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In an era of rapid environmental change and increasing human presence, researchers need efficient tools for tracking contaminants to monitor the health of Antarctic flora and fauna. Here, we examined the utility of leopard seal whiskers as a biomonitoring tool that reconstructs time-series of significant ecological and physiological biomarkers. Leopard seals (Hydrurga leptonyx) are a sentinel species in the Western Antarctic Peninsula due to their apex predator status and top-down effects on several Antarctic species. However, there are few data on their contaminant loads. We analyzed leopard seal whiskers (n = 18 individuals, n = 981 segments) collected during 2018-2019 field seasons to acquire longitudinal profiles of non-essential (Hg, Pb, and Cd) and essential (Se, Cu, and Zn) trace elements, stable isotope (ẟ 15 N and ẟ 13 C) values and to assess Hg risk with Se:Hg molar ratios. Whiskers provided between 46 and 286 cumulative days of growth with a mean ~ 125 days per whisker (n = 18). Adult whiskers showed variability in non-essential trace elements over time that could partly be explained by changes in diet. Whisker Hg levels were insufficient (<20 ppm) to consider most seals being at "high" risk for Hg toxicity. Nevertheless, maximum Hg concentrations observed in this study were greater than that of leopard seal hair measured two decades ago. However, variation in the Se:Hg molar ratios over time suggest that Se may detoxify Hg burden in leopard seals. Overall, we provide evidence that the analysis of leopard seal whiskers allows for the reconstruction of time-series ecological and physiological data and can be valuable for opportunistically monitoring the health of the leopard seal population and their Antarctic ecosystem during climate change., Competing Interests: Declaration of competing interest The authors declare there are no conflicts of interest., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Developmental increases in dive capacity have been reported in numerous species of air-breathing marine vertebrates. Previous studies in juvenile phocid seals suggest that increases in physiological dive capacity during the postweaning fast (PWF) are critical to support independent aquatic foraging. Although there is a strong relationship between size at weaning and PWF duration and body reserves at weaning vary considerably, few studies have considered whether such variation in body reserve magnitude promotes phenotypic modulation of dive capacity development during the PWF. Phenotypic modulation, a form of developmental plasticity in which rates and degrees of expression of the developmental program are modulated by environmental factors, may enhance diving capacity in weanlings with reduced PWF durations due to smaller body reserves at weaning if reduced body reserves promote accelerated development of dive capacity. We longitudinally measured changes in blood and muscle oxygen stores and muscle metabolic enzymes over the first 8 wk of the PWF in northern elephant seals and determined whether rates of change in these parameters varied with body reserves at weaning. We assessed whether erythropoietin (EPO), thyroid hormones, serum nonesterified fatty acid levels, and iron status influenced blood and muscle oxygen store development or were influenced by body reserves at weaning. Although mass-specific plasma volume and blood volume were relatively stable across the fast, both were elevated in animals with reduced body reserves. Surprisingly, hemoglobin and mean corpuscular hemoglobin concentrations declined over the PWF while hematocrit remained stable, and these variables were not associated with body reserves or EPO. Swimming muscle myoglobin and serum iron levels increased rapidly early in the PWF and were not related to body reserves. Patterns in maximal activities of muscle enzymes suggested a decline in total aerobic and anaerobic metabolic capacity over the PWF, despite maintenance of fat oxidation capacity. These results suggest that only development of blood volume is increased in smaller weanlings and that extended fasting durations in larger weanlings do not improve physiological dive capacity.

Northern elephant seals (Mirounga angustirostris) are extreme, hypoxia-adapted endotherms that rely largely on aerobic metabolism during extended breath-hold dives in near-freezing water temperatures. While many aspects of their physiology have been characterized to account for these remarkable feats, the contribution of adaptations in the aerobic powerhouses of muscle cells, the mitochondria, are unknown. In the present study, the ontogeny and comparative physiology of elephant seal muscle mitochondrial respiratory function was investigated under a variety of substrate conditions and respiratory states. Intact mitochondrial networks were studied by high-resolution respirometry in saponin-permeabilized fiber bundles obtained from primary swimming muscles of pup, juvenile and adult seals, and compared with fibers from adult human vastus lateralis. Results indicate that seal muscle maintains a high capacity for fatty acid oxidation despite a progressive decrease in total respiratory capacity as animals mature from pups to adults. This is explained by a progressive increase in phosphorylation control and fatty acid utilization over pyruvate in adult seals compared with humans and seal pups. Interestingly, despite higher indices of oxidative phosphorylation efficiency, juvenile and adult seals also exhibit a ~50% greater capacity for respiratory 'leak' compared with humans and seal pups. The ontogeny of this phenotype suggests it is an adaptation of muscle to the prolonged breath-hold exercise and highly variable ambient temperatures experienced by mature elephant seals. These studies highlight the remarkable plasticity of mammalian mitochondria to meet the demands for both efficient ATP production and endothermy in a cold, oxygen-limited environment., (© 2014. Published by The Company of Biologists Ltd.)

Myoglobin (Mb) is an oxygen-binding muscular hemeprotein regulated via Ca(2+)-signaling pathways involving calcineurin (CN), with Mb increases attributed to hypoxia, exercise, and nitric oxide. Here, we show a link between lipid supplementation and increased Mb in skeletal muscle. C2C12 cells were cultured in normoxia or hypoxia with glucose or 5% lipid. Mb assays revealed that lipid cohorts had higher Mb than control cohorts in both normoxia and hypoxia, whereas Mb Western blots showed lipid cohorts having higher Mb than control cohorts exclusively under hypoxia. Normoxic cells were compared with soleus tissue from normoxic rats fed high-fat diets; whereas tissue sample cohorts showed no difference in CO-binding Mb, fat-fed rats showed increases in total Mb protein (similar to hypoxic cells), suggesting increases in modified Mb. Moreover, Mb increases did not parallel CN increases but did, however, parallel oxidative stress marker augmentation. Addition of antioxidant prevented Mb increases in lipid-supplemented normoxic cells and mitigated Mb increases in lipid-supplemented hypoxic cells, suggesting a pathway for Mb regulation through redox signaling independent of CN., (Copyright © 2014 the American Physiological Society.)

Northern elephant seals (Mirounga angustirostris) (NES) are known to be deep, long-duration divers and to sustain long-repeated patterns of breath-hold, or apnea. Some phocid dives remain within the bounds of aerobic metabolism, accompanied by physiological responses inducing lung compression, bradycardia, and peripheral vasoconstriction. Current data suggest an absence of type IIb fibers in pinniped locomotory musculature. To date, no fiber type data exist for NES, a consummate deep diver. In this study, NES were biopsied in the wild. Ontogenetic changes in skeletal muscle were revealed through succinate dehydrogenase (SDH) based fiber typing. Results indicated a predominance of uniformly shaped, large type I fibers and elevated myoglobin (Mb) concentrations in the longissimus dorsi (LD) muscle of adults. No type II muscle fibers were detected in any adult sampled. This was in contrast to the juvenile animals that demonstrated type II myosin in Western Blot analysis, indicative of an ontogenetic change in skeletal muscle with maturation. These data support previous hypotheses that the absence of type II fibers indicates reliance on aerobic metabolism during dives, as well as a depressed metabolic rate and low energy locomotion. We also suggest that the lack of type IIb fibers (adults) may provide a protection against ischemia reperfusion (IR) injury in vasoconstricted peripheral skeletal muscle.

Muscle samples were collected from pup, juvenile and adult Weddell seals (Leptonychotes weddellii) near McMurdo Sound, Antarctica during the austral summer of 2006. Blubber samples were collected from juvenile and adult seals. Samples were analyzed for emerging and legacy persistent organic pollutants (POPs) including current and historic-use organochlorine pesticides, polychlorinated biphenyls (PCBs), and polybrominated diphenyl ethers (PBDEs). Of the 41 target analytes, 28 contaminants were recovered from the Weddell seal blubber, in this order of prevalence: p,p'-DDE, p,p'-DDT, trans-nonachlor, mirex, cis-nonachlor, PCB 153, PCB 138, dieldrin, heptachlor epoxide, nonachlor III, PCB 187, oxychlordane, cis-chlordane, PCB 118, PBDE 47, PCB 156, PCB 149, PCB 180, PCB 101, PCB 170, PCB 105, o,p'-DDT, PCB 99, trans-chlordane, PCB 157, PCB 167, PCB 189, and PCB 114. Fewer POPs were found in the muscle samples, but were similar in the order of prevalence to that of the blubber: p,p'-DDE, o,p'-DDT, trans-nonachlor, nonachlor III, oxychlordane, p,p'-DDT, dieldrin, mirex, cis-nonachlor, PCB 138, and PCB 105. Besides differences in toxicant concentrations reported between the muscle and blubber, we found differences in POP levels according to age class and suggest that differences in blubber storage and/or mobilization of lipids result in age class differences in POPs. To our knowledge, such ontogenetic associations are novel. Importantly, data from this study suggest that p,p'-DDT is becoming less prevalent temporally, resulting in an increased proportion of its metabolite p,p'-DDE in the tissues of this top predator. In addition, this study is among the first to identify a PBDE congener in Weddell seals near the McMurdo Station. This may provide evidence of increased PBDE transport and encroachment in Antarctic wildlife., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Diving mammals, are under extreme pressure to conserve oxygen as well as produce adequate energy through aerobic pathways during breath-hold diving. Typically a major source of energy, lipids participate in structural and regulatory roles and have an important influence on the physiological functions of an organism. At the stoichiometric level, the metabolism of polyunsaturated fatty acids (PUFAs) utilizes less oxygen than metabolizing either monounsaturated fatty acids or saturated fatty acids (SFAs) and yields fewer ATP per same length fatty acid. However, there is evidence that indicates the cellular metabolic rate is directly correlated to the lipid composition of the membranes such that the greater the PUFA concentration in the membranes the greater the metabolic rate. These findings appear to be incompatible with diving mammals that ingest and metabolize high levels of unsaturated fatty acids while relying on stored oxygen. Growing evidence from birds to mammals including recent evidence in Weddell seals also indicates that at the whole animal level the utilization of PUFAs to fuel their metabolism actually conserves oxygen. In this paper, we make an initial attempt to ascertain the beneficial adaptations or limitations of lipids constituents and potential trade-offs in diving mammals. We discuss how changes in Antarctic climate are predicted to have numerous different environmental effects; such potential shifts in the availability of certain prey species or even changes in the lipid composition (increased SFA) of numerous fish species with increasing water temperatures and how this may impact the diving ability of Weddell seals.

A key cellular adaptation to diving in Weddell seals is enhanced myoglobin concentrations in their skeletal muscles, which serve to store oxygen to sustain a lipid-based aerobic metabolism. The aim of this study was to determine whether seal muscle cells are inherently adapted to possess the unique skeletal muscle adaptations to diving seen in the whole animal. We hypothesized that the seal skeletal muscle cells would have enhanced concentrations of myoglobin de novo that would be greater than those from a C(2)C(12) skeletal muscle cell line and reflect the concentrations of myoglobin observed in previous studies. In addition we hypothesized that the seal cells would respond to environmental hypoxia similarly to the C(2)C(12) cells in that citrate synthase activity and myoglobin would remain the same or decrease under hypoxia and lactate dehydrogenase activity would increase under hypoxia as previously reported. We further hypothesized that β-hydroxyacyl CoA dehydrogenase activity would increase in response to the increasing amounts of lipid supplemented to the culture medium. Our results show that myoglobin significantly increases in response to environmental hypoxia and lipids in the Weddell seal cells, while appearing similar metabolically to the C(2)C(12) cells. The results of this study suggest the regulation of myoglobin expression is fundamentally different in Weddell seal skeletal muscle cells when compared with a terrestrial mammalian cell line in that hypoxia and lipids initially prime the skeletal muscles for enhanced myoglobin expression. However, the cells need a secondary stimulus to further increase myoglobin to levels seen in the whole animal.

Myoglobin is a well-characterized, cytoplasmic hemoprotein that is expressed primarily in cardiomyocytes and oxidative skeletal muscle fibers. However, recent studies also suggest low-level myoglobin expression in various non-muscle tissues. Prior studies incorporating molecular, pharmacological, physiological and transgenic technologies have demonstrated that myoglobin is an essential oxygen-storage hemoprotein capable of facilitating oxygen transport and modulating nitric oxide homeostasis within cardiac and skeletal myocytes. Concomitant with these studies, scientific investigations into the transcriptional regulation of myoglobin expression have been undertaken. These studies have indicated that activation of key transcription factors (MEF2, NFAT and Sp1) and co-activators (PGC-1alpha) by locomotor activity, differential intracellular calcium fluxes and low intracellular oxygen tension collectively regulate myoglobin expression. Future studies focused on tissue-specific transcriptional regulatory pathways and post-translational modifications governing myoglobin expression will need to be undertaken. Finally, further studies investigating the modulation of myoglobin expression under various myopathic processes may identify myoglobin as a novel therapeutic target for the treatment of various cardiac and skeletal myopathies.

Our objective was to elucidate age-related changes in lipids associated with skeletal muscle of Weddell seals and to suggest possible physiological implications. Muscle biopsies were collected from pups, juveniles and adults in McMurdo Sound, Antarctica and analyzed for intramuscular lipid (IML) and triacylglyceride (IMTG) amounts, fatty acid groups, as well as individual fatty acid profiles. The results from this study suggest a switch from primarily saturated fatty acids (SFAs) and monounsaturated fatty acids (MUFAs) in the skeletal muscle of young pups to increases in polyunsaturated fatty acids (PUFAs) as the percentage of blubber increases, resulting in possible thermoregulatory benefits. As Weddell pups continue to develop into juveniles, fatty acids associated with the skeletal muscle changes such that MUFA levels are relatively higher, which may be in response to energy depletion associated with their restricted diving ability and rapid growth. As juveniles transform into adults, a reduction in n-3 PUFA levels in the muscle as the percentage of blubber increases may be indicative of a trigger to prepare for deep diving or could be a mechanism for oxygen conservation during long-duration dives. We speculate that the observed change in lipids associated with the skeletal muscle of Weddell seals is related to ontogenetic differences in thermoregulation and locomotion.

As highly mobile marine predators, many pinniped species routinely encounter a wide range of water temperatures during foraging and in association with seasonal, geographical and climatic changes. To determine how such variation in environmental temperature may impact energetic costs in otariids, we determined the thermal neutral zone of adult and juvenile California sea lions (Zalophus californianus) by measuring resting metabolic rate using open-flow respirometry. Five adult female (body mass range =82.2-107.2 kg) and four juvenile (body mass=26.2-36.5 kg) sea lions were examined over experimental water temperatures ranging from 0 to 20 degrees C (adults) or 5 to 20 degrees C (juveniles). The metabolic rate of adult sea lions averaged 6.4+/-0.64 ml O(2) kg(-1) min(-1) when resting within the thermal neutral zone. The lower critical temperature of adults was 6.4+/-2.2 degrees C, approximately 4 degrees C lower than sea surface temperatures routinely encountered off coastal California. In comparison, juvenile sea lions did not demonstrate thermal neutrality within the range of water temperatures examined. Resting metabolic rate of the younger animals, 6.3+/-0.53 ml O(2) kg(-1) min(-1), increased as water temperature approached 12 degrees C, and suggested a potential thermal limitation in the wild. To determine whether muscle thermogenesis during activity could mitigate this limitation, we measured the active metabolic rate of juveniles swimming at water temperature (T(water))=5, 12 and 20 degrees C. No significant difference (F=0.377, P=0.583) in swimming metabolic rate was found among water temperatures, suggesting that thermal disadvantages due to small body size in juvenile sea lions may be circumvented by recycling endogenous heat during locomotor activity.

Myoglobin is an oxygen storage molecule that is selectively expressed in cardiac and slow-twitch skeletal muscles that have a high oxygen demand. Numerous studies have implicated hypoxia in the regulation of myoglobin expression as an adaptive response to hypoxic stress. However, the details of this relationship remain undefined. In the present study, adult mice exposed to 10% oxygen for periods up to 3 wk exhibited increased myoglobin expression only in the working heart, whereas myoglobin was either diminished or unchanged in skeletal muscle groups. In vitro and in vivo studies revealed that hypoxia in the presence or absence of exercise-induced stimuli reprograms calcium signaling and modulates myoglobin gene expression. Hypoxia alone significantly altered calcium influx in response to cell depolarization or depletion of endoplasmic reticulum calcium stores, which inhibited the expression of myoglobin. In contrast, our whole animal and transcriptional studies indicate that hypoxia in combination with exercise enhanced the release of calcium from the sarcoplasmic reticulum via the ryanodine receptors triggered by caffeine, which increased the translocation of nuclear factor of activated T-cells into the nucleus to transcriptionally activate myoglobin expression. The present study unveils a previously unrecognized mechanism where the hypoxia-mediated regulation of calcium transients from different intracellular pools modulates myoglobin gene expression. In addition, we observed that changes in myoglobin expression, in response to hypoxia, are not dependent on hypoxia-inducible factor-1 or changes in skeletal muscle fiber type. These studies enhance our understanding of hypoxia-mediated gene regulation and will have broad applications for the treatment of myopathic diseases.

Mammals balance heat dissipation with heat production to maintain core body temperatures independent of their environment. Thermal balance is undoubtedly most challenging for mammals born in polar regions because small body size theoretically results in high surface-area-to-volume ratios (SA:V), which facilitate heat loss (HL). Thus, we examined the ontogeny of thermoregulatory characteristics of an ice-breeding seal (Weddell seal Leptonychotes weddelli). Morphology, blubber thickness, rectal temperature (T(r)), muscle temperature (T(m)), and skin temperatures on the trunk (T(s)) and flipper (T(f)) in 3-5-wk-old pups, yearlings, and adults were measured. Adults maintained the thickest blubber layers, while yearlings had the thinnest; T(r) and T(m) fell within a narrow range, yet T(r) and T(m) decreased significantly with body length. All seals maintained skin temperatures lower than T(r), our index of core body temperature. The T(s)'s were positively correlated with environmental temperatures; conversely, T(f)'s were not. Although pups had the greatest proportion of blubber, their greater SA:V and limited ability to minimize body-to-environment temperature gradients led to the greatest calculated mass-specific HL. This implies that pups relied on elevated metabolic heat production to counter HL. Heat production in pups and yearlings may have been aided by nonshivering thermogenesis in the skeletal muscle via the enhanced muscle mitochondrial densities that have been observed in these segments of this population.

Our objective was to determine the ontogenetic changes in the skeletal muscles of Weddell seals that transform a non-diving pup into an elite diving adult. Muscle biopsies were collected from pups, juveniles and adults and analyzed for changes in fiber type, mitochondrial density, myoglobin concentrations and aerobic, lipolytic and anaerobic enzyme activities. The fiber type results demonstrated a decrease in slow-twitch oxidative (Type I) fibers and a significant increase in fast-twitch oxidative (Type IIA) fibers as the animals mature. In addition, the volume density of mitochondria and the activity of lipolytic enzymes significantly decreased as the seals matured. To our knowledge, this is the first quantitative account describing a decrease in aerobic fibers shifting towards an increase in fast-twitch oxidative fibers with a significant decrease in mitochondrial density as animals mature. These differences in the muscle physiology of Weddell seals are potentially due to their three very distinct stages of life history: non-diving pup, novice diving juvenile, and elite deep diving adult. During the first few weeks of life, pups are a non-diving terrestrial mammal that must rely on lanugo (natal fur) for thermoregulation in the harsh conditions of Antarctica. The increased aerobic capacity of pups, associated with increased mitochondrial volumes, acts to provide additional thermogenesis. As these future elite divers mature, their skeletal muscles transform to a more sedentary state in order to maintain the low levels of aerobic metabolism associated with long-duration diving.

Xin is a muscle-specific actin binding protein of which its role and regulation within skeletal muscle is not well understood. Here we demonstrate that Xin mRNA is robustly upregulated (>16-fold) within 12 h of skeletal muscle injury and is localized to the muscle satellite cell population. RT-PCR confirmed the expression pattern of Xin during regeneration, as well as within primary muscle myoblast cultures, but not other known stem cell populations. Immunohistochemical staining of single myofibers demonstrate Xin expression colocalized with the satellite cell marker Syndecan-4 further supporting the mRNA expression of Xin in satellite cells. In situ hybridization of regenerating muscle 5-7 days postinjury illustrates Xin expression within newly regenerated myofibers. Promoter-reporter assays demonstrate that known myogenic transcription factors [myocyte enhancer factor-2 (MEF2), myogenic differentiation-1 (MyoD), and myogenic factor-5 (Myf-5)] transactivate Xin promoter constructs supporting the muscle-specific expression of Xin. To determine the role of Xin within muscle precursor cells, proliferation, migration, and differentiation analysis using Xin, short hairpin RNA (shRNA) were undertaken in C2C12 myoblasts. Reducing endogenous Xin expression resulted in a 26% increase (P < 0.05) in cell proliferation and a 20% increase (P < 0.05) in myoblast migratory capacity. Skeletal muscle myosin heavy chain protein levels were increased (P < 0.05) with Xin shRNA administration; however, this was not accompanied by changes in myoglobin protein (another marker of differentiation) nor overt morphological differences relative to differentiating control cells. Taken together, the present findings support the hypothesis that Xin is expressed within muscle satellite cells during skeletal muscle regeneration and is involved in the regulation of myoblast function.

Recent studies have shown that harbor seals (Phoca vitulina) have an increased skeletal muscle mitochondrial volume density that may be an adaptation for maintaining aerobic metabolism during diving. However, these studies were based on single samples taken from locomotory muscles. In this study, we took multiple samples from a transverse section of the epaxial (primary locomotory) muscles and single samples from the m. pectoralis (secondary locomotory) muscle of five wild harbor seals. Average mitochondrial volume density of the epaxial muscles was 5.6%, which was 36.6% higher than predicted for a terrestrial mammal of similar mass, and most (82.1%) of the mitochondria were interfibrillar, unlike athletic terrestrial mammals. In the epaxial muscles, the total mitochondrial volume density was significantly greater in samples collected from the deep (6.0%) compared with superficial (5.0%) regions. Volume density of mitochondria in the pectoralis muscle was similar (5.2%) to that of the epaxial muscles. Taken together, these adaptations reduce the intracellular distance between mitochondria and oxymyoglobin and increase the mitochondrial diffusion surface area. This, in combination with elevated myoglobin concentrations, potentially increases the rate of oxygen diffusion into mitochondria and prevents diffusion limitation so that aerobic metabolism can be maintained under low oxygen partial pressure that develops during diving.

Cytoglobin (Cygb) is a novel tissue hemoprotein relatively similar to myoglobin (Mb). Because Cygb shares several structural features with Mb, we hypothesized that Cygb functions in the modulation of oxygen and nitric oxide metabolism or in scavenging free radicals within a cell. In the present study we examined the spatial and temporal expression pattern of Cygb during murine embryogenesis. Using in situ hybridization, RT-PCR, and Northern blot analyses, limited Cygb expression was observed during embryogenesis compared with Mb expression. Cygb expression was primarily restricted to the central nervous system and neural crest derivatives during the latter stages of development. In the adult mouse, Cygb is expressed in distinct regions of the brain as compared with neuroglobin (Ngb), another globin protein, and these regions are responsive to oxidative stress (i.e., hippocampus, thalamus, and hypothalamus). In contrast to Ngb, Cygb expression in the brain is induced in response to chronic hypoxia (10% oxygen). These results support the hypothesis that Cygb is an oxygen-responsive tissue hemoglobin expressed in distinct regions of thenormoxic and hypoxic brain and may play a key role in the response of the brain to ahypoxic insult.

Myoglobin is an abundant hemoprotein that is expressed in cardiomyocytes and oxidative skeletal myofibers of vertebrates. Elegant studies using physiological, biochemical and spectroscopic analyses support a role for myoglobin in facilitated oxygen transport and as a reservoir for oxygen in muscle of diving and hypoxia-adapted animals. In contrast, the functional role of myoglobin in terrestrial animals that function at ambient oxygen levels is a subject of debate. This debate was further fueled by the observation that genetically engineered mice that lack myoglobin are viable and capable of withstanding the hemodynamic stress associated with reproduction. Analysis of the myoglobin mutant striated muscle reveals a spectrum of adaptive mechanisms that partially compensate for the absence of myoglobin and further supports an important function for this hemoprotein in the maintenance of contractile function during exercise under ambient and hypoxic conditions. Future studies utilizing transgenic and gene deletional strategies will further enhance our understanding of myoglobin function under normoxic and hypoxic conditions and will impact our understanding of exercise physiology.

The successful use of myogenic progenitor cells for therapeutic applications requires an understanding of the intrinsic and extrinsic cues involved in their regulation. Herein we demonstrate the expression pattern and transcriptional regulation of Rad, a prototypical member of a family of novel Ras-related GTPases, during mammalian development and skeletal muscle regeneration. Rad was identified using microarray analysis, which revealed robust upregulation of its expression during skeletal muscle regeneration. Our current findings demonstrate negligible Rad expression with resting adult skeletal muscle; however, after muscle injury, Rad is expressed within the myogenic progenitor cell population. Rad expression is significantly increased and localized to the myogenic progenitor cell population during the early phases of regeneration and within the newly regenerated myofibers during the later phases of regeneration. Immunohistochemical analysis demonstrated that Rad and MyoD are coexpressed within the myogenic progenitor cell population of regenerating skeletal muscle. This expression profile of Rad during skeletal muscle regeneration is consistent with the proposed roles for Rad in the inhibition of L-type Ca(2+) channel activity and the inhibition of Rho/RhoA kinase activity. We also have demonstrated that known myogenic transcription factors (MEF2, MyoD, and Myf-5) can increase the transcriptional activity of the Rad promoter and that this ability is significantly enhanced by the presence of the Ca(2+)-dependent phosphatase calcineurin. Furthermore, this enhanced transcriptional activity appears to be dependent on the presence of a conserved NFAT binding motif within the Rad promoter. Taken together, these data define Rad as a novel factor within the myogenic progenitor cells of skeletal muscle and identify key regulators of its transcriptional activity.

When aquatic reptiles, birds and mammals submerge, they typically exhibit a dive response in which breathing ceases, heart rate slows, and blood flow to peripheral tissues is reduced. The profound dive response that occurs during forced submergence sequesters blood oxygen for the brain and heart while allowing peripheral tissues to become anaerobic, thus protecting the animal from immediate asphyxiation. However, the decrease in peripheral blood flow is in direct conflict with the exercise response necessary for supporting muscle metabolism during submerged swimming. In free diving animals, a dive response still occurs, but it is less intense than during forced submergence, and whole-body metabolism remains aerobic. If blood oxygen is not sequestered for brain and heart metabolism during normal diving, then what is the purpose of the dive response? Here, we show that its primary role may be to regulate the degree of hypoxia in skeletal muscle so that blood and muscle oxygen stores can be efficiently used. Paradoxically, the muscles of diving vertebrates must become hypoxic to maximize aerobic dive duration. At the same time, morphological and enzymatic adaptations enhance intracellular oxygen diffusion at low partial pressures of oxygen. Optimizing the use of blood and muscle oxygen stores allows aquatic, air-breathing vertebrates to exercise for prolonged periods while holding their breath.

Myoglobin-deficient mice are viable and have preserved cardiac function due to their ability to mount a complex compensatory response involving increased vascularization and the induction of the hypoxia gene program (hypoxia-inducible factor-1alpha, endothelial PAS, heat shock protein27, etc.). To further define and explore functional roles for myoglobin, we challenged age- and gender-matched wild-type and myoglobin-null mice to chronic hypoxia (10% oxygen for 1 day to 3 wk). We observed a 30% reduction in cardiac systolic function in the myoglobin mutant mice exposed to chronic hypoxia with no changes observed in the wild-type control hearts. The cardiac dysfunction observed in the hypoxic myoglobin-null mice was reversible with reexposure to normoxic conditions and could be prevented with treatment of an inhibitor of nitric oxide (NO) synthases. These results support the conclusion that hypoxia-induced cardiac dysfunction in myoglobin-null mice occurs via a NO-mediated mechanism. Utilizing enzymatic assays for NO synthases and immunohistochemical analyses, we observed a marked induction of inducible NO synthase in the hypoxic myoglobin mutant ventricle compared with the wild-type hypoxic control ventricle. These new data establish that myoglobin is an important cytoplasmic cardiac hemoprotein that functions in regulating NO homeostasis within cardiomyocytes.

Pinnipeds (seals and sea lions) have an elevated mitochondrial volume density [VV(mt)] and elevated citrate synthase (CS) and beta-hydroxyacyl-CoA dehydrogenase (HOAD) activities in their swimming muscles to maintain an aerobic, fat-based metabolism during diving. The goal of this study was to determine whether the heart, kidneys and splanchnic organs have an elevated VV(mt) and CS and HOAD activities as parallel adaptations for sustaining aerobic metabolism and normal function during hypoxia in harbor seals (Phoca vitulina). Samples of heart, liver, kidney, stomach and small intestine were taken from 10 freshly killed harbor seals and fixed in glutaraldehyde for transmission electron microscopy or frozen in liquid nitrogen for enzymatic analysis. Samples from dogs and rats were used for comparison. Within the harbor seal, the liver and stomach had the highest VV(mt). The liver also had the highest CS activity. The kidneys and heart had the highest HOAD activities, and the liver and heart had the highest lactate dehydrogenase (LDH) activities. Mitochondrial volume densities scaled to tissue-specific resting metabolic rate [VV(mt)/RMR] in the heart, liver, kidneys, stomach and small intestine of harbor seals were elevated (range 1.2-6.6x) when compared with those in the dog and/or rat. In addition, HOAD activity scaled to tissue-specific RMR in the heart and liver of harbor seals was elevated compared with that in the dog and rat (3.2x and 6.2x in the heart and 8.5x and 5.5x in the liver, respectively). These data suggest that organs such as the liver, kidneys and stomach possess a heightened ability for aerobic, fat-based metabolism during hypoxia associated with routine diving. However, a heightened LDH activity in the heart and liver indicates an adaptation for the anaerobic production of ATP on dives that exceed the animal's aerobic dive limit. Hence, the heart, liver, kidneys and gastrointestinal organs of harbor seals exhibit adaptations that promote an aerobic, fat-based metabolism under hypoxic conditions but can provide ATP anaerobically if required.

Myoglobin (Mb) is an intensely studied hemoprotein that is restricted mainly to the heart and oxidative myofibers in skeletal muscle. Previous physiologic and pharmacologic studies have supported a role for Mb in facilitated oxygen transport or as an oxygen reservoir in striated muscle. Transgenic and gene disruption technologies have been utilized to produce mice that lack Mb. Studies utilizing these transgenic mouse models support the notion that Mb may have multiple, diverse functions in the heart. Future studies using these emerging technologies will further enhance the understanding of the role of Mb and other hemoproteins in cardiovascular biology.

In contrast to terrestrial 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 function for the duration of a dive under progressive asphyxia, which is the combination of increasing hypoxia, hypercapnia and acidosis. Our previous studies on short-duration, shallow divers found marked adaptations in their skeletal muscles, which culminated in enhanced aerobic capacities that are similar to those of athletic terrestrial mammals. The purpose of the present study was to assess the aerobic capacity of skeletal muscles from long-duration divers. Swimming and non-swimming muscles were collected from adult Weddell seals, Leptonychotes weddelli, and processed for morphometric analysis, enzymology, myoglobin concentrations and fiber-type distribution. The results showed that the skeletal muscles of Weddell seals do not have enhanced aerobic capacities compared with those of terrestrial mammals but are adapted to maintain low levels of an aerobic lipid-based metabolism, especially under the hypoxic conditions associated with diving. The lower aerobic capacity of Weddell seal muscle as compared with that of shorter-duration divers appears to reflect their energy-conserving modes of locomotion, which enable longer and deeper dives.

Endurance exercise training promotes mitochondrial biogenesis in skeletal muscle and enhances muscle oxidative capacity, but the signaling mechanisms involved are poorly understood. To investigate this adaptive process, we generated transgenic mice that selectively express in skeletal muscle a constitutively active form of calcium/calmodulin-dependent protein kinase IV (CaMKIV*). Skeletal muscles from these mice showed augmented mitochondrial DNA replication and mitochondrial biogenesis, up-regulation of mitochondrial enzymes involved in fatty acid metabolism and electron transport, and reduced susceptibility to fatigue during repetitive contractions. CaMK induced expression of peroxisome proliferator-activated receptor gamma coactivator 1 (PGC-1), a master regulator of mitochondrial biogenesis in vivo, and activated the PGC-1 gene promoter in cultured myocytes. Thus, a calcium-regulated signaling pathway controls mitochondrial biogenesis in mammalian cells.

The purpose of this study was to examine muscle capillary supply in harbor seals. Locomotory and nonlocomotory muscles of four harbor seals (mass = 17.5-41 kg) were glutaraldehyde-perfusion fixed and samples processed for electron microscopy and analyzed by morphometry. Capillary-to-fiber number and surface ratios were 0.81 +/- 0.05 and 0.16 +/- 0.01, respectively. Capillary length and surface area per volume of muscle fiber were 1,495 +/- 83 mm/mm(3) and 22.4 +/- 1.6 mm(2)/mm(3), respectively. In the locomotory muscles, we measured capillary length and surface area per volume mitochondria (20.1 +/- 1.7 km/ml and 2,531 +/- 440 cm(2)/ml). All these values are 1.5-3 times lower than in muscles with similar or lower volume densities of mitochondria in dogs of comparable size. Compared with terrestrial mammals, the skeletal muscles of harbor seals do not match their increased aerobic enzyme capacities and mitochondrial volume densities with greater muscle capillary supply. They have a smaller capillary-to-fiber interface and capillary supply per fiber mitochondrial volume than terrestrial mammals of comparable size.

Unlike their terrestrial counterparts, marine mammals stop breathing and reduce their convective oxygen transport while performing activities (e.g. foraging, courtship, aggressive interactions, predator avoidance and migration) that require sustained power output during submergence. Since most voluntary dives are believed to remain aerobic, the goal of this study was to examine the potential importance of the dive response in optimizing the use of blood and muscle oxygen stores during dives involving different levels of muscular exertion. To accomplish this, we designed a numerical model based on Fick's principle that integrated cardiac output (Vb), regional blood flow, convective oxygen transport (Q(O2)), muscle oxymyoglobin desaturation and regional rates of oxygen consumption (VO2). The model quantified how the optimal matching or mismatching of QO2 to VO2 affected the aerobic dive limit (ADL). We chose an adult Weddell seal Leptonycotes weddellii on which to base our model because of available data on the diving physiology and metabolism of this species. The results show that the use of blood and muscle oxygen stores must be completed at the same time to maximize the ADL for each level of VO2. This is achieved by adjusting Vb (range 19-94 % of resting levels) and muscle QO2 according to the rate of muscle oxygen consumption (VMO2). At higher values of VMO2, Vb and muscle perfusion must increase to maintain an appropriate QO2/VO2 ratio so that available blood and muscle oxygen stores are depleted at the same time. Although the dive response does not sequester blood oxygen exclusively for brain and heart metabolism during aerobic dives, as it does during forced submersion, a reduction in Vb and muscle perfusion below resting levels is necessary to maximize the ADL over the range of diving VO2 (approximately 2-9 ml O2 min-1 kg-1). Despite the reduction in Vb, convective oxygen transport is adequate to maintain aerobic metabolism and normal function in the splanchnic organs, kidneys and other peripheral tissues. As a result, physiological homeostasis is maintained throughout the dive. The model shows that the cardiovascular adjustments known as the dive response enable the diving seal to balance the conflicting metabolic demands of (1) optimizing the distribution and use of blood and muscle oxygen stores to maximize the ADL over the normal range of diving VO2 and (2) ensuring that active muscle receives adequate oxygen as VMO2 increases.

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 = 27), Northern fur seals (Callorhinus ursinus, n = 5), and harbor seals (Phoca vitulina, n = 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 beta-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 beta-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.

The hunting behavior of a marine mammal was studied beneath the Antarctic fast ice with an animal-borne video system and data recorder. Weddell seals stalked large Antarctic cod and the smaller subice fish Pagothenia borchgrevinki, often with the under-ice surface for backlighting, which implies that vision is important for hunting. They approached to within centimeters of cod without startling the fish. Seals flushed P. borchgrevinki by blowing air into subice crevices or pursued them into the platelet ice. These observations highlight the broad range of insights that are possible with simultaneous recordings of video, audio, three-dimensional dive paths, and locomotor effort.