The maintenance of skeletal muscle is a complex task owing to the highly plastic nature of muscle metabolism and morphology. When a muscle is not used it can ‘waste away’ in a phenomenon known as muscle disuse atrophy (MDA). The process of MDA can be triggered by a variety of disuse conditions including denervation, spinal transection, extended bed rest, cast immobilisation, limb suspension, micro-gravity conditions, and prolonged dormancy. MDA can proceed via a plethora of biochemical pathways yet the end result of MDA is a downsizing of whole muscles and the muscle fibres, loss of muscle fibres, and transitions from one fibre type to another. A great deal of attention has been given to what physiological mechanism links the disuse condition with the pathways through which MDA proceeds. Wide spread support has existed for the role of a state of ‘oxidative stress’, resulting from an overwhelmingly high generation of reactive oxygen species (ROS). However, the exact role of oxidative stress in disuse and whether or not it is always a causal factor is controversial. MDA occurs to varying extents in different muscle types, different species, and different conditions of disuse, despite similar disuse timeframes. It has therefore been postulated that differences in rates of oxygen consumption between animals may provide insight into how oxidative stress and variable extents of MDA may intertwine.
The ROS that seem to be responsible for oxidative stress are produced during the process of the metabolic consumption of oxygen. Notably, species that undergo dormancy, and thus prolonged natural muscular disuse, show less MDA over an equivalent timeframe than artificially immobilised species that are not dormant. Since the hallmark of dormancy is a substantial metabolic depression it has been postulated that the extremely low rates of oxygen consumption, and concurrently low overall ROS generation relative to the active animal, exert a protective effect on disused muscle. However, the relationship between rate of oxygen consumption and ROS production remains to be fully elucidated for in vivo contexts. Low ROS generation presumably reduces the chance of an oxidative stress signal inducing MDA, translating to a slower rate of atrophy and a lower extent of atrophy over a given timeframe. The corollary of this is that a muscle can remain in disuse for a longer period of time yet incur less structural and functional deficit.
Preservation of disused muscle during dormancy is exhibited by both endothermic and ectothermic organisms. Elucidating the potential contribution of metabolic depression in slowing the rate of MDA is somewhat confounded by endothermic physiology. Periodic arousals with associated muscular activity likely contribute to signals stimulating the maintenance of muscle. Ectotherms undergoing dormancy appear to be free of these confounding factors. The intrinsic link between environmental temperature and biochemical rate processes of ectotherms means that metabolic depression could be susceptible to perturbation. Temperature-induced increases in metabolic rate during prolonged muscle disuse could therefore result in a greater extent of MDA.
This thesis evaluated the premise that the lower extent of MDA in dormant animals (relative to traditional laboratory models) is potentially contributed to by the substantial metabolic depression occurring with dormancy, by investigating whether high temperatures would perturb metabolic depression and exacerbate atrophy in a dormant ectotherm. The green-striped burrowing frog, Cyclorana alboguttata, enters aestivation (dry season dormancy) in response to dehydrating conditions in semi-arid regions of Queensland, Australia. The first aim of this study was to determine the effect of environmental temperature on the rate of oxygen consumption and the magnitude of the metabolic depression of C. alboguttata at both the whole-animal and muscle levels. Rate of oxygen consumption of the whole aestivating frog was significantly elevated when aestivating at 30oC, above that of frogs at 20oC and 24oC. In addition to the higher absolute rate of oxygen consumption the magnitude of the metabolic depression at 30oC was significantly restricted compared to aestivation at lower temperatures. Five skeletal muscles from the hind-limb of C. alboguttata all significantly depressed rate of oxygen consumption during dormancy at both 24oC and 30oC. However, muscle-specific differences were revealed both in absolute rates of oxygen consumption and the muscle’s response to aestivation at 30oC. Larger jumping muscles tended to have similar rates of oxygen consumption that were lower than smaller non-jumping muscles that also tended to have similar and higher rates of oxygen consumption. Three of five muscles showed significantly elevated rates of oxygen consumption during aestivation at 30oC compared to lower temperatures, primarily the large jumping muscles; suggesting that these muscles may be more susceptible to enhanced MDA during aestivation at high temperatures.
The second aim of this study was to determine the effect of temperature on the extent of MDA in the gastrocnemius (jumping muscle, metabolically temperature-sensitive) and iliofibularis (non-jumping muscle, metabolically temperature-insensitive) following six months of aestivation. Both muscles showed signs of atrophy at both temperatures. Surprisingly, aestivation at 30oC did not exacerbate the extent of atrophy evident in either muscle, compared to that evident at 24oC. However, patterns of atrophic morphological change were highly muscle-specific and appeared to correlate with rate of oxygen consumption; in that the iliofibularis with the inherently higher rate of oxygen consumption showed greater evidence of atrophy than the gastrocnemius. Thus, muscle-specific patterns of atrophy supported the potential for a connection between rate of oxygen consumption and extent of atrophy, yet the response of rate of oxygen consumption and MDA to higher aestivation temperature did not. These results suggested that either a sufficiently stressful oxidative threshold was not reached or the gastrocnemius is protected against enhanced MDA at higher temperatures via intervening biochemical means.
The third aim of this study was to assess the mobilisation of protective mechanisms and evidence of oxidative damage in both the gastrocnemius and iliofibularis muscles from frogs aestivating at 24oC and 30oC. The mobilisation of small molecule antioxidants, mitochondrial and cytosolic superoxide dismutase (SOD) and heat-shock proteins (HSPs) were highly muscle-specific as were the patterns and magnitudes of oxidative damage to lipids and proteins. Protective mechanisms were employed in both pre-emptive and stressor-responsive manners. Up-regulation of some protective mechanisms was induced by aestivation at 30oC in the gastrocnemius but not the iliofibularis. Notably, the regulation of antioxidants that work specifically to remove superoxide were up-regulated with temperature only in the gastrocnemius muscle which also showed an increase in rate of oxygen consumption during aestivation at 30oC. Oxidative damage to gastrocnemius lipids and proteins were not attenuated by the protective mechanisms but continued at control levels. The iliofibularis experienced significantly greater oxidative damage to proteins with aestivation but this was not increased by aestivation at 30oC compared to 24oC. The patterns of oxidative damage and regulation of protective mechanisms between the muscles appear to correlate with the extent of atrophy evident in each muscle type. However, it is clear that functionally different muscles use different biochemical strategies during aestivation in general and during aestivation at higher temperatures and that, in the gastrocnemius, aestivation at elevated temperatures appeared to represent an oxidative challenge that required biochemical defence. The mobilisation of protective strategies appears to be geared towards a preferential investment in muscles of important post-aestivation function (i.e. jumping muscles). At the temperature range used in this study the protective mechanisms appear to be successful in avoiding, or contributing to the avoidance of, a greater extent of MDA at higher aestivation temperatures.
An additional aim of this study was to characterise the environmental temperatures C. alboguttata are likely to experience in the field in order to garner an understanding of the ecological context for the physiological results of this study. Data from a variety of possible burrowing microhabitats showed that temperatures of around 30oC were towards the upper limit of the temperature range that C. alboguttata might experience. It is probable that C. alboguttata are suitably equipped to deal with 30oC however, the requirement to up-regulate biochemical defences against metabolically produced ROS at this temperature combined with whole animal temperature-insensitivity between 20oC and 24oC suggest that temperatures lower than 30oC may be more conducive to long-term preservation of disused muscle. However, in non-drought years C. alboguttata are probably unlikely to experience a constant temperature of 30oC for a duration of six months (as employed in this study). Therefore their ability to withstand enhanced atrophy under these conditions is remarkable.
Although high aestivation temperature did not result in an enhanced extent of atrophy in this study, the results indicate that it remains possible that high aestivation temperatures could exacerbate MDA during prolonged aestivation. Hence, the results of this study do not refute the premise that substantial metabolic depression during aestivation contributes to protecting disused muscle against MDA; metabolic depression is likely to be a contributor to the low extent of atrophy that occurs in dormant animals. However, this study also shows that the retention of a low extent of atrophy in disused muscles during prolonged aestivation is most certainly more complex than metabolic depression alone.