Myosin VI depletion delays neuromuscular junction maturation and exacerbates muscle performance

Myosin VI depletion delays neuromuscular junction maturation and exacerbates muscle performance | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Myosin VI depletion delays neuromuscular junction maturation and exacerbates muscle performance Jolanta Nowak, Paloma Alvarez-Suarez, Tomasz Włodarczyk, Renata Zakrzewska, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6965921/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 29 Dec, 2025 Read the published version in Scientific Reports → Version 1 posted 9 You are reading this latest preprint version Abstract Unconventional myosin VI (MVI) is an ATP-dependent actin-binding molecular motor that participates in numerous cellular and tissue functions, including striated muscle physiology. Lack of MVI expression significantly aberrates myogenesis and skeletal muscle metabolism, and alters myoblast adhesion, fusion, and cytoskeletal organisation. Concomitantly, MVI knockout mice display functional and structural cardiac defects. Here, for the first time, we investigate the impact of MVI on neuromuscular junctions (NMJs), the peripheral synapses crucial for skeletal muscle contraction. We show that MVI is enriched at the postsynaptic machinery of developing and adult NMJs. We analyse the morphology of NMJs of MVI knockout mice (Snell’s waltzer, SV) during development and show that MVI deficiency delays NMJ maturation in fast- and slow-twitch muscles. It also reduces the NMJ size of the soleus muscle, as demonstrated by the decreased morphological parameters of both presynaptic and postsynaptic compartments. Simultaneously, synaptic elimination remains unaffected after MVI knockout, suggesting that the observed phenotypes are innervation-independent. Lastly, depletion of MVI impairs the grip strength of both female and male SV/SV mice. In summary, our studies show that MVI is an important regulator of NMJ size and maturation, controls muscle performance, and its impact is independent of innervation and sex. Biological sciences/Developmental biology Biological sciences/Developmental biology/Organogenesis Biological sciences/Developmental biology/Organogenesis/Musculoskeletal development molecular motor myosin VI neuromuscular junction maturation skeletal muscle Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 INTRODUCTION Myosins are highly conserved actin-based molecular motors, expressed across Eukaryota [ 1 ] and participating in cytokinesis, cell migration, adhesion and fusion, endo- and exocytosis, intracellular trafficking, transcription, chromatin organisation and DNA damage repair [ 2 – 10 ]. Muscle myosin heavy chain isoforms serve as muscle fiber type-specific molecular markers, important to discern the mechanisms of myogenic development and to understand muscle function in health and disease. Based on their ability to form filaments and their origin, myosins are divided into conventional (class II) and unconventional, classified in humans into 11 classes (families) [ 11 – 13 ]. Out of forty genes encoding myosin heavy chains in humans, almost two-thirds encode unconventional ones, but their function is still less understood than conventional ones. Unconventional myosin VI (MVI) is a unique member of the myosin family that moves backwards, towards the minus end of actin filaments [ 14 , 15 ]. Snell’s waltzer (SV) mice are natural MVI knockouts carrying a spontaneous null mutation that causes deafness, circling, and hyperactivity [ 16 ]. Additionally, structural defects in kidneys, brain, and testes have been reported in SV/SV mice [ 17 – 19 ]. Given the multiple roles of MVI, it is localised in various cell compartments and organelles, such as the sarcoplasmic reticulum, Golgi apparatus, intercalated discs, cell nucleolus and nucleus, and around mitochondria [ 9 , 20 – 26 ]. Importantly, MVI is a crucial regulator of striated muscle development and physiology. It is involved in heart organisation and dysfunction of this protein entails left ventricular cardiomyopathy [ 22 , 27 – 29 ]. Growing evidence underlines the importance of MVI in myogenesis and skeletal muscle function. Studies of our research group show that loss of MVI results in aberrated myoblast adhesion, fusion, metabolism, and actin organisation in myoblasts in vitro [ 8 ]. MVI controls expression of crucial myogenic regulators, Pax7 (Paired Box 7), MyoD (Myogenic Differentiation 1), and myogenin, as well as adhesion proteins, and fusogens, myomaker and myomerger [ 8 ]. We also determined that lack of MVI increases the muscle/body weight ratio and significantly affects the morphology of the murine hindlimb muscles. Myosin VI knockout causes a 2.5-fold increase of thin muscle fibres with a cross-sectional area below 100 µm 2 . This effect, observed as early as at birth, is the most evident in the slow-twitch muscle soleus and is maintained throughout the animals’ life span [ 30 ]. Neuromuscular junctions (NMJs) are peripheral synapses connecting motoneurons and skeletal muscle fibers. Compromised NMJ integrity and function is a hallmark of neuromuscular disorders, such as Duchenne muscular dystrophy, myasthenia gravis, congenital myasthenic syndromes (CMS), Charcot-Marie-Tooth disease (CMTD) or amyotrophic lateral sclerosis (ALS) [ 31 – 37 ]. Despite the central role of NMJs in the onset and progression of neuromuscular disorders, the molecular mechanisms underlying these processes are still poorly understood. Thorough characterisation of the genetic models manifesting neuromuscular symptoms can bring valuable insights into their pathology and help to develop more effective therapies. We have previously identified MVI as a postsynaptic protein at NMJs in rats, however, the role of MVI in NMJ function remains unexplored [ 21 ]. Here we show that MVI is involved in NMJ development and maintenance. MVI is a protein abundant at NMJs during developmental remodelling and adulthood and is localised postsynaptically in both slow and fast-twitch muscles. The knockout of MVI delays NMJ maturation, however, independently of synaptic elimination. Moreover, loss of MVI decreases the size of both pre- and postsynaptic apparatuses, in accordance with the overall reduction of the body, muscle, and muscle fiber size in SV/SV mice [ 30 ]. The significantly diminished grip strength of both female and male MVI knockouts suggests that MVI depletion has universal functional consequences for muscle, regardless of sex. RESULTS MVI is enriched at NMJs during developmental remodelling and maintenance Mature neuromuscular junctions develop from simple oval acetylcholine receptor (AChR) assemblies during plaque-to-pretzel transition. After the first week of life, plaques become increasingly perforated and reshape into pretzel-like structures by P21, when maturation is mostly completed (Fig. 1 a) [ 38 ]. Subsequently, NMJs grow in size until the mouse reaches adulthood (P90). Another type of NMJ remodelling is induced by aging which starts around 14 months of age (Fig. 1 a) [ 39 , 40 ]. We assessed the localisation of MVI in fast-twitch tibialis anterior at the time of intense developmental remodelling (P10), and NMJ maintenance in mature adult (P120) and middle-aged (P365) mice. MVI was enriched at NMJs at all analysed stages, but distributed differently (Fig. 1 b). At the stage of NMJ maturation (P10) MVI was dispersed at the NMJ postsynaptic compartment (Fig. 1 b). At mature NMJs (P120, P365) MVI mainly occupied the domains between AChR-rich areas, and was also clustered at the vicinity of AChRs (Fig. 1 b, orthogonal views at P120 and Fig. 1 c). The enrichment of MVI along the entire postsynaptic machinery was characteristic for NMJs of adult muscles with different fiber composition and function: fast-twitch diaphragm and slow-twitch triangularis sterni and soleus (Supplementary Fig. 1). Our results show that MVI is localised postsynaptically at NMJs of various skeletal muscles throughout the mouse lifespan, during development and maintenance of NMJs. Knockout of MVI delays NMJ maturation in slow- and fast-twitch muscles Since MVI is present at NMJs during developmental remodelling, we tested its role in postsynaptic maturation. To this end, we determined the ratio of perforated NMJs upon MVI knockout at P10 in two types of muscles: fast-twitch tibialis anterior (TA) and slow-twitch soleus (Fig. 2 a-f). The number of maturing NMJs decreased in both analysed muscles by 34% (TA) and 36% (soleus) (Fig. 2 e and f, on the left). Simultaneously, we did not observe changes in the proportion of NMJs with different numbers of perforations (Fig. 2 f, on the right), which suggests that MVI expression rather affects the induction of postsynaptic maturation than its progression. Overall, these results show that MVI has a broad effect on NMJ development and the lack of this protein delays NMJ maturation both in fast- and slow-twitch muscles. Lack of MVI reduces the size of the NMJ pre- and postsynaptic domains, but does not affect innervation Postsynaptic maturation is tightly regulated by innervation, and pre- and postsynaptic compartments morphologically mirror each other to collaborate optimally. Initially, NMJs are polyinnervated, and during muscle development, they gradually lose surplus axonal inputs in a process called synaptic elimination, completed by the end of the second week after birth [ 41 – 44 ]. We assessed whether the observed delay in postsynaptic maturation upon MVI knockout at P10 is accompanied by changes in NMJ innervation (Fig. 3 a-d). Since soleus is the muscle where MVI expression is the most abundant [ 45 ] and its effect on NMJ maturation was more statistically significant (Fig. 2 f), we focused on this muscle in our subsequent analyses. We determined the ratio of mono- and polyinnervated NMJs, considered the presynaptic indicator of NMJ maturation (Fig. 3 a). A higher ratio of polyinnervated NMJs would indicate delayed synaptic elimination. We also assessed the number of denervated NMJs and NMJs with degenerating nerves, another two indicators of synapse elimination (Fig. 3 b). The overall number of NMJs was increased by 30% after MVI knockout (Fig. 3 c). However, there were no significant differences in innervation, suggesting that the delayed postsynaptic maturation was independent of synaptic elimination (Fig. 3 d). Thus, we analysed the morphology of the motoneuron terminals of SV/SV mice (Fig. 3 e-m). MVI depletion decreased nerve terminal perimeter and area by 14,5% and 27%, respectively (Fig. 3 f and g), as well as the total and average length of axonal branches by 19% (Fig. 3 k) and by 21% (Fig. 3 l), respectively. The changes in presynaptic morphology coincided with reduced size of the postsynaptic compartment and diminished SV/SV mice body weight (Fig. 4 a-m). MVI knockout decreased AChR area by 11% (Fig. 4 c), and endplate perimeter by 5% (Fig. 4 e), and area by 11% (Fig. 4 f). Since MVI knockout muscles are characterised by thinner muscle fibres, we verified whether the observed reduction in NMJ size is correlated with the muscle fibre diameter [ 30 ]. Indeed, endplate area when normalised to the muscle fibre diameter was similar in SV/SV and SV/+ mice, indicating that NMJs size is correlated with thinner muscle fibres (Supplementary Fig. 2). Parameters, such as number of AChR clusters, an indicator of endplate fragmentation, or overlap, showing endplate presynaptic coverage were similar (Fig. 4 j and l). This result supports our conclusion that the synaptic elimination is not impaired in MVI knockouts (Fig. 3 d). In summary, our observations confirm that MVI knockout impacts pre- and postsynaptic morphology of motor terminals, independently of denervation and degeneration characteristic for the loss of motoneurons during development. MVI is necessary to maintain proper muscle strength regardless of sex Next, we determined whether the observed changes in the NMJ morphology alter muscle performance. To this end, we performed grip strength test with adult SV/+ and SV/SV mice of both sexes (Fig. 5 a). Since both female and male MVI knockouts have reduced body weight [ 17 ], we normalised the obtained average force values to the body weight of mice used in our behavioral studies (Fig. 5 b and c). Both female and male SV/SV mice had weaker muscles in comparison to control animals, as shown by the 56% and 44% drop in their muscle strength, respectively (Fig. 5 b). Interestingly, the bigger impact of MVI knockout on female performance was not associated with the more pronounced decrease of their body weight, since MVI depletion caused more significant reduction of the male body weight (Fig. 5 c). DISCUSSION We show that MVI is ubiquitously expressed at NMJs of various muscles at different stages of the development (Fig. 1 and Supplementary Fig. 1). MVI is adjacent to AChRs and present in the domain devoid of AChRs. Given the broad role of MVI in endo- and exocytosis, it can be involved in synaptic signalling at NMJs [ 19 , 46 – 50 ]. In the central nervous system, MVI plays a role in synaptic transmission and plasticity [ 51 , 52 ]. MVI expression increases in slow- and fast-twitch muscles upon denervation, and the protein localises to the entire muscle fibre, in contrast to its peripheral localisation in innervated muscles [ 21 ]. This observation indicates that MVI expression is regulated by synaptic activity. Drosophila melanogaster MVI loss-of-function mutants display defects in NMJ morphology, synaptic vesicle distribution, and basal synaptic transmission, accompanied by the impaired locomotor activity of the mutant larvae [ 53 ]. Moreover, MVI is an important regulator of the proper organisation of synaptic vesicles at Drosophila NMJs and anchors them at the specific regions of synaptic domains [ 54 ]. MVI can play a similar role at mouse NMJs and participate in synaptic signalling during development. However, further studies identifying MVI molecular partners specific to peripheral synapses and the spatiotemporal regulation of their distribution at NMJs are necessary to unravel the function of this protein in the described context. The diminished percentage of perforated NMJs in both fast- and slow-twitch muscles of SV/SV mice indicates a wide-range delay in synapse maturation (Fig. 2 e and f). However, this impairment appears not to be caused by stalled synaptic elimination (Fig. 3 d and Fig. 4 i and l). One of the possible explanations is that MVI is required for AChR clustering and redistribution during the formation of synaptic perforations. It has been shown that MVI is a part of the complex regulating Rac1 (Rac Family Small GTPase 1) and Cdc42 (Cell Division Cycle 42) Rho GTPases, rearranging the actin cytoskeleton and vesicle trafficking, which can subsequently impact AChR turnover and degradation [ 55 – 57 ]. Moreover, Rho GEF ephexin1 is the effector protein of the Musk-Dok7 complex, crucial for AChR dispersal [ 57 , 58 ]. Thus, Rho GTPases are potential MVI downstream targets that control AChR recycling at the maturing NMJs. Simultaneously, MVI can support the formation of synaptic perforations by anchoring AChRs and restricting their localisation to certain NMJ areas, which facilitates the formation and propagation of perforations. MVI anchoring role at NMJs has been confirmed in studies of Drosophila MVI mutants. Normally, synaptic vesicles are concentrated at the outer ring of the synaptic boutons. Upon MVI knockout, this selective localisation is lost, and they occupy the entire area of the boutons [ 53 ]. Similarly, loss of MVI stabilisation can result in AChR dispersal and delay the formation of perforations at mouse NMJs. We observed the increase in the overall number of NMJs (Fig. 3 c). This confirms our previous observations showing that SV/SV mice are characterised by a higher number of muscle fibres [ 30 ]. Both pre- and postsynaptic endplate size parameters were reduced in MVI knockouts (Fig. 3 f, g, k and l and Fig. 4 c, e and f). It was shown previously by our group that soleus muscles of SV/SV mice are characterised by a larger number of fibres with decreased cross-sectional area [ 30 ]. In agreement with the impact of the nuclear content on the myofiber size, the number of myonuclei was also decreased after MVI knockout [ 30 , 59 ]. Endplate areas normalised to the corresponding muscle fibre diameters showed similar values for SV/+ and SV/SV mice (Supplementary Fig. 2), suggesting a relationship between NMJ and muscle fibre size. Some studies showed that these parameters are correlated [ 60 , 61 ], however, other groups reported that muscle fibre size is not a main contributor to NMJ morphology [ 62 ]. This discrepancy can be explained by the different developmental stages, muscle types and species analysed [ 60 , 62 – 64 ]. Another factor that can influence NMJ size is the fibre type [ 62 ]. Given the role of MVI in myogenesis and the significant increase in the number of thinner fibres in MVI knockout mice [ 8 , 30 ], it would be interesting to perform fibre type analyses and the measurements of the size of corresponding NMJs in these knockouts. It was recently reported that the absence of MVI expression in skeletal muscles causes glycolytic-to-oxidative fibre-type switch [ 45 ]. However, the relationship between the muscle fibre metabolic type and the size of NMJs is yet to be determined, due to mixed results from different types of muscles [ 62 , 65 , 66 ]. Nevertheless, impairments in muscle structure of MVI knockouts are accompanied by changes in their metabolism. The levels of phosphorylated PKA (Protein Kinase A) and CREB (cAMP Response Element-Binding) proteins, regulators of glucose and lipid metabolism and mitochondrial function, are decreased in MVI-devoid soleus muscles at birth [ 30 ]. In adult mice, loss of MVI impairs ATP production and mTOR-dependent signalling, major contributors to muscle growth [ 45 , 67 ]. Moreover, loss of MVI disrupts adhesion, fusion, and differentiation of myotubes in vitro [ 8 ]. Altogether, these alterations can delay muscle fibre development and growth, undermining the muscle’s ability to support the reorganisation of developing NMJs. The importance of the muscle intrinsic signals for NMJ remodelling and the role of MVI in muscle fibre development and myoblast fusion are well documented [ 8 , 30 , 68 – 71 ). Lack of MVI alters the formation of myotubes and decreases γ-actin, focal adhesion kinase (FAK), and M-cadherin expression, all of which have been implicated in synapse formation and function [ 8 , 72 – 75 ]. Expression of the key myogenic regulators, such as Pax7 and myogenin, is also reduced in MVI knockout primary myoblasts [ 8 ]. Myogenin has been identified as an important regulator of NMJ size [ 76 ], which is in accordance with the diminished pre- and postsynaptic area of the motor endplates observed in SV/SV mice (Fig. 3 f,g and Fig. 4 c, e and f). Importantly, myogenin is a myogenic regulatory factor (MRF) crucial for AChR clustering in myotubes in vitro , and its role cannot be replaced by other MRFs or AChR organisers, Musk and rapsyn [ 77 ]. Thus, myogenin should be a primary focus in future analyses aiming to explain MVI-dependent mechanisms regulating NMJ morphology. Future investigation should also assess the MVI impact on localisation and function of terminal Schwann cells (TSCs) which are crucial for NMJ maturation and maintenance [ 78 , 79 ]. TSCs regulate the expression of postsynaptic genes and if their function is impaired, NMJ maturation can be delayed even in the presence of unaltered innervation (Fig. 3 d). Altogether, these results and MVI emerging role in transcriptional regulation [ 9 , 80 , 81 ], suggest this protein could be a regulator of transcription factors that control the activity of NMJ-related genes. We show that MVI knockout causes a significant impairment in the muscle performance in both female and male mice (Fig. 5 b). This can be a result of the reduced endplate and nerve terminal size, which limit synaptic transmission and weaken the muscles which cannot be compensated by the higher number of NMJs (Figs. 3 and 4 ). The size of nerve terminals impacts neurotransmitter release at NMJs of various species [ 82 – 84 ]. The observed phenotype can also be a consequence of delayed maturation. A similar effect was reported for ephexin1 knockouts, where NMJs failed to acquire complex pretzel-like topology, which was accompanied by muscle weakening and impaired neurotransmission [ 58 ]. Finally, MVI binding partner Dock-7 (Dedicator of Cytokinesis 7) was shown to participate in the neuregulin-ErbB2 pathway, which regulates neurotransmission [ 85 – 87 ]. Moreover, recent analyses performed by our research group show impaired mitochondria respiration and reduced ATP production upon MVI knockout which can also cause muscle weakening [ 45 ]. Future analyses should determine how lack of MVI affects synaptic transmission and neurotransmitter release, neurotransmitter receptor expression and function, as well as downstream signalling pathways controlling muscle performance and fatigability. METHODS Ethics declarations Procedures involving animals were approved by the 1st Local Ethical Committee for Experiments on Animals in Warsaw (resolutions 1311/2022 and 1639/2024) and were performed in accordance with the Act on the Protection of Animals Used for Scientific or Educational Purposes (2015) from the European Communities Council directives approved by the Polish Parliament. The researchers had individual permissions for the work involving mice granted by the Director of the Nencki Institute of Experimental Biology [218(W)/2024/IBD, 396(U)/2019/IBD, 396W/2020/IBD, 194P/2019/IBD, 194W/2020/IBD, 194/2021/IBD, 17(W)/2020/IBD, 27(W;P)/2020/IBD]. All experiments were performed in accordance with relevant guidelines and regulations and are reported according to ARRIVE guidelines. Snell’s waltzer (SV) mice husbandry and genotyping Snell's waltzer (SV) mice (C57BL/6J genetic background), a gift from Dr. Folma Buss (Cambridge Institute of Medical Research, UK), and C57BL/6J mice were maintained in the animal house of the Nencki Institute of Experimental Biology. All analyses were performed on male mice except the grip strength test, which was performed using both sexes. The age of mice is reported in relevant description of the methods, figures and figure legends. The weight of mice representative of the age P10 (morphometric NMJ analysis) and P90 (grip strength test) is presented in Fig. 4 m and Fig. 5 c, respectively. Mice were euthanised with a lethal dose of isoflurane followed by cervical dislocation. SV mice tail clips (2–4 mm 2 ) were used for genotyping with PCR Master Mix (Thermo Scientific) or KAPA Mouse Genotyping Kit (KAPA Biosystems) per manufacturer’s instructions. Primers for genotyping were as follows: SV1 5′-CTGACCCTGATCACTTAGCAGAGTTG-3′; SV2 5′-CATTGGGCCAGGTCACAGAAGTAAGC-3′; SV3 5′-GGTCCTCTGAAAGAGTAACC-3′ (SV/+ 318 and 230 bp, SV/SV 318 bp). Sex-matched littermates (SV/+) were used as controls for the phenotype assessment of MVI knockouts (SV/SV). Whole-mount muscle fibre immunostaining Soleus or tibialis anterior (TA) hindlimb muscles were isolated at time points reflecting different stages of NMJ development and maintenance (P10, P120, P365 for C57BL/6J and P10 for SV mice). Muscles were fixed in 4% paraformaldehyde in phosphate buffer saline (PBS) at room temperature (RT) for 15–80 min., depending on the muscle size and washed three times for 15 min. in PBS. For each analysis, muscle fibers were isolated from randomly chosen parts spanning the whole muscle. Pieces comprising approximately 10–30 muscle fibers were dissected, incubated for 30 min. in 0,1 M glycine in PBS, and rinsed in PBS and 0,5% Triton X-100. Muscles were incubated for minimum 30 min. to overnight at RT in blocking buffer [2–3% bovine serum albumin, 2–5% normal goat serum, 0.05–0.5% Triton X-100, 0.02% NaN 3 in PBS]. Then, fibers were incubated with primary antibodies diluted in blocking buffer at 4°C overnight in a sample shaker, and washed three times with PBS for 5 min. After washing, specimens were incubated with secondary antibodies diluted in blocking buffer, washed with PBS, and stained with α-bungarotoxin (BTX) diluted in PBS at RT for 15 min. Control stainings omitting primary antibody were performed to test secondary antibody non-specific binding. Whole-mount preparations were mounted in Fluoromount Aqueous Mounting Medium (Sigma) with DAPI (4′,6-diamidino-2-phenylindole) or Vectashield Plus Antifade Mounting Medium with DAPI (Vector Laboratories). Antibodies and fluorescent reagents used are listed in Supplementary Table S1 . Fluorescence intensity was measured using ZEN Blue 3.1 software. Confocal imaging Images were collected using an Axio Observer Z.1 inverted microscopes: Spinning Disc equipped with CSU-X1 spinning disc unit (Yokogawa, Japan) and Evolve 512 EMCCD camera (Photometrics, USA) or LSM780 (Zeiss, Germany) using 40×/1.2 Water and 63×/1.4 Oil Plan Apochromat DIC objectives. Optical sections (1024 pixels×1024 pixels×8 or 12-Bit/pixel) were acquired at 0.5 µm Z-spacing with ZEN Blue 2012, 2.3 or 3.1 software (Zeiss, Germany). Images were further processed using FijiJ distribution of ImageJ software [ 88 ] or ZEN Blue software. NMJ morphology analysis Maturation of NMJs was assessed independently by two researchers using the ZEN 2012 and Blue 3.1 software. BTX-labelled NMJs were analysed from maximum intensity projections (0.5-1 µm interval). On average, 76 NMJs per mouse were analyzed from 9 mice per genotype for soleus (1290 NMJs total) and 7 mice per genotype for TA (1094 NMJs total). For pre- and postsynaptic morphometric analysis, NMJs were co-labelled with anti-neurofilament (2H3) and anti-synaptophysin antibodies or α-BTX, respectively. ImageJ software ( https://imagej.nih.gov/ij/ ) combined with the aNMJ-morph macro [ 89 ] was used to measure 20 individual pre- and postsynaptic morphological parameters (‘core variables’, ‘derived variables’, and ‘associated nerve and muscle variables’). Nerve terminal ‘complexity’ was calculated as log 10 (number of terminal branches x number of branch points x total length of branches). Endplate ‘compactness’ was calculated as (AChR area/endplate area) x 100. The ‘overlap’ of presynaptic and postsynaptic structures was calculated as (area of synaptic contact/total area of AChRs) x 100. On average, 24 en face NMJs with clearly visible pre-synaptic axons and terminals were assessed per mouse. Thirteen mice were analysed per genotype (631 NMJs total). All analyses were performed using maximum intensity projections. Two thresholding methods (‘ Huang ’ and default method) provided the most accurate binary representation of the original raw NMJ images (Supplementary Fig. 2). The ‘ Huang ’ method was used for 96,4% of NMJs, and the default method for 3,6% of NMJs. For the measurement of muscle fibre diameter, fibres were labelled with anti-dystrophin antibody as described above and analysed using ZEN Blue 2.3 software. Innervation analysis The analysis of NMJ innervation and the morphology of motoneuron terminals was performed in a blinded manner, using ZEN 2012 Blue software. Maximum intensity projections (0.5 µm interval) of BTX-, 2H3- and synaptophysin-labelled NMJs were used to count mono- and polyinnervated NMJs, NMJs with ruptured or absent nerve (denervated) and NMJs with degenerated nerves. Nine mice per genotype were scored with an average of 184 NMJs per mouse (3319 NMJs analysed total). Western blotting Muscles were homogenised with a Pro200 Double insulated tissue homogenizer (Bioeko) in 50 volumes of ice-cold lysis buffer per muscle weight [0,1 M K 2 HPO 4 , 0,1 M KH 2 PO 4 pH 7.2, 1mM PMSF] and samples were boiled in Laemmli buffer for 10 min. Twenty micrograms of protein/well were separated with SDS-PAGE through 10% polyacrylamide gels and transferred onto nitrocellulose membranes. Membranes were blocked in 3% non-fat milk in TBST [Tris-buffered saline (TBS) with 0.2% Triton X-100] at RT for 1 h, followed by overnight incubation with primary antibodies diluted in blocking buffer. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a protein loading control. Then, membranes were incubated with HRP-conjugated (horseradish peroxidase) secondary antibodies at RT for 1h. The list of antibodies used can be found in Supplementary Table S1 . The bands were visualised using Immobilon Western Chemiluminescent HRP substrate (Merck) per manufacturer’s instructions. Grip strength The forelimb grip strength of P90 male and female SV/SV mice was tested using a force meter (Bioseb, France) in a blinded manner. Prior to performing tests mice were habituated to the researchers and the environment. On the day of the test, mice were held closely to the grid of the force meter, allowing them to grasp it, and then they were pulled away horizontally. The force meter measured the peak force when the animal lost its grip. Five consecutive trials were performed with a few-minute intervals between them. The final performance was assessed as a mean from all five measurements and is presented as values normalised to body weight. Statistical analysis All statistical tests were performed using GraphPad Prism 7 (CA, USA). Normality of the data was tested with the D’Agostino-Pearson omnibus normality test or the Shapiro-Wilk normality test, depending on the sample size. Datasets were analysed with unpaired t-test, Mann-Whitney test or two-way ANOVA followed by the Sidak’s or Tukey’s multiple comparisons test. Sample size was determined based on previous similar analyses [ 89 – 91 ], represents biological replicates and is reported in figure legends. Error bars depict standard deviation (SD), minimum to maximum or standard error of the mean (SEM) as stated in the figure legends. Declarations Competing interests The authors declare no competing interests. Funding This work was supported by the Preludium 15 grant UMO-2018/29/N/NZ3/02682 (awarded to P. A.-S.) and Miniatura 8 enabling award UMO-2024/08/X/NZ4/00609 (awarded to J. N.), from the National Science Centre, Poland. Polish Euro-BioImaging Node is supported by the project co-financed by the Minister of Education and Science based on contract No 2022/WK/05 (Polish Euro-BioImaging Node “Advanced Light Microscopy Node Poland”). Author Contribution P.M.B., M.J.R. and M.G. – Conceptualisation; J.N., P.A.-S. and M.G. – Investigation, Validation, Formal analysis; T.W. and R.Z. – Investigation; J.N., P.A.-S. and M.G. – Visualisation; M.J.R and M.G. – Supervision; J.N. and P.A.-S. – Project administration and funding acquisition; M.G. – Writing – original draft; J. N., P. A.-S., T. W., R. Z., P.M.B., M. J. R. and M. G. – Writing – review and editing. Acknowledgement Confocal imaging was performed at the Laboratory of Imaging Tissue Structure and Function, which serves as an imaging core facility at the Nencki Institute of Experimental Biology and is part of the infrastructure of the Polish Euro-BioImaging Node. The authors thank Artur Wolny from the Laboratory of Imaging Tissue Structure and Function for technical support of automated image analysis. The anti-neurofilament monoclonal antibody (#AB_2314897) developed by T. M. Jessell and J. Dodd was obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA 52242. Illustrations created with Biorender.com. Data Availability The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. References Redowicz, M. J. Unconventional myosins in muscle. Eur. J. Cell. Biol. 86 , 549–558. 10.1016/j.ejcb.2007.05.007 (2007). Buss, F., Arden, S. D., Lindsay, M., Luzio, J. P. & Kendrick-Jones, J. Myosin VI isoform localized to clathrin-coated vesicles with a role in clathrin-mediated endocytosis. EMBO J. 20 , 3676–3684. 10.1093/emboj/20.14.3676 (2001). Sahlender, D. A. et al. Optineurin links myosin VI to the Golgi complex and is involved in Golgi organization and exocytosis. J. Cell. Biol. 169 , 285–295. 10.1083/jcb.200501162 (2005). Jung, E. J., Liu, G., Zhou, W. & Chen, X. 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(a) \u003c/strong\u003eMaturation of murine NMJs. Immature NMJs take the form of unperforated plaques. After around a week from birth first perforations in AChR assemblies start to appear, and they expand and fuse to form mature pretzel-like NMJs by P21. This complex structure is maintained throughout adulthood (P90) until the aging-related degeneration occurs at NMJs (P547). Timepoints of the analysis are marked in pink. Created in BioRender. Gawor, M. (2025)\u003cu\u003e https://BioRender.com/uk9u03o\u003c/u\u003e \u003cstrong\u003e(b) \u003c/strong\u003eRepresentative\u003cstrong\u003e \u003c/strong\u003eMVI localisation at NMJs in tibialis\u003cem\u003e anterior\u003c/em\u003e muscle at time points depicted in (a). MVI is enriched at NMJs at all analysed stages and is specific to the postsynaptic machinery (orthogonal views in the lower and right panels at P120). P10 represents the stage of NMJ maturation, and P120 and P365\u003cstrong\u003e \u003c/strong\u003eNMJ\u003cstrong\u003e \u003c/strong\u003emaintenance during adulthood and middle-age, before the age-dependent structural changes appear. Postsynaptic AChRs (green) are labelled with bungarotoxin (a-BTX), and motoneurons (blue) with anti-neurofilament (NF) and anti-synaptophysin (Syn.) antibodies. Antibody control – control of secondary antibody unspecific binding. Scale bar = 20 µm. \u003cstrong\u003e(c) \u003c/strong\u003eMeasurement of the fluorescence\u003cstrong\u003e \u003c/strong\u003eintensity\u003cstrong\u003e \u003c/strong\u003efrom a single plane of the optical cross-section from the NMJ at P120 (on the right, white line). Red peaks show MVI labelling fluorescence intensity and green and blue AChRs and motoneuron, respectively. Maximal intensity for MVI (black arrowheads) overlaps with the lowest for AChRs, confirming that MVI is abundant between AChR-rich areas, as shown in (b).\u003c/p\u003e","description":"","filename":"11.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6965921/v1/50a593f743977e544d6e3744.jpg"},{"id":88370858,"identity":"9dadec43-1e41-491d-8aa3-b90cfb46fe73","added_by":"auto","created_at":"2025-08-05 19:07:33","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":519546,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNMJ maturation is delayed in the absence of MVI. \u003c/strong\u003eEffective MVI knockout validated with animal morphology assessment \u003cstrong\u003e(a)\u003c/strong\u003e, genotyping\u003cstrong\u003e (b) \u003c/strong\u003eand Western blotting\u003cstrong\u003e (c). (a)\u003c/strong\u003e Body size of SV/SV mice at P10 is reduced, analogous to the results shown for other ages [\u003csup\u003e19,29,30,92\u003c/sup\u003e]. \u003cstrong\u003e(b) \u003c/strong\u003eRepresentative cropped gel showing SV mice genotyping\u003cstrong\u003e. \u003c/strong\u003eSV/+, controls, heterozygotes\u003cstrong\u003e \u003c/strong\u003e(WT allele = 230 bp, SV allele = 318 bp).\u003cstrong\u003e \u003c/strong\u003eSV/SV, MVI knockouts, homozygotes (SV allele = 318 bp). M, DNA molecular weight marker. Original gel presented in Supplementary Fig.3. \u003cstrong\u003e(c)\u003c/strong\u003e MVI protein expression is effectively knocked out in the soleus muscles of SV/SV mice. Representative cropped blot showing MVI protein levels in SV/SV an SV/+ mice. MVI and GAPDH protein levels are from different regions of the same blot. Original blot presented in Supplementary Fig.4. \u003cstrong\u003e(d)\u003c/strong\u003e Representative images of NMJ classes analysed in (f): immature, unperforated plaques and perforated NMJs (1, 2 and \u0026gt;2 perforations). Arrowheads depict perforated NMJs. Scale bar = 10 µm. \u003cstrong\u003e(e) \u003c/strong\u003eRepresentative images of NMJs in tibialis\u003cem\u003e \u003c/em\u003eanterior (TA, fast-twitch, top panel) and soleus (slow-twitch, bottom panel) muscles of SV/+ and SV/SV mice at P10. NMJs are labelled with a-BTX. Arrowheads show maturing NMJs. Scale bar = 10 µm. \u003cstrong\u003e(f)\u003c/strong\u003e MVI knockout decreases the percentage of maturing NMJs in both TA and soleus\u003cem\u003e \u003c/em\u003emuscles. Quantification of the percentage of plaques vs. perforated NMJs (left) and three classes of perforated NMJs (1, 2 and \u0026gt;2 perforations, right) in TA\u003cem\u003e \u003c/em\u003e(top) and soleus\u003cem\u003e \u003c/em\u003e(bottom) of SV/+ and SV/SV mice. Two-way ANOVA with Sidak’s multiple comparisons test, N = 7 (TA) and N = 9 (soleus), ±SD (left), ±SEM (right),* p£0.05, *** p£0.001.\u003c/p\u003e","description":"","filename":"12.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6965921/v1/3b08185881edc83224589e6f.jpg"},{"id":88371507,"identity":"13c72d20-fecd-43e0-9762-6bc19ac10ddf","added_by":"auto","created_at":"2025-08-05 19:15:33","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":447206,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMVI knockout impairs the size of motoneuron terminals, but not innervation. (a) \u003c/strong\u003eRepresentative images of lower motoneurons (green) labelled with anti-neurofilament (NF) and anti-synaptophysin (Syn.) antibodies and postsynaptic AChRs (magenta) labelled with a-BTX (soleus). Full arrowheads show monoinnervated NMJs, asterisks polyinnervated NMJs, and empty arrowhead NMJ that undergoes synaptic elimination. Magnification of the presynaptic compartment from the area marked with a dotted line is shown in (e). Scale bar = 10 µm. \u003cstrong\u003e(b) \u003c/strong\u003eRepresentative images of denervated NMJs and NMJs with degenerating nerve. Scale bar =\u003cstrong\u003e \u003c/strong\u003e3,5 µm. \u003cstrong\u003e(c)\u003c/strong\u003e Soleus muscles of the SV/SV mice are characterised by a higher number of NMJs at P10. Two-tailed unpaired \u003cem\u003et\u003c/em\u003e-test, N = 9, ±SD, * p£0.05. \u003cstrong\u003e(d)\u003c/strong\u003eMVI has no impact on NMJ innervation at P10 (soleus). Quantification of the NMJ classes presented in (a) and (b). Two-way ANOVA with Sidak’s multiple comparisons test, N = 9, ±SEM, not significant. \u003cstrong\u003e(e) \u003c/strong\u003eRepresentative image of the motor nerve terminals (green, axon identified by full arrowhead and axon terminal by empty arrowhead) presented in (a). \u003cstrong\u003e(f-m)\u003c/strong\u003e Motoneuron terminals of SV/SV mice\u003cstrong\u003e \u003c/strong\u003eare smaller with shorter branches (P10, soleus), as shown by the morphometric analysis. Two-tailed unpaired \u003cem\u003et\u003c/em\u003e-test, N = 13, ±SD, * p£0.05, ** p£0.01.\u003c/p\u003e","description":"","filename":"13.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6965921/v1/2efbe13df6cb6a663a265288.jpg"},{"id":88370087,"identity":"00d32b8f-6166-4edb-acf7-403abd82f9b8","added_by":"auto","created_at":"2025-08-05 18:51:33","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":242748,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLoss of MVI expression reduces the size of the postsynaptic machinery. (a) \u003c/strong\u003eMagnification of the postsynaptic AChRs (magenta) corresponding to the NMJs shown in Fig.3e. \u003cstrong\u003e(b-m) \u003c/strong\u003eMVI regulates the size of motor endplates\u003cstrong\u003e \u003c/strong\u003e(P10, soleus), as shown by the morphometric analysis of the postsynaptic apparatus. \u003cstrong\u003e(m) \u003c/strong\u003eReduced body weight of SV/SV mice at the stage of NMJ maturation (P10). Two-tailed unpaired \u003cem\u003et\u003c/em\u003e-test, N = 13, ±SD, * p£0.05, **** p£0.0001.\u003c/p\u003e","description":"","filename":"14.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6965921/v1/b4afd542de72619a4a6e6b13.jpg"},{"id":88370580,"identity":"8f27997b-fac4-435a-8ea9-2a4add9debc9","added_by":"auto","created_at":"2025-08-05 18:59:33","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":165336,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSV mice exhibit reduced muscle strength. (a) \u003c/strong\u003eExperimental design.\u003cstrong\u003e \u003c/strong\u003eThe\u003cstrong\u003e \u003c/strong\u003egrip strength of adult SV/+ and SV/SV mice (males and females) was\u003cstrong\u003e \u003c/strong\u003etested at P90 (marked in pink). The time when NMJ maturation is mostly completed (P21) is marked in grey. Created in BioRender. Gawor, M. (2025) \u003cu\u003ehttps://BioRender.com/eyshv76\u003c/u\u003e \u003cstrong\u003e(b) \u003c/strong\u003eLack of MVI expression alters muscle performance. The force of the grip (N) normalised to body weight (g) shown at \u003cstrong\u003e(c)\u003c/strong\u003e dropped for both female and male SV/SV mice. Two-way ANOVA with Tukey’s multiple comparisons test, N = 11 (females, SV/+ and SV/SV) and N = 8 (male, SV/+), and N = 9 (male, SV/SV), whiskers show min. to max. (b) or ±SD (c),* p£0.05, *** p£0.001, **** p£0.0001.\u003c/p\u003e","description":"","filename":"15.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6965921/v1/36896914337d42d8b38bb50f.jpg"},{"id":99545523,"identity":"53e3aef9-77c7-4d15-893c-e496a8be910d","added_by":"auto","created_at":"2026-01-05 16:08:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2702774,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6965921/v1/c2a4194c-4cf3-4cc8-bbf1-7011efa1dac3.pdf"},{"id":88370088,"identity":"d64cd9d6-f086-4742-af9e-faa85c029302","added_by":"auto","created_at":"2025-08-05 18:51:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":873441,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryinformation.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6965921/v1/c571551d15a9c5b366b230e5.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Myosin VI depletion delays neuromuscular junction maturation and exacerbates muscle performance","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eMyosins are highly conserved actin-based molecular motors, expressed across \u003cem\u003eEukaryota\u003c/em\u003e [\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e] and participating in cytokinesis, cell migration, adhesion and fusion, endo- and exocytosis, intracellular trafficking, transcription, chromatin organisation and DNA damage repair [\u003csup\u003e\u003cspan additionalcitationids=\"CR3 CR4 CR5 CR6 CR7 CR8 CR9\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e]. Muscle myosin heavy chain isoforms serve as muscle fiber type-specific molecular markers, important to discern the mechanisms of myogenic development and to understand muscle function in health and disease. Based on their ability to form filaments and their origin, myosins are divided into conventional (class II) and unconventional, classified in humans into 11 classes (families) [\u003csup\u003e\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e]. Out of forty genes encoding myosin heavy chains in humans, almost two-thirds encode unconventional ones, but their function is still less understood than conventional ones.\u003c/p\u003e\u003cp\u003eUnconventional myosin VI (MVI) is a unique member of the myosin family that moves backwards, towards the minus end of actin filaments [\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e]. Snell\u0026rsquo;s waltzer (SV) mice are natural MVI knockouts carrying a spontaneous null mutation that causes deafness, circling, and hyperactivity [\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e]. Additionally, structural defects in kidneys, brain, and testes have been reported in SV/SV mice [\u003csup\u003e\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e]. Given the multiple roles of MVI, it is localised in various cell compartments and organelles, such as the sarcoplasmic reticulum, Golgi apparatus, intercalated discs, cell nucleolus and nucleus, and around mitochondria [\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan additionalcitationids=\"CR21 CR22 CR23 CR24 CR25\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e]. Importantly, MVI is a crucial regulator of striated muscle development and physiology. It is involved in heart organisation and dysfunction of this protein entails left ventricular cardiomyopathy [\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e]. Growing evidence underlines the importance of MVI in myogenesis and skeletal muscle function. Studies of our research group show that loss of MVI results in aberrated myoblast adhesion, fusion, metabolism, and actin organisation in myoblasts \u003cem\u003ein vitro\u003c/em\u003e [\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e]. MVI controls expression of crucial myogenic regulators, Pax7 (Paired Box 7), MyoD (Myogenic Differentiation 1), and myogenin, as well as adhesion proteins, and fusogens, myomaker and myomerger [\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e]. We also determined that lack of MVI increases the muscle/body weight ratio and significantly affects the morphology of the murine hindlimb muscles. Myosin VI knockout causes a 2.5-fold increase of thin muscle fibres with a cross-sectional area below 100 \u0026micro;m\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. This effect, observed as early as at birth, is the most evident in the slow-twitch muscle soleus and is maintained throughout the animals\u0026rsquo; life span [\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e].\u003c/p\u003e\u003cp\u003eNeuromuscular junctions (NMJs) are peripheral synapses connecting motoneurons and skeletal muscle fibers. Compromised NMJ integrity and function is a hallmark of neuromuscular disorders, such as Duchenne muscular dystrophy, myasthenia gravis, congenital myasthenic syndromes (CMS), Charcot-Marie-Tooth disease (CMTD) or amyotrophic lateral sclerosis (ALS) [\u003csup\u003e\u003cspan additionalcitationids=\"CR32 CR33 CR34 CR35 CR36\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e]. Despite the central role of NMJs in the onset and progression of neuromuscular disorders, the molecular mechanisms underlying these processes are still poorly understood. Thorough characterisation of the genetic models manifesting neuromuscular symptoms can bring valuable insights into their pathology and help to develop more effective therapies.\u003c/p\u003e\u003cp\u003eWe have previously identified MVI as a postsynaptic protein at NMJs in rats, however, the role of MVI in NMJ function remains unexplored [\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e]. Here we show that MVI is involved in NMJ development and maintenance. MVI is a protein abundant at NMJs during developmental remodelling and adulthood and is localised postsynaptically in both slow and fast-twitch muscles. The knockout of MVI delays NMJ maturation, however, independently of synaptic elimination. Moreover, loss of MVI decreases the size of both pre- and postsynaptic apparatuses, in accordance with the overall reduction of the body, muscle, and muscle fiber size in SV/SV mice [\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e]. The significantly diminished grip strength of both female and male MVI knockouts suggests that MVI depletion has universal functional consequences for muscle, regardless of sex.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cem\u003eMVI is enriched at NMJs during developmental remodelling and maintenance\u003c/em\u003e\u003c/p\u003e\u003cp\u003eMature neuromuscular junctions develop from simple oval acetylcholine receptor (AChR) assemblies during plaque-to-pretzel transition. After the first week of life, plaques become increasingly perforated and reshape into pretzel-like structures by P21, when maturation is mostly completed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea) [\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e]. Subsequently, NMJs grow in size until the mouse reaches adulthood (P90). Another type of NMJ remodelling is induced by aging which starts around 14 months of age (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea) [\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e]. We assessed the localisation of MVI in fast-twitch tibialis anterior at the time of intense developmental remodelling (P10), and NMJ maintenance in mature adult (P120) and middle-aged (P365) mice. MVI was enriched at NMJs at all analysed stages, but distributed differently (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). At the stage of NMJ maturation (P10) MVI was dispersed at the NMJ postsynaptic compartment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). At mature NMJs (P120, P365) MVI mainly occupied the domains between AChR-rich areas, and was also clustered at the vicinity of AChRs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, orthogonal views at P120 and Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). The enrichment of MVI along the entire postsynaptic machinery was characteristic for NMJs of adult muscles with different fiber composition and function: fast-twitch diaphragm and slow-twitch triangularis sterni and soleus (Supplementary Fig.\u0026nbsp;1). Our results show that MVI is localised postsynaptically at NMJs of various skeletal muscles throughout the mouse lifespan, during development and maintenance of NMJs.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eKnockout of MVI delays NMJ maturation in slow- and fast-twitch muscles\u003c/em\u003e\u003c/p\u003e\u003cp\u003eSince MVI is present at NMJs during developmental remodelling, we tested its role in postsynaptic maturation. To this end, we determined the ratio of perforated NMJs upon MVI knockout at P10 in two types of muscles: fast-twitch tibialis anterior (TA) and slow-twitch soleus (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea-f). The number of maturing NMJs decreased in both analysed muscles by 34% (TA) and 36% (soleus) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee and f, on the left). Simultaneously, we did not observe changes in the proportion of NMJs with different numbers of perforations (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef, on the right), which suggests that MVI expression rather affects the induction of postsynaptic maturation than its progression. Overall, these results show that MVI has a broad effect on NMJ development and the lack of this protein delays NMJ maturation both in fast- and slow-twitch muscles.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eLack of MVI reduces the size of the NMJ pre- and postsynaptic domains, but does not affect innervation\u003c/em\u003e\u003c/p\u003e\u003cp\u003ePostsynaptic maturation is tightly regulated by innervation, and pre- and postsynaptic compartments morphologically mirror each other to collaborate optimally. Initially, NMJs are polyinnervated, and during muscle development, they gradually lose surplus axonal inputs in a process called synaptic elimination, completed by the end of the second week after birth [\u003csup\u003e\u003cspan additionalcitationids=\"CR42 CR43\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e]. We assessed whether the observed delay in postsynaptic maturation upon MVI knockout at P10 is accompanied by changes in NMJ innervation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-d). Since soleus is the muscle where MVI expression is the most abundant [\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e] and its effect on NMJ maturation was more statistically significant (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef), we focused on this muscle in our subsequent analyses. We determined the ratio of mono- and polyinnervated NMJs, considered the presynaptic indicator of NMJ maturation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). A higher ratio of polyinnervated NMJs would indicate delayed synaptic elimination. We also assessed the number of denervated NMJs and NMJs with degenerating nerves, another two indicators of synapse elimination (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). The overall number of NMJs was increased by 30% after MVI knockout (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). However, there were no significant differences in innervation, suggesting that the delayed postsynaptic maturation was independent of synaptic elimination (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). Thus, we analysed the morphology of the motoneuron terminals of SV/SV mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee-m). MVI depletion decreased nerve terminal perimeter and area by 14,5% and 27%, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef and g), as well as the total and average length of axonal branches by 19% (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ek) and by 21% (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003el), respectively. The changes in presynaptic morphology coincided with reduced size of the postsynaptic compartment and diminished SV/SV mice body weight (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea-m). MVI knockout decreased AChR area by 11% (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec), and endplate perimeter by 5% (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee), and area by 11% (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef). Since MVI knockout muscles are characterised by thinner muscle fibres, we verified whether the observed reduction in NMJ size is correlated with the muscle fibre diameter [\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e]. Indeed, endplate area when normalised to the muscle fibre diameter was similar in SV/SV and SV/+ mice, indicating that NMJs size is correlated with thinner muscle fibres (Supplementary Fig.\u0026nbsp;2). Parameters, such as number of AChR clusters, an indicator of endplate fragmentation, or overlap, showing endplate presynaptic coverage were similar (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ej and l). This result supports our conclusion that the synaptic elimination is not impaired in MVI knockouts (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). In summary, our observations confirm that MVI knockout impacts pre- and postsynaptic morphology of motor terminals, independently of denervation and degeneration characteristic for the loss of motoneurons during development.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eMVI is necessary to maintain proper muscle strength regardless of sex\u003c/em\u003e\u003c/p\u003e\u003cp\u003eNext, we determined whether the observed changes in the NMJ morphology alter muscle performance. To this end, we performed grip strength test with adult SV/+ and SV/SV mice of both sexes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Since both female and male MVI knockouts have reduced body weight [\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e], we normalised the obtained average force values to the body weight of mice used in our behavioral studies (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb and c).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eBoth female and male SV/SV mice had weaker muscles in comparison to control animals, as shown by the 56% and 44% drop in their muscle strength, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). Interestingly, the bigger impact of MVI knockout on female performance was not associated with the more pronounced decrease of their body weight, since MVI depletion caused more significant reduction of the male body weight (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec).\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eWe show that MVI is ubiquitously expressed at NMJs of various muscles at different stages of the development (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Supplementary Fig.\u0026nbsp;1). MVI is adjacent to AChRs and present in the domain devoid of AChRs. Given the broad role of MVI in endo- and exocytosis, it can be involved in synaptic signalling at NMJs [\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan additionalcitationids=\"CR47 CR48 CR49\" citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e]. In the central nervous system, MVI plays a role in synaptic transmission and plasticity [\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e,\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e]. MVI expression increases in slow- and fast-twitch muscles upon denervation, and the protein localises to the entire muscle fibre, in contrast to its peripheral localisation in innervated muscles [\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e]. This observation indicates that MVI expression is regulated by synaptic activity. \u003cem\u003eDrosophila melanogaster\u003c/em\u003e MVI loss-of-function mutants display defects in NMJ morphology, synaptic vesicle distribution, and basal synaptic transmission, accompanied by the impaired locomotor activity of the mutant larvae [\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e]. Moreover, MVI is an important regulator of the proper organisation of synaptic vesicles at \u003cem\u003eDrosophila\u003c/em\u003e NMJs and anchors them at the specific regions of synaptic domains [\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e]. MVI can play a similar role at mouse NMJs and participate in synaptic signalling during development. However, further studies identifying MVI molecular partners specific to peripheral synapses and the spatiotemporal regulation of their distribution at NMJs are necessary to unravel the function of this protein in the described context.\u003c/p\u003e\u003cp\u003eThe diminished percentage of perforated NMJs in both fast- and slow-twitch muscles of SV/SV mice indicates a wide-range delay in synapse maturation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee and f). However, this impairment appears not to be caused by stalled synaptic elimination (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed and Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ei and l). One of the possible explanations is that MVI is required for AChR clustering and redistribution during the formation of synaptic perforations. It has been shown that MVI is a part of the complex regulating Rac1 (Rac Family Small GTPase 1) and Cdc42 (Cell Division Cycle 42) Rho GTPases, rearranging the actin cytoskeleton and vesicle trafficking, which can subsequently impact AChR turnover and degradation [\u003csup\u003e\u003cspan additionalcitationids=\"CR56\" citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e]. Moreover, Rho GEF ephexin1 is the effector protein of the Musk-Dok7 complex, crucial for AChR dispersal [\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e,\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e]. Thus, Rho GTPases are potential MVI downstream targets that control AChR recycling at the maturing NMJs. Simultaneously, MVI can support the formation of synaptic perforations by anchoring AChRs and restricting their localisation to certain NMJ areas, which facilitates the formation and propagation of perforations. MVI anchoring role at NMJs has been confirmed in studies of \u003cem\u003eDrosophila\u003c/em\u003e MVI mutants. Normally, synaptic vesicles are concentrated at the outer ring of the synaptic boutons. Upon MVI knockout, this selective localisation is lost, and they occupy the entire area of the boutons [\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e]. Similarly, loss of MVI stabilisation can result in AChR dispersal and delay the formation of perforations at mouse NMJs.\u003c/p\u003e\u003cp\u003eWe observed the increase in the overall number of NMJs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). This confirms our previous observations showing that SV/SV mice are characterised by a higher number of muscle fibres [\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e]. Both pre- and postsynaptic endplate size parameters were reduced in MVI knockouts (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef, g, k and l and Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec, e and f). It was shown previously by our group that soleus muscles of SV/SV mice are characterised by a larger number of fibres with decreased cross-sectional area [\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e]. In agreement with the impact of the nuclear content on the myofiber size, the number of myonuclei was also decreased after MVI knockout [\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e]. Endplate areas normalised to the corresponding muscle fibre diameters showed similar values for SV/+ and SV/SV mice (Supplementary Fig.\u0026nbsp;2), suggesting a relationship between NMJ and muscle fibre size. Some studies showed that these parameters are correlated [\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e,\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e], however, other groups reported that muscle fibre size is not a main contributor to NMJ morphology [\u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e]. This discrepancy can be explained by the different developmental stages, muscle types and species analysed [\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e,\u003cspan additionalcitationids=\"CR63\" citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e]. Another factor that can influence NMJ size is the fibre type [\u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e]. Given the role of MVI in myogenesis and the significant increase in the number of thinner fibres in MVI knockout mice [\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e], it would be interesting to perform fibre type analyses and the measurements of the size of corresponding NMJs in these knockouts. It was recently reported that the absence of MVI expression in skeletal muscles causes glycolytic-to-oxidative fibre-type switch [\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e]. However, the relationship between the muscle fibre metabolic type and the size of NMJs is yet to be determined, due to mixed results from different types of muscles [\u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e,\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e,\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e]. Nevertheless, impairments in muscle structure of MVI knockouts are accompanied by changes in their metabolism. The levels of phosphorylated PKA (Protein Kinase A) and CREB (cAMP Response Element-Binding) proteins, regulators of glucose and lipid metabolism and mitochondrial function, are decreased in MVI-devoid soleus muscles at birth [\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e]. In adult mice, loss of MVI impairs ATP production and mTOR-dependent signalling, major contributors to muscle growth [\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e,\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e]. Moreover, loss of MVI disrupts adhesion, fusion, and differentiation of myotubes \u003cem\u003ein vitro\u003c/em\u003e [\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e]. Altogether, these alterations can delay muscle fibre development and growth, undermining the muscle\u0026rsquo;s ability to support the reorganisation of developing NMJs.\u003c/p\u003e\u003cp\u003eThe importance of the muscle intrinsic signals for NMJ remodelling and the role of MVI in muscle fibre development and myoblast fusion are well documented [\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan additionalcitationids=\"CR69 CR70\" citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/sup\u003e). Lack of MVI alters the formation of myotubes and decreases γ-actin, focal adhesion kinase (FAK), and M-cadherin expression, all of which have been implicated in synapse formation and function [\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan additionalcitationids=\"CR73 CR74\" citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e\u003c/sup\u003e]. Expression of the key myogenic regulators, such as Pax7 and myogenin, is also reduced in MVI knockout primary myoblasts [\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e]. Myogenin has been identified as an important regulator of NMJ size [\u003csup\u003e\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e\u003c/sup\u003e], which is in accordance with the diminished pre- and postsynaptic area of the motor endplates observed in SV/SV mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef,g and Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec, e and f). Importantly, myogenin is a myogenic regulatory factor (MRF) crucial for AChR clustering in myotubes \u003cem\u003ein vitro\u003c/em\u003e, and its role cannot be replaced by other MRFs or AChR organisers, Musk and rapsyn [\u003csup\u003e\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e\u003c/sup\u003e]. Thus, myogenin should be a primary focus in future analyses aiming to explain MVI-dependent mechanisms regulating NMJ morphology. Future investigation should also assess the MVI impact on localisation and function of terminal Schwann cells (TSCs) which are crucial for NMJ maturation and maintenance [\u003csup\u003e\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e,\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e\u003c/sup\u003e]. TSCs regulate the expression of postsynaptic genes and if their function is impaired, NMJ maturation can be delayed even in the presence of unaltered innervation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). Altogether, these results and MVI emerging role in transcriptional regulation [\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e,\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e\u003c/sup\u003e], suggest this protein could be a regulator of transcription factors that control the activity of NMJ-related genes.\u003c/p\u003e\u003cp\u003eWe show that MVI knockout causes a significant impairment in the muscle performance in both female and male mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). This can be a result of the reduced endplate and nerve terminal size, which limit synaptic transmission and weaken the muscles which cannot be compensated by the higher number of NMJs (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The size of nerve terminals impacts neurotransmitter release at NMJs of various species [\u003csup\u003e\u003cspan additionalcitationids=\"CR83\" citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e\u003c/sup\u003e]. The observed phenotype can also be a consequence of delayed maturation. A similar effect was reported for ephexin1 knockouts, where NMJs failed to acquire complex pretzel-like topology, which was accompanied by muscle weakening and impaired neurotransmission [\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e]. Finally, MVI binding partner Dock-7 (Dedicator of Cytokinesis 7) was shown to participate in the neuregulin-ErbB2 pathway, which regulates neurotransmission [\u003csup\u003e\u003cspan additionalcitationids=\"CR86\" citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e\u003c/sup\u003e]. Moreover, recent analyses performed by our research group show impaired mitochondria respiration and reduced ATP production upon MVI knockout which can also cause muscle weakening [\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e]. Future analyses should determine how lack of MVI affects synaptic transmission and neurotransmitter release, neurotransmitter receptor expression and function, as well as downstream signalling pathways controlling muscle performance and fatigability.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cp\u003e\u003cb\u003eEthics declarations\u003c/b\u003e\u003c/p\u003e\u003cp\u003e Procedures involving animals were approved by the 1st Local Ethical Committee for Experiments on Animals in Warsaw (resolutions 1311/2022 and 1639/2024) and were performed in accordance with the Act on the Protection of Animals Used for Scientific or Educational Purposes (2015) from the European Communities Council directives approved by the Polish Parliament. The researchers had individual permissions for the work involving mice granted by the Director of the Nencki Institute of Experimental Biology [218(W)/2024/IBD, 396(U)/2019/IBD, 396W/2020/IBD, 194P/2019/IBD, 194W/2020/IBD, 194/2021/IBD, 17(W)/2020/IBD, 27(W;P)/2020/IBD]. All experiments were performed in accordance with relevant guidelines and regulations and are reported according to ARRIVE guidelines.\u003c/p\u003e\u003cp\u003e\u003cb\u003eSnell\u0026rsquo;s waltzer (SV) mice husbandry and genotyping\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSnell's waltzer (SV) mice (C57BL/6J genetic background), a gift from Dr. Folma Buss (Cambridge Institute of Medical Research, UK), and C57BL/6J mice were maintained in the animal house of the Nencki Institute of Experimental Biology. All analyses were performed on male mice except the grip strength test, which was performed using both sexes. The age of mice is reported in relevant description of the methods, figures and figure legends. The weight of mice representative of the age P10 (morphometric NMJ analysis) and P90 (grip strength test) is presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003em and Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec, respectively. Mice were euthanised with a lethal dose of isoflurane followed by cervical dislocation. SV mice tail clips (2\u0026ndash;4 mm\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e) were used for genotyping with PCR Master Mix (Thermo Scientific) or KAPA Mouse Genotyping Kit (KAPA Biosystems) per manufacturer\u0026rsquo;s instructions. Primers for genotyping were as follows: SV1 5\u0026prime;-CTGACCCTGATCACTTAGCAGAGTTG-3\u0026prime;; SV2 5\u0026prime;-CATTGGGCCAGGTCACAGAAGTAAGC-3\u0026prime;; SV3 5\u0026prime;-GGTCCTCTGAAAGAGTAACC-3\u0026prime; (SV/+ 318 and 230 bp, SV/SV 318 bp). Sex-matched littermates (SV/+) were used as controls for the phenotype assessment of MVI knockouts (SV/SV).\u003c/p\u003e\u003cp\u003e\u003cb\u003eWhole-mount muscle fibre immunostaining\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSoleus or tibialis anterior (TA) hindlimb muscles were isolated at time points reflecting different stages of NMJ development and maintenance (P10, P120, P365 for C57BL/6J and P10 for SV mice). Muscles were fixed in 4% paraformaldehyde in phosphate buffer saline (PBS) at room temperature (RT) for 15\u0026ndash;80 min., depending on the muscle size and washed three times for 15 min. in PBS. For each analysis, muscle fibers were isolated from randomly chosen parts spanning the whole muscle. Pieces comprising approximately 10\u0026ndash;30 muscle fibers were dissected, incubated for 30 min. in 0,1 M glycine in PBS, and rinsed in PBS and 0,5% Triton X-100. Muscles were incubated for minimum 30 min. to overnight at RT in blocking buffer [2\u0026ndash;3% bovine serum albumin, 2\u0026ndash;5% normal goat serum, 0.05\u0026ndash;0.5% Triton X-100, 0.02% NaN\u003csub\u003e3\u003c/sub\u003e in PBS]. Then, fibers were incubated with primary antibodies diluted in blocking buffer at 4\u0026deg;C overnight in a sample shaker, and washed three times with PBS for 5 min. After washing, specimens were incubated with secondary antibodies diluted in blocking buffer, washed with PBS, and stained with α-bungarotoxin (BTX) diluted in PBS at RT for 15 min. Control stainings omitting primary antibody were performed to test secondary antibody non-specific binding. Whole-mount preparations were mounted in Fluoromount Aqueous Mounting Medium (Sigma) with DAPI (4\u0026prime;,6-diamidino-2-phenylindole) or Vectashield Plus Antifade Mounting Medium with DAPI (Vector Laboratories). Antibodies and fluorescent reagents used are listed in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. Fluorescence intensity was measured using ZEN Blue 3.1 software.\u003c/p\u003e\u003cp\u003e\u003cb\u003eConfocal imaging\u003c/b\u003e\u003c/p\u003e\u003cp\u003eImages were collected using an Axio Observer Z.1 inverted microscopes: Spinning Disc equipped with CSU-X1 spinning disc unit (Yokogawa, Japan) and Evolve 512 EMCCD camera (Photometrics, USA) or LSM780 (Zeiss, Germany) using 40\u0026times;/1.2 Water and 63\u0026times;/1.4 Oil Plan Apochromat DIC objectives. Optical sections (1024 pixels\u0026times;1024 pixels\u0026times;8 or 12-Bit/pixel) were acquired at 0.5 \u0026micro;m Z-spacing with ZEN Blue 2012, 2.3 or 3.1 software (Zeiss, Germany). Images were further processed using FijiJ distribution of ImageJ software [\u003csup\u003e\u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e\u003c/sup\u003e] or ZEN Blue software.\u003c/p\u003e\u003cp\u003e\u003cb\u003eNMJ morphology analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eMaturation of NMJs was assessed independently by two researchers using the ZEN 2012 and Blue 3.1 software. BTX-labelled NMJs were analysed from maximum intensity projections (0.5-1 \u0026micro;m interval). On average, 76 NMJs per mouse were analyzed from 9 mice per genotype for soleus (1290 NMJs total) and 7 mice per genotype for TA (1094 NMJs total).\u003c/p\u003e\u003cp\u003eFor pre- and postsynaptic morphometric analysis, NMJs were co-labelled with anti-neurofilament (2H3) and anti-synaptophysin antibodies or α-BTX, respectively. ImageJ software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://imagej.nih.gov/ij/\u003c/span\u003e\u003cspan address=\"https://imagej.nih.gov/ij/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) combined with the aNMJ-morph macro [\u003csup\u003e\u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e\u003c/sup\u003e] was used to measure 20 individual pre- and postsynaptic morphological parameters (\u0026lsquo;core variables\u0026rsquo;, \u0026lsquo;derived variables\u0026rsquo;, and \u0026lsquo;associated nerve and muscle variables\u0026rsquo;). Nerve terminal \u0026lsquo;complexity\u0026rsquo; was calculated as log\u003csub\u003e10\u003c/sub\u003e (number of terminal branches x number of branch points x total length of branches). Endplate \u0026lsquo;compactness\u0026rsquo; was calculated as (AChR area/endplate area) x 100. The \u0026lsquo;overlap\u0026rsquo; of presynaptic and postsynaptic structures was calculated as (area of synaptic contact/total area of AChRs) x 100. On average, 24 \u003cem\u003een face\u003c/em\u003e NMJs with clearly visible pre-synaptic axons and terminals were assessed per mouse. Thirteen mice were analysed per genotype (631 NMJs total). All analyses were performed using maximum intensity projections. Two thresholding methods (\u0026lsquo;\u003cem\u003eHuang\u003c/em\u003e\u0026rsquo; and default method) provided the most accurate binary representation of the original raw NMJ images (Supplementary Fig.\u0026nbsp;2). The \u0026lsquo;\u003cem\u003eHuang\u003c/em\u003e\u0026rsquo; method was used for 96,4% of NMJs, and the default method for 3,6% of NMJs. For the measurement of muscle fibre diameter, fibres were labelled with anti-dystrophin antibody as described above and analysed using ZEN Blue 2.3 software.\u003c/p\u003e\u003cp\u003e\u003cb\u003eInnervation analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe analysis of NMJ innervation and the morphology of motoneuron terminals was performed in a blinded manner, using ZEN 2012 Blue software. Maximum intensity projections (0.5 \u0026micro;m interval) of BTX-, 2H3- and synaptophysin-labelled NMJs were used to count mono- and polyinnervated NMJs, NMJs with ruptured or absent nerve (denervated) and NMJs with degenerated nerves. Nine mice per genotype were scored with an average of 184 NMJs per mouse (3319 NMJs analysed total).\u003c/p\u003e\u003cp\u003e\u003cb\u003eWestern blotting\u003c/b\u003e\u003c/p\u003e\u003cp\u003eMuscles were homogenised with a Pro200 Double insulated tissue homogenizer (Bioeko) in 50 volumes of ice-cold lysis buffer per muscle weight [0,1 M K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e, 0,1 M KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e pH 7.2, 1mM PMSF] and samples were boiled in Laemmli buffer for 10 min. Twenty micrograms of protein/well were separated with SDS-PAGE through 10% polyacrylamide gels and transferred onto nitrocellulose membranes. Membranes were blocked in 3% non-fat milk in TBST [Tris-buffered saline (TBS) with 0.2% Triton X-100] at RT for 1 h, followed by overnight incubation with primary antibodies diluted in blocking buffer. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a protein loading control. Then, membranes were incubated with HRP-conjugated (horseradish peroxidase) secondary antibodies at RT for 1h. The list of antibodies used can be found in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. The bands were visualised using Immobilon Western Chemiluminescent HRP substrate (Merck) per manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\u003cp\u003e\u003cb\u003eGrip strength\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe forelimb grip strength of P90 male and female SV/SV mice was tested using a force meter (Bioseb, France) in a blinded manner. Prior to performing tests mice were habituated to the researchers and the environment. On the day of the test, mice were held closely to the grid of the force meter, allowing them to grasp it, and then they were pulled away horizontally. The force meter measured the peak force when the animal lost its grip. Five consecutive trials were performed with a few-minute intervals between them. The final performance was assessed as a mean from all five measurements and is presented as values normalised to body weight.\u003c/p\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eAll statistical tests were performed using GraphPad Prism 7 (CA, USA). Normality of the data was tested with the D\u0026rsquo;Agostino-Pearson omnibus normality test or the Shapiro-Wilk normality test, depending on the sample size. Datasets were analysed with unpaired t-test, Mann-Whitney test or two-way ANOVA followed by the Sidak\u0026rsquo;s or Tukey\u0026rsquo;s multiple comparisons test. Sample size was determined based on previous similar analyses [\u003csup\u003e\u003cspan additionalcitationids=\"CR90\" citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e91\u003c/span\u003e\u003c/sup\u003e], represents biological replicates and is reported in figure legends. Error bars depict standard deviation (SD), minimum to maximum or standard error of the mean (SEM) as stated in the figure legends.\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis work was supported by the Preludium 15 grant UMO-2018/29/N/NZ3/02682 (awarded to P. A.-S.) and Miniatura 8 enabling award UMO-2024/08/X/NZ4/00609 (awarded to J. N.), from the National Science Centre, Poland. Polish Euro-BioImaging Node is supported by the project co-financed by the Minister of Education and Science based on contract No 2022/WK/05 (Polish Euro-BioImaging Node \u0026ldquo;Advanced Light Microscopy Node Poland\u0026rdquo;).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eP.M.B., M.J.R. and M.G. \u0026ndash; Conceptualisation; J.N., P.A.-S. and M.G. \u0026ndash; Investigation, Validation, Formal analysis; T.W. and R.Z. \u0026ndash; Investigation; J.N., P.A.-S. and M.G. \u0026ndash; Visualisation; M.J.R and M.G. \u0026ndash; Supervision; J.N. and P.A.-S. \u0026ndash; Project administration and funding acquisition; M.G. \u0026ndash; Writing \u0026ndash; original draft; J. N., P. A.-S., T. W., R. Z., P.M.B., M. J. R. and M. G. \u0026ndash; Writing \u0026ndash; review and editing.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eConfocal imaging was performed at the Laboratory of Imaging Tissue Structure and Function, which serves as an imaging core facility at the Nencki Institute of Experimental Biology and is part of the infrastructure of the Polish Euro-BioImaging Node. The authors thank Artur Wolny from the Laboratory of Imaging Tissue Structure and Function for technical support of automated image analysis. The anti-neurofilament monoclonal antibody (#AB_2314897) developed by T. M. Jessell and J. Dodd was obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA 52242. Illustrations created with Biorender.com.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eRedowicz, M. J. Unconventional myosins in muscle. \u003cem\u003eEur. J. Cell. Biol.\u003c/em\u003e \u003cb\u003e86\u003c/b\u003e, 549\u0026ndash;558. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.ejcb.2007.05.007\u003c/span\u003e\u003cspan address=\"10.1016/j.ejcb.2007.05.007\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2007).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBuss, F., Arden, S. D., Lindsay, M., Luzio, J. P. \u0026amp; Kendrick-Jones, J. 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Genet.\u003c/em\u003e \u003cb\u003e12\u003c/b\u003e, 2797\u0026ndash;2805. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/hmg/ddg308\u003c/span\u003e\u003cspan address=\"10.1093/hmg/ddg308\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2003).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"molecular motor, myosin VI, neuromuscular junction, maturation, skeletal muscle","lastPublishedDoi":"10.21203/rs.3.rs-6965921/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6965921/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eUnconventional myosin VI (MVI) is an ATP-dependent actin-binding molecular motor that participates in numerous cellular and tissue functions, including striated muscle physiology. Lack of MVI expression significantly aberrates myogenesis and skeletal muscle metabolism, and alters myoblast adhesion, fusion, and cytoskeletal organisation. Concomitantly, MVI knockout mice display functional and structural cardiac defects. Here, for the first time, we investigate the impact of MVI on neuromuscular junctions (NMJs), the peripheral synapses crucial for skeletal muscle contraction. We show that MVI is enriched at the postsynaptic machinery of developing and adult NMJs. We analyse the morphology of NMJs of MVI knockout mice (Snell\u0026rsquo;s waltzer, SV) during development and show that MVI deficiency delays NMJ maturation in fast- and slow-twitch muscles. It also reduces the NMJ size of the soleus muscle, as demonstrated by the decreased morphological parameters of both presynaptic and postsynaptic compartments. Simultaneously, synaptic elimination remains unaffected after MVI knockout, suggesting that the observed phenotypes are innervation-independent. Lastly, depletion of MVI impairs the grip strength of both female and male SV/SV mice. In summary, our studies show that MVI is an important regulator of NMJ size and maturation, controls muscle performance, and its impact is independent of innervation and sex.\u003c/p\u003e","manuscriptTitle":"Myosin VI depletion delays neuromuscular junction maturation and exacerbates muscle performance","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-05 18:51:28","doi":"10.21203/rs.3.rs-6965921/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-29T07:49:00+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-22T10:36:20+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"76773768159475206777887481122015223552","date":"2025-09-01T09:29:38+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-27T14:38:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"35155430545481762635715364281695352133","date":"2025-08-06T11:23:31+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-31T02:24:16+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-15T17:00:08+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-09T17:19:17+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-07-09T17:14:58+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"84d062a1-f055-4fbd-ad39-fb96c1442c9e","owner":[],"postedDate":"August 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":52576038,"name":"Biological sciences/Developmental biology"},{"id":52576039,"name":"Biological sciences/Developmental biology/Organogenesis"},{"id":52576040,"name":"Biological sciences/Developmental biology/Organogenesis/Musculoskeletal development"}],"tags":[],"updatedAt":"2026-01-05T16:05:56+00:00","versionOfRecord":{"articleIdentity":"rs-6965921","link":"https://doi.org/10.1038/s41598-025-28650-x","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-12-29 15:58:06","publishedOnDateReadable":"December 29th, 2025"},"versionCreatedAt":"2025-08-05 18:51:28","video":"","vorDoi":"10.1038/s41598-025-28650-x","vorDoiUrl":"https://doi.org/10.1038/s41598-025-28650-x","workflowStages":[]},"version":"v1","identity":"rs-6965921","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6965921","identity":"rs-6965921","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}