Epitranscriptomics as a New Layer of Regulation of Gene Expression in Skeletal Muscle: Known Functions and Future Perspectives.

Epitranscriptomics refers to post-transcriptional regulation of gene expression via RNA modifications and editing that affect RNA functions. Many kinds of modifications of mRNA have been described, among which are N6-methyladenosine (m6A), N1-methyladenosine (m1A), 7-methylguanosine (m7G), pseudouridine (Ψ), and 5-methylcytidine (m5C). They alter mRNA structure and consequently stability, localization and translation efficiency. Perturbation of the epitranscriptome is associated with human diseases, thus opening the opportunity for potential manipulations as a therapeutic approach. In this review, we aim to provide an overview of the functional roles of epitranscriptomic marks in the skeletal muscle system, in particular in embryonic myogenesis, muscle cell differentiation and muscle homeostasis processes. Further, we explored high-throughput epitranscriptome sequencing data to identify RNA chemical modifications in muscle-specific genes and we discuss the possible functional role and the potential therapeutic applications.

Corrigendum: Histone deacetylase functions and therapeutic implications for adult skeletal muscle metabolism.

[This corrects the article DOI: 10.3389/fmolb.2023.1130183.].

Histone deacetylase functions and therapeutic implications for adult skeletal muscle metabolism.

Skeletal muscle is a highly adaptive organ that sustains continuous metabolic changes in response to different functional demands. Healthy skeletal muscle can adjust fuel utilization to the intensity of muscle activity, the availability of nutrients and the intrinsic characteristics of muscle fibers. This property is defined as metabolic flexibility. Importantly, impaired metabolic flexibility has been associated with, and likely contributes to the onset and progression of numerous pathologies, including sarcopenia and type 2 diabetes. Numerous studies involving genetic and pharmacological manipulations of histone deacetylases (HDACs) in vitro and in vivo have elucidated their multiple functions in regulating adult skeletal muscle metabolism and adaptation. Here, we briefly review HDAC classification and skeletal muscle metabolism in physiological conditions and upon metabolic stimuli. We then discuss HDAC functions in regulating skeletal muscle metabolism at baseline and following exercise. Finally, we give an overview of the literature regarding the activity of HDACs in skeletal muscle aging and their potential as therapeutic targets for the treatment of insulin resistance.

Amyotrophic Lateral Sclerosis as a Systemic Disease.

The goal of this Special Issue is to report new research progress and reviews concerning amyotrophic lateral sclerosis (ALS) [...].

Histone Deacetylases: Molecular Mechanisms and Therapeutic Implications for Muscular Dystrophies.

Histone deacetylases (HDACs) are enzymes that regulate the deacetylation of numerous histone and non-histone proteins, thereby affecting a wide range of cellular processes. Deregulation of HDAC expression or activity is often associated with several pathologies, suggesting potential for targeting these enzymes for therapeutic purposes. For example, HDAC expression and activity are higher in dystrophic skeletal muscles. General pharmacological blockade of HDACs, by means of pan-HDAC inhibitors (HDACi), ameliorates both muscle histological abnormalities and function in preclinical studies. A phase II clinical trial of the pan-HDACi givinostat revealed partial histological improvement and functional recovery of Duchenne Muscular Dystrophy (DMD) muscles; results of an ongoing phase III clinical trial that is assessing the long-term safety and efficacy of givinostat in DMD patients are pending. Here we review the current knowledge about the HDAC functions in distinct cell types in skeletal muscle, identified by genetic and -omic approaches. We describe the signaling events that are affected by HDACs and contribute to muscular dystrophy pathogenesis by altering muscle regeneration and/or repair processes. Reviewing recent insights into HDAC cellular functions in dystrophic muscles provides new perspectives for the development of more effective therapeutic approaches based on drugs that target these critical enzymes.

Sex Differences in Inflammation and Muscle Wasting in Aging and Disease.

Only in recent years, thanks to a precision medicine-based approach, have treatments tailored to the sex of each patient emerged in clinical trials. In this regard, both striated muscle tissues present significant differences between the two sexes, which may have important consequences for diagnosis and therapy in aging and chronic illness. In fact, preservation of muscle mass in disease conditions correlates with survival; however, sex should be considered when protocols for the maintenance of muscle mass are designed. One obvious difference is that men have more muscle than women. Moreover, the two sexes differ in inflammation parameters, particularly in response to infection and disease. Therefore, unsurprisingly, men and women respond differently to therapies. In this review, we present an up-to-date overview on what is known about sex differences in skeletal muscle physiology and disfunction, such as disuse atrophy, age-related sarcopenia, and cachexia. In addition, we summarize sex differences in inflammation which may underly the aforementioned conditions because pro-inflammatory cytokines deeply affect muscle homeostasis. The comparison of these three conditions and their sex-related bases is interesting because different forms of muscle atrophy share common mechanisms; for instance, those responsible for protein dismantling are similar although differing in terms of kinetics, severity, and regulatory mechanisms. In pre-clinical research, exploring sexual dimorphism in disease conditions could highlight new efficacious treatments or recommend implementation of an existing one. Any protective factors discovered in one sex could be exploited to achieve lower morbidity, reduce the severity of the disease, or avoid mortality in the opposite sex. Thus, the understanding of sex-dependent responses to different forms of muscle atrophy and inflammation is of pivotal importance to design innovative, tailored, and efficient interventions.

PGC-1α in the myofibers regulates the balance between myogenic and adipogenic progenitors affecting muscle regeneration.

Skeletal muscle repair is accomplished by satellite cells (MuSCs) in cooperation with interstitial stromal cells (ISCs), but the relationship between the function of these cells and the metabolic state of myofibers remains unclear. This study reports an altered proportion of MuSCs and ISCs (including adipogenesis-regulatory cells; Aregs) induced by the transgenic overexpression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) in the myofibers (MCK-PGC-1α mice). Although PGC-1α-driven increase of MuSCs does not accelerate muscle regeneration, myogenic progenitors isolated from MCK-PGC-1α mice and transplanted into intact and regenerating muscles are more prone to fuse with recipient myofibers than those derived from wild-type donors. Moreover, both young and aged MCK-PGC-1α animals exhibit reduced perilipin-positive areas when challenged with an adipogenic stimulus, demonstrating low propensity to accumulate adipocytes within the muscle. Overall, these results unveil that increased PGC-1α expression in the myofibers favors pro-myogenic and anti-adipogenic cell populations in the skeletal muscle.

Functional Nutrients to Ameliorate Neurogenic Muscle Atrophy.

Neurogenic muscle atrophy is a debilitating condition that occurs from nerve trauma in association with diseases or during aging, leading to reduced interaction between motoneurons and skeletal fibers. Current therapeutic approaches aiming at preserving muscle mass in a scenario of decreased nervous input include physical activity and employment of drugs that slow down the progression of the condition yet provide no concrete resolution. Nutritional support appears as a precious tool, adding to the success of personalized medicine, and could thus play a relevant part in mitigating neurogenic muscle atrophy. We herein summarize the molecular pathways triggered by denervation of the skeletal muscle that could be affected by functional nutrients. In this narrative review, we examine and discuss studies pertaining to the use of functional ingredients to counteract neurogenic muscle atrophy, focusing on their preventive or curative means of action within the skeletal muscle. We reviewed experimental models of denervation in rodents and in amyotrophic lateral sclerosis, as well as that caused by aging, considering the knowledge generated with use of animal experimental models and, also, from human studies.

Determinants of epigenetic resistance to HDAC inhibitors in dystrophic fibro-adipogenic progenitors.

Pharmacological treatment of Duchenne muscular dystrophy (DMD) with histone deacetylase inhibitors (HDACi) is currently being tested in clinical trials; however, pre-clinical studies indicated that the beneficial effects of HDACi are restricted to early stages of disease. We show that FAPs from late-stage mdx mice exhibit aberrant HDAC activity and genome-wide alterations of histone acetylation that are not fully reversed by HDACi. In particular, combinatorial H3K27 and/or H3K9/14 hypo-acetylation at promoters of genes required for cell cycle activation and progression, as well as glycolysis, are associated with their downregulation in late-stage mdx FAPs. These alterations could not be reversed by HDACi, due to a general resistance to HDACi-induced H3K9/14 hyperacetylation. Conversely, H3K9/14 hyper-acetylation at promoters of Senescence Associated Secretory Phenotype (SASP) genes is associated with their upregulation in late-stage mdx FAPs; however, HDACi could reduce promoter acetylation and blunt SASP gene activation. These data reveal that during DMD progression FAPs develop disease-associated features reminiscent of cellular senescence, through epigenetically distinct and pharmacologically dissociable events. They also indicate that HDACi might retain anti-fibrotic effects at late stages of DMD.

Histone Deacetylases as Modulators of the Crosstalk Between Skeletal Muscle and Other Organs.

Skeletal muscle plays a major role in controlling body mass and metabolism: it is the most abundant tissue of the body and a major source of humoral factors; in addition, it is primarily responsible for glucose uptake and storage, as well as for protein metabolism. Muscle acts as a metabolic hub, in a crosstalk with other organs and tissues, such as the liver, the brain, and fat tissue. Cytokines, adipokines, and myokines are pivotal mediators of such crosstalk. Many of these circulating factors modulate histone deacetylase (HDAC) expression and/or activity. HDACs form a numerous family of enzymes, divided into four classes based on their homology to their orthologs in yeast. Eleven family members are considered classic HDACs, with a highly conserved deacetylase domain, and fall into Classes I, II, and IV, while class III members are named Sirtuins and are structurally and mechanistically distinct from the members of the other classes. HDACs are key regulators of skeletal muscle metabolism, both in physiological conditions and following metabolic stress, participating in the highly dynamic adaptative responses of the muscle to external stimuli. In turn, HDAC expression and activity are closely regulated by the metabolic demands of the skeletal muscle. For instance, NAD+ levels link Class III (Sirtuin) enzymatic activity to the energy status of the cell, and starvation or exercise affect Class II HDAC stability and intracellular localization. SUMOylation or phosphorylation of Class II HDACs are modulated by circulating factors, thus establishing a bidirectional link between HDAC activity and endocrine, paracrine, and autocrine factors. Indeed, besides being targets of adipo-myokines, HDACs affect the synthesis of myokines by skeletal muscle, altering the composition of the humoral milieu and ultimately contributing to the muscle functioning as an endocrine organ. In this review, we discuss recent findings on the interplay between HDACs and circulating factors, in relation to skeletal muscle metabolism and its adaptative response to energy demand. We believe that enhancing knowledge on the specific functions of HDACs may have clinical implications leading to the use of improved HDAC inhibitors for the treatment of metabolic syndromes or aging.

Cytoplasmic HDAC4 regulates the membrane repair mechanism in Duchenne muscular dystrophy.

BACKGROUND: Histone deacetylase 4 (HDAC4) is a stress-responsive factor that mediates multiple cellular responses. As a member of class IIa HDACs, HDAC4 shuttles between the nucleus and the cytoplasm; however, HDAC4 cytoplasmic functions have never been fully investigated. Duchenne muscular dystrophy (DMD) is a genetic, progressive, incurable disorder, characterized by muscle wasting, which can be treated with the unspecific inhibition of HDACs, despite this approach being only partially effective. More efficient strategies may be proposed for DMD only after the different HDAC members will be characterized. METHODS: To fully understand HDAC4 functions, we generated dystrophic mice carrying a skeletal muscle-specific deletion of HDAC4 (mdx;KO mice). The progression of muscular dystrophy was characterized in mdx and age-matched mdx;KO mice by means of histological, molecular, and functional analyses. Satellite cells (SCs) from these mice were differentiated in vitro, to identify HDAC4 intrinsic functions influencing the myogenic potential of dystrophic SCs. Gain-of-function experiments revealed the cytoplasmic functions of HDAC4 in mdx;KO muscles. RESULTS: Histone deacetylase 4 increased in the skeletal muscles of mdx mice (~3-fold; P < 0.05) and of DMD patients (n = 3, males, mean age 13.3 ± 1.5 years), suggesting that HDAC4 has a role in DMD. Its deletion in skeletal muscles importantly worsens the pathological features of DMD, leading to greater muscle fragility and degeneration over time. Additionally, it impairs SC survival, myogenic potential, and muscle regeneration, ultimately compromising muscle function (P < 0.05-0.001). The impaired membrane repair mechanism in muscles and SCs accounts for the mdx;KO phenotype. Indeed, the ectopic expression of Trim72, a major player in the membrane repair mechanism, prevents SC death (~20%; P < 0.01) and increases myogenic fusion (~40%; P < 0.01) in vitro; in vivo it significantly reduces myofibre damage (~10%; P < 0.005) and improves mdx;KO muscle function (P < 0.05). The mdx;KO phenotype is also fully rescued by restoring cytoplasmic levels of HDAC4, both in vitro and in vivo. The protective role of HDAC4 in the cytoplasm of mdx;KO muscles is, in part, independent of its deacetylase activity. HDAC4 expression correlates with Trim72 mRNA levels; furthermore, Trim72 mRNA decays more rapidly (P < 0.01) in mdx;KO muscle cells, compared with mdx ones. CONCLUSIONS: Histone deacetylase 4 performs crucial functions in the cytoplasm of dystrophic muscles, by mediating the muscle repair response to damage, an important role in ensuring muscle homeostasis, probably by stabilizing Trim72 mRNA. Consequently, the cytoplasmic functions of HDAC4 should be stimulated rather than inhibited in muscular dystrophy treatments, a fact to be considered in future therapeutic approaches.

Sex and HDAC4 Differently Affect the Pathophysiology of Amyotrophic Lateral Sclerosis in SOD1-G93A Mice.

Amyotrophic Lateral Sclerosis (ALS) is a devastating adult-onset neurodegenerative disease, with ineffective therapeutic options. ALS incidence and prevalence depend on the sex of the patient. Histone deacetylase 4 (HDAC4) expression in skeletal muscle directly correlates with the progression of ALS, pointing to the use of HDAC4 inhibitors for its treatment. Contrarily, we have found that deletion of HDAC4 in skeletal muscle worsened the pathological features of ALS, accelerating and exacerbating skeletal muscle loss and negatively affecting muscle innervations in male SOD1-G93A (SOD1) mice. In the present work, we compared SOD1 mice of both sexes with the aim to characterize ALS onset and progression as a function of sex differences. We found a global sex-dependent effects on disease onset and mouse lifespan. We further investigated the role of HDAC4 in SOD1 females with a genetic approach, and discovered morpho-functional effects on skeletal muscle, even in the early phase of the diseases. The deletion of HDAC4 decreased muscle function and exacerbated muscle atrophy in SOD1 females, and had an even more dramatic effect in males. Therefore, the two sexes must be considered separately when studying ALS.

Metabolic Remodeling in Skeletal Muscle Atrophy as a Therapeutic Target.

Skeletal muscle is a highly responsive tissue, able to remodel its size and metabolism in response to external demand. Muscle fibers can vary from fast glycolytic to slow oxidative, and their frequency in a specific muscle is tightly regulated by fiber maturation, innervation, or external causes. Atrophic conditions, including aging, amyotrophic lateral sclerosis, and cancer-induced cachexia, differ in the causative factors and molecular signaling leading to muscle wasting; nevertheless, all of these conditions are characterized by metabolic remodeling, which contributes to the pathological progression of muscle atrophy. Here, we discuss how changes in muscle metabolism can be used as a therapeutic target and review the evidence in support of nutritional interventions and/or physical exercise as tools for counteracting muscle wasting in atrophic conditions.

HDAC4 degradation during senescence unleashes an epigenetic program driven by AP-1/p300 at selected enhancers and super-enhancers.

BACKGROUND: Cellular senescence is a permanent state of replicative arrest defined by a specific pattern of gene expression. The epigenome in senescent cells is sculptured in order to sustain the new transcriptional requirements, particularly at enhancers and super-enhancers. How these distal regulatory elements are dynamically modulated is not completely defined. RESULTS: Enhancer regions are defined by the presence of H3K27 acetylation marks, which can be modulated by class IIa HDACs, as part of multi-protein complexes. Here, we explore the regulation of class IIa HDACs in different models of senescence. We find that HDAC4 is polyubiquitylated and degraded during all types of senescence and it selectively binds and monitors H3K27ac levels at specific enhancers and super-enhancers that supervise the senescent transcriptome. Frequently, these HDAC4-modulated elements are also monitored by AP-1/p300. The deletion of HDAC4 in transformed cells which have bypassed oncogene-induced senescence is coupled to the re-appearance of senescence and the execution of the AP-1/p300 epigenetic program. CONCLUSIONS: Overall, our manuscript highlights a role of HDAC4 as an epigenetic reader and controller of enhancers and super-enhancers that supervise the senescence program. More generally, we unveil an epigenetic checkpoint that has important consequences in aging and cancer.

Displaced Myonuclei in Cancer Cachexia Suggest Altered Innervation.

An idiopathic myopathy characterized by central nuclei in muscle fibers, a hallmark of muscle regeneration, has been observed in cancer patients. In cancer cachexia skeletal muscle is incapable of regeneration, consequently, this observation remains unaccounted for. In C26-tumor bearing, cachectic mice, we observed muscle fibers with central nuclei in the absence of molecular markers of bona fide regeneration. These clustered, non-peripheral nuclei were present in NCAM-expressing muscle fibers. Since NCAM expression is upregulated in denervated myofibers, we searched for additional makers of denervation, including AchRs, MUSK, and HDAC. This last one being also consistently upregulated in cachectic muscles, correlated with an increase of central myonuclei. This held true in the musculature of patients suffering from gastrointestinal cancer, where a progressive increase in the number of central myonuclei was observed in weight stable and in cachectic patients, compared to healthy subjects. Based on all of the above, the presence of central myonuclei in cancer patients and animal models of cachexia is consistent with motor neuron loss or NMJ perturbation and could underlie a previously neglected phenomenon of denervation, rather than representing myofiber damage and regeneration in cachexia. Similarly to aging, denervation-dependent myofiber atrophy could contribute to muscle wasting in cancer cachexia.

Thyroid Hormone Protects from Fasting-Induced Skeletal Muscle Atrophy by Promoting Metabolic Adaptation.

Thyroid hormones regulate a wide range of cellular responses, via non-genomic and genomic actions, depending on cell-specific thyroid hormone transporters, co-repressors, or co-activators. Skeletal muscle has been identified as a direct target of thyroid hormone T3, where it regulates stem cell proliferation and differentiation, as well as myofiber metabolism. However, the effects of T3 in muscle-wasting conditions have not been yet addressed. Being T3 primarily responsible for the regulation of metabolism, we challenged mice with fasting and found that T3 counteracted starvation-induced muscle atrophy. Interestingly, T3 did not prevent the activation of the main catabolic pathways, i.e., the ubiquitin-proteasome or the autophagy-lysosomal systems, nor did it stimulate de novo muscle synthesis in starved muscles. Transcriptome analyses revealed that T3 mainly affected the metabolic processes in starved muscle. Further analyses of myofiber metabolism revealed that T3 prevented the starvation-mediated metabolic shift, thus preserving skeletal muscle mass. Our study elucidated new T3 functions in regulating skeletal muscle homeostasis and metabolism in pathological conditions, opening to new potential therapeutic approaches for the treatment of skeletal muscle atrophy.

Interplay between Metabolites and the Epigenome in Regulating Embryonic and Adult Stem Cell Potency and Maintenance.

The environment surrounding stem cells has the ability to elicit profound, heritable epigenetic changes orchestrated by multiple epigenetic mechanisms, which can be modulated by the level of specific metabolites. In this review, we highlight the significance of metabolism in regulating stem cell homeostasis, cell state, and differentiation capacity, using metabolic regulation of embryonic and adult muscle stem cells as examples, and cast light on the interaction between cellular metabolism and epigenetics. These new regulatory networks, based on the dynamic interplay between metabolism and epigenetics in stem cell biology, are important, not only for understanding tissue homeostasis, but to determine in vitro culture conditions which accurately support normal cell physiology.

The Mechanical Stimulation of Myotubes Counteracts the Effects of Tumor-Derived Factors Through the Modulation of the Activin/Follistatin Ratio.

Activin negatively affects muscle fibers and progenitor cells in aging (sarcopenia) and in chronic diseases characterized by severe muscle wasting (cachexia). High circulating activin levels predict poor survival in cancer patients. However, the relative impact of activin in mediating muscle atrophy and hampered homeostasis is still unknown. To directly assess the involvement of activin, and its physiological inhibitor follistatin, in cancer-induced muscle atrophy, we cultured C2C12 myotubes in the absence or in the presence of a mechanical stretching stimulus and in the absence or presence of C26 tumor-derived factors (CM), so as to mimic the mechanical stimulation of exercise and cancer cachexia, respectively. We found that CM induces activin release by myotubes, further exacerbating the negative effects of tumor-derived factors. In addition, mechanical stimulation is sufficient to counteract the adverse tumor-induced effects on muscle cells, in association with an increased follistatin/activin ratio in the cell culture medium, indicating that myotubes actively release follistatin upon stretching. Recombinant follistatin counteracts tumor effects on myotubes exclusively by rescuing fusion index, suggesting that it is only partially responsible for the stretch-mediated rescue. Therefore, besides activin, other tumor-derived factors may play a significant role in mediating muscle atrophy. In addition to increasing follistatin secretion mechanical stimulation induces additional beneficial responses in myotubes. We propose that in animal models of cancer cachexia and in cancer patients purely mechanical stimuli play an important role in mediating the rescue of the muscle homeostasis reported upon exercise.

The JAK/STAT Pathway in Skeletal Muscle Pathophysiology.

The Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway is a key intracellular mediator of a variety of metabolically relevant hormones and cytokines, including the interleukin-6 (IL-6) family of cytokines. The JAK/STAT pathway transmits extracellular signals to the nucleus, leading to the transcription of genes involved in multiple biological activities. The JAK/STAT pathway has been reported to be required for the homeostasis of different tissues and organs. Indeed, when deregulated, it promotes the initiation and progression of pathological conditions, including cancer, obesity, diabetes, and other metabolic diseases. In skeletal muscle, activation of the JAK/STAT pathway by the IL-6 cytokines accounts for opposite effects: on the one hand, it promotes muscle hypertrophy, by increasing the proliferation of satellite cells; on the other hand, it contributes to muscle wasting. The expression of IL-6 and of key members of the JAK/STAT pathway is regulated at the epigenetic level through histone methylation and histone acetylation mechanisms. Thus, manipulation of the JAK/STAT signaling pathway by specific inhibitors and/or drugs that modulate epigenetics is a promising therapeutic intervention for the treatment of numerous diseases. We focus this review on the JAK/STAT pathway functions in striated muscle pathophysiology and the potential role of IL-6 as an effector of the cross talk between skeletal muscle and other organs.