James G. Ryall

Cellular and molecular mechanisms underlying age-related skeletal muscle wasting and weakness

Some of the most serious consequences of ageing are its effects on skeletal muscle. The term ‘sarcopenia’ describes the slow but progressive loss of muscle mass with advancing age and is characterised by a deterioration of muscle quantity and quality leading to a gradual slowing of movement and a decline in strength. The loss of muscle mass and strength is thought to be attributed to the progressive atrophy and loss of individual muscle fibres associated with the loss of motor units, and a concomitant reduction in muscle ‘quality’ due to the infiltration of fat and other non-contractile material. These age-related changes in skeletal muscle can be largely attributed to the complex interaction of factors affecting neuromuscular transmission, muscle architecture, fibre composition, excitation–contraction coupling, and metabolism. Given the magnitude of the growing public health problems associated with sarcopenia, there is considerable interest in the development and evaluation of therapeutic strategies to attenuate, prevent, or ultimately reverse age-related muscle wasting and weakness. The aim is to review our current understanding of some of the cellular and molecular mechanisms responsible for age-related changes in skeletal muscle.

The NAD+-Dependent SIRT1 Deacetylase Translates a Metabolic Switch into Regulatory Epigenetics in Skeletal Muscle Stem Cells

Stem cells undergo a shift in metabolic substrate utilization during specification and/or differentiation, a process that has been termed metabolic reprogramming. Here, we report that during the transition from quiescence to proliferation, skeletal muscle stem cells experience a metabolic switch from fatty acid oxidation to glycolysis. This reprogramming of cellular metabolism decreases intracellular NAD(+) levels and the activity of the histone deacetylase SIRT1, leading to elevated H4K16 acetylation and activation of muscle gene transcription. Selective genetic ablation of the SIRT1 deacetylase domain in skeletal muscle results in increased H4K16 acetylation and deregulated activation of the myogenic program in SCs. Moreover, mice with muscle-specific inactivation of the SIRT1 deacetylase domain display reduced myofiber size, impaired muscle regeneration, and derepression of muscle developmental genes. Overall, these findings reveal how metabolic cues can be mechanistically translated into epigenetic modifications that regulate skeletal muscle stem cell biology.

Polycomb EZH2 controls self-renewal and safeguards the transcriptional identity of skeletal muscle stem cells

Satellite cells (SCs) sustain muscle growth and empower adult skeletal muscle with vigorous regenerative abilities. Here, we report that EZH2, the enzymatic subunit of the Polycomb-repressive complex 2 (PRC2), is expressed in both Pax7+/Myf5⁻ stem cells and Pax7+/Myf5+ committed myogenic precursors and is required for homeostasis of the adult SC pool. Mice with conditional ablation of Ezh2 in SCs have fewer muscle postnatal Pax7+ cells and reduced muscle mass and fail to appropriately regenerate. These defects are associated with impaired SC proliferation and derepression of genes expressed in nonmuscle cell lineages. Thus, EZH2 controls self-renewal and proliferation, and maintains an appropriate transcriptional program in SCs.

AMPK-independent pathways regulate skeletal muscle fatty acid oxidation.

The activation of AMP‐activated protein kinase (AMPK) and phosphorylation/inhibition of acetyl‐CoA carboxylase 2 (ACC2) is believed to be the principal pathway regulating fatty acid oxidation. However, during exercise AMPK activity and ACC Ser‐221 phosphorylation does not always correlate with rates of fatty acid oxidation. To address this issue we have investigated the requirement for skeletal muscle AMPK in controlling aminoimidazole‐4‐carboxymide‐1‐β‐d‐ribofuranoside (AICAR) and contraction‐stimulated fatty acid oxidation utilizing transgenic mice expressing a muscle‐specific kinase dead (KD) AMPK α2. In wild‐type (WT) mice, AICAR and contraction increased AMPK α2 and α1 activities, the phosphorylation of ACC2 and rates of fatty acid oxidation while tending to reduce malonyl‐CoA levels. Despite no activation of AMPK in KD mice, ACC2 phosphorylation was maintained, malonyl‐CoA levels were reduced and rates of fatty acid oxidation were comparable between genotypes. During treadmill exercise both KD and WT mice had similar values of respiratory exchange ratio. These studies suggested the presence of an alternative ACC2 kinase(s). Using a phosphoproteomics‐based approach we identified 18 Ser/Thr protein kinases whose phosphorylation was increased by greater than 25% in contracted KD relative to WT muscle. Utilizing bioinformatics we predicted that extracellular regulated protein‐serine kinase (ERK1/2), inhibitor of nuclear factor (NF)‐κB protein‐serine kinase β (IKKβ) and protein kinase D (PKD) may phosphorylate ACC2 at Ser‐221 but during in vitro phosphorylation assays only AMPK phosphorylated ACC2. These data demonstrate that AMPK is not essential for the regulation of fatty acid oxidation by AICAR or muscle contraction.

The Orphan Nuclear Receptor, NOR-1, a Target of β-Adrenergic Signaling, Regulates Gene Expression that Controls Oxidative Metabolism in Skeletal Muscle

beta 1-3-Adrenoreceptor (AR)-deficient mice are unable to regulate energy expenditure and develop diet-induced obesity on a high-fat diet. We determined previously that beta2-AR agonist treatment activated expression of the mRNA encoding the orphan nuclear receptor, NOR-1, in muscle cells and plantaris muscle. Here we show that beta2-AR agonist treatment significantly and transiently activated the expression of NOR-1 (and the other members of the NR4A subgroup) in slow-twitch oxidative soleus muscle and fast-twitch glycolytic tibialis anterior muscle. The activation induced by beta-adrenergic signaling is consistent with the involvement of protein kinase A, MAPK, and phosphorylation of cAMP response element-binding protein. Stable cell lines transfected with a silent interfering RNA targeting NOR-1 displayed decreased palmitate oxidation and lactate accumulation. In concordance with these observations, ATP production in the NOR-1 silent interfering RNA (but not control)-transfected cells was resistant to (azide-mediated) inhibition of oxidative metabolism and expressed significantly higher levels of hypoxia inducible factor-1alpha. In addition, we observed the repression of genes that promote fatty acid oxidation (peroxisomal proliferator-activated receptor-gamma coactivator-1alpha/beta and lipin-1alpha) and trichloroacetic acid cycle-mediated carbohydrate (pyruvate) oxidation [pyruvate dehydrogenase phosphatase 1 regulatory and catalytic subunits (pyruvate dehydrogenase phosphatases-1r and -c)]. Furthermore, we observed that beta2-AR agonist administration in mouse skeletal muscle induced the expression of genes that activate fatty acid oxidation and modulate pyruvate use, including PGC-1alpha, lipin-1alpha, FOXO1, and PDK4. Finally, we demonstrate that NOR-1 is recruited to the lipin-1alpha and PDK-4 promoters, and this is consistent with NOR-1-mediated regulation of these genes. In conclusion, NOR-1 is necessary for oxidative metabolism in skeletal muscle.

β2‐Agonist administration reverses muscle wasting and improves muscle function in aged rats

The β2‐adrenoceptor agonist (β2‐agonist) fenoterol has potent anabolic effects on rat skeletal muscle. We conducted an extensive dose–response study to determine the most efficacious dose of fenoterol for increasing skeletal muscle mass in adult rats and used this dose in testing the hypothesis that fenoterol may have therapeutic potential for ameliorating age‐related muscle wasting and weakness. We used adult (16‐month‐old) rats that had completed their growth and development, and old (28‐month‐old) rats that exhibited characteristic muscle wasting and weakness, and treated them daily with either fenoterol (1.4 mg kg−1, i.p), or saline vehicle, for 4 weeks. Following treatment, functional characteristics of fast‐twitch extensor digitorum longus (EDL) and predominantly slow‐twitch soleus muscles of the hindlimb were assessed in vitro. Untreated old rats exhibited a loss of skeletal muscle mass and a decrease in force‐producing capacity, in both fast and slow muscles, compared with adult rats (P < 0.05). However, there was no age‐associated decrease in skeletal muscle β‐adrenoceptor density, nor was the muscle response to chronic β‐agonist stimulation reduced with age. Thus, muscle mass and force‐producing capacity of EDL and soleus muscles from old rats treated with fenoterol was equivalent to, or greater than, untreated adult rats. The increase in mass and strength was attributed to a non‐selective increase in the cross‐sectional area of all muscle fibre types, in both the EDL and soleus. Fenoterol treatment caused a small increase in fatiguability due to a decrease in oxidative metabolism in both EDL and soleus muscles, with some cardiac hypertrophy. Further studies are needed to fully separate the desirable effects on skeletal muscle and the undesirable effects on the heart. Nevertheless, our results demonstrate that fenoterol is a powerful anabolic agent that can restore muscle mass and strength in old rats, and provide preliminary evidence of therapeutic potential for age‐related muscle wasting and weakness.

Systemic administration of β2‐adrenoceptor agonists, formoterol and salmeterol, elicit skeletal muscle hypertrophy in rats at micromolar doses

β2‐Adrenoceptor agonists provide a potential therapy for muscle wasting and weakness, but their use may be limited by adverse effects on the heart, mediated in part, by β1‐adrenoceptor activation. Two β2‐agonists, formoterol and salmeterol, are approved for treating asthma and have an extended duration of action and increased safety, associated with greater β2‐adrenoceptor selectivity. The pharmacological profiles of formoterol and salmeterol and their effects on skeletal and cardiac muscle mass were investigated in 12‐week‐old, male F344 rats. Formoterol and salmeterol were each administered via daily i.p. injection at one of seven doses (ranging from 1 to 2000 μg kg−1 day−1), for 4 weeks. Rats were anaesthetised and the EDL and soleus muscles and the heart were excised and weighed. Dose–response curves were constructed based on skeletal and cardiac muscle hypertrophy. Formoterol was more potent than salmeterol, with a significantly lower ED50 in EDL muscles (1 and 130 μg kg−1 day−1, P <0.05), whereas salmeterol had greater intrinsic activity than formoterol in both EDL and soleus muscles (12% greater hypertrophy than formoterol). The drugs had similar potency and intrinsic activity in the heart, with a smaller leftward shift for formoterol than seen in skeletal muscle. A dose of 25 μg kg−1 day−1 of formoterol elicited greater EDL and soleus hypertrophy than salmeterol, but resulted in similar β‐adrenoceptor downregulation. These results show that doses as low as 1 μg kg−1 day−1 of formoterol can elicit significant muscle hypertrophy with minimal cardiac hypertrophy and provide important information regarding the potential therapeutic use of formoterol and salmeterol for muscle wasting.

Examination of ‘lipotoxicity’ in skeletal muscle of high‐fat fed and ob/ob mice

Excess lipid accumulation resulting from an elevated supply of plasma fatty acids is linked to the pathogenesis of the metabolic syndrome and heart disease. The term ‘lipotoxicity’ was coined to describe how lipid accumulation leads to cellular dysfunction and death in non‐adipose tissues including the heart, pancreas and liver. While lipotoxicity has been shown in cultured skeletal muscle cells, the degree of lipotoxicity in vivo and the functional consequences are unresolved. We studied three models of fatty acid overload in male mice: 5 h Intralipid® and heparin infusion, prolonged high fat feeding (HFF) and genetic obesity induced by leptin deficiency (ob/ob mice). Markers of apoptosis, proteolysis and autophagy were assessed as readouts of lipotoxicity. The Intralipid® infusion increased caspase 3 activity in skeletal muscle, demonstrating that enhancing fatty acid flux activates pro‐apoptotic pathways. HFF and genetic obesity increased tissue lipid content but did not influence apoptosis. Gene array analysis revealed that HFF reduced the expression of 31 pro‐apoptotic genes. Markers of autophagy (LC3β and beclin‐1 expression) were unaffected by HFF and were associated with enhanced Bcl2 protein expression. Proteolytic activity was similarly unaffected by HFF or in ob/ob mice. Thus, contrary to our previous findings in muscle culture in vitro and in other non‐adipose tissues in vivo, lipid overload did not induce apoptosis, autophagy or proteolysis in skeletal muscle. A broad transcriptional suppression of pro‐apoptotic proteins may explain this resistance to lipid‐induced cell death in skeletal muscle.

The potential and the pitfalls of β-adrenoceptor agonists for the management of skeletal muscle wasting

The beta-adrenergic signaling pathway represents a novel therapeutic target for skeletal muscle wasting and weakness due to its role in the mechanisms controlling protein synthesis and degradation and in modulating fiber type. Stimulation of the pathway with beta-adrenoceptor agonists (beta-agonists) has therapeutic potential for muscle wasting disorders including: sarcopenia, cancer cachexia, disuse and inactivity, unloading or microgravity, sepsis and other metabolic disorders, denervation, burns, HIV-AIDS, chronic kidney or heart failure, and neuromuscular diseases. However, there are also pitfalls associated with beta-agonist administration and clinical applications have so far been limited, largely because of cardiovascular side effects. In rats and mice, newer generation beta-agonists (such as formoterol) can elicit an anabolic response in skeletal muscle even at very low doses, with reduced effects on the heart and cardiovascular system compared with older generation beta-agonists (such as fenoterol and clenbuterol). However, the potentially deleterious cardiovascular side effects of beta-agonists have not been obviated completely and so it is important to refine their development and therapeutic approach in order to overcome these obstacles. This review describes the therapeutic potential of stimulating the beta-adrenergic signaling pathway with beta-agonists, highlighting the beneficial effects on skeletal muscle structure and function and identifying some of the pitfalls associated with short- and long-term beta-agonist administration. The review also identifies some important, but as yet unanswered questions, regarding the importance of beta-adrenoceptor signaling in muscle health and disease and the strategies needed to improve the efficacy and safety of beta-agonists for muscle wasting disorders.

Expression profiling of skeletal muscle following acute and chronic β2-adrenergic stimulation: implications for hypertrophy, metabolism and circadian rhythm

BackgroundSystemic administration of β-adrenoceptor (β-AR) agonists has been found to induce skeletal muscle hypertrophy and significant metabolic changes. In the context of energy homeostasis, the importance of β-AR signaling has been highlighted by the inability of β1-3-AR-deficient mice to regulate energy expenditure and susceptibility to diet induced obesity. However, the molecular pathways and gene expression changes that initiate and maintain these phenotypic modulations are poorly understood. Therefore, the aim of this study was to identify differential changes in gene expression in murine skeletal muscle associated with systemic (acute and chronic) administration of the β2-AR agonist formoterol.ResultsSkeletal muscle gene expression (from murine tibialis anterior) was profiled at both 1 and 4 hours following systemic administration of the β2-AR agonist formoterol, using Illumina 46K mouse BeadArrays. Illumina expression profiling revealed significant expression changes in genes associated with skeletal muscle hypertrophy, myoblast differentiation, metabolism, circadian rhythm, transcription, histones, and oxidative stress. Differentially expressed genes relevant to the regulation of muscle mass and metabolism (in the context of the hypertrophic phenotype) were further validated by quantitative RT-PCR to examine gene expression in response to both acute (1-24 h) and chronic administration (1-28 days) of formoterol at multiple timepoints. In terms of skeletal muscle hypertrophy, attenuation of myostatin signaling (including differential expression of myostatin, activin receptor IIB, phospho-Smad3 etc) was observed following acute and chronic administration of formoterol. Acute (but not chronic) administration of formoterol also significantly induced the expression of genes involved in oxidative metabolism, including hexokinase 2, sorbin and SH3 domain containing 1, and uncoupling protein 3. Interestingly, formoterol administration also appeared to influence some genes associated with the peripheral regulation of circadian rhythm (including nuclear factor interleukin 3 regulated, D site albumin promoter binding protein, and cryptochrome 2).ConclusionThis is the first study to utilize gene expression profiling to examine global gene expression in response to acute β2-AR agonist treatment of skeletal muscle. In summary, systemic administration of a β2-AR agonist had a profound effect on global gene expression in skeletal muscle. In terms of hypertrophy, β2-AR agonist treatment altered the expression of several genes associated with myostatin signaling, a previously unreported effect of β-AR signaling in skeletal muscle. This study also demonstrates a β2-AR agonist regulation of circadian rhythm genes, indicating crosstalk between β-AR signaling and circadian cycling in skeletal muscle. Gene expression alterations discovered in this study provides insight into many of the underlying changes in gene expression that mediate β-AR induced skeletal muscle hypertrophy and altered metabolism.