The Growth Differentiation Factor 11 (GDF11) and Myostatin (MSTN) in tissue specific aging

Accepted Manuscript Title: The growth differentiation factor 11 (GDF11) and myostatin (MSTN) in tissue specific aging Authors: X.L. Fan, U. Gaur, L. Sun, D.Y. Yang, M. Yang PII: DOI: Reference:

S0047-6374(16)30170-1 http://dx.doi.org/doi:10.1016/j.mad.2017.04.009 MAD 10954

To appear in:

Mechanisms of Ageing and Development

Received date: Revised date: Accepted date:

20-9-2016 18-4-2017 28-4-2017

Please cite this article as: Fan, X.L., Gaur, U., Sun, L., Yang, D.Y., Yang, M., The growth differentiation factor 11 (GDF11) and myostatin (MSTN) in tissue specific aging.Mechanisms of Ageing and Development http://dx.doi.org/10.1016/j.mad.2017.04.009 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

The Growth differentiation factor 11 (GDF11) and myostatin (MSTN) in tissue specific aging Fan XL¶1, Gaur U¶1, Sun L2, Yang DY 1and Yang M*1 1, Institute of Animal Genetics and Breeding, Sichuan Agricultural University, Chengdu, 611130, P. R. China 2, College of Pharmacy，Yancheng Institute of Health Science, Yancheng, 224000, P.R. China ¶ These authors contributed equally to this work. * Corresponding author: Yang M (Mingyao Yang) Institute of Animal Genetics and Breeding, Sichuan Agricultural University, Chengdu 611130, P. R. China Tel: (86) 28 82783043 Fax: (86) 28 86290987 [email protected] Highlights: 

Growth differentiation factor 11 (GDF11) and myostatin (MSTN) are evolutionarily conserved homologues proteins

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GDF11 expression level is 500 times less than MSTN

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Both proteins can regulate brain, muscle and cardiovascular aging

Abstract Growth differentiation factor 11 (GDF11) and myostatin (MSTN) are evolutionarily conserved homologues proteins which are closely related members of the transforming growth factor β superfamily. They are often perceived to serve similar or overlapping roles. Recently, GDF11 has been

identified as playing a role during aging, however there are conflicting reports as to the nature of this role. In this review, we will discuss the literature regarding functions of GDF11 and myostatin in the heart, brain, and skeletal muscle during aging. Consequently we expect to develop a deeper understanding about the function of these two proteins in organismal aging and disease.

Key words: Aging, GDF11, MSTN, Organismal aging Introduction Aging in multicellular organisms involves intensive signaling cascades between cells, tissues, and organ systems. Multiple studies on model organisms have provided sufficient evidence of unexpected connections between local and systemic aging (Rando, 2006). Recently, heart, brain, and muscle are considered as the most efficient model systems or tissues with the ability to affect aging and lifespan (Gouspillou et al., 2014; Rubio-Ruiz et al., 2014; Vida et al., 2014). Many epidemiological studies have already established the links between these model systems and morbidity/mortality of various age-related diseases, such as Alzheimer's disease (Tolppanen et al., 2012), hypertension, and Parkinson's disease (Compta et al., 2012). Transforming growth factor (TGF)-secreted factor superfamily consists of more than 30 members. This family can be subdivided into 3

subclasses;

the

TGFβs,

BMPs,

and

activin/MSTNs.

Growth

differentiation factor 11 (GDF11) and MSTN (MSTN also known as GDF8) belong to the activin/MSTN subclass, and share 90% sequence identity within their mature, signaling domain (Attisano and Wrana, 2002). The mature peptide sequences of GDF11 and MSTN are highly identical in vertebrate model animals. Although mature GDF11 and MSTN ligands share substantial sequence identity, their prodomains are only 52% identical (Walker et al., 2016). X-ray crystallography has revealed that GDF11 and MSTN have differences in the type I receptor binding epitope. Signaling induced by the TGF family ligands are necessary for multiple processes during vertebrate development, tissue homeostasis, and repair (Massague and Chen, 2000; Vizan et al., 2013; Wu et al., 2003). The TGFβ regulated mechanism to maintain cell homeostasis works by inducing cell protective proteins such as hemeoxygenases-1 (Ho-1) and NADPH ash or nitric oxide synthase activity and their interaction. Studies have shown that because of inflammation and repair response modulation (Bujak and Frangogiannis, 2007; Dobaczewski et al., 2011), TGF-β plays an important role in the pathogenesis of myocardial infarction. Similar to the other activin-type ligands, both GDF11 and MSTN predominantly use the type II receptors activin receptor kinaseII-A, type II receptors activin receptor kinase II-B, type I receptors activin receptor-like kinase 4 (ALK4), and ALK5 to elicit signal transduction via SMADs 2 and 3

(Attisano and Wrana, 2002; Budi et al., 2016; Nadjar-Boger et al., 2012). This leads to phosphorylation of SMAD2 and SMAD3 (SMAD2/3) transcription factors by type I receptor. Subsequent accumulation of SMAD2/3 in the nucleus results in regulation of GDF8 and GDF11 responsive genes (Fig1). Notably, GDF11 is more potent than MSTN and MSTN activated SMAD3. with a seven fold higher EC50 compared to GDF11 (45.7 nM versus 6.5 nM, separately) in A204 cell line (Walker et al., 2017). The furin type protease cleaves the peptide bond and the nTerminal peptide and mature growth factor domain remain associated on the disulfide bond, forming a potential complex known as the inactive complex. This may be freed from potential compounds through degradation by various proteases to release the active peptide of mature growth factor (Kondas et al., 2008). GDF11 has been shown to be involved in mesoderm formation and neurogenesis during embryonic development in mice and xenopus (Essalmani et al., 2008). The tissue specific expression pattern of the 407 amino acids long GDF11 peptide was measured in different tissues. It was found to be expressed in the pancreas, small intestine, kidney, skeletal muscle, and the developing nervous system (McPherron, 2010). The GDF11 knockout in mice resulted in perinatal lethality (McPherron et al., 2009). The homozygous targeted deletion of GDF11 in mice displayed extensive axial skeletal patterning defects, as well as renal and palate

abnormalities (McPherron et al., 1999; Oh et al., 2002). Notably, GDF11 is also related to human colon cancers, which is possibly due to the proangiogenic properties of GDF11 and most TGF-beta family ligands (Jakobsson and van Meeteren, 2013). MSTN is expressed in skeletal muscle, but is also expressed, to a lesser extent, in adipose tissue and cardiomyocytes (McPherron et al., 1997; Sharma et al., 1999b). The MSTN gene is thought to negatively regulate skeletal muscle growth (Lee, 2007) and the inhibition of MSTN protein function in adult mice increased muscle mass (Patel and Amthor, 2005). The mstn-/- mice had reduced fat mass, were resistant to obesity (Zhao et al., 2005), and developed insulin resistance. Both GDF11 and MSTN are found in the blood and their universal presence implies that these proteins may work as hormonal signals. Due to having high sequence similarity, functional overlap was also expected of these two ligands. Recent high profile studies have reported conflicting data regarding GDF11 and MSTN regulated muscle regeneration effects and age-related changes (Egerman et al., 2015; Katsimpardi et al., 2014; Loffredo et al., 2013; Sinha et al., 2014). Accumulating evidence suggests that these two ligands can have distinct functions under different conditions. GDF11 is important for mammalian development and has been suggested to regulate aging in multiple tissues, whereas MSTN is described as a negative regulator of postnatal skeletal and cardiac muscle mass that also moderates

metabolic processes. In this short review we will discuss the role of GDF11 and MSTN's in the aging process, which might help us to understand the aging reversal phenomenon by young blood.

Regulation of the cardiac and vascular aging Cardiovascular functions can decline with aging, with such decline always leading to heart failure (Schocken et al., 2008) and increased mortality risk under any given infarction event (Maggioni et al., 1993). Conjoined in a mouse model, Francesco et al found that the GDF11 blood level decreases with aging and leads to pathological cardiac hypertrophy (PCH). However, if the GDF11 levels are restored to normal, it can rescue the PCH in aged mice (Loffredo et al., 2013). It was also observed that after a stroke event, GDF11 levels decline in mice (Chauhan et al., 2016). Another group reported that daily injection of rGDF11 significantly improved the rGDF11 levels in the blood of old mice. However, they did not observe any improvement in the size of heart or cardiac myocyte, nor did they observe any changes in cardiac performance. They also found that GDF11 did not reduce neonatal rat ventricular myocyte hypertrophy; on the contrary, more GDF11 induced hypertrophy (Smith et al., 2015). Meanwhile, when GDF11 was injected into the mouse model, it increased the endogenous levels of GDF11 and resulted in overall bodily wasting, specifically with evidence of cardiac and skeletal muscle atrophy (Liang,

2016). More recent studies indicate that GDF11 circulating levels do not decline with age, body weight, echocardiographic variables, or the severity of chronic mitral valve insufficiency-induced heart failure in dogs (Ahn et al., 2016). By using a specifically developed LC-M/MS assay to quantify GDF11, Schafer et al found that the GDF11 levels did not decline in healthy men throughout aging, and that individuals with higher GDF11 levels were likely to be more frail and had diabetes or prior cardiac conditions (Schafer et al., 2016). When GDF11 levels were analyzed in paired samples of serum, plasma and platelet lysate (PL) from 23 volunteers, the results revealed that GDF11 was highly concentrated in platelets (Bueno et al., 2016). Multiple reports have shown that GDF11 level will not decline with aging (Egerman et al., 2015; Schafer et al., 2016). In mouse model, GDF11 daily administration did not extend lifespan of premature-aged mice (Freitas-Rodriguez et al., 2016). More evidence is required to confirm if GDF11 can reverse aging dependent cardiac pathological structural and functional remodeling. MSTN is primarily expressed in the skeletal muscle, but is also expressed in significant amounts in adipose tissue and the heart (Sharma et al., 1999a). Nadine et al demonstrated that MSTN represses the activation of AMP-activated kinase, thereby inhibiting a metabolic switch to maintain cardiac health steady and to prevent cardiac hypertrophy (Biesemann et al.,

2014). Furthermore, the activin type IIB receptor knockout mice displayed serious heart defects, pin-pointing the importance of MSTN-activin typeIIB receptor signaling axis in maintaining cardiac functions (Oh and Li, 1997). Cardiac MSTN levels were elevated in heart failure (HF) patients and during left ventricular assist device (LVAD) support (George et al., 2010). It was found by ELISA assay that mstn declines with age (Schafer et al., 2016). The gdf11 level is almost 500 times lower than mstn, which means that circulating GDF11 has little physiological relevance as it cannot contend with myostatin for ActRIIB binding sites (Rodgers and Eldridge, 2015). Many studies reported that MSTN levels increased in serum (Gruson et al., 2011), heart (Fernandez-Sola et al., 2011), and skeletal muscle (Lenk et al., 2012) of adult patients with HF. However, Breitbart (Breitbart et al., 2013) found no increase in serum MSTN in patients with stable chronic HF. Looking at the collective evidence, it can be firmly stated that MSTN may play a regulatory role in cardiac aging.

Regulation of brain aging Brain dysfunction could occur during the process of aging and atherosclerosis, causing a reduced blood supply to the neurons and leading to an increase in the number of age-related neurodegenerative and neurovascular diseases (Grinberg and Thal, 2010; Reed and Edelberg, 2004). In the beginning, GDF11 during neurogenesis serves to limit the

numbers of neural stem cells (Gokoffski et al., 2011; Wu et al., 2003). Katsimpardi et al showed that GDF11 can improve brain blood vessels and enhance the neurogenesis. In this study, the factors present in the young blood induced vascular restructuring, leading to enhanced neurogenesis and improved olfactory system in aging mice (Katsimpardi et al., 2014). Old mice treated with GDF11 had improved olfactory perception, improved cerebral vasculature, and neural stem cell function. All of these can protect the central nervous system, leading to an increase in the coping potential for age-related challenges. The role of MSTN in brain aging is not very well explored, with minimal reports observing the expression of MSTN in certain brain regions (that is, the mitral cells in the olfactory bulb and in the olfactory cortex neurons) (Moriwaki et al., 2013). Although the direct significance of MSTN expression in brain regions is not clear, most MSTN −/− mice had larger brains than wild type (WT) mice, suggesting the involvement of MSTN in brain size (Jeffery and Mendias, 2014).

Regulation of muscle aging Aging is associated with reduced quality and function of the skeletal muscles, as well as reduced muscle mass. One such disability, Sarcopenia, is the loss of muscle mass and strength with age. It affects general quality of life in the elderly, and can be used as a reliable predictor of death

independent of other risk factors (Roubenoff and Hughes, 2000). Skeletal muscle regeneration is a highly programmed event where muscle injury or some pharmacological intervention leads to the activation of muscle stem cells, or "satellite cells" (Jang et al., 2011). Old muscles exhibit reduced satellite cells, satellite cell dysfunction, and reduced regeneration potential (Brack et al., 2007; Chakkalakal et al., 2012; Cosgrove et al., 2014; Jang et al., 2011). Sinha et al found that systemic GDF11 levels typically decline with age. Dysfunction and the integrity of aged muscle satellite cells can be restored by heterochronic conjoined (Conboy et al., 2013) or systemic delivery of recombinant proteins and supplements. Increased GDF11 levels in older mice was found to improve muscle structure and function, as well as increase strength and endurance (Sinha et al., 2014). However Egerman et al reported contradictory results, observing an increase in the GDF11 levels in the serum of aged rats and humans. GDF11 mRNA expression was found to increase with age in rat muscles, which inhibited the muscle regeneration and reduced the expansion of satellite cells (Egerman et al., 2015). The role of GDF11 in muscle regeneration and in aging thus appears to be unclear. It was recently reported that the concentrations of GDF11 in non-diabetic people decreased with aging, but increased significantly in patients with Type 2 Diabetes and cardiovascular disease. (Schafer et al., 2016). It was also reported that Supraphysiological levels of GDF11 caused wasting of

both skeletal and cardiac muscle, with some reports even suggesting that GDF11 should be viewed as a potential deleterious biomarker in muscle wasting diseases (Hammers et al., 2017). Of particular note, although GDF11 mRNA levels increased with age, the levels of MSTN mRNA significantly reduced (Brun and Rudnicki, 2015; Rodgers and Eldridge, 2015). Since muscle tissue produces MSTN, the decline in MSTN protein may reflect the loss of muscle mass during aging (Conboy et al., 2015). As MSTN suppresses skeletal muscle growth and differentiation (McPherron et al., 1997), deletion and loss of function mutation in the MSTN gene leads to skeletal muscle cell hyperplasia and hypertrophy. Mendias found age-related muscle decline in mass and contractility (Mendias et al., 2015) in MSTN+/- and MSTN-/- mice. In a recent study, a short decorin protein peptide inhibited MSTN/Smad signaling pathways (El Shafey et al., 2016), which could help to combat muscle mass loss in sarcopenia and cachexia. Additionally, treatment with anti-MSTN antibodies increased muscle mass and strength in both young and old mice, and also increased insulin sensitivity in old mice (Camporez et al., 2016), attributed to increased insulin-stimulated skeletal muscle glucose update. In Drosophila, RNAi of the Myoglianin (a MSTN/GDF11 homologue) in muscles shortened the lifespan and increased the percentage of agerelated climbing defects, whereas overexpression had opposite effects

(Demontis et al., 2014; Patel and Demontis, 2014). In mice, the skeletal muscle-specific deletion of the GDF11 gene on the MSTN+/+ or MSTN-/background did not increase muscle fiber number or fiber types. The phenotype was similar to conditional KO MSTN, where no increase in phenotypic severity occurred (McPherron et al., 2009). In contrast, the double transgenes of MSTN and GDF11 propeptides lead to further growth and muscle mass enhancement. These reports clearly suggest that muscle growth inhibitor MSTN and GDF11 have redundant functions in skeletal pattern, and GDF11 may play a further role in muscle growth. In a clinical study, GDF11+MSTN levels significant decreased in older patients as compared to younger patients, for both males and females (Olson et al., 2015). In support, a separate study quantified circulating MSTN by mass spectrometry in humans without chronic diseases, and found that the MSTN level had sex-specific differences in aging humans. Similar results were seen for GDF11 as well (Bergen et al., 2015). The methods used in these studies were unable to distinguish GDF11 from MSTN. Last year Bergen’s group developed the LC-MS/MS assay (which can distinguish the two proteins) to measure GDF11 and MSTN serum protein level in humans aged between 21 and 93. This found that GDF11 levels did not statistically differ as a function of age or sex; MSTN levels were highest in men in their 20s, statistically declining throughout the succeeding lifespan (Schafer et al., 2016). Together, these studies reveal a

potential influence of health, age, and sex on GDF11+MSTN levels.

Concluding remarks In conclusion, GDF11 and MSTN may serve as evolutionarily conserved age-dependent factors in multiple organ systems. Mature GDF11 and MSTN can regulate the JNK, P38, RAS, and AKT pathways (Walker et al., 2016) (Figure1), and most of these pathway have been demonstrated to play an important role in aging. This increases the possibilities for using these signaling pathways to preserve or restore the function of aging tissues. Despite some conflicting results, there is an agreement between many independent groups (Kim et al., 2005; Wu et al., 2003) that GDF11 and MSTN activate identical signaling pathways (Figure 1). The difference in phenotype is that the two proteins have different expression patterns. With GDF11 appearing to act more broadly, regulating many organs and tissue development. There is also a difference in the ligand activity and structure listed (Table 1). The lack of equivalence in function of the two ligands may be partially due to differences in expression level, in concentrations of protein or antagonist, or in receptor types for different tissues. Future exploration of GDF11 and MSTN biology and biochemistry will elucidate the similarities and differences in the functions of these two proteins, and enhance our understanding of organismal aging and disease.

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Figure 1. The main pathway which GDF11 and MSTN activated in the cell.

Table 1. The differences between GDF11 and MSTN by phenotypes Phenotype

MSTN

Gdf11

Expression pattern

Skeletal

cardiomyocytes

developing nervous system.

Mutants

Mstn-/- mice can survive into adulthood

The gdf11-/- mice is embryonic lethal

muscle,

adipose

tissue,

Pancreas, intestine, kidney, skeletal muscle,

but have a hypermuscular phenotype

Structure

Apo-GDF8 is positoned in a “closed”

Apo-GDF11 is adopted “open” conformation

conformation

Receptors

ALK4, ALK5

ALK4,ALK,ALK7

Concentration in the human serum pool

Plasma MSTN concentrations ranged from

Plasma GDF11 concentrations ranged from

0.64-6.27ng/ml in the 96 adults with CVD

224-841pg/ml in the 96 adults with CVD age

age 65-94.

65-94.

Ligand activity

EC50 is 0.48nM and 5.4nM respectively in

More potent than MSTN.

the HEK293 and HepG2 cells, and the

3.4nM respectively in the HEK293 and HepG2

EC50 is 45.7nM in A204 cells to activate

cells, and the EC50 is 6.5 nM in A204 cells to

the SMAD3.

activate the SMAD3.

The MSTN−/− mice have larger brains

Activated of ALK5, that leads to increase adult

compared with wild-type mice.

neurogenesis ; increase neural activity, and

Brain

EC50 is 0.08nM and

improve performance in memory tests.

Cardiac and vascular

Myostatin participates in cardiac growth

GDF11 can regulate cardiomyocyte size and

and metabolism

hypertrophy

Muscle

Negatively inhibited hypertrophy.

regulate mstn

muscle induced

mass,

Conflicting results on the muscle aging, muscle

muscle

fiber–specific deletion of GDF11 showed no phenotype on muscle mass.