INTRODUCTION

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THE FUNCTIONAL CAPACITY of the respiratory pathway to deliver oxygen is matched to the oxygen requirements of the tissues in terrestrial mammals and birds (2, 11- 13, 20, 21). The large aerobic scopes of terrestrial animal athletes result from both high mitochondrial volume densities in skeletal muscles and cardiorespiratory adaptations that enhance convective and diffusive oxygen transport (e.g., increased capillary densities, capillary length, and surface area per volume mitochondria; Ref. 14). Terrestrial mammals increase ventilation and cardiac output during exercise. In contrast, 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 (5, 7), and appears to limit aerobic scope (4, 6, 7, 16).

Analogies have been drawn between high-altitude-adapted animals and marine mammals in terms of possible adaptations in skeletal muscles to maintain aerobic metabolism under hypoxic conditions (9, 10, 16). Contrary to high-altitude animals that function under hypoxia 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 half of those found in terrestrial mammals of comparable size to maintain oxidative metabolism under the hypoxic conditions associated with diving (10, 12). However, the results of our previous study indicated that pinniped skeletal muscles have oxidative capacities that are enhanced compared with terrestrial mammals of comparable size and aerobic scopes but similar to athletic terrestrial mammals of comparable size (16).

In marine mammals, dive time is inversely related to metabolic rate (1). Thus, during diving, the regulation of blood flow serves the conflicting functions of oxygen consumption and conservation. In contrast, there is a direct relationship between perfusion and cellular metabolism in terrestrial mammals (1). The capillary supply of terrestrial mammals is significantly correlated with the oxidative capacity of the skeletal muscles (13, 14, 29).

The purpose of this study was to determine muscle capillary supply in harbor seals (Phoca vitulina) and whether it is matched to the role of oxygen consumption or conservation in marine mammals. We examined both locomotory and nonlocomotory muscles of the seals compared with hindlimb muscles of dogs (Canis familaris) of comparable size. Specifically, we wanted to test the hypothesis that there is no difference in muscle capillary supply between marine and terrestrial mammals with comparable aerobic capacities.

	MATERIALS AND METHODS

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Four subadult harbor seals (Phoca vitulina, mass = 29.6 ± 5.8 kg) and two dogs (Canis familaris, mass = 17 ± 3 kg) were anesthesized and perfusion fixed. The entire vasculature was perfused with saline. Perfusion fixation followed with a 6.25% solution of glutaraldehyde in 0.1 M sodium cacodylate buffer (total osmolarity of the fixative: 1,100 mosM; pH 7.4) for 10 min. Muscle samples were taken from both locomotory (longissimus dorsi and latissimus dorsi) and nonlocomotory (diaphragm, gastrocnemius, vastus profundus, vastus lateralis, and semitendinosus) muscles from harbor seals and from the hindlimb muscles of dogs (Table 1). All samples were cut into thin longitudinal strips, stored in glutaraldehyde fixative, and processed for electron microscopy, as previously described (27).

From each muscle sample, four transverse and four longitudinal sections were collected from 4-8 blocks by using a LKB Ultrotome III. They were stained with 0.1% aqueous toluidine blue solution, and sarcomere length (l_o) was measured in longitudinal sections (27). Ultrathin sections (50-70 nm) were cut transversely to the muscle fiber axis in four blocks from each sample. They were contrasted with uranyl acetate and bismuth subnitrate, and electron micrographs for morphometry were taken on 70-mm film by use of a Zeiss 10 transmission electron microscope.

Capillary counts per fiber sectional area (i.e., capillary densities; Q_A) were obtained by morphometry on transverse [Q_A(0)] and longitudinal [Q_A(/2)] sections, using a 100-point square-grid test eyepiece at a magnification of ×400 on a light microscope. An average of 11 ± 1 (mean ± SE) fields/sample in transverse and longitudinal sections was measured.

The capillary anisotropy coefficient c(K,0), a relative measure of capillary orientation, was estimated as described previously (26). Capillary length per fiber volume [J_V(c,f)] was calculated as the product of Q_A(0) and c(K,0). Capillary diameter [ (c)], number of capillaries around a fiber (N_CAF), fiber cross-sectional area [(f)], and fiber perimeter were measured by use of an image analyzer (Videometric 150, American Innovision) on the same transverse sections used to estimate Q_A(0). An average of 247 ± 23 randomly selected fibers was measured per sample. The (c) was determined as the shorter axis of close to circular profiles only (<20% difference between the shorter and the longer diameters), which assumes capillary cross-sectional circularity (25). An average of 92 ± 7 capillary profiles per sample was measured.

The (f) and capillary density were normalized to l_o = 2.1 µm, a value chosen because it is close to the l_o at which maximal tension is developed in skeletal muscles (25). Capillary-to-fiber ratio [N_N(c,f)] was calculated as the product of Q_A(0) and (f). Capillary-to-fiber perimeter ratio [B_B(0)] was estimated in transverse sections at a magnification of ×1,000 by using a 100-point square-grid test eyepiece system. An average of 23 ± 2 fields per sample was measured. Capillary surface per fiber volume was measured by intersection counting on longitudinal sections with an O(6 × 6) eyepiece cycloid grid (Cruz-Orive, University of Berne, Switzerland) on an average of 20 ± 2 fields per sample at ×1,000 magnification.

The volume density of mitochondria, myofibrils, and lipid droplets per volume of muscle fiber was estimated by point counting at a final magnification of ×24,000. Contact prints of the electron microscope films were projected on a 144-point square-grid test of a microfilm reader (Documator DL 2, Jenoptic, Jena, Germany). Mitochondrial volume per micrometer of fiber length at 2.1 µm l_o was calculated as the product of total volume density of mitochondria [V_V(mt,f)] and (f) normalized to 2.1 µm.

Results are expressed as means ± SE. Statistical comparisons between species were made using t-tests from Microsoft Excel Analysis Tool Pak (P < 0.05).

	RESULTS

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Figure 1 shows light micrographs of transverse sections of the longissimus dorsi of a harbor seal and the sartorius muscle of a dog. The seal muscle has a higher volume density of mitochondria (8.7 ± 0.0 vs. 7.4 ± 0.5%; Table 3) but a lower capillary density (Table 1 and Fig. 1) than the dog. Values for l_o in the harbor seal muscles ranged from 1.77 to 3.17 µm, and group means (2.37 ± 0.10 µm) were similar to those in dog muscles (2.27 ± 0.20 µm). The (c) values were also similar in the two species (4.49 ± 0.14 and 4.16 ± 0.07 µm, Table 2). Capillary densities in transverse sections corrected for 2.1-µm l_o were 42% greater in dogs (1,617 ± 104 mm²) than in seals (1,138 ± 81 mm², P < 0.01; Table 1). N_N(c,f) and N_CAF in the harbor seal muscles were 0.81 ± 0.05 and 2.68 ± 0.13, respectively (Table 2). These values are 3 and 2.2 times lower than in dog muscles, respectively, whereas (f) and perimeter were significantly less in the harbor seals (780 ± 91 µm² and 107 ± 6 µm) than the dogs (1,530 ± 125 µm² and 171 ± 14 µm, P < 0.01). The average number of fibers sharing one capillary [SF = N_CAF/N_N(c,f)] was 35% greater in harbor seals (3.35 ± 0.12) than in dogs (2.48 ± 0.1, P < 0.01).

View larger version (106K): [in this window] [in a new window]   Fig. 1.   Light micrographs of transverse sections of harbor seal longissimus dorsi (A) and the sartorius of the dog (B). Capillaries are empty after vascular perfusion fixation. Note the greater capillary density in the sartorius of the dog compared with the longissimus of the seal.

The c(K,0) was 1.20 ± 0.04 in the harbor seals, which was similar to that measured in the dogs (1.23 ± 0.10), indicating a comparable contribution of tortuosity and branching to capillary length in the muscles (Table 1). Group mean capillary length and capillary surface per fiber volume in the harbor seals were 1,495 ± 83 mm² and 22.4 ± 1.6 mm¹, respectively (Tables 1 and 2). These values were 28% and 34% lower than in the dog.

Because the capillary orientation coefficient was <1.53 in all but one of the samples, the coefficient c'(K',0) was 1, and B_B(0) was a direct estimate of capillary-to-fiber surface ratio [S_S(c,f)] in those muscles (25). In the superficial medial gastrocnemius muscle of dog D3, the c(K,0) was 1.64 (Table 1), yielding a c'(K',0) = 1.05, i.e., an S_S(c,f) value 5% > B_B(0) in that muscle. Overall, S_S(c,f) was 1.75 times greater in the muscles of dogs than seals.

Table 3 summarizes the data on fiber ultrastructure in the locomotory muscles of the seals and the hindlimb muscles of the dogs. The volume density of mitochondria was significantly greater in the locomotory muscles of harbor seals (8.1 ± 0.5%) than the hindlimb muscles of dogs (5.9 ± 0.5%, P < 0.01). Figure 2 shows the plot of J_V(c,f) against the mitochondrial volume density, compared with previous data from rat and bat muscles. The values of J_V(c,f) in harbor seal locomotory muscles are lower than in the dog over their range of mitochondrial volume densities. They are also lower than would be predicted by the regression of J_V(c,f) vs. mitochondrial volume density from Mathieu-Costello et al. (27). Capillary length and surface area per unit volume mitochondria were 1.9 and 2.4 times lower in harbor seals (19.4 ± 1.9 km/ml and 2,463 ± 467 cm²/ml) than in dogs (35.9 ± 1.7 km/ml and 5,817 ± 202; Table 4).

View larger version (20K): [in this window] [in a new window]   Fig. 2.   A: plot of capillary length per fiber volume against mitochondria per fiber volume in longissimus dorsi of harbor seals (), latissimus dorsi of harbor seals () and the hindlimb muscles of the dog (). Group mean values and linear relationship [y = (249x) + 142; r = 0.993] in bat pectoralis and hindlimb and rat soleus (dotted line; Ref. 27) are shown for comparison. B: expanded view of inset from A.

The volume of mitochondria per unit length of fiber at 2.1 µm l_o [(mit)_2.1], was 32% lower in seals than in dogs (Table 4). Figure 3 shows that for any given (mit)_2.1 the seals have a lower N_CAF value than the dogs.

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Plot of capillary number around a fiber (NCAF) and mitochondrial volume per unit fiber length at 2.1 µm sarcomere (sarc.) length in the locomotory muscles of the harbor seals (, longissimus dorsi; , latissimus dorsi) and the hindlimb of the dog (). Linear relationship [y = (0.017x) + 3.5; r = 0.76] and average values in bat pectoralis and hindlimb and rat soleus are shown for comparison (Ref. 23).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

In this study, we show that the muscles of harbor seals have a substantially smaller capillary supply than the hindlimb muscles of dogs of comparable size. Capillary density, length, and surface area per fiber volume were 30%, 28%, and 34% lower, respectively, in the muscles of harbor seals than dogs, whereas the contribution of tortuosity and branching to capillary length was similar in the two species. Mitochondrial volume per fiber volume was significantly greater in the locomotory muscles of seals, whereas average (f) and perimeter were 49% and 37% less, respectively, in seals than in dogs. The average N_CAF at a given mitochondrial volume per unit fiber length was ~44% less in the seals, and capillary length and surface area per unit mitochondrial volume were 1.8 and 2.3 times lower in the seals.

The aerobic scope of seals, expressed as multiples of total body oxygen consumption required during maximum muscular effort, is relatively low compared with that of terrestrial mammals. It amounts to less than ten times the resting level whereas that of athletic dogs is 20-30 times (4, 14-16). Cardiac muscle of the seal has fewer mitochondria per gram of tissue, lower respiratory rate, and lower cytochrome content than dog heart (31, 32), which is consistent with the lower maximum oxygen consumption in seals. The results reported here, indicating lesser capillarity in skeletal muscles of the harbor seal, are consistent with the lower aerobic scope but contrary to the enhanced aerobic capacity of the locomotory muscles.

O₂ delivery and O₂ stores. In skeletal muscles of terrestrial animals, it has been shown that the capillary bed is designed primarily to meet fiber oxygen demands (15, 17, 29) and that capillary supply is directly correlated to the volume density of mitochondria (3, 13, 18, 22, 24, 29). On the basis of this relationship, the swimming muscles of harbor seals should have a capillary supply comparable to that of athletic terrestrial mammals of similar size. However, the results of this study show that there is a mismatch between capillary supply and fiber oxidative capacity or fiber mitochondrial volume in the skeletal muscles of seals. To our knowledge, there has only been one other study that has measured capillary density in muscles of marine mammals (30). In histochemical preparations, Reed et al. (30) found even lower values for capillary density, ranging from 352 capillaries/mm² in harbor seals to 639 capillaries/mm² in Antarctic fur seals. Some of the differences between the two studies could be related to sample preparation, because Reed et al. used histochemistry of frozen biopsy material whereas we used perfusion-fixed tissue in this study. In addition, Reed et al. studied adult free-ranging animals, whereas the harbor seals in this study were subadults. The (f) increases with maturation, and the lower capillary densities measured by Reed et al. may relate to larger (f) values in their samples. These, however, were not reported. Despite the differences in approach, the results of both studies support the hypothesis that capillary supply in pinnipeds may not scale in similar proportion to fiber mitochondrial volume as in terrestrial mammals, due to the conflicting requirements of oxygen conservation and consumption during diving.

Delivery of substrate. In addition to their role in oxygen delivery, the capillaries also play a role in the delivery of substrates to the active skeletal muscles. Davis et al. (4) found that plasma glucose, lactate, and free fatty acids accounted for only 3, 7, and 18%, respectively, of the ATP produced in harbor seals at rest and only 23% of the total ATP utilized during swimming exercise at 35 and 50% of maximum oxygen consumption. These relative contributions of blood-borne substrates to the energy metabolism of the muscles are very similar to those found in terrestrial animals with submaximal exercise. Capillary supply of oxidative substrates appears to be maximal at 40% of maximal oxygen consumption, and, as exercise intensity increases, the muscles begin to rely more on intracellular stores of substrates, such that at maximum oxygen consumption over 80% of the fuel is supplied by glycogen catabolism (15). A similar situation appears to hold true for harbor seals with one exception. In harbor seals, energy is derived mainly from a lipid-based metabolism, with intramuscular stores of lipids at least one order of magnitude greater than in terrestrial mammals of comparable size and fiber oxidative capacity (16). Assuming a swimming muscle metabolism of approximately two times resting metabolic rate, Kanatous et al. (16) found that the intramuscular stores of energy in harbor seals are sufficient to sustain routine swimming activity for ~11 h. As in terrestrial animals, the majority of oxidative substrates are derived from intramuscular stores as opposed to capillary delivery during exercise; however, the increased intracellular stores of energy in harbor seals compared with terrestrial mammals may reflect the greater reliance of harbor seals on intramuscular stores of substrate during diving.

Adaptations to hypoxia. As mentioned earlier, similarities have been found between high-altitude-adapted animals and marine mammals in terms of possible adaptations in skeletal muscles to maintain aerobic metabolism under hypoxic conditions (9, 10, 16, 19). Studies in terrestrial mammals have documented increases in N_N(c,f), myoglobin, and fiber oxidative capacity with chronic exercise at altitude or in normobaric hypoxia, whereas hypoxia alone without concomitant exercise training or chronic exposure to cold does not induce formation of new capillaries in skeletal muscles of adult terrestrial mammals (25). Marine mammals such as the harbor seal, which exercise under the hypoxic conditions imposed by breath-hold diving, do show similar intramuscular adaptations, such as the increased fiber oxidative capacities and myoglobin levels, but they do not have the concomitant increase in capillarity. In contrast to 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 begin their dives with only a finite amount of oxygen and continue to function and maintain aerobic metabolism with a continually shrinking oxygen supply throughout the entire duration of a dive.