The role of extracellular vesicles in skeletal muscle wasting

Abstract

Skeletal muscle wasting is a complicated metabolic syndrome accompanied by multiple diseases ranging from cancer to metabolic disorders and infectious conditions. The loss of muscle mass significantly impairs muscle function, resulting in poor quality of life and high mortality of associated diseases. The fundamental cellular and molecular mechanisms inducing muscle wasting have been well established, and those related pathways can be activated by a variety of extracellular signals, including inflammatory cytokines and catabolic stimuli. As an emerging messenger of cell‐to‐cell communications, extracellular vesicles (EVs) also get involved in the progression of muscle wasting by transferring bioactive cargoes including various proteins and non‐coding RNAs to skeletal muscle. Like a double‐edged sword, EVs play either a pro‐wasting or anti‐wasting role in the progression of muscle wasting, highly dependent on their parental cells as well as the specific type of cargo they encapsulate. This review aims to illustrate the current knowledge about the biological function of EVs cargoes in skeletal muscle wasting. Additionally, the potential therapeutic implications of EVs in the diagnosis and treatment of skeletal muscle wasting are also discussed. Simultaneously, several outstanding questions are included to shed light on future research.

Introduction

Skeletal muscle accounts for 40% of body weight and comprises 50–70% of body proteins in humans. ¹ It functions as an important locomotor system to generate force and power, as well as a significant metabolic organ to store amino acids and carbohydrates. ² Thus, it is critical for skeletal muscle to maintain homeostasis by keeping a balance between protein synthesis and degradation. However, a wide variety of diseases including cancer, organ failure, chronic infection and unhealthy aging are accompanied by muscle wasting, which severely deteriorates the integrity and function of skeletal muscle. ³ The most common disease indication with muscle wasting is cancer‐associated cachexia. Almost 80% of patients with cancer inevitably endure a fast loss of skeletal muscle mass in the advanced stage of cancer, which impairs the mobility of patients and compromises cancer therapies. ⁴ , ⁵ Furthermore, as muscle wasting can hardly be reversed by nutritional supplements, rapid weight loss is generally associated with poor quality of life and unsatisfactory prognosis, inducing a low survival rate in cancer patients. ⁶ Patients with cardiac disease, ⁷ chronic kidney disease, ⁸ chronic obstructive pulmonary disease ⁹ and sepsis ¹⁰ also endure profound muscle wasting that exacerbates the prognosis of those diseases. Muscle loss also occurs in elderly people who suffer from sarcopenia, which significantly reduces physical activity and increases the risk of disability. ¹¹ Currently, the countermeasure of muscle wasting is relatively ineffective as the only validated clinical treatment is exercise, and it can only limit the rate of wasting progress. ¹² The development of novel drug therapies is of great importance and will have extensive benefits to those patients suffering from muscle wasting.

Growing shreds of evidence indicate that extracellular vesicles (EVs), including exosomes (one type of EVs with a diameter of around 100 nm), can transfer biological molecules such as nucleic acids and proteins to the target cells through the circulation system, resulting in phenotypical changes in recipient cells. ¹³ , ¹⁴ Based on their characteristics and functions, EVs can be utilized as diagnostic biomarkers in diverse diseases, particularly in various types of cancer. ¹⁵ , ¹⁶ Furthermore, they can also be exploited and engineered for therapeutic strategies in the treatment of a wide variety of diseases. ¹⁷ , ¹⁸ Because of the profound involvement of EVs in both normal and abnormal physiological processes of different organs, it is fascinating to presume that EVs are involved in the maintenance and degeneration of skeletal muscle tissue. Indeed, there have been multiple studies recently highlighting the role of EVs in the progression of muscle wasting. In this article, we describe and discuss EVs as a potential route to influence skeletal muscle homeostasis and their potential therapeutic implications in the treatment of muscle wasting.

Mechanisms of skeletal muscle wasting

The skeletal muscle is composed of muscle fibres and associated connective tissues. Single muscle fibres are multinucleated and nearly 80% of the content is protein without the consideration of water. ¹ And each muscle fibre consists of thousands of myofibrils made up of orderly interconnected thick and thin myofilaments. Groups of muscle fibres are arranged in bundles and wrapped by a layer of connective tissue called perimysium. ¹ Bunches of muscle fibre bundles are then surrounded by another layer of dense connective tissue known as the epimysium to form the whole muscle. ¹ The perimysium and epimysium as well as the endomysium around each muscle fibre form the extracellular matrix of skeletal muscle. Extracellular matrix is composed of three major proteins including collagen, non‐collagen and proteoglycan. ¹⁹

Despite the fact that muscle wasting is accompanied by a large number of diverse diseases, the underlying mechanisms inducing muscle loss are highly conserved across different disease conditions. Generally, the loss of skeletal muscle mass results from the destructed balance between protein synthesis and proteolysis. Most studies focused on the importance of proteolysis, indicating that proteolysis of myofilaments accounts for the predominant factor contributing to muscle wasting. The fundamental mechanism of the wasting process is an increase in muscle protein degradation induced by activated ubiquitin‐proteasome and autophagy‐lysosome systems. ²⁰ Intact myofibrillar proteins are initially degraded to release the myofilaments from the myofibrillar for subsequent degradation of myofilaments. ²¹ The cleavage of myofilaments from the myofibrillar is calpain‐dependent, and the degradation of proteins that anchor myofilaments to the Z‐disc allows for the separation of myofilaments. ²²

Deterioration of myofilaments by activated ubiquitin‐proteasome and autophagy‐lysosome pathways consequently leads to muscle wasting. And multiple inflammatory cytokines such as tumour necrosis factor α, interleukin 6 (IL‐6), IL‐1α and interferon‐γ produced by tumour or host cells can act as triggers of these two pathways. ²³ , ²⁴ , ²⁵ These factors activate respective downstream pathways including the nuclear factor kappa‐B (NF‐κB) and the p38 MAPK (mitogen‐activated protein kinase) pathways, followed by the activation of forkhead family transcription factors of crucial E3 ubiquitin ligase genes such as MuRF‐1 (Muscle RING Finger containing protein 1, also known as TRIM63) and atrogin‐1 (muscle atrophy F‐box, also known as atrogin‐1) which mediates proteasomal degradation of muscle proteins and inhibition of muscle protein synthesis. ²⁶ Autophagy genes can also be activated through signalling cascades of these cytokines. ²⁴ In addition to inflammatory cytokines, several TGF‐β family members, including activin A and myostatin, have been proposed to be able to promote muscle wasting through the myostatin receptor ActRIIB. ¹² , ²⁷ Apart from the direct transduction of those cytokine signals, mounting evidence demonstrates that EV‐associated factors can also activate the catabolic status in skeletal muscle, which is discussed further below.

Another essential contributor to muscle wasting is mitochondrial dysfunction in various muscle wasting disorders. ²⁸ , ²⁹ Dysfunctional mitochondria contribute to skeletal muscle wasting by elevated ROS emissions, decreased ATP production and the release of proapoptotic mitochondrial proteins. ²⁸ , ³⁰ It has been reported that the dysfunction of mitochondria exists before the initiation of cancer‐induced muscle loss in animal models. ³¹ Penna et al. have proposed a scheme depicting the pathogenesis of muscle wasting under cancer circumstances, where mitochondrial dysfunction also precedes the cachectic phenotype and acts as a key antagonistic hallmark in the muscle wasting progress. ³² Despite of these consensus models, the mechanisms responsible for mitochondrial dysfunction under skeletal muscle wasting remain largely unknown. Therefore, the mechanism underlying the loss of skeletal muscle mass is multifactorial. Of note, most literature we discussed here applies to animal models only.

Extracellular vesicles: Biogenesis and composition

EVs comprise a large heterogeneous population of lipid bilayer membrane‐bounded vesicles that are generated by various types of cells, including prokaryotic and eukaryotic cells. ³³ Ectosome and exosome are two predominant subpopulations of EVs based on their formation process. Ectosomes (100–1000 nm in diameter) are presumably generated by the direct outward budding from the plasma membrane, while exosomes (30–150 nm in diameter) are produced by the fusion of intracellular multivesicular bodies with the plasma membranes. ³⁴ , ³⁵ The biogenesis of exosomes can be either endosomal sorting complex required for transport‐dependent or endosomal sorting complex required for transport)‐independent, while the biogenesis of ectosomes highly relies on several small GTPases. ³³ , ³⁶ , ³⁷ , ³⁸ There is an overlap between the size range of these two types of EVs, and current isolation methods cannot separate them effectively in the overlapped size ranges as they share the same properties in size, density as well as membrane topology. ³⁹ Despite their distinct sizes and subcellular origins, both types of EVs contain functional biomolecules which can be transferred to recipient cells to change cellular behaviour. ⁴⁰ , ⁴¹ The contents of EVs are diversified depending on their donor cells. Basically, EVs can contain various proteins, nucleic acids, lipids and metabolites. ⁴² Proteins and nucleic acids are the most abundant cargoes; thus, the majority of studies are focused on these two types of molecular cargoes. EV‐containing proteins include membrane proteins, cytosolic proteins, and nuclear proteins while EV‐associated nucleic acids include mRNA, distinct noncoding RNA species, and DNA fragments as well. ⁴³

The biological regulation of EV cargoes in skeletal muscle wasting

It is now well recognized that EVs can deliver their cargoes to various organs including the skeletal muscle, having a profound influence on these tissues and playing a pivotal role in tissue‐to‐tissue communication. The most studied EV cargoes related to skeletal muscle wasting are noncoding RNA species and proteins, and they can play either a pro‐wasting or anti‐wasting role in the process of skeletal muscle wasting (Figure 1).

Figure 1.

EV cargoes and skeletal muscle wasting. EVs derived from parental cells carry diversified functional biomolecules, exerting either pro‐wasting or anti‐wasting role upon assimilation by the skeletal muscle cells. These cargoes are mainly in the form of miRNAs and proteins, packaged into the lumen or loaded on the surface of EVs. Once delivered to the acceptant cells, they can activate or deactivate selective transcription factors through receptor‐mediated or non‐receptor‐mediated signalling transduction. These transcription factors then promote or inhibit the expression of genes encoding components of the ubiquitin‐proteasome system, autophagy system, or apoptosis system, leading to the protection or destruction of myofibrillar proteins. Loss of myofibrillar proteins results in muscle wasting eventually. IL‐6, Interleukin‐6; HMGB1, High Mobility Group Protein B1; PAI‐1, Plasminogen Activator Inhibitor‐1; GDF‐15, Growth Differentiation Factor‐15; Hsp, Heat shock protein.

Extracellular vesicle‐associated mRNAs in skeletal muscle wasting

EV‐encapsulated mRNAs can be protected from degradation by nuclease, and they can be directly translated upon entering the cytosol of recipient cells. ⁴⁰ A previous finding indicates that circulating EVs from xenograft tumours of human lung cancer in nude mice contain human glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) mRNA. ⁴⁴ It clearly reveals that tumour‐derived EVs in vivo encapsulate mRNA, but whether these EVs can be taken up by distal organs remains to be elucidated. Another study confirms that mRNA‐containing EVs can be taken up by skeletal muscle and induce functional alteration. It demonstrates that Klotho mRNA could be preferentially encapsulated and transferred to aged skeletal muscle through circulating EVs from young animals. And the EV‐associated Klotho mRNAs can rejuvenate aged cell mitochondrial function and enhance skeletal muscle regeneration. ⁴⁵ However, the cellular origin of the Klotho mRNA containing EVs remains elusive. Beyond that, little attention is paid to the EV‐associated mRNA relating to muscle wasting. So further studies are needed to figure out more about EV‐encapsulated mRNAs in the functional modulation of skeletal muscle.

Extracellular vesicle‐associated miRNAs in skeletal muscle wasting

Like EV‐associated mRNAs, EV‐enclosed miRNAs can be protected against nuclease digestion. However, miRNAs attract much more attention than mRNA within the context of EV‐induced skeletal muscle wasting, possibly due to their simple structure and direct regulation of gene expression. And plenty of studies establish that EV‐associated miRNAs can play a pivotal role in regulating skeletal muscle function. Recently, we have reported that breast cancer induces skeletal muscle mass decline through EVs encapsulated miR‐122. ⁴⁶ We find that miR‐122 presenting in breast cancer cell‐derived EVs, targets O‐GlcNAc transferase and mediates its downregulation, which promotes O‐GlcNAcylation of ryanodine receptor (RYR1), thus decreasing its ubiquitination and degradation. Increased RYR1 protein level leads to higher cytosolic Ca²⁺ levels and the activation of calpain proteases, which induces muscle protein cleavage and myofibrillar destruction and thereby results in skeletal muscle wasting of tumour‐bearing mice. ⁴⁶ Another elegant study by Waning et al. reports that the loss of muscle mass and function results from TGF‐β induced‐oxidation of RYR1 in the context of bone metastasis in murine model. ⁴⁷ Due to the low prevalence of cachexia in breast cancer and even some breast cancer patients may endure loss of muscle mass while gaining body weight, ²⁰ it should be noted that the term ‘muscle wasting’ applied here means literally the loss of muscle mass rather than the whole body weight loss.

Considering the heterogeneity characteristic of EV cargo population, distinct miRNAs might be encapsulated together into EVs and cooperate following taken up by target cells. For instance, Miao et al. demonstrate that miR‐195a‐5p and miR‐125b‐1‐3p enriched in colon cancer cell‐secreted EVs could induce muscle wasting in vitro and in vivo. ⁴⁸ Both miR‐195a‐5p and miR‐125b‐1‐3p can target the apoptosis inhibitor protein Bcl‐2 and the downregulated Bcl‐2 level induces the activation of apoptosis signalling pathway in EV‐treated C2C12 myotubes and in the gastrocnemius muscle (GA) of tumour‐bearing mice, which results in muscle wasting. ⁴⁸ This study reveals another mechanism‐the apoptotic pathway beyond the common ubiquitin‐proteasome and the autophagy‐lysosome pathway that contributes to muscle proteolysis. However, results in this study did not exclude the possibility that TUNEL reactivity derived from mononuclear cells localized outside the myofibers. It is also worth noting that the effect of EV‐associated miRNAs on skeletal muscle might also depend on the parental cells. In the same study, the researchers found that EVs from C26, rather than another colon cancer cell MC38 could induce muscle loss. The reason is that the levels of miR‐195a‐5p and miR‐125b‐1‐3p in C26 EVs were much higher than those in MC38 EVs, as confirmed by miRNA‐seq data. ⁴⁸ In another study, the same group reported another colon cancer cell‐derived EV‐associated miRNA named miR‐183‐5p could also induce myotube protein degradation. ⁴⁹ By targeting FHL1, miR183‐5p activates the myostatin/Smad3 pathway, thus promoting muscle proteolysis mediated by Atrogin‐1 and MuRF‐1, canonical regulators of the ubiquitin‐proteasome pathway. ⁴⁹ In agreement with the above studies, Fabrizio et al. found that EVs from C26 tumour cells could recapitulate the phenotype of skeletal muscle wasting when injected into healthy animals. The mediator was supposed to be EV‐transferred sncRNAs including miRNAs and snoRNAs. ⁵⁰ He et al. also demonstrated the participation of the apoptosis signalling pathway triggered by EV‐transferred miR‐21 in muscle mass wasting. ⁵¹ MiR‐21 containing EVs derived from lung cancer and pancreatic cancer cells targets TLR7 receptors on murine progenitor myoblasts and stimulates their apoptosis regulated by c‐Jun N‐terminal kinase activity. ⁵¹ EVs isolated from the serum of pancreatic adenocarcinoma patients showed the same myoblast‐killing activity, proving that mechanisms of muscle wasting induced by EV‐associated miRNAs apply to humans as well. ⁵¹ Qiu et al. reported in another study that oral squamous cell carcinoma cells can transfer endoplasmic reticulum stress to muscle cells through EV containing miR‐181a‐3p both in vivo and in vitro. ⁵² They suggested that miR‐181a‐3p regulates Grp78 and thus activates the endoplasmic reticulum stress pathway, which induces muscle cell apoptosis and muscle wasting consequently. ⁵²

While most of the reported EV‐associated miRNAs play promotive roles in the process of muscle wasting, a few studies focus on the inhibitive functions of those miRNAs. For instance, miR‐145‐5p present in EVs of tonsil‐derived mesenchymal stem cells (T‐MSCs) was found to be able to rescue muscle wasting both in vitro and in vivo. ⁵³ MiR‐145‐5p can target two of the activin A receptors, ACVR2A and ACVR1B, therefore inhibiting the binding of activin A and its downstream processes, which regulate gene transcriptions of proteolytic enzymes. ⁵³ And in this way, EV‐transferred miR‐145‐5p can inhibit muscle proteolysis and rescue skeletal muscle wasting induced by activin A. ⁵³ In another study, Li et al. demonstrated that miR‐486‐5p internalized by bone marrow mesenchymal stem cell (BMSC)‐EVs attenuated muscle wasting caused by dexamethasone by downregulating the nuclear translocation of FoxO1, which is a potent regulator of muscle proteolysis, suggesting that EV‐associated miRNA can play a protective role in muscle wasting. ⁵⁴ These findings show that EV‐associated miRNAs may have positive or discouraging functions relating to skeletal muscle wasting, depending on their particular targets (Table 1). Based on these limited studies, it can be presumably surmised that EV‐associated miRNAs derived from cancer cells most likely play a pro‐wasting role while that derived from other cell types especially the mesenchymal stem cells could act an anti‐wasting role in skeletal muscle wasting. Therefore, it is critical to investigate the specific mechanism by which EV‐associated miRNAs induce phenotypical changes in skeletal muscle.

Table 1.

EV‐associated miRNAs in skeletal muscle wasting

miRNA in EV	EV sources	Target genes	Biological functions	References
miR‐122	Breast cancer cells	OGT	Increased RYR1 → calpain proteases activation→ muscle protein cleavage and myofibrillar destruction	46
miR‐195a‐5p and miR‐125b‐1‐3p	Colon cancer cells	Bcl‐2	activation of apoptosis signalling pathway	48
miR‐183‐5p	Colon cancer cells	FHL1	Activation of myostatin/Smad3 pathway → activation of ubiquitin‐proteasome pathway (Atrogin‐1 and MuRF‐1)	49
miR‐21	Lung cancer and pancreatic cancer cells	TLR7	Activation of apoptosis signalling pathway regulated by c‐Jun N‐terminal kinase	51
miR‐181a‐3p	Oral squamous cell carcinoma cells	Grp78	Activation of endoplasmic reticulum stress pathway → muscle cell apoptosis	52
miR‐145‐5p	Tonsil‐derived mesenchymal stem cells	ACVR2A and ACVR1B	Inhibition of activin A → inhibition of muscle proteolysis	53
miR‐486‐5p	Bone marrow mesenchymal stem cells	FoxO1	Inhibition of nuclear translocation of FoxO1 → inhibition of muscle proteolysis	54

Extracellular vesicle‐associated proteins in skeletal muscle wasting

The protein content of EVs makes up a large proportion of their total cargoes, and growing evidence indicates that EV‐associated proteins can also play a fundamental role in the modulation of skeletal muscle function. Proteins on the membrane of EVs can directly bind and activate their respective receptors on the recipient cells, triggering downstream signalling pathways relating to muscle wasting. For example, Zhang et al. reported that Hsp70 and Hsp90 loaded on the surface of tumour‐released EVs promote muscle wasting by triggering TLR4 on muscle cells. ⁵⁵ TLR4 activates the p38 MAPK, which subsequently activates both the ubiquitin‐proteasome pathway and the autophagy‐lysosome pathway as evidenced by the upregulation of E3 ligases atrogin1 and UBR2 and the autophagy marker LC3. ⁵⁵ Overall, EV‐associated Hsp70 and Hsp90 proteins induce muscle wasting by activating the TLR4‐p38‐MAPK signalling cascade. ⁵⁵ The function of EVs containing Hsp70 and Hsp90 has also been verified by another study, where these EVs result in muscle loss by activating p38 MAPK and inducing the upregulation of F‐box protein 32 and UBR2 in myotubes. ⁵⁶ Another study related to EV‐induced muscle loss indicates that TLR4 on muscle cells can be activated by other EV‐capsulated proteins as well. Li et al. demonstrated that EVs containing HMGB1 induce muscle wasting by activating TLR4/NF‐κB signalling pathway. ⁵⁷ EVs derived from C26 colon cancer cells enrich HMGB1, thus inducing proteolysis of C2C12 myotubes in vitro, while knockdown of HMGB1 in C26 cells produces HMGB1‐low expressed EVs, which alleviates muscle wasting in vivo. ⁵⁷ Mechanically, EV‐transferred HMGB1 binds to its receptor TLR4, which then activates the ubiquitin‐proteasome system through the NF‐κB signalling pathway. ⁵⁷ In accordance, they also identified one HMGB1 inhibitor named Glycyrrhizin which could be used as a potential drug to alleviate muscle wasting. ⁵⁷ Proteins enveloped by EVs can also be assimilated by recipient cells via non‐receptor‐mediated endocytosis, thus playing a role in the downstream intracellular pathways. Recently, Shin et al. show that ionizing radiation‐treated glioblastoma increases the delivery of plasminogen activator inhibitor‐1, known as PAI‐1, through EVs to the skeletal muscle and induces muscle wasting. ⁵⁸ They find that EV‐derived PAI‐1 protein can promote the phosphorylation of STAT3 and induce muscle wasting by upregulating the transcription of ubiquitin ligases such as MuRF1 and Atrogin‐1. ⁵⁸ And similar to the above study, they also propose an inhibitor named TM5441 as a promising drug to decrease muscle loss induced by PAI‐1. ⁵⁸

Another EV‐encapsulated protein known as growth differentiation factor 15 mediates muscle wasting by activating the apoptosis pathway. ⁵⁹ EVs produced by C26 colon tumour cells enrich growth differentiation factor 15, could induce muscle loss directly by activating the Bcl‐2/caspse‐3 pathway in either myotubes cells or in GA tissues from C26 tumour‐bearing mice. ⁵⁹ Additional TUNEL staining to detect apoptosis in GA tissues could make this study more conceivable. It is intriguing to point out that the same group also reported the same tumour‐derived EVs could induce muscle wasting by activation of the apoptosis pathway via EV‐associated miR‐195a‐5p and miR‐125b‐1‐3p. ⁴⁸ Thus, it is highly possible that different miRNAs and proteins can be encapsulated simultaneously into EVs and trigger the same downstream signalling pathway together. If that is the case, more attention should be paid to EV itself as a whole functional unit rather than to a single molecule inside of it. In addition to canonical proteins, some special proteins such as diverse growth factors and cytokines, can also be transported to skeletal muscle via EVs. For example, the pro‐inflammatory factor IL‐6 could be encapsulated into Lewis lung carcinoma cell‐derived EVs and induces protein degradation of C2C12 myotubes by stimulating the STAT3 signalling pathway. ⁶⁰ It has been well established that circulating IL‐6 has a significant role in the process of muscle wasting. ⁶¹ , ⁶² , ⁶³ The above study provides another possibility of IL‐6 transference and functioning. Transportation through EVs might protect this cytokine from degradation and facilitate targeting more precisely in its downstream tissues, such as the skeletal muscle. All the findings demonstrate that EV‐associated proteins play a critical role in the process of muscle wasting (Table 2). However, almost all those studies focused on the provocative role of EV proteins on muscle wasting, while the inhibitory regulatory mechanism of EV proteins on muscle wasting needs to be further investigated.

Table 2.

EV‐associated proteins in skeletal muscle wasting.

Protein in EV	EV sources	Target genes	Biological functions	References
Hsp70 and Hsp90	Lewis lung carcinoma cells	TLR4	Activation of p38‐MAPK pathway → activation of ubiquitin‐proteasome pathway (Atrogin‐1 and UBR2) and autophagy‐lysosome pathway (LC3)	55 , 56
HMGB1	Colon cancer cells	TLR4	Activation of NF‐κB signalling pathway → activation of ubiquitin‐proteasome pathway (Atrogin‐1 and MuRF‐1)	57
PAI‐1	Glioblastoma cells	STAT3	Phosphorylation of STAT3 → activation of ubiquitin‐proteasome pathway (Atrogin‐1 and MuRF‐1)	58
GDF‐15	Colon cancer cells	Bcl‐2	Activation of apoptosis signalling pathway	59
IL‐6	Lewis lung carcinoma cells	STAT3	Activation of ubiquitin‐proteasome pathway (Atrogin‐1 and FOXO3)	60

Therapeutic implications of extracellular vesicles in skeletal muscle wasting

With the advancement of the above fundamental studies, researchers have also made much progress in the therapeutic implications of EVs relating to skeletal muscle wasting (Figure 2).

Figure 2.

Therapeutic applications of EVs in skeletal muscle wasting. (A) Cargoes of the pro‐wasting EVs are utilized as diagnostic biomarkers. (B) Exogenous EVs bearing anti‐wasting biomolecules in the treatment of skeletal muscle wasting. (C) Engineered EVs function as anti‐wasting drug delivery vehicles. (D) Chemicals to block EV biogenesis can be exploited as potential therapeutic drugs by inhibiting the production of pro‐wasting EVs. IL‐6ST, Interleukin‐6 Signal Transducer; sActRIIB, soluble Activin Receptor type‐2B; Hsp, Heat shock protein.

Most studies exploit the therapeutic potentiality of stem cell‐derived EVs, especially those from mesenchymal stem cells. Engineered MSC‐derived EVs which express IL6 signal transducer (IL‐6ST) decoy receptors on the EV membrane can specifically inhibit the activation of the IL‐6 trans‐signalling pathway, thus effectively decreasing the STAT3 phosphorylation level in C2C12 myoblasts and myotubes. ⁶⁴ Moreover, subcutaneous administration of IL‐6ST‐EVs in mice can also reduce the phosphorylation of STAT3 in multiple skeletal muscle tissues. ⁶⁴ As IL‐6 is a significant mediator of skeletal muscle loss, ⁶⁰ , ⁶¹ , ⁶² , ⁶³ , ⁶⁵ the inhibition of IL‐6 through IL‐6ST decoy receptor loaded by EVs demonstrates great therapeutic potential. Besides the IL‐6 decoy receptor, another promising drug named physiactisome for treating muscle wasting has been proposed recently. ⁶⁶ The functional molecules are actually Hsp60 contained by EVs derived from Hsp60‐overexpressing C2C12 cell lines. Treatment with Hsp60 containing EVs could induce the expression of PGC‐1α in naïve C2C12 cells, thus promoting mitochondria biogenesis and counteracting muscle wasting. ⁶⁶ It was said that physiactisome is the first and only patented EV‐based drug for the treatment of muscle wasting. ⁶⁶ In addition to EV‐encapsulated proteins, proteins loaded on the surface of EVs can also present therapeutic potential. For example, EV‐mediated delivery of myostatin propeptide anchored on the surface of EVs derived from murine fibroblast showed augmented inhibitory ability to the circulating serum mature myostatin and alleviated muscle wasting. ⁶⁷ Myostatin is a critical growth factor that negatively regulates skeletal muscle mass; thus, targeting the myostatin signalling pathway represents a promising approach to alleviate muscle wasting. ⁶⁸ By inserting the inhibitory domain of propeptide to the second extracellular loop of CD63 (a canonical EV marker), myostatin propeptide could be loaded on the surface of EVs and functions as a potent blockader of serum myostatin after intravenous injection into adult mice, and the inhibition of mature myostatin by propeptide results in improved skeletal muscle function. ⁶⁷ However, one phase II trial using an anti‐myostatin antibody LY2495655 in patients with pancreatic cancer did not get improvements related to muscle wasting, with possible reasons being that this drug did not inhibit other ligands of myostatin receptor such as activin A and that the circulating levels of myostatin in these patients were already lower than that of initial disease conditions so that anti‐myostatin strategy may be more effectual at preventing muscle wasting than reversing it. ⁶⁸ , ⁶⁹

As miRNAs also play a critical role in the process of muscle wasting, numerous studies have focused on the therapeutic implications of EV‐encapsulated miRNAs. For instance, intramuscular injection of EV‐encapsulated miR‐29 derived from muscle satellite cells could attenuate muscle loss in mice by repressing the upregulation of muscle proteolytic proteins TRIM63/MuRF1 and FBXO32/atrogin‐1. ⁷⁰ The same group also reported another EV‐encapsulated miRNA (miR‐26a) derived from muscle satellite cells that could limit skeletal muscle loss in mice by targeting FoxO1. ⁷¹ It is worth noting that they construct a vector by fusing EV membrane protein Lamp2b with muscular surface targeting peptide SKTFNTHPQSTP to increase the muscle targeting ability of miR‐26a containing EVs. ⁷¹ Furthermore, another study demonstrates that miR‐26a containing EVs derived from HEK293 cells could also prevent muscle wasting by inhibiting FoxO1. ⁷² Besides transferring biomolecules, EVs are ideal carriers of drugs due to their biocompatible property and organotrophic ability. ⁷³ , ⁷⁴ , ⁷⁵ However, there are currently few drugs available to counteract muscle wasting. We previously reported an angiotensin II receptor antagonist named losartan could enhance the chemotherapy efficacy of ovarian cancer. ⁷⁶ And it was found that skeletal muscle wasting could also be induced by angiotensin II, so the FDA‐approved drug losartan could be used to provide protection against muscle wasting. ⁷⁷ , ⁷⁸ Carnosol, an active component of the herbal medicine rosemary, has the ability to attenuate muscle loss induced by EVs. ⁴⁹ Glycyrrhizin, an inhibitor of HMGB1, can be used as a potential drug to alleviate muscle wasting. ⁵⁷ Similarly, a myostatin receptor ActRIIB antagonism sActRIIB has been reported to be able to ameliorate muscle wasting. ⁷⁹ And another agonist of the ghrelin receptor named anamorelin has entered a phase 3 trial for the treatment of muscle loss associated with non‐small‐cell lung cancer. ⁸⁰ Whether these medicines can be loaded by EVs and enable their increased delivery and therapeutic efficacy requires further investigation.

Given that EVs play a significant provocative role in most cases during the process of muscle wasting, inhibiting EV release might represent another therapeutic strategy for treating muscle wasting. For example, serving as an old drug for the treatment of hypertension, amiloride has been found to be able to inhibit EV release from cells. ⁸¹ , ⁸² More importantly, it is found that amiloride treatment could prevent skeletal muscle loss mechanistically by blocking tumour‐derived EV release and thus significantly downregulating the levels of Atrogin‐1 and MuRF‐1 in skeletal muscle. ⁸³ Based on the same mechanism as amiloride, other EV inhibitory compounds such as GW4869 ⁸⁴ , ⁸⁵ or simvastatins ⁸⁶ could also be investigated and exploited as potential therapeutic drugs for ameliorating skeletal muscle wasting.

Conclusions and perspectives

Skeletal muscle wasting definitely affects the quality of life as it contributes to muscle weakness and reduced mobility of patients. Various molecular mechanisms and distinct factors mediating muscle wasting have been investigated in the past few decades. And multiple studies have identified that EVs play a pivotal role in the progression of muscle wasting. Much attention was paid to the EV‐encapsulated miRNAs and proteins. Indeed, different types of miRNAs and proteins enveloped by EVs can be transferred to skeletal muscle and induce pro‐wasting or anti‐wasting effects. In most cases, EV‐associated miRNAs and proteins act as a pro‐wasting role in the development of muscle loss by activating the ubiquitin‐proteasome or autophagy‐lysosome pathways upon assimilation by muscle cells. And these miRNAs and proteins can function separately or collectively depending on their donor cells and targeting genes. While most related studies focus on the function of EV‐associated miRNAs and proteins, the role of other EV cargoes such as other types of sncRNAs, lipids and metabolites remains to be unfolded. In addition, current researches mainly focus on tumour‐derived EVs in the crosstalk between tumour and skeletal muscle. More studies are required to unravel the role of EVs in muscle wasting associated with other diseases such as organ failure, sepsis and sarcopenia. Further clarification of the role of EV cargoes in the process of muscle wasting will facilitate the progress of enabling EVs as diagnostic biomarkers. EV‐based liquid biopsies will contribute to the early detection of disease, thereby initiating intervention therapies as early as possible in the development of muscle wasting. Likewise, improved knowledge concerning the function of EVs in muscle wasting will also promote the development of novel treatment strategies. The protective lipid bilayer membrane and the intrinsic ability of tissue targeting as well as biocompatibility, enable EVs to be utilized as ideal drug carriers and delivery systems. Plenty of studies have proven that exogenous EVs bearing biomolecules and EV‐based drug delivery have the potential to attenuate muscle wasting. However, most of these studies are pre‐clinical. Methodology aiming at improving the productivity and therapeutic efficacy of exogenous EVs requires further investigations. Developing inhibitory chemicals targeting EV‐associated pro‐wasting cargoes or even EV biogenesis itself is also a promising strategy for counteracting EV‐induced muscle wasting. Please see the outstanding questions below.

Outstanding questions:

1. What is the function of other types of EV cargoes, such as mRNAs, sncRNAs and metabolites, in skeletal muscle wasting?

2. Whether and how EV cargoes function in other muscle‐related diseases besides cancer cachexia?

3. Are there any EV biomarkers to direct them assimilated by skeletal muscle rather than other organs?

4. Is there any specific biomarker to dissect skeletal or cardiac muscle‐derived EV?

5. What are the major characteristics or membrane markers for pro‐wasting and anti‐wasting EVs?

6. How to improve the productivity and therapeutic efficacy of anti‐wasting EVs, and EV‐based anti‐wasting drug delivery systems for better engineering EVs?

Funding

This research related to extracellular vesicles and skeletal muscle was funded by National Natural Science Foundation of China to Wei Yan, grant numbers 32270827 and 82203590.

Conflicts of interest

The authors declare no conflict of interest.

Zhang X, Zhao Y, Yan W. 2023; The role of extracellular vesicles in skeletal muscle wasting. Journal of Cachexia, Sarcopenia and Muscle, 14, 2462–2472, 10.1002/jcsm.13364