Mechanisms underlying inhibition of muscle disuse atrophy during aestivation in the green-striped burrowing frog, Cyclorana alboguttata Beau Daniel Reilly Bachelor of Marine Studies (Hons.) A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2014 School of Biological Sciences Abstract In most mammals, extended inactivity or immobilisation of skeletal muscle (e.g. bed- rest, limb-casting or hindlimb unloading) results in muscle disuse atrophy, a process which is characterised by the loss of skeletal muscle mass and function. In stark contrast, animals that experience natural bouts of prolonged muscle inactivity, such as hibernating mammals and aestivating frogs, consistently exhibit limited or no change in either skeletal muscle size or contractile performance. While many of the factors regulating skeletal muscle mass are known, little information exists as to what mechanisms protect against muscle atrophy in some species. Green-striped burrowing frogs (Cyclorana alboguttata) survive in arid environments by burrowing underground and entering into a deep, prolonged metabolic depression known as aestivation. Throughout aestivation, C. alboguttata is immobilised within a cast-like cocoon of shed skin and ceases feeding and moving. Remarkably, these frogs exhibit very little muscle atrophy despite extended disuse and fasting. The overall aim of the current research study was to gain a better understanding of the physiological, cellular and molecular basis underlying resistance to muscle disuse atrophy in C. alboguttata. The first aim of this study was to develop a genomic resource for C. alboguttata by sequencing and functionally characterising its skeletal muscle transcriptome, and to conduct gene expression profiling to identify transcriptional pathways associated with metabolic depression and maintenance of muscle function in aestivating burrowing frogs. A transcriptome was assembled using next-generation short read sequencing followed by a comparison of gene expression patterns between active and four-month aestivating C. alboguttata. This identified a complex suite of gene expression changes that occur in muscle during aestivation and provides evidence that aestivation in burrowing frogs involves transcriptional regulation of genes associated with cytoskeletal remodelling, avoidance of oxidative stress, energy metabolism, the cell stress response, cell death and survival and epigenetic modification. In particular, the expression levels of genes encoding cell cycle regulatory-, pro-survival and chromatin remodelling proteins, such as serine/threonine-protein kinase Chk1, cell division protein kinase 2, survivin, vesicular overexpressed in cancer prosurvival protein 1 and histone-binding protein RBBP4, were upregulated in aestivators. The second aim of this study was to examine the potential role of mitochondrial ROS in the regulation of muscle mass and function during aestivation in C. alboguttata. In II mammals, muscle disuse atrophy has been associated with oxidative damage due to increased mitochondrial ROS production. C. alboguttata reduced skeletal muscle mitochondrial respiration by approximately 50% following four months of aestivation, while mitochondrial ROS production was more than 80% lower in aestivating skeletal muscle relative to controls when mitochondrial substrates were present at physiologically-relevant concentrations. In contrast to skeletal muscle, cardiac muscle of aestivating frogs must remain relatively active to still maintain adequate perfusion of organs. Aestivating frogs maintained cardiac mitochondrial respiration and ROS production at levels similar to those of control animals. Accelerated protein degradation in mammalian skeletal muscle has been linked to increased mitochondrial ROS production and oxidative stress. When ROS are in excess, a number of proteolytic pathways appear to play a pivotal role in the development of atrophy in inactive muscle fibres including the cytosolic calcium-dependent calpains. The aim of the final chapter was to determine if aestivating C. alboguttata are able to resist disuse-induced atrophy as a consequence of the downregulation of calpain proteases in skeletal muscle. The enzyme activity, protein abundance and gene expression levels of calpain isoforms were examined in skeletal muscle of aestivating and control C. alboguttata. There was no decrease in the protein abundances of calpain 1 or calpain 2 in aestivating C. alboguttata muscle relative to controls. Similarly, gene expression and enzyme activity levels of calpain 1 and 2 were unaffected by aestivation. The protein abundance of ‘muscle-specific’ calpain 3, which is consistently downregulated during atrophic conditions, was also examined in aestivating muscle. Western blotting indicated that calpain 3 may be autolysed (and hence activated) in skeletal muscle of both active and aestivating frogs. Results from the current study suggest that the relative inhibition of muscle atrophy in aestivating C. alboguttata is multifactorial in origin. ATP-dependent chromatin remodelling appears to be an important mechanism to actively regulate gene expression throughout aestivation, while elevated expression of anti-apoptotic genes is likely to be critical in preventing premature apoptotic muscle fibre degradation. In addition, decreased rates of skeletal muscle mitochondrial respiration during aestivation allows energy savings to be maximised. Low levels of hydrogen peroxide production suggests that ROS can be suppressed in immobilised skeletal muscles of aestivating frogs, which in combination with bolstering antioxidant defences may protect against potential oxidative stress and preserve skeletal muscle structure during aestivation and during arousal. While it is difficult to III determine the specific function of calpain 3 in C. alboguttata muscle, the maintenance (rather than an increase) of pre-aestivation enzyme activity, protein and mRNA abundances of calpains is consistent with the protection of muscle against uncontrolled proteolysis throughout aestivation. IV Declaration by author This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis. I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my research higher degree candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award. I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School. I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis. V Publications during candidature Reilly, B.D., Schlipalius, D.I., Cramp, R.L., Ebert, P.R. and Franklin, C.E. (2013). Frogs and estivation: transcriptional insights into metabolism and cell survival in a natural model of extended muscle disuse. Physiological Genomics 45, 377-388. Reilly, B.D., Hickey, A.J.R., Cramp, R.L. and Franklin, C.E. (2013). 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. The Journal of Experimental Biology 217, 1087-1093. Reilly, B.D., Cramp, R.L. and Franklin, C.E. (2014). Activity, abundance and expression of Ca2+-activated proteases in skeletal muscle of the aestivating frog, Cyclorana alboguttata. Journal of Comparative Physiology B – Biochemical, Systemic and Environmental Physiology 185, 243-255. VI Publications included in this thesis Reilly, B.D., Schlipalius, D.I., Cramp, R.L., Ebert, P.R. and Franklin, C.E. (2013). Frogs and estivation: transcriptional insights into metabolism and cell survival in a natural model of extended muscle disuse. Physiological Genomics 45, 377-388 ‒ Incorporated as Chapter 2. Contributor Statement of contribution Conception and design (50%), data collection (100%), analysis Reilly, B.D. (90%), interpretation (80%), writing and drafting (60%). Conception and design (5%), analysis (5%), interpretation (5%), Schlipalius, D.I. writing and drafting (10%). Conception and design (20%), writing and drafting (10%). Cramp, R.L. Conception and design (5%), analysis (5%), interpretation (5%), Ebert, P.R. writing and drafting (10%). Conception and design (20%), interpretation (10%), writing and Franklin, C.E. drafting (10%). Reilly, B.D., Hickey, A.J.R., Cramp, R.L. and Franklin, C.E. (2013). Decreased hydrogen peroxide production and mitochondrial respiration in skeletal muscle but