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Adaptive plasticity of skeletal muscle energetics in hibernating frogs: mitochondrial proton leak during metabolic depression

Robert G. Boutilier^1,* and Julie St-Pierre^2,

¹ Department of Zoology, Downing Street, University of Cambridge, Cambridge CB2 3EJ, UK
² MRC Dunn Human Nutrition Unit, Hills Road, Cambridge CB2 2XY, UK
Present address: Dana-Farber Cancer Institute, One Jimmy Fund Way, SM958, Boston, MA 02115, USA

View larger version (19K): [in a new window]   Fig. 1. Oxygen-dependence of respiration at 20°C for superfused sartorius muscle and for skeletal muscle mitochondria isolated from the frog Rana temporaria. Measurements were made using high-resolution respirometry (Oroboros Oxygraph, Paar, Garz, Austria) that enables sensitive measurements of oxygen kinetics at low oxygen partial pressures (see St-Pierre et al., 2000c). Mitochondria show strict oxyregulation over a broad range of O2 tensions, while skeletal muscle begins to oxyconform at PO2 levels that are far in excess of the Km of isolated mitochondria. Although oxyconformation is seldom seen in isolated cell preparations (see, however, Brand et al., 2000; Bishop and Brand, 2000; Guppy et al., 2000; Bishop et al., 2002), it does operate at the level of intact skeletal muscle (Hochachka and Guppy, 1987; West and Boutilier, 1998). One possibility is that the PO2 of localised (hypoperfused) regions of tissue might fall below the critical PO2 (Pcrit) at which diffusion of oxygen to the mitochondria begins to limit oxidative phosphorylation. The metabolic rate of such localised regions could therefore become suppressed even though the mixed venous blood continues to exit the tissue at PO2 levels higher than the Pcrit. This so-called `diffusion limitation' could be one explanation for the well-known oxyconformation response seen in the intact skeletal muscle of cat (Whalen et al., 1973) and frog (Boutilier et al., 1997; West and Boutilier, 1998). Alternatively, oxyconformation could occur through some oxygen-sensing elements that trigger a reduction in the rate of mitochondrial respiration. From Boutilier (2001) with permission.

View larger version (20K): [in a new window]   Fig. 2. Kinetics of the mitochondrial proton leak at 25 °C for hypoxic submerged frogs (Rana temporaria). The proton conductance of the mitochondrial inner membrane is assayed by measuring the rate of proton cycling (measured as rate of oxygen consumption) through non-coupled pathways at defined values of the proton-motive force (measured as membrane potential). Stepwise changes in respiration rate and membrane potential were achieved by adding increasing amounts of malonate. The control, 1-month-submerged and 4-month-submerged groups of frogs are represented by black, grey and white circles, respectively. Values are means ± S.E.M.; N=5 for control and 1-month-submerged frogs, N=4 for 4-month-submerged frogs. From St-Pierre et al. (2000a).

View larger version (37K): [in a new window]   Fig. 3. (A) Measurements of whole-animal oxygen consumption of Rana temporaria under control conditions (in water but with access to the surface for air-breathing) and after 90 days of cold-submergence in normoxic (PO2=21 kPa) or hypoxic (PO2=8 kPa) water at 3 °C. (B) Respiration rates of skeletal muscle mitochondria isolated from frogs during normoxic and hypoxic hibernation, in state 4 and at 125 mV. Frog mitochondrial proton leak rate was measured at 25 °C because the respiration rate of frog mitochondria was so low at 3 °C that it was impossible to carry out accurate measurements with the apparatus used (St-Pierre et al., 2000a). Since that time, Q10 values between 3 and 20 °C have been determined for the state 4 respiration rate of mitochondria (St-Pierre et al., 2000c). As the Q10 values do not differ between control and 4-month-hypoxic-submerged frogs (St-Pierre et al., 2000c), the proton leak rate comparisons at 25 °C between the two groups of frogs should be adequate. Values are means ± S.E.M.; N=5 for control frogs, N=4 for 4-month-submerged frogs. From Boutilier (2001) with permission.

View larger version (15K): [in a new window]   Fig. 4. Kinetics of frog skeletal muscle mitochondrial proton leak during normoxia and in anoxia. During normoxia, the membrane potential is generated by the electron-transport chain; however, during anoxia, the electron-transport chain is inhibited, and the ATP synthase functions as an ATPase, pumping protons from the matrix to the cytosol to preserve a certain membrane potential. The normoxic and anoxic conditions are represented by filled and open circles, respectively. Values are means ± S.E.M.; N=6 for both normoxia and anoxia. *Resting proton leak rate and membrane potential. Significant difference from the resting proton leak rate during normoxia (P<0.05; Student's t-test). Significant difference from the resting membrane potential during normoxia (P<0.05; Student's t-test). From St-Pierre et al. (2000b) with permission.

View larger version (25K): [in a new window]   Fig. 5. Kinetics of frog mitochondrial oxygen consumption rates in the low oxygen range at 3 °C for hypoxic submerged frogs. The kinetics for state 3 (A) and state 4 (B) respiration are shown. Solid line, control; dashed line, 1-month-submerged; dotted line, 4-month-submerged groups of frogs. The insets show how P50 was calculated for each group of frogs. These curves were generated using the equation O2=100[PO2/(P50+PO2)], where O2 (as a percentage of maximum) is the rate of oxygen consumption for state 3 or state 4. The state 3 and state 4 values from the control group of frogs were set to 100%, and those for the 1- and 4- month-submerged groups of frogs were expressed relative to the controls. To convert PO2 to µmol, kPa must be multiplied by a factor of 11.3 and mmHg by a factor of 1.5. To convert kPa to mmHg, kPa must be multiplied by a factor of 7.51. From St-Pierre et al. (2000c) with permission.

View larger version (20K): [in a new window]   Fig. 6. Mitochondrial and glycolytic enzyme activities at 3 °C for frog skeletal muscle at different stages during hypoxic hibernation. Mitochondrial enzymes: citrate synthase (CS), cytochrome c oxidase (CCO) and NADH dehydrogenase (NDH). Glycolytic enzyme: lactate dehydrogenase (LDH). Values are means + S.E.M.; N=6 for control frogs, N=4 for 1-month-submerged frogs and N=5 for 4-month-submerged frogs. Statistically significant differences (P<0.05) between groups of frogs are represented by different letters (one-way ANOVA and a posteriori Tukey test). From St-Pierre and Boutilier (2001) with permission.