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Am J Physiol Regul Integr Comp Physiol 292: R827-R836, 2007. First published October 12, 2006; doi:10.1152/ajpregu.00379.2006 Free Article
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Sex Differences in Renal and Cardiovascular Function: Physiology and Pathophysiology

Sex differences in energy metabolism and performance of teleost cardiac tissue

Pavan K. Battiprolu, Kelli J. Harmon, and Kenneth J. Rodnick

Department of Biological Sciences, Idaho State University, Pocatello, Idaho

Submitted 1 June 2006 ; accepted in final form 4 October 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This study examined the effects of different oxygenation levels and substrate availability on cardiac performance, metabolism, and biochemistry in sexually immature male and female rainbow trout (Oncorhynchus mykiss). Ventricle strips were electrically paced (0.5 Hz, 14°C) in hyperoxic or hypoxic Ringer solution. Our results demonstrate that 1) males sustain isometric force production (F) longer than females under hyperoxia (PO2 = 640 mmHg) with exogenous glucose present; 2) contractility is not maintained under moderate (PO2 = 130 mmHg) or severe hypoxia (PO2 = 10–20 mmHg) with glucose in either sex; however, following reoxygenation, F is higher in females compared with males; and 3) female tissue has higher lactate levels, net lactate efflux, and lactate dehydrogenase activity than males, whereas males have higher glycogen, citrate synthase, and beta-hydroxy acyl-CoA dehydrogenase activities, and greater inotropic responses to exogenous glucose and octanoate. No sex differences were detected in responsiveness to epinephrine and inhibitors of glucose transport or activities of hexokinase and pyruvate kinase. We conclude that sex differences exist in rainbow trout cardiac tissue: females appear to prefer glycolysis for ATP production, whereas males have a higher capacity for aerobic and lipid metabolism.

glucose; glycogen; lactate; oxygenation; rainbow trout

ALTHOUGH SEX DIFFERENCES IN mammalian cardiovascular function and disease have received considerable attention (30), very few studies of nonmammalian species exist. Our laboratory (10) and others (5, 22) have documented a selective two- to threefold enlargement of the ventricle and ventricular myocytes of male rainbow trout (Oncorhynchus mykiss) during reproductive maturation. Not surprisingly, elevated androgens appear to promote sex-dependent ventricular hypertrophy in trout (12). Male trout are also hypertensive and hypervolemic at this time (10), and male salmon display higher heart rates (1) and activity (26) on the spawning grounds than females. Whether there are functional differences between hearts from male and female trout remains unanswered. To the best of our knowledge, even less is known about potential sex differences in cardiac energy metabolism in fishes.

The trout ventricle, similar to many active teleosts, is a composite organ. It consists of a compact epicardium receiving well-oxygenated blood from the gills via a coronary system and a spongy, avascular endocardium receiving luminal venous blood. The energy metabolism of fish hearts, like that of mammalian hearts, is supported by oxidation of glucose, lactate, and fatty acids (13, 15, 47). More specifically, hearts of trout can rapidly metabolize glucose and lactate (29). There is also some evidence that the heart of salmonid fishes exhibits transmural gradients in energy metabolism: the endocardium prefers oxidative carbohydrate and glycolytic metabolism, whereas oxidative capacity for fatty acids is greater in the epicardium (17, 19, 37). Unfortunately, the sex of fishes was neither documented nor considered an important variable in these studies.

Unlike the healthy mammalian heart, it is common for the fish ventricle (epicardium and endocardium) to be hypoxic due to low environmental PO2. However, similar to the mammalian heart, the trout heart is hypoxia-intolerant and experiences pronounced decrements in contractile function (2, 24, 33). During these periods, oxidation of fatty acids will be limited and ATP production is likely to be supported by anaerobic glycolysis. As a result, the fish heart may depend more on carbohydrate as an energy fuel source than the mammalian heart (41, 42). Studies conducted on the mammalian heart (41) and trout heart (18) have also shown that exogenous glucose improves contractile activity under different conditions, especially hypoxia (24). We recently demonstrated that sex differences exist in the inotropic effects of exogenous glucose on cardiac performance in rainbow trout during hyperoxia (18). Glucose promoted greater inotropism in males, and yet female cardiac tissue required physiological concentrations (5 mM) of glucose to maintain resting tension and presumably Ca2+ homeostasis. Although several studies of trout cardiac performance have been conducted under hypoxic or anoxic conditions, sex was either not mentioned (33, 34, 36), only one sex was studied (20), or comparisons were not made between males and females (2, 23, 24). The purpose of this study was to compare cardiac biochemistry, metabolism, and performance in male and female rainbow trout at different oxygenation levels.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Experimental Animals

Sexually immature, 10- to 12-mo-old male and female rainbow trout were obtained from Clear Springs Foods (Buhl, ID). Fish were transported to the Aquatics Research Facility at Idaho State University and held in 1,000-liter circular tanks containing filtered, dechlorinated water at 14 ± 1°C. Fish were fed commercial trout pellets (1% of body weight every other day), exposed to a constant 12:12-h light-dark photoperiod, and held for at least 1 wk before experiments. All experiments were conducted in accordance with the National Institutes of Health Guidelines, Department of Health Education and Welfare Publication No. National Institutes of Health 78–23 (1978), and were approved by the Animal Welfare Committee at Idaho State University. All chemicals were purchased from Sigma-Aldrich (St. Louis, MO) and were of analytical grade.

Ventricle Strip Preparation

Fish were netted rapidly and killed by a sharp blow to the head. The ventricle was quickly excised, weighed, and placed in ice-cold, modified teleost Ringer solution containing in mM: 111 NaCl, 5 KCl, 0.5 NaH2PO4, 10 NaHCO3, 1.5 CaCl2, 1.0 MgSO4, and 5.0 glucose, was gassed with 99.5% O2-0.5% CO2, and had a pH of 7.6 at 14°C. Concentrations reported for all chemicals are final values in the tissue baths. The sex of each fish was determined by visual or microscopic examination of the gonads, and gonads were weighed. A portion (~50 mg) of each ventricle was immediately frozen using aluminum clamps precooled in liquid nitrogen and stored at –80°C until biochemical assays were conducted.

Four uniform strips (weighing 15–25 mg, approximate dimensions: 4–5 mm long x 0.7–1.0 mm wide) were cut from each ventricle using a single-edge razor blade. For each treatment, we used just one ventricle strip per animal and considered this an independent data point. Multiple, dependent data points were recorded for each strip during time course experiments. Strips were vertically mounted, clamped at its base, tied at the other end with surgical silk (3–0), and attached to a Kent isometric transducer (Model TRN002, Litchfield, CT). Strips were suspended in open 30-ml tissue baths containing Ringer solution with or without 5 mM glucose, between platinum electrodes, and gassed with 99.5% O2-0.5% CO2 for 60 min (equilibration period). Temperature of the tissue baths was maintained at 14°C with a refrigerated recirculating bath. Strips were field stimulated with a voltage that elicited full contraction (60 V, two- to threefold higher than threshold) at a physiological frequency (0.5 Hz) with 5-ms square wave pulses (Grass S88 Stimulator, Grass Medical Instruments, Quincy, MA). The length of each strip was increased gradually until maximum isometric force production (Lmax) was achieved, and then muscle length was reduced to 90% Lmax to avoid damage to the preparation. After equilibration to allow for recovery from tissue cutting and stabilization of twitch force (F) at 90% Lmax, we measured F, time to peak force (tp), time to 80% relaxation (t0.8r), and resting tension using a data acquisition system (BioPac MP100, Santa Barbara, CA) and software (AcqKnowledge v 3.5.5, BioPac). For experiments with reduced oxygenation, we also calculated the time required to reduce F to 50% of initial values (F50%).

Effects of Extended Contractile Periods on Cardiac Performance

During the 60-min equilibration period, ventricle strips from both sexes were electrically stimulated at 0.5 Hz in Ringer solution containing either glucose (5 mM) or no glucose. Incubations continued for another 150 min without changing the media and maintaining hyperoxic (99.5% O2-0.5% CO2) conditions. Baseline F (set as 100% original twitch force) was established after the 60-min equilibration period and defined time 0 for performance comparisons. Our preliminary experiments demonstrated that ventricle strips under these conditions can maintain F at >40% of baseline values for at least 360 min.

Role of Facilitated Diffusion of Glucose for Contractile Performance

After equilibration with glucose for 60 min, contracting ventricle strips were exposed to fresh Ringer solution without glucose for another 60 min. Strips were then pretreated with inhibitors of glucose transport (25 µM cytochalasin B, from an ethanol stock solution, or 100 µM phloretin, stock solution containing Ringer solution) for 20 min. Cytochalasin B and phloretin block facilitated sugar transport across the cell membrane of many tissues from diverse animals. Using tracer methods, we have shown previously that cytochalasin B (11, 38) and phloretin (11) partially inhibit 2-deoxy-D-glucose transport in fish cardiac tissue and presumably target glucose transporter proteins (GLUTs). These compounds, used at similar concentrations to the present study, blocked 2-deoxy-D-glucose uptake by Xenopus oocytes expressing the rainbow trout GLUT1 (OnmyGLUT1, 43). Although not studied in fish GLUTs, the putative binding sites and mechanism of actions for cytochalasin B and phloretin on human GLUT1 have been modeled: cytochalasin B binds to the intracellular side and interferes with glucose passage through the endofacial channel opening, whereas phloretin interferes competitively with glucose for the exofacial glucose docking site (40). It is also possible that inhibition by phloretin occurs at both exofacial and endofacial sites (40).

To determine specific effects of glucose transport inhibitors, controls remained glucose free or received glucose (5 mM) for an additional 60 min without exchanging the media. Strips serving as controls for cytochalasin B experiments were also exposed to appropriate concentrations of ethanol. For comparisons of contractile performance, baseline F (set as 100% original twitch force and defining time 0) was established after 120 min. We also measured postrest potentiation (PRP) of F at the end of experiments to assess sarcoplasmic reticulum (SR) Ca2+ storage and subsequent release (16). PRP measurements were conducted as described previously (18). Briefly, electrical stimulation of ventricle strips was discontinued for 5 min, and F was recorded for the first contraction following the resumption of stimulation. PRP was defined as the ratio of F after 5 min of rest vs. F prior to rest and was expressed as a percent change.

Effects of Oxygenation, Contraction Frequency, Glucose and Epinephrine on Cardiac Performance

These experiments examined cardiac performance under severe hypoxia (PO2 = 10–20 mmHg, Ringer solution gassed with 99.5% N2-0.5% CO2), moderate hypoxia (PO2 = 130 mmHg, gassed with 21% O2-0.5% CO2-78.5% N2) and hyperoxia (PO2 = 640 mmHg, gassed with 99.5% O2-0.5% CO2). Measurable levels of PO2 were observed during severe hypoxia because tissue baths were open to atmospheric air. For severe hypoxia, ventricle strips were stimulated initially at 0.5 Hz with 5 mM glucose under hyperoxia. At the end of equilibration, fresh Ringer solution was added to the tissue bath, and contracting strips were gassed with 99.5% N2-0.5% CO2 and either 1) an increase in exogenous glucose (final concentration 5, 10, or 20 mM), 2) a decrease in stimulation frequency (0.2 Hz), 3) epinephrine (final concentration 1 or 10 µM), or 4) a combination of the above. The goal was to facilitate F under severe hypoxia and elevate epinephrine, which increases during physiological hypoxia (36). We have recently shown that the presence or absence of glucose does not affect epinephrine-induced inotropism in trout ventricle strips (18). We also conducted preliminary trials and found that epinephrine-induced inotropism during severe hypoxia is independent of exogenous glucose. Therefore, all of our experiments with severe hypoxia and epinephrine were conducted with glucose present. Although the concentration of epinephrine used in the current study is outside the physiological range, it gives a well-defined and close-to-maximal adrenergic stimulation of twitch force in rainbow trout cardiac tissue (31, 33). Ventricle strips exposed to moderate hypoxia or hyperoxia were equilibrated with 5 mM glucose for 60 min, then received either fresh Ringer solution with glucose (treatments) or without glucose (controls). All strips were stimulated to contract at 0.5 Hz. At the end of experiments, strips were freeze-clamped, and Ringer solution (1 ml) was collected and deproteinized (see Biochemical Characteristics of Cardiac Tissue), and both were stored at –80°C. We also evaluated the effects of reoxygenation with 99.5% O2-0.5% CO2 on cardiac performance after severe hypoxia. Only contractile variables were measured in these experiments.

Effects of a Medium-Chain Fatty Acid on Contractile Performance

Similar to exogenous glucose, preliminary experiments showed that octanoate exerted positive inotropic effects in ventricle strips from both sexes. After equilibration with 5 mM glucose for 60 min, ventricle strips were exposed to fresh Ringer solution containing either 5 mM glucose (controls) or different concentrations (0.1, 0.25, 0.5, or 1.0 mM) of octanoate without glucose for an additional 60 min. On the basis of preliminary experiments, maximal inotropic effects of octanoate were observed within 30–45 min at 0.5 mM for both males and females. We, therefore, used this concentration to compare the effect of octonoate vs. glucose treatment on contractile performance. At the end of experiments, strips were freeze-clamped and kept frozen for lactate measurements.

Biochemical Characteristics of Cardiac Tissue

Frozen samples of ventricle tissue were homogenized in ice-cold extraction medium using a motorized Duall-22 ground-glass homogenizer. The extraction medium and final dilution of the homogenate were dependent on the enzyme/substrate being assayed. Enzyme activities were measured at 15 ± 0.5°C under saturating substrate concentrations using a Perkin-Elmer Lambda 6 UV/VIS spectrophotometer (Norwalk, CT) with a thermostatically controlled recirculating water bath and water-jacketed cuvette holder. Myocardial glycogen and free-glucose concentrations were measured using acidified tissue extracts and established techniques (35). Values are expressed as milligrams per gram of tissue. Myocardial lactate concentration was measured using deproteinized tissue extracts and standard enzymatic methods (21). Lactate concentration is expressed as micromoles per gram of tissue. Net release of lactate from ventricle strips was measured from deproteinized [equal volume of 6% (vol/vol) perchloric acid] Ringer solution samples using published methods (21). Lactate release is expressed as nanomoles per minute per gram of tissue.

Hexokinase [HK; Enzyme Commission (EC) enzyme classification number 2.7.1.1 [EC] ], pyruvate kinase (PK; EC 2.7.1.40 [EC] ), Citrate synthase (CS; EC 4.1.3.7 [EC] ), and beta-hydroxyacyl-CoA dehydrogenase (HOAD; EC 1.1.1.35 [EC] ) activities were measured in whole tissue homogenates of frozen tissue using published techniques (9). We also measured lactate dehydrogenase (LDH; EC 1.1.1.27 [EC] ) activity and kinetics using a procedure modified from a previous study (9). Specifically, tissue samples (~20 mg) were homogenized in 9 vol of buffer (50 HEPES, 1 EDTA, and 2 dithiothreitol; pH 7.5 at 15°C), centrifuged for 5 min at 15,000 g, 4°C, and the supernatant was assayed. The reaction mixture contained (in mM): 50 HEPES, 1 KCN, 0.17 NADH and either 0, 0.025, 0.05, 0.1, 0.25, 0.5, or 1 pyruvate (omitted for controls); pH 7.5 at 15°C. Kinetic analyses were performed using Eadie-Hofstee plots. Activities are expressed as micromoles per minute per gram of tissue.

Data Analysis

For all measurements, one independent data point was taken per each ventricle strip. Performance variables (F, tp, t0.8r, and resting tension) were averaged for five consecutive waveforms at 5-min intervals after the equilibration period. In some cases, only Fmax or F50% was compared between sexes. Data are expressed as means ± SE of either absolute values or percent change (F and resting tension). Comparisons of cardiac performance or select biochemical variables (glycogen and lactate) were assessed by two-way (effect of sex and treatment) ANOVA with Bonferroni and LSD post hoc corrections using SAS, software (Cary, NC). Student's t-test and repeated-measures ANOVA were used to compare the means of metabolic enzyme activities and cardiac performance over extended periods, respectively. Significant differences (P < 0.05) are indicated in the text, tables, and figures.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Physical Characteristics of Experimental Animals

Body weight, fork length, ventricle mass, relative ventricular mass (RVM), gonad mass, and gonadosomatic index (GSI) were not different between male and female rainbow trout (Table 1).


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  		Table 1. Physical characteristics of experimental rainbow trout (Oncorhynchus mykiss)

 
Glucose Defines Sex Differences and Similarities in Cardiac Performance Over Time

When ventricle strips were continuously incubated in Ringer solution containing 5 mM glucose and well oxygenated (99.5% O2-0.5% CO2), male tissue selectively maintained absolute (0.950 ± 0.119 to 0.928 ± 0.123 g) and relative F (Fig. 1A), whereas female values decreased between 60 and 90 min (from 0.729 ± 0.080 to 0.627 ± 0.095 g, Fig. 1A, P = 0.036), and continued to drop over time (to 0.563 ± 0.090 g, 80% of original F at 150 min, P = 0.005). In oxygenated ventricle strips not receiving glucose, F decreased dramatically over 150 min in males (from 0.676 ± 0.078 to 0.475 ± 0.065 g) and females (from 0.662 ± 0.115 to 0.447 ± 0.113 g, Fig. 1B, males to 70%, P = 0.002; females to 68%, P = 0.003). These findings highlight the importance of physiological levels of glucose for sustaining contractile activity in vitro.


Figure 1
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  		Fig. 1. Twitch force (F) in ventricle strips from both sexes under hyperoxia in the presence (A) or absence (B) of glucose (5 mM) during 150 min of stimulation at 0.5 Hz. *P < 0.05, **P < 0.01, denotes significant difference from time 0 for both sexes. Values are means ± SE; n = 6–16 independent data points for males and females.

 
Cytochalasin B and Phloretin Reduce F and PRP in Both Sexes

Pretreatment of ventricle strips with cytochalasin B or phloretin completely blocked the positive inotropism induced by 5 mM glucose (Fig. 2A, cytochalasin B, ANOVA, F2,18 = 27.32, P = 0.004 and ANOVA, F2,20 = 31.34, P = 0.002 with phloretin). Cytochalasin B and phloretin also reduced PRP in both males and females compared with glucose controls (cytochalasin B, males: 131 ± 6%, females: 126 ± 8; phloretin, males: 120 ± 9%, females: 118 ± 6% vs. glucose, males: 175 ± 6% and females: 160 ± 3%, P < 0.01). However, lactate release was only reduced by phloretin (~70%) and not cytochalasin B (Fig. 2B, ANOVA, F2,20 = 12.21, P = 0.032).


Figure 2
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  		Fig. 2. Effects of cytochalasin B and phloretin on twitch force (A) and net lactate efflux (B). Plus (+) and minus (–) denote presence and absence, respectively, of specific compounds in the incubation medium. {dagger}P < 0.05 vs. females in the absence of inhibitors (controls), *P < 0.01 vs. inhibitors, §P < 0.05 vs. controls. Values are means ± SE; n = 4–6 independent data points for males and females.

 
Differential Responses to Oxygenation State: Female Ventricle Strips Depend More on Exogenous Glucose and Glycolysis for Contraction and Relaxation

Exogenous glucose maintained resting tension in both sexes under hyperoxia, and moderate or severe hypoxia (Table 2, ANOVA, F2,32 = 4.26, P = 0.223). The absence of glucose resulted in increased resting tension in females under all conditions, but only during hypoxia in males (Table 2, ANOVA, F2,32 ,= 20.17, P = 0.041). Under hyperoxia, glucose increased absolute and relative F when compared with glucose-free controls and more in males than females (Fig. 3, A and B, Table 2, ANOVA, F2,32 = 29.41, P = 0.022). F decreased to 50–60% of initial values in both sexes under moderate hypoxia and to a greater extent under severe hypoxia (Table 2, Fig. 3, A and B, ANOVA, F2,32 = 109.42, P = 0.0012). However, glucose did not help maintain F under moderate or severe hypoxia. Under all conditions at 0.5 Hz, tp was significantly longer in male compared with female ventricle strips (Table 2, ANOVA, F2,32 = 21.23, P = 0.037) and tp was longer in females in the presence vs. absence of glucose (Table 2, ANOVA, F2,32 = 26.72, P = 0.028). t0.8r also increased in males and females with hypoxia, except in female without glucose (Table 2, ANOVA, F2,32 = 28.12, P = 0.024). Glucose decreased t0.8r under hyperoxia and moderate hypoxia (Table 2, ANOVA, F2,32 = 17.89, P = 0.044); and female strips had shorter t0.8r under hypoxia compared with males, with or without glucose (Table 2, ANOVA, F2,32 = 21.03, P = 0.041).


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  		Table 2. Contractile variables (absolute values) under different oxygenated conditions, stimulated at 0.5 Hz, 60 V, 14°C with (+) or without (–) glucose

 

Figure 3
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  		Fig. 3. Effects of exogenous glucose (5 mM) on twitch force (F) under hyperoxia and severe hypoxia. In males (A) and females (B), incubations contained either glucose (G) or remained glucose free (GF). Dissimilar letters denote significant differences between sexes, P < 0.05). *Significant differences compared with time 0, P < 0.05. Values are means ± SE; n = 8–12 independent data points for males and females.

 
Sex Differences in Cardiac Tissue Lactate, Lactate Release, Glycogen, and Glycogen Depletion

Initial concentrations of lactate in freshly excised cardiac tissue were not different between males and females (P = 0.312). However, lactate was about 10-fold higher in females when compared with males under hyperoxia and severe hypoxia (Fig. 4A, ANOVA, F4,16 = 28.23, P < 0.001). In females, tissue lactate was also higher in the presence vs. absence of glucose under hyperoxia and severe hypoxia (Fig. 4A, ANOVA, F4,16 = 18.71, P = 0.006). Conversely, the presence or absence of glucose did not affect lactate levels in male ventricle strips. Similar results were observed with respect to net lactate efflux from ventricle strips during contraction. Under all conditions, lactate efflux was much greater in females compared with males (Fig. 4B, ANOVA, F5,32 = 25.63, P = 0.003). In females only, glucose promoted greater lactate release during hyperoxia (Fig. 4B, ANOVA, F5,32 = 16.83, P = 0.038).


Figure 4
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  		Fig. 4. Effects of oxygenation state on tissue lactate (A), lactate efflux (B), and glycogen levels (C) in ventricle strips with (+) or without (–) glucose (5 mM). §P < 0.001 vs. males during hyperoxia and severe hypoxia, **P < 0.01 vs. males, *P < 0.05 vs. females at initial value only, {dagger}P < 0.05 vs. all treatment groups in males. Values are means ± SE; for males and females, n = 6–12 independent data points per treatment.

 
The initial concentration of glycogen was higher in males compared with females (Fig. 4C, ANOVA, F6,30 = 24.59, P = 0.029). Interestingly, only male ventricle strips demonstrated significant glycogen depletion under different oxygenated conditions (Fig. 4C, ANOVA, F6,30 = 19.32, P = 0.037). The presence of glucose did not reduce glycogen depletion in males (Fig. 4C, ANOVA, F6,30 = 1.46, P = 0.631), suggesting that exogenous glucose does not protect cardiac glycogen stores.

Under severe hypoxia, epinephrine increases F in both males and females. After reoxygenation, female cardiac tissue maintains higher F than males.

In both sexes, epinephrine (10 µM) increased %F in ventricle strips and maintained elevated F for about 10 min during severe hypoxia (Fig. 5, ANOVA, F3,20 = 17.86, P = 0.031). However, unlike epinephrine, increasing concentrations of glucose (5 to 20 mM) did not restore or maintain F under moderate or severe hypoxia (data not shown). Although not statistically significant, stimulating ventricle strips at 0.2 Hz during severe hypoxia also appeared to maintain F compared with controls at 0.5 Hz (data not shown). After brief (15 min) reoxygenation (severe hypoxia to hyperoxia), F was ~30% less than its hyperoxia control in males but returned to prehypoxia levels in females (Fig. 5, ANOVA, F3,20 = 9.31, P = 0.043).


Figure 5
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  		Fig. 5. Effects of epinephrine (Epi, 10 µM) under severe hypoxia and hyperoxic reoxygenation after exposure to severe hypoxia for 60 min on twitch force (F) in ventricle strips receiving glucose (5 mM). Dissimilar letters denote significant differences between sexes and treatments, P < 0.05. Values are means ± SE; n = 8–10 independent data points for males and females.

 
Octanoate Promotes Higher Inotropism and Lower Tissue Lactate in Males Compared With Females

Octanoate (0.5 mM) increased F in both sexes compared with glucose controls (P < 0.05), and males responded more than females (165 ± 12% vs. 135 ± 10%, n = 4–6 independent data points for each sex, P = 0.039). Resting tension also increased in both sexes compared with glucose controls (males: 133 ± 7% vs. 103 ± 4% and females: 151 ± 9% vs. 106 ± 6% with octanoate vs. glucose, respectively, n = 4–6 per group, P = 0.003). Similar to our previous findings, tissue lactate was much lower in males (0.39 ± 0.27 µmoles/g) compared with females (2.72 ± 0.13 µmoles/g, P < 0.001) at the end of experiments.

Male Trout Hearts Have Higher Oxidative Capacity and Female Trout Hearts May Prefer Glycolysis for Energy Production

Maximal activities of CS (Fig. 6C) and HOAD (Fig. 6D) were about 40% higher in cardiac tissue from males (P < 0.01), whereas LDH activity (Fig. 6E) was about 30% higher in females. These differences may reflect a larger mitochondrial compartment in the male ventricle and, together with higher tissue lactate concentration (Fig. 4A) and net release (Fig. 4B), may indicate a greater reliance on anaerobic glycolysis by females. On the other hand, the apparent affinity of LDH for pyruvate was comparable in males (Km = 0.164 ± 0.01 mM) and females (0.145 ± 0.01 mM), and maximal activities of HK (Fig. 6A) and pyruvate kinase (Fig. 6B) were similar between sexes.


Figure 6
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  		Fig. 6. Maximal activites of metabolic enzymes in trout cardiac tissue. Hexokinase (HK; A), pyruvate kinase (PK; B), citrate synthase (CS; C), beta-hydroxy acyl-CoA dehydrogenase (HOAD; D) and lactate dehydrogenase (LDH; E). *P < 0.05 vs. males, **P < 0.01 vs. females. V, reaction velocity, S, substrate (pyruvate). Values are means ± SE; n = 10–15 independent data points for males and females.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Unlike previous studies of teleost cardiac physiology, we compared male and female myocardial biochemistry, energy metabolism, and function. Similar to the mammalian heart, we detected significant sex differences. These results were both novel and noteworthy because they occurred in sexually immature animals, when circulating sex steroids are very low or not detectable. The fact that we used identical-size ventricle strips for males and females and they developed similar isometric F values under control conditions makes our comparisons more meaningful.

Sex Differences in Cardiac Tissue Function and Biochemistry Under Hyperoxia and Hypoxia

The current paper and previous studies (18, 24) suggest that there is something unique about exogenous glucose and the glycolytic pathway for maintaining mechanical performance and possibly Ca2+ homeostasis in trout cardiac tissue under aerobic conditions. We recently demonstrated that substituting glucose with isomolar or higher concentrations of lactate or pyruvate results in reduced or absent inotropic effects in both sexes (18). In addition, resting tension was selectively increased for 1) ventricle strip preparations from females without glucose or with other substrates [pyruvate, lactate, or octanoate (current data)], and 2) for male strips exposed to octanoate compared with one supplied with physiological (5 mM) glucose.

Functionally, we now demonstrate for the first time that 1) male ventricle strips maintain F longer than females in the presence of exogenous glucose and well-oxygenated conditions (Fig. 1A) and 2) glucose promoted greater inotropism in males compared with females after 60 min of incubation in glucose free Ringer solution (Fig. 3A). This suggests that male cardiac tissue may have a higher capacity for oxidizing exogenous glucose or endogenous substrates (glycogen and or triglyceride) than females. This idea is supported by our observations that 1) initial glycogen levels were higher in males compared with females (Fig. 4C), 2) glycogen depletion occurred in males but not females (Fig. 4C), 3) lactate levels (Fig. 4A) and net efflux (Fig. 4B) were dramatically higher in females, and 4) similar to glucose, octanoate exerted greater inotropism in males. Under hyperoxia and hypoxia, tissue lactate concentrations for female ventricle strips and lactate efflux for both sexes are consistent with studies that do not mention sex of the fish (3, 25, 32, 33). Despite sex differences in tissue glycogen levels, we cannot ascertain whether female cardiac tissue uses similar amounts of glycogen as males during contractile activity in vitro or in vivo. To do so will require more definitive studies involving stable isotopes or radioisotopes. It is possible that females maintain higher rates of glycogen synthesis than males during contraction. In addition, it is important to note that our "hypoxic" experimental conditions are not anoxic. Male ventricle strips—with a higher aerobic capacity—may maintain higher rates of aerobic metabolism and, therefore, produce less lactate than females, even though force production is reduced and comparable between sexes.

Importance of Glucose Uptake for Cardiac Contractile Characteristics and Metabolism in Both Sexes

Results of this study suggest that facilitated diffusion of glucose through a specific glucose transporter protein is related to the inotropism induced by glucose after glucose free incubation. This mechanism appears to be important for both male and female rainbow trout. Preincubation of ventricle strips with pharmacological agents cytochalasin B and phloretin reduced F (Fig. 2A) and PRP to a similar degree after glucose was added. We have shown previously that cytochalasin B (11, 38) and phloretin (11) inhibit glucose uptake in teleost cardiac muscle, presumably by interacting with glucose transporter proteins (40). The fact that net lactate release was decreased by phloretin but not cytochalasin B (Fig. 2B), may reflect a greater reduction in facilitated glucose transport and glycolytic flux with phloretin compared with cytochalasin B. Ultimately, despite comparable binding and inhibition of GLUTs, it is also possible that cytochalasin B and phloretin exert different "nonspecific effects" on trout cardiac metabolism. What remains to be determined is the relative importance of simple diffusion directly through the sarcolemma vs. glucose transporter-mediated uptake of glucose for maintenance of energy metabolism, Ca2+ homeostasis, and contractile function in both sexes.

Previous studies in mammals (41) and fish (18) show that exogenous glucose and glycolysis support cardiac Ca2+ homeostasis via the SR under aerobic conditions. Our present findings that resting tension was increased selectively in ventricle strips from female rainbow trout (under all conditions tested) not receiving glucose (Table 2) agrees with previous work (18) and extends the hypothesis that fish exhibit sex differences in cardiac energy metabolism. In this case, the limitation for cardiac function in females could be due to lower glycogen levels and an absolute requirement for exogenous glucose to support anaerobic and aerobic glycolysis. We further show that resting tension increased in males under hypoxia without glucose (Table 2). Earlier work by Bailey and colleagues (6) also reported that glucose was required to maintain resting tension of ventricle strips from American eel (Anguilla rostrata, sex not noted) under anoxia or normoxia, with elevated extracellular Ca2+. The presence of exogenous glucose is clearly beneficial to the trout heart under working conditions, and apparently more important to females than males.

Epinephrine, But Not Glucose or Reduced Stimulation Frequency, Exerts Sex-Independent Inotropism During Hypoxia

Interestingly, ventricle strips from either sex exposed to physiological (Fig. 3, A and B and Table 2) or higher concentrations of glucose and or stimulated at a lower frequency (0.2 Hz, data not shown) could not maintain F during hypoxia. This finding of hypoxia intolerance is consistent with previous studies on trout hearts (3, 33). However, a high concentration of epinephrine (10 µM) significantly improved F in both males and females for ~10 min (Fig. 5), supporting the consensus that this hormone helps to maintain cardiac function in vivo during hypoxic conditions (19, 27). In contrast to some mammalian studies, results from our study do not show a sex difference in epinephrine-induced inotropism under hypoxia. For example, Vizgirda and colleagues (44) found a greater response in male rats to beta-adrenergic stimulation due to increased transsarcolemmal Ca2+ influx and possible higher beta-adrenergic receptor density. Conversely, Chu and colleagues (8) did not detect sex differences in the functional response of right papillary muscles to isoproterenol. It is worth noting that these studies were performed under oxygenated conditions.

Sex Differences in Contractile Measures of Ca2+ Homeostasis, Recovery From Hypoxia and Biochemical Markers of Cardiac Energy Metabolism

Our observation that PRP was higher in males supports our previous hypothesis that males store more Ca2+ in SR than females (18). A longer tp in males, despite similar F values as females, may also reflect sex differences in Ca2+ flux. During hypoxia, t0.8r increased in both sexes (Table 2), suggesting that SR Ca2+ uptake was compromised. However, exogenous glucose reduced t0.8r without affecting F, suggesting that there was adequate ATP production to maintain SR Ca2+ uptake but not isometric force production.

On the basis of our reoxygenation studies, cardiac tissue from female rainbow trout appears more resistant to the negative effects of hypoxia than males. These findings are novel for fishes and agree with several mammalian studies (2, 4, 7, 45) and yet disagree with others (14, 28, 39). Our complementary findings that ventricle strips from female trout 1) rely more on exogenous glucose; 2) have higher lactate levels, lactate efflux, and maximal LDH activity; 3) have lower aerobic capacity (CS activity); and 4) exhibit a reduced preference for fatty acid oxidation (lower HOAD activity and reduced inotropism in response to exogenous octanoate) vs. males, provide possible metabolic explanations for enhanced hypoxia tolerance in females. This idea is consistent with studies on rats reporting that glycolysis was higher in female nonhypertrophied hearts than in males (39) and studies on monkeys showing a decreased expression of glycolytic enzymes in older males compared with young males, and females (46).

To the best of our knowledge, our study is the first to report sex differences in biochemical characteristics in a nonmammalian species. Males had higher prestimulation glycogen levels and increased catalytic potential for mitochondrial oxidation (CS and HOAD). These data are consistent with males having a preference for glucose and fatty acid oxidation and females relying more on glycolysis for ATP production. More definitive studies are warranted to define whether these sex differences occur in one or both myocardial layers. It is possible that immature males and females have different epicardium to endocardium ratios and different metabolic enzyme activities in each layer. With ventricular hypertrophy, sexually maturing male rainbow trout show higher epicardial but not endocardial activities of CS and HOAD (9). On the basis of previous studies of transmural gradients in energy metabolism (17, 19, 37), immature males may have higher epicardial oxidative capacity for fatty acids than females. Finally, the functional consequences of sex differences in cardiac function and biochemistry measured in vitro are unknown. We also realize that our studies conducted in vitro do not mimic in vivo conditions. However, the observed differences between male and female trout should raise new questions about our current understanding of teleost and mammalian cardiac physiology. Studies of cardiac energy metabolism in vivo and performance should be performed to further our understanding of sex differences in fish hearts. It also remains to be determined whether the observed differences between male and female rainbow trout apply to other fish species.

Perspectives

Our study highlights the importance of documenting sex and sexual maturity of fishes when conducting studies of cardiac physiology. Given that cardiac energy metabolism differs between sexually immature, male and female rainbow trout in vitro, the possibility exists for relevant sex differences in vivo under different environmental conditions. In athletic fishes like the rainbow trout, the heart supports axial skeletal muscle and sustained swimming activity. However, reductions in aquatic PO2 will have proportional and compromising effects on tissue PO2. In addition, swimming activity of trout in even well-oxygenated water will lower venous PO2, elevate cardiac output, and thereby affect myocardial energy demands and performance. Whether lower capacities for aerobic metabolism and Ca2+ storage and a greater dependence on glycolysis for energy production in female vs. male ventricles result in sex differences in cardiac function in vivo and swimming performance remains to be determined. Ultimately, subtle differences in circulating sex steroids or other factors may promote physiologically relevant changes in the trout heart during development, well before sexual maturation.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
The project described was supported in part by National Institutes of Health Grant P20 RR016454 from the INBRE Program of the National Center for Research, the NSF-Idaho EPSCoR Program and by the National Science Foundation under award numbers EPS-0447689 and IOB-517669, the MSTI/MSMRI Research Institute of the St. Luke's Regional Medical Center, the Department of Biological Sciences, and the Graduate Student Research and Scholarship Committee at Idaho State University.


   ACKNOWLEDGMENTS
 
We would like to thank Tracy Becker, Adam Goddard, Olav Sorensen, and Gayathri Ananthakrishnan for their assistance in tissue sampling and processing. Animals and assistance were generously provided by personnel at Clear Springs Food, Buhl, Idaho.


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. J. Rodnick, Dept. of Biological Sciences, Idaho State Univ., Pocatello, ID 83209–8007 (e-mail: rodnkenn{at}isu.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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