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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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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

View larger version (26K): [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.)

View larger version (21K): [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.)

View larger version (14K): [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Time courses for fractional efflux of 22Na+ (mean ± s.e.m.) in isolated frog sartorius (T. G. West and R. G. Boutilier, unpublished observations) at 20°C during normoxia (98% O2:2% CO2; open symbols) and anoxia (98% N2:2% CO2; closed symbols).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.)

View larger version (13K): [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.)

View larger version (18K): [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.)

View larger version (17K): [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 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.)