Relationship between red blood cell lifespan and endogenous carbon monoxide in the common bottlenose dolphin and beluga.

Certain deep-diving marine mammals [i.e., northern elephant seal (Mirounga angustirostris), Weddell seal (Leptonychotes weddellii)] have blood carbon monoxide (CO) levels that are comparable with those of chronic cigarette smokers. Most CO produced in humans is a byproduct of heme degradation, which is released when red blood cells (RBCs) are destroyed. Elevated CO can occur in humans when RBC lifespan decreases. The contribution of RBC turnover to CO concentrations in marine mammals is unknown. Here, we report the first RBC lifespans in two healthy marine mammal species with different diving capacities and heme stores, the shallow-diving bottlenose dolphin (Tursiops truncatus) and deep-diving beluga whale (Delphinapterus leucas), and we relate the lifespans to the levels of CO in blood and breath. The belugas, with high blood heme stores, had the longest mean RBC lifespan compared with humans and bottlenose dolphins. Both cetacean species were found to have three times higher blood CO content compared with humans. The estimated CO production rate from heme degradation indicates some marine mammals may have additional mechanisms for CO production, or delay CO removal from the body, potentially from long-duration breath-holds.NEW & NOTEWORTHY This is the first study to determine the red blood cell lifespan in a marine mammal species. High concentrations of carbon monoxide (CO) were found in the blood of bottlenose dolphins and in the blood and breath of belugas compared with healthy humans. Red blood cell turnover accounted for these high levels in bottlenose dolphins, but there may be alternative mechanisms of endogenous CO production that are contributing to the CO concentrations observed in belugas.

Intraspecific variation in muscle growth of two distinct populations of Port Jackson sharks under projected end-of-century temperatures.

Although pervasive, the effects of climate change vary regionally, possibly resulting in differential behavioral, physiological, and/or phenotypic responses among populations within broadly distributed species. Juvenile Port Jackson sharks (Heterodontus portusjacksoni) from eastern and southern Australia were reared at their current (17.6 °C Adelaide, South Australia [SA]; 20.6 °C Jervis Bay, New South Wales [NSW]) or projected end-of-century (EOC) temperatures (20.6 °C Adelaide, SA; 23.6 °C Jervis Bay, NSW) and assessed for morphological features of skeletal muscle tissue. Nearly all skeletal muscle properties including cellularity, fiber size, myonuclear domain, and satellite cell density did not differ between locations and thermal regimes. However, capillary density was significantly influenced by thermal treatment, where Adelaide sharks raised at current temperatures had a lower capillarity than Jervis Bay sharks raised at ambient or projected EOC temperatures. This may indicate higher metabolic costs at elevated temperatures. However, our results suggest that regardless of the population, juvenile Port Jackson sharks may have limited acclimatory potential to alter muscle metabolic features under a temperature increase, which may make this species vulnerable to climate change.

Microvascular anatomy suggests varying aerobic activity levels in the adipose tissues of diving tetrapods.

Adipose tissue has many important functions including metabolic energy storage, endocrine functions, thermoregulation and structural support. Given these varied functions, the microvascular characteristics within the tissue will have important roles in determining rates/limits of exchange of nutrients, waste, gases and molecular signaling molecules between adipose tissue and blood. Studies on skeletal muscle have suggested that tissues with higher aerobic capacity contain higher microvascular density (MVD) with lower diffusion distances (DD) than less aerobically active tissues. However, little is known about MVD in adipose tissue of most vertebrates; therefore, we measured microvascular characteristics (MVD, DD, diameter and branching) and cell size to explore the comparative aerobic activity in the adipose tissue across diving tetrapods, a group of animals facing additional physiological and metabolic stresses associated with diving. Adipose tissues of 33 animals were examined, including seabirds, sea turtles, pinnipeds, baleen whales and toothed whales. MVD and DD varied significantly (P < 0.001) among the groups, with seabirds generally having high MVD, low DD and small adipocytes. These characteristics suggest that microvessel arrangement in short duration divers (seabirds) reflects rapid lipid turnover, compared to longer duration divers (beaked whales) which have relatively lower MVD and greater DD, perhaps reflecting the requirement for tissue with lower metabolic activity, minimizing energetic costs during diving. Across all groups, predictable scaling patterns in MVD and DD such as those observed in skeletal muscle did not emerge, likely reflecting the fact that unlike skeletal muscle, adipose tissue performs many different functions in marine organisms, often within the same tissue compartment.

A higher mitochondrial content is associated with greater oxidative damage, oxidative defenses, protein synthesis and ATP turnover in resting skeletal muscle.

An unavoidable consequence of aerobic metabolism is the production of reactive oxygen species (ROS). Mitochondria have historically been considered the primary source of ROS; however, recent literature has highlighted the uncertainty in primary ROS production sites and it is unclear how variation in mitochondrial density influences ROS-induced damage and protein turnover. Fish skeletal muscle is composed of distinct, highly aerobic red muscle and anaerobic white muscle, offering an excellent model system in which to evaluate the relationship of tissue aerobic capacity and ROS-induced damage under baseline conditions. The present study used a suite of indices to better understand potential consequences of aerobic tissue capacity in red and white muscle of the pinfish, Lagodon rhomboides. Red muscle had a 7-fold greater mitochondrial volume density than white muscle, and more oxidative damage despite also having higher activity of the antioxidant enzymes superoxide dismutase and catalase. The dominant protein degradation system appears to be tissue dependent. Lysosomal degradation markers and autophagosome volume density were greater in white muscle, while ubiquitin expression and 20S proteasome activity were significantly greater in red muscle. However, ubiquitin ligase expression was significantly higher in white muscle. Red muscle had a more than 2-fold greater rate of translation and total ATP turnover than white muscle, results that may be due in part to the higher mitochondrial density and the associated increase in oxidative damage. Together, these results support the concept that an elevated aerobic capacity is associated with greater oxidative damage and higher costs of protein turnover.

Endosomal-lysosomal dysfunction in metabolic diseases and Alzheimer's disease.

The endosomal-lysosomal pathways and related autophagic processes are responsible for proteostasis, involving complexes between lysosomes and autophagosomes. Lysosomes are a key component of homeostasis, involved in cell signaling, metabolism, and quality control, and they experience functional compromise in metabolic diseases, aging, and neurodegenerative diseases. Many genetic mutations and risk factor genes associated with proteinopathies, as well as with metabolic diseases like diabetes, negatively influence endocytic trafficking and autophagic clearance. In contrast, health-improving exercise induces autophagy-lysosomal degradation, perhaps promoting efficient digestion of injured organelles so that undamaged organelles ensure cellular healthiness. Reductions in lysosomal hydrolases are implicated in Alzheimer's, Parkinson's, and lysosomal storage diseases, as well as obesity-related pathology, and members of the cathepsin enzyme family are involved in clearing both Aß42 and α-synuclein. Upregulation of cathepsin hydrolases improves synaptic and memory functions in models of dementia and in exercising humans, thus identifying lysosomal-related systems as vital for healthy cognitive aging.

Low-dose caffeine administration increases fatty acid utilization and mitochondrial turnover in C2C12 skeletal myotubes.

Caffeine has been shown to directly increase fatty acid oxidation, in part, by promoting mitochondrial biogenesis. Mitochondrial biogenesis is often coupled with mitophagy, the autophagy-lysosomal degradation of mitochondria. Increased mitochondrial biogenesis and mitophagy promote mitochondrial turnover, which can enhance aerobic metabolism. In addition, recent studies have revealed that cellular lipid droplets can be directly utilized in an autophagy-dependent manner, a process known as lipophagy. Although caffeine has been shown to promote autophagy and mitochondrial biogenesis in skeletal muscles, it remains unclear whether caffeine can increase lipophagy and mitochondrial turnover in skeletal muscle as well. The purpose of this study was to determine the possible contribution of lipophagy to caffeine-dependent lipid utilization. Furthermore, we sought to determine whether caffeine could increase mitochondrial turnover, which may also contribute to elevated fatty acid oxidation. Treating fully differentiated C2C12 skeletal myotubes with 0.5 mM oleic acid (OA) for 24 hr promoted an approximate 2.5-fold increase in cellular lipid storage. Treating skeletal myotubes with 0.5 mM OA plus 0.5 mM caffeine for an additional 24 hr effectively returned cellular lipid stores to control levels, and this was associated with an increase in markers of autophagosomes and autophagic flux, as well as elevated autophagosome density in TEM images. The addition of autophagy inhibitors 3-methyladenine (10 mM) or bafilomycin A1 (10 µM) reduced caffeine-dependent lipid utilization by approximately 30%. However, fluorescence and transmission electron microscopy analysis revealed no direct evidence of lipophagy in skeletal myotubes, and there was also no lipophagy-dependent increase in fatty acid oxidation. Finally, caffeine treatment promoted an 80% increase in mitochondrial turnover, which coincided with a 35% increase in mitochondrial fragmentation. Our results suggest that caffeine administration causes an autophagy-dependent decrease in lipid content by increasing mitochondrial turnover in mammalian skeletal myotubes.

Locomotor muscle morphology of three species of pelagic delphinids.

The locomotor muscle morphology of diving mammals yields insights into how they utilize their environment and partition resources. This study examined a primary locomotor muscle, the longissimus, in three closely related, similarly sized pelagic delphinids (n = 7-9 adults of each species) that exhibit different habitat and depth preferences. The Atlantic spotted dolphin (Stenella frontalis) is a relatively shallow diver, inhabiting continental shelf waters; the striped (Stenella coeruleoalba) and short-beaked common (Delphinus delphis) dolphins are sympatric, deep-water species that dive to different depths. Based upon comparative data from other divers, it was hypothesized that the locomotor muscle of the deepest-diving S. coeruleoalba would exhibit a higher percentage of slow oxidative fibers, larger fiber diameters, a higher myoglobin concentration [Mb], and a lower mitochondrial density than that of the shallow-diving S. frontalis, and that the muscle of D. delphis would display intermediate values for these features. As expected, the locomotor muscle of S. coeruleoalba exhibited a significantly higher proportion of slow (57.3 ± 3.9%), oxidative (51.7 ± 2.5%) fibers and higher [Mb] (8.2 ± 0.7 g/100 g muscle) than that of S. frontalis (41.3 ± 3.9%, 31.0 ± 3.2%, 4.7 ± 0.05 g/100 g muscle, respectively). There were no differences in fiber size or mitochondrial density among these species. Like other deep divers, S. coeruleoalba displayed locomotor muscle features that enhance oxygen storage capacity and metabolic efficiency but did not display features that limit aerobic capacity. These results suggest a previously undescribed muscle design for an active, small-bodied, deep-diving cetacean. HIGHLIGHTS: The locomotor muscle features displayed by the striped dolphin, which are unique among deep divers, enhance oxygen stores but do not limit aerobic capacity. This novel muscle design may facilitate the active lifestyle of this small-bodied deep diver.

ß-guanidinopropionic acid and metformin differentially impact autophagy, mitochondria and cellular morphology in developing C2C12 muscle cells.

The serine/threonine kinase AMP-activated protein kinase (AMPK) is a drug target for the treatment of obesity and type 2 diabetes (T2D). Metformin, a widely prescribed anti-hyperglycemic agent, and ß-guanidinopropionic acid (ß-GPA), a dietary supplement and creatine analog, have been shown to increase activity of AMPK. Macroautophagy is an intracellular degradation pathway for aggregated proteins and dysfunctional organelles, which can be mediated by AMPK. The present study sought to elucidate how metformin and ß-GPA affect cell morphology, AMPK activity, autophagy and mitochondrial morphology and function in developing C2C12 myotubes. ß-GPA reduced myotube diameter and increased length throughout differentiation, while metformin increased myotube diameter only at the 48 h time point. ß-GPA treatment enhanced AMPK signaling and expression of autophagy-related proteins. ß-GPA treatment also increased the density of autophagosomes, autolysosomes, and lysosomes. Metformin also increased activation of AMPK after 48 h, but in contrast to ß-GPA, led to a dramatic reduction in the density of autophagosomes and lysosomes. Both metformin and ß-GPA reduced the mitochondrial oxygen consumption rate, and differentially altered mitochondrial morphology. Obesity and T2D have been shown to increase mitochondrial dysfunction and reduce autophagic flux in skeletal muscle cells. Therefore, ß-GPA may help to alleviate the effects of metabolic disease by increasing autophagic flux in skeletal muscle cells. In contrast, the reduction of autophagy by metformin may lead to dysregulation of mitochondrial maintenance, as well as muscle development.

The effects of dietary ß-guanidinopropionic acid on growth and muscle fiber development in juvenile red porgy, Pagrus pagrus.

ß-guanidinopropionic acid (ß-GPA) has been used in mammalian models to reduce intracellular phosphocreatine (PCr) concentration, which in turn lowers the energetic state of cells. This leads to changes in signaling pathways that attempt to re-establish energetic homeostasis. Changes in those pathways elicit effects similar to those of exercise such as changes in body and muscle growth, metabolism, endurance and health. Generally, exercise effects are beneficial to fish health and aquaculture, but inducing exercise in fishes can be impractical. Therefore, this study evaluated the potential use of supplemental ß-GPA to induce exercise-like effects in a rapidly growing juvenile teleost, the red porgy (Pagrus pagrus). We demonstrate for the first time that ß-GPA can be transported into teleost muscle fibers and is phosphorylated, and that this perturbs the intracellular energetic state of the cells, although to a lesser degree than typically seen in mammals. ß-GPA did not affect whole animal growth, nor did it influence skeletal muscle fiber size or myonuclear recruitment. There was, however, an increase in mitochondrial volume within myofibers in treated fish. GC/MS metabolomic analysis revealed shifts in amino acid composition of the musculature, putatively reflecting increases in connective tissue and decreases in protein synthesis that are associated with ß-GPA treatment. These results suggest that ß-GPA modestly affects fish muscle in a manner similar to that observed in mammals, and that ß-GPA may have application to aquaculture by providing a more practical means of generating some of the beneficial effects of exercise in fishes.

Transparent anemone shrimp (Ancylomenes pedersoni) become opaque after exercise and physiological stress in correlation with increased hemolymph perfusion.

Whole-body transparency, an effective camouflage strategy in many aquatic species, can be disrupted by environmental and/or physiological stressors. We found that tail-flip escape responses temporarily disrupt the transparency of the anemone shrimp Ancylomenes pedersoni After as few as three tail flips, the previously transparent abdominal muscle became cloudy. Eliciting additional tail flips to the point of exhaustion (16±1 s.e.m.; n=23) resulted in complete opacity, though the original transparency returned after 20-60 min of inactivity. We hypothesized that an exercise-induced increase in blood volume between muscle fibers creates regions of low refractive index fluid between high refractive index muscles, thereby increasing light scattering. We documented pre- and post-contraction perfusion by injecting Alexa Fluor 594 wheat germ agglutinin that labeled sarcolemmal surfaces and endothelial cells in contact with hemolymph and found more hemolymph perfused through the abdominal tissue post-exercise, presumably owing to more capillaries opening. In addition, we altered salinity (to 55‰ and 8‰), perforated the abdomen and injected a vasodilator. All three treatments increased both perfusion and opacity, lending further support to our hypothesis that increased hemolymph perfusion to the abdomen is one mechanism that can disrupt a shrimp's transparency. The fact that transparent shrimp at rest have little to no evidence of perfusion to their abdominal musculature (unlike the opaque shrimp Lysmata pederseni, which had more perfusion even at rest) indicates that they may experience significant physiological trade-offs in order to maintain their transparency; specifically, limiting blood flow and thereby reducing oxygen delivery may result in reduced performance.

ß-GPA treatment leads to elevated basal metabolic rate and enhanced hypoxic exercise tolerance in mice.

Treatments that increase basal metabolic rate (BMR) and enhance exercise capacity may be useful therapeutic approaches for treating conditions such as type 2 diabetes, obesity, and associated circulatory problems. ß-guanidinopropionic acid (ß-GPA) supplementation decreases high-energy phosphate concentrations, such as ATP and phosphocreatine (PCr) resulting in an energetic challenge that is similar to both exercise programs and hypoxic conditions. In this study, we administered ß-GPA to mice for 2 or 6 weeks, and investigated the effect on muscle energetic status, body and muscle mass, muscle capillarity, BMR, and normoxic and hypoxic exercise tolerance (NET and HET, respectively). Relative [PCr] and PCr/ATP ratios significantly decreased during both treatment times in the ß-GPA fed mice compared to control mice. Body mass, muscle mass, and muscle fiber size significantly decreased after ß-GPA treatment, whereas muscle capillarity and BMR were significantly increased in ß-GPA fed mice. NET significantly decreased in the 2-week treatment, but was not significantly different in the 6-week treatment. HET significantly decreased in 2-week treatment, but in contrast to NET, significantly increased in the 6-week-treated mice compared to control mice. We conclude that ß-GPA induces a cellular energetic response in skeletal muscle similar to that of chronic environmental hypoxia, and this energetic perturbation leads to elevated BMR and increased hypoxic exercise capacity in the absence of hypoxic acclimation.

Dietary supplementation of ß-guanidinopropionic acid (ßGPA) reduces whole-body and skeletal muscle growth in young CD-1 mice.

Increased AMP-activated protein kinase (AMPK) activity leads to enhanced fatty acid utilization, while also promoting increased ubiquitin-dependent proteolysis (UDP) in mammalian skeletal muscle. ß-guanidinopropionic acid (ßGPA) is a commercially available dietary supplement that has been shown to promote an AMPK-dependent increase in fatty acid utilization and aerobic capacity in mammals by compromising creatine kinase function. However, it remains unknown if continuous ßGPA supplementation can negatively impact skeletal muscle growth in a rapidly growing juvenile. The current study was conducted to examine the effect of ßGPA supplementation on whole-body and skeletal muscle growth in juvenile and young adult mice. Three-week old, post weanling CD-1 mice were fed a standard rodent chow that was supplemented with either 2% (w/w) α-cellulose (control) or ßGPA. Control and ßGPA-fed mice (n = 6) were sampled after 2, 4, and 8 weeks. Whole-body and hindlimb muscle masses were significantly (P < 0.05) reduced in ßGPA-fed mice by 2 weeks. The level of AMPK (T172) phosphorylation increased significantly (P < 0.05) in the gastrocnemius of ßGPA-fed versus control mice at 2 weeks, but was not significantly different at the 4- and 8-week time points. Further analysis revealed a significant (P < 0.05) increase in the skeletal muscle-specific ubiquitin ligase MAFbx/Atrogin-1 protein and total protein ubiquitination in the gastrocnemius of ßGPA versus control mice at the 8-week time point. Our data indicate that feeding juvenile mice a ßGPA-supplemented diet significantly reduced whole-body and skeletal muscle growth that was due, at least in part, to an AMPK-independent increase in UDP.

Effects of resveratrol on growth and skeletal muscle physiology of juvenile southern flounder.

Resveratrol is a naturally occurring antioxidant that has been widely studied in mammals due to its potential to extend lifespan. However, antioxidants may also limit protein damage and therefore reduce rates of protein degradation, providing a potential avenue for enhancing growth in an aquaculture setting. The present study tested the hypotheses that in Southern flounder, Paralichthys lethostigma, resveratrol would decrease protein carbonylation and 4-HNE (indicators of protein and lipid oxidative damage, respectively), levels of ubiquitinylation and LC3 (indicators of non-lysosomal and lysosomal protein degradation, respectively), while having no effect on S6K activation (indicator of protein synthesis). These effects were predicted to increase growth rate. Mitochondrial volume density was also examined since resveratrol may lead to the proliferation of mitochondria, which are the principal source of reactive oxygen species (ROS) that cause oxidative damage. Juvenile fish (n=142) were fed a control diet or a diet supplemented with 600 µg resveratrol per g of food for 16 weeks. Fish treated with resveratrol had a 9% greater length and 33% greater body mass than control fish after 16 weeks. Additionally, there was lower protein carbonylation and lipid 4-HNE within the muscle tissues of treated fish, indicating decreased oxidative damage, and reduced protein ubiquitinylation in the resveratrol fed flounder, indicating less protein degradation. However, there was not a significant difference in LC3, S6K activation, or mitochondrial volume density. These results suggest that resveratrol has positive effects on growth due to its antioxidant properties that reduce non-lysosomal protein degradation.

Large fibre size in skeletal muscle is metabolically advantageous.

Skeletal muscle fibre size is highly variable, and while diffusion appears to limit maximal fibre size, there is no paradigm for the control of minimal size. The optimal fibre size hypothesis posits that the reduced surface area to volume in larger fibres reduces the metabolic cost of maintaining the membrane potential, and so fibres attain an optimal size that minimizes metabolic cost while avoiding diffusion limitation. Here we examine changes during hypertrophic fibre growth in metabolic cost and activity of the Na⁺-K⁺-ATPase in white skeletal muscle from crustaceans and fishes. We provide evidence for a major tenet of the optimal fibre size hypothesis by demonstrating that larger fibres are metabolically cheaper to maintain, and the cost of maintaining the membrane potential is proportional to fibre surface area to volume. The influence of surface area to volume on metabolic cost is apparent during growth in 16 species spanning a 20-fold range in fibre size, suggesting that this principle may apply widely.

Locomotor muscle profile of a deep (Kogia breviceps) versus shallow (Tursiops truncatus) diving cetacean.

When a marine mammal dives, breathing and locomotion are mechanically uncoupled, and its locomotor muscle must power swimming when oxygen is limited. The morphology of that muscle provides insight into both its oxygen storage capacity and its rate of oxygen consumption. This study investigated the m. longissimus dorsi, an epaxial swimming muscle, in the long duration, deep-diving pygmy sperm whale (Kogia breviceps) and the short duration, shallow-diving Atlantic bottlenose dolphin (Tursiops truncatus). Muscle myoglobin content, fiber type profile (based upon myosin ATPase and succinate dehydrogenase assays), and fiber size were measured for five adult specimens of each species. In addition, a photometric analysis of sections stained for succinate dehydrogenase was used to create an index of mitochondrial density. The m. longissimus dorsi of K. breviceps displayed significantly a) higher myoglobin content, b) larger proportion of Type I (slow oxidative) fibers by area, c) larger mean fiber diameters, and d) lower indices of mitochondrial density than that of T. truncatus. Thus, this primary swimming muscle of K. breviceps has greater oxygen storage capacity, reduced ATP demand, and likely a reduced rate of oxygen consumption relative to that of T. truncatus. The locomotor muscle of K. breviceps appears able to ration its high onboard oxygen stores, a feature that may allow this species to conduct relatively long duration, deep dives aerobically.

Ring bands in fish skeletal muscle: reorienting the myofibrils and microtubule cytoskeleton within a single cell.

Skeletal muscle cells (fibers) contract by shortening their parallel subunits, the myofibrils. Here we show a novel pattern of myofibril orientation in white muscle fibers of large black sea bass, Centropristis striata. Up to 48% of the white fibers in fish >1168 g had peripheral myofibrils undergoing an ∼90(o) shift in orientation. The resultant ring band wrapped the middle of the muscle fibers and was easily detected with polarized light microscopy. Transmission electron microscopy showed that the reoriented myofibrils shared the cytoplasm with the central longitudinal myofibrils. A microtubule network seen throughout the fibers surrounded nuclei but was mostly parallel to the long-axis of the myofibrils. In the ring band portion of the fibers the microtubule cytoskeleton also shifted orientation. Sarcolemmal staining with anti-synapsin was the same in fibers with or without ring bands, suggesting that fibers with ring bands have normal innervation and contractile function. The ring bands appear to be related to body-mass or age, not fiber size, and also vary along the body, being more frequent at the midpoint of the anteroposterior axis. Similar structures have been reported in different taxa and appear to be associated with hypercontraction of fibers not attached to a rigid structure (bone) or with fibers with unusually weak links between the sarcolemma and cytoskeleton, as in muscular dystrophy. Fish muscle fibers are attached to myosepta, which are flexible and may allow for fibers to hypercontract and thus form ring bands. The consequences of such a ring band pattern might be to restrict the further expansion of the sarcolemma and protect it from further mechanical stress.

Nuclear DNA content variation associated with muscle fiber hypertrophic growth in fishes.

Muscle fiber hypertrophic growth can lead to an increase in the myonuclear domain (MND), leading to greater diffusion distances within the cytoplasmic volume that each nucleus services. We tested the hypothesis that hypertrophic growth in the white muscle of fishes was associated with increases in the mean DNA content of nuclei, which may be a strategy to offset increasing diffusion constraints. DAPI-stained chicken erythrocytes standards and image analysis were used to estimate nuclear DNA content in erythrocytes and muscle fibers from 17 fish species. Mean diploid (2C) values in fish erythrocytes ranged from 0.78 to 7.2 pg. Erythrocyte 2C values were used to determine ploidy level in muscle tissue of small and large size classes of each species. Within each species, mean muscle fiber diameter was greater in the large size class than the small size class, and MND was significantly greater in larger fibers for 11 of the 17 species. Nuclear DNA content per species in muscle ranged from 2 to 64C. Fiber-size dependent increases in ploidy were observed in nine species, which is consistent with our hypothesis and indicates that endoreduplication is occurring during fiber growth. However, two species exhibited significantly lower ploidy in the larger size class, and the mechanistic basis and potential advantage of this ploidy shift is unclear. These results suggest that increases in ploidy may be a common mechanism to compensate for increases in MND associated with fiber hypertrophy in fishes, although it is likely that other factors also affect ploidy changes that occur in muscle during animal growth.

An evaluation of muscle maintenance costs during fiber hypertrophy in the lobster Homarus americanus: are larger muscle fibers cheaper to maintain?

Large muscle fiber size imposes constraints on muscle function while imparting no obvious advantages, making it difficult to explain why muscle fibers are among the largest cell type. Johnston and colleagues proposed the 'optimal fiber size' hypothesis, which states that some fish have large fibers that balance the need for short diffusion distances against metabolic cost savings associated with large fibers. We tested this hypothesis in hypertrophically growing fibers in the lobster Homarus americanus. Mean fiber diameter was 316±11 µm in juveniles and 670±26 µm in adults, leading to a surface area to volume ratio (SA:V) that was 2-fold higher in juveniles. Na(+)/K(+)-ATPase activity was also 2-fold higher in smaller fibers. (31)P-NMR was used with metabolic inhibitors to determine the cost of metabolic processes in muscle preparations. The cost of Na(+)/K(+)-ATPase function was also 2-fold higher in smaller than in larger diameter fibers. Extrapolation of the SA:V dependence of the Na(+)/K(+)-ATPase over a broad fiber size range showed that if fibers were much smaller than those observed, maintenance of the membrane potential would constitute a large fraction of whole-animal metabolic rate, suggesting that the fibers grow large to reduce maintenance costs. However, a reaction-diffusion model of aerobic metabolism indicated that fibers in adults could attain still larger sizes without diffusion limitation, although further growth would have a negligible effect on cost. Therefore, it appears that decreased fiber SA:V makes larger fibers in H. americanus less expensive to maintain, which is consistent with the optimal fiber size hypothesis.

Growth patterns and nuclear distribution in white muscle fibers from black sea bass, Centropristis striata: evidence for the influence of diffusion.

This study investigated the influence of fiber size on the distribution of nuclei and fiber growth patterns in white muscle of black sea bass, Centropristis striata, ranging in body mass from 0.45 to 4840 g. Nuclei were counted in 1 µm optical sections using confocal microscopy of DAPIand Acridine-Orange-stained muscle fibers. Mean fiber diameter increased from 36±0.87 µm in the 0.45 g fish to 280±5.47 µm in the 1885 g fish. Growth beyond 2000 g triggered the recruitment of smaller fibers, thus significantly reducing mean fiber diameter. Nuclei in the smaller fibers were exclusively subsarcolemmal (SS), whereas in larger fibers nuclei were more numerous and included intermyofibrillar (IM) nuclei. There was a significant effect of body mass on nuclear domain size (F=118.71, d.f.=3, P<0.0001), which increased to a maximum in fish of medium size (282-1885 g) and then decreased in large fish (>2000 g). Although an increase in the number of nuclei during fiber growth can help preserve the myonuclear domain, the appearance of IM nuclei during hypertrophic growth seems to be aimed at maintaining short effective diffusion distances for nuclear substrates and products. If only SS nuclei were present throughout growth, the diffusion distance would increase in proportion to the radius of the fibers. These observations are consistent with the hypothesis that changes in nuclear distribution and fiber growth patterns are mechanisms for avoiding diffusion limitation during animal growth.

Molecules in motion: influences of diffusion on metabolic structure and function in skeletal muscle.

Metabolic processes are often represented as a group of metabolites that interact through enzymatic reactions, thus forming a network of linked biochemical pathways. Implicit in this view is that diffusion of metabolites to and from enzymes is very fast compared with reaction rates, and metabolic fluxes are therefore almost exclusively dictated by catalytic properties. However, diffusion may exert greater control over the rates of reactions through: (1) an increase in reaction rates; (2) an increase in diffusion distances; or (3) a decrease in the relevant diffusion coefficients. It is therefore not surprising that skeletal muscle fibers have long been the focus of reaction-diffusion analyses because they have high and variable rates of ATP turnover, long diffusion distances, and hindered metabolite diffusion due to an abundance of intracellular barriers. Examination of the diversity of skeletal muscle fiber designs found in animals provides insights into the role that diffusion plays in governing both rates of metabolic fluxes and cellular organization. Experimental measurements of metabolic fluxes, diffusion distances and diffusion coefficients, coupled with reaction-diffusion mathematical models in a range of muscle types has started to reveal some general principles guiding muscle structure and metabolic function. Foremost among these is that metabolic processes in muscles do, in fact, appear to be largely reaction controlled and are not greatly limited by diffusion. However, the influence of diffusion is apparent in patterns of fiber growth and metabolic organization that appear to result from selective pressure to maintain reaction control of metabolism in muscle.