activin pathway antagonism: Molecular basis and therapeutic potential

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ARTICLE IN PRESS The International Journal of Biochemistry & Cell Biology xxx (2013) xxx–xxx

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Review

Myostatin/activin pathway antagonism: Molecular basis and therapeutic potential夽 H.Q. Han a,∗ , Xiaolan Zhou a , William E. Mitch b , Alfred L. Goldberg c a b c

Metabolic Disorders Department, Amgen, Thousand Oaks, CA, USA Nephrology Division, Baylor College of Medicine, Houston, TX, USA Department of Cell Biology, Harvard Medical School, Boston, MA, USA

a r t i c l e

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Article history: Available online xxx Keywords: Myostatin/activin-ActRIIB signaling pathway Ubiquitin–proteasome system Ubiquitin ligases Protein breakdown Muscle wasting diseases

a b s t r a c t Muscle wasting is associated with a wide range of catabolic diseases. This debilitating loss of muscle mass and functional capacity reduces the quality of life and increases the risks of morbidity and mortality. Major progress has been made in understanding the biochemical mechanisms and signaling pathways regulating muscle protein balance under normal conditions and the enhanced protein loss in atrophying muscles. It is now clear that activation of myostatin/activin signaling is critical in triggering the accelerated muscle catabolism that causes muscle loss in multiple disease states. Binding of myostatin and activin to the ActRIIB receptor complex on muscle cell membrane leads to activation of Smad2/3-mediated transcription, which in turn stimulates FoxO-dependent transcription and enhanced muscle protein breakdown via ubiquitin–proteasome system and autophagy. In addition, Smad activation inhibits muscle protein synthesis by suppressing Akt signaling. Pharmacological blockade of the myostatin/activin-ActRIIB pathway has been shown to prevent or reverse the loss of muscle mass and strength in various disease models including cancer cachexia and renal failure. Moreover, it can markedly prolong the lifespan of animals with cancer-associated muscle loss. Furthermore, inhibiting myostatin/activin actions also improves insulin sensitivity, reduces excessive adiposity, attenuates systemic inflammation, and accelerates bone fracture healing in disease models. Based on these exciting advances, the potential therapeutic benefits of myostatin/activin antagonism are now being tested in multiple clinical settings. This article is part of a Directed Issue entitled: Molecular basis of muscle wasting. © 2013 Published by Elsevier Ltd.

Contents 1. 2. 3. 4.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Disorders associated with muscle wasting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Signaling pathways regulating muscle protein balance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Myostatin/activin signaling in muscle wasting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Myostatin biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Activin A biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Myostatin/activin signal transduction in muscle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. Regulation of protein balance by myostatin/activin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5. Activation of myostatin/activin signaling pathway in disease states . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pharmacologic inhibition of myostatin/activin signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Overview of preclinical findings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Potential therapeutic benefits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.1. Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.2. Chronic kidney disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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夽 This article is part of a Directed Issue entitled: Molecular basis of muscle wasting. ∗ Corresponding author at: Metabolic Disorders Department, Amgen, One Amgen Center Drive, Thousand Oaks, CA 91320, USA. Tel.: +1 805 447 4770; fax: +1 805 480 1329. E-mail address: [email protected] (H.Q. Han). 1357-2725/$ – see front matter © 2013 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.biocel.2013.05.019

Please cite this article in press as: Han HQ, et al. Myostatin/activin pathway antagonism: Molecular basis and therapeutic potential. Int J Biochem Cell Biol (2013), http://dx.doi.org/10.1016/j.biocel.2013.05.019

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5.2.3. Congestive heart failure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.4. Neuromuscular diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.5. Sarcopenia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.6. Other metabolic diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Skeletal muscle comprises approximately 40–50% of body weight and, by mass, is the largest organ in the human body. Muscle health is vital in maintaining energy balance, exercise capacity and respiratory function. Systemic muscle wasting is associated with weakness, fatigue, frailty, insulin resistance, bone fracture, hospitalization, disability and death. Epidemiologic and clinical investigations have documented that muscle wasting increases the risks of morbidity and mortality in the elderly as well as patients with various catabolic conditions, such as cancer, chronic kidney disease, congestive heart failure, chronic obstructive pulmonary disease, muscular dystrophy, motor neuron disease, neurodegenerative disease, trauma, immobilization, infections, rheumatoid arthritis and diabetes (Anker et al., 1997; Evans et al., 2008; Fouque et al., 2008; Griffiths, 1996; Huang et al., 2010; Inagaki et al., 1974; Plank and Hill, 2000; Schols et al., 2005; Tisdale, 2002; von Haehling et al., 2012; Han and Mitch, 2011). Despite its medical importance, agents that combat muscle wasting remain a large unmet need, as presently there is no approved therapy to prevent or treat this debilitating and life-threatening disorder. In recent years, exciting insights have been gained into the molecular mechanisms of muscle atrophy. In addition, consensus definitions have been established for clinical diagnosis of muscle wasting in certain disease conditions, including cachexia (Evans et al., 2008; Fearon et al., 2011), protein-energy wasting associated with chronic renal disease (Fouque et al., 2008) and sarcopenia (Morley et al., 2011; Fielding et al., 2011). These advances provide new hope for the discovery and development of safe and effective therapies for treatment or prevention of muscle wasting. In this review, we summarize our current understanding of the signaling mechanisms that regulate muscle protein breakdown and synthesis and especially, the role of myostatin/activin signaling in the pathogenesis of muscle wasting. We also highlight the promising recent evidence that antagonism of the myostatin/activin pathway offers clear potential benefits in treating this debilitating loss of muscle mass. 2. Disorders associated with muscle wasting Loss of muscle mass is a physiological response to fasting or malnutrition, but also is a characteristic feature of numerous disease conditions that include (1) Neuromuscular diseases, such as muscular dystrophy, ALS, spinal cord injury, stroke and myasthenia gravis, (2) Systemic catabolic diseases, such as cancer, chronic kidney disease, congestive heart failure, chronic obstructive pulmonary disease, Cushing’s disease, excess glucocorticoids, and diabetes, (3) Inflammatory and autoimmune diseases, including rheumatoid arthritis and multiple sclerosis, (4) Infectious diseases, such as AIDS, tuberculosis, and sepsis, (5) Trauma, such as motorcycle accident, surgery, and burns, (6) Neurodegenerative diseases, such as Alzheimer’s disease, Parkinson’s disease and Huntington’s disease, (7) Disuse atrophy, such as occurs with prolonged bed-rest, immobilization, and spaceflight, and (8) Age-related sarcopenia, the systematic loss of muscle in aged populations. These various conditions have been best studied in rodent models as distinct conditions, but in patients, these multiple stimuli for atrophy

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often function together to drive muscle loss. For example, trauma patients often are immobilized, septic, have high glucocorticoid levels, and receive limited nutritional intake, all of which are stimuli for muscle wasting. Although their etiologies appear vastly different, muscle wasting disorders exhibit several common features – inactivity, inflammation, high glucocorticoids and insulin resistance – which are characteristic of a hyper-catabolic disease state and are all contributors to the pathogenesis of muscle loss. First, the lack of contractile activity, as occurs with disuse or denervation, stimulates protein degradation and leads to muscle atrophy (Evans, 2010; Slimani et al., 2012). Second, systemic inflammation is often present in wasting conditions and can contribute to the atrophy process as proinflammatory cytokines somehow activate muscle protein breakdown (Argiles et al., 1992; Spate and Schulze, 2004; Kwak et al., 2004). Third, high glucocorticoids as frequently seen in stressful diseases exacerbate muscle atrophy by reducing muscle protein synthesis and promoting proteolysis (Goldberg and Goodman, 1969; Schakman et al., 2008). Fourth, insulin resistance or low insulin levels are common characteristics of wasting diseases and can promote muscle atrophy due to diminished response to insulin (Honors and Kinzig, 2012; Wang et al., 2006) and to IGF-1, which enhances muscle protein synthesis and inhibits proteolysis. These signaling mechanisms are best understood in fasting in which the net breakdown of muscle protein (i.e. reduced protein synthesis and enhanced proteolysis) provides amino acids for energy metabolism in the muscle (Chang and Goldberg, 1978) or hepatic gluconeogenesis. This response in fasting (or untreated diabetes) is signaled by the fall in insulin and increased glucocorticoid levels; clearly, similar signaling cellular mechanisms are functioning in these acute disease states. 3. Signaling pathways regulating muscle protein balance Muscle mass is dynamically regulated by various extracellular signals, which activate distinct intracellular signaling processes that alter protein balance in the muscle fibers, as well as influencing proliferation of muscle stem cells (see below). As shown in Fig. 1, insulin, IGF-1, testosterone and ␤2adrenergic agonist have anabolic actions on skeletal muscle. Binding of insulin and IGF-1 to membrane receptors leads to activation of Akt/mTOR-mediated signal transduction and thereby promotes muscle protein synthesis and inhibits proteolysis leading eventually to fiber hypertrophy (Glass, 2005; Schiaffino and Mammucari, 2011). Catabolic signals, such as the glucocorticoids, which function as hormones (Menconi et al., 2007; Schakman et al., 2008), and a number of proinflammatory cytokines (TNF-␣, IL-6, IL-1 and IFN-␥) (Reid and Li, 2001) have been reported to trigger muscle wasting, but the precise contributions of these latter factors remain controversial and uncertain in different conditions. There is now strong evidence that members of the TGF-␤ family, myostatin, activin A and TGF-␤ are powerful catabolic stimuli that inhibit muscle growth normally and can promote muscle protein loss in disease states (Zhou et al., 2010; Zimmers et al., 2002; Zugmaier et al., 1991). Binding of these TGF-␤ ligands to muscle surface receptors lead to activation of Smad2/3-mediated signal transduction, thereby stimulating net proteolysis and fiber atrophy

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Fig. 1. Signaling pathways regulating muscle protein balance. Various extracellular stimuli achieve their catabolic and anabolic effects on muscle homeostasis by converging upon the intracellular signaling pathways in muscle cell mediated by Smad, NF-␬B, FoxO and Akt/mTOR transcription systems. Smad, NF-␬B, and FoxO mediate transcription of the set of atrogenes and enhance net muscle protein breakdown in part via activation of ubiquitin–proteasome system (UPS), whereas Akt/mTOR transcription system promotes muscle protein synthesis. Generally, catabolic and anabolic processes are regulated in a reciprocal manner (e.g. by IGF1 or mTOR) (see text).

(Sartori et al., 2009). It has now become clear that the Smad, FoxO and NF-␬B transcription factors in muscle are critical in promoting atrophy and together with the PI3K/Akt/mTOR anabolic pathway, determine overall protein balance and hence muscle mass. Activation of one or more of these interconnected signaling pathways can lead to muscle proteolysis and wasting. In muscle, these pathways influence each other’s activity (Fig. 1). For instance, activation of Akt anabolic signaling by IGF-1 or insulin inactivates FoxO-dependent transcription (Sandri et al., 2004; Schiaffino and Mammucari, 2011; Stitt et al., 2004). Also, synthesis and degradation of proteins are often regulated coordinately. Thus, Smad activation by myostatin and activin or NF-␬B activation by proinflammatory cytokines or glucocorticoids both activate FoxOs and inhibit protein translation and anabolic activity of Akt. Also, mTOR stimulates translation initiation and also suppresses proteolysis by both autophagy and the ubiquitin–proteasome system (Sartori et al., 2009; Trendelenburg et al., 2009). Thus, while growth is triggered by enhanced PI3K/Akt/mTOR signaling, decreased Akt/mTOR activity has catabolic effects. In fact, the pathogenesis of muscle wasting originates from an imbalance in the regulation of protein synthesis and degradation by these multiple signaling pathways. The ubiquitin–proteasome system (UPS) is the major proteolytic pathway responsible for the breakdown of muscle proteins, especially myofibrillar proteins (Attaix et al., 2005; Lecker et al., 2006; Cohen et al., 2009, 2012; Piccirillo and Goldberg, 2012), and its activity in muscle is constantly regulated by activities of Smad, NF␬B, and FoxO. Total activity of the UPS activity in muscle has been shown to increase in muscles upon fasting or denervation (Mitch and Goldberg, 1996; Cohen et al., 2009) and in various catabolic states (e.g. acidosis or cancer cachexia) (Acharyya et al., 2004;

Workeneh and Mitch, 2010; Kwak et al., 2004; Attaix et al., 2008). Similarly, activation of the UPS occurs sharply upon activation of Smad by myostatin and activin (Zhou et al., 2010), by proinflammatory cytokines (Acharyya et al., 2004; Kwak et al., 2004) or by glucocorticoids (Zheng et al., 2010; Sandri et al., 2004). In all these conditions, there is a common set of transcriptional changes in a group of atrophy-related genes, often termed now “atrogenes”, of which over 100 have been identified (Lecker et al., 2004; Sacheck et al., 2007). Furthermore, impaired insulin/IGF-1 signaling (Glass, 2010; Hu et al., 2009) or androgen deficiency (Pires-Oliveira et al., 2010; Schakman et al., 2008) weakens PI3K/Akt pathway and as a result triggers FoxO-mediated activation of protein ubiquitination and breakdown via the proteasome (Zhao et al., 2007). The increase in UPS activity as seen in catabolic states depends on transcriptional activation of the muscle-specific ubiquitin ligases, Atrogin-1/MAFbx (Gomes et al., 2001), and MuRF1 (Bodine et al., 2001; Koyama et al., 2008; Cohen et al., 2009; Polge et al., 2011), as well as ubiquitin and proteasome subunits (Lecker et al., 2006; Mitch and Goldberg, 1996). Degradation of key regulatory proteins by Atrogin-1/MAFbx is primarily important in inhibiting protein synthesis during atrophy (Lagirand-Cantaloube et al., 2008) but may have other roles (Lokireddy et al., 2012a,b), while MuRF1 is of special importance in atrophying muscle because it catalyzes the most of the increased polyubiquitination of major muscle proteins, especially components of the thick filaments (Cohen et al., 2009, 2012), which leads to their rapid degradation by the 26S proteasome. Not surprisingly, therefore, increased expression of Atrogin-1/MAFbx and MuRF1 is often considered synonymous in many studies with activation of proteolysis and the atrogene program, and a close correlation has been shown between Atrogin1/MAFbx mRNA and rates of proteolysis in cultured myotubes

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(Sacheck et al., 2004). Indeed, the expression of these E3s in muscle has also been shown to be markedly upregulated in various wasting disease conditions including cancer, diabetes, renal failure, starvation and denervation (Pickering et al., 2002; Sacheck et al., 2007; Lecker et al., 2006). Under these catabolic conditions, additional non-transcriptional mechanisms, such as substrate phosphorylation, also function to activate ubiquitination and breakdown of thin filament and cytoskeletal proteins by the constitutive ubiquitin ligase, Trim32 (Cohen et al., 2012). In addition, muscle cells seem to contain back-up mechanisms to activate muscle wasting even in the absence of Trim32 (Kudryashova et al., 2012). While the UPS is of primary importance in the breakdown of the bulk of muscle proteins, the cell’s other main proteolytic pathway, the autophagic lysosomal pathway is also important in the protein loss in atrophying muscle (Bechet et al., 2005; Zhao et al., 2007; Mammucari et al., 2007). In addition to genes for the UPS, FoxO3 also increases the transcription of many genes for autophagy, which triggers lysosomal breakdown of organelles, especially mitochondria (Zhao et al., 2007; Mammucari et al., 2007). In addition, FoxO causes induction of the ubiquitin ligase Mul1, which ubiquitinates mitochondrial components and triggers the specific clearance of these organelles (Lokireddy et al., 2012a,b). Coordinated activation of the UPS and autophagy also occurs on a more rapid time scale with decreases in mTOR activity (Sengupta et al., 2010). Although best studied in fasting and denervation, activation of autophagy presumably plays a similar role in loss of muscle mass in other catabolic conditions, where loss of mitochondria by this pathway is likely to contribute to reduced exercise capacity, while loss of myofibrillar mass by the UPS leads to decreased strength.

4. Myostatin/activin signaling in muscle wasting 4.1. Myostatin biology Myostatin, also referred to as growth differentiation factor-8 (GDF-8), belongs to the transforming growth factor-beta (TGF␤) family of secreted factors. It is predominantly expressed in skeletal muscle, although cardiac muscle and adipose tissue also express it at low levels. Myostatin is secreted from muscle cells and acts in a paracrine/autocrine fashion by binding to its muscle surface receptor complex, triggering a downstream signaling cascade that leads to translocation of Smad2/3 into the nucleus and changes in gene expression (see below). Myostatin is a disulfidelinked dimeric protein, which is produced as larger precursor (pre-promyostatin). Proteolytic cleavage of this precursor generates an amino-terminal propeptide and the mature, biologically active carboxy-terminal ligand. The activity of myostatin is controlled not only by transcription, but also post-translationally. After cleavage from the promyostatin, most of the mature myostatin is stored in a latent complex in the extracellular space (Lee and McPherron, 2001; Thies et al., 2001; Anderson et al., 2008), where it is bound to the propetide that prevents it from binding to the receptor (Lee and McPherron, 2001; Zimmers et al., 2002). Activation of the latent complex releases the mature myostatin and allows it to interact with its receptor. Several factors including low pH, free radicals and proteases have been shown to activate the latent complex (Annes et al., 2003; Wolfman et al., 2003). However, the precise mechanisms that regulate myostatin release from its latent complex under physiological and pathological conditions remain an important subject for further investigation. Besides the propeptide, several circulating proteins, including follistatin and the follistatin-like proteins, are known to bind and inhibit myostatin (Hemmati-Brivanlou et al., 1994; Sidis et al., 2006). However, unlike the propeptide that selectively binds myostatin, the follistatins also bind and inhibit multiple other TGF-␤ family ligands

including activins, GDF11 and TGF-␤ (see below), and their precise physiological roles are also presently uncertain. Myostatin has now been widely recognized as an important negative regulator of skeletal muscle growth. Our understanding of myostatin function began in 1997 with the identification of the myostatin gene by Lee and colleagues (McPherron et al., 1997), who first cloned it and constructed the myostatin-null mice that exhibit an astonishing “double-muscle phenotype”. Upon knockout of the myostatin gene, the mice exhibited a dramatic increase in the size and number of skeletal muscle fibers and physical strength. Numerous genetic investigations followed this seminal work and corroborated these original findings. These investigators subsequently showed that transgenic mice overexpressing a dominant negative myostatin receptor or a propeptide or other proteins that sequester myostatin also yield phenotypes with a dramatic increase in muscle mass (Lee and McPherron, 2001). Besides these mouse models, cattle, sheep, dogs and a human with a loss-of-function myostatin mutation exhibit large increases in muscle mass and strength (McPherron and Lee, 1997; Schuelke et al., 2004; Clop et al., 2006; Mosher et al., 2007). Furthermore, myostatin mutations have been shown to influence athletic performance. For example, whippet dogs bearing a single copy of the myostatin mutation are among the fastest, although whippets with two copies of the same mutation are overmuscled and fail to outrun sibling controls (Mosher et al., 2007). Additionally, myostatin polymorphisms in elite thoroughbred horses also reveal a strong association with racing outcomes (Binns et al., 2010; Bower et al., 2012). These compelling findings demonstrate conclusively that myostatin is a key negative regulator of muscle growth and its deficiency results in muscle hypertrophy and even improved physical performance. One mechanism by which myostatin negatively regulates muscle growth is through inhibiting myogenesis during development. In vitro, myostatin suppresses myoblast growth and also inhibits myogenic differentiation in part by down-regulation of MyoD (Langley et al., 2002) and upregulation of p21 (McFarlane et al., 2011). Moreover, in adult muscles, myostatin negatively regulates satellite cell activation and self-renewal (McCroskery et al., 2003). Satellite cells (Mauro, 1961) are muscle stem cells, which are normally quiescent but can proliferate rapidly in response to exercise and injury and are necessary for fiber repair and regeneration after injury (Dhawan and Rando, 2005; Kuang et al., 2008). In response to anabolic stimuli, these satellite cells proliferate and fuse with hypertrophing muscle fibers, apparently to keep the protein to nuclei ratio low. Remarkably, myostatin gene ablation or myostatin blockade in both normal and atrophying muscles have been shown to cause satellite cell activation and proliferation (McCroskery et al., 2003; Zhou et al., 2010). Thus, myostatin plays a critical role in maintaining satellite cell quiescence. This inhibitory effect of myostatin on satellite cells is mediated in part by PAX7, a transcription factor required for the specification of myogenic satellite cells (McFarlane et al., 2008; Seale et al., 2000). Interestingly, PAX7-null mutant mice suffer severe wasting with muscle loss by nearly 50% and die prematurely; but, blocking myostatin/activin signaling in the PAX7-null mice still induced a marked gain of muscle mass by 37–52% (although this effect was less than that seen in wildtype mice) (Lee et al., 2012). Thus, even in the absence of PAX7 and hence, without usual changes in satellite cell function, blocking myostatin/activin signaling is still effective in eliciting skeletal muscle hypertrophy, apparently by directly stimulating protein synthesis and inhibiting protein breakdown in the muscle fiber (see below). 4.2. Activin A biology Activins belong to the activins-inhibins subfamily of TGF-␤ family of secreted proteins. Activins were originally purified from

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gonadal fluids as glycoproteins that stimulated follicle stimulating hormone (FSH) release from the pituitary gland (Bilezikjian et al., 1993, 2004; Bilezikjian and Vale, 2011; Blumenfeld and Ritter, 2001). Like myostatin, activins are disulfide-linked dimeric proteins produced as larger precursors. An amino-terminal propeptide is cleaved to release the mature carboxy-terminal bioactive ligand. Activins are homodimers or heterodimers of the various ␤ subunit isoforms referred to as activin A (␤A–␤A), activin B (␤B–␤B) or activin AB (␤A–␤B) (Vale et al., 2004). Activin A is a major form of the activins. Under physiological conditions, it is primarily expressed in gonadal tissues and functions as an endocrine factor to stimulate FSH biosynthesis and secretion in the pituitary gland (Woodruff et al., 1993). Hence, activin A is well known for its role in the regulation of gonadal functions (Luisi et al., 2001; Muttukrishna et al., 2000; Woodruff and Mayo, 1990). Beyond its classic role in reproductive biology, activin A can also function as a paracrine/autocrine factor in non-gonadal tissues, including skeletal and heart muscles. Activin A levels are upregulated in many catabolic disease states, suggesting its involvement in pathogenesis of wasting (see below). Activin A mediates a wide range of biological activities including wound healing (Bamberger et al., 2005; Hubner et al., 1999), cell proliferation and differentiation (Clotman and Lemaigre, 2006; Ota et al., 2003), immune response (Yu and Dolter, 1997) and angiogenesis (Poulaki et al., 2004). Of special interest is the fact that in muscle, activin A and myostatin bind to the same surface receptor complex and activate the same signaling cascade that leads to Smad2/3 translocation into the nucleus. The activity of activin A is regulated by its latent complex, where its propeptide remains associated and prevents its binding to its receptor, and also by follistatin and follistatin-like proteins, which inhibit its activity, as they do to myostatin (Tsuchida et al., 2009). These follistatins, as mentioned above, are also capable of binding and neutralizing several TGF-␤ family members, including myostatin, GDF11 and TGF-␤. One additional mechanism that negatively control the activity of the activins are the inhibins, which antagonize activin A signaling by interfering with the recruitment of Type-I activin receptor by the Type-II receptors, thus silencing type I receptor activation and Smad2/3 signaling (Gray et al., 2001; Lewis et al., 2000). Inhibin-deficiency is associated with activin hyperactivity due to activin deregulation (Matzuk et al., 1994).

4.3. Myostatin/activin signal transduction in muscle Myostatin and activin A exert their biological activities through binding to the same heterodimeric complex of the membrane spanning serine-threonine receptor kinases designated type-I and type-II activin receptors (Bilezikjian et al., 2006). This complex is composed of two type-II receptors, referred to as ActRIIA and ActRIIB, and two type-I receptors, known as activin receptor-like kinase 4 (ALK 4) and activin receptor-like kinase 5 (ALK5). ActRIIB is enriched in muscle (compared to ActRIIA) and exhibits higher affinity for myostatin (Lee and McPherron, 2001). The downstream signaling cascades of myostatin and activin A are similar to those of TGF-␤ (Massague, 2000, 2012). Binding of myostatin or activin A to these high-affinity type-II receptor ActRIIB or ActRIIA causes its dimerization on the membrane, which leads to recruitment and activation of the type-I receptor transmembrane kinase ALK4 or ALK5 into the receptor complex. This in turn causes phosphorylation of Smad2 and Smad3 in the cytosol and recruitment of Smad4 into a Smad2/3/4 complex. The Smad complex then enters the nucleus and elicits gene transcription, ultimately leading to muscle wasting (Fig. 2).

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4.4. Regulation of protein balance by myostatin/activin In addition to causing Smad2/3 activation, which by itself can trigger muscle loss (Sartori et al., 2009), myostatin and activin A also inhibit Akt and consequently activate FoxO signaling and thus, upregulate critical components of UPS and autophagy gene to induce muscle protein catabolism (Zhou et al., 2010; Lee et al., 2011; Lokireddy et al., 2011). Binding of myostatin or activin A to its muscle surface receptor complex suppresses Akt activity leading to reduction of phospho-FoxOs (FoxO1, FoxO3, and FoxO4) and accumulation of dephospho-FoxOs. The precise role of each in muscle wasting remains uncertain. Dephospho-FoxOs are the active forms that regulate gene transcription. When Akt activity is decreased, the FoxOs are dephosphorylated and this form can enter the nucleus to activate transcription of atrophy-specific genes, such as ubiquitin ligases Atrogin-1/MAFbx and MuRF1, leading to ubiquitination and subsequent degradation by the proteasome of muscle proteins, including contractile proteins (Fig. 2). Recent evidence demonstrates that in animal models of wasting diseases, increased myostatin/activin-Smad signaling inhibits Akt activity in muscle, increases FoxO activity and upregulates critical atrogenes (e.g. Atrogin-1/MAFbx and MuRF1). These changes in turn result in accelerated proteolysis and depressed protein synthesis in muscle and cause wasting. Thus, blocking myostatin/activin signaling eliminates most, if not all, of these changes and can reverse muscle wasting (Zhou et al., 2010; Zhang et al., 2011b). Furthermore, besides regulating Smad, FoxO and Akt, myostatin signaling can also cross-talk with the exercise-induced transcription coactivator peroxisome proliferator-activated receptor-␥ coactivator-1␣ (PGC1-␣) in muscle. Inhibition of myostatin has been shown to up-regulate PGC1-␣ activity in muscle, which enhances mitochondria biogenesis (LeBrasseur et al., 2009), binds to FoxOs, and inhibits their transcriptional activity (Mammucari et al., 2008; Brault et al., 2010) and increases AMPK levels in muscle, which should raise insulin sensitivity and responsiveness (Zhang et al., 2011a). Recently, a new isoform of PGC1-␣, referred to as PCG1-␣4 (Ruas et al., 2012), has been described by Spiegelman and coworkers that is induced in muscles, especially by isometric exercise and seems to inhibit atrophy and induce hypertrophy, rather than promote mitochondrial biogenesis and oxidative metabolism, as does PGC1-␣ or PGC1-␤ (Brault et al., 2010; Choi et al., 2008). Interestingly, overexpression of PCG1-␣4 in muscle down-regulates myostatin expression and at the same time up-regulates IGF-1 production, resulting in profound muscle hypertrophy as well as a marked increase in resistance to cancerinduced muscle wasting (Ruas et al., 2012). 4.5. Activation of myostatin/activin signaling pathway in disease states There is growing evidence that the activities of myostatin/activin pathway are upregulated in catabolic diseases and contribute to the development of muscle wasting. Myostatin levels in serum or expression in muscles have been reported to increase in patients with cancer (Aversa et al., 2012), AIDS (Gonzalez-Cadavid et al., 1998), renal failure (Sun et al., 2006), COPD (Ju and Chen, 2012; Plant et al., 2010) and heart failure (Breitbart et al., 2011; George et al., 2010; Gruson et al., 2011). Myostatin levels are also increased in aged subjects (Yarasheski et al., 2002) and in response to prolonged bed rest (Reardon et al., 2001; Zachwieja et al., 1999). Similar changes occur in rodent models of cancer cachexia (Costelli et al., 2008; Zhou et al., 2010), chronic kidney disease (Zhang et al., 2011b), glucocorticoid administration (Lang et al., 2001; Ma et al., 2003), burn injury (Lang et al., 2001), mechanical unloading and space flight (Allen et al., 2009; Carlson et al., 1999; Lalani et al., 2000; Wehling et al., 2000). In addition,

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Fig. 2. Myostatin/activin signaling in muscle. Myostatin or activin binds to type IIB activin receptor (ActRIIB) on the muscle membrane to cause its dimerization, which leads to recruitment and activation of type I activin receptor transmembrane kinase ALK4 or ALK5. This in turn causes phosphorylation of Smad2 and Smad3 and the recruitment of Smad4 into a Smad complex. The Smad complex translocates into the nucleus to elicit transcription changes of genes, which result in muscle wasting. Myostatin/activin binding to the receptor also reduces Akt activity and consequently diminishes FoxO phosphorylation. Dephosphorylated FoxOs enter the nucleus to activate transcription of atrophy-specific E3 ligases MuRF1, Atrogin-1/MAFbx, and other atrogenes, which cause muscle protein ubiquitination and degradation by the proteasome or autophagy. SBE, Smad-binding element.

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activin A has been shown to be markedly upregulated in various catabolic diseases in humans. Elevated activin A has been found in serum and tumor tissues of patients with various malignancies, including ovarian cancer (Cobellis et al., 2004; Harada et al., 1996; Lambert-Messerlian et al., 1999; Steller et al., 2005), endometrial adenocarcinoma (Otani et al., 2001; Tanaka et al., 2004), prostate cancer (Risbridger et al., 2001; Thomas et al., 1997; Zhang et al., 1997), multiple myeloma (Terpos et al., 2012; Vallet et al., 2010), colorectal cancer (Wildi et al., 2001), esophageal cancer (Yoshinaga et al., 2003), breast cancer (Reis et al., 2002), and pancreatic cancer (Kleeff et al., 1998). Furthermore, activin A overproduction has been associated with cancer progression (Chang et al., 2010; Hofland et al., 2012; Yoshinaga et al., 2008). In non-cancer settings, elevated activin A has been reported in patients with various catabolic disorders including chronic renal failure (Harada et al., 1996), heart failure (Yndestad et al., 2004), pulmonary hypertension (Yndestad et al., 2009), liver cirrhosis (Harada et al., 1996), hyperthyroidism (Harada et al., 1996), rheumatoid arthritis (Ota et al., 2003), inflammatory bowel disease (Hubner et al., 1997), and pre-eclampsia (D’Antona et al., 2000; Muttukrishna et al., 1997, 2006). Circulating activin A levels also appear to rise with human aging including in both elderly men and post-menopausal women, especially in the last decades of life (Baccarelli et al., 2001; Harada et al., 1996; Santoro et al., 1999). Experimentally, rising levels of activin A have been reported in rodents with cancer (Zhou et al., 2010), heart failure (Yndestad et al., 2004), hypertension (Larsen et al., 2011), nephritis (Peters et al., 2004) and inflammation (Jones et al., 2007; Mayer et al., 2012). A variety of observations have documented the profound abilities of myostatin and activin A to decrease rapidly muscle mass. For example, mice administered either myostatin (Zimmers et al., 2002) or activin A (Zhou et al., 2010) experienced approximately a 30% decrease in muscle mass and inhibin-deficient mice, which have marked increases in activin activity, suffer profound weight loss and muscle wasting. Based on their key roles in muscle atrophy, the question for therapeutic development is: can myostatin and activin hyperactivities be overcome by an acceptable treatment strategy? As outlined below, compelling evidence from a variety of experimental animal models demonstrates that antagonizing myostatin/activin pathway can effectively prevent and even reverse muscle wasting. 5. Pharmacologic inhibition of myostatin/activin signaling Myostatin/activin signaling blockade can be achieved by several types of proteins that interfere with ligand–receptor interactions. Examples of these large molecule inhibitors include antagonistic peptibody, antibody, decoy receptor and native binding protein, which prevent ligand binding to the transmembrane receptor complex on the muscle cell surface. A number of such agents have been shown to be efficacious in preclinical disease models (see Table 1). Another approach is to identify small molecule inhibitors against the serine/threonine kinase activity of the transmembrane receptors (e.g., ALK4/5) or inhibitors that can interfere with the intracellular signaling processes involving protein-protein or protein-DNA interactions. Notably, some small molecule inhibitors of TGF-␤ signaling have been identified for treatment of other diseases (e.g. cancer) and a number of them are now in clinical trials (Akhurst and Hata, 2012). However, a small molecule inhibitor able to selectively antagonize myostatin/activin signaling and stimulate muscle growth in vivo is yet to be discovered. 5.1. Overview of preclinical findings Extensive preclinical proof-of-concept studies have been conducted with various large molecule inhibitors that interfere with

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myostatin/activin signaling. Table 1 shows a list of published experiments concerning the effects of pharmacologic myostatin/activin blockade in a variety of animal models of wasting disease ranging from cancer cachexia to renal failure, heart failure, neuromuscular diseases, disuse atrophy, and sarcopenia, as well as a number of other disease models including bone fracture, obesity and diabetes. Below, we highlight the key findings of these preclinical investigations in the context of human disease. 5.2. Potential therapeutic benefits 5.2.1. Cancer Cancer cachexia is one of the most frequent and debilitating causes of muscle wasting. Up to 80% of patients with advanced cancers develop muscle wasting, and 25% of cancer-related deaths have even been proposed to result from the cachexia. However, very few therapeutic options are currently available for muscle wasting in cancer. The mechanism by which cancers provoke the loss of the host’s muscle mass is generally thought to be multifactorial (Tisdale, 2009; Fearon et al., 2013). As outlined above, various hormones, cytokines and tumor-derived factors influence muscle protein balance in normal and disease states through several major intracellular signal transduction systems. While activation of the PI3K/Akt/mTOR pathway is associated with muscle growth, in atrophying muscles, this pathway is less active and there is an activation of FoxO as well as NF-␬B and Smad transcription factors, which promote protein degradation (Glass, 2005; Sandri et al., 2004; Zhou et al., 2010). FoxO3 by itself induces a set of atrophy-related genes, especially the muscle-specific ubiquitin ligases Atrogin-1/MAFbx and MuRF1 (Cohen et al., 2009), which promotes breakdown of myofibrillar apparatus, and also genes for autophagy (Mammucari et al., 2007; Zhao et al., 2007). However, only recently has it become clear which of the extracellular signaling factors plays a dominant role in the pathogenesis of cancer-associated muscle loss. To that end, results of a number of recent preclinical studies demonstrate that the myostatin/activin-Smad signaling pathway plays a key role in cancer-induced muscle wasting. Administration of a soluble ActRIIB decoy receptor or a myostatin antibody was found to protect mice from muscle wasting induced by colon26 adenocarcinoma or Lewis lung carcinoma, resulting in marked improvements in muscle mass and function in the cancer-bearing animals (Zhou et al., 2010; Benny Klimek et al., 2010; Murphy et al., 2011a; Busquets et al., 2012). These findings have been extended by evidence for a clear prolongation of survival of the tumor-bearing mice despite the presence of cancer and no change in tumor growth rate. Zhou et al. (2010) administered the ActRIIB decoy receptor to mice with different cancers including colon-26 carcinoma, TOV-21G ovarian carcinoma and G361 melanoma as well as inhibin-deficient mice that spontaneously develop gonadal tumors. In each case, blocking myostatin/activin signaling was found to prevent completely or reverse muscle wasting (if administered after the cachexia was clearly evident). Strikingly, this blocking of cachexia led to a marked prolongation of survival of the tumor-bearing mice implanted with colon-26 cancer. This reversal of muscle wasting and dramatic extension of life span occurred even though the tumor continued to grow at the same rate as in untreated mice. Another important and unexpected benefit of these remarkable responses to myostatin/activin blockade includes the virtual elimination of cancer-induced cardiac atrophy (Zhou et al., 2010). Surprisingly, “atrophy of the heart” has remained an unappreciated phenomenon that has been largely ignored in both cardiac pathophysiology and cachexia research even though investigators over half a century ago (Hellerstein and SantiagoStevenson, 1950) had documented marked reductions in cardiac size in patients with cancer, malnutrition, as well as infections. Biochemically, administration of the ActRIIB decoy receptor resulted

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Table 1 Summary of preclinical proof-of-concept studies on myostatin/activin inhibition. Disease

Model

Inhibitor

Efficacy findings

Reference

Cancer (cancer cachexia)

C26 tumor-bearing mice

Soluble ActRIIB decoy receptor Soluble ActRIIB decoy receptor Anti-myostatin antibody

Reversed loss of muscle mass and strength; prolonged survival Prevented loss of muscle mass and strength Prevented loss of muscle mass and function Improved muscle mass and physical activity Reversed loss of muscle mass and strength; prolonged survival Mitigated muscle wasting; reduced systemic inflammation Increase muscle mass and strength Attenuated dystrophy and improved diaphragm pathology Increased muscle mass and strength; reversed muscle fibrosis

Zhou et al. (2010)

C26 tumor-bearing mice Lewis lung tumor-bearing mice Lewis lung tumor-bearing mice Inhibin-␣ KO mice Renal failure (PEW) Heart failure (cardiac cachexia) Duchenne Muscular Dystrophy (DMD)

CKD mice (5/6 nephrectomy) Heart failure mice (TAC) MDX mice

Soluble ActRIIB decoy receptor Soluble ActRIIB decoy receptor Anti-myostatin peptibody Anti-myostatin antibody Anti-myostatin antibody

MDX mice

Soluble ActRIIB decoy receptor

MDX mice Sgcd−/− and CAV3P104L mice Scgd−/− mice

Myostatin propeptide Soluble ActRIIB decoy receptor Anti-myostatin antibody

Spinal Muscular Atrophy (SMA)

SMN7 mice

Follistatin

X-Linked Myotubular Myopathy (XLMTM) Amyotrophic Lateral Sclerosis (ALS)

Mtm1␦4 mice

Soluble ActRIIB decoy receptor Anti-myostatin antibody

Limb-Girdle Muscular Dystrophy (LGMD)

G93A

SOD1

mice

SOD1G93A mice

Soluble ActRIIB decoy receptor

Limb-cast mice

Anti-myostatin antibody

Immobilization (disuse atrophy) Age-related muscle loss (Sarcopenia) Androgen deficiency

Aged mice

Anti-myostatin antibody

Orchiectomized mice

Trauma (bone fracture)

Fibula osteotomy mice

Soluble ActRIIB decoy receptor Myostatin propeptide

Metabolic diseases (obesity and diabetes)

Obesity mice (HFD) Obesity mice (HFD)

Soluble ActRIIB decoy receptor Soluble ActRIIB decoy receptor

Energy expenditure mice (cold-challenge)

Anti-ActRIIB antibody

Obesity mice (ob/ob)

Anti-myostatin antibody

Increased muscle mass and strength Prevented muscle atrophy Increased muscle mass in young mice; slowed disease progression Attenuated muscle loss and prevented early death Increased muscle mass and strength; extended survival Increase muscle mass and strength during early-stage disease Increased muscle size and grip strength in young mice; slowed disease progression Attenuated muscle atrophy and improve functional Increased muscle mass, reduced fatigue and improved strength Restored muscle mass, adiposity, and bone quality to normal level Accelerated bone fracture healing and enhanced muscle regeneration Decreased fat mass and enhanced insulin’s glucose-lowering action Reduced obesity and enhanced fatty acid oxidation; improved brown adipogenesis and thermogenesis Improved brown adiposeness and increased thermogenesis at ambient temperature Improved energy expenditure, excise capacity and glucose homeostasis

Benny Klimek et al. (2010) Benny Klimek et al. (2010), Murphy et al. (2011a) Busquets et al. (2012) Zhou et al. (2010) Zhang et al. (2011b) Heineke et al. (2010) Bogdanovich et al. (2002), Murphy et al. (2010b) Bo Li et al. (2012), George Carlson et al. (2011), Morine et al. (2010), Pistilli et al. (2011) Bogdanovich et al. (2005) Ohsawa et al. (2006) Parsons et al. (2006) Rose et al. (2009) Lawlor et al. (2011) Holzbaur et al. (2006) Morrison et al. (2009)

Murphy et al. (2011b) LeBrasseur et al. (2009), Murphy et al. (2010a) Koncarevic et al. (2010) Hamrick et al. (2010) Akpan et al. (2009) Zhang et al. (2012)

Fournier et al. (2012)

Bernardo et al. (2010)

PEW, protein-energy wasting. TAC, transverse aortic constriction. HFD, high fat diet.

in an increase in phospho-Akt as well as phospho-FoxO3a in muscle, which could explain the suppression of Atrogin-1/MAFbx and MuRF1 and hence, the reduced ubiquitin conjugation to muscle proteins in the treated animals (see above). Additionally, blocking myostatin/activin signaling enhanced the ability of satellite cells to proliferate (see above). Together, these changes presumably contribute to the rapid reversal of muscle atrophy following inhibition of myostatin/activin signaling. Finally, treatment with the ActRIIB decoy receptor reversed muscle loss in colon-26 cancerbearing mice even though the circulating levels of TNF-␣, IL-1 and IL-6 remained elevated, all of which had been proposed as factors that mediate the muscle wasting (as discussed above). Thus in this widely studied model, myostatin/activin signaling must play the dominant role in the regulation of muscle homeostasis (Zhou et al., 2010). These findings clearly underscore the importance of

activation of the myostatin/activin pathway in the pathogenesis of skeletal and cardiac muscle wasting in this cancer, although the importance of this mechanism in various human cancers awaits rigorous study. 5.2.2. Chronic kidney disease Chronic kidney disease (CKD) is a chronic catabolic condition characterized by muscle wasting (McIntyre et al., 2006; Rajan and Mitch, 2008; Workeneh and Mitch, 2010; Bonanni et al., 2011; Mak et al., 2011, 2012). Protein-energy wasting (PEW) is a new clinical terminology for the wasting condition in patients with CKD (Fouque et al., 2008) to describe the “state of decreased body stores of protein and energy fuels” (i.e. diminished muscle and adipose mass), which can be separated into different categories according to disease severity with cachexia being the most severe form.

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PEW is a reliable predictor for risk of death in patients with CKD. A strong PEW-mortality association has been documented in patients on maintenance dialysis as well ones before dialysis was initiated (Kovesdy et al., 2009). In rodent models of chronic kidney disease, insulin resistance, inflammation and glucocorticoid excess develop. These abnormalities are important because they have been linked to the loss of muscle mass that occurs frequently in patients with CKD (Bailey et al., 2006; Zhang et al., 2009; May et al., 1987). Besides CKD, these characteristics are also present in complications of CKD, including metabolic acidosis, diabetes and an excess of angiotensin II (Mitch et al., 1994; Bailey et al., 1996; Price et al., 1996; Wang et al., 2006; Song et al., 2005; Zhang et al., 2009). Although these associations are well documented, the mechanisms accounting for them are unsettled. What is established, however, is that these abnormalities activate caspase-3 and the ubiquitin–proteasome system (UPS) and caspase-3 and the UPS act together to increase the degradation of muscle proteins. Stimulation of caspase-3 promotes protein degradation in the UPS in two ways: first, caspase-3 cleaves the complex structure of muscle proteins to provide substrates for the UPS; and second, caspase-3 cleaves Rpt2 and Rpt6 subunits of the 19S particle of the 26S proteasome leading to an increase in proteasome-mediated proteolysis (Du et al., 2004; Lee et al., 2004; Bailey et al., 1996; Price et al., 1996). Another response to acidosis, diabetes or inflammation is an increase in glucocorticoid production. This is relevant because high circulating or “stress” levels of glucocorticoids are necessary but not sufficient to activate proteolysis by the UPS (May et al., 1986; Mitch et al., 1999). The mechanism for the dependence of protein degradation on glucocorticoids was uncovered when it was shown that activation of the glucocorticoid receptor resulted in an interaction between PI3K and insulin substrate-1 (IRS-1). This interaction resulted in an impairment in insulin-stimulated intracellular signaling. This in turn reduced phosphorylation of Akt and led to a decrease in phosphorylation of forkhead transcription factors (FoxOs). With low values of phospho-FoxO1 or phospho-FoxO3, FoxO1 or FoxO3 can enter the nuclei to stimulate the expression of muscle-specific E3 ubiquitin ligases to raise the degradation of muscle proteins by the UPS. It should be pointed out that pharmacologic doses of glucocorticoids also activate protein degradation in muscle but the mechanism by which this occurs is not settled (Sandri, 2008). Since impaired insulin and IGF-1 intracellular suppress phosphorylation of Akt, muscle protein synthesis and inhibition of proteolysis will cause loss of muscle mass. Other conditions causing interference with insulin signaling will stimulate progressive loss of muscle mass. For example, inflammation arising from an excess of angiotensin II impairs insulin signaling which in turn, serves to stimulate muscle proteolysis (Song et al., 2005; Zhang et al., 2009). In addition, there is evidence that CKD causes resistance to insulin or IGF-1 intracellular signaling in muscle resulting in abnormal protein metabolism plus defects in the function of satellite cells (Bailey et al., 2006; Price et al., 1996; Wang et al., 2006; Zhang et al., 2010). These reports lead to the conclusion that catabolic signals, such as inflammatory cytokines, metabolic acidosis or excess glucocorticoids impair intracellular insulin signaling, resulting in activation of caspase-3 and the UPS to cause muscle wasting. Presently, there are no approved therapies that satisfactorily block the development of PEW in CKD patients; clinical interventions are currently limited to nutritional supplementation plus exercise. These strategies are limited for two reasons: (1) correcting PEW occurs infrequently; and (2) uremic symptoms appear if the diet or dietary supplements contain an excess of protein. To evaluate whether myostatin stimulates muscle wasting in CKD, Zhang et al. (2011b) studied a mouse model of CKD (subtotal nephrectomy plus a high protein diet to mimic the laboratory

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values of patients with CKD). The blood urea nitrogen (BUN) of the mice was >80 mg/dL and they had metabolic acidosis. Mice with CKD were paired for BUN and age and pair-fed for 4 weeks. One mouse in each pair received subcutaneous injections of an antimyostatin peptibody every other day while the uremic, control mouse was injected with the diluent. The peptibody treatment suppressed myostatin levels in muscle and reversed the loss of body weight. The latter was due in part to improvement in muscle mass as the weights of individual muscles had increased. The increase in muscle mass was due to an improvement in protein synthesis and a decrease in protein degradation and there were improved functions of satellite cells. One remarkable finding was that the levels of circulating inflammatory cytokines were decreased when myostatin was inhibited by the peptibody. This response includes a decrease in circulating IL-6, a response that is especially relevant because an increase in IL-6 has been shown to impair insulinstimulated cell signaling in muscle leading to an increase in muscle protein degradation (Zhang et al., 2009). These findings are consistent with a protein catabolic pathway: impaired insulin or IGF-1 signaling causes a decrease in phospho-Akt and a low phospho-Akt reduces the phosphorylation of FoxOs. In this case, FoxO1 or FoxO3 enters the nucleus to increase the expression of muscle-specific E3 ubiquitin ligases (Atrogin-1/MAFbx and MuRF1). The result of this response is to stimulate muscle protein degradation. Besides this pathway of muscle protein losses, changes in IL-6 are also of interest because inflammation stimulates the expression of IL-6 and an acute phase protein, serum Amyloid A. The combination of IL-6 and serum Amyloid A acts synergistically to stimulate muscle protein losses (Zhang et al., 2009). Finally, clues to the perpetuation of muscle protein losses were suggested when results of cultured muscle cells were examined. When the muscle cells were treated with TNF-␣, the production of myostatin increased but when they were treated with myostatin the production of IL-6 increased (Zhang et al., 2011b). These results suggest that an inflammatory condition with high levels of TNF-␣ not only raises myostatin resulting in loss of muscle protein but also can stimulate IL-6 to perpetuate itself. In summary, mechanisms causing muscle atrophy in CKD could be present in other catabolic conditions which are characterized by insulin or IGF-1 resistance, increased circulating levels of inflammatory cytokines and an increase in glucocorticoid production. Besides offering myostatin as a target for therapeutic efforts, other strategies could be developed to combat insulin resistance, inflammation and/or glucocorticoid excess. 5.2.3. Congestive heart failure A major fraction of patients with chronic heart failure (CHF) suffer profound weight loss and muscle wasting, leading to weakness, extreme fatigue and shortened survival. These complications are together often referred to as cardiac cachexia (Anker et al., 2004; Coats, 2002). The etiology of cardiac cachexia may involve neurohormonal abnormalities, inflammation and metabolic disturbances, but as with cancer or CKD, increased myostatin/activin signaling has been implicated in the pathogenesis of cardiac cachexia (Gruson et al., 2011; Breitbart et al., 2011; George et al., 2010). In animal models of heart failure, it has been reported that myostatin expression occurs in the peri-infarct zone and in the ventricular muscle in response to cardiac overload (Sharma et al., 1999; Shyu et al., 2006). In a mouse model of aortic pressure overload, heart failure is accompanied by skeletal muscle wasting, which is attenuated by administration of a myostatin antibody (Heineke et al., 2010). Remarkably, these investigators demonstrated that a cardiac-specific knockout of myostatin prevented skeletal muscle atrophy in this model of heart failure. The interpretation of these results in mice is that heart-derived myostatin might, in addition to influencing cardiac size and function, be promoting loss of skeletal muscle mass (Heineke et al., 2010). On the other hand, reduced

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physical activity associated with CHF by itself can induce myostatin expression in skeletal muscles to promote wasting (Lenk et al., 2012). Which interpretation is correct is unsettled but congestive heart failure like cancer or renal failure could be another wasting condition in which myostatin/activin signaling plays a critical role. Therefore, inhibiting myostatin/activin signaling may provide potential therapeutic benefits for treating cardiac cachexia, possibly through both direct effects on the heart and systemic effects on muscle mass. 5.2.4. Neuromuscular diseases Muscle wasting is associated with numerous neuromuscular diseases, including various genetic disorders primarily affecting muscle (generally termed myopathies) and motor neuron diseases (e.g. Amyotrophic Lateral Sclerosis) which lead to fiber denervation. Many of these conditions are highly debilitating and some are life threatening. Evidence from recent preclinical investigations (see Table 1) demonstrate that blocking myostatin/activin signaling improves muscle mass and functional capacity in animal models of Duchenne Muscular Dystrophy (DMD) (Bogdanovich et al., 2002; Murphy et al., 2010b; Bo Li et al., 2012; George Carlson et al., 2011; Morine et al., 2010; Pistilli et al., 2011; Bogdanovich et al., 2005), Limb-Girdle Muscular Dystrophy (LGMD) (Ohsawa et al., 2006; Parsons et al., 2006), Spinal Muscular Atrophy (SMA) (Rose et al., 2009), X-Linked Myotubular Myopathy (XLMTM) (Lawlor et al., 2011), and Amyotrophic Lateral Sclerosis (ALS) (Holzbaur et al., 2006; Morrison et al., 2009). In particular, Bo Li et al. (2012) revealed that administration of ActRIIB decoy receptor to MDX mice, a model of Duchenne Muscular Dystrophy, was able to reverse pre-existent muscle fibrosis by inducing apoptosis of myofibroblasts in the dystrophic muscle. Because fibrosis is a major problem in muscular dystrophy, this finding is encouraging as it raises the possibility that blocking myostatin/activin signaling could potentially improve muscle quality in dystrophic patients. Rose et al. blocked myostatin/activin signaling with recombinant follistatin in SMN7 mice, a model of Spinal Muscular Atrophy, a deadly childhood motor neuron disease resulting from loss of function mutation of the SMN gene (Hsieh-Li et al., 2000). Besides gain in muscle mass, the treatment led surprisingly to increases in the number and sizes of ventral horn neurons, improved motor function and delayed early deaths. Since these conditions involve very different disease etiologies, it is remarkable that these various wasting models, some of which involve primary defects in the muscle and some in the neurons, all consistently responded to myostatin/activin signaling blockade with a favorable preclinical efficacy. 5.2.5. Sarcopenia Sarcopenia refers to the progressive loss of skeletal muscle mass and function during aging (Morley et al., 2001; Morley, 2012; Rosenberg, 2011). Loss of muscle mass and strength in the elderly leads to weakness, fatigue, frailty and loss of physical function and independence. Most importantly, frailty increases the risk of fall, fracture, hospitalization and death. It has been reported that approximately 25% of men and women above the age of 65 was afflicted with sarcopenia (Iannuzzi-Sucich et al., 2002) and between 70 and 80 years of age, maximal voluntary contractile strength is decreased by 20–40% for both men and women in proximal and distal muscles (Doherty, 2003). With its high prevalence and the increase in numbers of the aged population, sarcopenia has emerged as a growing public health concern. The direct healthcare costs of sarcopenia in US in the year of 2000 were estimated to be as high as $26.2 billion dollars (Janssen et al., 2004). Multiple factors during aging such as disuse, insulin resistance, decreased growth hormone production, hypogonadism, inflammation and nutritional deficiency can lead to reduced muscle protein synthesis, increased proteolysis, and impaired muscle progenitor cell function. In

addition, with age, there is a decline in motor neuron innervation leading to sarcopenia. The importance of these various contributing factors vs. inherent changes with aging is unclear and difficult to resolve. There is evidence of elevated myostatin and activin A in elderly men and women (Yarasheski et al., 2002; Baccarelli et al., 2001; Harada et al., 1996; Santoro et al., 1999), suggesting a possible role of these TGF-␤ family ligands in age-related muscle loss. Therefore, it is important to determine whether pharmacologic inhibition of myostatin/activin can reverse sarcopenia given its multifactorial etiology. Several preclinical studies have attempted to address this question using different approaches. LeBrasseur et al. (2009) demonstrated that administration of myostatin antibody to old mice (24 month of age) enhanced the effect of exercise on performance and increased treadmill running time and distance to exhaustion. Murphy et al. (2010a,b) showed that administration of myostatin antibody to old mice (18 month of age) for 14 weeks prevented the age-related decline in muscle mass and enhanced maximum in situ force of hindlimb muscle, as well as muscle oxidative enzyme content. This treatment also led to reductions in apoptosis markers and caspase-3 transcript levels in muscle, suggesting an inhibitory effect on muscle apoptosis in the aged mice. In a separate experiment, Murphy et al. (2011b) showed that treatment with a myostatin antibody treatment attenuated the loss of muscle mass and function in mice subjected to hindlimb casting. Thus, blocking myostatin signaling was effective in counteracting disuse atrophy induced by immobilization. This result is relevant to sarcopenia, because decreased activity is a major component contributing to age-related muscle wasting. In addition, Koncarevic et al. (2010) reported that treatment of castrated male mice with an ActRIIB decoy receptor fully restored muscle mass to control levels despite the androgen deficiency. Thus, blocking myostatin/activin signaling is effective in stimulating muscle growth under condition of hypogonadism, which also occurs with aging, as well as prostate cancer in patients, which often are treated by orchiectomy. Collectively, these findings suggest that inhibiting myostatin/activin signaling is a promising approach to treat age-associated muscle loss and counters the multiple factors that must contribute to sarcopenia. 5.2.6. Other metabolic diseases Although most myostatin/activin pathway research has focused on muscle biology, there have been several intriguing studies investigating its biological impact on non-muscle tissues. As shown in Table 1, blockade of myostatin/activin signaling has been found to be also efficacious in improving fracture healing, glucose homeostasis and fat metabolism in relevant disease animal models. Hamrick et al. (2010) demonstrated that inhibition of myostatin with recombinant propeptide accelerated healing of bone fractures and enhanced muscle regeneration in mice with fibula osteotomy. In mice on a high fat diet as well as in ob/ob mice, it has been found that blocking myostatin/activin reduces obesity, enhances insulin sensitivity, increases fatty acid oxidation and fat browning and improves thermogenesis (Akpan et al., 2009; Zhang et al., 2012; Bernardo et al., 2010; Fournier et al., 2012; Shan et al., 2013). These promising findings suggest that pharmacologic inhibition of myostatin/activin may have potential additional benefits beyond muscle wasting in countering obesity, the metabolic syndrome, and recovery from trauma. 6. Conclusions The dramatic growth of interest in pharmacological use of myostatin/activin antagonists has now established several clear conclusions. Although several signaling pathways regulate muscle

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homeostasis, the myostatin/activin-ActRIIB signaling pathway is a dominant pathway whose inhibition can override the influence of other pathways and lead to a prevention or reversal of muscle loss in diverse disease states. Compelling proof-of-concept data from preclinical animal models strongly suggest potential benefits of myostatin/activin antagonism in multiple human diseases, including cancer cachexia, muscular dystrophy, myosities, disuse atrophy, COPD, sarcopenia and frailty. Consequently, multiple early-stage clinical trials are currently ongoing or being initiated to test the safety and effectiveness of myostatin or myostatin/activin inhibition for treating these various wasting disorders (www.clinicaltrials.gov).

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