High aerobic capacities in the skeletal muscles of pinnipeds: adaptations to diving hypoxia SHANE B. KANATOUS,1 LEONARD V. DIMICHELE,2 DANIEL F. COWAN,3 AND RANDALL W. DAVIS2 1Department of Medicine, University of California at San Diego, La Jolla, California, 92092-0623A; 2Department of Wildlife and Fisheries Science, Texas A & M University, College Station, Texas 77845; and 3Department of Pathology, University of Texas Medical Branch, Galveston, Texas 77555-0588 Kanatous, Shane B., Leonard V. DiMichele, Daniel F. Cowan, and Randall W. Davis. High aerobic capacities in the skeletal muscles of pinnipeds: adaptations to diving hypoxia. J. Appl. Physiol. 86(4): 1247–1256, 1999.—The objective was to assess the aerobic capacity of skeletal muscles in pinnipeds. Samples of swimming and nonswimming muscles were collected from Steller sea lions (Eumetopias jubatus, n 5 27), Northern fur seals (Callorhinus ursinus, n 5 5), and harbor seals (Phoca vitulina, n 5 37) by using a needle biopsy technique. Samples were either immediately fixed in 2% glutaraldehyde or frozen in liquid nitrogen. The volume density of mitochondria, myoglobin concentration, citrate synthase activity, and b-hydroxyacyl-CoA dehydrogenase was determined for all samples. The swimming muscles of seals had an average total mitochondrial volume density per volume of fiber of 9.7%. The swimming muscles of sea lions and fur seals had average mitochondrial volume densities of 6.2 and 8.8%, respectively. These values were 1.7- to 2.0-fold greater than in the nonswimming muscles. Myoglobin concentration, citrate synthase activity, and b-hydroxyacyl-CoA dehydrogenase were 1.1- to 2.3-fold greater in the swimming vs. nonswimming muscles. The swimming muscles of pinnipeds appear to be adapted for aerobic lipid metabolism under the hypoxic conditions that occur during diving. mitochondria; citrate synthase; b-hydroxyacyl-coenzyme A dehydrogenase; myoglobin of laboratory simulated-dive studies (8), it was first believed that marine mammals relied on an enhanced anaerobic capacity to maintain ATP synthesis under the hypoxic conditions of diving. It was later shown that marine mammals did not possess unusually high anaerobic enzyme activities compared with terrestrial mammals (3). With the development of dive recorders, information became available on the free-ranging diving behavior of marine mammals, and it was found that the majority of dives remained within the aerobic dive limit of the animals. The aerobic dive limit is defined as the longest breath-hold dive that is possible without an increase in lactic acid concentration in the blood during or after the dive. During these dives, marine mammals appear to maintain an overall low metabolic rate and rely principally on oxygen stored in ON THE BASIS 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. http://www.jap.org the blood and muscles in order to maintain aerobic metabolism and physiological homeostasis (25). In terrestrial mammals, the functional capacity of the respiratory pathway to deliver oxygen is matched to the oxygen requirements of the tissues (15, 18, 29). For example, the large aerobic scopes of athletic animals, such as dogs and racehorses, result from both high mitochondrial volume densities in skeletal muscles and cardiorespiratory adaptations that enhance convective and diffusive oxygen transport (18, 24). Unlike terrestrial mammals that increase ventilation and cardiac output during exercise, marine mammals stop breathing, reduce cardiac output, and limit peripheral blood flow during diving. This dive response (i.e., apnea, bradycardia, and peripheral vasoconstriction) reduces convective oxygen transport to muscles resulting in tissue hypoxia (8) and appears to limit aerobic scope (6). However, studies on the free-ranging diving behavior of marine mammals indicate that they still maintain aerobic metabolism during the majority of their dives (6, 13, 25). How, then, do the skeletal muscles of pinnipeds maintain aerobic metabolism under such adverse conditions? Analogies have been drawn between high-altitudeadapted animals and marine mammals in terms of possible adaptations in skeletal muscles to maintain aerobic metabolism under hypoxic conditions (12). Unlike high-altitude animals that function under hypoxic conditions but display the typical exercise response of increasing ventilation and cardiac output, marine mammals exercise under a different form of hypoxic stress. They must function for the duration of a dive with only a finite amount of oxygen. A number of studies have postulated a possible downregulation of oxidative metabolism, to levels approximately one-half those found in terrestrial mammals of comparable size, to maintain oxidative metabolism under the hypoxic conditions associated with diving (12, 13). The objective of this study was to assess the aerobic capacity of pinniped skeletal muscle by measuring the volume density of mitochondria, the activity of key oxidative enzymes, and the concentration of myoglobin in swimming and nonswimming muscles. Our results indicate that pinniped skeletal muscles have an enhanced oxidative capacity to maintain aerobic lipid metabolism under the hypoxic conditions associated with diving and that these adaptations are more pronounced in swimming than in nonswimming muscles. 8750-7587/99 $5.00 Copyright r 1999 the American Physiological Society 1247 1248 HIGH AEROBIC CAPACITIES IN PINNIPED SKELETAL MUSCLE MATERIALS AND METHODS Animals. Muscle samples from healthy adult female Steller sea lions (Eumetopias jubatus; n 5 27, mean body mass 260 kg) and healthy adult harbor seals (Phoca vitulina; n 5 37, mean body mass 61 kg) were collected over a 3-yr period from central and southeast Alaska as part of ongoing studies conducted by the Alaska Department of Fish and Game and the National Marine Fisheries Service. Samples from male juvenile Northern fur seals (Callorhinus ursinus; n 5 5, estimated body mass 29 kg) were obtained immediately after death during a native subsistence hunt in the Pribilof Islands, AK. Biopsy procedure. The sea lions were darted with Telezol (2.0 mg/kg, tiletamine HCl and zolazepam HCl; Fort Dodge Laboratories, Fort Dodge, IA), intubated, and maintained with isoflourane anesthesia by using large-animal anesthesia equipment and monitoring techniques (11). The harbor seals were captured with entangling nets and sedated with a mixture of Ketalar (2.0 mg/kg ketamine HCl, Parke-Davis, Morris Plains, NJ) and Valium (0.1 mg/kg diazepam, Roche Products, Manati, PR). Two muscle samples of ,50 mg each were collected from a single biopsy site with a 6-mm biopsy cannula (Depuy, Warsaw, IN) from both the swimming and nonswimming muscles of both species. For the harbor seals, biopsies of swimming and nonswimming muscles were taken from the longissimus dorsi and pectoralis muscles, respectively (Fig. 1). Samples from the swimming and nonswimming muscles of sea lions and fur seals were taken from the pectoralis muscle and hindlimb complex, respectively. Control samples for electron microscopy were collected from the soleus muscle, a predominantly slow-twitch oxidative muscle, of laboratory rats (Sprague Dawley) that were killed by cervical dislocation after 2–3 min of carbon dioxide anesthesia. Control samples for citrate synthase (CS) and myoglobin assays were collected from the vastus medialis muscle of free-ranging cotton rats (Sigmodon hispidus) that were killed by 2- to 4-min exposure to methane gas. The cotton rats were trapped as part of a study to assess the distribution and health status of small mammals in the wetlands of Texas. Tissue preparation. Muscle samples were either placed into 2% glutaraldehyde and 2% paraformaldehyde fixative in 0.1 M cacodylate buffer or were frozen in liquid nitrogen immediately after they were collected. Samples remained in the fixative for 2–14 days before being transferred and stored in 0.1 M cacodylate buffer, pH 7.4, for 21–90 days. Frozen samples were stored in liquid nitrogen until they were returned to Texas A & M University and then stored at 270°C until analysis for CS activity and myoglobin concentration. Electron microscopy. Fixed muscle samples were rinsed in 0.1 M cacodylate buffer and postfixed for 2 h in a 1% solution of osmium tetroxide. They were stained en bloc with 2% uranyl acetate overnight at 5°C. After dehydration with increasing concentrations of ethanol (50–100%), they were passed through propylene oxide and increasing concentrations of epoxy (50–100%). The samples were finally embedded in fresh epoxy and allowed to polymerize overnight at 60°C. Semithick sections (1 µm) were cut with a Leica Ultratome and stained with toluidine blue to check for fiber orientation. Ultrathin (50–70 nm) transverse sections from four randomly chosen blocks per muscle were cut and contrasted with lead citrate. Micrographs were taken with a Phillips 201 transmission electron microscope. The number of micrographs per muscle that were analyzed was between 25 and 40, yielding relative SE of total mitochondrial volume density [Vv(mt,f)] estimates of ,10% in all muscles. Determination of the volume densities per volume of muscle fiber of mitochondria, Fig. 1. Skeletal muscle anatomy of seals and sea lions. Biopsy samples of swimming muscles were obtained from longissimus dorsi in harbor seals and pectoralis in fur seals and sea lions. Samples of nonswimming muscles were collected from hindlimb complex in fur seals and seal lions and from pectoralis in harbor seals. [Redrawn from A. B. Howell (Ref. 20a) by M. Cooley.] myofibrils [Vv(fi,f)], and lipid droplets [Vv(li,f)] were performed by using standard point-counting procedures (18, 29). Enzyme and myoglobin assays. Frozen muscle samples were thawed, blotted, weighed, and immediately homogenized at 0°C in 1 ml of buffer containing (in mM): 1 EDTA, 2 MgCl2, and 75 Tris · HCl, pH 7.6, at 25°C (33). The homogenates were spun at 2,900 g for 30 min at 4°C. A 500-ml aliquot from the supernatant was prepared for myoglobin assays, and the rest was used for the CS and b-hydroxyacyl-CoA dehydrogenase (HAD) assays. CS was assayed on a Beckman DU series 64 spectrophotometer according to the method of Reed et al. (33). Assay temperature was maintained at 37°C by using a constant-temperature water bath and a waterjacketed cuvette holder. The assay conditions for CS (EC 4.1.3.7) were (in mM): 50 imidazole, 0.25 5,58-dithiobis(2nitrobenzoic acid), 0.4 acetyl CoA, and 0.5 oxaloacetate, at pH 7.5; DA412, e412 5 13.6, where DA indicates absorbance HIGH AEROBIC CAPACITIES IN PINNIPED SKELETAL MUSCLE wavelength and e is exinction coefficient. Assay conditions for HAD (EC 1.1.1.35) were (in mM): 50 imidazole, 1 EDTA, 0.1 acetoacetyl CoA, and 0.15 NADH, pH 7.0, at 37°C; DA340, e340 5 6.22 (33). Enzyme activities, in micromoles per min per gram wet mass muscle, were calculated from the rate of change in absorbance at the maximum linear slope. Myoglobin was assayed according to the method of Reynafarje (34), with the following modifications. A portion (500 µl) of the homogenate was further diluted with 1 ml of phosphate buffer (0.04 M, pH 6.6). The resulting mixture was centrifuged for 50 min at 28,000 g at 4°C. The supernatant was bubbled with carbon monoxide for 3 min. Spectrophotometric absorbance (Abs) was measured at 538 and 568 nm, and the concentration of myoglobin, in milligrams per gram wet mass of muscle, was calculated as (Abs538 2 Abs568) 3 5.865[(1.5/0.5) 3 (mass of sample)] Statistical analysis. Results are expressed as means 6 SE. Statistical comparisons were made between species by using an ANOVA (Bonferroni-adjusted P value # 0.01), and comparisons between swimming and nonswimming muscles within a species were made by paired sample t-test (P # 0.05). Correlations between oxidative enzymes and the volume density of mitochondria were determined, and significance was assessed at P # 0.05. RESULTS Skeletal muscle morphology. The general morphology of the skeletal muscle fibers was similar among the three species of pinnipeds and was typical for mammals (Fig. 2). Electron micrographs of transverse sections showed irregularly shaped myofibrils separated by sarcoplasmic reticulum, mitochondria, and lipid droplets. The mean volume density of myofibrils per volume of muscle fiber was not significantly different between the swimming (76.2 6 1.8 to 81.4 6 2.0%) and nonswimming muscles (79.5 6 1.7 to 82.1 6 2.4%) in all three species (Table 1). In each of the three species, the volume densities of total mitochondria (interfibrillar and subsarcolemmal) per volume of muscle fiber were significantly greater (1.7- to 2.0-fold) in swimming than in nonswimming muscles (Table 1). Most of the mitochondria (mean, 95 6 0.67%; range, 93–96.8%) were interfibrillar, and the remainder were subsarcolemmal. Harbor seals and fur seals had significantly higher Vv(mt,f) in their swimming and nonswimming muscles than did Steller sea lions (ANOVA, P 0.01; Table 1). Roughly spherical lipid droplets ,0.25 µm in diameter were distributed in the interfibrillar space. The Vv(li,f) for both the swimming and nonswimming muscles was generally ,1% (Table 1). The swimming muscles of fur seals had a significantly greater Vv(li,f) than did nonswimming muscles, whereas there were no significant differences between the swimming and nonswimming muscles of harbor seals and Steller sea lions. The Vv(li,f) in the swimming muscle of the fur seals was significantly greater than in the muscles of the harbor seals and Steller sea lions. Oxidative enzymes. CS activities were 1.1–1.5 times greater in the swimming muscles than in the nonswimming muscles and were significantly correlated (P , 0.001) to the Vv(mt,f) in all three species (Fig. 3A). 1249 Differences between swimming and nonswimming muscles were significant in the Steller sea lions and the Northern fur seals (paired sample t-test, P value # 0.05). The activity of CS was significantly greater in the swimming muscles of the harbor seals and Northern fur seals compared with the swimming muscle of the Steller sea lions and the vastus medialis of the cotton rat (ANOVA, P # 0.01; Table 2). The activities of HAD were 1.0–1.3 times greater in the swimming muscles than in the nonswimming muscles and were also significantly correlated (P , 0.001) to the volume density of mitochondria in all three species (Fig. 3B). HAD values were significantly different in the harbor seals and Steller sea lions (ANOVA, P # 0.05). They were approximately twofold greater in the swimming muscles of the harbor seals and Northern fur seals compared with the Steller sea lions, and they were significantly greater in the muscles of all pinnipeds compared with the vastus medialis of the cotton rat (ANOVA, P # 0.01; Table 2). The CS/HAD ratio was calculated as an index of the amount of total aerobic metabolism supported by the oxidation of fatty acids. This ratio ranged from 0.59 to 0.97 and was similar between all three species of pinnipeds for both swimming and nonswimming muscles (Table 2). These ratios were two- to threefold lower than in the rat. Myoglobin. The concentration of myoglobin was significantly greater in the swimming muscles than in the nonswimming muscles in all three species of pinnipeds (paired samples t-test; P # 0.05). The values in the swimming muscles were significantly greater in the harbor seals than in the Northern fur seals and Steller sea lions (ANOVA, P # 0.01). The concentration of myoglobin in the muscles of the pinnipeds was 10- to 22-fold greater than those measured in the vastus medialis of the cotton rat (Table 3). DISCUSSION The Vv(fi,f) is inversely related to the oxidative capacity of the skeletal muscles: the higher the oxidative capacity of the muscle, the lower the Vv(fi,f) (17, 18). Our results for pinnipeds ranged from 76.2 to 82.1%. They are similar to the value of ,80% reported in the locomotory muscles of athletic terrestrial animals (17). The Vv(mt,f) in pinniped swimming muscles was 1.7- to 2.2-fold greater than the predicted values for terrestrial mammals of comparable size but was similar to that in athletic dogs and ponies (Fig. 4). However, when one compares the volume density of interfibrillar mitochondria [Vv(mi,f)] in pinniped swimming muscles with that in the locomotory muscles of athletic terrestrial animals of similar size, values in the swimming muscles were greater (Table 4). In pinnipeds’ muscles, ,7% of their total mitochondrial volume was located near the sarcolemma. In contrast, ,28% of the total mitochondria in athletic terrestrial mammals are subsarcolemmal (24). Subsarcolemmal mitochondria are thought to play an important role in reducing the diffusion distance for blood-borne substrates and oxygen in terrestrial mammals (5). The increased 1250 HIGH AEROBIC CAPACITIES IN PINNIPED SKELETAL MUSCLE Fig. 2. Representative electron micrographs of swimming muscles of different species. A: harbor seal; B: Steller sea lion; C: Northern fur seal. M, mitochondria; L, lipid droplet; F, myofibril. Vv(mi,f) and low volume density of subsarcolemmal mitochondria [Vv(ms,f)] in the skeletal muscles of pinnipeds may reflect their reduced reliance on bloodborne oxygen and fuel substrates during diving due to the reduced peripheral perfusion brought about by their natural dive response. It is also interesting to note that a preferential increase in the Vv(mi,f) is also found in muscles with increased activity under hypoxic condi- 1251 HIGH AEROBIC CAPACITIES IN PINNIPED SKELETAL MUSCLE Table 1. Summary data for the electron microscopy of swimming and nonswimming muscles of harbor seals, Steller sea lions, and Northern fur seals Species Harbor seals (Phoca vitulina) Mass, kg 61 6 5 Volume Density, % n Muscle Vv(mt,f ) Vv(mi,f ) Vv(ms,f ) Vv(fi,f ) Vv(li,f ) 10 10 Swimming (longissimus) Nonswimming (pectoralis) 9.7 6 0.5‡ 5.7 6 0.4†‡ 9.1 6 0.3‡ 5.3 6 0.3† 0.6 6 0.3 0.4 6 0.2 76.2 6 1.8‡ 79.5 6 1.7 0.13 6 0.07 0.08 6 0.06 1.7 1.7 1.5 0.96 1.6 6.2 6 0.5 3.1 6 0.4† 6.0 6 0.4 2.9 6 0.4† 0.2 6 0.1 0.2 6 0.1 78.9 6 1.6 81.5 6 1.1 0.10 6 0.05 0.03 6 0.01 2.0 2.1 1.0 0.96 3.3 8.8 6 0.5‡ 4.4 6 0.3†‡ 8.5 6 0.5‡ 4.2 6 0.3† 0.3 6 0.2 0.2 6 0.2 81.4 6 2.0 82.1 6 2.4 1.0 6 0.20§ 0.2 6 0.06† 2.0 2.0 1.5 0.99 3.0 Ratio of swimming to nonswimming Steller sea lions (Eumetopias jubatus) 240 6 10 14 13 Swimming (pectoralis) Nonswimming (hindlimb) Ratio of swimming to nonswimming Northern fur seals (Callorhinus ursinus) 29* 5 5 Swimming (pectoralis) Nonswimming (hindlimb) Ratio of swimming to nonswimming Values are means 6 SE; n, no. of animals. Vv(mt,f ), volume density of total mitochondria; Vv(mi,f ), volume density of interfibrillar mitochondria; Vv(ms,f ), volume density of subsarcolemmal mitochondria; Vv (fi,f ), volume density of myofibrils; and Vv(li,f ), volume density of intracellular lipid droplets; all quantities expressed per fiber volume. Soleus of the rat (Sprague-Dawley; n 5 5) was used as control [Vv(mt,f ) 4.5 6 0.2] and agreed with previously published values (30). * Estimated mass from Baker et al. (1). † Significantly different from swimming muscle, P # 0.05. ‡ Significantly different from Steller sea lion, P # 0.01. § Significantly different from Steller sea lion and harbor seal, P , 0.01. tions (4, 16, 30), in contrast to training in normoxia in which a greater increase occurs in the Vv(ms,f) (30). It has been found that Vv(mt,f) scales in direct proportion to the capillary density in skeletal muscle (29). On the basis of this relationship, the swimming muscles should have capillary densities (800–1,000 capillaries/mm2 ) comparable to those measured in athletic terrestrial mammals of similar size (5, 23). Capillary densities in the swimming muscles in pinnipeds have only been measured in a limited number of samples from harbor seals, grey seals, and Antarctic fur seals (33). Reed et al. (33) found values ranging from 352 to 639 capillaries/mm2 in harbor seals and Antarctic fur seals, respectively. These values are lower than would be predicted from the regression of Conley (5) if these animals had similar Vv(mt,f) to those of the harbor seals and Northern fur seals measured in this study. Capillary density may not scale in direct proportion to the total density of mitochondria, because pinnipeds reduce cardiac output and peripheral blood flow during diving. All of the harbor seals and Steller sea lions sampled in this study were adult animals, whereas the Northern fur seals were 2-yr-old juveniles. Previous research has found that there are no significant differences in hemoglobin concentrations, hematocrit, red blood cell concentrations, or muscle myoglobin concentrations between elephant seals (Mirounga angustirostris) and Galapagos fur seals (Arctocephalus galapagoenis) older than 6 mo and adult animals (19). However, the pinnipeds examined in this study use different modes of underwater propulsion and vary greatly in body mass (29–240 kg). Harbor seals swim by using alternate lateral sweeps of their hind flippers, with thrust generated by the muscles of the back (e.g., longissimus dorsi muscle; Ref. 33). In contrast, sea lions and fur seals use their fore flippers as paddles and generate thrust with the chest (pectoralis muscle) and shoulder muscles (e.g., deltoideus muscle; Ref. 33). To correct the volume densities of mitochondria for differences in metabolic rate due to body mass, the absolute volume of mitochondria was calculated for the swimming muscles (2, 18) and divided by the animal’s body mass0.75 (28) by using the following equation 5(M)(Ms)[Vv(mt,f)/Dmus]/M 0.756 where M represents body mass in kilograms, Ms represents the percent of total body mass that is swimming muscle, and Dmus is the density of muscle. Despite differences in the mode of propulsion, there were no significant differences in the body-mass-adjusted mitochondrial volume densities of swimming muscles among harbor seals, fur seals, or sea lions (16.5 6 0.5, 13.1 6 0.8, and 15.5 6 1.1 ml of mitochondria/kg0.75, respectively). In other words, the mitochondrial volume density in the propulsive muscles appeared to be independent of swimming mode. The activities of oxidative enzymes were measured as indicators of total aerobic capacity. As in terrestrial animals, the enzyme activities were found to be significantly correlated to the Vv(mt,f) (Fig. 3). The values obtained in pinnipeds were higher than those previously reported for the same species (3, 13, 33), whereas the values for our control agreed well with previously published values (33). The differences in the enzyme activities between the present study and previous studies may have resulted from a different collection procedure. We collected samples from live animals or from animals immediately after death (i.e., 10–15 min postmortem) and placed them directly into liquid nitrogen. The immediate freezing of the samples should have prevented any degradation of the samples associated with other freezing techniques. The activities of aerobic enzymes such as CS normally scale inversely 1252 HIGH AEROBIC CAPACITIES IN PINNIPED SKELETAL MUSCLE Fig. 3. A: citrate synthase activity is significantly correlated to volume density of mitochondria between species (Pearson correlation, r 5 0.71; P , 0.001). Values for emperor penguins (not included in correlation analysis) are from Ponganis et al. (32). B: b-hydroxyacyl-CoA dehydrogenase (HAD) activity is significantly correlated to volume density of total mitochondria [Vv(mt,f)] between species (Pearson correlation, r 5 0.68; P , 0.001). Table 2. Enzyme activities of citrate synthase (CS) and b-hydroxyacyl-CoA dehydrogenase (HAD) in swimming and nonswimming muscles of harbor seals, Steller sea lions, and Northern fur seals Species Harbor seals Mass, kg CS Activity, IU/g wet wt muscle HAD, IU/g wet wt muscle CS/HAD Ratio Swimming (longissimus dorsi) Nonswimming (pectoralis) 37.0 6 1.7§ 34.1 6 1.7§ 1.1 51.9 6 2.4§ 40.5 6 2.5†§ 1.3 0.71 0.84 27 27 Swimming (pectoralis) Nonswimming (hindlimb) 21.9 6 1.2‡ 15.0 6 1.0† 1.5 22.5 6 1.3‡§ 18.0 6 1.0† 1.3 0.97 0.83 5 5 Swimming (pectoralis) Nonswimming (hindlimb) 39.3 6 2.9§ 28.3 6 2.8† 1.4 47.5 6 7.0§ 48.0 6 8.3§ 1.0 0.83 0.59 5 Vastus medialis 24.0 6 1.4 11.9 6 1.7 2.0 6 0.3 n Muscle 61 6 5 37 37 260 6 8 29* Ratio of swimming to nonswimming Steller sea lions Ratio of swimming to nonswimming Northern fur seals Ratio of swimming to nonswimming Cotton rat (Sigmodon hispidus) 0.107 formed · min21 · g Values are expressed as means 6 SE. CS and HAD activities are expressed as µmol product wet wt * Estimated body mass from Baker et al. (1). † Significantly different from swimming muscles of seals (paired sample t-test, P # 0.05). ‡ Significantly different from harbor seal and fur seal (ANOVA, P # 0.01). § Significantly different from rat (ANOVA, P # 0.01). muscle21. 1253 HIGH AEROBIC CAPACITIES IN PINNIPED SKELETAL MUSCLE Table 3. Concentration of myoglobin in swimming and nonswimming muscles of harbor seals, Steller sea lions, and Northern fur seals Species Harbor seals n Mass, kg 37 37 61 6 5 27 27 260 6 10 Muscle Swimming (longissimus dorsi) Nonswimming (pectoralis) 37.4 6 1.7§ 24.1 6 0.8†§ 1.6 Swimming (pectoralis) Nonswimming (hindlimb) 28.7 6 1.5§ 20.0 6 1.3†§ 1.4 Swimming (pectoralis) Nonswimming (hindlimb) 22.4 6 2.5§ 16.0 6 0.9§ 1.4 Ratio of swimming to nonswimming Steller sea lions Ratio of swimming to nonswimming Northern fur seals 5 5 29* Ratio of swimming to nonswimming Cotton rat 5 0.107 Myoglobin Concentration, mg/g wet wt muscle Vastus medialis 1.7 6 0.2 Values are means 6 SE. * Estimated body mass from Baker et al. (1). † Significantly different from swimming muscles in seals (paired sample t-test, P # 0.05). § Significantly different from rat, P # 0.01. with body mass (14) unless a species exhibits some adaptive deviation. The activities of CS in the swimming muscles were either similar (Steller sea lions) or significantly greater (harbor seals and fur seals) than those measured in the rat, whereas HAD activities were significantly greater in the muscles of all pinnipeds compared with our control. Because the pinnipeds were three to four orders of magnitude greater in mass than the control animal, these results indicate an adaptive increase in aerobic enzyme capacity in the swimming muscles compared with locomotory muscles from terrestrial mammals. The correlations between the oxidative enzymes and the volume density of mitochondria (Fig. 3) are higher than those found in terrestrial mammals (20). This might indicate a difference in the mitochondria between terrestrial and marine mammal muscle. This Fig. 4. Percent Vv(mt,f) of locomotory muscles in relation to body mass. Regression line (y 5 6.75 2 1.34 [log x], r2 5 0.70) shows relationship for vastus medialis, a primary locomotory muscle, for a wide variety of terrestrial mammals ranging in size from 0.04 kg (dwarf mongoose, Helogale pervula) to 450 kg (steer, Bos taurus) (24, 29). Dashed lines, 99% confidence intervals for the regression. s, Pinnipeds in this study; r, terrestrial animal athletes (17). %Volume densities of mitochondria in swimming muscles of sea lions, fur seals, and seals were 1.7, 1.9, and 2.1 times greater, respectively, than values predicted on the basis of their body size. %Vv(mt,f) in vastus medialis of dog (Canis familiaris, 10.7%) and pony (Equus caballus, 6.8%) were 2.2 and 1.8 times greater, respectively, than predicted values and were similar to those of comparably sized pinnipeds. would contrast with the idea that all mitochondria are the same regardless of the muscle type or species (35). However, recent studies have found differences in the respiratory rate of mitochondria isolated from different species [i.e., hummingbirds (Selasphorus rufus); Ref. 37] or fiber types (21). In addition, data from the swimming muscle of another diving animal, the emperor penguin (Aptenodytes forsteri), show an even greater CS activity for the volume density of mitochondria than was found in pinnipeds (Fig. 3) (32). This suggests that there may be differences between mitochondria from the muscles of diving species as compared with the muscles of terrestrial animals. In the skeletal muscles of pinnipeds, the CS/HAD ratios ranged from 0.59 to 0.97 and were substantially less than those observed in the vastus medialis of the rat (2.0 6 0.3). CS/HAD ratios of 1.25 to 2.6 have been reported in the gluteal muscle, a major locomotory muscle in horses and steers (22). The ratios measured in this study agree with previously published values for the longissimus muscle of the harbor seals (33) and indicate that essentially all of the aerobic metabolism of pinniped skeletal muscles could be supported by the oxidation of fatty acids. These findings agree with earlier studies that found respiratory quotients of pinnipeds to be ,0.74, thus indicating that fat was the primary fuel source during exercise (6). Available data on fiber types in pinniped swimming muscles indicate that they consist of a mixed composition of fiber types: slow oxidative, fast oxidative glycolytic, and fast glycolytic fibers (33). Similarly, we found a fiber type population distribution of 20% slow oxidative, 27% fast oxidative glycolytic, and 53% fast glycolytic in a single Steller sea lion (S. B. Kanatous, unpublished observations). Reed et al. (33) found that harbor seals have a higher oxidative capacity and greater proportion of slow oxidative fibers than do Antarctic fur seals. However, our data indicated that the swimming muscles of harbor seals and Northern fur seals had a greater aerobic capacity than did Steller sea lions, on the basis of the volume density of mitochondria and oxidative enzyme activities. 1254 HIGH AEROBIC CAPACITIES IN PINNIPED SKELETAL MUSCLE Table 4. Comparison of volume density of mitochondria in the locomotory muscles of pinnipeds and terrestrial animal athletes n Mass, kg Muscle Vv(mt,f ) Vv(mi,f ) Harbor seals Dog (Canus familiaris) Ratio of seals to dogs 10 3 61 28 Longissimus Vastus medialis 9.7 6 0.5 10.7† 0.91 9.1 6 0.2 7.3† 1.25 Stellar sea lion Pony (Equus caballus) Ratio of sea lions to ponies 14 3 240 175 Pectoralis Vastus medialis 6.2 6 0.5 6.8† 0.91 6.0 6 0.4 5.2† 1.2 Pectoralis Vastus medialis 8.8 6 0.5 10.7† 0.82 8.5 6 0.5 7.3† 1.2 Species Fur seals Dog Ratio of fur seals to dogs 5 3 29* 28 Values are means 6 SE; n, no. of animals. * Estimated body mass from Baker et al. (1). † Values from Hoppeler et al. (17). In terrestrial mammals, intracellular stores of oxygen are small, and the transport of oxygen from the capillaries to the mitochondria is critical to sustain oxidative phosphorylation during exercise. In contrast, intracellular stores of oxygen are substantially larger in marine mammals and provide an important source of oxygen for the mitochondria, as convective oxygen transport decreases during the dive (33). In addition, myoglobin plays an important role, not only as an oxygen source but also in the facilitated diffusion of oxygen throughout the muscle, especially under hypoxic conditions (10, 33, 39). The myoglobin concentrations measured in this study agree with those previously reported for harbor seals and fur seals; there are no published myoglobin values for Steller sea lions (3, 33). Like high-altitude-adapted mammals, the pinnipeds in this study have enhanced concentrations of myoglobin in their skeletal muscles compared with sea-level-adapted terrestrial mammals. The combination of an increased myoglobin concentration and volume density of mitochondria will have the apparent effect of increasing the intracellular diffusing capacity of the skeletal muscles (26). The Vv(li,f) per volume of muscle fiber in the pinnipeds was generally ,1%. The values obtained in the swimming muscles of harbor seals and Steller sea lions (0.13 6 0.07 and 0.1 6 0.05%, respectively) were approximately an order of magnitude greater than in the vastus medialis of comparably sized dogs and ponies (0.03 6 0.02 and 0.01 6 0.01%, respectively) (17). The mean value in fur seals was ,37 times greater than for a comparably size dog. Unlike the findings in fur seals, there were no differences in the Vv(li,f) between swimming and nonswimming muscles in harbor seals and Steller sea lions. Typically, locomotory muscles in terrestrial mammals have a greater store of energy than do nonlocomotory muscles (24). However, previous studies have found in the muscles of freeranging mammals considerable variation in the Vv(li,f) that was not related to either body size or behavior (29). Studies of harbor seals led to the hypothesis that endogenous stores of triglycerides may play an important role in fuel homeostasis, especially during diving (6). In terrestrial mammals, intramuscular stores of triglycerides act as a fuel reservoir when the rate of delivery of plasma free fatty acids is lower than the rate of utilization in the muscles, especially during submaximal exercise (36). Although the Vv(li,f) values in the skeletal muscles of Steller sea lions and harbor seals were an order of magnitude greater than in comparably sized terrestrial mammals, the level of Vv(li,f) was significantly less than in the swimming muscles of the juvenile Northern fur seals. In the Steller sea lions, this level may have resulted from samples being taken during the postpartum fast and lactation. Fasting has been found to decrease the content of intramuscular triglycerides in terrestrial mammals (36). The stress of parturition and fasting may have diminished the lipid droplet densities to levels less than would normally be seen in actively feeding animals, such as the juvenile Northern fur seals. However, the relatively low levels of lipid droplets observed in the harbor seals compared with the Northern fur seals were more difficult to understand. Samples were collected from both females and males, during the spring, early summer and fall, when it was believed the animals were actively feeding. However, the population of harbor seals in Prince William Sound, AK, has been declining since 1984 (9). This decline appears to be a non-density-dependent response, possibly attributed to a decreased carrying capacity of the environment due to food limitations (9). This may be a cause of the Vv(li,f) in the skeletal Table 5. Lipid stores in swimming muscles of harbor seals, Steller sea lions, and Northern fur seals Species Mass, kg n Muscle Vv(li,f ) Energy from Lipid, kJ/kg Harbor seals Steller sea lions Northern fur seals 61 6 5 260 6 10 29* 37 28 5 Swimming Swimming Swimming 0.2 6 0.1 0.1 6 0.0 1.1 6 0.3 64 32 321 Values are means 6 SE; n, no. of animals. Values for Vv(li,f ) are from Table 1. * Estimated body mass from Baker et al. (1). HIGH AEROBIC CAPACITIES IN PINNIPED SKELETAL MUSCLE muscles of the harbor seals compared with levels in the fur seals. How much energy is stored in the form of triglycerides in the skeletal muscles? The mass of triglycerides per kilogram of swimming muscle was estimated from the Vv(li,f) and then converted into kilojoules of energy per kilogram of swimming muscle (Table 5). The energy within the lipid droplets was estimated with the following assumptions: the lipid droplets consisted entirely of triglycerides, 94% of the triglycerides mass was fatty acids, and the density of the lipids was 0.92 (determined from cod liver oil). Based on these calculations, there were 321, 44, and 32 kJ of energy from lipid per kilogram swimming muscle in Northern fur seals, harbor seals, and Steller sea lions, respectively. If we assume a resting muscle metabolic rate similar to those measured in humans [,1.6 ml O2 · min21 · kg21 (33)], these endogenous stores of triglycerides would supply enough energy for 171, 22, and 17 h in the fur seals, harbor seals, and Steller sea lions at rest, respectively. Considering the low CS/HAD ratios, these results support the hypothesis that endogenous triglyceride stores in the muscles are an important source of energy in marine mammals. Because the aerobic scope of an organism is proportional to the volume density of mitochondria (17, 24), an apparent paradox is the low aerobic scope of pinnipeds relative to the elevated mitochondrial volume densities in their swimming muscles. The maximum aerobic scope measured for pinnipeds swimming in a water flume is approximately ninefold their resting metabolic rate (6), which is less than the 30-fold aerobic scope reported for dogs and horses with similar mitochondrial volume densities (17). The lower aerobic scope of pinnipeds may result from the smaller percentage of total muscle mass used for swimming compared with that used for terrestrial locomotion (2). In addition, the dive response, which decreases convective oxygen transport to muscles, may limit the aerobic scope to levels less than the observed maximum. The effects of hypoxia on muscle structure and function remain controversial. Studies of humans exposed to chronic hypoxia show significant reductions in muscle mass, volume densities of mitochondria, and oxidative capacity (4, 16, 20). In contrast, studies of birds and mammals active at high altitude, including humans trained under discontinuous hypoxia, display a vastly different response. Under these conditions, skeletal muscles show an increase in the volume density of mitochondria, with larger increases in interfibrillar than subsarcolemmal mitochondria, enhanced aerobic enzyme activities, and increased concentrations of myoglobin compared with sea level controls (8, 15, 16, 27, 30, 38). The results of this study are consistent with the findings of the second group of studies and suggest that the increased aerobic capacity in the skeletal muscles of pinnipeds is not an adaptation to hypoxia alone but an adaptation to activity under hypoxic conditions. In conclusion, the swimming muscles of pinnipeds appear to be adapted for aerobic lipid metabolism even under the hypoxic conditions that occur during diving. 1255 Our results are consistent with the hypothesis that increased mitochondrial volume densities, aerobic enzyme capacities, and myoglobin concentrations are adaptations that facilitate aerobic metabolism under hypoxic conditions. The high aerobic capacity of pinniped swimming muscles, as indicated by the high oxidative enzyme activities, volume densities of mitochondria, and myoglobin concentrations, may have less to do with increasing aerobic scope and more to do with maintaining oxygen flux through the respiratory pathway under hypoxic conditions. Because the mitochondrial uptake of oxygen under diving conditions is likely to be diffusion limited, the increased mitochondrial densities may serve to increase the oxygen sink and decrease the diffusional distance from extra- and intracellular oxygen stores. These results also indicate that the overall aerobic capacity of skeletal muscle is enhanced to a similar degree in mammals adapted to either low oxygen availability (high-altitude and marine mammals) or high oxygen demand (athletic terrestrial mammals). We thank the following for their support: Ukeavik Inupiat Corporation, Northern Arctic Research Laboratory, Arctic Research Foundation, J. Wen, V. Han, D. Calkins, K. Frost, L. Llowry, T. Loughlin, T. Spraker, D. Bradley, G. Antonelis, O. Mathieu-Costello, A. E. Bull, E. A. Brandon, T. C. Adams, K. D. Dudzinski, C. Hice, J. L. Respess, and T. D. Sparks. We also thank M. A. Castellini, W. E. Evans, M. Horning, O. Mathieu-Costello, C. Ribic, T. M. Williams, G. A. J. Worthy, and B. Wursig for their reviews and help with this manuscript. Special thanks are extended to Dr. G. A. Brooks and two anonymous reviewers whose comments vastly improved the manuscript. This work was supported by funds from the Alaska Department of Fish and Game, National Marine Mammal Lab, National Marine Fisheries Service, and Texas A & M University. Address for reprint requests and other correspondence: S. B. Kanatous, Dept. of Medicine 0623A, Univ. of California at San Diego, La Jolla, CA 92092-0623A (E-mail: skanatou@ucsd.edu). Received 11 May 1998; accepted in final form 2 December 1998. REFERENCES 1. Baker, J. D., C. W. Fowler, and G. A. Antonelis. Mass changes in fasting male Northern fur seals. Can. J. Zool. 72: 326–329, 1994. 2. Bryden, M. M. Relative growth of the major body components of the southern elephant seal, Mirounga leonina. Aust. J. Zool. 17: 153–177, 1969. 3. Castellini, M. A., G. N. Somero, and G. L. Kooyman. Glycolytic enzyme activities in tissues of marine and terrestrial mammals. Physiol. Zool. 54: 242–252, 1981. 4. Cerretelli, P., and H. Hoppeler. Morphologic and metabolic response to chronic hypoxia: the muscle system. 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