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First published online March 17, 2006
Journal of Experimental Biology 209, 1159-1168 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02101

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Review Article

Tribute to R. G. Boutilier: The role for skeletal muscle in the hypoxia-induced hypometabolic responses of submerged frogs

T. G. West^1,*, P. H. Donohoe², J. F. Staples³ and G. N. Askew⁴

¹ Imperial College London, Biomedical Sciences, Biological Nanoscience Section, SAF-Building, South Kensington, London, SW7 2AZ, UK
² Department of Physiology, Otago School of Medical Sciences, Dunedin, New Zealand
³ Department of Biology, University of Western Ontario, London, ON, N6A 5B7, Canada
⁴ Institute of Integrative and Comparative Biology, University of Leeds, Leeds, LS2 9JT, UK

^* Author for correspondence (e-mail: t.west{at}imperial.ac.uk)

Accepted 17 January 2006

	Summary

TOP Summary Introduction A model of anoxic... Frogs and their muscles... Muscle function during... Conclusion Appendix References

 
Much of Bob Boutilier's research characterised the subcellular, organ-level and in vivo behavioural responses of frogs to environmental hypoxia. His entirely integrative approach helped to reveal the diversity of tissue-level responses to O₂ lack and to advance our understanding of the ecological relevance of hypoxia tolerance in frogs. Work from Bob's lab mainly focused on the role for skeletal muscle in the hypoxic energetics of overwintering frogs. Muscle energy demand affects whole-body metabolism, not only because of its capacity for rapid increases in ATP usage, but also because hypometabolism of the large skeletal muscle mass in inactive animals impacts so greatly on in vivo energetics. The oxyconformance and typical hypoxia-tolerance characteristics (e.g. suppressed heat flux and preserved membrane ion gradients during O₂ lack) of skeletal muscle in vitro suggest that muscle hypoperfusion in vivo is possibly a key mechanism for (i) downregulating muscle and whole-body metabolic rates and (ii) redistributing O₂ supply to hypoxia-sensitive tissues. The gradual onset of a low-level aerobic metabolic state in the muscle of hypoxic, cold-submerged frogs is indeed important for slowing depletion of on-board fuels and extending overwintering survival time. However, it has long been known that overwintering frogs cannot survive anoxia or even severe hypoxia. Recent work shows that they remain sensitive to ambient O₂ and that they emerge rapidly from quiescence in order to actively avoid environmental hypoxia. Hence, overwintering frogs experience periods of hypometabolic quiescence interspersed with episodes of costly hypoxia avoidance behaviour and exercise recovery.

In keeping with this flexible physiology and behaviour, muscle mechanical properties in frogs do not deteriorate during periods of overwintering quiescence. On-going studies inspired by Bob Boutilier's integrative mindset continue to illuminate the cost–benefit(s) of intermittent locomotion in overwintering frogs, the constraints on muscle function during hypoxia, the mechanisms of tissue-level hypometabolism, and the details of possible muscle atrophy resistance in quiescent frogs.

Key words: Rana spp., hypoxia, skeletal muscle, resting metabolism, contraction

	Introduction

TOP Summary Introduction A model of anoxic... Frogs and their muscles... Muscle function during... Conclusion Appendix References

 
One of Bob Boutilier's outstanding contributions to the field of comparative biology is his body of research into amphibian physiology, encompassing areas as diverse as subcellular metabolic regulation to the effects of environmental change on amphibian behaviour. Bob's integrative mindset was a major influence on his research group investigating how frogs (Rana temporaria L.) cope with acute and chronic environmental O₂ lack. Research from the Zoology lab at Cambridge University during the period 1993–2003 addressed (i) cellular, molecular and mitochondrial responses to hypoxia/anoxia in skeletal muscle, cardio-myocytes and hepatocytes (St Pierre et al., 2000; Currie and Boutilier, 2001; Court and Boutilier, 2005), (ii) patterns of O₂-dependent heat and ion fluxes in isolated intact skeletal muscles (West and Boutilier, 1998; Donohoe et al., 2000), (iii) in vivo metabolism in quiescent overwintering frogs (Donohoe and Boutilier, 1998; Donohoe et al., 1998) and (iv) activity patterns of free-moving frogs submerged in temperature and O₂ gradients (Tattersall and Boutilier, 1997, 1999). Studies that characterise the integrative responses to environmental O₂ lack are important for guiding researchers to relevant (e.g. tissue- and species-specific) mechanistic hypotheses of cellular hypoxia-tolerance and for increasing our understanding of the energetics and physiological ecology of hypoxia-tolerant species.

View larger version (26K): [in this window] [in a new window]   Fig. 1. Boutilier's hypothetical time course of cellular ATP-turnover during anoxia or hypothermia includes the events that promote energy imbalance and cause cell death (red line; Boutilier, 2001a). Neither hypoxia-tolerant nor hypoxia-sensitive cells can survive prolonged energy imbalance, but cells of hypoxia-tolerant animals are adapted to avoid energy imbalance through the coordinated suppression of energy demand and energy supply (inset). Reversible metabolic suppression thus greatly extends hypoxic survival time. (From Boutilier, 2001a.)

Integrative studies of energy partitioning are essential for assessing the cost–benefit of intermittent locomotion in heterogeneous environments, especially in overwintering conditions where air access may be cut off for months. Improved understanding of energy supply/demand relationships in cold-submerged frogs will also help to predict the impact of water quality and environmental change on survival and on the capacity for strenuous reproductive activities upon emergence from hibernation. The aim of this tribute paper is to review studies that have helped to characterise the hypoxia/anoxia tolerance capacity of skeletal muscle in R. temporaria and to highlight some of the on-going work into the ionic homeostasis and contractile performance of frog muscle during acute and chronic O₂ lack.

	A model of anoxic cell death

TOP Summary Introduction A model of anoxic... Frogs and their muscles... Muscle function during... Conclusion Appendix References

 
Much has been written about the causes of anoxia induced energy imbalances in vertebrate cells and about the proposed mechanisms needed to avoid the catastrophic events that lead to cell death (see Hochachka et al., 1996; Boutilier, 2001a; Boutilier, 2001b; Hochachka and Lutz, 2001). Fig. 1 is a hypothetical time course for anoxia-induced cell death, as presented originally by Boutilier (Boutilier, 2001a) in a timely review of the hypoxia–hypothermia field. One feature of this model is that the danger period for all cells begins as anaerobic ATP supply starts to fall short of the ATP demanded by various cellular ATPases. Irreversible damage begins essentially as ion gradients across cellular and subcellular membranes fail; the ensuing osmosis and proteolysis cause membrane rupture and necrotic cell death. A second feature of the model is that anoxia-tolerant animals and cells are not any less susceptible to mismatches in ATP supply/demand than are anoxia-sensitive systems. However, anoxia-tolerant animals are able to avoid energy imbalances by entering into `regulated hypometabolic states' (see inset, Fig. 1) when environmental O₂ is limiting. Energetic homeostasis is achieved through the coordinated downregulation of ATP supply and demand. Hypometabolism, together with the large on-board reserves of tissue glycogen in anoxia-tolerant animals, provide the capacity for the organism to endure natural cycles of environmental O₂ availability and thus prevent the onset of necrotic cell death.

The shoulder of the anoxic time course in Fig. 1, where energy imbalance leads directly to failure of membrane ion gradients, highlights the critical role for membrane–metabolic coupling in cell survival of O₂ lack. Mechanisms that promote hypoxia-induced hypometabolism and membrane–metabolic coupling in different vertebrate systems are discussed in detail elsewhere (Hochachka et al., 1996; Boutilier, 2001a; Boutilier, 2001b; Hochachka and Lutz, 2001; Lutz and Nilsson, 2004). The general events that have been identified and/or proposed are; (i) reallocation of ATP supply away from `dispensable' energy consumers such as protein and RNA/DNA synthesis (Land and Hochachka, 1994; Buttgereit and Brand, 1995) and toward the ATP-dependent ion pumps; (ii) reduction in the rates of ion pumping; (iii) reduction of membrane ion-leak; (iv) reduction of glycolytic ATP production to match overall anaerobic energy demand; and (v) the capacity for rapid return to normoxic rates of energy supply/demand when O₂ availability increases. Ion pumping is a dominant energy sink during anoxia because pump activities are not inhibited as completely as other processes (e.g. see Buck and Hochachka, 1993). In cells where anoxia does not affect transmembrane ion gradients there must be a reduction in passive channel-mediated ion fluxes to match the suppression of ATP-dependent pump rates (Hochachka et al., 1996; Hochachka and Lutz, 2001). This so-called channel- and pump-arrest may be mediated by reversible inhibition and/or changes in density of membrane channel proteins (Perez-Pinzon et al., 1992). There are important species and tissue-type variations on this theme, for example, the discussion on anoxia-tolerance in vertebrate brains (Lutz and Nilsson, 2004), but it is generally true that hypometabolic homeostasis requires coordinated stabilization of cytosolic ATP concentration/turnover and transmembrane ion gradients.

	Frogs and their muscles as models of hypoxia tolerance

TOP Summary Introduction A model of anoxic... Frogs and their muscles... Muscle function during... Conclusion Appendix References

 
The common frog (Rana temporaria) possesses only modest hypoxia/anoxia tolerance capacity in comparison to better known anoxia-tolerant models, like some freshwater turtles (e.g. Chrysemys picta) and the crucian carp Carassius carassius (Bradford, 1983; Wegener and Krause, 1993; Boutilier et al., 1997; Lutz and Nilsson, 2004). Frogs survive severe hypoxia for hours to days depending on ambient temperature, whereas turtles and carp tolerate total anoxia for several weeks or even months. Nevertheless, interest in the frog model has grown, largely from the desire to understand the physiological and behavioural mechanisms that frogs without air-access must exploit in order to survive in waterways that are naturally heterogenous with respect to temperature and dissolved O₂.

It has been argued previously (e.g. Boutilier et al., 1997) that the modest hypoxia-tolerance capacity of frogs may mean that they express a wide range of tissue-level hypoxia tolerances; from true anoxia-tolerance to O₂-dependence. We hypothesised that the skeletal muscle of R. temporaria displays the typical characteristics of cellular anoxia-tolerance, whereas the core organs like liver, brain and heart require a minimal level of continuous O₂ delivery. Periods of muscular quiescence are certainly important for energy conservation in overwintering frogs, not only because of the shut-down of ATP demand for contraction, but also because of the overall savings brought about by hypometabolism of the large skeletal muscle mass (making up 40% of total body mass). Similarly, non-hibernating frogs at summer temperatures will need to conserve energy if they have to evade predators by remaining submerged in hypoxic water. It may be that hypoperfusion of skeletal muscle accounts for the bulk of the whole-body metabolic adjustment to hypoxia by lowering muscle energy demand and sparing blood O₂ and substrates for tissues that tolerate hypoxia to a lesser degree.

Room temperature anoxic responses
It has been known for more than 70 years (Feng, 1932) that the anoxic heat flux of frog sartorius is stable at 20–30% of the normoxic rate at 20°C. Hypometabolism in sartorius was reversible and repeatable (Feng, 1932), and the excess recovery heat after re-oxygenation was exquisitely responsive to the level of metabolic load imposed on the muscle during anoxia. Recent experiments have further demonstrated that (i) step changes in O₂ availability cause step changes in the stable metabolic heat production of isolated skeletal muscle (West and Boutilier, 1998; Boutilier, 2001b) and of the whole animal (Schulz et al., 1991); (ii) the extent of suppression of muscle heat flux (to 20–30% of normal; Fig. 2) matches the reduction in muscle ATP synthesis as well as the anoxia-induced change in whole-animal metabolism (Schulz et al., 1991; West and Boutilier, 1998; Vezzoli et al., 2003; Vezzoli et al., 2004); and (iii) muscle ATP concentration is protected during anoxia. Anaerobic lactate accumulation is relatively low in anoxic sartorius (West and Boutilier, 1998) and gastrocnemius (Vezzoli et al., 2004) at rest. This may partly reflect the fact that lactate efflux is favoured in isolated muscle preparations that are perifused with lactate-free solution (Boutilier et al., 1986). However, the chief reason seems to be that glycolytic activation is minimal in non-contracting anoxic muscle (Hsu and Dawson, 2003). Interestingly, Vezzoli et al. (Vezzoli et al., 2004) observed that the time course of suppression of ATP synthesis rate was essentially paralleled by changes in the phosphocreatine (PCr) breakdown rate, perhaps indicating that anaerobic energy turnover in resting and/or hypometabolic muscle is controlled to a large extent by ATP supply from PCr hydrolysis. Glycolysis clearly has a role in energy provision, particularly at higher temperatures (15°C) and when PCr is exhausted (Vizzoli et al., 2004), but the low level of glycolytic activation overall is consistent with the so-called reversed Pasteur effect that is characteristic of so many other anoxia-tolerant tissues (Hochachka et al., 1996; Hochachka and Lutz, 2001). The apparent dependence of anoxic muscle on the limited on-board PCr reserves emphasises the fundamental importance of hypometabolism for prolonging energetic homeostasis.

View larger version (21K): [in this window] [in a new window]   Fig. 2. The effect of a cycle of normoxia, anoxia and re-oxgenation on heat output and interstitial lactate and K+ concentrations (means ± s.e.m.) in frog sartorius muscle at 20°C. Interstitial lactate and K+ levels were determined from microdialysis effluents of muscle interstitial space during continuous micro-calorimetric monitoring of heat flux. The lack of change in interstitial [K+] suggests that anoxia and re-oxygenation did not increase K+ loss from skeletal muscle. (From West and Boutilier, 1998.)

There is also evidence of tight membrane–metabolic coupling during acute room temperature anoxia in frog muscle. Interstitial K⁺ concentration is stable during anoxia and reoxygenation (Fig. 2), indicating that maintenance of membrane integrity does not depend on O₂ availability (West and Boutilier, 1998). Total (i.e. not pump-specific) fractional efflux of ²²Na⁺ is also suppressed during anoxia at 20°C (Fig. 3; T. G. West and R. G. Boutilier, unpublished observations). The pattern of fractional efflux loosely parallels that of total metabolic heat flux (Fig. 2) during anoxia and reoxygenation, possibly indicating that Na⁺ homeostasis is closely coupled to muscle energy economy. Preliminary data on a small number of sartorius preparations (N=2; T. G. West and R. G. Boutilier, unpublished observations) suggests that Na⁺ uptake may also be suppressed during anoxia. Reduced leakiness of sartorius membranes could directly account for reduced effluxes, but further studies are needed to quantify (i) the significance of any reduced Na⁺ channel leak and (ii) Na⁺-pump specific (i.e. ouabain-dependent) changes in ion fluxes in cycles of normoxia–anoxia–reoxygenation.

View larger version (14K): [in this window] [in a new window]   Fig. 3. Time courses for fractional efflux of 22Na+ (mean ± s.e.m.) in isolated frog sartorius (T. G. West and R. G. Boutilier, unpublished observations) at 20°C during normoxia (98% O2:2% CO2; open symbols) and anoxia (98% N2:2% CO2; closed symbols). Fractional efflux was determined using methods modified from Overgaard et al. (Overgaard et al., 1997). Briefly, whole sartorius muscles were first preloaded with 22Na+ (2 µCi ml–1 for 30 min at 20°C in oxygenated Ringer solution). Then the muscles were washed (4x 10 min) in ice-cold Na-free Ringer; a process expected to remove extracellular 22Na+ (see Overgaard et al., 1997). Finally, the muscles were transferred through 2 ml volumes of normal Ringer at 20°C with the treatments and time courses shown. Fractional efflux was calculated as the amount of 22Na+ released to the bathing medium during each time interval divided by the total amount of 22Na+ loaded into the muscle. The total 22Na+ load was the cumulative sum of 22Na+ c.p.m. released plus the c.p.m. remaining in the muscle at the end of the experiment (measured in trichloroacetic acid extracts). Fractional efflux after 30 min anoxia was significantly lower (t-test; t=4.16, P=0.005) than that of normoxic controls at the same time point (i.e. 70 min into the time courses). The ouabain-sensitive component of total fractional efflux (i.e. giving the portion of efflux attributed specifically to pump activity) could not be assessed in these studies because uncontrolled muscle twitches were always induced within 10 min by the addition of 1 mmol l–1 ouabain, possibly owing to rising cytosolic Ca2+ mediated by increased Na+/Ca2+ exchange after Na+-pump blockade. (From T. G. West and R. G. Boutilier, unpublished observations.)

Another proposed mechanism of pump-arrest is the anoxia-induced switch from Na⁺/H⁺ exchange to Na⁺-dependent Cl^–/HCO₃^–/H⁺ exchange (Vezzoli et al., 2004). With increased dependence on the multi-ion exchanger there is reduced uptake of Na⁺ needed for regulating intracellular pH (pH_i). Thus the amount of ATP used by the Na⁺K⁺-ATPase mol^–1 H⁺ removed from muscle is suppressed (Reipschläger and Pörtner, 1996; Vezzoli et al., 2004). This mechanism could be important in vivo because anoxia-induced reductions in extracellular pH (pH_e) are thought to inhibit Na⁺/H⁺ exchange. However, the low anaerobic heat flux and stable ionic characteristics of isolated muscle during continuous-flow anoxic perifusions, in the absence of direct manipulation of pH_e (Fig. 2; West and Boutilier, 1998), suggest that frog muscle has other intrinsic mechanisms for anoxic ion homeostasis and reversible metabolic suppression.

Regulated hypoperfusion of skeletal muscle is possibly an important mechanism of in vivo metabolic suppression and whole-body O₂ conservation. However, it should be emphasized that there is currently only indirect evidence that this mechanism operates in hypoxic frogs. Skeletal muscle clearly displays typical anoxia tolerance characteristics, but a key point is that the room-temperature oxyconformation response in isolated muscle (West and Boutilier, 1998) was observed in perifused, not cannulated or perfused, muscle. The onset of a true (i.e. physiological) oxyconformance response is likely to be overestimated because the gradient of O₂ into the core of a perifused muscle will initiate hypometabolic responses in some fibres at an apparently high P_O2. The sensitivity of resting muscle to changes in blood or O₂ flow, and the possibility that O₂ acts directly to trigger suppression of channel and pump activities, needs to be assessed in more physiological circumstances. Similarly, there needs to be further study of in vivo blood flow patterns to strengthen the idea that muscle hypoperfusion is linked directly to muscle quiescence and whole-body oxyconformance at room temperature and to the hypometabolic rescue response seen in cold-submerged frogs (discussed in the next section).

Hypoxia responses during cold-submergence
Frog skeletal muscle expresses the typical characteristics of hypoxia-tolerance and metabolic suppression. This capacity is almost certainly exploited in frogs that overwinter without air-access and gradually suppress their whole-body metabolic rate by up to 75% of normal (Donohoe et al., 1998). Greater understanding of the precise role of skeletal muscle in overwintering frogs has come from laboratory studies into how the metabolic and ionic characteristics of muscle change as whole-animal hypometabolism progresses during normoxic and hypoxic cold-submergence (Donohoe and Boutilier, 1998; Donohoe et al., 1998; Donohoe et al., 2000).

Stable normoxic conditions impose no apparent anaerobic stress on quiescent overwintering animals; body glycogen stores remain relatively high (Donohoe and Boutilier, 1998) and there is no change in muscle and plasma lactate levels throughout 16 weeks of continuous cold-submergence (Fig. 4). Muscle ATP and PCr concentrations are also unchanged during normoxic submergence.

View larger version (13K): [in this window] [in a new window]   Fig. 4. Plasma lactate changes (means ± s.e.m.) in normoxic (air-equilibrated water) and hypoxic (60 mmHg O2) cold-submerged (16 weeks, 4°C) frogs. (From Donohoe and Boutilier, 1998.)

Remarkably, whole-body O₂ consumption in hypoxic cold-submerged frogs approaches a phase of deep suppression before muscle glycogen stores become depleted. Donohoe and Boutilier (Donohoe and Boutilier, 1998) refer to this response as `aerobic hypometabolic rescue' because the lower metabolic demands of the animal can at this point be supported entirely aerobically. Continued muscle quiescence is of course essential for minimising perturbations in whole-animal metabolism. Indeed, the time course of plasma lactate concentration (Fig. 4) supports the view that frogs switch from an initial hypoxic, high-activity state to a phase of prolonged aerobic hypometabolic quiescence. Long-term energy supply to skeletal muscle during chronic environmental hypoxia seems to be largely shared between anaerobic glycolysis and the aerobic oxidation of mixed fuel. It is perhaps expected that muscle PCr should participate in the early phases of these gradual metabolic transitions, but the metabolite time courses presented by Donohoe and Boutilier (Donohoe and Boutilier, 1998) indicate that PCr concentration remained essentially stable in hypoxic cold-submerged frogs. Future studies that relate more detailed metabolite time courses specifically to episodes of muscle contraction and/or ischemia (i.e. when PCr hydrolysis is expected to be important) may possibly reveal acute fluctuations in PCr level. Clearly, having flexibility in energy supply is important for frogs that overwinter in environments where ambient O₂ is heterogeneous and when periods of quiescence are interspersed with costly hypoxia avoidance behaviour. Moreover, hypometabolic rescue is critical for extending the time that on-board fuel reserves can support all of the complex behaviours of overwintering frogs (Boutilier et al., 1997).

Two characteristics of ion balance processes in cold-submerged frogs support the hypothesis that channel- and pump-arrest help to lower muscle energy demands. Firstly, membrane permeability of Na⁺ and K⁺ is gradually reduced in sartorius from both normoxic and hypoxic cold-submerged frogs, and the ouabain-dependent ²²Na⁺ efflux from sartorius muscle is also significantly suppressed (Donohoe et al., 2000). The time courses for the reduction of Na⁺ and K⁺ permeability are similar in normoxic and hypoxic frogs, but there is greater reduction of Na⁺K⁺-ATPase activity in the hypoxic frogs. The time courses of changes in muscle ion transport properties in cold-submerged frogs are in good accord with the onset of aerobic hypometabolic rescue. Reduced costs for muscle ion pumping will help to rescue the tissue by ensuring that energy imbalance due to any O₂ limitation is not prolonged (Boutilier, 2001b), but it remains unclear whether controlled hypoperfusion of muscle triggers the downregulation of channel and pump densities/activities.

View larger version (18K): [in this window] [in a new window]   Fig. 5. Changes in intracellular [K+] (means ± s.e.m.) in muscle and heart (A) and extracellular [Na+] (B) in frogs during 16 weeks of cold-submerged (4°C) normoxia (air equilibrated water; open symbols) and hypoxia (60 mmHg; closed symbols). (From Donohoe et al., 2000.)

	Muscle function during hypoxia/anoxia

TOP Summary Introduction A model of anoxic... Frogs and their muscles... Muscle function during... Conclusion Appendix References

 
While it is undoubtedly true that muscle quiescence is an important energy-saving feature of submerged frogs, it is nevertheless clear that skeletal muscle must remain continuously poised for rapid and perhaps even strenuous contractions. Energy provision for skeletal muscle must be flexible in order to support the range of behaviours characteristic of submerged frogs: from the slow exploratory behaviour of natural O₂ and thermal gradients to the burst behaviour needed for predator avoidance. How might O₂-dependent changes of muscle metabolic/ionic properties constrain muscle function in submerged frogs?

Metabolite changes during hypoxia/anoxia
Frogs in continuous normoxic cold-submergence show low accumulation of lactate together with stable levels of muscle pH_i, adenylates, PCr, creatine (Donohoe and Boutilier, 1998) and presumably inorganic phosphate (P_i). Hence, during continuous normoxic cold-submergence there seems to be no substantial changes in metabolite levels that might limit muscle contraction. In frogs confined to uniformly hypoxic cold-submerged conditions (Fig. 4), the time course of lactate changes suggests that muscle tissue recruits glycolysis to defend energetic homeostasis in the early stages of O₂ lack. It is possible that the initial phase of glycolysis protects PCr and ATP levels, and that the end products of anaerobic glycolysis are removed from the muscle for disposal in other tissues (as discussed previously). Hence, even before the phase of aerobic hypometabolic rescue, there appear to be no drastic changes in muscle metabolite levels. Key aspects of the aerobic `hypometabolic rescue' response therefore seem to be the preservation of muscle fuel reserves and, possibly, the inhibition of muscle atrophy (see next section), both of which will be important for powering the animals' movements if ambient water quality deteriorates.

As discussed above, energy provision during acute anoxia/ischaemia in quiescent isolated skeletal muscle (over the temperature range 4–25°C) is highly dependent on PCr hydrolysis (Hsu and Dawson, 2003; Vezzoli et al., 2004). Complete and sustained muscle anoxia or ischemia is likely to be a rare occurrence in vivo because of the frogs' need to avoid severe environmental hypoxia. Even so, skeletal muscles of frogs do express typical anoxia-tolerant characteristics. In natural waterways, quiescent muscle anoxia/ischaemia could conceivably arise in situations where overwintering frogs are not able to retreat from progressive environmental hypoxia. One might expect that the accumulation of P_i, which generally mirrors the depletion of PCr, could limit the mechanical function of skeletal muscle. A growing view, however, is that there are few direct metabolic consequences of P_i build-up at physiological temperature. For example, glycolytic activation is associated more with contractions than with increases in P-metabolites (Hsu and Dawson, 2004). Moreover, at physiological temperatures elevated P_i seems to have minimal effect on the capacity for skinned muscle fibres to produce isometric force (Coupland et al., 2001; Debold et al., 2004). Recent work on intact dogfish fibres at physiological/acclimation temperature (12°C) indicates that P_i build up after 3.5 s of isometric contraction reduces ATP usage by actomyosin ATPase to 20% of initial values, while only diminishing plateau force to 97.5% (West et al., 2004; West et al., 2005). Hence, there is a significant effect of P_i accumulation on energy release of intact fibres at physiological temperature, but not on the generation and maintenance of isometric force. It would be of interest to examine these relationships at physiological temperature in the skeletal muscle of overwintering frogs to determine, for example, whether anoxia- or ischemia-induced increases in P_i concentration affect subsequent contraction mechanics and energetics. Effects of muscle pH_i are also important to consider because it believed that there are only minor effects of increased H⁺ concentration on muscle function at physiological temperatures (Westerblad et al., 2000). Further studies are needed to determine the effects of specific metabolites on shortening velocity, Ca²⁺ kinetics and work output in frog skeletal muscle at overwintering tempeatures. Given that submerged frogs would normally exploit ambient thermal gradients and risk temporary `hypothermia' in order to avoid anaerobiosis, close attention to the thermal and O₂ histories and to the activity levels of overwintering frogs will be important for assessing the physiological/ecological significance of any interactive effects of temperature and metabolite levels on muscle contractile properties.

View larger version (17K): [in this window] [in a new window]   Fig. 6. Force–velocity relationships in frog sartorius muscle taken from (4°C) air-access control frogs, normoxic cold-submerged frogs (12–16 weeks in air equilibrated water) and hypoxic cold-submerged frogs (12–16 weeks in 60 mmHg O2) determined using isovelocity contractions (see Appendix for experimental details). Muscle force (P) is normalised to maximal isometric force (P0). Velocity (V) is in muscle lengths (L) s–1. The lines are hyperbolic-linear fits for each treatment of the form V=B(1–P/P0)/(A+P/P0)+C(1–P/P0) (Marsh and Bennett, 1986), calculated using the mean values for the constants A, B and C determined for each muscle for each group. Maximum shortening velocity was significantly different for the three groups (one-way ANOVA: F1,8=4.47, P=0.05). The curvature of the force–velocity relationship (F1,8=0.745, P=0.51) and maximum power output (F1,8=0.832, P=0.47) were not significantly different between the three groups. (G. N. Askew and R. G. Boutilier, unpublished observations.)

View larger version (13K): [in this window] [in a new window]   Fig. 7. Power-cycle frequency relationships (means ± s.e.m.) at 4°C for sartorius and external oblique muscles taken from (4°C) air-access control frogs, normoxic cold-submerged frogs (12–16 weeks in air equilibrated water) and hypoxic cold-submerged frogs (12–16 weeks in 60 mmHg O2). A general linear model was used to test for changes in power with cycle frequency and treatment, considering power as the dependent variable and frequency and treatment as independent variables. Power was significantly affected by cycle frequency but there was no significant effect of treatment (Frequency F6,40=7.208, P<0.001; Treatment F2,40=1.381, P=0.265). (G. N. Askew and R. G. Boutilier, unpublished observations.)

Preservation of muscle mechanical properties is in keeping with the active hypoxia avoidance strategy of overwintering frogs. Since frogs will engage in strenuous mate-calling and reproductive behaviours soon after emergence from their overwintering hibernaculum (Boutilier et al., 1997), it is likely that their muscles resist any dramatic deterioration or fibre-type remodelling for the entire overwintering season. At present, however, it is difficult to conclude that muscles of overwintering R. temporaria express true atrophy-resistance, as is the case with aestivating frogs Cyclorana alboguttata (Hudson and Franklin, 2002). On the one hand, R. temporaria overwintering in natural waterways are clearly not victims of their changing environments (Boutilier, 2001a; Boutilier, 2001b) since they can, and must, react to dwindling ambient O₂ levels. In contrast, muscular quiescence is sustained for months in C. alboguttata without any sign of fibre atrophy (Hudson and Franklin, 2002). On the other hand, constant O₂ levels were provided in our laboratory studies of cold-submerged R. temporaria and these stable conditions should promote extended quiescence, particularly during normoxia. The preservation of mechanical properties in these circumstances is indeed remarkable.

Future systematic analysis of muscle fibre-types and fibre dimensions in conjunction with in vitro mechanical assays will help determine how cold-submerged R. temporaria compare with known atrophy-resistant hibernation and aestivation models. Direct comparison of mechanical and energetic properties of intact and skinned muscle fibres from R. temporaria can further help to characterise any changes in actomyosin function and sensitivity to metabolites/ions during time-courses of hypoxic and normoxic submergence. Plasma Ca²⁺ levels were also shown to be reduced by 30–40% in cold-adapted R. temporaria (Sinsch, 1991). Mechanical and energetic properties of intact muscle fibres could be affected, particularly if intramuscular Ca²⁺ stores follow the pattern of changes in plasma Ca²⁺ levels. It may be important in future work to incorporate changes in extracellular ion concentrations (Fig. 5) into in vitro protocols to determine (i) the impact of reduced ion gradients on membrane excitability and energy demand, (ii) Ca²⁺ sensitivity of mechanical properties and (iii) ionic-strength dependence of mechanical properties. West et al. (West et al., 2005) note that, like the effects of P_i on muscle force, alterations in ionic strength seem to perturb peak isometric force and energy demand minimally when measured at physiological, or acclimation, temperatures. Even so, it is important to determine how force production, power output and energy demand might be affected by ions and metabolites over an ecologically relevant winter thermal range, in which relatively small shifts of body temperature can have potentially large impacts on rate processes.

	Conclusion

TOP Summary Introduction A model of anoxic... Frogs and their muscles... Muscle function during... Conclusion Appendix References

 
Characterisation of frog hypoxia/anoxia responses has begun to verify that frogs express a range of tissue-level modes of hypometabolism (Boutilier et al., 1997); from the true anoxia-tolerance capacity of skeletal muscle, to the intermediate, and probably O₂-dependent, hypometabolic capacity of brain (Lutz and Nilsson, 2004). Skeletal muscle physiology is possibly the focal point of an integrated physiological and behavioural hypoxia defense strategy in frogs. Hypometabolism in the large, possibly oxy-conforming and often quiescent skeletal muscle mass contributes significantly to (i) whole animal hypometabolism and (ii) the conservation of O₂ supply for use by more hypoxia-sensitive core tissues. Metabolic and ionic responses of skeletal muscle are consistent with the channel/pump arrest hypothesis (Hochachka and Lutz, 2001), although a possibly unique feature of membrane energetics during cold-submergence may arise from the alteration of muscle Na⁺ and K⁺ gradients and plasma Ca²⁺ level to new and stable set-points (Sinsch, 1991; Donohoe et al., 2001). The lack of effect of prolonged normoxic and hypoxic cold-submergence on muscle mechanical properties highlights the readiness of frogs to move away from severe hypoxia in natural waterways. More work on muscle dimensions and energetics/mechanics during overwintering time courses is needed to assess (i) how changes in metabolites and ionic strength affect contraction and (ii) the quiescent cold-submerged frog as a model of muscle atrophy-resistance.

	Appendix

TOP Summary Introduction A model of anoxic... Frogs and their muscles... Muscle function during... Conclusion Appendix References

 
Animals
Frogs (Rana temporaria L.) collected from wild populations in the British Isles and Ireland were purchased from commercial suppliers (Blades Biological, Cowden, UK). The animals were acclimated in the laboratory for 2–3 weeks in two 3400 l `Living Streams' (LS900, Frigid Units Inc, Toledo, OH, USA), at a water temperature of 4°C. During this time, the frogs had access to air-breathing and the water was held at air-saturated levels of oxygen (i.e. `normoxic conditions'). Two randomly selected groups of animals were submerged in water-filled perspex boxes placed below the water surface of both tanks to prevent any further air-breathing and to induce hibernation. The water of one Living Stream was held at air-saturated partial pressures of oxygen (P_O2 150 mmHg), while the water of the other was adjusted with mixed gases to achieve an ambient P_O2 of 60 mmHg (Donohoe and Boutilier, 1998). A control group of animals was held in normoxic conditions with air access.

Muscle physiology
In vitro muscle experiments were made at 4°C after 3–4 months for control and normoxic and hypoxic cold submergence frogs. The animals were killed and a sartorius muscle and a fascicle bundle dissected from the external oblique muscle were removed. One muscle was used immediately, while the other was pinned out at approximately resting length in oxygenated Ringer's solution (composition after Fischmeister and Hartzell, 1987).

For physiological measurements, the muscle was attached using aluminium foil clips between a force transducer (SensoNor AE801, Horten, Norway) and an electrodymanic shaker (Ling Dynamic Systems V201, Royston, UK) and bathed in oxygenated Ringer's solution at 4°C. Custom written software controlled the shaker and a stimulator (model S4, Grass-Telefactor, West Warwick, RI, USA) and was used to impose length changes and phasic stimulation on the muscle. Muscle force and length were recorded onto a personal computer at 5 kHz via a data acquisition card (Keithley Instruments model DAS1802AO, Theale, UK).

Following a recovery period of approximately 30 min, a series of isometric twitches was used to set the length of the muscle to that at which twitch force was maximal (L₀). At this length maximum isometric tetanic force was determined using a 500 ms train of stimuli at 50 Hz. Following the initial optimisation of muscle length, the work loop technique (Josephson, 1985) was used to measure muscle power output during sinusoidal length changes. Cycle frequency was varied between 0.5 and 5 Hz using a constant strain of 0.1 L₀, symmetrical around L₀. The net power output of the muscle was calculated from the force and differentiated strain trajectory data (i.e. velocity). The onset and duration of stimulation were optimised to maximise net power output.

Force–velocity characteristics were determined for a number of muscles during isovelocity contractions imposed during the plateau of an isometric tetanus. Tetani were performed at regular intervals to allow for correction for the decline in muscle performance. A hyperbolic linear regression equation (Marsh and Bennett, 1986) was fitted to the data using the non-linear curve fitting routine in the application Igor (version 5.0, Wavemetrics, Portland, OR, USA), and the maximum velocity of shortening estimated by extrapolation to zero force.

	Acknowledgments

 
The authors dedicate this paper to the memory of Bob Boutilier; a true friend, a talented mentor, and a gifted comparative biologist. This work was supported by the Biotechnology and Biological Sciences Research Council (to R.G.B.), the Natural Environment Research Council (to R.G.B. and G.N.A.), and the Wellcome Trust (to T.G.W.).

	References

TOP Summary Introduction A model of anoxic... Frogs and their muscles... Muscle function during... Conclusion Appendix References

 

Balog, E. M. and Fitts, R. H. (1996). Effects of fatiguing stimulation of intracellular Na⁺ and K⁺ in frog skeletal muscle. J. Appl. Physiol. 81,679 -685.[Abstract/Free Full Text]

Boutilier, R. G. (2001a). Mechanisms of cell survival in hypoxia and hypothermia. J. Exp. Biol. 204,3171 -3181.

Boutilier, R. G. (2001b). Mechanisms of metabolic defense against hypoxia in hibernating frogs. Respir. Physiol. 128,365 -377.[CrossRef][Medline]

Boutilier, R. G., Emilo, M. G. and Shelton, G. (1986). Aerobic and anaerobic correlates of mechanical work by gastrocnemius muscle of Xenopus laevis. J. Exp. Biol. 122,223 -235.

Boutilier, R. G., Donohoe, P. H., Tattersall, G. J. and West, T. G. (1997). Hypometabolic homeostasis in overwintering aquatic amphibians. J. Exp. Biol. 200,387 -400.[Abstract]

Bradford, D. F. (1983). Winterkill, oxygen relations and energy metabolism of a submerged dormant amphibian. Ecology 64,1171 -1183.[CrossRef]

Buck, L. T. and Hochachka, P. W. (1993). Anoxic suppression of Na⁺K⁺-ATPase and constant membrane potential in hepatocytes: support for channel arrest. Am. J. Physiol. 265,R1020 -R1025.

Buttgereit, F. and Brand, M. D. (1995). A hierarchy of ATP-consuming processes in mammalian cells. Biochem. J. 312,163 -167.

Coupland, M. E., Puchert, E. and Ranatunga, K. W. (2001). Temperature dependence on active tension in mammalian (rabbit psoas) muscle fibres: effect of inorganic phosphate. J. Physiol. 536,879 -891.[Abstract/Free Full Text]

Court, E. L. and Boutilier, R. G. (2005). Survival mechanisms in the liver of the overwintering frog, Rana temporaria. Comp. Biochem. Physiol. 141A, S188.

Currie, S. and Boutilier, R. G. (2001). Strategies of hypoxia and anoxia tolerance in cardiomyocytes from the overwintering common frog, Rana temporaria. Physiol. Biochem. Zool. 74,420 -428.

Debold, E. P., Dave, H. and Fitts, R. H. (2004). Fibre type and temperature dependence of inorganic phosphate: implications for fatigue. Am. J. Physiol. 287,C673 -C681.

Donohoe, P. H. and Boutilier, R. G. (1998). The protective effects of metabolic rate depression in hypoxic cold submerged frogs. Respir. Physiol. 111,325 -336.[CrossRef][Medline]

Donohoe, P. H., West, T. G. and Boutilier, R. G. (1998). Respiratory and metabolic correlates of aerobic metabolic rate reduction in overwintering frogs. Am. J. Physiol. 43,R704 -R710.

Donohoe, P. H., West, T. G. and Boutilier, R. G. (2000). Factors affecting membrane permeability and ionic homeostasis in the cold-submerged frog. J. Exp. Biol. 203,405 -414.[Abstract]

Feng, T. P. (1932). The effect of length on the resting metabolism of muscle. J. Physiol. 74,441 -454.[Free Full Text]

Fischmeister, R. and Hartzell, H. C. (1987). Cyclic guanosine-3',5'-monophosphate regulates the calcium current in single cells from frog ventricle. J. Physiol., Lond. 387,453 -472.[Abstract/Free Full Text]

Hochachka, P. W. and Lutz, P. L. (2001). Mechanism, origin and evolution of anoxia tolerance in animals. Comp. Biochem. Physiol. 130B,435 -459.[CrossRef][Medline]

Hochachka, P. W., Buck, L., Doll, C. and Land, S. (1996). Unifying theory of of hypoxia tolerance: molecular/metabolic defense and rescue mechanisms for surviving oxygen lack. Proc. Natl. Acad. Sci. USA 93,9493 -9498.[Abstract/Free Full Text]

Hsu, A. C. and Dawson, M. J. (2003). Muscle glycogenolysis is not activated by changes in cytosolic P-metabolites: A ³¹P and ¹H MRS demonstration. Magn. Reson. Med. 49,626 -631.[CrossRef][Medline]

Hudson, N. J. and Franklin, C. E. (2002). Maintaining muscle mass during extended disuse: Aestivating frogs as a model species. J. Exp. Biol. 205,2297 -2303.[Abstract/Free Full Text]

Josephson, R. K. (1985). Mechanical power output from striated-muscle during cyclic contractions. J. Exp. Biol. 114,493 -512.[Abstract/Free Full Text]

Land, S. C. and Hochachka, P. W. (1994). Protein turnover during metabolic arrest in turtle hepatocytes: role and energy dependence of proteolysis. Am. J. Physiol. 266,C1028 -C1036.

Lutz, P. L. and Nilsson, G. E. (2004). Vertebrate brains at the pilot light. Respir. Physiol Neurobiol. 141,285 -296.[CrossRef][Medline]

Marsh, R. L. and Bennet, A. F. (1986). Thermal dependence of contractile properties of skeletal muscle from the lizard Sceloporus occidentalis with comments on methods for fitting and comparing force–velocity curves. J. Exp. Biol. 126, 63-77.[Abstract/Free Full Text]

Overgaard, K., Nielsen, O. B. and Clausen, T. (1997). Effects of reduced electrochemical Na⁺ gradient on contractility in skeletal muscle: role for the Na⁺-K⁺ pump. Eur. J. Physiol. 434,457 -465.[CrossRef][Medline]

Peréz-Pinzon, M. A., Rosenthal, M., Sick, T. J., Lutz, P. L., Pablo, J. and Mash, D. (1992). Downregulation of sodium channels during anoxia: a putative survival strategy of turtle brain. Am. J. Physiol. 262,R712 -R715.

Reipschläger, A. and Pörtner, H. O. (1996). Metabolic depression during environmental stress: the role of extracellular versus intracellular pH in Sipunculus nudus. J. Exp. Biol. 199,1801 -1807.

Schulz, C., Thuy, M. and Wegner, G. (1991). Heat production of frogs under normoxic and hypoxic conditions: a microcalorimetric study a gas flow system. Thermochim. Acta 187,71 -78.[CrossRef]

Sinsch, U. (1991). Cold acclimation in frogs (Rana): Microhabitat choice, osmoregulation, and hydromineral balance. Comp. Biochem. Physiol. 98A,469 -477.[CrossRef]

St-Pierre, J., Brand, M. D. and Boutilier, R. G. (2000). Mitochondria as ATP consumers: Cellular treason in anoxia. Proc. Natl. Acad. Sci. USA 97,8670 -8674.[Abstract/Free Full Text]

Tattersall, G. J. and Boutilier, R. G. (1997). Balancing hypoxia and hypothermia in cold-submerged frogs. J. Exp. Biol. 200,1031 -1038.[Abstract]

Tattersall, G. J. and Boutilier, R. G. (1999). Behavioural oxyregulation by cold-submerged frogs in heterogeneous oxygen environments. Can. J. Zool. 77,843 -850.[CrossRef]

Vezzoli, A., Gussoni, M., Greco, F. and Zetta, L. (2003). Effects of temperature and extracellular pH on metabolites: kinetics of anaerobic metabolism in resting muscle by ³¹P- and ¹H-NMR spectroscopy. J. Exp. Biol. 206,3043 -3052.[Abstract/Free Full Text]

Vezzoli, A., Gussoni, M., Greco, F., Zetta, L. and Cerretelli, P. (2004). Temperature and pH dependence of energy balance by ³¹P- and ¹H-MRS in anaerobic frog muscle. J. Exp. Biol. 206,3043 -3052.

Wegener, G. and Krause, U. (1993). Environmental and exercise anaerobiosis in frogs. In Surviving Hypoxia (ed. P. W. Hochachka, P. L. Lutz, T. Sick, M. Rosenthal and G. van den Thillart), pp. 217-236. Boca Raton: CRC Press.

West, T. G. and Boutilier, R. G. (1998). Metabolic suppression in anoxic frog muscle. J. Comp. Physiol. B 168,273 -280.[CrossRef][Medline]

West, T. G., Curtin, N. A., Ferenczi, M. A., He, Z. H., Sun, Y. B., Irving, M. and Woledge, R. C. (2004). Actomyosin energy turnover declines while force remains constant during isometric muscle contraction. J. Physiol. 555, 27-43.[Abstract/Free Full Text]

West, T. G., Ferenczi, M. A., Woledge, R. C. and Curtin, N. A. (2005). Influence of ionic strength on the time course of force development and phosphate release by dogfish muscle fibres. J. Physiol. 567,989 -1000.[Abstract/Free Full Text]

Westerblad, H., Allen, D. G. and Lännergren, J. (2002). Muscle fatigue: lactic acid or inorganic phosphate the major cause? News Physiol. Sci. 17, 17-21.[Abstract/Free Full Text]

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