Regulatory role of N6-Methyladenosine on skeletal muscle development in Hu sheep.

N6-Methyladenosine (m6A) RNA modification plays an essential role in many biological processes. To investigate the regulatory role of m6A on the skeletal muscle development in Hu sheep, this study took newborn Hu sheep (b_B Group) and six-month-old Hu sheep (s_B Group) as the objects. MeRIP-seq and RNA-Seq analysis techniques were used to detect differentially methylated genes (DMGs) and differentially expressed genes (DEGs) in the longissimus dorsi muscle of Hu sheep at different months of age. Then, conjoint analysis was further employed to screen for key genes involved in skeletal muscle development that are modified by m6A and expressed by mRNA. According to the results of the MeRIP-seq analysis, there were 285 m6A differentially methylated peaks (DMPs) in total between b_B Group and s_B Group, with 192 significant upregulated peaks and 93 significant downregulated peaks. GO and KEGG analysis revealed that DMGs are mainly enriched in actin-binding, cellular transport, and metabolic pathways. According to the results of the RNA-seq analysis, there were 4,349 DEGs in total between b_B Group and s_B Group, with 2010 upregulated genes and 2,339 downregulated genes. DEGs are found to be mainly enriched in the regulation of actin cytoskeleton tissue, AMPK and FoxO signaling pathways, etc. The conjoint analysis demonstrated that 283 genes were both modified by m6A and expressed by mRNA. Among them, three genes relevant to muscle growth (RGMB, MAPK8IP3, and RSPO3) were selected as candidates for quantitative validation, and the results were in line with the sequencing results. The results mentioned above all suggest that m6A plays a certain role in the skeletal muscle development in Hu sheep.

Joint segmentation of sternocleidomastoid and skeletal muscles in computed tomography images using a multiclass learning approach.

Deep-learning-based methods can improve robustness against individual variations in computed tomography (CT) images of the sternocleidomastoid muscle, which is a challenge when using conventional methods based on probabilistic atlases are used for automatic segmentation. Thus, this study proposes a novel multiclass learning approach for the joint segmentation of the sternocleidomastoid and skeletal muscles in CT images, and it employs a two-dimensional U-Net architecture. The proposed method concurrently learns and segmented segments the sternocleidomastoid muscle and the entire skeletal musculature. Consequently, three-dimensional segmentation results are generated for both muscle groups. Experiments conducted on a dataset of 30 body CT images demonstrated segmentation accuracies of 82.94% and 92.73% for the sternocleidomastoid muscle and entire skeletal muscle compartment, respectively. These results outperformed those of conventional methods, such as the single-region learning of a target muscle and multiclass learning of specific muscle pairs. Moreover, the multiclass learning paradigm facilitated a robust segmentation performance regardless of the input image range. This highlights the method's potential for cases that present muscle atrophy or reduced muscle strength. The proposed method exhibits promising capabilities for the high-accuracy joint segmentation of the sternocleidomastoid and skeletal muscles and is effective in recognizing skeletal muscles, thus, it holds promise for integration into computer-aided diagnostic systems for comprehensive musculoskeletal analysis. These findings are expected to enhance medical image analysis techniques and their applications in clinical decision support systems.

Valsartan Rescues Suppressed Mitochondrial Metabolism during Insulin Resistance in C2C12 Myotubes.

Elevated circulating branched-chain amino acids (BCAA) have been linked with the severity of insulin resistance across numerous populations, implicating heightened BCAA metabolism as a potential therapy for insulin resistance. Recently, the angiotensin II type 1 receptor (AT1R) inhibitor Valsartan (VAL) was identified as a potent inhibitor of branched-chain alpha-keto acid dehydrogenase kinase (BCKDK), a negative regulator of BCAA metabolism. This work investigated the effect of VAL on myotube metabolism and insulin sensitivity under both insulin sensitive and insulin resistant conditions. C2C12 myotubes were treated with or without VAL at 8 µM for 24 h, both with and without hyperinsulinemic-induced insulin resistance. Oxygen consumption and extracellular acidification were used to measure mitochondrial and glycolytic metabolism, respectively. Gene expression was assessed via qRT-PCR, and insulin sensitivity was assessed via Western blot. Insulin resistance significantly reduced both basal and peak mitochondrial function which were rescued to control levels by concurrent VAL. Changes in mitochondrial function occurred without substantial changes in mitochondrial content or related gene expression. Insulin sensitivity and glycolytic metabolism were unaffected by VAL, as was lipogenic signaling and lipid content. Additionally, both VAL and insulin resistance depressed Bckdha expression. Interestingly, an interaction effect was observed for extracellular isoleucine, valine, and total BCAA (but not leucine), suggesting VAL may alter BCAA utilization in an insulin sensitivity-dependent manner. Insulin resistance appears to suppress mitochondrial function in a myotube model which can be rescued by VAL. Further research will be required to explore the implications of these findings in more complex models.

Functional study of the ST6GAL2 gene regulating skeletal muscle growth and development.

ST6GAL2, a member of the sialoglycosyltransferase family, primarily localizes within the cellular Golgi apparatus. However, the role of the ST6GAL2 gene in skeletal muscle growth and development remains elusive. In this study, the impact of the ST6GAL2 gene on the proliferation, differentiation, and apoptosis of primary chicken myoblasts at the cellular level was investigated. Quantitative fluorescent PCR was used to measure the expression levels of genes. Subsequently, using gene knockout mice, we assessed its effects on skeletal muscle growth and development in vivo. Our findings reveal that the ST6GAL2 gene promotes the expression of cell cycle and proliferation-related genes, including CCNB2 and PCNA, and apoptosis-related genes, such as Fas and Caspase-9. At the individual level, double knockout of ST6GAL2 inhibited the formation of both fast and slow muscle fibers in the quadriceps, extensor digitorum longus, and tibial anterior muscle, while promoting their formation in the gastrocnemius and soleus. These results collectively demonstrate that the ST6GAL2 gene facilitates the proliferation, apoptosis, and fusion processes of primary chicken myoblasts. Additionally, it promotes the enlargement of cross-sectional muscle fiber areas and regulates the formation of fast and slow muscle fibers at the individual level, albeit inhibiting muscle fusion. This study provides valuable insights into the role of the ST6GAL2 gene in promoting proliferation of skeletal muscle.

A Skeletal Muscle-Mediated Anticontractile Response on Vascular Tone: Unraveling the Lactate-AMPK-NOS1 Pathway in Femoral Arteries.

The regulation of vascular tone by perivascular tissues is a complex interplay of various paracrine factors. Here, we investigate the anti-contractile effect of skeletal muscle surrounding the femoral and carotid arteries and its underlying mechanisms. Using male and female Wistar rats, we demonstrated that serotonin, phenylephrine, and U-46619 induced a concentration-dependent vasoconstrictor response in femoral artery rings. Interestingly, this response was diminished in the presence of surrounding femoral skeletal muscle, irrespective of sex. No anti-contractile effect was observed when the carotid artery was exposed to its surrounding skeletal muscle. The observed effect in the femoral artery persisted even in the absence of endothelium and when the muscle was detached from the artery. Furthermore, the skeletal muscle surrounding the femoral artery was able to promote an anti-contractile effect in three other vascular beds (basilar, mesenteric, and carotid arteries). Using inhibitors of lactate dehydrogenase and the 1/4 monocarboxylate transporter, we confirmed the involvement of lactate, as both inhibitors were able to abolish the anti-contractile effect. However, lactate did not directly promote vasodilation; rather, it exerted its effect by activating 5' AMP-activated protein kinase (AMPK) and neuronal nitric oxide synthase (NOS1) in the skeletal muscle. Accordingly, Nω-propyl L-arginine, a specific inhibitor of NOS1, prevented the anti-contractile effect, as well as lactate-induced phosphorylation of NOS1 at the stimulatory serine site (1417) in primary skeletal muscle cells. Phosphorylation of NOS1 was reduced in the presence of Bay-3827, a selective AMPK inhibitor. In conclusion, femoral artery-associated skeletal muscle is a potent paracrine and endocrine organ that influences vascular tone in both sexes. Mechanistically, the anti-contractile effect involves muscle fiber type and/or its anatomical location but not the type of artery or its related vascular endothelium. Finally, the femoral artery anti-contractile effect is mediated by the lactate-AMPK-phospho-NOS1Ser1417-NO signaling axis.

Subcutaneous adipose tissue and skeletal muscle mitochondria following weight loss.

Obesity is a major global health issue with various metabolic complications. Both bariatric surgery and dieting achieve weight loss and improve whole-body metabolism, but vary in their ability to maintain these improvements over time. Adipose tissue and skeletal muscle metabolism are crucial in weight regulation, and obesity is linked to mitochondrial dysfunction in both tissues. The impact of bariatric surgery versus dieting on adipose tissue and skeletal muscle mitochondrial metabolism remains to be elucidated. Understanding the molecular pathways that modulate tissue metabolism following weight loss holds potential for identifying novel therapeutic targets in obesity management. This narrative review summarizes current knowledge on mitochondrial metabolism following bariatric surgery and diet-induced weight loss in adipose tissue and skeletal muscle, and sheds light on their respective effects.

Regulatory role of lncMD1 in goat skeletal muscle satellite cell differentiation via miR-133a-3p and miR-361-3p targeting.

Skeletal muscle satellite cells (SMSCs) are pivotal in skeletal muscle development and are influenced by numerous regulatory factors. This study focuses on the regulatory and functional mechanism roles of lncMD1, a muscle-specific long non-coding RNA, in the proliferation and differentiation of goat SMSCs. Employing in vitro cultured goat SMSCs, this study demonstrated that lncMD1, functions as a decoy for miR-133a-3p and miR-361-3p. This interaction competitively binds these microRNAs to modulate the expression of dynactin subunit 2 (DCTN2) and dynactin subunit 1 (DCTN1), thereby affects SMSCs proliferation and differentiation. These findings enhance the understanding of non-coding RNAs in goat SMSCs growth cycles and offer a theoretical foundation for exploring the molecular processes of goat skeletal muscle myogenic development.

Myotube Guidance: Shaping up the Musculoskeletal System.

Myofibers are highly specialized contractile cells of skeletal muscles, and dysregulation of myofiber morphogenesis is emerging as a contributing cause of myopathies and structural birth defects. Myotubes are the myofiber precursors and undergo a dramatic morphological transition into long bipolar myofibers that are attached to tendons on two ends. Similar to axon growth cones, myotube leading edges navigate toward target cells and form cell-cell connections. The process of myotube guidance connects myotubes with the correct tendons, orients myofiber morphology with the overall body plan, and generates a functional musculoskeletal system. Navigational signaling, addition of mass and volume, and identification of target cells are common events in myotube guidance and axon guidance, but surprisingly, the mechanisms regulating these events are not completely overlapping in myotubes and axons. This review summarizes the strategies that have evolved to direct myotube leading edges to predetermined tendon cells and highlights key differences between myotube guidance and axon guidance. The association of myotube guidance pathways with developmental disorders is also discussed.

Ginkgolic acid regulates myogenic development by influencing the proliferation and differentiation of C2C12 myoblast cells.

Ginkgolic acid (GA), isolated from the leaves and seed coats of Ginkgo biloba, exerts several biological effects, including antitumor, antibacterial, anti­HIV and anti­inflammatory effects. However, the effects of GA on C2C12 myoblasts remain unclear. The present study assessed cell viability with the MTT assay and evaluated colony formation through crystal violet staining. Flow cytometry was used to analyze apoptosis with Annexin V/7­AAD staining, proliferation with Ki67 staining and cell cycle arrest. Western blotting detected myogenic markers and other relevant proteins. Myotube formation was examined by immunofluorescence, and autophagy was measured using an LC3 antibody­based kit via flow cytometry. The present study showed that treatment of C2C12 cells with GA significantly inhibited their viability and colony formation capacity but did not trigger apoptosis, as indicated by Annexin V/7­AAD staining. However, Ki67 staining indicates that GA exerted dose­dependent antiproliferative effects. Further analysis revealed that GA partially inhibited the growth of C2C12 cells via cell cycle arrest in S phase, highlighting its role in the disruption of cell proliferation. Furthermore, treatment with GA impaired myoblast differentiation, as evidenced by a reduction in the expression of the myogenesis markers, the myosin­heavy chain, myoblast determination protein 1 and myogenin, and suppressed myotube formation. Notably, during C2C12 cell differentiation, GA promoted apoptosis without affecting cell cycle progression or Ki67 expression. Mechanistically, GA could suppress nuclear extracellular signal­regulated kinase phosphorylation, suggesting that it modulates cell proliferation pathways. Moreover, GA triggered autophagy in differentiated C2C12 cells, as confirmed by elevated LC3 II levels. These findings highlight the multifaceted effects of GA on C2C12 cells.

Muscle endurance, neuromuscular fatigability, and cognitive control during prolonged dual-task in people with chronic obstructive pulmonary disease: a case-control study.

PURPOSE: Recent studies suggest that, compared to healthy individuals, people with chronic obstructive pulmonary disease (pwCOPD) present a reduced capacity to perform cognitive-motor dual-task (CMDT). However, these studies were focused on short-duration CMDT offering limited insight to prolonged CMDT inducing fatigue, which can be encountered in daily life. The present study aimed to explore the effect of adding a cognitive task during repeated muscle contractions on muscle endurance, neuromuscular fatigability, and cognitive control in pwCOPD compared to healthy participants. METHODS: Thirteen pwCOPD and thirteen age- and sex-matched healthy participants performed submaximal isometric contractions of the knee extensors until exhaustion in two experimental sessions: (1) without cognitive task and (2) with a concurrent working memory task (i.e., 1-back task). Neuromuscular fatigability (as well as central and peripheral components measured by peripheral magnetic stimulation), cognitive performance, and perceived muscle fatigue were assessed throughout the fatiguing tasks. RESULTS: Independently to the experimental condition, pwCOPD exhibited lower muscle endurance compared to healthy participants (p = 0.039), mainly explained by earlier peripheral fatigue and faster attainment of higher perceived muscle fatigue (p < 0.05). However, neither effect of cognitive task (p = 0.223) nor interaction effect (group × condition; p = 0.136) was revealed for muscle endurance. Interestingly, cognitive control was significantly reduced only in pwCOPD at the end of CMDT (p < 0.015), suggesting greater difficulty for patients with dual tasking under fatigue. CONCLUSION: These findings provide novel insights into how and why fatigue develops in COPD in dual-task context, offering a rationale for including such tasks in rehabilitation programs.

Diffusion Tensor Imaging of Rat Rotator Cuff Muscle with Histopathological Correlation: An Exploratory Study.

Diffusion tensor imaging (DTI) is a magnetic resonance imaging (MRI) technique that can be used to assess microstructural features of skeletal muscle that are related to tissue function. Although widely used, direct correlations between DTI derived metrics such as fractional anisotropy and spatially matched tissue microstructure assessed with histology have not been performed. This study investigated the relationship between scalar-based DTI measurements and histologically derived muscle microstructural measurements in rat rotator cuff muscles. Despite meticulous co-localization of MRI and histology data, negligible correlations were found between DTI metrics and histological measurements including muscle fiber diameter, cross-sectional area, and surface-to-volume ratio. These findings highlight the challenges in validating DTI with histology due to requirements in anatomical co-localization, necessity of high-quality histology, and consideration of diffusion measurement scales. Our findings underscore the need for further research with optimized imaging parameters to enhance our knowledge regarding the sensitivity of DTI to important features of muscle microstructure.

Effects of thyroid hormones in skeletal muscle protein turnover.

Thyroid hormones (THs) are critical regulators of muscle metabolism in both healthy and unhealthy conditions. Acting concurrently as powerful anabolic and catabolic factors, THs are endowed with a vital role in muscle mass maintenance. As a result, thyroid dysfunctions are the leading cause of a wide range of muscle pathologies, globally identified as myopathies. Whether muscle wasting is a common feature in patients with hyperthyroidism and is mainly caused by THs-dependent stimulation of muscle proteolysis, also muscle growth is often associated with hyperthyroid conditions, linked to THs-dependent stimulation of muscle protein synthesis. Noteworthy, also hypothyroid status negatively impacts on muscle physiology, causing muscle weakness and fatigue. Most of these symptoms are due to altered balance between muscle protein synthesis and breakdown. Thus, a comprehensive understanding of THs-dependent skeletal muscle protein turnover might facilitate the management of physical discomfort or weakness in conditions of thyroid disease. Herein, we describe the molecular mechanisms underlying the THs-dependent alteration of skeletal muscle structure and function associated with muscle atrophy and hypertrophy, thus providing new insights for targeted modulation of skeletal muscle dynamics.

The androgen receptor in mesenchymal progenitors regulates skeletal muscle mass via Igf1 expression in male mice.

Androgens exert their effects primarily by binding to the androgen receptor (AR), a ligand-dependent nuclear receptor. While androgens have anabolic effects on skeletal muscle, previous studies reported that AR functions in myofibers to regulate skeletal muscle quality, rather than skeletal muscle mass. Therefore, the anabolic effects of androgens are exerted via nonmyofiber cells. In this context, the cellular and molecular mechanisms of AR in mesenchymal progenitors, which play a crucial role in maintaining skeletal muscle homeostasis, remain largely unknown. In this study, we demonstrated expression of AR in mesenchymal progenitors and found that targeted AR ablation in mesenchymal progenitors reduced limb muscle mass in mature adult, but not young or aged, male mice, although fatty infiltration of muscle was not affected. The absence of AR in mesenchymal progenitors led to remarkable perineal muscle hypotrophy, regardless of age, due to abnormal regulation of transcripts associated with cell death and extracellular matrix organization. Additionally, we revealed that AR in mesenchymal progenitors regulates the expression of insulin-like growth factor 1 (Igf1) and that IGF1 administration prevents perineal muscle atrophy in a paracrine manner. These findings indicate that the anabolic effects of androgens regulate skeletal muscle mass via, at least in part, AR signaling in mesenchymal progenitors.

Sexual Dimorphism in the Musculoskeletal System: Sex Hormones and Beyond.

Mounting evidence indicates that whereas some fundamental aspects of bone cell differentiation and function are similar in females and males, there is a clear contribution of sex/gender on the effects of signaling molecules on bone mass and strength and, consequently, on the effects of pharmacologic approaches to treat skeletal disorders. However, until recently, most studies were designed and performed using only 1 sex, resulting in a scarcity of published information on sexual dimorphism of the musculoskeletal system, including the mandible/masticatory muscles and the axial and appendicular bones and skeletal muscles. Further, it is now recognized that scientific rigor requires the study of both males and females. Therefore, there is an increasing need to understand the molecular and cellular basis for the differential outcomes of genetic manipulations and therapeutic agent administration depending on the sex of the experimental animals. Studies have shown higher muscle mass, cancellous bone mass, and long bone width in males compared with females as well as different traits in the pelvis and the skull, which are usually used for gender identification in forensic anthropology. Yet, most reports focus on the role of sex hormones, in particular, the consequences of estrogen deficiency with menopause in humans and in ovariectomized animal models. In addition, emerging data is starting to unveil the effects of gender-affirming hormonal therapy on the musculoskeletal system. We summarize here the current knowledge on the sex/gender-dependent phenotypic characteristics of the bone and skeletal muscles in humans and rodents, highlighting studies in which side by side comparisons were made.

Exercise and nutrition benefit skeletal muscle: From influence factor and intervention strategy to molecular mechanism.

Sarcopenia is a progressive systemic skeletal muscle disease induced by various physiological and pathological factors, including aging, malnutrition, denervation, and cardiovascular diseases, manifesting as the decline of skeletal muscle mass and function. Both exercise and nutrition produce beneficial effects on skeletal muscle growth and are viewed as feasible strategies to prevent sarcopenia. Mechanisms involve regulating blood flow, oxidative stress, inflammation, apoptosis, protein synthesis and degradation, and satellite cell activation through exerkines and gut microbiomes. In this review, we summarized and discussed the latest progress and future development of the above mechanisms for providing a theoretical basis and ideas for the prevention and treatment of sarcopenia.

Bibliometric analysis of skeletal muscle ischemia/reperfusion (I/R) research from 1986 to 2022.

Introduction: Tissue damage due to ischemia and reperfusion is a critical medical problem worldwide. Studies in this field have made remarkable advances in understanding the pathogenesis of ischemia/reperfusion (I/R) injury and its treatment with new and known drugs. However, no bibliometric analysis exists in this area of research. Methods: Research articles and reviews related to skeletal muscle I/R from 1986 to 2022 were retrieved from the Web of Science Core Collection. Bibliometric analysis was performed using Microsoft Excel 2019, VOSviewer (version 1.6.19), Bibliometrix (R-Tool for R-Studio), and CiteSpace (version 6.1.R5). Results: A total of 3682 research articles and reviews from 2846 institutions in 83 countries were considered in this study. Most studies were conducted in the USA. Hobson RW (UMDNJ-New Jersey Medical School) had the highest publication, and Korthuis RJ (Louisiana State University) had the highest co-citations. Our analysis showed that, though the Journal of Surgical Research was most favored, the Journal of Biological Chemistry had the highest number of co-citations. The pathophysiology, interventions, and molecular mechanisms of skeletal muscle I/R injury emerged as the primary research areas, with "apoptosis," "signaling pathway," and "oxidative stress" as the main keywords of research hotspots. Conclusions: This study provides a thorough overview of research trends and focal points in skeletal muscle I/R injury by applying bibliometric and visualization techniques. The insights gained from our findings offer a profound understanding of the evolving landscape of skeletal muscle I/R injury research, thereby functioning as a valuable reference and roadmap for future investigations.

Advance in the mechanism and clinical research of myalgia in long COVID.

As severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve, mortality rates of coronavirus disease 2019 (COVID-19) have significantly decreased. However, a variable proportion of patients exhibit persistent prolonged symptoms of COVID-19 infection (long COVID). This virus primarily attacks respiratory system, but numerous individuals complain persistent skeletal muscle pain or worsening pre-existing muscle pain post COVID-19, which severely affects the quality of life and recovery. Currently, there is limited research on the skeletal muscle pain in long COVID. In this brief review, we review potential pathological mechanisms of skeletal muscle pain in long COVID, and summarize the various auxiliary examinations and treatments for skeletal muscle pain in long COVID. We consider abnormal activation of inflammatory response, myopathy, and neurological damages as pivotal pathological mechanisms of skeletal muscle pain in long COVID. A comprehensive examination is significantly important in order to work out effective treatment plans and relieve skeletal muscle pain. So far, rehabilitation interventions for myalgia in long COVID contain but are not limited to drug, nutraceutical therapy, gut microbiome-targeted therapy, interventional therapy and strength training. Our study provides a potential mechanism reference for clinical researches, highlighting the importance of comprehensive approach and management of skeletal muscle pain in long COVID. The relief of skeletal muscle pain will accelerate rehabilitation process, improve activities of daily living and enhance the quality of life, promoting individuals return to society with profound significance.

Muscle aging and sarcopenia: The pathology, etiology, and most promising therapeutic targets.

Sarcopenia is a progressive muscle wasting disorder that severely impacts the quality of life of elderly individuals. Although the natural aging process primarily causes sarcopenia, it can develop in response to other conditions. Because muscle function is influenced by numerous changes that occur with age, the etiology of sarcopenia remains unclear. However, recent characterizations of the aging muscle transcriptional landscape, signaling pathway disruptions, fiber and extracellular matrix compositions, systemic metabolomic and inflammatory responses, mitochondrial function, and neurological inputs offer insights and hope for future treatments. This review will discuss age-related changes in healthy muscle and our current understanding of how this can deteriorate into sarcopenia. As our elderly population continues to grow, we must understand sarcopenia and find treatments that allow individuals to maintain independence and dignity throughout an extended lifespan.

Automated Microfluidics-Assisted Hydrogel-Based Wet-Spinning for the Biofabrication of Biomimetic Engineered Myotendinous Junction.

The muscle-tendon junction (MTJ) plays a pivotal role in efficiently converting the muscular contraction into a controlled skeletal movement through the tendon. Given its complex biomechanical intricacy, the biofabrication of such tissue interface represents a significant challenge in the field of musculoskeletal tissue engineering. Herein, a novel method to produce MTJ-like hydrogel yarns using a microfluidics-assisted 3D rotary wet-spinning strategy is developed. Optimization of flow rates, rotational speed, and delivery time of bioinks enables the production of highly compartmentalized scaffolds that recapitulate the muscle, tendon, and the transient MTJ-like region. Additionally, such biofabrication parameters are validated in terms of cellular response by promoting an optimal uniaxial alignment for both muscle and tendon precursor cells. By sequentially wet-spinning C2C12 myoblasts and NIH 3T3 fibroblasts, a gradient-patterned cellular arrangement mirroring the intrinsic biological heterogeneity of the MTJ is successfully obtained. The immunofluorescence assessment further reveals the localized expression of tissue-specific markers, including myosin heavy chain and collagen type I/III, which demonstrate muscle and tenogenic tissue maturation, respectively. Remarkably, the muscle-tendon transition zone exhibits finger-like projection of the multinucleated myotubes in the tenogenic compartment, epitomizing the MTJ signature architecture.