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

Bone mass and skeletal muscle mass are controlled by factors such as genetics, diet and nutrition, growth factors and mechanical stimuli. Whereas increased mechanical loading of the musculoskeletal system stimulates an increase in the mass and strength of skeletal muscle and bone, reduced mechanical loading and disuse rapidly promote a decrease in musculoskeletal mass, strength and ultimately performance (i.e. muscle atrophy and osteoporosis). In stark contrast to artificially immobilised laboratory mammals, animals that experience natural, prolonged bouts of disuse and reduced mechanical loading, such as hibernating mammals and aestivating frogs, consistently exhibit limited or no change in musculoskeletal performance. What factors modulate skeletal muscle and bone mass, and what physiological and molecular mechanisms protect against losses of muscle and bone during dormancy and following arousal Understanding the events that occur in different organisms that undergo natural periods of prolonged disuse and suffer negligible musculoskeletal deterioration could not only reveal novel regulatory factors but also might lead to new therapeutic options. Here, we review recent work from a diverse array of species that has revealed novel information regarding physiological and molecular mechanisms that dormant animals may use to conserve musculoskeletal mass despite prolonged inactivity. By highlighting some of the differences and similarities in musculoskeletal biology between vertebrates that experience disparate modes of dormancy, it is hoped that this Review will stimulate new insights and ideas for future studies regarding the regulation of atrophy and osteoporosis in both natural and clinical models of muscle and bone disuse

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

Suppression of disuse-induced muscle atrophy has been associated with altered mitochondrial reactive oxygen species (ROS) production in mammals. However, despite extended hindlimb immobility, aestivating animals exhibit little skeletal muscle atrophy compared with artificially immobilised mammalian models. Therefore, we studied mitochondrial respiration and ROS (H2O2) production in permeabilised muscle fibres of the green-striped burrowing frog, Cyclorana alboguttata. Mitochondrial respiration within saponin-permeabilised skeletal and cardiac muscle fibres was measured concurrently with ROS production using high-resolution respirometry coupled to custom-made fluorometers. After 4 months of aestivation, C. alboguttata had significantly depressed whole-body metabolism by similar to 70% relative to control (active) frogs, and mitochondrial respiration in saponin-permeabilised skeletal muscle fibres decreased by almost 50% both in the absence of ADP and during oxidative phosphorylation. Mitochondrial ROS production showed up to an 88% depression in aestivating skeletal muscle when malate, succinate and pyruvate were present at concentrations likely to reflect those in vivo. The percentage ROS released per O-2 molecule consumed was also similar to 94% less at these concentrations, indicating an intrinsic difference in ROS production capacities during aestivation. We also examined mitochondrial respiration and ROS production in permeabilised cardiac muscle fibres and found that aestivating frogs maintained respiratory flux and ROS production at control levels. These results show that aestivating C. alboguttata has the capacity to independently regulate mitochondrial function in skeletal and cardiac muscles. Furthermore, this work indicates that ROS production can be suppressed in the disused skeletal muscle of aestivating frogs, which may in turn protect against potential oxidative damage and preserve skeletal muscle structure during aestivation and following arousal

Effects of eight neuropsychiatric copy number variants on human brain structure

Many copy number variants (CNVs) confer risk for the same range of neurodevelopmental symptoms and psychiatric conditions including autism and schizophrenia. Yet, to date neuroimaging studies have typically been carried out one mutation at a time, showing that CNVs have large effects on brain anatomy. Here, we aimed to characterize and quantify the distinct brain morphometry effects and latent dimensions across 8 neuropsychiatric CNVs. We analyzed T1-weighted MRI data from clinically and non-clinically ascertained CNV carriers (deletion/duplication) at the 1q21.1 (n = 39/28), 16p11.2 (n = 87/78), 22q11.2 (n = 75/30), and 15q11.2 (n = 72/76) loci as well as 1296 non-carriers (controls). Case-control contrasts of all examined genomic loci demonstrated effects on brain anatomy, with deletions and duplications showing mirror effects at the global and regional levels. Although CNVs mainly showed distinct brain patterns, principal component analysis (PCA) loaded subsets of CNVs on two latent brain dimensions, which explained 32 and 29% of the variance of the 8 Cohen’s d maps. The cingulate gyrus, insula, supplementary motor cortex, and cerebellum were identified by PCA and multi-view pattern learning as top regions contributing to latent dimension shared across subsets of CNVs. The large proportion of distinct CNV effects on brain morphology may explain the small neuroimaging effect sizes reported in polygenic psychiatric conditions. Nevertheless, latent gene brain morphology dimensions will help subgroup the rapidly expanding landscape of neuropsychiatric variants and dissect the heterogeneity of idiopathic conditions

Activity, abundance and expression of Ca2+-activated proteases in skeletal muscle of the aestivating frog, Cyclorana alboguttata

In most mammals, prolonged muscle disuse (e.g. bed-rest, limb casting or spaceflight) results in atrophy of muscle fibres which is largely due to unregulated proteolysis. Although numerous proteolytic pathways are known to participate in muscle disuse atrophy, recent evidence suggests that activation of Ca2+-dependent cysteine proteases (calpains) is required for disuse atrophy in limb skeletal muscles. In contrast to typical models of muscle disuse (humans and rodents), animals that experience natural bouts of chronic muscle inactivity, such as hibernating mammals and aestivating frogs, consistently exhibit limited or no change in skeletal muscle size. In the current study, we examined enzyme activity, protein abundance and gene expression levels of calpain isoforms in gastrocnemius muscle of the aestivating frog, Cyclorana alboguttata. We predicted that in aestivating C. alboguttata there would be a downregulation of the abundance, activity and gene expression of calpain 1 and calpain 2. In contrast to our hypothesis, there was no significant decrease in the enzyme activity levels or the relative protein abundances of calpain 1 and calpain 2. Similarly, gene expression assays (both qRT-PCR and RNA Seq data) indicated that calpains were unaffected by aestivation. Western blotting of ‘muscle-specific’ calpain 3, which is consistently downregulated during atrophic conditions, indicated that this isoform is present in C. alboguttata muscle where it appears to be in its autolysed state. The absence of any increase in enzyme activity, protein and mRNA abundance of calpains in aestivators is consistent with the protection of gastrocnemius muscle against uncontrolled proteolysis throughout aestivation

Branchial osmoregulation in the euryhaline bull shark, 'Carcharhinus leucas': a molecular analysis of ion transporters

Bull sharks, 'Carcharhinus leucas', are one of only a few species of elasmobranchs that live in both marine and freshwater environments. Osmoregulation in euryhaline elasmobranchs is achieved through the control and integration of various organs (kidney, rectal gland and liver) in response to changes in environmental salinity. However, little is known regarding the mechanisms of ion transport in the gills of euryhaline elasmobranchs and how they are affected by osmoregulatory challenges. This study was conducted to gain insight into the branchial ion and acid-base regulatory mechanisms of 'C. leucas' by identifying putative ion transporters and determining whether their expression is influenced by environmental salinity. We hypothesised that expression levels of the Na⁺/K⁺-ATPase (NKA) pump, Na⁺/H⁺ exchanger 3 (NHE3), vacuolar-type H⁺-ATPase (VHA) and anion exchanger pendrin (PDN) would be upregulated in freshwater (FW) 'C. leucas'. Immunohistochemistry was used to localise all four ion transporters in gills of bull sharks captured in both FW and estuarine/seawater (EST/SW) environments. NHE3 immunoreactivity occurred in the apical region of cells with basolateral NKA expression whereas PDN was apically expressed in cells that also exhibited basolateral VHA immunoreactivity. In accordance with our hypotheses, quantitative real-time PCR showed that the mRNA expression of NHE3 and NKA was significantly upregulated in gills of FW-captured 'C. leucas' relative to EST/SW-captured animals. These data suggest that NHE3 and NKA together may be important in mediating branchial Na+ uptake in freshwater environments, whereas PDN and VHA might contribute to Cl⁻/HCO₃⁻ transport in marine and freshwater bull shark gills

Frogs and estivation: transcriptional insights into metabolism and cell survival in a natural model of extended muscle disuse

Green-striped burrowing frogs (Cyclorana alboguttata) survive in arid environments by burrowing underground and entering into a deep, prolonged metabolic depression known as estivation. Throughout estivation, C. alboguttata is immobilized 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. Little is known about the transcriptional regulation of estivation or associated mechanisms that may minimize degradative pathways of atrophy. To investigate transcriptional pathways associated with metabolic depression and maintenance of muscle function in estivating burrowing frogs, we assembled a skeletal muscle transcriptome using next-generation short read sequencing and compared gene expression patterns between active and 4 mo estivating C. alboguttata. This identified a complex suite of gene expression changes that occur in muscle during estivation and provides evidence that estivation in burrowing frogs involves transcriptional regulation of genes associated with cytoskeletal remodeling, avoidance of oxidative stress, energy metabolism, the cell stress response, and apoptotic signaling. In particular, the expression levels of genes encoding cell cycle and prosurvival proteins, such as serine/threonine-protein kinase Chk1, cell division protein kinase 2, survivin, and vesicular overexpressed in cancer prosurvival protein 1, were upregulated during estivation. These data suggest that estivating C. alboguttata are able to regulate the expression of genes in several major cellular pathways critical to the survival and viability of cells, thus preserving muscle function while avoiding the deleterious consequences often seen in laboratory models of muscle disuse