Mitochondrial ROS Produced by Skeletal Muscle Mitochondria Promote the Decisive Signal for UPRmt Activation.

The mitochondrial unfolded protein response (UPRmt) can repair and remove misfolded or unfolded proteins in mitochondria and enhance mitochondrial protein homeostasis. Reactive oxygen species (ROS) produced by regular exercise is a crucial signal for promoting health, and skeletal muscle mitochondria are the primary source of ROS during exercise. To verify whether UPRmt is related to ROS produced by mitochondria in skeletal muscle during regular exercise, we adapted MitoTEMPO, mitochondrially targeted antioxidants, and ROS production by mitochondria. Our results showed that mitochondrial ROS is the key factor for activating UPRmt in different pathways.

Perspectives on hiPSC-Derived Muscle Cells as Drug Discovery Models for Muscular Dystrophies.

Muscular dystrophies are a heterogeneous group of inherited diseases characterized by the progressive degeneration and weakness of skeletal muscles, leading to disability and, often, premature death. To date, no effective therapies are available to halt or reverse the pathogenic process, and meaningful treatments are urgently needed. From this perspective, it is particularly important to establish reliable in vitro models of human muscle that allow the recapitulation of disease features as well as the screening of genetic and pharmacological therapies. We herein review and discuss advances in the development of in vitro muscle models obtained from human induced pluripotent stem cells, which appear to be capable of reproducing the lack of myofiber proteins as well as other specific pathological hallmarks, such as inflammation, fibrosis, and reduced muscle regenerative potential. In addition, these platforms have been used to assess genetic correction strategies such as gene silencing, gene transfer and genome editing with clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), as well as to evaluate novel small molecules aimed at ameliorating muscle degeneration. Furthermore, we discuss the challenges related to in vitro drug testing and provide a critical view of potential therapeutic developments to foster the future clinical translation of preclinical muscular dystrophy studies.

A multiscale sliding filament model of lymphatic muscle pumping.

The lymphatics maintain fluid balance by returning interstitial fluid to veins via contraction/compression of vessel segments with check valves. Disruption of lymphatic pumping can result in a condition called lymphedema with interstitial fluid accumulation. Lymphedema treatments are often ineffective, which is partially attributable to insufficient understanding of specialized lymphatic muscle lining the vessels. This muscle exhibits cardiac-like phasic contractions and smooth muscle-like tonic contractions to generate and regulate flow. To understand the relationship between this sub-cellular contractile machinery and organ-level pumping, we have developed a multiscale computational model of phasic and tonic contractions in lymphatic muscle and coupled it to a lymphangion pumping model. Our model uses the sliding filament model (Huxley in Prog Biophys Biophys Chem 7:255-318, 1957) and its adaptation for smooth muscle (Mijailovich in Biophys J 79(5):2667-2681, 2000). Multiple structural arrangements of contractile components and viscoelastic elements were trialed but only one provided physiologic results. We then coupled this model with our previous lumped parameter model of the lymphangion to relate results to experiments. We show that the model produces similar pressure, diameter, and flow tracings to experiments on rat mesenteric lymphatics. This model provides the first estimates of lymphatic muscle contraction energetics and the ability to assess the potential effects of sub-cellular level phenomena such as calcium oscillations on lymphangion outflow. The maximum efficiency value predicted (40%) is at the upper end of estimates for other muscle types. Spontaneous calcium oscillations during diastole were found to increase outflow up to approximately 50% in the range of frequencies and amplitudes tested.

Forward and backward locomotion patterns in C. elegans generated by a connectome-based model simulation.

Caenorhabditis elegans (C. elegans) can produce various motion patterns despite having only 69 motor neurons and 95 muscle cells. Previous studies successfully elucidate the connectome and role of the respective motor neuron classes related to movement. However, these models have not analyzed the distribution of the synaptic and gap connection weights. In this study, we examined whether a motor neuron and muscle network can generate oscillations for both forward and backward movement and analyzed the distribution of the trained synaptic and gap connection weights through a machine learning approach. This paper presents a connectome-based neural network model consisting of motor neurons of classes A, B, D, AS, and muscle, considering both synaptic and gap connections. A supervised learning method called backpropagation through time was adapted to train the connection parameters by feeding teacher data composed of the command neuron input and muscle cell activation. Simulation results confirmed that the motor neuron circuit could generate oscillations with different phase patterns corresponding to forward and backward movement, and could be switched at arbitrary times according to the binary inputs simulating the output of command neurons. Subsequently, we confirmed that the trained synaptic and gap connection weights followed a Boltzmann-type distribution. It should be noted that the proposed model can be trained to reproduce the activity patterns measured for an animal (HRB4 strain). Therefore, the supervised learning approach adopted in this study may allow further analysis of complex activity patterns associated with movements.

An hourglass circuit motif transforms a motor program via subcellularly localized muscle calcium signaling and contraction.

Neural control of muscle function is fundamental to animal behavior. Many muscles can generate multiple distinct behaviors. Nonetheless, individual muscle cells are generally regarded as the smallest units of motor control. We report that muscle cells can alter behavior by contracting subcellularly. We previously discovered that noxious tastes reverse the net flow of particles through the C. elegans pharynx, a neuromuscular pump, resulting in spitting. We now show that spitting results from the subcellular contraction of the anterior region of the pm3 muscle cell. Subcellularly localized calcium increases accompany this contraction. Spitting is controlled by an 'hourglass' circuit motif: parallel neural pathways converge onto a single motor neuron that differentially controls multiple muscles and the critical subcellular muscle compartment. We conclude that subcellular muscle units enable modulatory motor control and propose that subcellular muscle contraction is a fundamental mechanism by which neurons can reshape behavior.

Selective Inhibition of Genomic and Non-Genomic Effects of Thyroid Hormone Regulates Muscle Cell Differentiation and Metabolic Behavior.

Thyroid hormones (THs) are key regulators of different biological processes. Their action involves genomic and non-genomic mechanisms, which together mediate the final effects of TH in target tissues. However, the proportion of the two processes and their contribution to the TH-mediated effects are still poorly understood. Skeletal muscle is a classical target tissue for TH, which regulates muscle strength and contraction, as well as energetic metabolism of myofibers. Here we address the different contribution of genomic and non-genomic action of TH in skeletal muscle cells by specifically silencing the deiodinase Dio2 or the ß3-Integrin expression via CRISPR/Cas9 technology. We found that myoblast proliferation is inversely regulated by integrin signal and the D2-dependent TH activation. Similarly, inhibition of the nuclear receptor action reduced myoblast proliferation, confirming that genomic action of TH attenuates proliferative rates. Contrarily, genomic and non-genomic signals promote muscle differentiation and the regulation of the redox state. Taken together, our data reveal that integration of genomic and non-genomic signal pathways finely regulates skeletal muscle physiology. These findings not only contribute to the understanding of the mechanisms involved in TH modulation of muscle physiology but also add insight into the interplay between different mechanisms of action of TH in muscle cells.

Gap Junctional Communication via Connexin43 between Purkinje Fibers and Working Myocytes Explains the Epicardial Activation Pattern in the Postnatal Mouse Left Ventricle.

The mammalian ventricular myocardium forms a functional syncytium due to flow of electrical current mediated in part by gap junctions localized within intercalated disks. The connexin (Cx) subunit of gap junctions have direct and indirect roles in conduction of electrical impulse from the cardiac pacemaker via the cardiac conduction system (CCS) to working myocytes. Cx43 is the dominant isoform in these channels. We have studied the distribution of Cx43 junctions between the CCS and working myocytes in a transgenic mouse model, which had the His-Purkinje portion of the CCS labeled with green fluorescence protein. The highest number of such connections was found in a region about one-third of ventricular length above the apex, and it correlated with the peak proportion of Purkinje fibers (PFs) to the ventricular myocardium. At this location, on the septal surface of the left ventricle, the insulated left bundle branch split into the uninsulated network of PFs that continued to the free wall anteriorly and posteriorly. The second peak of PF abundance was present in the ventricular apex. Epicardial activation maps correspondingly placed the site of the first activation in the apical region, while some hearts presented more highly located breakthrough sites. Taken together, these results increase our understanding of the physiological pattern of ventricular activation and its morphological underpinning through detailed CCS anatomy and distribution of its gap junctional coupling to the working myocardium.

miR-130b inhibits proliferation and promotes differentiation in myocytes via targeting Sp1.

Muscle regeneration after damage or during myopathies requires a fine cooperation between myoblast proliferation and myogenic differentiation. A growing body of evidence suggests that microRNAs play critical roles in myocyte proliferation and differentiation transcriptionally. However, the molecular mechanisms underlying the orchestration are not fully understood. Here, we showed that miR-130b is able to repress myoblast proliferation and promote myogenic differentiation via targeting Sp1 transcription factor. Importantly, overexpression of miR-130b is capable of improving the recovery of damaged muscle in a freeze injury model. Moreover, miR-130b expression is declined in the muscle of muscular dystrophy patients. Thus, these results indicated that miR-130b may play a role in skeletal muscle regeneration and myopathy progression. Together, our findings suggest that the miR-130b/Sp1 axis may serve as a potential therapeutic target for the treatment of patients with muscle damage or severe myopathies.

Identification of key pathways and hub genes in the myogenic differentiation of pluripotent stem cell: a bioinformatics and experimental study.

BACKGROUND: The regeneration of muscle cells from stem cells is an intricate process, and various genes are included in the process such as myoD, mf5, mf6, etc. The key genes and pathways in the differentiating stages are various. Therefore, the differential expression of key genes after 4 weeks of differentiation were investigated in our study. METHOD: Three published gene expression profiles, GSE131125, GSE148994, and GSE149055, about the comparisons of pluripotent stem cells to differentiated cells after 4 weeks were obtained from the Gene Expression Omnibus (GEO) database. Common differentially expressed genes (DEGs) were obtained for further analysis such as protein-protein interaction (PPI) network, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and GSEA analysis. After hub genes and key pathways were obtained, we manipulated in vitro cell research for substantiation such as immunohistochemical staining and semi-quantitative analysis and quantitative real-time PCR. RESULTS: A total of 824 DEGs including 350 upregulated genes and 474 downregulated genes were identified in the three GSEs. Nineteen hub genes were identified from the PPI network. The GO and KEGG pathway analyses confirmed that myogenic differentiation at 4 weeks was strongly associated with pathway in cancer, PI3K pathway, actin cytoskeleton regulation and metabolic pathway, biosynthesis of antibodies, and cell cycle. GSEA analysis indicated the differentiated cells were enriched in muscle cell development and myogenesis. Meanwhile, the core genes in each pathway were identified from the GSEA analysis. The in vitro cell research revealed that actin cytoskeleton and myoD were upregulated after 4-week differentiation. CONCLUSIONS: The research revealed the potential hub genes and key pathways after 4-week differentiation of stem cells which contribute to further study about the molecular mechanism of myogenesis regeneration, paving a way for more accurate treatment for muscle dysfunction.

Amiodarone inhibits arrhythmias in hypertensive rats by improving myocardial biomechanical properties.

The prevalence of arrhythmia in patients with hypertension has gradually attracted widespread attention. However, the relationship between hypertension and arrhythmia still lacks more attention. Herein, we explore the biomechanical mechanism of arrhythmia in hypertensive rats and the effect of amiodarone on biomechanical properties. We applied micro-mechanics and amiodarone to stimulate single ventricular myocytes to compare changes of mechanical parameters and the mechanism was investigated in biomechanics. Then we verified the expression changes of genes and long non-coding RNAs (lncRNAs) related to myocardial mechanics to explore the effect of amiodarone on biomechanical properties. The results found that the stiffness of ventricular myocytes and calcium ion levels in hypertensive rats were significantly increased and amiodarone could alleviate the intracellular calcium response and biomechanical stimulation. In addition, experiments showed spontaneously hypertensive rats were more likely to induce arrhythmia and preoperative amiodarone intervention significantly reduced the occurrence of arrhythmias. Meanwhile, high-throughput sequencing showed the genes and lncRNAs related to myocardial mechanics changed significantly in the spontaneously hypertensive rats that amiodarone was injected. These results strengthen the evidence that hypertension rats are prone to arrhythmia with abnormal myocardial biomechanical properties. Amiodarone effectively inhibit arrhythmia by improving the myocardial biomechanical properties and weakening the sensitivity of mechanical stretch stimulation.

Burst mitofusin activation reverses neuromuscular dysfunction in murine CMT2A.

Charcot-Marie-Tooth disease type 2A (CMT2A) is an untreatable childhood peripheral neuropathy caused by mutations of the mitochondrial fusion protein, mitofusin (MFN) 2. Here, pharmacological activation of endogenous normal mitofusins overcame dominant inhibitory effects of CMT2A mutants in reprogrammed human patient motor neurons, reversing hallmark mitochondrial stasis and fragmentation independent of causal MFN2 mutation. In mice expressing human MFN2 T105M, intermittent mitofusin activation with a small molecule, MiM111, normalized CMT2A neuromuscular dysfunction, reversed pre-treatment axon and skeletal myocyte atrophy, and enhanced axon regrowth by increasing mitochondrial transport within peripheral axons and promoting in vivo mitochondrial localization to neuromuscular junctional synapses. MiM111-treated MFN2 T105M mouse neurons exhibited accelerated primary outgrowth and greater post-axotomy regrowth, linked to enhanced mitochondrial motility. MiM111 is the first pre-clinical candidate for CMT2A.

Charcot-Marie-Tooth disease type 2A is a rare genetic childhood disease where dying back of nerve cells leads to muscle loss in the arms and legs, causing permanent disability. There is no known treatment. In this form of CMT, mutations in a protein called mitofusin 2 damage structures inside cells known as mitochondria. Mitochondria generate most of the chemical energy to power a cell, but when mitofusin 2 is mutated, the mitochondria are less healthy and are unable to move within the cell, depriving the cells of energy. This particularly causes problems in the long nerve cells that stretch from the spinal cord to the arm and leg muscles. Now, Franco, Dang et al. wanted to see whether re-activating mitofusin 2 could correct the damage to the mitochondria and restore the nerve connections to the muscles. The researchers tested a new class of drug called a mitofusin activator on nerve cells grown in the laboratory after being taken from people suffering from CMT2A, and also from a mouse model of the disease. Mitofusin activators improved the structure, fitness and movement of mitochondria in both human and mice nerve cells. Franco, Dang et al. then tested the drug in the mice with a CMT2A mutation and found that it could also stimulate nerves to regrow and so reverse muscle loss and weakness. This is the first time scientists have succeeded to reverse the effects of CMT2A in nerve cells of mice and humans. However, these drugs will still need to go through extensive testing in clinical trials before being made widely available to patients. If approved, mitofusin activators may also be beneficial for patients suffering from other genetic conditions that damage mitochondria.

The Rho-GEF PIX-1 directs assembly or stability of lateral attachment structures between muscle cells.

PIX proteins are guanine nucleotide exchange factors (GEFs) that activate Rac and Cdc42, and are known to have numerous functions in various cell types. Here, we show that a PIX protein has an important function in muscle. From a genetic screen in C. elegans, we found that pix-1 is required for the assembly of integrin adhesion complexes (IACs) at borders between muscle cells, and is required for locomotion of the animal. A pix-1 null mutant has a reduced level of activated Rac in muscle. PIX-1 localizes to IACs at muscle cell boundaries, M-lines and dense bodies. Mutations in genes encoding proteins at known steps of the PIX signaling pathway show defects at muscle cell boundaries. A missense mutation in a highly conserved residue in the RacGEF domain results in normal levels of PIX-1 protein, but a reduced level of activated Rac in muscle, and abnormal IACs at muscle cell boundaries.

Rose Bengal Crosslinking to Stabilize Collagen Sheets and Generate Modulated Collagen Laminates.

For medical application, easily accessible biomaterials with tailored properties are desirable. Collagen type I represents a biomaterial of choice for regenerative medicine and tissue engineering. Here, we present a simple method to modify the properties of collagen and to generate collagen laminates. We selected three commercially available collagen sheets with different thicknesses and densities and examined the effect of rose bengal and green light collagen crosslinking (RGX) on properties such as microstructure, swelling degree, mechanical stability, cell compatibility and drug release. The highest impact of RGX was measured for Atelocollagen, for which the swelling degree was reduced from 630% (w/w) to 520% (w/w) and thickness measured under force application increased from 0.014 mm to 0.455 mm, indicating a significant increase in mechanical stability. Microstructural analysis revealed that the sponge-like structure was replaced by a fibrous structure. While the initial burst effect during vancomycin release was not influenced by crosslinking, RGX increased cell proliferation on sheets of Atelocollagen and on Collagen Solutions. We furthermore demonstrate that RGX can be used to covalently attach different sheets to create materials with combined properties, making the modification and combination of readily available sheets with RGX an attractive approach for clinical application.

Proposing Urothelial and Muscle In Vitro Cell Models as a Novel Approach for Assessment of Long-Term Toxicity of Nanoparticles.

Many studies evaluated the short-term in vitro toxicity of nanoparticles (NPs); however, long-term effects are still not adequately understood. Here, we investigated the potential toxic effects of biomedical (polyacrylic acid and polyethylenimine coated magnetic NPs) and two industrial (SiO2 and TiO2) NPs following different short-term and long-term exposure protocols on two physiologically different in vitro models that are able to differentiate: L6 rat skeletal muscle cell line and biomimetic normal porcine urothelial (NPU) cells. We show that L6 cells are more sensitive to NP exposure then NPU cells. Transmission electron microscopy revealed an uptake of NPs into L6 cells but not NPU cells. In L6 cells, we obtained a dose-dependent reduction in cell viability and increased reactive oxygen species (ROS) formation after 24 h. Following continuous exposure, more stable TiO2 and polyacrylic acid (PAA) NPs increased levels of nuclear factor Nrf2 mRNA, suggesting an oxidative damage-associated response. Furthermore, internalized magnetic PAA and TiO2 NPs hindered the differentiation of L6 cells. We propose the use of L6 skeletal muscle cells and NPU cells as a novel approach for assessment of the potential long-term toxicity of relevant NPs that are found in the blood and/or can be secreted into the urine.

DNA methylation across the genome in aged human skeletal muscle tissue and muscle-derived cells: the role of HOX genes and physical activity.

Skeletal muscle tissue demonstrates global hypermethylation with age. However, methylome changes across the time-course of differentiation in aged human muscle derived cells, and larger coverage arrays in aged muscle tissue have not been undertaken. Using 850K DNA methylation arrays we compared the methylomes of young (27 ± 4.4 years) and aged (83 ± 4 years) human skeletal muscle and that of young/aged heterogenous muscle-derived human primary cells (HDMCs) over several time points of differentiation (0, 72 h, 7, 10 days). Aged muscle tissue was hypermethylated compared with young tissue, enriched for; pathways-in-cancer (including; focal adhesion, MAPK signaling, PI3K-Akt-mTOR signaling, p53 signaling, Jak-STAT signaling, TGF-beta and notch signaling), rap1-signaling, axon-guidance and hippo-signalling. Aged cells also demonstrated a hypermethylated profile in pathways; axon-guidance, adherens-junction and calcium-signaling, particularly at later timepoints of myotube formation, corresponding with reduced morphological differentiation and reductions in MyoD/Myogenin gene expression compared with young cells. While young cells showed little alterations in DNA methylation during differentiation, aged cells demonstrated extensive and significantly altered DNA methylation, particularly at 7 days of differentiation and most notably in focal adhesion and PI3K-AKT signalling pathways. While the methylomes were vastly different between muscle tissue and HDMCs, we identified a small number of CpG sites showing a hypermethylated state with age, in both muscle tissue and cells on genes KIF15, DYRK2, FHL2, MRPS33, ABCA17P. Most notably, differential methylation analysis of chromosomal regions identified three locations containing enrichment of 6-8 CpGs in the HOX family of genes altered with age. With HOXD10, HOXD9, HOXD8, HOXA3, HOXC9, HOXB1, HOXB3, HOXC-AS2 and HOXC10 all hypermethylated in aged tissue. In aged cells the same HOX genes (and additionally HOXC-AS3) displayed the most variable methylation at 7 days of differentiation versus young cells, with HOXD8, HOXC9, HOXB1 and HOXC-AS3 hypermethylated and HOXC10 and HOXC-AS2 hypomethylated. We also determined that there was an inverse relationship between DNA methylation and gene expression for HOXB1, HOXA3 and HOXC-AS3. Finally, increased physical activity in young adults was associated with oppositely regulating HOXB1 and HOXA3 methylation compared with age. Overall, we demonstrate that a considerable number of HOX genes are differentially epigenetically regulated in aged human skeletal muscle and HDMCs and increased physical activity may help prevent age-related epigenetic changes in these HOX genes.

Gα/GSA-1 works upstream of PKA/KIN-1 to regulate calcium signaling and contractility in the Caenorhabditis elegans spermatheca.

Correct regulation of cell contractility is critical for the function of many biological systems. The reproductive system of the hermaphroditic nematode C. elegans contains a contractile tube of myoepithelial cells known as the spermatheca, which stores sperm and is the site of oocyte fertilization. Regulated contraction of the spermatheca pushes the embryo into the uterus. Cell contractility in the spermatheca is dependent on actin and myosin and is regulated, in part, by Ca2+ signaling through the phospholipase PLC-1, which mediates Ca2+ release from the endoplasmic reticulum. Here, we describe a novel role for GSA-1/Gαs, and protein kinase A, composed of the catalytic subunit KIN-1/PKA-C and the regulatory subunit KIN-2/PKA-R, in the regulation of Ca2+ release and contractility in the C. elegans spermatheca. Without GSA-1/Gαs or KIN-1/PKA-C, Ca2+ is not released, and oocytes become trapped in the spermatheca. Conversely, when PKA is activated through either a gain of function allele in GSA-1 (GSA-1(GF)) or by depletion of KIN-2/PKA-R, the transit times and total numbers, although not frequencies, of Ca2+ pulses are increased, and Ca2+ propagates across the spermatheca even in the absence of oocyte entry. In the spermathecal-uterine valve, loss of GSA-1/Gαs or KIN-1/PKA-C results in sustained, high levels of Ca2+ and a loss of coordination between the spermathecal bag and sp-ut valve. Additionally, we show that depleting phosphodiesterase PDE-6 levels alters contractility and Ca2+ dynamics in the spermatheca, and that the GPB-1 and GPB-2 Gß subunits play a central role in regulating spermathecal contractility and Ca2+ signaling. This work identifies a signaling network in which Ca2+ and cAMP pathways work together to coordinate spermathecal contractions for successful ovulations.

Distribution of data in cellular electrophysiology: Is it always normal?

The distribution of data presented in many electrophysiological studies is presumed to be normal without any convincing evidence. To test this presumption, the cell membrane capacitance and magnitude of inward rectifier potassium currents were recorded by the whole-cell patch clamp technique in rat atrial myocytes. Statistical analysis of the data showed that these variables were not distributed normally. Instead, a positively skewed distribution appeared to be a better approximation of the real data distribution. Consequently, the arithmetic mean, used inappropriately in such data, may substantially overestimate the true mean value characterizing the central tendency of the data. Moreover, a large standard deviation describing the variance of positively skewed data allowed 95% confidence interval to include unrealistic negative values. We therefore conclude that the normality of the electrophysiological data should be tested in every experiment and, if rejected, the positively skewed data should be more accurately characterized by the median and interpercentile range or, if justified (namely in the case of log-normal and gamma data distribution), by the geometric mean and the geometric standard deviation.

Muscle cell differentiation and development pathway defects in Emery-Dreifuss muscular dystrophy.

Emery-Dreifuss muscular dystrophy (EDMD) is a rare genetic disorder characterised by the early development of muscle contractures, progressive muscle weakness, and heart abnormalities. The latter may result in serious complications, or in severe cases, sudden death. Currently, there are very few effective treatment options available for EDMD and so there is a high clinical need for new therapies. Various genetic mutations have been identified in the development and causation of EDMD, each encoding proteins that are components of the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex, which spans the nuclear envelope and serves to connect the nuclear lamina to the cytoskeleton. Within this review, we examine how mutations in the genes encoding these proteins, including lamins A/C, emerin, nesprins 1/2, FHL1, and SUN1/2 lead to muscle cell differentiation and development pathway defects. Further work to identify conserved molecular pathways downstream of these defective proteins may reveal potential targets for therapy design.

Pre-innervated tissue-engineered muscle promotes a pro-regenerative microenvironment following volumetric muscle loss.

Volumetric muscle loss (VML) is the traumatic or surgical loss of skeletal muscle beyond the inherent regenerative capacity of the body, generally leading to severe functional deficit. Formation of appropriate somato-motor innervations remains one of the biggest challenges for both autologous grafts as well as tissue-engineered muscle constructs. We aim to address this challenge by developing pre-innervated tissue-engineered muscle comprised of long aligned networks of spinal motor neurons and skeletal myocytes on aligned nanofibrous scaffolds. Motor neurons led to enhanced differentiation and maturation of skeletal myocytes in vitro. These pre-innervated tissue-engineered muscle constructs when implanted in a rat VML model significantly increased satellite cell density, neuromuscular junction maintenance, graft revascularization, and muscle volume over three weeks as compared to myocyte-only constructs and nanofiber scaffolds alone. These pro-regenerative effects may enhance functional neuromuscular regeneration following VML, thereby improving the levels of functional recovery following these devastating injuries.