Stephen T. Kinsey

Physiological effects of short-term salinity changes on Ruppia maritima

Changes in Ruppia maritimaL. leaf-tissue osmolality, compatible solute synthesis in leaf tissues, and maximum effective quantum yield in response to short-term changes in salinity were investigated. Plants cultured at 20‰ S were exposed to 0‰ S, 10‰ S (half-ambient), 20‰ S (ambient), and 40‰ S (twice-ambient) salinities. Total and non-vacuolar leaf osmolality for cultured plants significantly decreased (total: from 1464 ± 266 to 712± 210 mmol kg −1 ; non-vacuolar: from 880 ± 108 to 257 ± 80 mmol kg −1 ) or increased (total: from 1464 to 2532 ± 673 mmol kg −1 , non-vacuolar from 880 to 1168 ± 15 mmol kg −1 ), within 1 min of exposure to 0 and 40‰ S, respectively. After the initial rapid change in leaf osmolality, values were relatively constant for the first 180 min of exposure. Osmolality then changed again over the period from 1 to 2 days post-treatment with values again increasing (40‰ S: total = 3152 ± 335 mmol kg −1 , non-vacuolar = 1967 ± 103 mmol kg −1 )o r decreasing (0‰ S: total = 357± 46 mmol kg −1 , non-vacuolar = 74± 32 mmol kg −1 ) with salinity. Soluble and total carbohydrates in leaf tissues responded differently to changing salinity. Total carbohydrates decreased by 65%, while soluble levels increased by 34%, in high salinity. Total and soluble proline levels increased (63 and 18%, respectively), and decreased (−36 and −20% for 10‰ S; −72 and −32% for 0‰ S, respectively), with salinity. These results suggest that both proline and soluble carbohydrates act as compatible solutes. Maximum quantum yields (Fv/Fm) were measured over a 48 h period in response to changes in medium salinity and inorganic carbon (ambient and ∼2.0 mM bicarbonate-equilibrium treatments). Fv/Fm exhibited significant variation in response to salinity, bicarbonate-level and time as main effects, with significant interactions. Quantum yields were lowest in the 0 and 40‰ S treatments; the 10 and 20‰ S treatments had significantly higher quantum yields. These short-term responses indicated that both increases and reductions of external ion concentrations are initially stressful for R. maritima, but that it can physiologically adjust after several days.

Diffusional anisotropy is induced by subcellular barriers in skeletal muscle

The time‐ and orientational‐dependence of phosphocreatine (PCr) diffusion was measured using pulsed‐field gradient nuclear magnetic resonance (PFG‐NMR) as a means of non‐invasively probing the intracellular diffusive barriers of skeletal muscle. Red and white skeletal muscle from fish was used because fish muscle cells are very large, which facilitates the examination of diffusional barriers in the intracellular environment, and because they have regions of very homogeneous fiber type. Fish were cold‐acclimated (5°C) to amplify the contrast between red and white fibers. Apparent diffusion coefficients, D, were measured axially, D∥, and radially, D⟂, in small muscle strips over a time course ranging from 12 to 700 ms. Radial diffusion was strongly time dependent in both fiber types, and D⟂ decreased with time until a steady‐state value was reached at a diffusion time ≊ 100 ms. Diffusion was also highly anisotropic, with D∥ being higher than D⟂ for all time points. The time scale over which changes in D⟂ occurred indicated that the observed anisotropy was not a result of interactions with the thick and thin filament lattice of actin and myosin or restriction within the cylindrical sarcolemma, as has been previously suggested. Rather, the sarcoplasmic reticulum (SR) and mitochondria appear to be the principal intracellular structures that inhibit mobility in an orientation‐dependent manner. This work is the first example of diffusional anisotropy induced by readily identifiable intracellular structures. Copyright

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.

Metabolic influences of fiber size in aerobic and anaerobic locomotor muscles of the blue crab, Callinectes sapidus.

SUMMARY Diameters of some white locomotor muscle fibers in the adult blue crab, Callinectes sapidus, exceed 500 μm whereas juvenile white fibers are <100 μm. It was hypothesized that aerobically dependent processes, such as metabolic recovery following burst contractions, will be significantly impeded in the large white fibers. In addition, dark aerobic fibers of adults, which rely on aerobic metabolism for both contraction and recovery, grow as large as the white fibers. These large aerobic fibers are subdivided, however, thus decreasing the effective diameter of each metabolic functional unit and enabling aerobic contraction. The two goals of this study were: (1) to characterize the development of subdivisions in the dark levator muscle fibers and (2) to monitor post-contractile metabolism as a function of fiber size in aerobic and anaerobic levator muscles. Dark levator muscle fibers from crabs ranging from <0.1 g to >190 g were examined with transmission electron microscopy to determine the density of mitochondria and subdivision diameters. Across all size classes, there was a constant mitochondrial fractional area (25% of the total subdivision area) and subdivision size (mean diameter of 36.5±2.7 μm). Thus, blue crab dark levator fibers are unusual in having metabolic functional units (subdivisions) that do not increase in size during development while the contractile functional units (fibers) grow hypertrophically. The body mass scaling of post-contractile lactate dynamics was monitored during recovery from anaerobic, burst exercise in white and dark muscle, and in hemolymph. There were no differences among size classes in lactate accumulation during exercise in either muscle. However, in white fibers from large crabs, lactate continued to increase after exercise, and lactate removal from tissues required a much longer period of time relative to smaller crabs. Differences in lactate removal among size classes were less pronounced in dark fibers, and post-contractile lactate accumulation was significantly higher in white than in dark fibers from large animals. These data suggest that the large white fibers invoke anaerobic metabolism following contraction to accelerate certain phases of metabolic recovery that otherwise would be overly slow. This implies that, in addition to the typical mass-specific decrease in oxidative capacity that accompanies increases in animal mass, aerobic metabolic processes become increasingly limited by surface area to volume and intracellular diffusion constraints in developing white muscle fibers.

The influence of oxygen and high-energy phosphate diffusion on metabolic scaling in three species of tail-flipping crustaceans

SUMMARY We examined the influence of intracellular diffusion of O2 and high-energy phosphate (HEP) molecules on the scaling with body mass of the post-exercise whole-animal rate of O2 consumption (V̇O2) and muscle arginine phosphate (AP) resynthesis rate, as well as muscle citrate synthase (CS) activity, in three groups of tail-flipping crustaceans. Two size classes in each of three taxa (Palaemonetes pugio, Penaeus spp. and Panulirus argus) were examined that together encompassed a 27,000-fold range in mean body mass. In all species, muscle fiber size increased with body mass and ranged in diameter from 70±1.5 to 210±8.8 μm. Thus, intracellular diffusive path lengths for O2 and HEP molecules were greater in larger animals. The body mass scaling exponent, b, for post-tail flipping V̇O2 (b=–0.21) was not similar to that for the initial rate of AP resynthesis (b=–0.12), which in turn was different from that of CS activity (b=0.09). We developed a mathematical reaction–diffusion model that allowed an examination of the influence of O2 and HEP diffusion on the observed rate of aerobic flux in muscle. These analyses revealed that diffusion limitation was minimal under most conditions, suggesting that diffusion might act on the evolution of fiber design but usually does not directly limit aerobic flux. However, both within and between species, fibers were more diffusion limited as they grew larger, particularly when hemolymph PO2 was low, which might explain some of the divergence in the scaling exponents of muscle aerobic capacity and muscle aerobic flux.

The effects of rapid salinity change on in vivo arginine kinase flux in the juvenile blue crab, Callinectes sapidus

The effect of acclimation salinity and salinity changes on the concentration of high-energy phosphate metabolites and arginine kinase (AK) flux was examined in vivo in juvenile blue crabs using 31P-nuclear magnetic resonance (NMR). Crabs were acclimated for 7 days to a salinity of 5 or 35 per thousand and then placed in a flow apparatus that could sustain the animals while NMR spectra were acquired. Crabs were subjected to either hyperosmotic salinity changes, where an animal acclimated to 5 per thousand was exposed to a salinity of 35 per thousand, or hyposmotic changes, which involved the reciprocal exchange. Neither acclimation salinity nor salinity change had a significant effect on the concentrations of arginine phosphate, inorganic phosphate or ATP. 31P-NMR saturation transfer experiments were used to determine the effect of salinity on the forward and reverse flux of the AK reaction. There was no significant effect of acclimation salinity or salinity change on the flux rate through this reaction. This is in contrast to previous results, which showed that AK flux in isolated muscle was sensitive to prevailing osmotic conditions (Holt and Kinsey, J. Exp. Biol. 205 (2002) 1775-1785). The present study indicates that the integrated osmoregulatory capacity of the intact animal is sufficient to preserve cellular energy status and enzyme function during acute salinity changes.

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.

Novel locomotor muscle design in extreme deep-diving whales

SUMMARY Most marine mammals are hypothesized to routinely dive within their aerobic dive limit (ADL). Mammals that regularly perform deep, long-duration dives have locomotor muscles with elevated myoglobin concentrations that are composed of predominantly large, slow-twitch (Type I) fibers with low mitochondrial volume densities (Vmt). These features contribute to extending ADL by increasing oxygen stores and decreasing metabolic rate. Recent tagging studies, however, have challenged the view that two groups of extreme deep-diving cetaceans dive within their ADLs. Beaked whales (including Ziphius cavirostris and Mesoplodon densirostris) routinely perform the deepest and longest average dives of any air-breathing vertebrate, and short-finned pilot whales (Globicephala macrorhynchus) perform high-speed sprints at depth. We investigated the locomotor muscle morphology and estimated total body oxygen stores of several species within these two groups of cetaceans to determine whether they (1) shared muscle design features with other deep divers and (2) performed dives within their calculated ADLs. Muscle of both cetaceans displayed high myoglobin concentrations and large fibers, as predicted, but novel fiber profiles for diving mammals. Beaked whales possessed a sprinters fiber-type profile, composed of ~80% fast-twitch (Type II) fibers with low Vmt. Approximately one-third of the muscle fibers of short-finned pilot whales were slow-twitch, oxidative, glycolytic fibers, a rare fiber type for any mammal. The muscle morphology of beaked whales likely decreases the energetic cost of diving, while that of short-finned pilot whales supports high activity events. Calculated ADLs indicate that, at low metabolic rates, both beaked and short-finned pilot whales carry sufficient onboard oxygen to aerobically support their dives.

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

SUMMARY 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. 31P-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.