the formation of reactive oxygen species (ROS) and anaerobic end products (Sommer et al., 1997; Hardewig et al., 1999; Frederich and Pörtner, 2000; Hilton et al., 2010; Iftikar and Hickey, 2013; Strobel et al., 2013). Thus, biochemical constraints associated with mitochondrial energy metabolism and ATP production may ultimately define physiological performance at high temperatures.

http://dx.doi.org/10.1016/j.jtherbio.2014.02.012 0306-4565 & 2014 Elsevier Ltd. All rights reserved.

Please cite this article as: Rodnick, K.J., et al., Temperature and sex dependent effects on cardiac mitochondrial metabolism in Atlantic cod (Gadus morhua L.). J. Thermal Biol. (2014), http://dx.doi.org/10.1016/j.jtherbio.2014.02.012i

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Cardiac output (the product of heart rate and stroke volume) is a key determinant of whole animal oxygen consumption and limitations on cardiac output contribute to establishing the upper thermal limits of fishes (Farrell, 2002, 2009; Sandblom et al., 2009; Farrell et al., 2009; Gamperl, 2011). Given that the heart of all but a few fishes relies predominantly on aerobic metabolism for ATP production, mitochondrial OXPHOS is a key process in cardiac energy metabolism (Driedzic, 1992), and a possible constraint to cardiac function at elevated temperatures. To maintain or increase contractile performance of the heart there must be a continuous production of intracellular ATP via aerobic metabolism and a balance established between energy demand and mitochondrial OXPHOS. Fish cardiomyocytes have a high mitochondrial volume density (Santer, 1985; Clark and Rodnick, 1998), but face kinetic challenges when exposed to changes in ambient temperature. For example, studies show that cardiac mitochondria of both New Zealand triplefin fishes (Hilton et al., 2010) and Atlantic wolfish (Anarhichas lupus, Lemieux et al., 2010) exhibit decreased efficiency (P:O ratios) and an inability to maintain OXPHOS with increasing temperature. However, they also suggest that the effects of temperature on mitochondrial respiration vary between species, and differences in OXPHOS at elevated temperatures may be related to the geographical range of the species, maximal habitat temperature, and possibly, exposure to environmental hypoxia. The thermal biology of Atlantic cod (Gadus morhua) has attracted considerable interest (Gollock et al., 2006; Pérez-Casanova et al., 2008 a, b, Hori et al., 2013), and this species appears to be appropriate for investigating the hypothesis that high temperatures impair cardiac mitochondrial function and energy transduction in fishes. Specifically, Atlantic cod prefer temperatures of 8–15 1C (Schurmann and Stefensen, 1992; Petersen and Steffensen, 2003), yet experience temperatures up to 20 1C and daily temperature fluctuations of up to 10 1C during the summer months (Gollock et al., 2006; Righton et al., 2010). These environmental temperatures are at, or very close, to the critical thermal maximum (CTM) for this species ( 22 1C; Gollock et al., 2006; Pérez-Casanova et al., 2008b). Lastly, Gollock et al. (2006) report that the metabolic rate and cardiac output of 10 1C acclimated Newfoundland cod plateaued at approximately 20 1C, and that negative influences of elevated temperatures on cardiac function (i.e. arrhythmias) begin at  18 1C. In view of the key role that mitochondria have in defining cardiac energy metabolism and performance, the main goal of the current study was to examine the thermal sensitivity of cardiac mitochondrial respiration and enzyme activity in Atlantic cod. “Maximal” mitochondrial function has traditionally been assessed using saturating ADP concentrations to define cellular respiratory capacity. However, this approach may not accurately reflect conditions in vivo, where ADP provision is thought to limit mitochondrial respiration (Brown, 1982; Saks et al., 1995). Therefore, we measured mitochondrial function at a number of submaximal ADP concentrations (25–200 μM). Based upon evidence that high environmental temperature negatively affects cardiac function, we hypothesized that ventricular mitochondrial function would be compromised at temperatures approaching the physiological limit for this species. Further, given recent findings of sex differences in cardiac energy metabolism (Battiprolu et al., 2007; Harmon et al., 2011) and thermal sensitivity in salmonid fishes (Jeffries et al., 2012; Martins et al., 2012), we also wanted to determine whether such differences exist in Atlantic cod.

2. Materials and methods 2.1. Experimental animals Sexually immature, male and female Atlantic cod weighing 300–450 g were reared from fertilized eggs for  2 years at the

Dr. Joe Brown Aquatic Research Building (Ocean Sciences Center, Memorial University of Newfoundland, St. John's, Canada). These fish were maintained in 3000 l circular tanks provided with aerated seawater at 10–11 1C and a 12 h light:12 h dark photoperiod, and fed a commercial cod diet (EWOS Canada Ltd., Surrey, BC, Canada) daily. All studies were conducted in accordance with the guidelines of the Canadian Council on Animal Care, and approved by the Institutional Animal Care Committee of Memorial University of Newfoundland. 2.2. Isolation of ventricular mitochondria Individual fish were rapidly killed by a blow to the head, measured for length and body mass, and had their sex determined by visual inspection of the gonads. The atrium and ventricle were then quickly excised and weighed. The entire ventricle was placed in ice-cold isolation medium [230 mM mannitol, 75 mM sucrose, 20 mM HEPES, 1 mM EGTA, and 2% (wt/vol) fatty acid free bovine serum albumin (BSA, product A6003, Sigma), pH 7.4 at 20 1C] before being cut into small pieces ( o1 mm3) with fine scissors. These pieces were subsequently rinsed twice in isolation medium to remove trapped blood cells, and homogenized in 10 volumes of isolation medium for 2 min in an ice-cooled glass homogenizer with a motor driven Teflon pestle. The homogenate was centrifuged at 500g for 10 min at 4 1C, and the resulting supernatant was centrifuged at 10,000g for 10 min at 4 1C. The mitochondrial pellet was then re-suspended in isolation medium and centrifuged at 10,000g for 10 min at 4 1C, and the final pellet was re-suspended in 5 volumes of ice-cold incubation medium [160 mM KCl, 30 mM HEPES, 10 mM KH2PO4, 2 mM EDTA, 1 mM MgCl2, and 1% fatty acid free BSA, pH 7.4 at 20 1C] at a concentration of  1–2 mg mitochondrial protein ml  1. All preparative procedures were conducted at 4 1C and the total time for mitochondrial isolation was  50–60 min. Given the temperature coefficient of HEPES buffer (dpKa/dT¼  0.014/1C), the actual pH of the isolation medium was 7.62 at 4 1C and the incubation medium ranged from 7.53 at 10 1C to 7.34 at 24 1C. All chemicals were obtained from Sigma Chemical Co. (St. Louis, MO, USA). 2.3. Measurement of mitochondrial respiration Mitochondrial respiration was measured at 10, 16, 20, or 24 1C in randomized order under air-saturated conditions (always 480%) in separate, closed, thermostatted glass incubation chambers (2 or 3 ml volume). These temperatures were selected because they extend from the acclimation temperature to just above the Atlantic cod's CTM (22.2 1C; Gollock et al., 2006). The solubility of oxygen in the air-saturated incubation medium was 368 nmol O2 ml  1 at 10 1C and 260 nmol O2 ml  1 at 20 1C. Incubation chambers were equipped with oxygen sensitive spots and a fiber-optic light guide that was connected to an oxygen meter (Model FIBOX 3 LCD, PreSens, Germany); the oxygen meter interfaced with a laptop computer running Oxyviews software (Version LCDPST3, PreSens). Respiration measurements on isolated mitochondria were conducted randomly and expressed in nmoles O2 consumed min  1 mg protein  1. We defined State 2 respiration as oxygen consumption in the presence of substrates without ADP added. State 3 and State 4 respiration, and the respiratory control ratio (RCR; State 3/State 4), were defined according to established terminology (Chance and Williams, 1955). Specifically, State 3 occurs when ADP is added to the substrates and was defined as ADP-stimulated respiration rate in the presence of saturating levels of pyruvate and malate. State 4 is achieved when ADP is exhausted, with substrates still present, and there is a decrease in respiratory rate that reflects a lack of ATP

Please cite this article as: Rodnick, K.J., et al., Temperature and sex dependent effects on cardiac mitochondrial metabolism in Atlantic cod (Gadus morhua L.). J. Thermal Biol. (2014), http://dx.doi.org/10.1016/j.jtherbio.2014.02.012i

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synthesis. State 4 largely represents O2 consumption associated with proton leak and is suggestive of mitochondrial uncoupling. Mitochondria (0.1–0.2 mg protein ml  1 final concentration) were added to incubation medium containing saturating L-malate (1 mM) and pyruvate (10 mM). The substrate combination of Lmalateþpyruvate activates mitochondrial dehydrogenases (with reduction of NAD) and feeds electrons into Complex I (NADH: ubiquinone oxidoreductase) of the oxidative phosphorylation system. After reaching a stable rate of oxygen consumption (State 2, 5–10 min), ADP (200 mM) was added to initiate State 3 respiration and to determine the P:O ratio (ATP produced/O consumed; see Fig. 1). Finally, after all the ADP was phosphorylated to ATP during mitochondrial incubations, oxygen consumption was recorded for another 5–10 min, and this is reported as uncoupled (State 4) respiration. To examine whether sex influences the thermal sensitivity of mitochondrial respiration, this experiment was conducted on hearts from 6 female and 6 male fishes. In a separate group of fish (5 males and 4 females), we also assessed the ADP sensitivity of mitochondrial respiration using sequential, but randomized, additions of submaximal (25, 50, 100, and 200 mM) ADP at two incubation temperatures (10 and 20 1C) (see Fig. 1). In this experiment, P:O ratios, State 3 respiration and RCR were calculated as above. Cytosolic free ADP concentrations in teleost skeletal muscle range from 20 to 100 μM (Van Waarde et al., 1990) and studies in human soleus muscle demonstrate that ADP concentrations can double (from 15 to 30 μM) during periods of high ATP turnover (Hochachka and McClelland, 1997). Thus, in 100

%O2Saturation

50 μM ADP

200 μM ADP State 2

100 μM ADP

90

#

*

State 3

85 State 4

80 10

the current study, we used what we estimated to be a physiological range of ADP concentrations to investigate the interactive effects of an acute temperature increase and sex on ventricular mitochondrial function. 2.4. Determination of citrate synthase activity and protein concentration The remaining mitochondrial suspension was placed in a plastic cryovial, frozen at the temperature of liquid N2, and stored at  80 1C for later measurements of citrate synthase (CS) activity and mitochondrial protein concentration. Citrate synthase activity has been shown to correlate well with mitochondrial volume densities in fish oxidative skeletal muscle (Egginton and Sidell, 1989) and has served as a mitochondrial marker enzyme in fish hearts (Treberg et al., 2007). Aliquots of mitochondrial suspensions were freeze-thawed twice before being used for enzymatic analysis. Maximal activities of CS [EC 4.1.3.7, the initial enzyme of the tricarboxylic acid (TCA) cycle] were measured at 10, 16, 20, and 24 1C over 5 min according to previously published methods (Rodnick and Sidell, 1994) using a UV/vis spectrophotometer (Model 8413, Agilent Technologies, Santa Clara, CA, USA) containing a water-jacketed 8-cell cuvette holder connected to a recirculating refrigerated water bath. Protein concentration of the mitochondrial suspension was determined by the Bradford method (Bradford, 1976) with BSA as the standard, and used to normalize values of mitochondrial O2 consumption. 2.5. Data and statistical analyses

25 μM ADP

95

3

20

30

40

50

Time (Minutes) Fig. 1. Original recording of oxygen consumption by ventricular mitochondria from Atlantic cod when provided with different concentrations of ADP. The arrow with the # above it indicates when the chamber was opened for a few minutes to allow O2 levels to increase prior to the last addition of ADP. The ‘n’ indicates the O2 consumption that was used to calculate the P:O ratio, based on the points of intersection between State 3 respiration with that of States 2 and 4. Note: the order of the 4 ADP additions was randomized for each preparation.

The mitochondrial respiration data was analyzed by transferring the Oxyviews data files into Logger Pro (Version 3.4, Vernier Software and Technology, Beaverton, OR, USA) and measuring the rate of oxygen decline in each incubation chamber. We used Q10 as an index of the effect of increasing temperature on respiration rates and CS activity. Q10 values were calculated using the following equation: Q 10 ¼ ðRatetemp2 =Ratetemp1 Þ½10=ðtemp2  temp1Þ The possible influence of sex on morphometric parameters, mitochondrial protein content, and CS activity at 10 1C was analyzed on a dataset containing all fish in this study (11 males and 10 females) using one-way analyses of variance (ANOVA, see Table 1). Differences in States 2, 3 and 4 respiration rates, RCR, and CS activity with temperature were initially examined for statistical significance using two-way ANOVAs with sex and assay temperature as main effects. This analysis was then followed by Bonferroni

Table 1 Physical characteristics of 10 1C-acclimated Atlantic cod used for ventricular mitochondrial isolation and measurements of oxygen consumption, and data for mitochondrial protein content and citrate synthase activity. Values are means 7S.E. and are based on all the fish used in this study (11 males and 10 females). RVM ¼relative ventricular mass [(ventricle mass/body mass)  100]. RAM¼ relative atrial mass. Condition factor was calculated as [(body mass/length3)  100]. None of the parameters were different between male and female Atlantic cod (P 40.05).

Mass (g) Length (cm) Condition factor Ventricle mass (mg) RVM (%) Atrium mass (mg) RAM (%) μg Mitochondrial protein/mg heart Citrate synthase (mU/mg heart) Citrate synthase (mU/μg mitochondrial protein)

Female

Male

351.5 711.6 29.1 70.3 1.43 70.04 247.5 78.3 0.071 70.001 70.972.8 0.020 70.001

387.5 714.5 29.5 70.4 1.50 70.03 280.5 714.3 0.072 70.001 75.3 73.6 0.019 70.001

5.9 70.6 7.3 70.2 1.4 70.2

7.1 70.7 6.9 70.2 1.1 70.1

Please cite this article as: Rodnick, K.J., et al., Temperature and sex dependent effects on cardiac mitochondrial metabolism in Atlantic cod (Gadus morhua L.). J. Thermal Biol. (2014), http://dx.doi.org/10.1016/j.jtherbio.2014.02.012i

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post-hoc tests to examine (1) if there were sex differences at each test temperature (10, 16, 20 and 24 1C) and (2) within each sex, if temperature had a significant effect on any of these parameters. The effects of increasing ADP concentration on P:O ratio and State 3 respiration at 10 and 20 1C were initially analyzed using a 3-way repeated measures ANOVA with temperature, sex and [ADP] as main effects. For the P:O ratio, there was a significant interaction between temperature and sex, and thus, two separate two-way repeated measures ANOVAs, with [ADP] and temperature as main effects, were run for female and male fishes. In contrast, sex was not shown to have an effect on the [ADP] vs. temperature (10 and 20 1C) relationship, and thus, a two-way repeated measures ANOVA was subsequently used to identify statistical differences. These analyses were followed by Holm-Sidak multiple comparison procedures to identify specific [ADP] and temperature (10 vs. 20 1C) effects on both parameters. The apparent Km for ADP was determined for individual fish at 10 and 20 1C using Lineweaver–Burk plots, and a 2  2 ANOVA (temperature and sex as main effects) was used to test for statistical differences. Statistical analyses were performed using GraphPad (Version 5, GraphPad Software Inc. La Jolla, CA) and SigmaPlot (Version 12.0, Systat Software Inc., San Jose CA, USA) and Po0.05 was used as the level of statistical significance. Values presented in the figures, and throughout the text, are means71 standard error of the mean (S.E.).

3. Results 3.1. Experimental animals and the functional integrity of isolated mitochondria There were no sex differences in fish size, condition factor, relative or absolute atrial and ventricular mass, ventricular protein content, or the yield of mitochondrial protein for the 10 1Cacclimated Atlantic cod (Table 1). States 3 and 4 respiration rates (both sexes combined) at 10 1C and 200 μM ADP averaged 109 7 14.2, and 11.071.7 nmol O2 min  1 mg protein  1, respectively, and RCR values and P:O ratios were approx. 11.3 and 1.5, respectively (Fig. 2D and E). These values are characteristic of intact mitochondria with a broad metabolic scope and tight coupling between oxygen consumption and ATP synthesis. 3.2. Temperature and sex-dependent effects on mitochondrial respiration and function When the complete data set is analyzed, State 3 respiration increased by 71% between 10 and 24 1C (Q10 ¼1.46, Fig. 2B) and did not plateau. Corresponding States 2 and 4 respiration values increased much more than State 3 between 20 and 24 1C (Fig. 2A–C). This resulted in Q10 values of 2.10 for State 2 and 1.94 for State 4 between 10 and 24 1C, and a State 4 that represented approximately 16% of State 3 respiration at 24 1C (Fig. 2B and C). As such, the large increase in State 4 (by 70%) that occurred between 20 and 24 1C resulted in an RCR value at 24 1C ( 7.5) that was significantly lower as compared to that measured at 10 1C ( 11.3) (Fig. 2D). Interestingly, there were clear sex differences in how temperature affected mitochondrial respiration. State 3 respiration at 200 μM ADP increased by 73% in mitochondria from female Atlantic cod between 10 and 24 1C (Q10 ¼1.48, Fig. 3B) and did not plateau. In sharp contrast, the increase in State 3 was small, and not significant (P4 0.05) in mitochondria from male fish. As a result, State 3 values were significantly lower in male as compared to female fish at 20 and 24 1C (47 and 41% lower, respectively, Fig. 3B). This reduced temperature-dependence of State 3 respiration in males was also reflected in State 2 and State 4 values (Fig. 3A and C). There was no significant increase in State

2 respiration in mitochondria from male fish, and State 4 respiration at 24 1C was significantly less in males as compared to females. Thus, the drop in RCR between 10 and 24 1C was of a similar magnitude in both sexes (  26%) (Fig. 3D). State 3 respiration was only significantly higher in 20 vs. 10 1C preparations at 100 and 200 μM ADP (Fig. 4). This [ADP]-dependent effect on the temperature sensitivity of State 3 respiration was not sex dependent, but was also reflected in Q10 and Km values. The Q10 value for State 3 was significantly lower at 25 μM ADP (1.17) vs. 200 μM (1.57), and the Km value for the [ADP]-State 3 relationship was 687 10 μM at 10 1C vs. 105 7 7 μM at 20 1C (P ¼0.013, Fig. 4). ADP concentration also influenced the efficiency of OXPHOS (i.e. the P:O ratio), but in this case the effect was more pronounced at both 20 1C and in male cod (Fig. 5). In females, although mitochondrial P:O was not significantly affected by [ADP] at 10 1C (range 1.45–1.73, P 40.05), it increased by 33% (from 1.46 to 1.94) between 25 and 100 μM ADP at 20 1C (P o0.05). In males, the P:O ratio increased significantly (from 1.49 to 1.76, Po0.05) at 10 1C between 25 and 200 μM ADP. Further, the P:O value was only 1.05 at 25 μM ADP at 20 1C, and increased by  70% (P 40.05) at 100 μM ADP. The large [ADP]-dependency at 20 1C in male fish resulted in significantly lower P:O ratios at 20 vs. 10 1C at 25 and 50 μM, and a significantly lower P:O ratio for male fish at 25 μM and 20 1C as compared to female fish (Fig. 5). 3.3. Effect of temperature on maximal CS activity There was no effect of sex on the temperature-sensitivity of maximal CS activity in the isolated mitochondrial preparations (P ¼0.71). CS activity (N ¼8; 4 of each sex) at 10 1C was 0.957 0.08 mU mg mitochondrial protein  1 and increased to 1.18 70.10 at 16 1C, to 1.42 70.11 at 20 1C and to 1.69 70.13 at 24 1C. The Q10 value for the 10–20 1C interval (1.497 0.02) was not different from the 20–24 1C range (1.54 70.07), suggesting that the activity of this enzyme was conserved over the entire temperature range studied for both sexes.

4. Discussion The thermal tolerance of fishes appears to be largely determined by the effects of temperature on the capacity of the heart to deliver blood to the tissues (Farrell, 2002, 2009; Wang and Overaard, 2007; Farrell et al., 2009; Keen and Gamperl, 2012), and some authors (Hilton et al., 2010; Iftikar and Hickey, 2013) suggest that compromised mitochondrial respiration is a primary mechanism leading to diminished cardiac performance or failure at high temperatures. In Atlantic cod, we found that measures of mitochondrial function (States 2, 3 and 4 respiration, RCR, P:O ratios, and CS activity) at 10 1C were similar between the sexes and indicative of mitochondria that were coupled (e.g., RCR 10) and functioning well (Table 1; Fig. 2). However, acutely increasing temperature to 20, and then 24 1C, revealed diminished respiratory activity in mitochondria from males vs. females, and provided evidence for increased proton leak, reduced coupling and mitochondrial dysfunction in both sexes. These data suggest that oxidative metabolism may be compromised in the Atlantic cod heart at high temperatures, a time when ATP turnover and kinetic demands on cardiomyocytes increase substantially. State 3 respiration is controlled by ATP synthesis and substrate oxidation, whereas both States 2 and 4 respiration are largely determined by the permeability of the inner mitochondrial membrane to protons (Hafner et al., 1990; Brand and Nicholls, 2011). Many studies on vertebrate mitochondria have shown that increasing temperature within the normal physiological range increases mitochondrial respiration (e.g., Cassuto, 1971; Guderley

Please cite this article as: Rodnick, K.J., et al., Temperature and sex dependent effects on cardiac mitochondrial metabolism in Atlantic cod (Gadus morhua L.). J. Thermal Biol. (2014), http://dx.doi.org/10.1016/j.jtherbio.2014.02.012i

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nmol O2· min-1· mg protein-1

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Temperature (°C) Fig. 2. The effect of increasing temperature on States 2, 3 and 4 respiration of isolated ventricular Atlantic cod mitochondria at 10, 16, 20 and 24 1C, and corresponding RCR values and P:O ratios. Values are means 7S.E. (n ¼12). Values without a letter in common are significantly different (Po 0.05). ‘n’ Indicates that State 3 respiration was different between 10 and 16 1C at Po 0.10.

and Johnston 1996; Berner, 1999; Blier and Lemieux, 2001). Further, it is apparent that acute temperature changes impact State 3 respiration through effects on the substrate oxidation system, including the tricarboxylic acid cycle, the electron transport chain, metabolite transporters, F1F0-ATP synthase and adenine nucleotide translocase (ANT) (Chamberlin, 2004). In Atlantic cod ventricle (sexes combined), the submaximal State 3 respiration (i.e. at 200 mM ADP) of isolated mitochondria and the in vitro catalytic capacity of CS both increased between 10 and 24 1C, and the thermal sensitivity of State 3 respiration (Q10 ¼1.46) closely matched that of CS (Q10 ¼1.51) (Table 1, Fig. 2B). Using isolated mitochondria from the red muscle of Arctic charr (Salvelinus

alpinus), Blier and Lemieux (2001) also found comparable thermal sensitivities for mitochondrial oxidation of pyruvate and malate, and cytochrome c oxidase (COX) activity, over a wide temperature range (1–18 1C). Between 12 and 18 1C, the Q10 values were 1.35 and 1.50 for State 3 and COX activity, respectively. Our results are also similar to studies on saponin-skinned cardiomyocytes from Atlantic cod (Birkedal and Gesser, 2003) showing that State 4 respiration and mitochondrial coupling were significantly higher and lower, respectively, at 20 versus 10 1C. However, the same authors reported that the higher temperature (20 1C) decreased the mitochondria's apparent Km for ADP (i.e., increased ADP affinity) (Birkedal and Gesser, 2003), which contrasts with the

Please cite this article as: Rodnick, K.J., et al., Temperature and sex dependent effects on cardiac mitochondrial metabolism in Atlantic cod (Gadus morhua L.). J. Thermal Biol. (2014), http://dx.doi.org/10.1016/j.jtherbio.2014.02.012i

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Fig. 3. The effect of increasing temperature (10–24 1C) on States 2, 3 and 4 respiration, and RCR, in isolated ventricular mitochondria from male and female Atlantic cod (n¼ 6 per sex). Effects of temperature, within each sex, without a letter in common are statistically significant (Po 0.05). ‘n’ Indicates a significant difference between male and female fish at a particular temperature. Values are mean 7 S.E.

Q 10 = 1.57

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ADP Concentration (µM) Fig. 4. Relationship between ADP concentration and State 3 respiration in Atlantic cod ventricular mitochondria at 10 and 20 1C. The asterisk (n) indicates a significant difference (Po 0.05) between 10 and 20 1C at a particular [ADP]. Dissimilar lower and upper case letters indicate significant differences between [ADP] at 10 and 20 1C, respectively. The reported Q10 values are specific to each [ADP]. Km values, as determined using the data for individual fish and Lineweaver–Burk plots, were 68 7 10 μM at 10 1C and 1057 7 μM at 20 1C (P ¼0.013). Note: data were combined for male and female fish as the [ADP]–State 3 relationship was not significantly different between sexes. Values are means 7S.E. (n¼ 9).

current study on isolated mitochondria. The mechanism(s) that increase or decrease mitochondrial ADP affinity following an acute temperature increase in Atlantic cod are unknown. However, a decrease in ADP affinity at higher temperatures (current data) could compromise the balance between cardiac ATP supply and demand, and result in increased ROS production (Korshunov et al., 1997) as this species approaches its thermal limits. Mitochondrial proton leak can occur via simple diffusion across the membrane, or through the ANT, uncoupling proteins, the dicarboxylate carrier or the aspartate/glutamate antiporter (Brookes et al., 1997; Samartsev et al., 1997; Wieckowski and

Wojtczak, 1997; Cadenas et al., 2000; Stuart et al., 2001; Hoerter et al., 2004; Brand et al., 2005). State 4 respiration (and presumably the proton leakiness of Atlantic cod mitochondria) was significantly elevated at and above 20 1C, and both States 2 and 4 respiration increased dramatically (by  1.4 and 1.7-fold, respectively) between 20 and 24 1C (Fig. 2). Further, this was concomitant with a 25% decrease in RCR (Fig. 2D), due mainly to the large increase in State 4 with little (female) or no (male) change in State 3 respiration. RCR is considered to be a very informative measure of function in isolated mitochondria (Brand and Nicholls, 2011). The observed increase in State 4 respiration and decrease in RCR for both sexes of Atlantic cod at high temperatures could be due to (1) a loss of mitochondrial membrane integrity by way of a temperature-induced phase transition of membrane lipids (Dahlhoff and Somero, 1993); (2) membrane damage associated with elevated ROS production (Iftikar and Hickey, 2013), and/or (3) mitochondrial substrate (ADP) futile cycling (Sazanov and Jackson, 1994; and below). The temperature range of 20–24 1C spans the critical thermal maximum of Atlantic cod acclimated to 10 1C (  22 1C; Gollock et al., 2006), and thus, our findings are consistent with Hilton et al. (2010) and Iftikar and Hickey (2013) who report that mitochondrial function in permeabilized cardiomyocytes was compromised just a few degrees Celsius below the upper temperature limits for triplefin (various species) and the New Zealand Spotty (Notolabrus celidotus), respectively. The nature of mitochondrial dysfunction at elevated temperatures has not been addressed in Atlantic cod, but Iftikar and Hickey (2013) suggest that increased ROS production, and a loss of inner and outer mitochondrial membrane integrity (i.e. increased permeability), are related to the depression of RCR and the increase in States 2 and 4 respiration in fish mitochondria at high temperatures. A more complete understanding of the bioenergetic processes responsible for the mitochondrial dysfunction observed at elevated temperatures in

Please cite this article as: Rodnick, K.J., et al., Temperature and sex dependent effects on cardiac mitochondrial metabolism in Atlantic cod (Gadus morhua L.). J. Thermal Biol. (2014), http://dx.doi.org/10.1016/j.jtherbio.2014.02.012i

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Fig. 5. The effect of temperature (10 vs. 20 1C) and ADP concentration on the efficiency of mitochondrial phosphorylation (P:O) in ventricular mitochondria from female (A) and male (B) cod. # Indicates a significant difference (P o 0.05) between male and female fish at a particular [ADP] and temperature. n Indicates a significant difference between 10 and 20 1C in male or female fish at a particular [ADP]. Dissimilar letters indicate significant differences between [ADP] in male and female fish within a temperature. Values are mean7 S.E. (n¼9).

fish will require additional studies that include measurements of mitochondrial membrane potential/proton leak, ROS formation and futile substrate cycling, along with oxygen consumption and phosphorylation rate. However, the above data collectively suggest that high, yet environmentally relevant, temperatures impair cardiac mitochondrial function in fishes, and that this is a contributing factor in determining heart failure and upper thermal tolerance. This study on Atlantic cod provides the first evidence for sex differences in cardiac mitochondrial function in fishes. Most physiological studies on fish do not compare sexes and measurements of mitochondrial respiration are usually conducted using saturating ADP levels. Our finding that P:O ratios decreased at elevated temperature (20 1C) and physiological concentrations of ADP (25 and 50 μM) to a greater extent in males may be due to less efficient shuttling of ADP in to the mitochondria by the ANT, and explain their lower State 3 respiration as compared to females at 20 and 24 1C (Hickey et al., 2009). However, there are a number of alternate explanations. The futile cycling of ATP may have been a major contributor to mitochondrial dysfunction in males. State 4 respiration increased by 57% between 20 and 24 1C without a significant increase in State 2 respiration (Fig. 2A and C). Hilton et al. (2010) suggest that differences in lipid composition, membrane-associated protein function, and/or cytochrome c

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oxidase flux may account for the enhanced coupling in cardiac mitochondria in Bellapiscis medius permeabilized cardiomyocytes as compared with two less thermally tolerant triplefin species (Forsterygion varium and F. malcolmi). Finally, based on data from spontaneously hypertensive rat hearts (Hickey et al., 2009), it is possible that the combination of elevated temperature, a reduced supply of ADP and decreased ADP sensitivity (higher Km value, current study) might increase superoxide (ROS) production, and consequently proton leak in ventricular mitochondria from Atlantic cod. Overall, our results suggest that acute exposure to high temperatures has the potential to selectively limit the respiratory activity of cardiomyocytes, and thus, decrease cardiovascular performance in male Atlantic cod. However, there does not appear to be a consistent pattern amongst fishes in how sex affects cardiovascular performance and physiology when exposed to high temperatures. Adult male sockeye salmon (Oncorhynchus nerka) are more thermally tolerant than females when exposed to 19 1C for 10 d (Jeffries et al., 2012) or when migrating fish experience warm (19 1C) river temperatures (Martins et al., 2012). Male rainbow trout (Oncorhynchus mykiss) also have higher ventricular oxidative capacity, glycogen content, and can utilize mitochondrial oxidation of fatty acids to a greater degree than females (Battiprolu et al., 2007, Harmon et al., 2011). In contrast, there were no sex differences in cardiovascular variables (heart rate, stroke volume, and cardiac output) measured in vivo as quiescent, sexuallyimmature, rainbow trout were acutely warmed (by 2 1C h  1) from 14 to 24 1C (Gamperl et al., 2011). This inconsistency between studies may be due to lower than maximal metabolic rates and mitochondrial respiratory flux in inactive rainbow trout, and the requirement of significantly elevated cardiac function to unmask sex differences in mitochondrial function. In this study, and those of Hilton et al. (2010) and Iftikar and Hickey (2013), mitochondrial respiration was measured at relatively high oxygen levels (  200 mM, 21% O2, 160 mm Hg) and not at the significantly lower O2 levels experienced by cardiomyocytes in vivo. For example, the ventricle of Atlantic cod does not receive oxygenated blood via a coronary artery (Farrell and Jones, 1992), and venous PO2 in 10 1C-acclimated Atlantic cod is approx. 36 mm Hg at 10 1C and 10–20 mm Hg at 19 1C (Lannig et al., 2004). Furthermore, at increased body temperatures the metabolic demand of tissues is greater, and there can be uncompensated decreases in tissue oxygen levels that impose limits to maintaining elevated rates of myocardial mitochondrial respiration. For example, mitochondrial respiration has been shown to be inhibited by hypoxia, and it has been reported that proton leak is reduced in cardiac mitochondria exposed to decreased oxygen concentrations (Gnaiger et al., 2000; Casey et al., 2002). Both of these issues may impact the extent to which the mitochondrial dysfunction we report for Atlantic cod mitochondria at high temperatures would be observed at physiological (i.e. in vivo) oxygen levels. Clearly, future studies investigating the effect of high temperatures on mitochondrial function should be conducted at physiological O2 and ADP levels (see above), and using a wider range of substrates (e.g., glutamate, succinate, and palmitoylcarnitine). In addition, because we only exposed isolated mitochondria from Atlantic cod to different temperatures for approximately 60 min, it will also be important to define the temporal effects of elevated temperatures on mitochondrial metabolism and to determine whether damage exists and is reversible. In mammalian cells, mitochondria are one of the main organelles harmed by heat, and exposure of isolated rat heart mitochondria to a febrile temperature (40 1C) for just 3 min increased a marker of oxidative damage of mitochondrial lipids (Zukiene et al., 2010). Finally, it would be of significant value to (1) define the mechanism(s) responsible for compromised mitochondrial function in Atlantic cod, particularly

Please cite this article as: Rodnick, K.J., et al., Temperature and sex dependent effects on cardiac mitochondrial metabolism in Atlantic cod (Gadus morhua L.). J. Thermal Biol. (2014), http://dx.doi.org/10.1016/j.jtherbio.2014.02.012i

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in males, at warm temperatures ( 420 1C); and (2) confirm whether our current in vitro measurements of sex differences in mitochondrial function are relevant to heart and swimming performance, and the upper thermal tolerance of Atlantic cod and other fish species.

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Please cite this article as: Rodnick, K.J., et al., Temperature and sex dependent effects on cardiac mitochondrial metabolism in Atlantic cod (Gadus morhua L.). J. Thermal Biol. (2014), http://dx.doi.org/10.1016/j.jtherbio.2014.02.012i