Being the most abundant tissue, skeletal muscle comprises 40–50% of the total mass in healthy-weight individuals and regarded as the protein reservoir in the human body. It acts as a vital organ for metabolic homeostasis, physical performance and for maintaining a high quality of life. To estimate health loss from death and disability due to musculoskeletal disorders, the Global Burden of Disease (GBD) 2020 data revealed that musculoskeletal disorders were the second-highest cause of non-fatal disability, and more than 1.63 billion people live with musculoskeletal impairments worldwide [1]. Skeletal muscle atrophy is a global health burden and highly prevalent in several chronic pathophysiological conditions, such as obesity, diabetes, cancer cachexia, chronic obstructive pulmonary disease, cardiovascular diseases, renal failure, and aging. Impairment in skeletal muscle homeostasis and decline in skeletal muscle strength and function negatively affect everyday life with increased morbidity and mortality. Physical activity, exercises, healthy diet and nutritional supplements are widely acknowledged to prevent skeletal muscle atrophy but till date there is no promising treatment strategy. Therefore, it is very crucial to discover novel therapeutics to combat skeletal muscle diseases.

Skeletal muscle undergoes remodeling from birth until death to adapt stimuli such as contractile activity, resistance, exercise, endurance training, innervation, denervation, and hypoxia. This plasticity nature allows physiological changes to alter the structural and functional properties of skeletal muscle to adapt itself for the imposed environmental conditions. Skeletal muscle has high regenerative capacity to repair damaged tissue and regenerate new muscle fibers upon injury or consequence of degenerative diseases. Muscle stem cells or satellite cells in adult skeletal muscle differentiate into myofibers during muscle growth, repair (exercise or trauma), injury or aging due to their high proliferative and myogenic nature (Figure 1). Recently, skeletal muscle as secretory organ is an emerging area of research as the muscle fiber releases hormones, myokines, proteins, peptides, miRNAs, and metabolites to exert its effects through autocrine/paracrine and/or endocrine mechanisms.

Figure 1. Role of miRNA and myokines in regulating key pathways in skeletal muscle.

Skeletal muscle secretes myokines (cytokines or peptides) which crosstalk within itself or with other organs such as brain, liver, skin, bone, adipose tissue, etc. Myokines regulate oxidative stress, redox homeostasis, inflammation, mitochondrial function, and metabolic balance across multiple organs [2]. Irisin, decorin, FGF21, BDNF, myonectin are myokines which positively regulate muscle mass, strength and function by stimulating PI3K/Akt/mTOR pathway, have been evidenced to promote hypertrophy whereas myostatin, IL-6, IL-15 negatively regulate the muscle mass by stimulating Smad2/3 or FoxO pathway to induce atrophy [3].

Exercise induces PGC-1α expression in skeletal muscle which in turn stimulates the secretion of irisin from skeletal muscle into the bloodstream. The physiological level of irisin decreases with age but irisin expression is reported to be higher in elderly high-fitness men than in elderly low-fitness men [4]. Exercise-induced irisin and supplementation of recombinant irisin might be a successful strategy to ameliorate muscle atrophy, obesity, osteoporosis, liver injury, cardiovascular diseases, age-associated sarcopenia and metabolic dysfunction [5,6]. Irisin induces myogenic differentiation and myoblast fusion by increasing expression of a number of pro-myogenic and exercise response genes in myotubes via activation of IL-6 signaling pathway. In addition, irisin supplementation induces hypertrophy, enhances grip strength of uninjured muscle, repairs damaged fibers, rescues loss of skeletal muscle mass in case of muscle injury and improves regeneration by enhancing protein synthesis pathway (Akt/mTOR and Erk1/2 pathway), satellite cell activation and reducing protein degradation (ubiquitin proteosome pathway, including Atrogin-1 and MuRF-1) pathway [7].

Myostatin downregulates PI3K/Akt/mTOR pathway and decreases skeletal muscle protein synthesis. It negatively regulates the myogenic differentiation, proliferation, growth and development by stimulating myodegradation mediated by ubiquitin, autophagy-lysosomal system, FoxO-dependent, and Smad2/3/4 mediated protein degradation pathway [8]. Myostatin induces the expression of atrogin-1, MuRF-1, and several atrogenes that inhibits the development of skeletal muscle fibers by limiting the number and size of muscle fibers. Myostatin level is found to be elevated in the serum of individuals suffering from obesity, type 2 diabetes mellitus, cancer cachexia, sarcopenia and heart failure [9]. Regulating skeletal muscle mass through myostatin inhibition is emerging as a therapeutic tool for the treatment of skeletal muscle loss/atrophy. Both preclinical and clinical approaches that inhibit the myostatin signaling pathway can enhance the muscle mass and combat muscle wasting associated with cancer and other disorders. Many interventions have been exploited to block myostatin either by directly binding to myostatin itself or by indirectly binding to its receptor complex [10]. Myostatin inhibition induces skeletal muscle hypertrophy, improves the muscle strength, and increases the mass and the cross-sectional area of muscle fibers [11]. Our findings on healthy Indian participants revealed that myostatin level was significantly increased and irisin level was significantly decreased during chronic exposure as compared to sea level participants, indicating a shift in muscle homeostasis and increased muscle degradation in hypobaric hypoxia (HH) environments [12]. After 2-week exposure to HH, there was significant decline in body mass, BMI, fat free mass, decreased irisin and increased myostatin serum concentrations in high-altitude male climbers [13]. Therefore, understanding the signaling pathways/mechanisms regulating muscle growth, regeneration as well as repair is very essential to develop novel strategies to ameliorate skeletal muscle atrophy.

Another key mediator which governs skeletal muscle development and repair are miRNAs, the small non-coding (approximately 18–25 nucleotides) RNAs. Recent research in miRNA indicates their role as biomarkers and therapeutic targets in skeletal muscle diseases [14]. The most studied myomiRs (miR-1, miR-206, miR-133) and a subset of non-muscle-specific miRNAs (miR-29 family, miR-320 miR-23a/b clusters, miR-486-5p) are differentially expressed during developmental stages, differentiation, proliferation, repair, and pathological diseases such as muscular dystrophy, cancer cachexia, sarcopenia [15–17]. Myokines and miRNA also have a crucial role in high-altitude induced skeletal muscle loss [18–20]. Thus, recent evidence highlights a complex and bidirectional interplay between skeletal muscle myokines and miRNAs. This article explores the novel strategy of myokines and miRNA to develop therapeutics for various skeletal muscle-related diseases.

Recently, miRNA therapeutics such as miRNA mimics (agomiRs) and molecules targeting/inhibiting the miRNAs (antimiRs or antagomiRs) have shown promising results in targeting the key proteins regulating skeletal muscle diseases. The antagomiR-based inhibition of miR-1/miR-206 resulted in decreased expression of PAX3 (target gene), loss of myogenin expression and delayed myogenesis [21]. Also, the expression of miR-127 and miR-22-3p gets upregulated and downregulated respectively to promote myoblast proliferation in vitro. The repeated intramuscular administration of miR-127 mimic (agomiR-127) and antagomiR-22-3p upregulated mTOR expression to promote protein synthesis in vivo [22].

Hence, this therapeutic strategy offers completely new possibilities for gene modification, cell therapy, and drug development for the treatment of diseases.

In this article, miRNAs (miR-1 and miR-320) that play major role in maintaining skeletal muscle homeostasis have been shortlisted. Using miRDB 6.0, miRWalk 2.0, miRTarBase 10.0, and TargetScan 8.0 version databases, gene targets of miR-1 and miR-320 were searched till February 2026 and retrieved in Homo sapiens and Rattus norvegicus (Table 1). Using Literature survey, known myokine genes were screened. Out of total number of gene targets, myokines have been screened for hsa-miR-1, rno-miR-1, hsa-miR-320 and rno-miR-320 as shown in Tables 2 and 3. Schematic workflow for the identification and screening of myokine-targeting miRNAs is shown in Figure 2. After thorough scrutiny, 20 myokines for hsa-miR-1, 16 myokines for rno-miR-1, 33 myokines for hsa-miR-320 and 29 myokines for rno-miR-320 were found as target genes of their respective regulating miRNAs (Table 3). The interactions were imported into Cytoscape 3.10.2 version software for regulatory network construction to identify highly regulated myokines by miR-1 and miR-320 (Figure 3). This crosstalk of miRNAs (miR-1 and miR-320) and myokines would in turn help to decipher novel therapeutic targets for skeletal muscle diseases.

Figure 2. Bioinformatics workflow for the identification and screening of myokine-targeting miRNAs.

Figure 3. Crosstalk of miRNAs (miR-1 and miR-320) and myokines to decipher novel therapeutic targets for skeletal muscle diseases using Cytoscape 3.10.2 version software. (a) Visualization of hsa-miR-1 and 20 myokines network. (b) Representation of rno-miR-1 and 16 myokines network. (c) Visualization of hsa-miR-320 and 33 myokines network. (d) Representation of rno-miR-320 and 29 myokines using Cytoscape v.3.10.2.

Using in silico tools, we observed FNDC5 (irisin, a positive regulator of skeletal muscle mass) as target gene for hsa-miR-1 and hsa-miR-320; and MSTN (myostatin, a negative regulator of skeletal muscle mass) as target gene for hsa-miR-1 and rno-miR-320, which might have regulatory mechanisms to uplift or suppress compromised muscle maintenance. The miRNAs regulating crucial myokines, irisin and myostatin, might play dynamic role in maintaining skeletal muscle homeostasis. Further, experimental validation is required to decode the significant role of these candidate myokines (targets of miR-1 and miR-320) using quantitative real-time PCR (qRT-PCR), luciferase reporter assays, and gain-/loss-of-function experiments employing miRNA mimics or antagomiRs.

Further, our earlier studies have experimentally validated miRNA-myokine interactions under normal physiological conditions as well as stress environments. We observed significantly increased expressions of miR-1, miR-320, myostatin, IL-6, IL-15, and decreased expressions of irisin, apelin, decorin, osteocrin, Meteorin-like myokines under HH [18,19]. The altered expressions of miRNAs and myokines under HH negatively affected muscle adaptive responses, modulated myogenesis, metabolic signaling and upregulated protein degradation contributing to muscle loss.

Despite extensive knowledge of miRNA function, the gene-mediated regulation of miRNA expression or precise gene-level mechanisms governing miRNA transcription and maturation under tissue-specific and stress-induced conditions remain insufficiently studied. A deeper understanding of miRNA-myokine interactions might offer promising opportunities for therapeutic interventions and biomarker development. This attempt might unravel various ways to discover novel therapeutics against skeletal muscle diseases, and to counteract metabolic dysfunction, inflammation, skeletal muscle atrophy and impaired muscle degeneration.

Although the myokines and miRNA are novel candidates for therapeutics, another important aspect of research is, miRNA-myokine interaction and regulation. Can a miRNA target myokine? Can a myokine regulate the expression of a particular miRNA?

This hypothetical idea can unravel a new paradigm in skeletal muscle research with relevant experimental validation (Figure 4). Myokines and miRNAs act as potent modulators of metabolism, muscle homeostasis, regeneration, skeletal muscle remodeling, inflammation, and inter-organ communication. This myokine-miRNA network may be associated with various molecular responses that promote or alter metabolic flexibility and reinforce or suppress pathological signaling to govern systemic health. Despite the recognized role of miRNAs (or myomiRs) and myokines in skeletal muscle homeostasis, the gene-level and epigenetic mechanisms regulating their expressions under hypoxia and atrophy-associated conditions remain largely unresolved. Several bioinformatic approaches help in screening the targets regulated by miRNAs, predict the miRNA-mRNA binding sites, affinity, detect the true miRNA-mRNA interactions, and determine miRNA-based gene regulatory effects. This may bridge a gap between computational tools and experimental validation to understand the precise function of miRNAs.

Figure 4. Crosstalk of miRNA and myokines to decipher novel therapeutic targets for skeletal muscle diseases.

Also, in silico human and rat miRNA–myokine interaction networks reveal that several miRNAs target the same myokines across both species, while some miRNAs exhibit species-specific targeting patterns. These differences likely arise from a combination of genuine biological divergence, disparities in database annotation and coverage, and the inherent limitations of computational target prediction algorithms. At the same time, human miRNA databases are generally more comprehensive and experimentally validated than those for rat, leading to differences in the number and confidence of reported interactions. Nevertheless, the identification of conserved miRNA–myokine interactions across species is particularly valuable, as these are more likely to represent fundamental regulatory mechanisms preserved through evolution. Such conserved interactions provide stronger candidates for biomarker discovery and therapeutic intervention than species-specific predictions. Based on earlier reported preclinical studies, irisin and myostatin regulating miRNAs have been listed in Table 4.

We have comprehensively reviewed the role of myokines in redox homeostasis, muscle proteostasis and mitochondrial dynamics affecting the skeletal muscle health and function [23]. Irisin and myostatin represent two complementary therapeutic targets for miRNA-based intervention in skeletal muscle disorders. Irisin, a cleaved product of the FNDC5 gene, promotes mitochondrial biogenesis, myogenesis, angiogenesis, and oxidative metabolism, making it an attractive target for treating sarcopenia, cachexia, muscular dystrophy, obesity, and metabolic diseases. Several miRNAs, including miR-214-3p, miR-665-3p, miR-150, miR-758, miR-668, miR-199a, miR-135a-5p, miR-337-3p, miR-34a, have been reported to suppress FNDC5/irisin expression; therefore, inhibition of these miRNAs using antagomiRs may restore irisin levels and improve muscle regeneration and metabolic function [24–32]. In contrast, myostatin is a potent negative regulator of skeletal muscle growth, and therapeutic inhibition of myostatin signaling has emerged as a promising strategy for preventing muscle wasting. miRNAs such as miR-1, miR-133a/b, miR-206, miR-27a/b, miR-486, directly or indirectly suppress myostatin expression or its downstream signaling, thereby enhancing muscle hypertrophy, satellite cell activation, and protein synthesis [33–38]. Targeting miRNA-regulating myokines or myokine-regulating miRNAs has emerged as a promising therapeutic strategy for skeletal muscle disorders; however, several translational challenges must translation depends on overcoming challenges related to tissue-specific delivery, improving the safety and efficiency of viral and non-viral delivery systems, minimizing off-target effects, toxicity, and identifying miRNAs with high specificity for beneficial myokine regulatory networks. Advances in muscle-targeted lipid nanoparticles, engineered viral vectors, multi-omics technologies, and precision medicine approaches are expected to facilitate the development of safer and more effective miRNA-based therapies for skeletal muscle atrophy, sarcopenia, cachexia, and other muscle-related disorders. These pleiotropic effects highlight the need for comprehensive transcriptomic, proteomic, pharmacokinetics, immune activation, and toxicological studies to evaluate safety before clinical application. Most miRNA-based approaches remain in the preclinical stage while several myostatin-targeting biologics (monoclonal antibodies and ActRII receptor inhibitors) have advanced into Phase I-III clinical trials for conditions such as obesity, spinal muscular dystrophy, sarcopenia, and muscle-wasting disorders. This highlights the role of myostatin as the most clinically advanced myokine target. Current status on development of irisin- and myostatin-based therapies has been summarized in Table 5.

As myokines are key players in myogenesis and myodegradation, miRNAs which regulate these myokines are very crucial for developing therapeutics. Hence, miRNA and myokine interaction is a pivotal research area to be exploited. The field of miRNA-based therapeutics still faces key challenges in establishing specificity and sensitivity towards their intended targets. Further, optimal dosing, stability, off-target effects and targeted delivery with minimal toxicity are also crucial concerns to be addressed. Future research should focus on integrating artificial intelligence (AI)-assisted target prediction of miRNA with multi-omics approaches to improve the discovery of clinically relevant miRNA–myokine regulatory networks. Advanced machine learning and deep learning algorithms can integrate RNA secondary structure, binding affinity of myokines and miRNAs, sequence characteristics, evolutionary conservation, and experimentally validated interactions to prioritize high-confidence miRNA–myokine pairs while minimizing false-positive predictions. Therefore, understanding the pivotal role of miRNA–myokine network in skeletal muscle is essential for identifying novel therapeutic targets for chronic diseases such as sarcopenia, cachexia, and muscular dystrophy to reduce global health burden and better quality of life.

The authors declare no conflicts of interest.