MicroRNA regulatory networks in the pathogenesis of sarcopenia

Abstract Sarcopenia is an age‐related disease characterized by disturbed homeostasis of skeletal muscle, leading to a decline in muscle mass and function. Loss of muscle mass and strength leads to falls and fracture, and is often accompanied by other geriatric diseases, including osteoporosis, frailty and cachexia, resulting in a general decrease in quality of life and an increase in mortality. Although the underlying mechanisms of sarcopenia are still not completely understood, there has been a marked improvement in the understanding of the pathophysiological changes leading to sarcopenia in recent years. The role of microRNAs (miRNAs), especially, has been clearer in skeletal muscle development and homeostasis. miRNAs form part of a gene regulatory network and have numerous activities in many biological processes. Intervention based on miRNAs may develop into an innovative treatment strategy to conquer sarcopenia. This review is divided into three sections: firstly, the latest understanding of the pathogenesis of sarcopenia is summarized; secondly, increasing evidence for the involvement of miRNAs in the regulation of muscle quantity or quality and muscle homeostasis is highlighted; and thirdly, the possibilities and limitations of miRNAs as a treatment for sarcopenia are explored.

tary sequences within mRNA molecules, miRNAs silence mRNA by inhibiting translation or allowing degradation of mRNA. miRNAs thus act as powerful regulators of gene expression at the post-transcriptional level. Each individual miRNA is capable of targeting diverse mRNAs and, additionally, each individual mRNA can be modulated by various miRNAs. 5 Large numbers of miRNAs have been identified over recent years by comprehensive analyses of mammalian transcripts. miRNAs act as part of a gene regulatory network and control numerous biological processes, as well as showing abnormal expression in many disease states. 6 The in-depth studies of miRNAs are of great importance for the diagnosis, treatment and prognosis of diseases, and recently, miRNAs have been confirmed to be closely associated with the occurrence and development of sarcopenia. 7 Sarcopenia is an important manifestation of ageing and causes a gradual decline in physiological functions in the elderly, leading to decreased quality of life and even reduced life expectancy. Because of confused definitions and inaccurate screening tools, sarcopenia remains undiagnosed in most cases. Crucially, other than increasing exercise and improving nutrition, which may provide some benefit, there is no safe and effective intervention for sarcopenia. Effective biomarkers and treatments are thus urgently needed to overcome sarcopenia. With the development of high-throughput sequencing technology, a large number of miRNA molecules, which differ in sequence, structure, expression and function, have now been associated with sarcopenia. This review will summarize the latest understanding of the pathophysiological changes that take place in sarcopenia and highlight the increasing evidence associating miR-NAs with sarcopenia (Table 1).

| PATHOG ENE S IS OF SARCOPENIA
Sarcopenia is an age-related disease that is influenced by both en- and result in a transition of type II muscle fibres to type I muscle fibres. This is accompanied by intramuscular and intermuscular fat infiltration, which leads to abnormal skeletal muscle structure and, eventually, develops into sarcopenia 8 (Figure 1).

| Abnormalities of satellite cells and regenerative capacity
A reduction of approximately 65% in the number of satellite cells, which are also known as adult muscle-specific stem cells, 9 and abnormal characteristics of these cells is observed in skeletal muscle of older individuals. This leads to impaired muscle regeneration, reduced antioxidant capacity, increased DNA damage and changes in gene expression. 10 The process of muscle repair/regeneration is mainly mediated by the activation of satellite cells, which causes the cells to enter the cell cycle, thereby leading to proliferation of myogenic precursor cells, which eventually differentiate and fuse with existing muscle fibres or form new muscle fibres. 11,12 Paired box-protein-7 (PAX7), paired box-protein-3 (PAX3) and myogenesis regulator factors (MRFs) constitute key regulatory networks in this process. PAX7, which is the most important marker gene of satellite cells, determines the proliferation and differentiation of satellite cells by regulating genes such as myogenic differentiation (MYOD) and myogenic factor 5 (MYF5), which play important roles in repair following muscle injury. 11,13,14 Quiescent satellite cells begin proliferation mainly by expression of PAX7 rather than MYOD, MYF5 or other MRFs. Self-renewal or differentiation of proliferating satellite cells is achieved by maintaining expression of PAX7 and inducing expression of MYOD. By inducing expression of myogenin and down-regulating expression of PAX7, some of these satellite cells are committed to differentiation. Other satellite cells commit to self-renewal by inhibiting expression of MYOD. These cells return to the quiescent state to complement the existing quiescent satellite pool, thus ensuring that sufficient numbers of quiescent satellite cells continue to be activated to facilitate subsequent rounds of muscle repair. The intricate transcriptional mechanisms that regulate the quiescence and activation of satellite cells involve about 500 functionally diverse expressed genes. The majority of these are controlled transcriptionally and post-transcriptionally, 15 suggesting that changes in satellite cells are key to the development of sarcopenia. It should, however, be noted that some studies have shown that satellite cells may be indispensable in muscle hypertrophy 16 and other researchers even believe that satellite cells play a negligible role in sarcopenia. 17

| Imbalance of muscle protein homeostasis
Imbalance in protein synthesis and degradation is a major characteristic of muscle wasting in the elderly. It is indisputable that the most important signalling pathway for muscle protein synthesis is the phosphoinositide 3-kinase/serine-threonine protein kinase/ mammalian target of rapamycin (PI3K/AKT/mTOR) signalling pathway. 18 Simultaneously, this pathway can indirectly promote protein and lead to protein synthesis. 8 In addition to reduction of anabolic signals, UPS and autophagy are two main protein degradation pathways and play significant roles in sarcopenia. 21 The UPS induces protein degradation by stimulating expression of muscle atrophy F-box (MAFBX), and muscle ring finger 1 (MURF1) has been confirmed in some sarcopenia models. 22,23 The expression of MAFBX and MURF1 is mainly regulated by the transcription factors, nuclear factor-κB (NF-κB) and FOXO family members, which are induced by inflammatory factors or hormones. 24 Another important mechanism for muscle wasting is autophagy, which leads to degradation of monoubiquitinated proteins and is mainly induced by FOXO transcription factors and the AMP-dependent protein kinase (AMPK) signalling pathway 21 ( Figure 2).

| Muscle fibre transformation
During ageing, muscle fibres, especially type II fibres, atrophy and the resulting transition from type II muscle fibres to type I muscle fibres are a prominent feature of sarcopenia. 8 26 There is a general consensus that muscles in elderly individuals contain more slow muscle fibres, 27,28 which reduces contractility ability. Some studies, however, have shown no alteration in the type of muscle fibres with ageing, 29 and some have even reported an increase in the proportion of fast fibres. 30

| Mitochondrial dysfunction and ROS imbalance
The elderly often suffer from mitochondrial dysfunction and overproduction of reactive oxygen species (ROS), which are important causes of sarcopenia. [31][32][33] Mitochondria are the primary regulators of energy metabolism in the cell and supply 90% of the energy of the cell via oxidative phosphorylation. Mitochondria thus play a central role in cellular homeostasis. 34 The senescence-associated secretory phenotype (SASP) of senescent cells, which is characterized by a significant decline in mitochondrial function, an imbalance of metabolism and disrupted homeostasis, leads to 'signalling noise' and muscle wasting. 35 Mitochondrial dysfunction is also closely associated with redox imbalance and overproduction of ROS, which is a product of signalling pathway, 38 deregulate expression of the cell proliferation inhibitor, p16INK4a, 39 and be associated with defective autophagy 40 in aged satellite cells. An imbalance in ROS may thus be responsible F I G U R E 2 MicroRNA involvement in the homeostasis of muscle protein for age-related changes in proliferation and differentiation properties. ROS can also accelerate degeneration of skeletal muscle and lead to sarcopenia by activating the UPS and muscle proteases. 41,42 Excessive accumulation of ROS can damage the structure and function of mitochondria and induce myocyte apoptosis, which leads to dysfunction of the mitochondrial oxidative respiratory chain and increases the production of ROS, eventually forming a vicious cycle. 43

| Neuromuscular degeneration
Normal function of motor neurons is essential for the survival of muscle fibres, and loss of α-MNs is a key factor in the pathogenesis of sarcopenia. The number of α-MNs was found to be reduced by up to 50% after the age of 70, which significantly affected the function of muscle. 44 In the elderly, significant loss of MNs and motor units directly leads to decreased muscle coordination and muscle strength. 44

| Fat infiltration
Ageing is often accompanied by increased body adiposity and this increase in fat mass is associated with deposition of ectopic fat in tissues, including muscle. 49 This age-related fat accumulation seems to have synergistic effects with sarcopenia. The ageing process should be considered as a physiological degradation process, which can be accelerated by concomitant lipotoxic insults. 49 Several studies have shown that the increase in ectopic fat in muscles of the elderly can lead to reduced strength and performance, which are independently associated with metabolic abnormalities, such as insulin resistance. 50,51 Several studies have found lipid redistribution and increased lipid deposition in muscle of rats fed a high-fat diet. A strong negative correlation was shown between myosteatosis and muscle volume, suggesting that 'lipotoxicity' may lead to the accumulation of lipids in muscle cells, reduce the rate of protein synthesis and accelerate the development of sarcopenia. 51,52 Increased catabolism of muscle protein was also seen in rats fed a high-fat diet, which may be related to increased levels of free fatty acids and decreased levels of adiponectin in plasma. 53 Fat accumulation in muscle can also affect the function of mitochondria, leading to reduced capacity for oxidation of fatty acids, which has a negative impact on protein metabolism. 51 Thus, when homeostatic control of adipose tissue is lost, infiltration of fat into skeletal muscle may have a detrimental effect on muscle function and the balance between protein synthesis and degradation. suggesting that miRNAs might be used as a therapeutic strategy against muscle damage. 61 In mice, miR-27 can directly target Pax3

| miRNAs in the regulation of satellite cells and regenerative capacity
to inhibit proliferation and migration and promote differentiation of satellite cells. 62 It has also been shown that miR-27 can promote proliferation of satellite cells and muscle fibre hypertrophy by down-regulating myocyte enhancer factor 2C (Mef2C). 63 In cytoplasm, miR-31 and Myf5 mRNA can associate with proteins to form messenger ribonucleoprotein granules, thereby inhibiting post-transcriptional translation of Myf5 and keeping skeletal muscle satellite cells quiescent. 64 Additionally, miR-195 and miR-497 target Cdc25 and Ccnd genes and miR-489 targets DEK genes to maintain the skeletal muscle satellite cells in a quiescent state. 65,66 In another study, Let-7b/e were up-regulated during ageing of skeletal muscle, possibly affecting the expression of Pax7 through repression of cell cycle regulators, and impeding satellite cell self-renewal. 67

| miRNAs in muscle protein homeostasis
The PI3K/AKT/mTOR and TGF-β/myostatin/BMP pathways increase protein synthesis and are vital for myogenic differentiation. In addition to targeting IGF-1, miR-199-3p has also been found to target mTOR and ribosomal protein S6 kinase (RPS6K), two significant factors in the PI3K/AKT/mTOR pathway. 70 miR-21 also targets transforming growth factor-beta-induced (TGFβI) and suppresses PI3K/AKT signalling, thereby regulating skeletal muscle development. 78 By directly targeting Akt3, miR-29 appears to participate in regulating the same pathway, leading to inhibition of protein synthesis. 79 The TGF-β/myostatin/BMP pathway is also regulated by several miRNAs. For example, TGF-βI and Smad1 are targeted by miR-21 and miR-26a, respectively, 78 89 Although specific targets and molecular mechanisms of these differentially expressed miRNAs remain to be explored, they have been demonstrated to play a vital role in muscle protein homeostasis (Figure 2).

| miRNAs in different types of muscle fibre
Muscle fibres often change with age, and the principal difference between the different types of fibre is the composition of MyHC isoforms. 90 Since these MyHC isoforms are encoded by independent genes located at specific genomic loci in humans, it was believed that the composition of different types of muscle fibre was regulated mainly at the transcriptional level, 90

| miRNAs in mitochondria and ROS
Mitochondrial dysfunction and oxidative damage are key processes underlying the majority of age-related diseases, including sarcopenia. The role of mitochondria in sarcopenia is very suggestive of their role in energy metabolism. miR-1 not only inhibits cytoplasmic gene expression by targeting HDAC4 and Hand2 but also promotes the expression of mitochondrial genes by targeting mtCox1, mtNd1, mtCytb, mtCox3 and mtAtp8. 95 This suggests that some miRNAs can coordinate the concurrent translation of nuclear-encoded and mitochondrial-encoded mitochondrial proteins. miR-696 negatively affects fatty acid oxidation and mitochondrial function by targeting the transcription factor peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), a master regulator of mitochondrial biogenesis and ROS removal. 96 Another study showed that deficiency of miR-133a in mice led to low levels of Pgc-1α and nuclear respiratory factor-1(Nrf1), and lower mitochondrial mass and exercise tolerance. 97 This phenotype is similar to the sarcopenia phenotype, suggesting that miR-133a has a significant role in maintaining skeletal muscle mitochondrial dynamics. Overexpression of miR-23 in amyotrophic lateral sclerosis (ALS) patients also represses the expression of PGC-1α, resulting in mitochondrial dysfunction. 98

| miRNAs in fat infiltration
In sarcopenia, muscle wasting is commonly associated with fat infil-  119 Additionally, miR-143 promoted synthesis of triglycerides, both in humans and rodents, which may contribute to infiltration of adipocytes. 120 These data suggest that miRNAs may participate in accumulation of fat during sarcopenia.  124 Ensuring target specificity and limiting unnecessary off target effects and toxicity are also significant hurdles for miRNA drugs. 125 Other issues that need to be urgently addressed include the development of appropriate delivery systems to provide safety and stability, understanding the long-term effects of regulating miRNA activity, establishing the safety and efficacy of miRNA-based therapeutics and modelling the pharmacodynamics and pharmacokinetics of these miRNA drugs.

| PROS PEC TS OF MIRNA THER APY FOR SARCOPENIA
With ageing, deregulation of miRNAs in muscle is closely associated with sarcopenia, suggesting that miRNAs may be a viable therapeutic target for sarcopenia. Compared with studies of miRNA in diseases such as cancer and heart disease, the study of miRNA function in skeletal muscle, especially in sarcopenia, is still in its infancy and the roles and specific mechanisms of miRNAs in sarcopenia need to be further elucidated. Efficient delivery systems and stable therapeutic molecules will be key to the development of miRNA-based therapy for muscle diseases, including sarcopenia. Increasing numbers of clinical trials of miRNA-based therapies are under way, and these should provide proof of principle for the use of miRNA to fight refractory diseases. The development of miRNA-based therapy for sarcopenia will inevitably face challenges but, once these obstacles are overcome, miRNA-based therapy may provide a welcome strategy for the treatment of sarcopenia.

| CON CLUS IONS
Loss of skeletal muscle is a major problem associated with ageing.
The decline in muscle quantity and quality leads to decreased quality of life and increased mortality. Many studies have sought to identify the intrinsic and external factors that cause muscle ageing and to discover targeted interventions for age-related muscle atrophy. miRNAs in mammalian genomes are essential for the development and function of life, and a large number of miRNAs are important regulators. Their expression profiles change with age, functionally contributing to ageing-related loss of muscle quantity and quality.
With continuous updating of technology to detect miRNAs, the understanding of the roles of miRNAs as a whole will become more comprehensive. This review article describes how, by negatively regulating the expression of target genes, miRNAs modulate the processes involved in sarcopenia and also discusses the prospect of miRNAs as a treatment for sarcopenia. In summary, miRNAs are promising small molecule therapeutics and it is hoped that, through both large scale validation research and carefully designed functional research using in vitro and in vivo systems, miRNAs will, in the future, become an effective means of treating refractory diseases.