Kristin M. Hardy

The long and winding road: influences of intracellular metabolite diffusion on cellular organization and metabolism in skeletal muscle.

SUMMARY A fundamental principle of physiology is that cells are small in order to minimize diffusion distances for O2 and intracellular metabolites. In skeletal muscle, it has long been recognized that aerobic fibers that are used for steady state locomotion tend to be smaller than anaerobic fibers that are used for burst movements. This tendency reflects the interaction between diffusion distances and aerobic ATP turnover rates, since maximal intracellular diffusion distances are ultimately limited by fiber size. The effect of diffusion distance on O2 flux in muscle has been the subject of quantitative analyses for a century, but the influence of ATP diffusion from mitochondria to cellular ATPases on aerobic metabolism has received much less attention. The application of reaction–diffusion mathematical models to experimental measurements of aerobic metabolic processes has revealed that the extreme diffusion distances between mitochondria found in some muscle fibers do not necessarily limit the rates of aerobic processes per se, as long as the metabolic process is sufficiently slow. However, skeletal muscle fibers from a variety of animals appear to have intracellular diffusion distances and/or fiber sizes that put them on the brink of diffusion limitation. Thus, intracellular metabolite diffusion likely influences the evolution of muscle design and places limits on muscle function.

A skeletal muscle model of extreme hypertrophic growth reveals the influence of diffusion on cellular design

Muscle fibers that power swimming in the blue crab Callinectes sapidus are <80 microm in diameter in juveniles but grow hypertrophically, exceeding 600 microm in adults. Therefore, intracellular diffusion distances become progressively greater as the animals grow and, in adults, vastly exceed those in most cells. This developmental trajectory makes C. sapidus an excellent model for characterization of the influence of diffusion on fiber structure. The anaerobic light fibers, which power burst swimming, undergo a prominent shift in organelle distribution with growth. Mitochondria, which require O2 and rely on the transport of small, rapidly diffusing metabolites, are evenly distributed throughout the small fibers of juveniles, but in the large fibers of adults they are located almost exclusively at the fiber periphery where O2 concentrations are high. Nuclei, which do not require O2, but rely on the transport of large, slow-moving macromolecules, have the inverse pattern: they are distributed peripherally in small fibers but are evenly distributed across the large fibers, thereby reducing diffusion path lengths for large macromolecules. The aerobic dark fibers, which power endurance swimming, have evolved an intricate network of cytoplasmically isolated, highly perfused subdivisions that create the short diffusion distances needed to meet the high aerobic ATP turnover demands of sustained contraction. However, fiber innervation patterns are the same in the dark and light fibers. Thus the dark fibers appear to have disparate functional units for metabolism (fiber subdivision) and contraction (entire fiber). Reaction-diffusion mathematical models demonstrate that diffusion would greatly constrain the rate of metabolic processes without these developmental changes in fiber structure.

Does intracellular metabolite diffusion limit post-contractile recovery in burst locomotor muscle?

SUMMARY Post-metamorphic growth in the blue crab entails an increase in body mass that spans several orders of magnitude. The muscles that power burst swimming in these animals grow hypertrophically, such that small crabs have fiber diameters that are typical of most cells (<60 μm) while in adult animals the fibers are giant (>600 μm). Thus, as the animals grow, their muscle fibers cross and greatly exceed the surface area to volume ratio (SA:V) and intracellular diffusion distance threshold that is adhered to by most cells. Large fiber size should not impact burst contractile function, but post-contractile recovery may be limited by low SA:V and excessive intracellular diffusion distances. A number of changes occur in muscle structure, metabolic organization and metabolic flux during development to compensate for the effects of increasing fiber size. In the present study, we examined the impact of intracellular metabolite diffusive flux on the rate of post-contractile arginine phosphate (AP) resynthesis in burst locomotor muscle from small and large animals. AP recovery was measured following burst exercise, and these data were compared to a mathematical reaction–diffusion model of aerobic metabolism. The measured rates of AP resynthesis were independent of fiber size, while simulations of aerobic AP resynthesis yielded lower rates in large fibers. These contradictory findings are consistent with previous observations that there is an increased reliance on anaerobic metabolism for post-contractile metabolic recovery in large fibers. However, the model results suggest that the interaction between mitochondrial ATP production rates, ATP consumption rates and diffusion distances yield a system that is not particularly close to being limited by intracellular metabolite diffusion. We conclude that fiber SA:V and O2 flux exert more control than intracellular metabolite diffusive flux over the developmental changes in metabolic organization and metabolic fluxes that characterize these muscles.

Gene transcripts encoding hypoxia-inducible factor (HIF) exhibit tissue- and muscle fiber type-dependent responses to hypoxia and hypercapnic hypoxia in the Atlantic blue crab, Callinectes sapidus

Hypoxia inducible factor (HIF) is a transcription factor that under low environmental oxygen regulates the expression of suites of genes involved in metabolism, angiogenesis, erythropoiesis, immune function, and growth. Here, we isolated and sequenced partial cDNAs encoding hif-α and arnt/hif-β from the Atlantic blue crab, Callinectes sapidus, an estuarine species that frequently encounters concurrent hypoxia (low O(2)) and hypercapnia (elevated CO(2)). We then examined the effects of acute exposure (1h) to hypoxia (H) and hypercapnic hypoxia (HH) on relative transcript abundance for hif-α and arnt/hif-β in different tissues (glycolytic muscle, oxidative muscle, hepatopancreas, gill, and gonads) using quantitative real-time RT-PCR. Our results indicate that hif-α and arnt/hif-β mRNAs were constitutively present under well-aerated normoxia (N) conditions in all tissues examined. Further, H and HH exposure resulted in both tissue-specific and muscle fiber type-specific effects on relative hif-α transcript abundance. In the gill and glycolytic muscle, relative hif-α mRNA levels were significantly lower under H and HH, compared to N, while no change (or a slight increase) was detected in oxidative muscle, hepatopancreas and gonadal tissues. H and HH did not affect relative transcript abundance for arnt/hif-β in any tissue or muscle fiber type. Thus, in crustaceans the HIF response to H and HH appears to involve changes in hif transcript abundance, with variation in hif-α and arnt/hif-β transcriptional dynamics occurring in both a tissue- and muscle fiber type-dependent manner.

Effect of hypercapnic hypoxia and bacterial infection (Vibrio campbellii) on protein synthesis rates in the Pacific whiteleg shrimp, Litopenaeus vannamei.

Estuarine species frequently encounter areas of simultaneously low dissolved O2 (hypoxia) and high CO2 (hypercapnia). Organisms exposed to hypoxia experience a metabolic depression that serves to decrease ATP utilization and O2 demand during stress. This downregulation is typically facilitated by a reduction in protein synthesis, a process that can be responsible for up to 60% of basal metabolism. The added effects of hypercapnia, however, are unclear. Certain decapods also exhibit a metabolic depression in response to bacterial challenges, leading us to hypothesize that protein synthesis may also be reduced during infection. In the present study, we examined the effects of hypoxia (H), hypercapnic hypoxia (HH), and bacterial infection (Vibrio campbellii) on tissue-specific (muscle and hepatopancreas) fractional protein synthesis rates (ks) in Litopenaeus vannamei. We observed a significant decrease in ks in muscle after 24 h exposure to both H and HH, and in hepatopancreas after 24 h exposure to HH. Thus ks is responsive to changes in O2, and the combined effect of hypercapnic hypoxia on ks is more severe than hypoxia alone. These reductions in ks appear to be driven by changes in RNA translational efficiency (kRNA), and not RNA capacity (Cs). Bacterial infection, however, had no significant effect on ks in either tissue. These results suggest that crustaceans reduce metabolic demand during environmental hypoxia by reducing global protein synthesis, and that this effect is magnified when hypercapnia is concomitantly present. Conversely, an immune-mediated metabolic depression is not associated with a decrease in overall protein production.

Endocrine and metabolic impacts of warming aquatic habitats: differential responses between recently isolated populations of a eurythermal desert pupfish

Imperilled desert fishes occupying isolated habitats that limit dispersal are vulnerable to temperature alterations because of a changing climate. Here, we identify differences in temperature effects on thyroid hormone signalling, anaerobic metabolism and thyroid hormone-mediated metabolic gene expression between recently isolated populations of a desert pupfish.