Lessons from an estivating frog: sparing muscle protein despite starvation and disuse

Abstract

Long (6- to 9-mo) bouts of estivation in green-striped burrowing frogs lead to 28% atrophy of cruralis oxidative fibers (P < 0.05) and some impairment of in vitro gastrocnemius endurance (P < 0.05) but no significant deficit in maximal twitch force production. These data suggest the preferential atrophy of oxidative fibers at a rate slower than, but comparable to, laboratory disuse models. We tested the hypothesis that the frog limits atrophy by modulating oxidative stress. We assayed various proteins at the transcript level and verified these results for antioxidant enzymes at the biochemical level. Transcript data for NADH ubiquinone oxidoreductase subunit 1 (71% downregulated, P < 0.05) and ATP synthase (67% downregulated, P < 0.05) are consistent with mitochondrial quiescence and reduced oxidant production. Meanwhile, uncoupling protein type 2 transcription (P = 0.31), which is thought to reduce mitochondrial leakage of reactive oxygen species, was maintained. Total antioxidant defense of water-soluble (22.3 +/- 1.7 and 23.8 +/- 1.5 microM/microg total protein in control and estivator, respectively, P = 0.53) and membrane-bound proteins (31.5 +/- 1.9 and 42.1 +/- 7.3 microM/microg total protein in control and estivator, respectively, P = 0.18) was maintained, equivalent to a bolstering of defense relative to oxygen insult. This probably decelerates muscle atrophy by preventing accumulation of oxidative damage in static protein reserves. Transcripts of the mitochondrially encoded antioxidant superoxide dismutase type 2 (67% downregulated, P< 0.05) paralleled mitochondrial activity, whereas nuclear-encoded catalase and glutathione peroxidase were maintained at control values (P = 0.42 and P = 0.231), suggesting a dissonance between mitochondrial and nuclear antioxidant expression. Pyruvate dehydrogenase kinase 4 transcription was fourfold lower in estivators (P = 0.11), implying that, in contrast to mammalian hibernators, this enzyme does not drive the combustion of lipids that helps spare hypometabolic muscle.

Decreased hydrogen peroxide production and mitochondrial respiration in skeletal muscle but not cardiac muscle of the green-striped burrowing frog, a natural model of muscle disuse

“…Previous studies have shown that aestivating C. alboguttata sustains hindlimb muscle mass until 6-9 months of aestivation (Hudson et al, 2006;Mantle et al, 2009). We suggest that decreased ROS production in 4 month aestivating skeletal muscle may represent a mechanism by which dormant C. alboguttata limit muscle fibre atrophy.…”

“…It has been shown that long periods of limb immobilisation stimulate increased ROS production in disused fibres (Min et al, 2011). Though many studies have investigated muscle antioxidant levels during dormancy and/or arousal in aestivators and hibernators (Ramos-Vasconcelos and Hermes-Lima, 2003;Hudson et al, 2006;Allan and Storey, 2012;James et al, 2013;Young et al, 2013), little is known about changes in mitochondrial ROS production and this has only recently received experimental attention in natural models of muscle disuse (Brown et al, 2012). Additionally, relatively few physiological studies examine mitochondrial function using permeabilised fibres, tissues or cells.…”

“…In C. alboguttata, aestivation for 4 months resulted in the induction of mRNA transcripts associated with skeletal muscle nuclear factor erythroid 2-related factor 2 (Nrf2), a regulator of the oxidative stress response (Reilly et al, 2013). Furthermore, water-soluble and membrane-bound antioxidants and gene expression levels of muscle catalase and glutathione peroxidase were shown to be maintained at control levels in muscles of dormant frogs (Hudson et al, 2006). These studies indicate that modulation of antioxidants in aestivating muscle might decrease the susceptibility of muscle fibres to the atrophic effects of oxidative stress.…”

Prevention of muscle wasting and osteoporosis: the value of examining novel animal models

“…In C. alboguttata, 6-9 months of aestivation was reported to have no effect on muscle mass, water content or myofibre number in gastrocnemius or cruralis muscles (Mantle et al, 2009). Similarly, 6 months of aestivation had no effect on the total cross-sectional area of cruralis muscle (Hudson et al, 2006;Mantle et al, 2009). Furthermore, aestivation for 9 months in this species had no effect on myofibre cross-sectional area in the gastrocnemius, a key muscle involved in power output during locomotion (Mantle et al, 2009).…”

“…Consequently, the green-striped burrowing frog (C. alboguttata) has recently been used in our laboratory to study the effects of aestivation on skeletal muscle and bone, and the associated physiological and molecular processes that may confer resistance to musculoskeletal deterioration (Hudson et al, 2004(Hudson et al, , 2006Hudson and Franklin, 2002a;Mantle et al, 2009;Reilly et al, 2013;Symonds et al, 2007;Young et al, 2013). Prolonged inactivity associated with aestivation has been shown to have negligible effects on muscle mass, whole-muscle cross-sectional area, myofibre number, in vitro force production, bone-bending strength and swimming performance in C. alboguttata (Hudson et al, 2004(Hudson et al, , 2006Hudson and Franklin, 2002a;Mantle et al, 2009). The apparent absence of musculoskeletal losses in Cyclorana is consistent with the patterns reported for periods of disuse in hibernators as discussed above, and it is evident that the responses of muscle and bone to disuse in disparate organisms that undergo dormancy are profoundly different from those of typical models of disuse.…”