Epigenetic control of skeletal muscle atrophy

Abstract

Skeletal muscular atrophy is a complex disease involving a large number of gene expression regulatory networks and various biological processes. Despite extensive research on this topic, its underlying mechanisms remain elusive, and effective therapeutic approaches are yet to be established. Recent studies have shown that epigenetics play an important role in regulating skeletal muscle atrophy, influencing the expression of numerous genes associated with this condition through the addition or removal of certain chemical modifications at the molecular level. This review article comprehensively summarizes the different types of modifications to DNA, histones, RNA, and their known regulators. We also discuss how epigenetic modifications change during the process of skeletal muscle atrophy, the molecular mechanisms by which epigenetic regulatory proteins control skeletal muscle atrophy, and assess their translational potential. The role of epigenetics on muscle stem cells is also highlighted. In addition, we propose that alternative splicing interacts with epigenetic mechanisms to regulate skeletal muscle mass, offering a novel perspective that enhances our understanding of epigenetic inheritance’s role and the regulatory network governing skeletal muscle atrophy. Collectively, advancements in the understanding of epigenetic mechanisms provide invaluable insights into the study of skeletal muscle atrophy. Moreover, this knowledge paves the way for identifying new avenues for the development of more effective therapeutic strategies and pharmaceutical interventions.

Introduction

Skeletal muscle, constituting approximately 40–50% of the total body mass in a healthy individual, is the most abundant tissue in the human body. It is indispensable for movement, heat generation, and metabolic equilibrium. Beyond these functions, skeletal muscle acts as a critical regulator of the body’s protein reserves, glucose, and lipid homeostasis [1, 2]. Skeletal muscle atrophy, a condition characterized by a decrease in muscle mass and strength, can result from a variety of factors, including genetic mutations [e.g., Duchenne muscular dystrophy (DMD), amyotrophic lateral sclerosis (ALS), and spinal muscular atrophy (SMA)], denervation, disuse, cancer cachexia, weightlessness, aging, and chronic diseases such as diabetes [3,4,5,6,7,8]. This condition not only impairs an individual’s mobility but also significantly affects their quality of life, imposing a considerable economic and social burden. Histologically, skeletal muscle atrophy is marked by a reduction in muscle fiber cross-sectional area, disintegration of myofilament structures, degradation of myogenic fibers, mitochondrial dysfunction, and increased mitochondrial autophagy. Despite the vital role of skeletal muscle and the severe consequences of its atrophy, it remains one of the least effectively treated tissues [3, 4]. Furthermore, skeletal muscle exhibits secretory functions; following exercise or injury, it releases cytokines into the bloodstream, influencing systemic health [9]. Given these facts, the need for comprehensive research into the pathogenesis and therapeutic approaches for skeletal muscle atrophy is both urgent and significant.

Under normal physiological conditions, skeletal muscle anabolic and catabolic metabolism are in dynamic balance and work together to maintain muscle quality. Skeletal muscle atrophy occurs when this balance is disrupted, specifically when catabolic processes predominate, leading to greater protein breakdown than synthesis. The principal pathways involved in this degradation are the ubiquitin–proteasome pathway, the calpain system, and the autophagy-lysosome pathway. A significant indicator of muscle atrophy is the overactivation of the ubiquitin–proteasome degradation system, highlighted by the upregulation of E3 ubiquitin ligases such as muscle atrophy F-box (MAFbx, also known as Atrogin-1), Muscle RING Finger-1(MuRF1, also known as Trim63), and TNF receptor associated factor 6 (Traf6) [10,11,12], which can be triggered by factors such as proinflammatory cytokines, oxidative stress, and fluctuations in energy levels that then stimulate the ubiquitin–proteasome proteolytic system through various downstream signaling cascades. This process begins with the ubiquitin-activating enzyme E1, which attaches ubiquitin to ATP, forming an activated ubiquitin molecule. The E2 ubiquitin-conjugating enzyme then transfers this activated ubiquitin to the E3 ubiquitin ligase, which in turn links the ubiquitin to the target protein, marking it for degradation. The complex nature and not fully understood mechanisms underlying skeletal muscle atrophy have hindered the development of effective pharmacological interventions.

Skeletal muscle is wrapped externally by the epimysium, and one layer inward is the perimysium, which surrounds the fascicles and blood vessels. Below the perimysium, satellite cells (SCs) are distributed between the basal lamina and the endomysium. The endomysium wraps around the muscle fibers and contains myofibers within each muscle fiber, which are made up of sarcomeres (Fig. 1). Individual muscle fibers constitute the smallest functional units of skeletal muscle. Based on the expression of myosin heavy chain (MyHC) isozymes, human skeletal muscle fibers are categorized into three types: type I, type IIa, and type IIx. Additionally, rodent skeletal muscles include type IIb fibers. Type I fibers, known as slow-twitch fibers, contract slowly and predominantly utilize oxidative phosphorylation for energy. In contrast, type IIb and IIx fibers, referred to as fast-twitch fibers, contract rapidly and primarily generate energy through the glycolytic pathway [13]. There are fibers that express multiple MyHC isoforms, called hybrid fibers. Hybrid fibers are common in humans and have variable proportions of MyHC isoforms that correspond to the high degree of muscle plasticity. Although both fast- and slow-twitch muscle fibers can undergo atrophy, the sensitivity to atrophy varies between these fiber types. Slow-twitch fibers are more prone to wasting and denervation-induced atrophy, while fast-twitch fibers are particularly vulnerable to atrophy associated with conditions such as cancer cachexia, diabetes, and aging [14,15,16]. The differential susceptibility of muscle fiber types to various forms of atrophy has been a subject of long-standing investigation. Despite extensive research, the specific atrophy signaling pathways that mediate these differences between fast and slow muscles remain debatable.

Fig. 1

Organization of skeletal muscle. The epimysium surrounds the entire skeletal muscle. The next inner layer is the perimysium, which surrounds the fascicles and blood vessels. Below the perimysium, between the basal lamina and the endomysium, satellite cells are distributed. The endomysium surrounds individual myofibers, each of which contains myofibrils. These myofibrils are made up of sarcomeres. This figure was created with https://www.BioRender.com

As a resident stem cell, the fate of SCs is critical for skeletal muscle injury repair. Under homeostatic conditions, SCs remain quiescent for extended periods of time. Upon receiving activation signals, SCs promptly exit their dormant state and commence cell cycle progression to proliferate. The proliferating SCs subsequently undergo differentiation to repair damaged tissue, while a subset of SCs reenters quiescence following self-renewal, maintaining a stable stem cell population [17]. This dynamic transition between states underpins the plasticity of skeletal muscle and constitutes a crucial mechanism for triggering regeneration following muscle atrophy. The transcription factor Paired-box 7 (Pax7) plays a pivotal role in sustaining SC quiescence and is instrumental in regulating both self-renewal and proliferation [18]. The myogenic regulatory factors (MRFs) family of transcription factors, including Myogenic Differentiation 1 (MyoD or MyoD1), Myogenin (Myog), Myogenic Factor 5 (Myf5), and Myogenic Regulatory Factor 4 (MRF4, also known as Myf6), act as molecular switches that determine the fate of SCs and skeletal muscle phenotype establishment [19, 20]. Discovering the drivers of MRF expression and elucidating the molecular mechanisms could help to understand the skeletal muscle atrophy process.

Increasing evidence indicates that epigenetic inheritance plays an important role in determining muscle stem cell fate and influencing the progression of skeletal muscle atrophy. Epigenetics involves the regulation of gene expression via chemical modifications to histones, DNA, or RNA, without altering the underlying gene sequence, thereby impacting the phenotype [21, 22]. The establishment and removal of these chemical marks are regulated by various epigenetic regulatory proteins, categorized as readers, writers, and erasers. Although genetic studies on various epigenetic regulatory proteins have affirmed their significance in maintaining skeletal muscle quality, comprehensive investigations into the underlying mechanisms remain sparse [23, 24]. Recent advancements in high-throughput sequencing technologies have propelled the field of epigenetics into detailed molecular investigations through approaches such as epigenomics, epitranscriptomics, proteomics, and single-cell epitranscriptomics. A recent study has demonstrated alterations in chromatin accessibility during skeletal muscle atrophy at the resolution of individual muscle fibers, offering novel insights into the epigenetic regulation of skeletal muscle atrophy [25].

Here, we summarize and provide a mechanistic perspective gained from recent research on the epigenetic control of skeletal muscle atrophy. First, we outline in turn the different types of modifications of DNA, histones, and RNA underpinning the molecular basis of skeletal muscle atrophy. Their dynamics during skeletal muscle atrophy, their role in skeletal muscle atrophy, and their role in satellite cells are of major interest. We discuss the molecular mechanisms of epigenetic regulation of skeletal muscle atrophy at the transcriptional, post-transcriptional, and post-translational levels and summarize the key downstream molecules. In addition, the co-regulation of skeletal muscle atrophy by alternative splicing and epigenetic crosslinking is an interesting new mechanism. Finally, we discuss neglected but important directions in current study and the therapeutic potential of epigenetic drugs for skeletal muscle atrophy.

The role of epigenetic in skeletal muscle atrophy

Epigenetic and epitranscriptomic modifications play crucial roles in the regulation of gene expression across transcriptional, post-transcriptional, and translational stages. These modifications, which include the addition or removal of molecular marks such as DNA methylation, histone modifications (e.g., acetylation, methylation), and RNA modifications (e.g., methylation, splicing), can either activate or inhibit gene expression, influencing cellular states and behaviors. In the context of disease, these epigenetic changes can persist through cell division, leading to long-lasting impacts on cellular functions and characteristics. Understanding epigenetic mechanisms provides valuable insights into the regulation of genes associated with muscle atrophy and the adaptative responses of muscle cells to functional demands. This knowledge opens new perspectives for exploring the origins and progression of skeletal muscle atrophy, offering novel targets for therapeutic intervention and management.

DNA modifications

DNA modifications and regulators

Chemical modifications of DNA bases were discovered as early as 1948, with DNA methylation being the first to be recognized [26]. Various forms of DNA methylation exist, such as 5-methylcytosine (5mC), N⁶-methyladenine (6 mA), N⁴-methylcytosine (4mC), and 5-hydroxymethylcytosine (5hmC) [27]. In prokaryotes and lower eukaryotes, 6mA methylation prevails, whereas 4mC methylation is found in certain bacteria. 5hmC is generally considered a reactive intermediate in the process of DNA demethylation and has been shown to play a pivotal role in this process. In mammals, 5mC constitutes the predominant form of DNA methylation.

DNA methylation is catalyzed by a family of DNA methyltransferases (DNMTs). In this process, S-adenosylmethionine (SAM) acts as a methyl donor, transferring the methyl group to the fifth carbon atom of cytosine through a covalent bond [28, 29]. The DNMT family in mammals comprises three main members: DNMT1, DNMT2, and DNMT3 (Table 1). DNMT1 acts as a maintenance methyltransferase, preserving DNA methylation patterns during DNA replication. DNMT2, despite possessing methyltransferase capabilities, is unique in that it targets tRNA rather than DNA owing to the absence of an N-terminal regulatory region, thus not contributing to DNA methylation [30]. DNMT3A and DNMT3B are key players in the de novo methylation of DNA, targeting unmethylated DNA on both strands [31, 32] (Table 1). These enzymes also play roles in maintaining methylation patterns and methylating repetitive sequences [33]. DNMT3C, a rodent-specific duplicate of DNMT3B, shares structural similarities with DNMT3A and DNMT3B but lacks the PWWP domain in its N-terminal regulatory region, which is essential for chromatin binding. The primary role of DNMT3C is to methylate and, thus, inhibit the activation of evolutionarily recent retrotransposons during sperm development, safeguarding genomic integrity [34].

Table 1 List of DNA modification regulators

DNA methylation and demethylation processes work together to maintain a dynamic balance in cells, ensuring genome stability. The demethylation of 5-methylcytosine (5mC) involves the action of the ten-eleven translocation (TET) family of enzymes, which includes TET1, TET2, and TET3 (Table 1). These enzymes catalyze the oxidation of 5mC into three intermediate forms: 5hmC, 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). Following this, the intermediate products undergo a thymine DNA glycosylase (TDG)-mediated base excision repair process, leading to the conversion of 5mC back to unmodified cytosine (C)[35].

DNA methylation dynamic in skeletal muscle atrophy

Skeletal muscle tissue or cells originating from skeletal muscle display distinct methylation profiles compared with other tissues [36, 37]. These muscle-specific methylation patterns highlight a notable feature: genes coding for proteins predominantly found in skeletal muscle—such as OBSCN, MYOT, and MYH7, which play important roles in sarcomere organization—are found to be hypomethylated in skeletal muscle [38,39,40]. This hypomethylation of genes encoding dominant muscle proteins suggests a reduced susceptibility to regulatory mechanisms, thereby promoting their consistent expression essential for the fundamental functions of skeletal muscle, including contraction. Nonetheless, the relationship between these methylation patterns and muscle atrophy remains to be elucidated.

As we age, skeletal muscles atrophy and lose strength and function, leading to sarcopenia. A genome-wide analysis on methylation patterns showed an increase in methylation within the skeletal muscles of older people and in rodent models [41,42,43]. One gene significantly affected by this methylation process is tubulin folding cofactor D (TBCD), which plays a crucial role in microtubule assembly and is observed predominantly in samples from older humans [41]. Interestingly, most of these methylation sites are found within the gene’s body, not in the promoter regions, correlating less with gene expression levels. Similar methylation alterations have been identified in the skeletal muscles of aged rats, aligning with human findings. Notably, there is a reported increase in Dnmt3b expression in these cases [44]. A comprehensive meta-analysis conducted by Sarah Voisin et al. examined age-related DNA methylation changes in human skeletal muscle, pooling data from ten separate studies. This analysis uncovered 6710 distinct methylation regions across 6367 unique genes. The findings indicated a significant increase in DNA methylation at polysome target genes and bivalent chromatin domains, whereas methylation at enhancer regions was significantly higher [45]. To facilitate broader access and analysis of these findings, the research team has introduced MetaMeth, a web-based tool designed for user-friendly interaction and efficient data sharing (https://sarah-voisin.shinyapps.io/MetaMeth/).

Progressive weight-bearing exercise has been shown to partially reverse the hypermethylation observed in aged skeletal muscle [46]. Notably, the changes in methylation and demethylation patterns following exercise are more significant in elderly individuals than in younger counterparts, suggesting that the benefits of exercise for skeletal muscle health and function may be more pronounced in older adults [47].

Compared with aging skeletal muscles, there has been limited systematic analysis on DNA methylation in other models of muscle atrophy. The question of whether skeletal muscle atrophy induced by different factors shares DNA methylation patterns remains open. Fisher et al. developed a denervated muscle atrophy model in rats by injecting tetrodotoxin (TTX) into the peripheral nerves [48] and used real-time quantitative PCR and pyrosequencing to investigate DNA methylation in the promoter regions of several atrophy-related genes (atrogenes) and discovered that Myog, MuRF1, MAFbx, and Chrna1 exhibited demethylation at critical points of denervated muscle atrophy, corresponding to increased gene expression. Upon cessation of TTX treatment and recovery of the skeletal muscles, the methylation status of these genes was significantly reversed, coinciding with reduced gene expression. The findings of Fisher et al. indicate that DNA methylation changes play a crucial role in atrogene activation. Additionally, muscle atrophy resulting from prolonged bed rest was examined by Lisa Van Dyck et al. in a study involving critically ill adult patients in the intensive care unit [49]. They identified two regions of hypomethylation associated with muscle regeneration and postsynaptic acetylcholine receptors in the patients.

The roles of DNA methylation regulators in skeletal muscle atrophy

In mouse models of denervation, aging, and disuse-induced muscle atrophy, a significant downregulation of Dnmt3a has been observed, while the levels of the other two methyltransferases, Dnmt1 and Dnmt3b, remain relatively unchanged [50]. This finding contrasts with the observed hypermethylation in aged skeletal muscle, implying the involvement of alternative regulators of DNA methylation or different mechanisms behind the increase in methylation [51]. Notably, Dnmt3a knockout (KO) mice demonstrated a substantial reduction in muscle regeneration capability following injury, with SCs appearing smaller (in terms of length and area) and exhibiting inhibited myotube differentiation [50]. SCs from mice with a conditional KO of Dnmt3a, driven by the Pax3 promoter (specific to muscle SCs), also displayed a marked decline in proliferation when cultured [52]. At the molecular level, Dnmt3a was shown to enhance the expression of growth differentiation factor 5 (Gdf5) and p57Kip2, a member of the Cip/Kip family of cyclin-dependent kinase inhibitors, through DNA methylation-dependent pathways [50, 52]. Intriguingly, compared with mice with a complete KO of Dnmt3a (induced by Cre recombinase under the human α-actin promoter), those with a muscle satellite cell-specific deletion of Dnmt3a exhibited more severe muscle mass loss. These findings collectively suggest that Dnmt3a-mediated DNA methylation changes play a crucial role not only in the proliferation and differentiation of muscle SCs but also in influencing various aspects of skeletal muscle atrophy, potentially impacting other cell types as well.

Beyond influencing genes involved in proliferation and differentiation, Dnmt3a directly modulates the transcription of genes associated with muscle atrophy. The receptor for TNF-like weak inducer of apoptosis (TWEAK), fibroblast growth factor-inducible receptor 14 (Fn14), experiences a significant upregulation during denervation-induced muscle atrophy. This upregulation activates the transcription factor NF-κB, leading to increased expression of MuRF1 [53] (Fig. 2 and Table 3). Subsequent research has revealed that denervation-induced muscle atrophy results in the hypomethylation of specific CpG sites within the Fn14 promoter. Overexpression of Dnmt3a leads to increased methylation at these sites, suppression of Fn14 expression, and mitigation of denervation-induced muscle atrophy [54]. Moreover, the reduction in Dnmt3a expression observed during denervation-induced atrophy aligns with the hypomethylation of various atrogenes [48]. Consequently, it is suggested that Dnmt3a could be a key regulator in the upregulation of atrophy gene expression across different models of skeletal muscle atrophy. This hypothesis was examined in a study utilizing a diabetes mellitus (DM) mouse model of skeletal muscle atrophy. The study demonstrated that continuous administration of Dnmt3a to the tibialis anterior muscle significantly counteracted streptozotocin (STZ)-induced DM muscle atrophy and enhanced myotube formation in C2C12 myoblasts. Crucially, Dnmt3a overexpression also reinstated the expression levels of atrophy genes such as MAFbx, MuRF1, MyoD, and Myog, both in vivo and in vitro [55].

Fig. 2

Epigenetic control of skeletal muscle atrophy. Epigenetic modifications regulate the expression of transcription factors such as FoxO3 at the transcriptional, post-transcriptional, and post-translational levels, which in turn activate the E3 ubiquitin ligases MuRF1 and MAFbx, ultimately leading to skeletal muscle atrophy

The expression patterns of DNA methylation writers exhibit contrasting trends in skeletal muscle atrophy models driven by genetic factors versus those induced by denervation and aging. In ALS mice, both 5mC levels and the expression of Dnmt1 and Dnmt3a were found to be increased within the spinal cord and skeletal muscle [56]. Notably, the use of DNMT inhibitors has been demonstrated to significantly alleviate muscle wasting symptoms and enhance motor functions, including body weight and grip strength, in mice suffering from inherited forms of amyotrophy [56, 57]. Further investigations across various animal models of ALS have revealed that Dnmt3a is localized to the outer mitochondrial membrane in muscle fibers. A decrease in mitochondrial Dnmt3a expression, alongside an increase in 5mC levels, is associated with mitochondrial loss in skeletal muscle [58]. These findings underscore the dynamic changes in DNA methylation during muscle atrophy due to different causative factors, highlighting the necessity for distinct studies on the expression of methylation writers.

While the expression of Dnmt1 and Dnmt3b remains relatively stable during muscle atrophy, implying a lack of significant change, this steadiness should not be interpreted as an absence of functionality [59]. They are implicated in important processes such as the differentiation of skeletal muscle stem cells and the switching between muscle types. There is a pressing need for further research to elucidate their specific roles and underlying mechanisms associated with skeletal muscle atrophy.

Contradictory reports exist regarding the viability of Tet1 KO mice. While some studies report that Tet1 KO results in embryonic lethality [60], others have observed that these mice are viable and fertile, though a proportion of the mutant offspring may exhibit reduced size [61, 62]. Gene expression analysis has identified hypermethylation and altered expression in the promoter regions of genes crucial for skeletal muscle development and contraction. Tet2 KO mice demonstrate a muscle atrophy phenotype, characterized by a marked decrease in body weight and a reduction in both size and cross-sectional area of the tibialis anterior muscle fibers at ages 2 and 8 weeks [63]. Research by Wang Hongye et al. revealed that Tet2 deficiency compromises the regenerative ability and myotube fusion in skeletal muscle [64]. Strategies aimed at enhancing Tet2 stability or ensuring its elevated expression have been shown to reduce the effects of denervation-induced muscle atrophy and improve skeletal muscle function in the context of obesity [65, 66]. Further investigation has shown that Tet2 deficiency disrupts the proliferation and differentiation of SCs, a process mediated by the calcium signaling gene Slc8a3 and Myog, a critical regulator of myogenesis [63, 64].

In differentiated myotubes cultured in vitro, an increase in the expression of Tet1 and Tet2 has been observed, accompanied by a significantly higher level of 5hmC compared with that in undifferentiated C2C12 myoblasts. The reduction of Tet2 through knockdown experiments has been shown to alter the methylation status and expression of genes critical to myogenesis, thereby impairing the differentiation process of myoblasts [67]. Further investigations have revealed that the differentiation of C2C12 cells is influenced by the phosphorylation of Tet2, a process regulated by AMPK [68]. However, the question of whether the phosphorylation of Tet2 is essential for the proliferation and differentiation of muscle SCs in vivo remains unanswered.

During the regeneration process following muscle atrophy, the expression levels of Tet1, Tet2, and Tet3 are all elevated. However, only the suppression of Tet2 expression significantly impacts myoblast differentiation [64]. This observation indicates that, despite their shared function as deacetylases, each Tet enzyme plays distinct roles in the context of skeletal muscle atrophy and regeneration. Supporting this notion, the knockdown of the methyltransferase Dnmt3a and the demethylase Tet2 exhibits similar effects on muscle SCs. Therefore, DNA methylation regulators can influence skeletal muscle atrophy not through broad changes in genomic methylation but rather through specific regulatory networks and targets, indicating the need for further investigation into their precise mechanisms of action.

Histone modifications

Histone modifications and regulators

DNA is wrapped around an octamer consisting of four core histones: H2A, H2B, H3, and H4. Modifications to these histones are critical in modulating chromatin accessibility, which in turn influences the initiation or repression of gene expression. A vast array of histone post-translational modifications (PTMs) has been identified, encompassing acylation (such as acetylation, benzoylation, butyrylation, crotonylation, glutarylation, lactylation), ADP-ribosylation, dopamine acylation, glycosylation, methylation, phosphorylation, serotonylation, sumoylation, and ubiquitination [69,70,71,72,73,74]. These modifications, located on the histone tails and within their globular core domains, play direct or indirect roles in the regulation of gene expression, DNA replication, and various other cellular functions by altering the structure of chromatin and the dynamics of protein interactions [75].

Histone acetylation was the first discovered form of histone acyl modification, and it is catalyzed by histone acetyltransferases (HATs). These enzymes catalyze the transfer of acetyl groups from acetyl coenzyme A to histones, forming ε-N-acetyl lysine. This addition of acetyl groups reduces the positive charge on histone proteins, weakening the interaction between histones and DNA and consequently leading to the relaxation of chromosomal DNA [76]. The process of histone acetylation enhances DNA accessibility, thereby facilitating the binding of transcription factors and RNA polymerases to specific regions of the chromatin. This facilitates the initiation of transcription, significantly altering chromatin structure and gene expression [77]. HATs are categorized into three main families: the Gcn5-associated N-acetyltransferase (GNAT) superfamily, which includes GCN5/KAT2A, PCAF/KAT2B, Ada, and SGAG; the p300/CBP family, which encompasses p300/KAT3B, and CBP/KAT3A; and the MYST family, which consists of members such as Tip60/KAT5, MOZ/KAT6A, MORF/KAT6B, HBO1/KAT7, MOF/KAT8, SAS2, and SAS3 [78, 79] (Table 2). Moreover, certain transcription factors, notably TFIIIC (a universal transcription factor for RNA polymerase III) and CLOCK (an epigenetic regulator of circadian rhythms in skeletal muscle) are recognized for their roles in histone acetylation [80, 81].

Table 2 List of histone modification regulators

Histone deacetylase (HDAC)-mediated deacetylation of histones results in the formation of closed chromatin structures. HDACs remove acetyl groups from histone proteins, thereby restoring their positive charge and enhancing the interaction between histones and DNA. Consequently, the chromatin structure becomes more condensed, which restricts DNA accessibility [77]. Therefore, HDACs are predominantly associated with the repression of transcription [82]. Eighteen HDACs have been identified in mammals and are categorized into four classes: Class I HDACs encompass HDACs 1, 2, 3, and 8; Class II HDACs are further divided into Class IIa, comprising HDACs 4, 5, 7, and 9, and Class IIb, including HDACs 6 and 10; Class III consists of the sirtuin family members SIRT1, 2, 3, 4, 5, 6, and 7; and Class IV is represented by HDAC11 (Table 2).

Histone methylation is a reversible reaction catalyzed by histone methyltransferase (HMT) and histone demethylase (HDM). Typical histone lysine methylation mainly occurs at sites such as H3K4, H3K9, H3K27, H3K36, H3K79, and H4K20, while the common sites for arginine methylation include H3R2, H3R8, H3R17, H3R26, H2AR3, H4R3, etc.[83]. Different lysine sites can generally undergo monomethylation (me1), dimethylation (me2), and trimethylation (me3) modifications. The process of histone methylation is dependent on specific enzymes that are known as histone methylases, which can be classified as histone lysine methylases and histone arginine methylases, depending on the site at which the modification occurs. Histone lysine methyltransferases (KMTs) are further divided into two categories: enzymes containing a SET domain and proteins without a SET domain, such as DOT1L [84]. Currently, more than 50 proteins containing SET structural domains have been identified in mammals, mainly including lysine methylation enzymes such as the SUV39 family, the mixed-lineage leukemia (MLL) family, the enhancer of zeste homolog (EZH) family, the SET and MYND domain-containing (SMYD) family, and the PRDM family [85, 86] (Table 2). It was found that methylation modifications on histones H3K4, H3K36 and H3K79 are enriched on euchromatin and positively correlate with gene expression, while methylation modifications on H3K9, H3K27, and H4K20 are negatively correlated with gene expression and are usually enriched on heterochromatin or facultative heterochromatin [87].

Histone demethylation (HDMs) involves the removal of methyl groups from histone proteins, converting methylated arginine or lysine residues back to their unmethylated form. This process is facilitated by various mechanisms involving distinct enzyme systems and regulatory pathways. HDMs are classified into two primary categories: the lysine-specific demethylase 1 (LSD1) family of demethylases, which resemble polyamine or monoamine oxidases, utilize flavin adenine dinucleotide (FAD) as a cofactor to demethylate mono- and dimethylated lysines, and the dioxygenase class, which resembles hydroxylases, contains the Jumonji C (JMJC) domain, and uses divalent iron ions and α-ketoglutarate to demethylate trimethylated lysines [73, 88, 89]. These demethylation activities play a crucial role in gene expression and cellular functions by modulating chromatin’s open state, enhancing gene accessibility for transcription, and influencing protein–chromatin interactions, thereby governing cellular processes.

Histone modifications dynamic in skeletal muscle atrophy

In aged rats, the gastrocnemius muscle shows a decline in global acetylation levels of histones H3K9 and H3K27, as well as a decrease in the trimethylation of H3K9, which may be associated with age-related skeletal muscle atrophy [90]. In contrast, histone acetylation appears to increase in conditions of muscle atrophy induced by hind limb cast immobilization in the soleus and plantaris muscles, as compared with healthy adult rat skeletal muscle [91]. Similarly, an increase in pan-acetylation of histone H3 is observed in skeletal muscle atrophy induced by denervation [92]. Despite these findings, it is premature to assert that the dynamics of histone acetylation are uniformly opposite between aging and other forms of muscle atrophy, partly due to methodological differences, such as the use of different antibodies (pan-acetyl-lysine antibody for unloading and denervation studies versus acetylated histone H3 (K9, K27) antibody for aging studies), and the examination of different muscle types. The complexities inherent in histone modifications, coupled with the limitations of available antibodies, currently preclude a clear understanding of how methylation and acetylation alterations contribute to skeletal muscle atrophy across different conditions. Although the use of transposases to assess chromatin’s open state could shed light on the dynamics of histone modification, this approach has yet to be widely applied in this context.

Muscle types exhibit distinct patterns of atrophy, a process that may be associated with histone modifications. Ramachandran et al. used chromatin immunoprecipitation sequencing for H3K4me2 and H3K27ac, along with transposase-accessible chromatin profiling, to generate epigenetic profiles for four distinct muscle groups [93]. Their findings indicate that each muscle group possesses unique cis-regulatory networks characterized by specific super-enhancers. Further research compared the epigenetic landscapes of the plantaris (a fast-twitch muscle) and the soleus (a slow-twitch muscle) in adult rats, using antibodies against H3K4me3 and pan-acetyl H3 [92]. This comparison revealed that genes in fast-twitch muscles respond more dynamically to physiological stimuli than those in slow-twitch muscles, a response linked to enhanced H3K4me3 and H3 acetylation. Conversely, the expression of functional genes in slow-twitch muscles seems less reliant on these histone modifications [92].

Endurance exercise can decrease the risk of muscle atrophy. Research indicates that just 4 weeks of running training can initiate histone turnover in the skeletal muscle fibers of mice, facilitating nucleosome relaxation and improving the muscle’s genetic response to exercise [94]. Following 6 weeks of endurance training, significant alterations in H3K4me1 and H3K27ac were observed in human skeletal muscle, with the H3K27ac mark being a crucial indicator of enhancer activity that influences the expression of numerous functional genes [95]. Furthermore, acute treadmill exercise in flounder muscle leads to increased histone H3 acetylation at the first exon of the peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) gene, thereby stimulating PGC-1α expression [96]. PGC-1α plays a vital role in enhancing mitochondrial oxidative metabolism in skeletal muscle and elevates the proportion of oxidative muscle fibers [97]. In addition, the expression of PGC-1α has been shown to decrease the level of FOXO3, which regulates the expression of the downstream ubiquitin ligases MuRF1 and MAFbx, contributing to the prevention of muscle atrophy [98].

Myofibers, the smallest functional units of skeletal muscle, are challenging to analyze due to the presence of hundreds of nuclei within each fiber [99]. Traditional bulk RNA sequencing, proteomics, and epigenomics aggregate signals from various myofibers and cell types, thus failing to accurately reflect the status of individual myofibers. Consequently, single-cell or single-nucleus sequencing methods are also inadequate for detailed myofiber analysis. To address this challenge, Soleimani et al. developed specific transcriptome and epigenome sequencing methods for individual muscle fibers [100, 101]. Through this innovative approach, the chromatin accessibility of single muscle fibers in both normal and CTX-damaged extensor digitorum longus (EDL) muscles was examined. The findings revealed that differences in chromatin accessibility between damaged and undamaged muscle fibers predominantly occur in the intron/distal intergenic region, also known as the enhancer region [25]. Notably, the ATAC peaks identified in most individual EDL fibers overlapped with H3K27ac peaks from whole EDL muscle, as reported by Ramachandran et al., indicating that histone acetylation mediates enhancer activation following muscle fiber injury [25, 93].

The roles of histone methylation regulators in skeletal muscle atrophy

Histone lysine methyltransferases are classified into two main categories: those with SET structural domains, including the SUV39, MLL, EZH, SMYD, and PRDM families, and the non-SET domain protein DOT1L. Members of the SUV39 family are known to inhibit the myogenic differentiation of muscle stem cells following skeletal muscle atrophy, primarily by modulating MyoD expression. However, research on skeletal muscle fibers in this context is scarce. In vitro experiments have demonstrated that SUV39H1 targets the MyoD promoter region, where its persistent methylation results in MyoD gene silencing and the inhibition of myogenic differentiation. This effect can be reversed by treating with SUV39H1 siRNA [102]. Further investigations have shown that p38α MAPK can disrupt the binding between MyoD and KMT1A (SUV39H1) by phosphorylating KMT1A, leading to a transition of the MyoD promoter from the transcriptionally repressive state marked by H3K9me3 to an active state indicated by H3K9 acetylation, thus promoting myogenic gene activation and differentiation [103]. G9a and GLP, which are highly homologous, account for the majority of the mono- and dimethylation of H3K9 in euchromatin [104]. In vivo experiments have revealed that the deletion of G9a and GLP in mice leads to embryonic lethality and a significant decrease in H3K9 mono- and dimethylation [105, 106]. Conditional KO mice lacking GLP exhibit developmental delays, hypotonia, and other abnormalities [107]. Similar to the roles of SUV39H1 and SUV39H2, G9a and GLP repress myogenic differentiation by negatively regulating the transcriptional activity of MyoD. Additionally, PRDM16, from the PRDM family of histone methyltransferases, interacts with G9a and GLP to regulate the deposition of H3K9me2 at the nuclear periphery, consequently silencing the key myogenic differentiation gene MyoD [108]. Despite these functions, studies have indicated that G9a might not be essential for muscle development and regeneration in vivo. Conditional KO mice for G9a exhibit a significant reduction in total H3K9me2 levels but do not display any noticeable developmental abnormalities in muscle size and appearance [109], suggesting the possibility that G9a and GLP form heterodimers, with GLP potentially compensating for the absence of G9a in vivo.

The EZH family, comprising Polycomb proteins EZH1 and EZH2, is another group of histone methyltransferases involved in regulating the transcription of muscle-specific genes. Both EZH1 and EZH2 possess a conserved SET structural domain, enabling them to act as methyltransferases targeting H3K27 [110]. Although EZH1 and EZH2 are paralogs, their functions diverge significantly. Following skeletal muscle injury, muscle stem cells are activated from their quiescent state, initiating muscle regeneration. Swarnali Acharyya et al. demonstrated that, in the dystrophic muscle environment of the DMD model, a high concentration of inflammatory mediators such as TNF-α acts as stimulants for the transcription factor NF-κB, thus activating the NF-κB signaling pathway and promoting muscle degeneration. Further elucidation of the underlying mechanism revealed that the increased levels of TNFα and NF-κB facilitate the recruitment of EZH2 and Dnmt3b to the promoter region of the Notch-1 gene, which leads to the epigenetic silencing of Notch-1, consequently impairing the regenerative capacity of SCs [111, 112]. Silvia Consalvi et al. demonstrated that the E3 ubiquitin ligase Praja1 mediates the degradation of EZH2 following p38α activation, facilitating skeletal myogenesis and highlighting EZH2’s involvement in skeletal muscle regeneration [113]. Apart from this pathway, inflammation has been identified as a factor that activates NF-κB, which in turn targets MyoD, thus inhibiting myogenesis [112]. In the absence of EZH1, muscle stem cells spontaneously exit their quiescent state. With repeated muscle injuries, the muscle stem cell pool is progressively depleted, leading to compromised muscle regeneration capabilities. EZH1 plays a crucial role in sustaining the expression of Notch signaling pathway genes in SCs, preventing their premature activation [114].

MLL1, which is upregulated in denervation-induced skeletal muscle atrophy, has been shown to activate the E3 ubiquitin ligases MuRF1 and MAFbx, along with the FOXO3a and NF-κB atrophy pathways. The specific mechanisms through which MLL1 influences skeletal muscle atrophy and its exact relationship with these atrophy pathways remain to be elucidated [115]. Similar to the role of the SUV39 family, MLL1 and its related family members, MLL2, MLL3, and MLL5, could be important in the self-renewal of SCs by regulating the expression of key MRFs such as MyoD, Pax7, or Myf5 [116, 117]. In vivo studies indicated that MLL1 and MLL2 contribute to the inhibition of Pax7 and MyoD expression by silencing the HIRA chaperone specific to histone H3.3, adversely affecting the regeneration and self-renewal capabilities of muscle stem cells and leading to decreased muscle cross-sectional area [118]. Notably, conditional KO of MLL1 in Pax7-positive muscle SCs in mice resulted in significant reductions in muscle mass, cross-sectional area, and muscle fiber diameter, further supporting this observation. However, it has been proposed that MLL2 might not be essential for activating Pax7 expression in SCs and myoblasts [116]. Pax7 interacts with MLL1 and recruits it to the Myf5 promoter to maintain H3K4me3 abundance and is required for muscle stem cell proliferation and muscle repair [116, 119]. MRF4/Myf6 also plays an important role in muscle differentiation, but whether its expression is regulated by epigenetic mechanisms remains elusive [120]. Additional findings reveal that MLL2 and MLL3 can be recruited to the Myog promoter region in nucleosomes via the p38 MAPK signaling in C2C12 cells [121]. Furthermore, skeletal muscle-specific KO of MLL4 in mice has been associated with a downregulation of slow oxidative myofiber gene programs, a decrease in type I myofibers, and diminished mitochondrial respiration, resulting in lowered muscle fatty acid utilization and reduced exercise endurance [122].

The SMYD family, comprising three members, plays a crucial role in muscle integrity, with their deletion leading to muscle atrophy, compromised myofibrillar integrity, and enhanced protein degradation [123, 124]. Conditional KO studies of SMYD1 in mice and zebrafish have shown a decrease in muscle mass and anomalies in myofibrillar structure [125]. SMYD2’s glutathionylation results in the disintegration of myofibrillar integrity and the degradation of sarcomeric proteins through the action of MMP-2 and calpain-1. This process occurs as SMYD2, upon glutathionylation or oxidation at Cys13, fails to interact with Hsp90 and the N2A domain of nebulin, compromising its sarcomeric stabilizing function [126]. In a mouse model of dexamethasone-induced skeletal muscle atrophy, the methyltransferase SMYD3 was found to exacerbate atrophy by upregulating genes associated with muscle wasting, such as myostatin and the c-Met genes [124]. SMYD3 specifically targets the regulatory regions of the myostatin and c-Met genes, facilitating the recruitment of the bromodomain-containing protein BRD4 through protein–protein interactions. This recruitment allows for the chromatin engagement of the pause-release factor p-TEFb and the elongation of Ser2-phosphorylated RNA polymerase II, ultimately diminishing the transcription of myostatin and c-Met and influencing glucocorticoid-induced myotube atrophy [124].

The DOT1L family, while not extensively studied in the context of skeletal muscle atrophy, has shown pivotal implications in embryonic mice where its absence was associated with a greater risk of mortality [127]. Additionally, in patients suffering from chronic obstructive pulmonary disease (COPD), a notable downregulation of the histone methyltransferase DOT1L has been observed in the vastus lateralis muscle. In vitro analyses further reveal that DOT1L gene suppression in human skeletal muscle SCs correlated significantly with increased cellular arrest and aging markers, specifically p21WAF1/Cip1/CDKN1A [128].

In addition to protein lysine methyltransferases, protein arginine methyltransferases (PRMTs) have been found to be important regulators of skeletal muscle metabolism and regeneration, and their roles in skeletal muscle have been summarized in the literature [129,130,131,132,133,134,135,136,137] (Table 2). PRMT family members primarily contribute to the regulation of skeletal muscle atrophy through mechanisms involving autophagy or degradation pathways mediated by E3 ubiquitin ligase, as well as pathways supporting muscle stem cell regeneration. These pathways include the regulation of MyoD expression, enhancement of myogenic differentiation, and facilitation of muscle regeneration [130, 138,139,140]. Specifically, mice with a skeletal muscle-specific KO of PRMT1 exhibited decreased body weight and muscle mass and elevated levels of FOXO3, the muscle-specific ubiquitin ligase MuRF1, and the autophagy marker LC3-II [135]. Further research indicates that PRMT1 modulates autophagy via the PRMT6–FOXO3 pathway and protein degradation mechanisms. During denervation, PRMT1 engages with AMPK and PGC-1α to establish the PRMT1–AMPK–PGC-1α signaling axis, potentially active in muscle remodeling scenarios [141]. Similarly, PRMT7 appears to influence PGC-1α expression through interaction with its upstream regulator p38 and activating transcription factor 2 [142, 143]. Conversely, CARM1 (alias of TRMT4) is integral to autophagy regulation. It modulates autophagy through the AMPK–SKP2–CARM1 signaling pathway. Under conditions of glucose abundance, nuclear CARM1 is degraded by the SKP2-containing E3 ubiquitin ligase SCF (SKP1-cullin1-F-box protein). In addition, glucose deprivation leads to nuclear accumulation of AMPK, which phosphorylates FOXO3, enhancing its transcriptional activity. FOXO3, in turn, transcriptionally represses SKP2, resulting in the stabilization of CARM1 protein and an increase in histone H3R17 dimethylation, triggering autophagy [144]. These findings indicate that PRMT family member expression induction during denervation might serve as a compensatory mechanism to restrict skeletal muscle atrophy. Nonetheless, the potential of their overexpression or continuous activation in preventing muscle atrophy and the involvement of common signaling pathways, such as those associated with PGC-1α or FOXO3, remains to be investigated.

HDMs, including members of the KDM and JMJC families (Table 2), are recognized for their role in the epigenetic regulation of muscle stem cells [88, 89, 145]. Similar to histone methyltransferases, HDMs primarily influence myogenic differentiation via the regulation of MyoD expression. However, certain studies have also identified their role in the atrophy mechanisms within skeletal muscle fibers. The depletion of LSD1 in skeletal muscle fibers amplifies glucocorticoid-induced atrophy in fast-twitch muscle fibers by diminishing the nuclear retention of the anti-autophagy transcription factor Foxk1, achieved through the inhibition of the Akt–mTORC1 axis [146]. This reduction in Foxk1 allows for the replacement of its target genes with those of FOXO3, enhancing the accessibility of atrophy-associated genes within muscle cells [147, 148]. Conversely, in the presence of abundant nutrients, the Akt–mTORC1 pathway fosters the nuclear presence of Foxk1, thereby suppressing autophagy and other catabolic processes [149]. Research into hypoxic conditions has revealed that the signaling cascade involving mechanistic target of rapamycin (mTOR), NF-κB, and TGF-β pathways can elevate the expression of hypoxia-inducible transcription factor-1α (HIF-1α). This, in turn, stimulates the expression of KDM3A, KDM4B, KDM4C, and KDM6B, facilitating gene expression through demethylation regulation [150, 151]. Furthermore, conditions such as oxidative stress, impaired energy metabolism, and inflammation associated with skeletal muscle atrophy are known to activate the mTOR, NF-κB, and TGF-β signaling pathways. Nevertheless, the specific regulatory interactions between HDMs and these pathways remain largely unexplored. While some HDMs may interact with the oxidative stress-responsive transcription factor FOXO [152], the exact signaling pathways they regulate and their impacts on muscle fibers in models of skeletal muscle atrophy warrant further investigation.

The mechanism of histone acetylation regulators controlling skeletal muscle atrophy

Research into protein acetylation is well documented, with numerous publications systematically detailing the influence of different histone acetylation regulators on skeletal muscle atrophy [79, 96, 171]. In this section, we provide insights into how several key regulators act on common myasthenic pathways leading to muscle atrophy.

P300/CBP-specific KOs in the skeletal muscle of transgenic mice exhibit no alterations in muscle structure, mass, fiber type, or locomotor activity [172]. A combined KO of P300 and CBP within skeletal muscle does not impact muscle mass or structure but significantly compromises muscle strength and contractility [173]. This finding does not suggest that P300/CBP primarily influences skeletal muscle physiology over structure and mass. In an in vitro muscle atrophy model, P300/CBP was found to enhance the acetylation levels of transcription factors critical for muscle mass regulation, including C/EBP, FOXO, and NF-κB/p65. Inhibition of P300/CBP-mediated acetylation of these transcription factors led to a decrease in the expression of the ubiquitin ligase MuRF1, subsequently mitigating myotube atrophy [174]. The function of P300/CBP in the atrophy of skeletal muscle primarily involves the modulation of FOXO family transcription factors [175]. CBP/p300-mediated non-histone acetylation facilitates the formation of a cysteine-thiol disulfide-dependent complex with FOXO transcription factors, leading to a negative regulation of FOXO activity at the post-translational level [176, 177]. Additionally, CBP/p300-mediated histone acetylation suppresses FOXO expression at the transcriptional level [164]. FOXO transcription factors target the promoter of MAFbx, triggering rapid transcription of MAFbx and resulting in significant atrophy of myotubes and myofibers [178] (Fig. 2 and Table 3). Inhibiting the transcriptional activity of FOXO or reducing its expression substantially mitigates skeletal muscle atrophy [179]. The supplementation of P300 in disuse atrophic skeletal muscle has been shown to decrease FOXO and MAFbx expression [164]. Recent studies in various models have broadened our understanding of the molecular mechanisms by which CBP/p300 contributes to skeletal muscle atrophy. In diabetes-induced atrophy, high fat and insulin resistance were shown to promote CBP/p300 phosphorylation. This modification enhances its acetyltransferase activity, subsequently activating autophagic processes and leading to atrophy-associated morphological and molecular alterations [180]. Further mechanistic insights have been gained from examining skeletal muscle atrophy in cancer cachexia, where it has been reported that cancer cells activate Toll-like receptor 4 in skeletal muscle, which then phosphorylates CBP/p300 via the p38β–MAPK pathway [181]. Within myotubes exposed to a pro-oncogenic environment, phosphorylated CBP/p300 increases the acetylation of the transcription factor C/EBPβ at the Lys39 residue, thereby activating it [162]. This activated C/EBPβ then binds to cis-elements in the promoter region of the MuRF1 and MAFbx gene independently of FOXO, leading to an upregulation of MuRF1 and MAFbx expression and consequent skeletal muscle atrophy [163].

Table 3 Functional epigenetic regulators of skeletal muscle atrophy and their targets

HDAC1, functioning oppositely to CBP/p300, similarly contributes to the induction of skeletal muscle atrophy. During skeletal muscle atrophy prompted by nutritional deprivation, the transcriptional activation of FOXO is contingent upon histone deacetylation. Changes in HDAC1 expression alone can activate FOXO, leading to the upregulation of MAFbx and the promotion of muscle wasting and fiber atrophy [165]. Furthermore, HDAC1 plays a role in the deacetylation of the zinc finger transcription factor Krüppel-like factor 5 (KLF5), which negatively impacts its protein level expression [166, 167]. KLF5 is a mediator in the early phase of skeletal muscle atrophy, with its muscle-specific deletion markedly mitigating atrophy induced by mechanical unloading. The mechanism underlying this involves KLF5’s interaction with FOXO1 and its co-regulatory effect on the transcriptional activity of MuRF1 and MAFbx [182]. Intriguingly, the removal of KLF5 also led to decreased H3K27ac levels at the MuRF1 promoter and enhancer regions [182]. Additionally, autophagy is targeted by HDAC1. Mice lacking HDAC1 and HDAC2, specifically in skeletal muscle, exhibited higher mortality rates along with ultrastructural defects, such as sarcomere disruption and mitochondrial abnormalities, yet without overt muscle atrophy [183]. Subsequent research indicated that HDAC1 and HDAC2 influence autophagosome formation, though it remains uncertain whether this effect relies on their deacetylase activity. HDAC1 has also been shown to complex with C/EBPβ, co-regulating downstream pathways including p53, SIRT1, and PGC1α, though their precise roles in skeletal muscle atrophy remain to be elucidated [168].

HDAC4 is one of the first epigenetic factors identified to directly regulate skeletal muscle atrophy. Its expression is upregulated in skeletal muscle atrophy induced by various factors such as denervation, wasting, and aging [184]. Notably, HDAC4 is primarily localized at the neuromuscular junction (NMJ), and upon denervation, it dissociates from this site, leading to a significant induction of its expression and subsequent nuclear accumulation [185], suggesting HDAC4’s increased sensitivity to neurogenic skeletal muscle atrophy. One of the key mechanisms via which HDAC4 mediates muscle atrophy is by inhibiting myogenic protein Myog, which acts as a transcription factor and directly targets the promoters of MuRF1 and MAFbx, thereby upregulating their transcriptional expression [169, 186]. The forced expression of Myog in skeletal muscles of mice lacking HDAC4 can reverse the muscle atrophy that ensues post-denervation. However, the precise mechanism of how HDAC4 suppresses Myog expression—whether through direct acetylation of the Myog protein or via transcriptional regulation through histone deacetylation—remains unclear. HDAC4 also targets other downstream molecules in skeletal muscle, particularly under conditions of denervation. For example, the transcription factor AP1 is a significant target, with HDAC4 activating AP1 not through its canonical transcriptional repression function but by deacetylating MAP kinase, thereby activating AP1 via the MAPK pathway [170]. A systematic analysis of the downstream molecules affected by HDAC4 KO in mouse skeletal muscle revealed that HDAC4 influences numerous noncoding RNAs and the competitive endogenous RNA (ceRNA) networks they form, within which lncRNAs such as XR_377582.2 and ENSMUST00000143649 have been identified as potentially crucial in regulating muscle atrophy in relation to HDAC4 [187]. HDAC4’s role in skeletal muscle atrophy is not initiated independently; rather, it is preceded by the activity of signaling pathways such as PKB/Akt and mTORC1 following nerve injury. These pathways promote the nuclear import of HDAC4, which in turn facilitates the expression changes in numerous genes associated with muscle atrophy [188].

It is worth noting that HDAC4 also plays an important role in the genetic neuromuscular disease ALS. In the skeletal muscle of ALS patients and mouse models, the level of HDAC4 has been reported to be much higher than in normal controls [189, 190]. Using a genetic strategy to knock out HDAC4 in the skeletal muscle of SOD1 mice was found to result in early onset of ALS, manifested by weight loss, muscle denervation and atrophy, and impaired muscle performance [191]. In contrast to nonhereditary muscle atrophy, catabolic pathways such as the ubiquitin–proteasome are not activated, and the specific pathways leading to atrophy remain unclear.

Several HDAC inhibitors (HDACIs), such as trichostatin A (TSA) and butyrate, have been utilized to treat skeletal muscle atrophy, effectively inhibiting the expression of MuRF1 and MAFbx and alleviating skeletal muscle atrophy in a mouse model [192, 193]. However, the application of HDACIs in treating hereditary skeletal muscular atrophy, including ALS, remains challenging. In diseases such as ALS, not only the skeletal muscle but also motor neurons and NMJs are involved, leading to different sensitivities to HDACIs. For instance, the intraspinal injection of the HDACI sodium valproate effectively protects motor neuron cell bodies but fails to prevent the degeneration of NMJs [194].

Similarly, while butyrate has shown efficacy in alleviating skeletal muscle atrophy, it has a limited effect on protecting motor neurons against chronic spinal motor neuron death induced by AMPA-mediated excitotoxicity in an ALS model [195]. Thus, more targeted treatment strategies are required for these complex genetic disorders. Additionally, despite the current classification of HDACs and HDACIs, the specific matching of these inhibitors to their appropriate substrates is not fully understood, adding uncertainty to the clinical application of HDACIs.

RNA modifications

RNA modifications and regulators

Similar to DNA, all forms of RNA—including messenger RNA (mRNA), noncoding RNA, ribosomal RNA (rRNA), and transfer RNA (tRNA)—undergo modifications that, while not altering their nucleotide sequence, significantly impact their function and destiny. This phenomenon, known as epitranscriptomics, highlights the role of RNA modifications in gene expression and cellular function. RNA modifications are diverse, with over 150 identified to date. Among these, N⁶-methylation of adenosine (m6A) is the most abundant in eukaryotes, accounting for more than half of all RNA methylation in cells [196, 197]. As the name suggests, this modification involves the addition of a methyl group to the sixth nitrogen atom of adenine. Other well-characterized RNA modifications include N¹-methyladenine (m1A) modification, 5-methylcytosine (m5C) modification, 3-methylcytosine(m3C) 7-methylguarine (m7G) modification, N⁶, 2′-O-dimethyladenine nucleoside (m6Am), pseudouridine (Ψ) modification, etc. [198] (Table 4).

Table 4 List of RNA modifications and regulators

The m6A modification is the most common form of RNA modification and is widely distributed in various RNAs. Studies have identified a nonuniform distribution of m6A across mRNA, showing a preference for the coding sequence (CDS) and the 3′ untranslated region (3′UTR), particularly in regions proximal to the stop codon. The occurrence of m6A is sequence dependent, predominantly appearing on adenines within the conserved RRACH motif (R = A or G; H = A, C, or U). This motif significantly overlaps with the binding sites of the SRSF splicing factor, suggesting that exons modified by m6A are more likely to be included in mRNA transcripts [199, 200]. Research on other RNA modifications, particularly in tRNAs and rRNAs, is limited due to their scarce presence and the challenges associated with detecting them with high sensitivity. The enzymatic process leading to m6A modification involves a methyltransferase complex that includes methyltransferase-like 3 (METTL3), METTL14, Wilms tumor 1 associated protein (WTAP), and Vir like m6A methyltransferase associated (VIRMA), collectively referred to as the “writers” of m6A. Conversely, the demethylation of m6A is facilitated by enzymes such as alpha-ketoglutarate-dependent dioxygenase Alk B homolog 5 (ALKBH5) and fat mass and obesity-associated protein (FTO), known as “erasers,” which can reverse m6A modifications. The dynamic interplay between writers and erasers contributes to a global modulation of m6A levels. The regulatory impact of m6A modifications on gene expression is mediated through its influence on mRNA stability or protein translation, which is accomplished by recruiting various m6A-binding proteins, or “readers.” Recognized readers include the YTH domain-containing proteins (YTHDF1, YTHDF2, YTHDF3, YTHDC1, and YTHDC2) and members of the heterogeneous nuclear ribonucleoprotein (HNRNP) family (HNRNPA2B1 and HNRNPC). It is proposed that the number of such readers may be considerably higher [201, 202]. The regulatory mechanisms for other RNA methylation modifications also involve distinct sets of writers and erasers, which are detailed in Table 4.

RNA modification regulators in skeletal muscle atrophy

RNA modification plays a crucial role in virtually all metabolic processes of RNA, from synthesis to degradation [203], with research confirming that m6A methylation is pivotal in various aspects of skeletal muscle physiology. Existing reviews have comprehensively summarized its significance in skeletal muscle development [204, 205], as well as in other skeletal muscle disorders [206, 207].

The aging of primate skeletal muscle is marked by a decline in METTL3 expression and a consequent reduction in m6A methylation. Deletion of METTL3 in myotubes derived from human pluripotent stem cells leads to a significant decrease in both myotube diameter and nuclei count. NPNT, a downstream target of METTL3, exhibits decreased mRNA and m6A levels in aging myoblast tubes, mirroring the phenotype observed with METTL3 deletion [153]. Experimental supplementation of METTL3 in vivo promotes skeletal muscle growth, whereas conditional genetic deletion of METTL3, specifically in myofibers, results in spontaneous muscle atrophy. These findings indicate that METTL3 expression levels are a critical determinant of skeletal muscle mass [208]. Furthermore, METTL3 influences the expression of numerous skeletal muscle-specific noncoding RNAs (ncRNAs) through m6A modification [209, 210], suggesting that ncRNAs, which play a crucial role in muscle atrophy, may represent one of the mechanisms by which METTL3 modulates skeletal muscle mass [211].

The promoting effect of METTL3 deletion on skeletal muscle atrophy may be linked to its impact on muscle SCs. Specific reduction in METTL3 expression within muscle SCs markedly suppresses the proliferation of these muscle stem cells and impairs muscle regeneration post-atrophy [212]. Conversely, overexpressing METTL3 significantly enhances both the proliferation and myogenic differentiation of C2C12 myoblasts [156]. In proliferating muscle SCs, METTL3 is essential for sustaining m6A levels in the 5′ untranslated region (5′UTR) of the myogenic transcription factor MyoD mRNA. MyoD mRNA levels are notably decreased following METTL3 knockdown [154]. Moreover, METTL3 plays a pivotal role in managing the transition of muscle SCs/myoblasts from a proliferative to a differentiated state. Suppressing METTL3 expression leads to premature differentiation of myoblasts and boosts the engraftment of muscle SCs after their initial transplantation [213].

m6A readers work in conjunction with METTL3 in an m6A-dependent manner. Specifically, YTHDF1/2 influences the mRNA stability of MRFs such as STK11, MyHC, MyoD and Myog, and plays a role in their translation [155, 156]. In some cases, these readers directly impact the myoblast function. For instance, during muscle regeneration following acute injury, YTHDC1 regulates SCs activation and proliferation. SCs lacking YTHDC1 fail to exit quiescence, and their regenerative capacity is significantly compromised [214, 215]. Mechanistically, YTHDC1 interacts with m6A-modified mRNAs to influence their alternative splicing, with these mRNAs primarily involved in pathways related to the regulation of actin filament-based processes and PI metabolism [214, 215].

Both our research and findings from another independent team indicate that loss of innervation in skeletal muscle leads to a reduction in global m6A levels, similar to the decline observed in aging skeletal muscle [157, 216], suggesting a reduction in m6A might be a universal characteristic of skeletal muscle atrophy. However, in denervated muscle atrophy, the decrease in m6A is predominantly due to demethylation, marked by the upregulation of the demethylases FTO and ALKBH5. Specifically, ALKBH5 increases the stability of HDAC4 mRNA through demethylation, activating the HDAC4-FOXO3 pathway and resulting in muscle atrophy [157] (Fig. 2 and Table 3). Our research further demonstrates that, during denervated muscle atrophy, m6A modification of several genes associated with the ubiquitin–proteasome pathway is reduced, leading to their elevated expression, indicating that reduced m6A is a contributing factor to the activation of the ubiquitin–proteasome pathway [216]. Furthermore, we also explored m6A’s role in skeletal muscle atrophy through pharmacological interventions. The results indicated that FTO inhibitor R-2-hydroxyglutarate (R-2HG) in denervated tibialis anterior muscle increased total m6A levels, ameliorating muscle atrophy, while treatment with 3-deazadenosine (Daa) in healthy tibialis anterior muscle reduced m6A levels, resulting in spontaneous muscle atrophy [216]. Importantly, R-2HG specifically inhibited FTO but not ALKBH5, while Daa did not selectively target m6A regulators. Despite both ALKBH5 and FTO recognizing m6A-modified RNAs, differences in their substrate preferences, cellular localization and reaction products exist [217,218,219]. These findings imply that the impact of m6A on skeletal muscle atrophy is related to the overall m6A level rather than specific regulatory proteins, which is further illustrated by the synergistic action of FTO and ALKBH5 in activating the FOXO pathway in some contexts [158].

The role of FTO in myoblasts presents a complex picture, challenging the notion of its straightforward impact on overall skeletal muscle atrophy. Research indicates that, while FTO is essential for myogenic differentiation, an excess of FTO does not enhance myotube formation [159]. It was observed that silencing FTO impaired mitochondrial biogenesis and energy production, attributable to the downregulation of the mTOR and PGC-1α [159]. Further investigation revealed that m6A modification of the 5′UTR of TGFβ1 mRNA facilitates its degradation, with FTO influencing these modifications to hinder myoblast differentiation [160]. Experiments in genetically engineered mice demonstrated that FTO overexpression did not alter skeletal muscle mass [220], whereas FTO deficiency compromised skeletal muscle development [159]. This discrepancy may be elucidated by considering two aspects: firstly, the differentiation of SCs into myotubes might differ between developmental processes and post-injury repair; secondly, m6A modifications may exert varying impacts across different cell types or subtypes, with their collective interactions influencing skeletal muscle atrophy [221, 222].

Research on other RNA modifications in the context of skeletal muscle atrophy remains limited, potentially due to challenges in detecting these modifications with high sensitivity. One study utilizing pigs as a model organism demonstrated a positive correlation between m5C mRNA levels in skeletal muscle and muscle mass, indicating a possible regulatory role of m5C in skeletal muscle atrophy [223]. The lack of functional studies on various RNA-modifying writers, readers, and erasers in human and rodent models hinders further advancement in the biomedical research of skeletal muscle atrophy.

Epifactors and alternative splicing crosslinks during muscle atrophy

Alternative splicing (AS) and epigenetic regulation are crucial mechanisms that steer gene expression. In certain physiological or pathological contexts, adjacent splice sites on pre-mRNAs compete, resulting in the generation of various splice isoforms. These isoforms can have differing or even opposing functions, significantly expanding the repertoire of functional proteins and enhancing cellular complexity [224]. Pre-mRNAs contain numerous cis-regulatory elements that interact with trans-acting factors [notably, splicing factors, primarily RNA-binding proteins (RBPs)] to collaboratively determine AS outcomes [225]. Furthermore, AS is modulated by alterations in RNA secondary structure [226, 227]. In patients with skeletal muscle atrophy and corresponding animal models, numerous aberrant AS events have been documented that contribute to muscle atrophy [228,229,230,231]. Moreover, significant skeletal muscle atrophy has been both induced and reversed by genetically manipulating RBPs [232,233,234,235,236,237,238,239,240]. Our research identified that, in a model of denervation-induced atrophy, AS modification of a broad array of structural and functional proteins associated with muscle atrophy aligns with the atrophic phenotype. Remarkably, we observed a substantial increase in several highly conserved exons (HCEs) of Obscn following three days of denervation [241]. Similar outcomes have been observed in the skeletal muscles of mice subjected to prolonged microgravity conditions in space [242]. The Obscn gene encodes obscurin, a large structural protein critical for myofibril assembly [243]. We propose that HCEs are rich in RNA secondary structures, specifically G-quadruplexes, which may trigger cytotoxicity and consequently lead to skeletal muscle atrophy [241].

In skeletal muscle tissue and muscle stem cells, the Obscn undergoes DNA methylation, predominantly characterized by hypermethylation [40]. Hypomethylated sites in the untranslated regions (UTRs) might correlate with gene expression, whereas the abundance of hypomethylated sites across exons could relate to the generation of numerous AS variants. Indeed, the processes of epigenetics and AS are intricately linked and jointly influence skeletal muscle atrophy. Epigenetic modifications and their enzymes (writers) can influence AS, while the mechanism of AS itself can prompt epigenetic regulators to produce variant isoforms with distinct functions (Fig. 3). Although we have extensively reviewed the dynamics of epigenetic changes in skeletal muscle atrophy and the roles of epigenetic writers, a direct connection between RNA AS and epigenetic inheritance in skeletal muscle has yet to be established. Below, we outline three hypothetical models that could shed light on the regulatory mechanisms governing skeletal muscle atrophy and regeneration.

Fig. 3

Epigenetic and alternative splicing are intertwined. A Regulatory mechanism of pre-mRNA alternative splicing. Splicing regulatory elements such as exonic splicing enhancers (ESEs), exonic splicing silencers (ESSs), intronic splicing enhancers (ISEs), and intronic splicing silencers (ISSs), etc., which are located around the splice site. Splicing factor SR proteins or hnRNPs can bind to them and recruit core splicing elements such as U2AF and U1 snRNP to regulate alternative splicing. B Histone or DNA modifications affect alternative splicing. Transcription and splicing occur almost simultaneously in the same space, and various epigenetic modifications can alter the rate of Pol II elongation during transcription, which can affect recognition of the splice site by the spliceosome and, thus, alternative splicing. C RNA m6A modifications affect alternative splicing. Many m6A readers are splicing factors themselves, which can regulate numbers of alternative splicing events. m6A regulators are also able to recruit splicing factors to participate in alternative splicing regulation, e.g., YTHDC1 binds to pre-mRNAs in an m6A-dependent manner, and it can recruit the splicing factor SRSF1 to regulate alternative splicing. D Alternative splicing regulates the function of epigenetic regulators. The expression of multiple epigenetic regulators is regulated by alternative splicing, which produces full-length isoforms and N-or C-terminally reduced protein products. These truncated isoforms differ in enzymatic activity, cellular localization, and interactions

Epigenetic modifications affect AS

Epigenetic modifications, particularly DNA methylation and histone modifications, have a direct, global and significant impact on AS [244]. Genome-wide, DNA methylation can alter the splicing of approximately 20% of alternative exons. Heterochromatin protein 1 (HP1) detects DNA methylation in a location-specific manner and functions as a linker protein, facilitating the recruitment of the splicing factor SRSF3 to methylated sites [245]. Histone modifications, including methylation and acetylation, also affect the recruitment of splicing regulators via chromatin-binding proteins, impacting the splicing outcomes of thousands of genes [246,247,248]. For instance, the histone modification H3K36me3, recognized by the MORF-related gene 15 (MRG15), influences the splicing of numerous variable exons by recruiting the splicing factor PTBP1 [246]. The removal of the H3K36me3 mark’s writer, SETD2, during skeletal myogenesis triggers simultaneous shifts in gene expression and AS, disrupting myotube formation through metabolic reprogramming [249].

Spliceosome assembly and transcription processes occur almost simultaneously within the same cellular compartments, and epigenetic modifications also affect a large number of AS by altering the rate of transcription elongation [250]. The DNA-binding protein CCCTC-binding factor (CTCF) is involved genome-wide in mediating local RNA polymerase II pauses to promote the inclusion of weakly upstream exons, whereas 5mC expels CTCF, leading to exon skipping. Inhibiting DNMT1 expression helps maintain the slow rate of RNA polymerase II and promotes co-transcriptional spliceosome assembly at weak splice sites [251]. Decreased TET levels can increase 5mC, leading to CTCF expulsion and alternative exon skipping [252]. Acetylation modification of histone H3K9 increases the rate of RNA polymerase II, whereas methylation modification decreases it, dynamically generating two distinct splicing patterns [253]. Numerous histone modifications can affect the speed of RNA polymerase II, as detailed in the literature [254] (Fig. 3B).

M6A modifications and regulatory recruitment of splicing factors regulate AS

Approximately 10% of the m6A signal is found in variable exons and adjacent introns, indicating a potential relationship between m6A modifications and AS [255]. The presence of m6A at splice sites, co-transcribed with mRNA, facilitates the splicing mechanism, leading to an increase in alternative exon exclusion [256]. Further analysis of existing epitranscriptomic datasets has uncovered the co-localization of G-quadruplex structures and m6A modifications, which are particularly enriched near splice sites [257] and align with the observation that nearly all known m6A regulators influence global AS patterns in an m6A-dependent fashion [258,259,260,261]. Among these regulators, YTHDC1 can bind several SRSF family members, recruiting them to m6A sites and thereby influencing a broad spectrum of splicing events [200, 262, 263]. Mice with reduced SRSF1 levels appeared normal at birth but exhibited significant skeletal muscle atrophy and died within 5 days, highlighting SRSF1’s critical role in satellite cell proliferation and the establishment of functional NMJs [264] (Fig. 3C).

Beyond YTHDC1, the m6A reader IGF2BP2 has been implicated in the activation of muscle SCs [265]. In the context of sarcopenia and in orchiectomized mice, downregulation of IGF2BP2 by miRNA Let-7e-5p was observed, which hindered myosin heavy chain development and energy metabolism, contributing to muscle atrophy [266]. However, it is not clear whether the function of IGF2BP2 is related to the AS.

AS of epigenetic regulators

Functional splice isoforms have been identified for all members of the DNA methyltransferase family [267]. A specific DNMT1 isoform is present in mouse skeletal muscle, distinct from those in most peripheral tissues. This isoform is truncated at the N-terminus and is specifically expressed in terminally differentiated muscle cells, potentially influencing de novo DNA methylation during the myogenesis process [268]. Numerous DNMT3b splice isoforms have been documented in humans and rodents [269,270,271]. These isoforms serve distinct functions: some alter the subcellular localization of DNMT3b, others affect its affinity for DNA, and some function as helper proteins that positively regulate de novo methylation by DNMT3a and DNMT3b, despite lacking catalytic activity [271,272,273]. Additionally, the DNA demethylase Tet1 and its truncated isoforms exhibit different nuclear localizations and target m5C at various positions, leading to altered cytosine modification levels in human and mouse cells [274].

Histone modification regulators frequently undergo AS. For skeletal muscle to regenerate following injury, it is necessary to remove repressive chromatin marks from the promoter of the Myog. As myoblasts differentiate into myotubes, the HDM JMJD2A/KDM4A transitions to an isoform devoid of the N-terminal demethylase domain (ΔN-JMJD2A), playing a crucial role in the demethylation of H3K9me2 and H3K9me3 at the Myog promoter [275] (Fig. 3). Recent research has shown that numerous genes encoding histone-modifying enzymes are subject to AS under specific physiological conditions. For instance, the AS of exon 9 in HDAC7 significantly affects global histone modifications and gene expression [276]. Further examples abound, providing valuable insights into the complexity of AS and its impact on gene regulation [244, 248, 277].

There are several splice variants of METTL3. Some variants are less catalytically active, leading to a reduction in the total amount of m6A in the cell and altering the ability of the cell to proliferate, migrate, and invade [278]. Not all AS variants have an effect on the m6A catalytic activity of METTL3. CRISPR/Cas9-based methods to remove METTL3 can remove only about half of m6A because other METTL3 splicing isoforms bypass the CRISPR/Cas9 mutation and produce functional methyltransferases [279]. A METTL3 isoform that retains introns 8 and 9 has also been found in human tissues. The extended 3′UTR inhibits the transport of this isoform’s mRNA to the cytoplasm, thereby inhibiting protein expression [280]. Additionally, evidence suggests that METTL14 undergoes AS, with the skipping or inclusion of exon 10 being regulated by the phosphorylation of SRSF5. Isoforms of METTL14 that retain exon 10 exhibits higher catalytic activity [281].

A review of public databases revealed that approximately half of the epifactors possess at least one splice isoform, suggesting the presence of numerous yet unidentified isoforms [282]. These epifactors undergo AS to generate variant transcripts, which exhibit distinct functions in skeletal muscle atrophy, varying by tissue type and disease state. This raises several key questions for future research: What shifts in epifactors isoforms occur during skeletal muscle atrophy or regeneration? How do these isoforms compare in terms of their contributions to epigenetic modifications and physiological roles? What mechanisms of AS regulate the generation of these isoforms?

Conclusion and perspectives

Understanding how atrophy genes or pathways are initiated following skeletal muscle atrophy is pivotal for developing strategies to delay this condition. Herein, we discussed the roles of epigenetic modifications of DNA, histones, and RNA, as well as their regulators, and provided insights into how epigenetic is an early signal that orchestrates the progression of skeletal muscle atrophy. Despite this, the functions of a limited number of epigenetic modifications and regulators have been systematically investigated in the context of skeletal muscle atrophy. A promising avenue for future research lies in elucidating the complex interplay between epigenetic mechanisms and alternative splicing and their collective impact on skeletal muscle atrophy. Moreover, the potential interactions between different forms of methylation (DNA, RNA, and histone) and between specific modifications such as 5mC and m6A in influencing skeletal muscle atrophy warrant further exploration [283, 284]. The interaction network of these epigenetic modifications in human skeletal muscle diseases and rodent models remains largely unclear.

Skeletal muscle exhibits considerable heterogeneity at various levels, including differences in the physiology and rate of atrophy between fast- and slow-twitch muscles, which may be under epigenetic control. For instance, the transcriptional states of type I, IIx, and IIb MyHC genes during MyHC conversion after muscle unloading show varying sensitivity to histone modifications [285]. Current insights into the epigenetic landscape of skeletal muscle are predominantly derived from bulk-omics data, potentially overlooking distinctions between fast and slow muscle myonuclei, muscle SCs, macrophages, and adipocytes. Recent attention to chromatin accessibility dynamics during skeletal muscle atrophy, at the resolution of single nuclei or fibers, represents a significant step forward. However, the dynamics of nuclear RNA and DNA methylation at such fine resolutions remain to be fully understood, offering a promising direction to unravel the complex regulation of skeletal muscle atrophy more profoundly [286].

With the advancement of epigenetics research, significant progress has been made in drug discovery and development [287]. The approval rate of epigenetic drugs for clinical use is increasing, with CRISPR–Cas-based epigenetic editing emerging as a groundbreaking approach in this field [288]. At present, most of these drugs function as inhibitors targeting epigenetic regulators, predominantly for cancer treatment, though a limited selection has been applied to combat skeletal muscle atrophy. Trichostatin A, derived from fungal metabolites and a potent histone deacetylase inhibitor, has demonstrated efficacy against skeletal muscle wasting and notable therapeutic outcomes in models of DMD, ALS, and SMA [192, 289]. Despite their potential, HDACI often encounter challenges in clinical development, primarily due to their wide-ranging effects and the lack of specificity in targeting downstream molecules, complicating the mitigation of pharmacological side effects. Thus, addressing this issue necessitates a comprehensive analysis of the molecular interaction networks of epigenetic drugs and a deeper understanding of their action mechanisms in specific physiological contexts.