Defining the Most Potent Osteoinductive Culture Conditions for MC3T3-E1 Cells Reveals No Implication of Oxidative Stress or Energy Metabolism.

The MC3T3-E1 preosteoblastic cell line is widely utilised as a reliable in vitro system to assess bone formation. However, the experimental growth conditions for these cells hugely diverge, and, particularly, the osteogenic medium (OSM)'s composition varies in research studies. Therefore, we aimed to define the ideal culture conditions for MC3T3-E1 subclone 4 cells with regard to their mineralization capacity and explore if oxidative stress or the cellular metabolism processes are implicated. Cells were treated with nine different combinations of long-lasting ascorbate (Asc) and ß-glycerophosphate (ßGP), and osteogenesis/calcification was evaluated at three different time-points by qPCR, Western blotting, and bone nodule staining. Key molecules of the oxidative and metabolic pathways were also assessed. It was found that sufficient mineral deposition was achieved only in the 150 µg.mL-1/2 mM Asc/ßGP combination on day 21 in OSM, and this was supported by Runx2, Alpl, Bglap, and Col1a1 expression level increases. NOX2 and SOD2 as well as PGC1α and Tfam were also monitored as indicators of redox and metabolic processes, respectively, where no differences were observed. Elevation in OCN protein levels and ALP activity showed that mineralisation comes as a result of these differences. This work defines the most appropriate culture conditions for MC3T3-E1 cells and could be used by other research laboratories in this field.

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.

Low protein intake during reproduction compromises the recovery of lactation-induced bone loss in female mouse dams without affecting skeletal muscles.

Lactation-induced bone loss occurs due to high calcium requirements for fetal growth but skeletal recovery is normally achieved promptly postweaning. Dietary protein is vital for fetus and mother but the effects of protein undernutrition on the maternal skeleton and skeletal muscles are largely unknown. We used mouse dams fed with normal (N, 20%) or low (L, 8%) protein diet during gestation and lactation and maintained on the same diets (NN, LL) or switched from low to normal (LN) during a 28 d skeletal restoration period post lactation. Skeletal muscle morphology and neuromuscular junction integrity was not different between any of the groups. However, dams fed the low protein diet showed extensive bone loss by the end of lactation, followed by full skeletal recovery in NN dams, partial recovery in LN and poor bone recovery in LL dams. Primary osteoblasts from low protein diet fed mice showed decreased in vitro bone formation and decreased osteogenic marker gene expression; promoter methylation analysis by pyrosequencing showed no differences in Bmpr1a, Ptch1, Sirt1, Osx, and Igf1r osteoregulators, while miR-26a, -34a, and -125b expression was found altered in low protein fed mice. Therefore, normal protein diet is indispensable for maternal musculoskeletal health during the reproductive period.

Age-related changes in skeletal muscle: changes to life-style as a therapy.

As we age, there is an age-related loss in skeletal muscle mass and strength, known as sarcopenia. Sarcopenia results in a decrease in mobility and independence, as well as an increase in the risk of other morbidities and mortality. Sarcopenia is therefore a major socio-economical problem. The mechanisms behind sarcopenia are unclear and it is likely that it is a multifactorial condition with changes in numerous important mechanisms all contributing to the structural and functional deterioration. Here, we review the major proposed changes which occur in skeletal muscle during ageing and highlight evidence for changes in physical activity and nutrition as therapeutic approaches to combat age-related skeletal muscle wasting.

Role of nerve-muscle interactions and reactive oxygen species in regulation of muscle proteostasis with ageing.

Skeletal muscle ageing is characterised by atrophy, a deficit in specific force generation, increased susceptibility to injury, and incomplete recovery after severe damage. The hypothesis that increased generation of reactive oxygen species (ROS) in vivo plays a key role in the ageing process has been extensively studied, but remains controversial. Skeletal muscle generates ROS at rest and during exercise. ROS can cause oxidative damage particularly to proteins. Indeed, products of oxidative damage accumulate in skeletal muscle during ageing and the ability of muscle cells to respond to increased ROS becomes defective. The aim of this review is to examine the evidence that ROS manipulation in peripheral nerves and/or muscle modifies mechanisms of proteostasis in skeletal muscle and plays a key role in initiating sarcopenia.

Ageing in relation to skeletal muscle dysfunction: redox homoeostasis to regulation of gene expression.

Ageing is associated with a progressive loss of skeletal muscle mass, quality and function-sarcopenia, associated with reduced independence and quality of life in older generations. A better understanding of the mechanisms, both genetic and epigenetic, underlying this process would help develop therapeutic interventions to prevent, slow down or reverse muscle wasting associated with ageing. Currently, exercise is the only known effective intervention to delay the progression of sarcopenia. The cellular responses that occur in muscle fibres following exercise provide valuable clues to the molecular mechanisms regulating muscle homoeostasis and potentially the progression of sarcopenia. Redox signalling, as a result of endogenous generation of ROS/RNS in response to muscle contractions, has been identified as a crucial regulator for the adaptive responses to exercise, highlighting the redox environment as a potentially core therapeutic approach to maintain muscle homoeostasis during ageing. Further novel and attractive candidates include the manipulation of microRNA expression. MicroRNAs are potent gene regulators involved in the control of healthy and disease-associated biological processes and their therapeutic potential has been researched in the context of various disorders, including ageing-associated muscle wasting. Finally, we discuss the impact of the circadian clock on the regulation of gene expression in skeletal muscle and whether disruption of the peripheral muscle clock affects sarcopenia and altered responses to exercise. Interventions that include modifying altered redox signalling with age and incorporating genetic mechanisms such as circadian- and microRNA-based gene regulation, may offer potential effective treatments against age-associated sarcopenia.

Neuron-specific expression of CuZnSOD prevents the loss of muscle mass and function that occurs in homozygous CuZnSOD-knockout mice.

Deletion of copper-zinc superoxide dismutase (CuZnSOD) in Sod1(-/-) mice leads to accelerated loss of muscle mass and force during aging, but the losses do not occur with muscle-specific deletion of CuZnSOD. To determine the role of motor neurons in the muscle decline, we generated transgenic Sod1(-/-) mice in which CuZnSOD was expressed under control of the synapsin 1 promoter (SynTgSod1(-/-) mice). SynTgSod1(-/-) mice expressed CuZnSOD in brain, spinal cord, and peripheral nerve, but not in other tissues. Sciatic nerve CuZnSOD content in SynTgSod1(-/-) mice was ~20% that of control mice, but no reduction in muscle mass or isometric force was observed in SynTgSod1(-/-) mice compared with control animals, whereas muscles of age-matched Sod1(-/-) mice displayed 30-40% reductions in mass and force. In addition, increased oxidative damage and adaptations in stress responses observed in muscles of Sod1(-/-) mice were absent in SynTgSod1(-/-) mice, and degeneration of neuromuscular junction (NMJ) structure and function occurred in Sod1(-/-) mice but not in SynTgSod1(-/-) mice. Our data demonstrate that specific CuZnSOD expression in neurons is sufficient to preserve NMJ and skeletal muscle structure and function in Sod1(-/-) mice and suggest that redox homeostasis in motor neurons plays a key role in initiating sarcopenia during aging.

Lifelong dietary protein restriction accelerates skeletal muscle loss and reduces muscle fibre size by impairing proteostasis and mitochondrial homeostasis.

The early life environment significantly affects the development of age-related skeletal muscle disorders. However, the long-term effects of lactational protein restriction on skeletal muscle are still poorly defined. Our study revealed that male mice nursed by dams fed a low-protein diet during lactation exhibited skeletal muscle growth restriction. This was associated with a dysregulation in the expression levels of genes related to the ribosome, mitochondria and skeletal muscle development. We reported that lifelong protein restriction accelerated loss of type-IIa muscle fibres and reduced muscle fibre size by impairing mitochondrial homeostasis and proteostasis at 18 months of age. However, feeding a normal-protein diet following lactational protein restriction prevented accelerated fibre loss and fibre size reduction in later life. These findings provide novel insight into the mechanisms by which lactational protein restriction hinders skeletal muscle growth and includes evidence that lifelong dietary protein restriction accelerated skeletal muscle loss in later life.

Aging increases the oxidation of dichlorohydrofluorescein in single isolated skeletal muscle fibers at rest, but not during contractions.

An increase in the activity of reactive oxygen species (ROS) has been implicated in the mechanisms of loss of skeletal muscle that occurs during aging, but few studies have attempted to directly assess activities in intact muscle fibers. The current project used the nonspecific fluorescent probe for ROS and reactive nitrogen species, 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein (CM-DCFH), in single, isolated, mature skeletal muscle fibers from adult and old mice in addition to biochemical measurements of key regulatory proteins for ROS in muscles of these animals. Data confirmed the changes in key regulatory processes for ROS (increased glutathione peroxidase 1 and catalase activities and reduced total glutathione content) previously reported in muscle from old mice and showed increased CM-DCFH oxidation in muscle fibers from old mice at rest and indicate that these changes are likely due to an increase in generation of oxidants rather than a lack of scavenging capacity. The increased CM-DCFH oxidation persisted even when cellular defenses against oxidants were increased by loading fibers from young and old mice with glutathione. During contractile activity, and in contrast to the increase observed in fibers from young mice, there was no further increase in CM-DCFH oxidation in muscle fibers from old mice. These data also suggest that the defect in short-term adaptations to contractions that occurs in old mice may be related to a diminished, or absent, increase in the muscle generation of ROS and/or reactive nitrogen species that normally accompanies contractile activity in young mice.

Is oxidative stress a physiological cost of reproduction? An experimental test in house mice.

Investment in reproduction is costly and frequently decreases survival or future reproductive success. However, the proximate underlying causes for this are largely unknown. Oxidative stress has been suggested as a cost of reproduction and several studies have demonstrated changes in antioxidants with reproductive investment. Here, we test whether oxidative stress is a consequence of reproduction in female house mice (Mus musculus domesticus), which have extremely high energetic demands during reproduction, particularly through lactation. Assessing oxidative damage after a long period of reproductive investment, there was no evidence of increased oxidative stress, even when females were required to defend their breeding territory. Instead, in the liver, markers of oxidative damage (malonaldehyde, protein thiols and the proportion of glutathione in the oxidized form) indicated lower oxidative stress in reproducing females when compared with non-reproductive controls. Even during peak lactation, none of the markers of oxidative damage indicated higher oxidative stress than among non-reproductive females, although a positive correlation between protein oxidation and litter mass suggested that oxidative stress may increase with fecundity. Our results indicate that changes in redox status occur during reproduction in house mice, but suggest that females use mechanisms to cope with the consequences of increased energetic demands and limit oxidative stress.

Small-RNA Sequencing Reveals Altered Skeletal Muscle microRNAs and snoRNAs Signatures in Weanling Male Offspring from Mouse Dams Fed a Low Protein Diet during Lactation.

Maternal diet during gestation and lactation affects the development of skeletal muscles in offspring and determines muscle health in later life. In this paper, we describe the association between maternal low protein diet-induced changes in offspring skeletal muscle and the differential expression (DE) of small non-coding RNAs (sncRNAs). We used a mouse model of maternal protein restriction, where dams were fed either a normal (N, 20%) or a low protein (L, 8%) diet during gestation and newborns were cross-fostered to N or L lactating dams, resulting in the generation of NN, NL and LN offspring groups. Total body and tibialis anterior (TA) weights were decreased in weanling NL male offspring but were not different in the LN group, as compared to NN. However, histological evaluation of TA muscle revealed reduced muscle fibre size in both groups at weaning. Small RNA-sequencing demonstrated DE of multiple miRs, snoRNAs and snRNAs. Bioinformatic analyses of miRs-15a, -34a, -122 and -199a, in combination with known myomiRs, confirmed their implication in key muscle-specific biological processes. This is the first comprehensive report for the DE of sncRNAs in nutrition-associated programming of skeletal muscle development, highlighting the need for further research to unravel the detailed molecular mechanisms.

Low steady-state oxidative stress inhibits adipogenesis by altering mitochondrial dynamics and decreasing cellular respiration.

Adipogenesis is a fundamental process of white adipose tissue function, supporting lipid storage and release, while avoiding its spillover and ectopic accumulation in tissues and organs. During aging adipogenesis is impaired and among other factors, oxidative stress contributes to this process. Adipogenesis requires functional and dynamic mitochondria; however, this organelle itself becomes dysfunctional during aging and accounts for most of reactive oxygen species (ROS) production. Here, we evaluated whether oxidative stress impairs adipogenesis through functional impairment of mitodynamics by utilizing hyperoxia as a continuous source of oxidative stress while maintaining cellular viability. This negatively impacted mitochondrial function, including respiration and dynamics and ultimately blocked adipogenesis. Interestingly, this state was reversible by using the antidiabetic drug, Rosiglitazone, which reduced oxidative stress, restored mitochondrial dynamics and respiration and augmented adipogenesis. Moreover, in vitro results were in agreement with in vivo models of oxidative stress and aging, in which mice depleted of the superoxide dismutase enzyme 1 (SOD1) and old wild-type C57BL/6JRj mice demonstrated the same trend of adipogenic potential. Importantly, in humans the results follow the same pattern, showing a downregulation of adipogenic markers during aging. Since the levels of oxidative stress and peripheral insulin resistance increase with age, while adipogenesis decreases during aging, our model helps to understand a possible way to overcome physiologically low, steady stress conditions and restore adipogenesis, avoiding accumulation of deleterious hypertrophic adipocytes in favor of beneficial hyperplasia.

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.

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.

Accelerated sarcopenia in Cu/Zn superoxide dismutase knockout mice.

Mice lacking Cu/Zn-superoxide dismutase (Sod1-/- or Sod1KO mice) show high levels of oxidative stress/damage and a 30% decrease in lifespan. The Sod1KO mice also show many phenotypes of accelerated aging with the loss of muscle mass and function being one of the most prominent aging phenotypes. Using various genetic models targeting the expression of Cu/Zn-superoxide dismutase to specific tissues, we evaluated the role of motor neurons and skeletal muscle in the accelerated loss of muscle mass and function in Sod1KO mice. Our data are consistent with the sarcopenia in Sod1KO mice arising through a two-hit mechanism involving both motor neurons and skeletal muscle. Sarcopenia is initiated in motor neurons leading to a disruption of neuromuscular junctions that results in mitochondrial dysfunction and increased generation of reactive oxygen species (ROS) in skeletal muscle. The mitochondrial ROS generated in muscle feedback on the neuromuscular junctions propagating more disruption of neuromuscular junctions and more ROS production by muscle resulting in a vicious cycle that eventually leads to disaggregation of neuromuscular junctions, denervation, and loss of muscle fibers.

Repeated bouts of aerobic exercise lead to reductions in skeletal muscle free radical generation and nuclear factor kappaB activation.

Chronic exercise improves endurance and skeletal muscle oxidative capacity. Despite the potential importance of reactive oxygen species (ROS) generated during exercise as regulators of these adaptations, the effect of repeated bouts of aerobic exercise on ROS generation by skeletal muscles during contractions has not been examined. Our aim was to establish the impact of repeated treadmill running exercise on muscle ROS generation and activation of redox-sensitive transcription factors. Following 8 weeks of treadmill running, mice displayed an improvement in running speed that was associated with an enhanced ability of gastrocnemius (GTN) muscles to maintain force during a protocol of isometric contractions. In contrast to GTN muscles of cage-sedentary (Sed) mice, muscles from exercised (Exer) mice did not release superoxide or nitric oxide during the isometric contractions. For male mice, basal levels of nuclear factor kappaB (NFkappaB) and activator protein-1 (AP-1) DNA binding were increased by treadmill running, and the contraction-induced activation of NFkappaB and AP-1 observed in muscles of Sed mice was absent in Exer muscles. Also in contrast to Sed muscles, Exer muscles displayed no reductions in glutathione or protein thiol levels in response to contraction. Our observations of decreases for Exer compared with Sed muscles in contraction-induced (i) ROS generation, (ii) activation of redox-sensitive signalling pathways, and (iii) ROS stress suggest that exercise conditioning enhances the ability of skeletal muscle to readily and rapidly detoxify ROS and/or reduces ROS generation, providing protection from ROS-induced damage and reducing signals that might act to mediate further unnecessary adaptations.

Comparison of Whole Body SOD1 Knockout with Muscle-Specific SOD1 Knockout Mice Reveals a Role for Nerve Redox Signaling in Regulation of Degenerative Pathways in Skeletal Muscle.

AIMS: Lack of Cu,Zn-superoxide dismutase (CuZnSOD) in homozygous knockout mice (Sod1-/-) leads to accelerated age-related muscle loss and weakness, but specific deletion of CuZnSOD in skeletal muscle (mSod1KO mice) or neurons (nSod1KO mice) resulted in only mild muscle functional deficits and failed to recapitulate the loss of mass and function observed in Sod1-/- mice. To dissect any underlying cross-talk between motor neurons and skeletal muscle in the degeneration in Sod1-/- mice, we characterized neuromuscular changes in the Sod1-/- model compared with mSod1KO mice and examined degenerative molecular mechanisms and pathways in peripheral nerve and skeletal muscle. RESULTS: In contrast to mSod1KO mice, myofiber atrophy in Sod1-/- mice was associated with increased muscle oxidative damage, neuromuscular junction degeneration, denervation, nerve demyelination, and upregulation of proteins involved in maintenance of myelin sheaths. Proteomic analyses confirmed increased proteasomal activity and adaptive stress responses in muscle of Sod1-/- mice that were absent in mSod1KO mice. Peripheral nerve from neither Sod1-/- nor mSod1KO mice showed increased oxidative damage or molecular responses to increased oxidation compared with wild type mice. Differential cysteine (Cys) labeling revealed a specific redox shift in the catalytic Cys residue of peroxiredoxin 6 (Cys47) in the peripheral nerve from Sod1-/- mice. Innovation and Conclusion: These findings demonstrate that neuromuscular integrity, redox mechanisms, and pathways are differentially altered in nerve and muscle of Sod1-/- and mSod1KO mice. Results support the concept that impaired redox signaling, rather than oxidative damage, in peripheral nerve plays a key role in muscle loss in Sod1-/- mice and potentially sarcopenia during aging. Antioxid. Redox Signal. 28, 275-295.

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.

Glutathione-peroxidase-1 null muscle progenitor cells are globally defective.

Mice lacking glutathione peroxidase-1 (Gpx1) have decreased resistance to systemically administered oxidants as well as infections, and sustain increased damage after ischemia-reperfusion injuries. However, stem or progenitor cell function in these animals has not been studied. We characterized patterns of proliferation, apoptosis, and differentiation of primary muscle progenitor cells (myoblasts) from Gpx1(-/-) mice. Myoblasts are the transit amplifying compartment of skeletal muscle. All aspects of myoblast biology are negatively affected by deletion of Gpx1. In particular, passaged, proliferating Gpx1(-/-) myoblasts, when induced to differentiate into fused multinucleated myotubes, show significant impairment, and form only a few immature myotubes. This defect occurs despite increased expression of the core regulators of muscle differentiation, the myogenic basic helix-loop-helix (bHLH) transcription factors, in the Gpx1(-/-) myoblasts. Furthermore, Gpx1(-/-) myoblasts exhibited decreased proliferation and increased apoptosis compared to wild-type cells. In vivo, muscle fiber areas are decreased in Gpx1(-/-) vs wild-type mice. These data suggest that Gpx1 is important for adult muscle progenitor cell function at many levels, is necessary for integrity of muscle differentiation, and that quiescent resident stem cell populations may be particularly vulnerable to peroxide-mediated damage.

HSF expression in skeletal muscle during myogenesis: implications for failed regeneration in old mice.

The ability of muscles of old mice to recover force generation following substantial damage is severely impaired, particularly during the late phase of regeneration. This inability to recover successfully may be associated with the attenuated ability of muscles of old mice to produce heat shock proteins (HSPs) in response to stress since muscles of old mice overexpressing HSP70 recover successfully following damage. The capacity of mature mammalian skeletal muscle to regenerate following damage is due to the presence of undifferentiated mononuclear myogenic precursor cells (satellite cells) at the periphery of mature skeletal muscle fibres. HSP expression is under the primary transcriptional control of heat shock factors 1 and 2 (HSF1 and HSF2). The aim of this study was to examine the expression of heat shock factors 1 and 2 by western blotting in mouse-derived C2C12 myoblasts as an experimental model system for investigating skeletal muscle regeneration. Data demonstrated that the HSF2 content of myotubes was significantly increased during the early stages of regeneration. In contrast, the HSF1 content of myotubes remained relatively low until late during regeneration. Thus, abnormal activation of HSF1 may play a role in the defective regeneration seen in muscles of old mice.