Magnetization transfer (MT) magnetic resonance imaging (MRI) can be used to estimate the fraction of water and macromolecular proton pools in tissues. MT modeling paired with ultrashort echo time acquisition (UTE-MT modeling) has been proposed to improve the evaluation of the myotendinous junction and fibrosis in muscle tissues, which the latter increases with aging. This study aimed to determine if the UTE-MT modeling technique is sensitive to age-related changes in the skeletal muscles of the lower leg. Institutional review board approval was obtained, and all recruited subjects provided written informed consent. The legs of 31 healthy younger (28.1 ± 6.1 years old, BMI = 22.3 ± 3.5) and 20 older (74.7 ± 5.5 years old, BMI = 26.7 ± 5.9) female subjects were imaged using UTE sequences on a 3 T MRI scanner. MT ratio (MTR), macromolecular fraction (MMF), macromolecular T2 (T2-MM), and water T2 (T2-W) were calculated using UTE-MT modeling for the anterior tibialis (ATM), posterior tibialis (PTM), soleus (SM), and combined lateral muscles. Results were compared between groups using the Wilcoxon rank sum test. Three independent observers selected regions of interest (ROIs) and processed UTE-MRI images separately, and the intraclass correlation coefficient (ICC) was calculated for a reproducibility study. Significantly lower mean MTR and MMF values were present in the older compared with the younger group in all studied lower leg muscles. T2-MM showed significantly lower values in the older group only for PTM and SM muscles. In contrast, T2-W showed significantly higher values in the older group. The age-related differences were more pronounced for MMF (-17 to -19%) and T2-W (+20 to 47%) measurements in all muscle groups compared with other investigated MR measures. ICCs were higher than 0.93, indicating excellent consistency between the ROI selection and MRI measurements of independent readers. As demonstrated by significant differences between younger and older groups, this research emphasizes the potential of UTE-MT MRI techniques in evaluating age-related skeletal muscle changes.

Morphological studies of skeletal muscle tissue provide insights into the architecture of muscle fibers, the surrounding cells, and the extracellular matrix (ECM). However, a spatial proteomics analysis of the skeletal muscle including the muscle-tendon transition zone is lacking. Here, we prepare cryotome muscle sections of the mouse soleus muscle and measure each slice using short liquid chromatography-mass spectrometry (LC-MS) gradients. We generate 3,000 high-resolution protein profiles that serve as the basis for a network analysis to reveal the complex architecture of the muscle-tendon junction. Among the protein profiles that increase from muscle to tendon, we find proteins related to neuronal activity, fatty acid biosynthesis, and the renin-angiotensin system (RAS). Blocking the RAS in cultured mouse tenocytes using losartan reduces the ECM synthesis. Overall, our analysis of thin cryotome sections provides a spatial proteome of skeletal muscle and reveals that the RAS acts as an additional regulator of the matrix within muscle-tendon junctions.

UNASSIGNED: A 72-year-old male presented for evaluation of a 2-wk history left buttock pain that began while playing pickleball. He sustained a left inversion ankle sprain while in a squatted position and landed on his left buttock. Four days after his injury, he developed extensive bruising involving his lower back, buttock, and left thigh. On examination, he had tenderness to palpation at the left side of the sacrum and in the region of the deep external rotators. Left hip range of motion was full in extension but limited to 90° of flexion, which reproduced left-sided buttock pain. External rotation provoked pain, but internal rotation was full and pain free. MRI of the pelvis demonstrated a grade 2 partial thickness tear of the left gluteus maximus muscle at its distal myotendinous junction with associated retraction and intramuscular hematoma. He was managed with compression with biking shorts, icing, acetaminophen, and physical therapy. He returned to pickleball approximately 4 wk after his injury, and at his 4-wk follow-up, he reported 99% improvement in his symptoms with the only remaining complaint being minimal discomfort with gluteal stretching.

Myotendinous junction (MTJ) injuries are prevalent in clinical practice, yet the treatment approaches are limited to surgical suturing and conservative therapy, exhibiting a high recurrence rate. Current research on MTJ tissue engineering is scarce and lacks in vivo evaluation of repair efficacy. Here, we developed a three-dimensional-printed bioactive fiber-reinforced hydrogel containing mesenchymal stem cells (MSCs) and Klotho for structural and functional MTJ regeneration. In a rat MTJ defect model, the bioactive fiber-reinforced hydrogel promoted the structural restoration of muscle, tendon, and muscle-tendon interface and enhanced the functional recovery of injured MTJ. In vivo proteomics and in vitro cell cultures elucidated the regenerative mechanisms of the bioactive fiber-reinforced hydrogel by modulating oxidative stress and inflammation, thus engineering an optimized microenvironment to support the survival and differentiation of transplanted MSCs and maintain the functional phenotype of resident cells within MTJ tissues, including tendon/muscle cells and macrophages. This strategy provides a promising treatment for MTJ injuries.

The myotendinous junction (MTJ) is a vulnerable region at the interface of skeletal muscle and tendon that forms an integrated mechanical unit. This study presents a technique for the spatially restrictive co-culture of human embryonic stem cell (hESC)-derived skeletal myocytes and primary tenocytes for two-dimensional modeling of the MTJ. Micropatterned lanes of extracellular matrix and a 2-well culture chamber define the initial regions of occupation. On day 1, both lines occupy less than 20 % of the initially vacant interstitial zone, referred to henceforth as the junction. Myocyte-tenocyte interdigitations are observed by day 7. Immunocytochemistry reveals enhanced organization and alignment of patterned myocyte and tenocyte features, as well as differential expression of multiple MTJ markers. On day 24, electrically stimulated junction myocytes demonstrate negative contractile strains, while positive tensile strains are exhibited by mechanically passive tenocytes at the junction. Unpatterned tenocytes distal to the junction experience significantly decreased strains in comparison to cells at the interface. Unpatterned myocytes have impaired organization and uncoordinated contractile behavior. These findings suggest that this platform is capable of inducing myocyte-tenocyte junction formation and mechanical coupling similar to the native MTJ, showing transduction of force across the cell-cell interface. STATEMENT OF SIGNIFICANCE: The myotendinous junction (MTJ) is an integrated structure that transduces force across the muscle-tendon boundary, making the region vulnerable to strain injury. Despite the clinical relevance, previous in vitro models of the MTJ lack the structure and mechanical accuracy of the native tissue and have difficulty transmitting force across the cell-cell interface. This study demonstrates an in vitro model of the MTJ, using spatially restrictive cues to inform human myocyte-tenocyte interactions and architecture. The model expressed MTJ markers and developed anisotropic myocyte-tenocyte integrations that resemble the native tissue and allow for force transduction from contracting myocytes to passive tenocyte regions. As such, this study presents a system capable of investigating development, injury, and pathology in the human MTJ.

The peripheral nerve injury (PNI) affects the morphology of the whole locomotor apparatus, which can reach the myotendinous junction (MTJ) interface. In the injury condition, the skeletal muscle satellite cells (SC) are triggered, activated, and proliferated to repair their structure, and in the MTJ, the telocytes (TC) are associated to support the interface with the need for remodeling; in that way, these cells can be associated with SC. The study aimed to describe the SC and TC relationship after PNI at the MTJ. Sixteen adult Wistar rats were divided into Control Group (C, n = 8) and PNI Group (PNI, n = 8), PNI was performed by the constriction of the sciatic nerve. The samples were processed for transmission electron microscopy and immunostaining analysis. In the C group was evidenced the arrangement of sarcoplasmic evaginations and invaginations, the support collagen layer with a TC inside it, and an SC through vesicles internally and externally to then. In the PNI group were observed the disarrangement of invaginations and evaginations and sarcomeres degradation at MTJ, as the disposition of telopodes adjacent and in contact to the SC with extracellular vesicles and exosomes in a characterized paracrine activity. These findings can determine a link between the TCs and the SCs at the MTJ remodeling. RESEARCH HIGHLIGHTS: Peripheral nerve injury promotes the myotendinous junction (MTJ) remodeling. The telocytes (TC) and the satellite cells (SC) are present at the myotendinous interface. TC mediated the SC activity at MTJ.

BACKGROUND: Hamstring muscles are the most frequently reported sites of muscle strain injuries, especially near the bi-articular muscles\' myotendinous junction, where aponeurosis provides a connective tissue network linking muscle fibers to the tendon. This study aimed to investigate the reliability and site-specific differences of hamstring aponeuroses under different conditions (formalin and urea) using MyotonPRO.
METHODS: Eight hamstring muscle groups were dissected from four human cadavers (two males and two females) aged 83-93 years. Measurements of the mechanical properties of the aponeuroses from the superficial and deep regions of biceps femoris long head, semitendinosus, and semimembranosus (after formalin solution immersion) were done using MyotonPRO (intra-rater reliability was examined within a 24-h interval), following which the hamstring aponeuroses were measured using a similar procedure after urea solution immersion.
RESULTS: Test-retest (intra-rater) results revealed that the MyotonPRO measurement of tone, stiffness, relaxation, and creep of cadaveric aponeuroses presented good to excellent reliability (ICC: 0.86 to 0.98). There were no significant differences in tone, stiffness, elasticity, relaxation, and creep among the six sites of hamstring aponeuroses under both formalin and urea conditions. Significant differences between formalin and urea conditions were found in the tone, stiffness, relaxation, and creep of hamstring aponeuroses (P < 0.05).
CONCLUSIONS: These results suggested that the biomechanical properties of hamstring aponeuroses showed homogeneity between the sites using MyotonPRO. Urea solution could potentially neutralize the effect of formalin on the biomechanical properties of cadaveric muscle-aponeurosis-tendon units. The present findings might influence the design of subsequent cadaveric studies on hamstring muscle strains.

Myofibres serve as the functional unit for locomotion, with the sarcomere as fundamental subunit. Running the entire length of this structure are hundreds of myonuclei, located at the periphery of the myofibre, juxtaposed to the plasma membrane. Myonuclear specialisation and clustering at the centre and ends of the fibre are known to be essential for muscle contraction, yet the molecular basis of this regionalisation has remained unclear. While the \'myonuclear domain hypothesis\' helped explain how myonuclei can independently govern large cytoplasmic territories, novel technologies have provided granularity on the diverse transcriptional programs running simultaneously within the syncytia and added a new perspective on how myonuclei communicate. Building upon this, we explore the critical cellular and molecular sources of transcriptional and functional heterogeneity within myofibres, discussing the impact of intrinsic and extrinsic factors on myonuclear programs. This knowledge provides new insights for understanding muscle development, repair, and disease, but also opens avenues for the development of novel and precise therapeutic approaches.

UNASSIGNED: Animal models of muscle injury have primarily relied on methods which do not mimic the chronic scarring that typically occurs adjacent to the myotendinous junction (MTJ). The goal of this study was three-fold: (i) to create a strain-induced in vivo model of rectus femoris MTJ injury in rats; (ii) to document clinical manifestations of injury using longitudinal tracking of individual animals via voluntary and compulsory (treadmill) mobility analyses and (iii) to validate and assess the model for persistent scarring through serial histologic assessment and development of a semi-quantitative grading scheme to characterize injury response over time.
UNASSIGNED: Strain-induced MTJ injury was generated in male Sprague Dawley rats via needle tension directed along the transverse axis between the rectus femoris muscle and distal tendon that attaches to the patella. Animals received mobility assessments (gait analysis using a DigiGait Treadmill System and weight bearing using a Tekscan Rodent Walkway System) at days 0, 1, 3, 6, 13, 20, and 27 of the experimental protocol. Rats were euthanized at 1, 3, 7, 14, and 28 days post-injury (n = 6 rats per time-point) and hindlimbs were processed for histology.
UNASSIGNED: Significant changes in locomotor parameters included injured and contralateral limb paw area, max dA/dt (limb deceleration/breaking time), stride time, stance time, force time impulse, and fore/hind symmetry, and injured limb maximum force. The most significant and consistent histologic finding was a pathologic fibrotic adhesive lesion at the muscle and tendon interface along the proximal aspect of the patella just distal to the injury site. This lesion was composed of reactive fibroblasts, disorganized collagen fibers, vascular profiles, and a myxomatous ground substance stroma.
UNASSIGNED: This work is the first to characterize the clinical and pathologic development of a chronic model of rectus femoris MTJ injury, which resulted in altered mobility likely caused by a strain-induced fibrotic scar along the anterior patella. Notably, both the functional and pathologic changes recapitulated the course of injury progression similar to what is described in humans. This work provides a unique model to study MTJ injury mechanisms for the identification of enhanced treatment options for patients who suffer from activity-related muscle conditions.

Skeletal muscle fiber is a large syncytium with multiple and evenly distributed nuclei. Adult subsynaptic myonuclei beneath the neuromuscular junction (NMJ) express specific genes, the products of which coordinately function in the maintenance of the pre- and post-synaptic regions. However, the gene expression profiles that promote the NMJ formation during embryogenesis remain largely unexplored. We performed single-nucleus RNA sequencing (snRNA-seq) analysis of embryonic and neonatal mouse diaphragms, and found that each myonucleus had a distinct transcriptome pattern during the NMJ formation. Among the previously reported NMJ-constituting genes, Dok7, Chrna1, and Chrnd are specifically expressed in subsynaptic myonuclei at E18.5. In the E18.5 diaphragm, ca. 10.7% of the myonuclei express genes for the NMJ formation (Dok7, Chrna1, and Chrnd) together with four representative β-catenin regulators (Amotl2, Ptprk, Fam53b, and Tcf7l2). Additionally, the temporal gene expression patterns of these seven genes are synchronized in differentiating C2C12 myoblasts. Amotl2 and Ptprk are expressed in the sarcoplasm, where β-catenin serves as a structural protein to organize the membrane-anchored NMJ structure. In contrast, Fam53b and Tcf7l2 are expressed in the myonucleus, where β-catenin serves as a transcriptional coactivator in Wnt/β-catenin signaling at the NMJ. In C2C12 myotubes, knockdown of Amotl2 or Ptprk markedly, and that of Fam53b and Tcf7l2 less efficiently, impair the clustering of acetylcholine receptors. In contrast, knockdown of Fam53b and Tcf7l2, but not of Amotl2 or Ptprk, impairs the gene expression of Slit2 encoding an axonal attractant for motor neurons, which is required for the maturation of motor nerve terminal. Thus, Amotl2 and Ptprk exert different roles at the NM compared to Fam53b and Tcf7l2. Additionally, Wnt ligands originating from the spinal motor neurons and the perichondrium/chondrocyte are likely to work remotely on the subsynaptic nuclei and the myotendinous junctional nuclei, respectively. We conclude that snRNA-seq analysis of embryonic/neonatal diaphragms reveal a novel coordinated expression profile especially in the Wnt/β-catenin signaling that regulate the formation of the embryonic NMJ.