The goal of this study was to determine the distribution of citrate synthase (CS), β-hydroxyacyl coenzyme A dehydrogenase (HOAD), and lactate dehydrogenase (LDH) activities and myoglobin (Mb) concentration in the locomotor muscles (epaxial muscles) and heart of harbor seals. The entire epaxial musculature, which produces most of the power for submerged swimming, was removed and weighed, and three transverse sections (cranial, middle, and caudal) were taken along the muscle bundle. Multiple samples were taken along points on a circular grid using a 6-mm biopsy. A single sample was taken from the left ventricle of the heart. Muscle groups of similar function were taken from three dogs as a control. Mean values were calculated for four roughly equal quadrants in each transverse section of the epaxial muscles. There were no significant differences among the quadrants within any of the transverse sections for the three enzymes or Mb. However, there were significant differences in the mean enzyme activities and Mb concentrations along the length of the muscle. The middle and caudal sections had significantly higher mean levels of CS, LDH, and Mb than the cranial section, which may be correlated with power production during swimming. The enzyme ratios CS/HOAD and LDH/CS exhibited no variation within transverse sections or along the length of the epaxial muscles. Relative to the dog, the epaxial muscles and heart of the harbor seal had higher HOAD levels and lower CS/HOAD, which, taken together, indicate an increased capacity for aerobic lipid metabolism during diving.
- citrate synthase
- lactate dehydrogenase
- β-hydroxyacyl coenzyme A dehydrogenase
to maintain aerobic metabolism during diving, pinnipeds (order Carnivora, suborder Pinnipedia: seals, sea lions, and walrus) generally exhibit a low metabolic rate and rely principally on oxygen stored in the blood and muscle (19). Reed et al. (27) found that muscle myoglobin (Mb) concentrations in harbor seals are significantly greater (20–27 times) than those of terrestrial mammals. Mb is a significant storage site for oxygen in the muscle and may also enhance the diffusion of intracellular oxygen, particularly under hypoxic conditions (12, 27, 37). Recently, Kanatous et al. (16) showed that the muscles of harbor seals have a higher mitochondrial volume density [Vv(mt)] and higher activities of citrate synthase (CS) and β-hydroxyacyl coenzyme A dehydrogenase (HOAD) relative to terrestrial mammals. The increased Vv(mt) is thought to facilitate aerobic metabolism under hypoxic diving conditions by decreasing the diffusion distance between mitochondria and intracellular oxygen stores (i.e., oxymyoglobin). Elevated activities of CS and HOAD indicate greater aerobic capacity and a significant contribution to energy metabolism from fatty acid oxidation. These morphological and enzymatic adaptations maintain an aerobic, fat-based metabolism in muscles as oxygen partial pressure decreases during a dive and tissues become hypoxic (6).
Earlier studies of marine mammal muscle morphology and function have generally relied on single biopsies or spot samples from dead animals (2, 3, 13, 16, 25, 27, 31). Mapping intramuscular enzyme activities and Mb concentration is a new approach for obtaining a better understanding of metabolic adaptations in the swimming muscles of marine mammals, but it requires a high-density sampling regime in muscle cross sections. Polasek and Davis (24) used a mathematical mapping technique for contouring Mb concentration in the locomotory muscles of cetaceans. This proved to be a successful technique for revealing the heterogeneity of oxygen stores within the muscle. In the present study, we used this approach to map the activities of CS, HOAD, and lactate dehydrogenase (LDH) and Mb concentration in transverse sections of the epaxial muscles (the primary locomotory muscles) of harbor seals. In addition, single samples were analyzed from the hindlimb complex (locomotory muscle), the pectoralis (nonlocomotory muscle), and the heart. For comparison, we analyzed locomotory and nonlocomotory muscles of the dog. Our null hypothesis was that CS, HOAD, and LDH activities and Mb concentrations are homogenously distributed throughout the epaxial muscles and that they do not differ significantly from a terrestrial control species (i.e., the dog). Our alternative hypothesis was that regional heterogeneity reflects differences in the oxygen consumption and work performed by different areas of the muscle and that elevations in the aerobic enzyme activities and Mb concentrations are adaptations that sustain aerobic, fat metabolism during dive-induced hypoxia.
MATERIALS AND METHODS
Animals and tissue samples.
Samples from the epaxial muscles, hindlimb complex, pectoralis muscle, and heart were obtained from eight female and two male harbor seals (Table 1) during a native subsistence harvest in Prince William Sound, Alaska. Based on standard length and body mass, the seals were subadults or adults (23). All samples were taken within 6 h of death. Two locomotory muscles were sampled. The primary locomotory musculature lies along the vertebral column (Fig. 1) and will be referred to as the epaxial muscles. This musculature is composed of three main muscle bundles that include the longissimus dorsi, iliocostalis lumborum, and the latissimus dorsi. The epaxial muscles on either side of the vertebral column alternately contract and stretch to produce the lateral spinal flexions that generate thrust by the hind flippers during swimming (8). The entire epaxial musculature along one side of the spine was removed. After the muscle was weighed, three transverse sections were taken in the cranial (CR), middle (MID), and caudal (CA) regions (Fig. 1). The CR section was taken at the 7th cervical vertebra, MID was taken at the 14th thoracic vertebra, and CA was from the lower lumbar region. Samples (∼0.5 g) were taken at points on a circular grid using a 6-mm stainless steel biopsy punch, averaging 20 samples per transverse section (Fig. 2). A spot sample was taken from the hindlimb muscle complex, which also contributes to locomotion. By hindlimb muscle complex, we are referring to muscles on the dorsal surface of the femur that include, but are not exclusive of, the gluteus maximus and the biceps femoris. Single samples were taken from the pectoralis muscle (a nonlocomotory muscle) and the left ventricle of the heart. All samples were stored in liquid nitrogen until they were returned to Texas A&M University, where they were stored at −70°C until analysis. Control samples were obtained from the gastrocnemius muscle (locomotory) and the intercostal muscle (nonlocomotory) of three female dogs killed for research purposes at Texas A&M University. Heart samples were collected from both the seal and the dog. In addition, methodological control samples were obtained from the heart of three male rats (Sprague-Dawley). Tissue samples were taken in accordance with guidelines for the humane treatment of animals and with the approval of the Institutional Animal Care and Use Committee Texas A&M University.
Muscle samples were thawed, blotted, weighed, and then homogenized on ice, in buffer containing 1 mmol/l EDTA, 2 mmol/l MgCl2, and 50 mmol/l imidazole, pH 7.6, at 37°C. In preliminary tests, we determined that sonication after homogenizing the sample was not necessary for complete enzyme extraction. Each homogenate was centrifuged for 4–5 min at 10,000 g, and the supernatant was used for the assays. Protein concentration was measured in all supernatants using a protein assay kit (Bio-Rad Hercules, CA), based on the Bradford method, with bovine gamma globulin as a standard (Bio-Rad). Optimal in vitro activities were measured at 37°C under conditions of saturating substrate concentrations for the following enzymes: CS (EC 188.8.131.52), which catalyzes the first reaction in the citric acid cycle and is a measure of aerobic capacity; HOAD (EC 184.108.40.206), which catalyzes one reaction in the β-oxidation of fatty acids and is a measure of the capacity for fatty acid catabolism; and LDH (EC 220.127.116.11), which catalyzes the reversible conversion of pyruvate to lactate and is a measure of muscle anaerobic capacity. All assays were performed using a SPECTRAmax 340 microplate reader (Molecular Devices, Sunnyvale, CA) in a final volume of 160 μl. The assay conditions were as follows: CS: 0.5 mmol/l oxaloacetate, 0.25 mmol/l 5,5′-dithiobis(2-nitrobenzoic acid), 0.4 mmol/l acetyl CoA, and 50 mmol/l imidazole, pH 7.5, at 37°C, change in 412-nm absorbance, 412-nm excitation = 13.6 cm2/μmol; HOAD: 0.1 mmol/l acetoacetyl CoA, 1 mmol/l EDTA, 0.15 mmol/l NADH, and 50 mmol/l imidazole, pH 7.0 at 37°C, change in 340-nm absorbance, 340-nm excitation = 6.22 cm2/μmol; and LDH: 1 mmol/l pyruvate, 0.3 mmol/l NADH, and 50 mmol/l imidazole, pH 7.0 at 37°C, change in 340-nm absorbance, 340-nm excitation = 6.22 cm2/μmol. Enzyme-specific activities [μmol of substrate converted to product·min (IU)−1·g wet mass−1] were calculated from the rate of change in absorbance of the assay, which was linear for the duration of the reaction. Path length was determined for each assay solution. Enzyme activity ratios (CS/HOAD, LDH/CS) were calculated to assess the relative importance of different metabolic pathways in the muscle. The CS/HOAD is an index of the capacity of β-oxidation to generate acetyl-CoA relative to the capacity of the citric acid cycle to oxidize it, and the LDH/CS is a measure of the relative anaerobic vs. aerobic metabolic capacity of the tissue. Methods for enzyme analysis were adapted from Reed et al. (27).
Aliquots from the same supernatants used for enzyme assays were diluted with phosphate buffer (0.04 mmol/l, pH 6.6), and the resulting mixture was centrifuged for 50 min at 28,000 g at 4°C. The method of Reynafarje (29) was used to determine Mb concentration. The supernatant was bubbled with 99.9% carbon monoxide for 5 min to convert the Mb to carboxymyoglobin (24). After bubbling, the absorbance of the supernatant at 538 and 568 nm was measured using a Bio-Tek PowerWave 340× microplate reader. A Mb standard (horse Mb, Sigma-Aldrich, St. Louis, MO) was run with each set of samples. The Mb concentration was calculated as described by Reynafarje (29) and expressed in milligrams per gram of wet tissue mass.
Contours and statistical analysis.
Due to the small sample size of animals and the fact that harbor seals are not extremely sexually dimorphic, the female and males seals were grouped for statistical analysis. Statistical comparisons were made within each transverse section of the seal epaxial muscles, between the three sections of the epaxial muscles, and among different muscles within the seals. Interspecific comparisons among seal and dog were made for muscles with similar function (e.g., locomotory or nonlocomotory). Maximum aerobic capacity and Vv(mt) of the muscle tissue both scale to body mass (21, 33). For seal muscle, Kanatous et al. (16) showed that CS and HOAD activities correlate with Vv(mt) and also scale allometrically with body mass, similar to maximum aerobic capacity and mass-specific resting metabolic rate (RMR). Enzyme activities were scaled to each tissue's calculated specific RMR to adjust for differences in body mass between the seal and the control species. Based on the work of Wang et al. (34), the scaling exponent for the RMR of individual tissues is more variable than for the whole body RMR. Therefore, instead of scaling enzyme activities with the whole body RMR, estimated from 70Mb−0.25 (32), where Mb is the body mass of the animal (kg), we used the estimated specific RMR for each tissue (34). The estimated tissue-specific RMR (kJ·kg−1·day−1) were as follows: heart RMR = 3725Mb−0.12 and muscle tissue RMR = 125Mb−0.17(this includes muscle, bone, and adipose tissue).
Enzyme activities (IU/g wet tissue mass) were first tested for correlations with supernatant protein concentration (mg soluble protein/g wet tissue mass), to assess possible variation among samples in tissue composition and preparation that would affect enzyme activity values. Pearson product-moment correlation coefficients were calculated for all samples within the epaxial muscle of each seal for each enzyme. Because there were no significant positive correlations [r values ranged from −0.018 to 0.326 (mean of 0.085); P > 0.05], all enzyme activities are expressed in IU per gram wet tissue mass, and these values were compared statistically. Analysis of variance, followed by a Tukey test, was used for all statistical comparisons using InStat 3 (GraphPad Software, San Diego, CA) and SYSTAT 10 (SPSS, Chicago, IL). Contour maps of enzyme activities and Mb concentration within the transverse sections of the three epaxial muscles were made using Surfer (Golden Software, Golden, CO). Kriging was used to contour the data because it generates the best interpretation of medium-to-small data sets (17). Values stated in the text are means ± SE, unless otherwise noted.
The harbor seals had a mean body mass of 46.1 ± 4.3 kg and a standard length of 126.2 ± 5.3 cm (Table 1). The epaxial muscles were 6.1 ± 0.4% of total body mass and 41.7 ± 0.9% of standard length. The epaxial muscles are approximately circular in shape at their origin along the cervical vertebrae, but extend ventrally along the thoracic vertebrae to cover part of the rib cage before tapering back to a circular shaped along the lumbar vertebrae (Fig. 1). The muscle is dark red in color due to its high Mb content.
We tested for the homogeneity of enzyme activities within each transverse section of the epaxial muscles using three comparisons. Due to variations in size of the seals and the epaxial muscles, tissue cores within each of the transverse sections were not taken at identical locations among animals. Clustering data was the best way to determine statistical variation within the muscle. First, each section was divided into roughly four equal quadrants (dorsal, ventral, right, and left) (Fig. 2), and mean enzyme activities were calculated for each quadrant. There were no significant differences among the quadrants within any of the transverse sections for CS, HOAD, or LDH activities (Fig. 3). Second, each transverse section was divided into four approximately equal quadrants along a vertical and horizontal plane (dorsal right, dorsal left, ventral right, and ventral left). Again, there were no significant differences among the quadrants within any of the transverse sections for the three enzymes. Finally, the transverse sections were divided into thirds along the horizontal axis, and no significant differences were found among the three regions. Although low levels of heterogeneity within the transverse sections of the epaxial muscles were visually apparent in contour maps of the activities of CS, HOAD, and LDH, no statistically significant pattern was detected because of interanimal variation. As a result, average enzyme activities were calculated for each transverse section.
There were significant differences in CS, HOAD, and LDH activities along the length of the epaxial muscles (Fig. 3). The average CS and LDH activities in the MID and CA sections were significantly greater (P < 0.001) than the CS and LDH activities in the CR section (Table 2). HOAD activity did not differ significantly among the three sections. The CS/HOAD and LDH/CS did not differ significantly along the length of the epaxial muscles (Table 2).
Average enzyme activities for the epaxial muscles were compared with activities in the hindlimb complex, pectoralis muscle, and heart in the harbor seals (Table 3). For CS activity, the heart was the only tissue that was significantly different (P < 0.01). The heart had 2.6–3.0 times higher CS activity than did the epaxial muscles, hindlimb, and pectoralis. HOAD activities in the hindlimb complex and the pectoralis muscle were significantly less (∼31%) than in the epaxial muscles (P < 0.001), but did not differ significantly in the heart and epaxial muscles. LDH activity was significantly lower in the seal heart than in the epaxial muscles (P < 0.01). There were no significant differences in LDH activities among the other tissues. The CS/HOAD in the epaxial muscles was significantly lower than in the other muscles and heart in the harbor seal (P < 0.001) (Table 3), indicating a high potential for oxidative fat metabolism. The harbor seal heart had a significantly lower LDH/CS than was found in the epaxial muscles. No significant difference was seen in the LDH/CS among the other muscles in the harbor seal.
Interspecific comparisons were made for muscles of similar function (Table 3). Both the seal and the dog locomotory and nonlocomotory muscles and heart tissue did not differ significantly in CS activity, but both had significantly lower CS than the rat heart (P < 0.01). The seal had significantly higher HOAD activity in the epaxial muscles than in the locomotory muscles of the dog (P < 0.01). However, HOAD activity in the locomotory hindlimb complex of the seal did not differ significantly from that of the locomotory muscles of the dog. All three species had similar HOAD activity in the heart. The seal had higher LDH activity than the dog in both the locomotory and nonlocomotory muscles (P < 0.01), but similar LDH activity in the heart. The rat heart had significantly higher LDH activity than the seal and dog hearts (P < 0.01).
The CS/HOAD was significantly lower in the seal epaxial muscles and the hindlimb complex than in the locomotory muscles of the dog (P < 0.01) (Table 3). There was no significant difference in the CS/HOAD between the seal pectoralis and dog intercostal muscles. For the heart, the CS/HOAD did not differ interspecifically. The seal locomotory and nonlocomotory muscles had significantly higher LDH/CS than did those of the dog (P < 0.01) (Table 3). The seal nonlocomotory muscles had significantly higher LDH/CS than the dog nonlocomotory muscles. No significant difference was seen in the LDH/CS for the heart of the three species.
To adjust for allometric differences due to body mass, we scaled the enzyme activities to the estimated tissue-specific RMR for each species (Table 4; Figs. 4, 5, and 6). There were no significant interspecific differences in the RMR ratios for the heart of the three species. There were no significant interspecific differences in the CS/RMR or the HOAD/RMR for the nonlocomotory muscles. The seal locomotor muscle had a significantly higher (∼6 times) HOAD/RMR than the dog gastrocnemius muscle (P < 0.001). The seal had significantly higher LDH/RMR than did the dog for both the locomotory and nonlocomotor muscles (∼3 times) (P < 0.001).
Similar to the enzyme activities, there were no statistically significant differences in the distribution of Mb concentrations within each of the transverse sections of the seal epaxial muscles, although heterogeneity was visually apparent on some contour maps (Fig. 7). However, there were significant differences along the length of the epaxial muscles (Fig. 7). The average Mb concentrations for the MID and CA sections were significantly greater (P = 0.001) than that of the CR section (Table 2). The epaxial muscles had a significantly higher Mb concentration (P < 0.001) than did the hindlimb complex and the pectoralis. Average Mb concentrations for all seal muscles were more than 20 times those measured in the dog muscles (Table 3). The seal heart Mb concentration was 10 times greater than that in the dog and rat heart.
No consistent patterns of distribution were evident in the activities of CS, HOAD, and LDH or the concentration of Mb within the CR, MID, and CA transverse sections of the seal epaxial muscles. These results are consistent with the homogeneous distribution of slow (type I)- and fast (type IIa)-twitch aerobic fibers within the respective transverse sections of the epaxial muscles (35). In contrast, there were significant differences in enzyme activities and Mb concentration among the transverse sections of the epaxial muscles that may be correlated with power production during swimming. Seals generate thrust with their hind flippers by contralateral contraction of the epaxial muscles that cause lateral flexions in the posterior half of their body (8). Because the CR section of the epaxial muscles is furthest from the hind flippers, the lower CS and LDH activities and Mb concentration in this region may reflect less of a contribution of the CR epaxial muscles to the generation of thrust during submerged swimming. In a study of four species of dolphins, Polasek and Davis (24) found that the highest concentrations of Mb were located in the CA region of the epaxial muscles where most of the thrust is generated during swimming (7, 22). However, the CR-CA gradient of Mb concentration in harbor seals in this study was not as pronounced as that in the faster swimming dolphins. It appears that the capacity for aerobic metabolism, oxygen storage, and glycolytic metabolism in the epaxial muscles reflects their potential contribution to thrust generation during submerged swimming.
Comparisons were also made among the epaxial muscles, the hindlimb complex, and pectoralis muscle of the harbor seals to test for differences between locomotory and nonlocomotory muscles. Although there were no significant differences in CS and LDH activities, HOAD activity and Mb concentration were significantly higher in the epaxial muscles than in the hindlimb complex and the pectoralis muscle. Kanatous et al. (16) observed similar results for the epaxial and pectoralis muscles in the harbor seal. In addition, the harbor seal heart had higher CS activity and lower LDH activity than the epaxial muscles, reflecting a higher aerobic potential. Seals use anaerobic ATP production in muscles only when they exceed the aerobic dive limit (ADL). Within the ADL, they rely on lipid-based aerobic metabolism and use oxygen stores in the muscle and blood. LDH becomes increasingly important when they exceed their ADL. Therefore, in seals, the same muscle needs to be able to use aerobic metabolic pathways for ATP production as effectively as possible within the ADL but also be capable of switching to anaerobic metabolism during prolonged dives. The similarity in aerobic and anaerobic capacities (as measured by CS and LDH activities, respectively) in the epaxial and pectoralis muscles reflects a parallel degree of adaptation for sustained aerobic metabolism under hypoxic conditions, as well as enhanced anaerobic metabolism under anoxic conditions. However, there was a higher capacity to provide acetyl-CoA from fatty acids and to store oxygen in the epaxial muscles than in the pectoralis and hindlimb muscles, which, we hypothesize, reflects their greater reliance on intracellular or intramuscular oxygen and fuel for locomotion during diving (due to vasoconstriction or reduction in blood flow to the muscles). A concurrent study on the same animals by Fuson et al. (9) also suggests that the liver, kidney, and stomach possess higher capacities for fat metabolism, which further supports the hypotheses for enhanced aerobic metabolism in the harbor seal.
After scaling for tissue RMR, we found no significant difference between the CS/RMR in the seal epaxial muscles and the dog gastrocnemius muscle. An enhanced oxidative capacity has been observed in animal athletes, such as dogs, and in high-altitude-adapted and acclimated mammals (1, 10, 14, 15, 20, 28). Kanatous et al. (16) found a greater Vv(mt) and a more homogeneous distribution of mitochondria within the muscle fibers of harbor seals than in those of terrestrial mammals; together, these characteristics reduce the distance for the intracellular diffusion of Mb-bound oxygen to mitochondria. These adaptations, along with the high-Mb concentration, sustain aerobic metabolism in the muscle during diving as the arterial partial pressure of oxygen declines to as low as 22 Torr (6, 26).
The HOAD/RMR in the seal epaxial muscles was six times greater than in the dog, suggesting that seal muscle has a higher capacity to oxidize fatty acids as a source of energy. This is further supported by the lower CS/HOAD (1.2) in the seal epaxial muscles, compared with the ratio (7.2) in the dog locomotory muscles. Similar results have been reported for harbor seals and other seal species (27). Moreover, metabolic studies on seals have found respiratory exchange ratios (CO2 produced/O2 consumed) of 0.75 at rest and 0.71 during exercise, which indicates that fat is the main source of fuel at rest and during submerged swimming (4). This dependence on lipid as an energy source in seals is probably related to their diet, which is rich in fat and protein but contains little carbohydrate (4, 18, 30). Studies of terrestrial mammals have shown that high-fat, low-carbohydrate diets increase the rate of lipid oxidation (30) and that this is accompanied by an increase in the concentration of enzymes involved in fatty acid oxidation in skeletal muscles (11, 30). A greater reliance on fatty acid oxidation within the locomotory muscles would also spare glucose for red blood cells and the central nervous system, which are obligate glucose metabolizers. Similar adaptations that spare glycogen and enhance lipid oxidation have been found in endurance-trained terrestrial mammals (1).
When scaled to RMR, LDH activity was greater in all of the skeletal muscles sampled in the seal than in the dog muscles. LDH activity is usually interpreted as an index of tissue anaerobic capacity, because it is required for redox balance during anaerobic ATP production in vertebrate muscle (by catalyzing the conversion of pyruvate to lactate). Elevated LDH levels, as well as the higher LDH/CS in all of the muscles of the seal compared with the dog, may support a parallel adaptation for both sustained aerobic metabolism for dives within the ADL and, when needed, an enhanced ability to produce ATP anaerobically for the occasional dive that exceeds the ADL, but this needs further investigation.
In conclusion, the greater CS and LDH enzyme activities and Mb concentrations measured in the MID and CA regions of the seal epaxial muscles are consistent with differences in the oxygen consumption and work performed by the locomotory muscles, during a dive when oxygen is limited. Similar to animal athletes such as the dog and pony (33, 36), harbor seals have high-oxidative enzyme activities that are an indicator of enhanced aerobic capacity. This enhanced aerobic capacity appears to be an adaptation for sustained, low-level metabolism under hypoxic conditions during diving rather than for high levels of exertion. The epaxial muscles of the harbor seal have high HOAD/RMR and relatively low CS/HOAD, which, taken together, suggest a substantial capacity for aerobic lipid metabolism during diving. Relative to the dog, the LDH/RMR in the seal muscle is elevated. Hence, the muscles of harbor seals exhibit adaptations that promote an aerobic, lipid-based metabolism under hypoxic conditions, but can provide ATP anaerobically, if required.
This study was conducted under Marine Mammal Permit No. 1021.
The research described in this paper was supported by the Exxon Valdez Oil Spill Trustee Council. However, the findings and conclusions presented by the authors are their own and do not necessarily reflect the views or position of the Trustee Council.
We thank the Alaska Native Harbor Seal Commission and F. Weltz for assistance in obtaining tissue samples. We also thank C. Ball, B. Dawe, and R. Prior for assistance with sample preparation.
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.
- Copyright © 2006 the American Physiological Society