Postnatal Protein Intake as a Determinant of Skeletal Muscle Structure and Function in Mice-A Pilot Study.

Sarcopenia is characterised by an age-related decrease in the number of muscle fibres and additional weakening of the remaining fibres, resulting in a reduction in muscle mass and function. Many studies associate poor maternal nutrition during gestation and/or lactation with altered skeletal muscle homeostasis in the offspring and the development of sarcopenia. The aim of this study was to determine whether the musculoskeletal physiology in offspring born to mouse dams fed a low-protein diet during pregnancy was altered and whether any physiological changes could be modulated by the nutritional protein content in early postnatal stages. Thy1-YFP female mice were fed ad libitum on either a normal (20%) or a low-protein (5%) diet. Newborn pups were cross-fostered to different lactating dams (maintained on a 20% or 5% diet) to generate three groups analysed at weaning (21 days): Normal-to-Normal (NN), Normal-to-Low (NL) and Low-to-Normal (LN). Further offspring were maintained ad libitum on the same diet as during lactation until 12 weeks of age, creating another three groups (NNN, NLL, LNN). Mice on a low protein diet postnatally (NL, NLL) exhibited a significant reduction in body and muscle weight persisting up to 12 weeks, unlike mice on a low protein diet only prenatally (LN, LNN). Muscle fibre size was reduced in mice from the NL but not LN group, showing recovery at 12 weeks of age. Muscle force was reduced in NLL mice, concomitant with changes in the NMJ site and changes in atrophy-related and myosin genes. In addition, µCT scans of mouse tibiae at 12 weeks of age revealed changes in bone mass and morphology, resulting in a higher bone mass in the NLL group than the control NNN group. Finally, changes in the expression of miR-133 in the muscle of NLL mice suggest a regulatory role for this microRNA in muscle development in response to postnatal diet changes. Overall, this data shows that a low maternal protein diet and early postnatal life low-protein intake in mice can impact skeletal muscle physiology and function in early life while postnatal low protein diet favours bone integrity in adulthood.

Redox Control of Signalling Responses to Contractile Activity and Ageing in Skeletal Muscle.

Research over almost 40 years has established that reactive oxygen species are generated at different sites in skeletal muscle and that the generation of these species is increased by various forms of exercise. Initially, this was thought to be potentially deleterious to skeletal muscle and other tissues, but more recent data have identified key roles of these species in muscle adaptations to exercise. The aim of this review is to summarise our current understanding of these redox signalling roles of reactive oxygen species in mediating responses of muscle to contractile activity, with a particular focus on the effects of ageing on these processes. In addition, we provide evidence that disruption of the redox status of muscle mitochondria resulting from age-associated denervation of muscle fibres may be an important factor leading to an attenuation of some muscle responses to contractile activity, and we speculate on potential mechanisms involved.

Neuron-specific deletion of CuZnSOD leads to an advanced sarcopenic phenotype in older mice.

Age-associated loss of muscle mass and function (sarcopenia) has a profound effect on the quality of life in the elderly. Our previous studies show that CuZnSOD deletion in mice (Sod1-/- mice) recapitulates sarcopenia phenotypes, including elevated oxidative stress and accelerated muscle atrophy, weakness, and disruption of neuromuscular junctions (NMJs). To determine whether deletion of Sod1 initiated in neurons in adult mice is sufficient to induce muscle atrophy, we treated young (2- to 4-month-old) Sod1flox/SlickHCre mice with tamoxifen to generate i-mn-Sod1KO mice. CuZnSOD protein was 40-50% lower in neuronal tissue in i-mn-Sod1KO mice. Motor neuron number in ventral spinal cord was reduced 28% at 10 months and more than 50% in 18- to 22-month-old i-mn-Sod1KO mice. By 24 months, 22% of NMJs in i-mn-Sod1KO mice displayed a complete lack of innervation and deficits in specific force that are partially reversed by direct muscle stimulation, supporting the loss of NMJ structure and function. Muscle mass was significantly reduced by 16 months of age and further decreased at 24 months of age. Overall, our findings show that neuronal-specific deletion of CuZnSOD is sufficient to cause motor neuron loss in young mice, but that NMJ disruption, muscle atrophy, and weakness are not evident until past middle age. These results suggest that loss of innervation is critical but may not be sufficient until the muscle reaches a threshold beyond which it cannot compensate for neuronal loss or rescue additional fibers past the maximum size of the motor unit.

Development of a Checklist to Prevent Reconstructive Errors Made By Undergraduate Dental Students.

PURPOSE: To design a checklist in order to reduce the frequency of reconstructive preventable errors (PE) performed by undergraduate dental students at McGill University. MATERIALS AND METHODS: The most common PE occurring at a university dental clinic were identified by three reviewers analyzing the refunded cases, and used to create a preliminary checklist. This checklist was then validated by a panel of dental educators to produce a finalized 20-item checklist. The 20-question checklist was then submitted to students in a cross-sectional survey-based study to evaluate its relevance to undergraduate clinical education needs. RESULTS: As many as 81% of students reported to have forgotten at least one item of the checklist during care of their last patient, and the most forgotten checklist items corresponded to the pretreatment stage. The students also reported that 17 of the 20 items in the checklist were relevant to a considerable extent or highly relevant. CONCLUSION: Common PE identified in the undergraduate clinic could be used to create a checklist of relevant items designed to reduce errors made by students and practitioners performing prosthodontic and reconstructive treatments. However, further studies are required to evaluate the implementation and efficiency of the checklist.

Redox responses in skeletal muscle following denervation.

Previous studies have shown a significant increase in the mitochondrial generation of hydrogen peroxide (H2O2) and other peroxides in recently denervated muscle fibers. The mechanisms for generation of these peroxides and how the muscle responds to these peroxides are not fully established. The aim of this work was to determine the effect of denervation on the muscle content of proteins that may contribute to mitochondrial peroxide release and the muscle responses to this generation. Denervation of the tibialis anterior (TA) and extensor digitorum longus (EDL) muscles in mice was achieved by surgical removal of a small section of the peroneal nerve prior to its entry into the muscle. An increase in mitochondrial peroxide generation has been observed from 7 days and sustained up to 21 days following denervation in the TA muscle fibers. This increased peroxide generation was reduced by incubation of skinned fibers with inhibitors of monoamine oxidases, NADPH oxidases or phospholipase A2 enzymes and the muscle content of these enzymes together with peroxiredoxin 6 were increased following denervation. Denervated muscle also showed significant adaptations in the content of several enzymes involved in the protection of cells against oxidative damage. Morphological analyses indicated a progressive significant loss of muscle mass in the TA muscle from 7 days up to 21 days following denervation due to fiber atrophy but without fiber loss. These results support the possibility that, at least initially, the increase in peroxide production may stimulate adaptations in an attempt to protect the muscle fibers, but that these processes are insufficient and the increased peroxide generation over the longer term may activate degenerative and atrophic processes in the denervated muscle fibers.

Aberrant redox signalling and stress response in age-related muscle decline: Role in inter- and intra-cellular signalling.

Age-associated frailty is predominantly due to loss of muscle mass and function. The loss of muscle mass is also associated with a greater loss of muscle strength, suggesting that the remaining muscle fibres are weaker than those of adults. The mechanisms by which muscle is lost with age are unclear, but in this review we aim to pull together various strands of evidence to explain how muscle contractions support proteostasis in non-muscle tissues, particularly focussed on the production and potential transfer of Heat Shock Proteins (HSPs) and how this may fail during ageing, Furthermore we will identify logical approaches, based on this hypothesis, by which muscle loss in ageing may be reduced. Skeletal muscle generates superoxide and nitric oxide at rest and this generation is increased by contractile activity. In adults, this increased generation of reactive oxygen and nitrogen species (RONS) activate redox-sensitive transcription factors such as nuclear factor κB (NFκB), activator protein-1 (AP1) and heat shock factor 1 (HSF1), resulting in increases in cytoprotective proteins such as the superoxide dismutases, catalase and heat shock proteins that prevent oxidative damage to tissues and facilitate remodelling and proteostasis in both an intra- and inter-cellular manner. During ageing, the ability of skeletal muscle from aged organisms to respond to an increase in ROS generation by increased expression of cytoprotective proteins through activation of redox-sensitive transcription factors is severely attenuated. This age-related lack of physiological adaptations to the ROS induced by contractile activity appears to contribute to a loss of ROS homeostasis, increased oxidative damage and age-related dysfunction in skeletal muscle and potentially other tissues.

Denervated muscle fibers induce mitochondrial peroxide generation in neighboring innervated fibers: Role in muscle aging.

Disruption of neuromuscular junctions and denervation of some muscle fibers occurs in ageing skeletal muscle and contribute to loss of muscle mass and function. Aging is associated with mitochondrial dysfunction and loss of redox homeostasis potentially occurs through increased mitochondrial generation of reactive oxygen species (ROS). No specific link between increased mitochondrial ROS generation and denervation has been defined in muscle ageing. To address this, we have examined the effect of experimental denervation of all fibers, or only a proportion of the fibers, in the mouse tibialis anterior (TA) muscle on muscle mitochondrial peroxide generation. Transection of the peroneal nerve of mice caused loss of pre-synaptic axons within 1-3 days with no significant morphological changes in post-synaptic structures up to 10 days post-surgery when decreased TA mass and fiber size were apparent. Mitochondria in the denervated muscle showed increased peroxide generation by 3 days post-transection. Use of electron transport chain (ETC) substrates and inhibitors of specific pathways indicated that the ETC was unlikely to contribute to increased ROS generation, but monoamine oxidase B, NADPH oxidase and phospholipase enzymes were implicated. Transection of one of the 3 branches of the peroneal nerve caused denervation of some TA muscle fibers while others retained innervation, but increased mitochondrial peroxide generation occurred in both denervated and innervated fibers. Thus the presence of recently denervated fibers leads to increased ROS generation by mitochondria in neighboring innervated fibers providing a novel explanation for the increased mitochondrial oxidative stress and damage seen with aging in skeletal muscles.

The effect of lengthening contractions on neuromuscular junction structure in adult and old mice.

Skeletal muscles of old mice demonstrate a profound inability to regenerate fully following damage. Such a failure could be catastrophic to older individuals where muscle loss is already evident. Degeneration and regeneration of muscle fibres following contraction-induced injury in adult and old mice are well characterised, but little is known about the accompanying changes in motor neurons and neuromuscular junctions (NMJs) following this form of injury although defective re-innervation of muscle following contraction-induced damage has been proposed to play a role in sarcopenia. This study visualised and quantified structural changes to motor neurons and NMJs in Extensor digitorum longus (EDL) muscles of adult and old Thy1-YFP transgenic mice during regeneration following contraction-induced muscle damage. Data demonstrated that the damaging contraction protocol resulted in substantial initial disruption to NMJs in muscles of adult mice, which was reversed entirely within 28 days following damage. In contrast, in quiescent muscles of old mice, ∼15 % of muscle fibres were denervated and ∼80 % of NMJs showed disruption. This proportion of denervated and partially denervated fibres remained unchanged following recovery from contraction-induced damage in muscles of old mice although ∼25 % of muscle fibres were completely lost by 28 days post-contractions. Thus, in old mice, the failure to restore full muscle force generation that occurs following damage does not appear to be due to any further deficit in the percentage of disrupted NMJs, but appears to be due, at least in part, to the complete loss of muscle fibres following damage.

Ageing-induced changes in the redox status of peripheral motor nerves imply an effect on redox signalling rather than oxidative damage.

Ageing is associated with loss of skeletal muscle fibres, atrophy of the remaining fibres and weakness. These changes in muscle are accompanied by disruption of motor neurons and neuromuscular junctions although the direct relationship between the nerve and muscle degeneration is not understood. Oxidative changes have been implicated in the mechanisms leading to age-related loss of muscle mass and in degeneration of the central nervous system, but little is known about age-related changes in oxidation in specific peripheral nerves that supply muscles that are affected by ageing. We have therefore examined the sciatic nerve of old mice at an age when loss of tibialis anterior muscle mass and function is apparent. Sciatic nerve from old mice did not show a gross increase in oxidative damage, but electron paramagnetic resonance (EPR) studies indicated an increase in the activity of superoxide and/or peroxynitrite in the nerves of old mice at rest that was further exacerbated by electrical stimulation of the nerve to activate muscle contractions. Proteomic analyses indicated that specific redox-sensitive proteins are increased in content in the nerves of old mice that may reflect an adaptation to regulate the increased superoxide/peroxynitrite and maintain redox homoeostasis. Analysis of redox active cysteines showed some increase in reversible oxidation in specific proteins in nerves of old mice, but this was not universally seen across all redox-active cysteines. Detailed analysis of the redox-active cysteine in one protein in the nerve of old mice that is key to redox signalling (Peroxiredoxin 6, Cys 47) showed a minor increase in reversible oxidation that would be compatible with a change in its redox signalling function. In conclusion, the data presented indicate that sciatic nerve from old mice does not show a gross increase in oxidative damage similar to that seen in the TA and other muscles that it innervates. Our results indicate an adaptation to increased oxidation with minor changes in the oxidation of key cysteines that may contribute to defective redox signalling in the nerve.

Measurement and computer modeling of temporary arrangements of polygonal actin structures in trabecular meshwork cells which consist of cross-linked actin networks and polygonal actin arrangements.

PURPOSE: In trabecular meshwork (TM) cells, actin geodesic arrangements were measured and then subjected to computational modeling to appreciate the response of different dome shapes to mechanical force. METHODS: Polygonal actin arrangements (PAAs) and cross-linked actin networks (CLANs) were induced and imaged by Alexa Flour(®) 488 Phalloidin in bovine TM and human TM cells. Masked images were examined for size, circularity, and spoke and hub dimensions using ImageJ. Finite element modeling was used to create idealized dome structures and "realistic" PAA and CLAN models. The models were subjected to different loads simulating concentrated force and distortion measured. RESULTS: We provide evidence that PAAs and CLANs are not identical. Both structures formed flattened domes but PAAs were 6 times larger than CLANs, significantly more circular and had greater height. The dimensions of the triangulations of hubs and spokes were, however, remarkably similar. Hubs were around 2 µm(2) in area, whereas spokes were about 5 µm in length. Our modeling showed that temporary arrangements of polygonal actin structures (TAPAS) were because of their flattened shape, more resistant to shearing than compression when compared with idealized domes. CLANs were marginally more resistant to shearing than PAAs but because of size much more resistant to compression. CONCLUSIONS: Evidence is provided that there are 2 types of actin icosahedrons in cultured TM cells we collectively call TAPAS. Modeling suggests that TAPAS have rigidity and are better at dealing with shearing than compression forces. The 2 types of TAPAS, PAAs, and CLANs, have much in common but there are size and mechanical response differences that need to be taken into account in future experimentation.

Inducers of cross-linked actin networks in trabecular meshwork cells.

PURPOSE: It is well established that the unusual actin arrangements known as cross-linked actin networks (CLANs) can be induced by dexamethasone (DEX) in trabecular meshwork (TM) cells. Recent work reporting their presence in elderly glaucomatous and nonglaucomatous tissue, however, has highlighted the presence of other inducers. In this study, the authors sought to identify CLAN induction agents that may be present within and around the outflow system. METHODS: Studies were conducted on confluent bovine TM (BTM) cells in culture, and actin was stained with Alexa-Fluor 488 phalloidin to identify CLANs in the target cells. The CLAN-inducing potential of aqueous humor was expanded and included investigation of transforming growth factor-beta 2 (TGF-ß2). The effect of decorin and fetal calf serum (FCS) on BTM cell cytoskeleton was also investigated, and all were compared with DEX with an exposure period of up to 7 days. RESULTS: CLAN numbers were increased after 7 days of exposure to TGF-ß2 (45%), aqueous humor (37%), and decorin (69%). Even FCS had some modest CLAN-inducing ability (reaching 12%) in BTM cells. Neutralization of TGF-ß2 reduced CLAN incidence in aqueous humor conditions to baseline (12%) levels. Blocking TGF-ß2 receptors reduced CLAN formation in TM cells by 25% to 30%, whereas the inhibition of Smad3 negated CLAN incidence. CONCLUSIONS: In this study the authors identified TGF-ß2 as a CLAN-inducing component present in aqueous humor. Decorin was also implicated as another CLAN-inducing agent and it was confirmed that FCS has CLAN-inducing properties.

Cross-linked actin networks (CLANs) are present in lamina cribrosa cells.

AIMS: A study was undertaken to determine and compare the F-actin staining patterns in the cells of the lamina cribrosa (LC) of normal, dexamethasone (DEX)-treated and glaucomatous dissected tissue and cell cultures. METHODS: About 30 dissected donor eyes and nine cell lines provided the human specimens; 25 eyes and 20 primary cell cultures provided the bovine material. Appropriate samples were exposed to 1×10⁻7 M DEX for up to 14 days. LC tissue and cells were stained with Phalloidin-Alexa 488 to identify F-actin, and all samples were examined by confocal or epifluorescent microscopy. RESULTS: Both in the LC tissue and LC cell cultures the dominant actin arrangement was bundles of stress fibres. However, cross-linked actin networks (CLANs) were identified in the tissue and in culture. These were markedly increased by steroid treatment and were particularly large and abundant in cultures from glaucoma donors. CLANs were not associated with optic nerve head astrocytes. CONCLUSIONS: The presence of abundant stress fibres in situ and in vitro highlights the biomechanical contribution of LC cells. However, the identification of CLANs in these cells shows that they are not exclusive to the trabecular meshwork, the only other place they have been found, and may have a role in glaucoma pathology.

Cross-linked actin networks (CLANs) in the trabecular meshwork of the normal and glaucomatous human eye in situ.

PURPOSE: A percentage of trabecular meshwork (TM) cells in tissue and organ culture have been shown to contain cross-linked actin networks (CLANs) when exposed to dexamethasone, as have TM cultures derived from glaucomatous individuals. The purpose of this study was to determine whether CLANs exist within TM cells in situ in tissue unmanipulated by culturing, thereby eliminating the possibility that CLANs are artifacts of culture conditions, and to determine their numbers and dimensions in normal and glaucoma TM cells. METHODS: Twelve human donor eyes (five normal and seven with glaucoma) provided the TM tissue. Each eye was dissected, and the TM tissue was exposed either by microdissection (qualitative studies) or cryo-sectioning (quantitative analysis). The actin cytoskeleton was visualized using a high-affinity probe and viewed using confocal microscopy. RESULTS: Qualitative examination of the microdissected tissue revealed that CLANs and CLAN-like structures were a common finding in the TM cells in every specimen, irrespective of whether they were from normal or glaucomatous eyes. CLAN size and phenotype were variable, with the same variations occurring in both normal and glaucomatous eyes. Quantitative analysis showed that there were more CLANs in glaucoma TM specimens than normal TM specimens, but this difference was not statistically significant. The mean number of CLANs/TM cell in our glaucoma tissue was estimated to be 1.03, while in the elderly normal controls it was 0.67. CONCLUSIONS: This study showed for the first time that CLANs exist in cells of TM tissues from both normal and glaucomatous eyes that have not been manipulated by either tissue or organ culture procedures. It also provides quantitative data on CLAN prevalence in organized TM tissue, which indicates that CLANs are far more common than predicted (even from tissue culture) and there may be one in every cell in the glaucomatous TM in situ.