Skeletal Muscle Regeneration Research Articles

Osteopontin May Improve Postinjury Muscle Repair Via Matrix Metalloproteinases And tgf-β Activation in Regular Exercise.

Skeletal muscle injuries are commonly observed during sports and trauma. Regular exercise promotes muscle repair; however, the underlying mechanisms require further investigation. In addition to exercise, osteopontin (OPN) contributes to skeletal muscle regeneration and fibrosis following injury. However, whether and how OPN affects matrix proteins to promote post-injury muscle repair remains uncertain. We recruited regular exercise (RE) and sedentary control (SC) groups to determine plasma OPN levels. Additionally, we developed a murine model of muscle contusion injury and compared the extent of damage, inflammatory state, and regeneration-related proteins in OPN knockout (OPN KO) and wild-type (WT) mice. Our results show that regular exercise induced the increase of OPN, matrix metalloproteinases (MMPs), and transforming growth factor-β (TGF-β) expression in plasma. Injured muscle fibers were repaired more slowly in OPN-KO mice than in WT mice. The expression levels of genes and proteins related to muscle regeneration were lower in OPN-KO mice after injury. OPN also promotes fibroblast proliferation, differentiation, and migration. Additionally, OPN upregulates MMP expression by activating TGF-β, which promotes muscle repair. OPN can improve post-injury muscle repair by activating MMPs and TGF-β pathways. It is upregulated by regular exercise. Our study provides a potential target for the treatment of muscle injuries and explains why regular physical exercise is beneficial for muscle repair.

Myonecrosis Induction by Intramuscular Injection of CTX.

Skeletal muscle, one of the most abundant tissue in the body, is a highly regenerative tissue. Indeed, compared to other tissues that are not able to regenerate after injury, skeletal muscle can fully regenerate upon mechanically, chemically, and infection-induced trauma. Several injury models have been developed to thoroughly investigate the physiological mechanisms regulating skeletal muscle regeneration. This protocol describes how to induce muscle regeneration by taking advantage of a cardiotoxin (CTX)-induced muscle injury model. The overall steps include CTX injection of tibialis anterior (TA) muscles of BL6N mice, collection of regenerating muscles at different time points after CTX injury, and histological characterization of regenerating muscles. Our protocol, compared with others such as those for freeze-induced injury models, avoids laceration or infections of the muscles since it involves neither surgery nor suture. In addition, our protocol is highly reproducible, since it causes homogenous myonecrosis of the whole muscle, and further reduces animal pain and stress. Graphical abstract.

Combinatorial extracellular matrix cues with mechanical strain induce differential effects on myogenesis in vitro.

Skeletal muscle regeneration remains a clinical unmet need for volumetric muscle loss and atrophy where muscle function cannot be restored to prior capacity. Current experimental approaches do not account for the complex microenvironmental factors that modulate myogenesis. In this study we developed a biomimetic tissue chip platform to systematically study the combined effects of the extracellular matrix (ECM) microenvironment and mechanical strain on myogenesis of murine myoblasts. Using stretchable tissue chips composed of collagen I (C), fibronectin (F) and laminin (L), as well as their combinations thereof, we tested the addition of mechanical strain regimens on myogenesis at the transcriptomic and translational levels. Our results show that ECMs have a significant effect on myotube formation in C2C12 murine myoblasts. Under static conditions, laminin substrates induced the longest myotubes, whereas fibronectin produced the widest myotubes. Combinatorial ECMs showed non-intuitive effects on myotube formation. Genome-wide analysis revealed the upregulation in actin cytoskeletal related genes that are suggestive of myogenesis. When mechanical strain was introduced to C + F + L combinatorial ECM substrates in the form of constant or intermittent uniaxial strain at low (5%) and high (15%) levels, we observed synergistic enhancements in myotube width, along with transcriptomic upregulation in myosin heavy chain genes. Together, these studies highlight the complex role of microenvironmental factors such as ECM interactions and strain on myotube formation and the underlying signaling pathways.

PGC-1α senses the CBC of pre-mRNA to dictate the fate of promoter-proximally paused RNAPII

PGC-1α is well established as a metazoan transcriptional coactivator of cellular adaptation in response to stress. However, the mechanisms by which PGC-1α activates gene transcription are incompletely understood. Here, we report that PGC-1α serves as a scaffold protein that physically and functionally connects the DNA-binding protein estrogen-related receptor α (ERRα), cap-binding protein 80 (CBP80), and Mediator to overcome promoter-proximal pausing of RNAPII and transcriptionally activate stress-response genes. We show that PGC-1α promotes pausing release in a two-arm mechanism (1) by recruiting the positive transcription elongation factor b (P-TEFb) and (2) by outcompeting the premature transcription termination complex Integrator. Using mice homozygous for five amino acid changes in the CBP80-binding motif (CBM) of PGC-1α that destroy CBM function, we show that efficient differentiation of primary myoblasts to myofibers and timely skeletal muscle regeneration after injury require PGC-1α binding to CBP80. Our findings reveal how PGC-1α activates stress-response gene transcription in a previously unanticipated pre-mRNA quality-control pathway.

Conductive self-healing biodegradable hydrogel based on hyaluronic acid-grafted-polyaniline as cell recruitment niches and cell delivery carrier for myogenic differentiation and skeletal muscle regeneration

Scaffold is one of the most important roles in skeletal muscle tissue engineering. Being capable of mimicking extracellular matrix components, having electrical conductivity, and recruiting cells proactively to meet the cell-free requests are the proper characteristics of tissue engineering scaffolds. In this work, a series of leucine (Leu) loaded self-healing conductive hydrogels based on aminated hyaluronic acid-graft-polyaniline (AHA-PANI) and oxidized hyaluronic (OHA) were prepared for skeletal muscle tissue engineering. Leu can promote skeletal myogenesis and hydrogel can provide a suitable growth environment for cells. They have self-healing properties, antioxidant activity, suitable modulus, good electrical conductivity, high porosity, and proper swelling ratio. Additionally, these hydrogels have good cytocompatibility and can provide a 3D culture environment for C2C12 and hADSC cells. The Leu-loaded hydrogel has recruiting ability of cells and can promote myogenic differentiation of C2C12 cells. Besides, these hydrogels enable sustained cell delivery, and have good in vivo degradability and exhibited promoting effect on skeletal muscle regeneration in the rat tibialis anterior muscle defect model with reduced post-repair inflammatory factor expression and enhanced myogenic differentiation-related gene expression. All the results indicated that these conductive hydrogels as self-healing cell recruitment niches are excellent biomaterial for the treatment of volumetric muscle loss injury.

Osteopontin - The stirring multifunctional regulatory factor in multisystem aging.

Osteopontin (OPN) is a multifunctional noncollagenous matrix phosphoprotein that is expressed both intracellularly and extracellularly in various tissues. As a growth regulatory protein and proinflammatory immunochemokine, OPN is involved in the pathological processes of many diseases. Recent studies have found that OPN is widely involved in the aging processes of multiple organs and tissues, such as T-cell senescence, atherosclerosis, skeletal muscle regeneration, osteoporosis, neurodegenerative changes, hematopoietic stem cell reconstruction, and retinal aging. However, the regulatory roles and mechanisms of OPN in the aging process of different tissues are not uniform, and OPN even has diverse roles in different developmental stages of the same tissue, generating uncertainty for the future study and utilization of OPN. In this review, we will summarize the regulatory role and molecular mechanism of OPN in different tissues and cells, such as the musculoskeletal system, central nervous system, cardiovascular system, liver, and eye, during senescence. We believe that a better understanding of the mechanism of OPN in the aging process will help us develop targeted and comprehensive therapeutic strategies to fight the spread of age-related diseases.

AGING-RELATED VEGF IMPAIRS MUSCLE REGENERATION

Abstract Purpose Aging is associated with frailty, a parameter that correlates with mortality and loss of muscle mass. The molecular mechanisms behind aging-associated impairment of muscle regeneration remain incompletely understood. We hypothesized VEGF-A with known role in angiogenesis and muscle progenitor differentiation to regulate regeneration in aged skeletal muscle. Methods Young C57BL/6 (10 weeks old) and old C57BL/6 mice (24 months old) were subjected to muscle cryoinjury to induce regeneration. Quantifications of cross-sectional area (CSA) of regenerating myofibers were performed. Tibialis anterior muscle lysates was used for quantifying VEGF-A. To evaluate the role of VEGF in muscle regeneration, a similar experiment was performed on VEGFlo mice with a 75% decrease in VEGF-A activity and littermate controls. ML228, a hypoxia signaling activator that increases VEGF-A levels, was injected into young and old mice as well as VEGFlo and littermate controls. Results Old mice exhibited marked reduction in the VEGF-A protein levels and regenerating myofiber CSA on DPI 10 (1250 vs. 833μm2, p<.001). Similarly, VEGFlo mice exhibited significantly smaller regenerating fiber CSA as compared to littermate controls on DPI 10 (541 vs. 238μm2, p=.0011). Pharmacological augmentation of VEGFA using ML228 increased muscle VEGF levels by 2 folds and skeletal muscle regeneration in both old mice (25% increase in regenerating fiber CSA, p<.01) and VEGFlo (20% increase in regenerating fiber CSA, p<.01) mice, but not young or littermate controls. Conclusions Muscle regeneration declines with aging in correlation with loss of VEGFA levels within skeletal muscle. Supplementation of VEGFA represents a therapeutic target for sarcopenia.

Soluble components from mesenchymal stromal cell processing exert anti-inflammatory effects and facilitate ischemic muscle regeneration

Background aimsSkeletal muscle regeneration after severe damage is reliant on local stem cell proliferation and differentiation, processes that are tightly regulated by macrophages. Peripheral artery disease is a globally prevalent cardiovascular disease affecting millions of people. Progression of the disease leads to intermittent claudication, subsequent critical limb ischemia and muscle injury. Tissue-derived and ex vivo–expanded mesenchymal stromal cells (MSCs) for skeletal muscle regeneration have been studied, but pre-clinical and clinical results have not been consistent. As a result, the potential therapeutic efficacy and associated repair mechanisms of MSCs remain unclear. Numerous studies have demonstrated the vulnerability of delivered MSCs, with a precipitous drop in cell viability upon transplantation. This has prompted investigation into the therapeutic benefit of apoptotic cells, microvesicles, exosomes and soluble signals that are released upon cell death. MethodsIn this study, we characterized various components produced by MSCs after cell death induction under different conditions. We discovered anti-inflammatory and pro-regenerative effects produced by cell components following a freeze and thaw (F&T) process on macrophage polarization in vitro. We further investigated the underlying mechanisms of macrophage polarization by those components resulting from severe cell death induction. ResultsWe found potent therapeutic effects from F&T-induced cell debris are dependent on the externalization of phosphatidylserine on the plasma membrane. In contrast, effects from the supernatant of F&T-induced cell death primarily depends on the released protein content. We then applied the F&T-induced cell supernatant to an animal model of peripheral artery disease to treat muscle injury caused by severe ischemia. Treatment with the F&T supernatant but not the vulnerable MSCs resulted in significantly improved recovery of muscle function, blood flow and morphology and inflammation resolution in the affected muscles 2 weeks after injury. ConclusionsThis study validates the therapeutic potential of F&T-induced supernatant obviating the need for a viable population from vulnerable MSCs to treat injury, thus providing a roadmap for cell-free therapeutic approaches for tissue regeneration.

Bioactive MXene Promoting Angiogenesis and Skeletal Muscle Regeneration through Regulating M2 Polarization and Oxidation Stress.

Complete repair of skeletal muscles caused by severe mechanical damage and muscle-related diseases remains a challenge. 2D Ti3 C2 Tx (MXene) possesses special photoelectromagnetic properties and has attracted considerable attention in materials science and engineering. However, the bioactive properties and potential mechanism of MXene in tissue engineering, especially in skeletal muscle regeneration, are unclear. Herein, the antioxidation and anti-inflammation activities of MXene and its effects on myogenic differentiation and regeneration of skeletal muscle in vivo are investigated. In vitro studies have shown that MXene has excellent antioxidation and anti-inflammatory properties, and promotes myogenic differentiation and angiogenesis. MXene can remove excess reactive oxygen species in macrophage cells to alleviate oxidative stress and induce the transformation of M1 macrophages into M2 macrophages to reduce excessive inflammation, which can significantly promote the proliferation and differentiation of myoblasts, as well as the proliferation, migration, and tube formation of endothelial cells. Animal experiments with rat tibial anterior muscle defects show that MXene can promote angiogenesis, muscle fiber formation, and skeletal muscle regeneration by regulating the cell microenvironment through anti-inflammatory and antioxidant pathways. The findings suggest that MXene can be used as a multifunctional bioactive material to enhance tissue regeneration through robust antioxidation, anti-inflammation, and angiogenesis activities.

Multidimensional analyses of the pathomechanism caused by the non-catalytic GNE variant, c.620A>T, in patients with GNE myopathy

GNE myopathy is a distal myopathy caused by biallelic variants in GNE, which encodes a protein involved in sialic acid biosynthesis. Compound heterozygosity of the second most frequent variant among Japanese GNE myopathy patients, GNE c.620A>T encoding p.D207V, occurs in the expected number of patients; however, homozygotes for this variant are rare; three patients identified while 238 homozygotes are estimated to exist in Japan. The aim of this study was to elucidate the pathomechanism caused by c.620A>T. Identity-by-descent mapping indicated two distinct c.620A>T haplotypes, which were not correlated with age onset or development of myopathy. Patients homozygous for c.620A>T had mildly decreased sialylation, and no additional pathogenic variants in GNE or abnormalities in transcript structure or expression of other genes related to sialic acid biosynthesis in skeletal muscle. Structural modeling of full-length GNE dimers revealed that the variant amino acid localized close to the monomer interface, but far from catalytic sites, suggesting functions in enzymatic product transfer between the epimerase and kinase domains on GNE oligomerization. In conclusion, homozygotes for c.620A>T rarely develop myopathy, while symptoms occur in compound heterozygotes, probably because of mildly decreased sialylation, due to partial defects in oligomerization and product trafficking by the mutated GNE protein.

Muscle stem cells and fibro-adipogenic progenitors in female pelvic floor muscle regeneration following birth injury

Pelvic floor muscle (PFM) injury during childbirth is a key risk factor for pelvic floor disorders that affect millions of women worldwide. Muscle stem cells (MuSCs), supported by the fibro-adipogenic progenitors (FAPs) and immune cells, are indispensable for the regeneration of injured appendicular skeletal muscles. However, almost nothing is known about their role in PFM regeneration following birth injury. To elucidate the role of MuSCs, FAPs, and immune infiltrate in this context, we used radiation to perturb cell function and followed PFM recovery in a validated simulated birth injury (SBI) rat model. Non-irradiated and irradiated rats were euthanized at 3,7,10, and 28 days post-SBI (dpi). Twenty-eight dpi, PFM fiber cross-sectional area (CSA) was significantly lower and the extracellular space occupied by immune infiltrate was larger in irradiated relative to nonirradiated injured animals. Following SBI in non-irradiated animals, MuSCs and FAPs expanded significantly at 7 and 3 dpi, respectively; this expansion did not occur in irradiated animals at the same time points. At 7 and 10 dpi, we observed persistent immune response in PFMs subjected to irradiation compared to non-irradiated injured PFMs. CSA of newly regenerated fibers was also significantly smaller following SBI in irradiated compared to non-irradiated injured PFMs. Our results demonstrate that the loss of function and decreased expansion of MuSCs and FAPs after birth injury lead to impaired PFM recovery. These findings form the basis for further studies focused on the identification of novel therapeutic targets to counteract postpartum PFM dysfunction and the associated pelvic floor disorders.

Growth arrest and DNA damage-inducible alpha regulates muscle repair and fat infiltration through ATP synthase F1 subunit alpha.

Skeletal muscle fat infiltration is a common feature during ageing, obesity and several myopathies associated with muscular dysfunction and sarcopenia. However, the regulatory mechanisms of intramuscular adipogenesis and strategies to reduce fat infiltration in muscle remain unclear. Here, we identified the growth arrest and DNA damage-inducible alpha (GADD45A), a stress-inducible histone folding protein, as a critical regulator of intramuscular fat (IMAT) infiltration. To explore the role of GADD45A on IMAT infiltration and muscle regeneration, the gain or loss function of GADD45A in intramuscular preadipocytes was performed. The adipocyte-specific GADD45A knock-in (KI) mice and high IMAT-infiltrated muscle model by glycerol injection (50μL of 50% v/v GLY) were generated. RNA-sequencing, histological changes, gene expression, lipid metabolism, mitochondrial function and the effect of dietary factor epigallocatechin-3-gallate (EGCG) treatment (100mg/kg) on IMAT infiltration were studied. The unbiased transcriptomics data analysis indicated that GADD45A expression positively correlates with IMAT infiltration and muscle metabolic disorders in humans (correlation: young vs. aged people, Gadd45a and Cebpa, r2 =0.20, P<0.05) and animals (correlation: wild-type [WT] vs. mdx mice, Gadd45a and Cebpa, r2 =0.38, P<0.05; NaCl vs. GLY mice, Gadd45a and Adipoq/Fabp4, r2 =0.80/0.71, both P<0.0001). In vitro, GADD45A overexpression promotes intramuscular preadipocyte adipogenesis, upregulating the expression of adipogenic genes (Ppara: +47%, Adipoq: +28%, P<0.001; Cebpa: +135%, Fabp4: +16%, P<0.01; Pparg: +66%, Leptin: +77%, P<0.05). GADD45A knockdown robustly decreased lipid accumulation (Pparg: -57%, Adipoq: -35%, P<0.001; Fabp4: -37%, P<0.01; Leptin: -28%, P<0.05). GADD45A KI mice exhibit inhibited skeletal muscle regeneration (myofibres: -40%, P<0.01) and enhanced IMAT infiltration (adipocytes: +20%, P<0.05). These KI mice have impaired exercise endurance and mitochondrial function. Mechanistically, GADD45A affects ATP synthase F1 subunit alpha (ATP5A1) ubiquitination degradation (ubiquitinated ATP5A1, P<0.001) by recruiting the E3 ubiquitin ligase TRIM25, which decreases ATP synthesis (ATP production: -23%, P<0.01) and inactivates the cAMP/PKA/LKB1 signalling pathway (cAMP: -36%, P<0.01; decreased phospho-PKA and phospho-LKB1 protein content, P<0.01). The dietary factor EGCG can protect against muscle fat infiltration (triglyceride: -64%, P<0.05) via downregulating GADD45A (decreased GADD45A protein content, P<0.001). Our findings reveal a crucial role of GADD45A in regulating muscle repair and fat infiltration and suggest that inhibition of GADD45A by EGCG might be a potential strategy to combat fat infiltration and its associated muscle dysfunction.

Functional Skeletal Muscle Regeneration Using Muscle Mimetic Tissue Fabricated by Microvalve-Assisted Coaxial 3D Bioprinting.

3D-printed artificial skeletal muscle, which mimics the structural and functional characteristics of native skeletal muscle, is a promising treatment method for muscle reconstruction. Although various fabrication techniques for skeletal muscle using 3D bio-printers are studied, it is still challenging to build a functional muscle structure. A strategy using microvalve-assisted coaxial 3D bioprinting in consideration of functional skeletal muscle fabrication is reported. The unit (artificial muscle fascicle: AMF) of muscle mimetic tissue is composed of a core filled with medium-based C2C12 myoblast aggregates as a role of muscle fibers and a photo cross-linkable hydrogel-based shell as a role of connective tissue in muscles that enhances printability and cell adhesion and proliferation. Especially, a microvalve system is applied for the core part with even cell distribution and strong cell-cell interaction. This system enhances myotube formation and consequently shows spontaneous contraction. A multi-printed AMF (artificial muscle tissue: AMT) as a piece of muscle is implanted into the anterior tibia (TA) muscle defect site of immunocompromised rats. As a result, the TA-implanted AMT responds to electrical stimulation and represents histologically regenerated muscle tissue. This microvalve-assisted coaxial 3D bioprinting shows a significant step forward to mimicking native skeletal muscle tissue.

CREG1 stimulates AMPK phosphorylation and glucose uptake in skeletal muscle cells

The cellular repressor of adenovirus early region 1A-stimulated gene 1 (CREG1) is a secreted glycoprotein involved in cell differentiation and energy metabolism. It also binds to insulin-like growth factor 2 receptor (IGF2R), a protein implicated in muscle regeneration. However, whether CREG1 regulates the regeneration and metabolism of skeletal muscles via IGF2R remains unclear. This study investigates the role of CREG1 in skeletal muscle regeneration and glucose uptake in C2C12 myotubes and a cardiotoxin (CTX)-induced mouse skeletal muscle regeneration model. CTX-treated skeletal muscle showed significantly higher levels of IGF2R, CREG1, phospho-AMPKα Thr172, and GLUT4 proteins. Similarly, treatment of myotubes with CREG1 also stimulated AMPKα phosphorylation and GLUT4 expression. CREG1-induced AMPKα phosphorylation and 2DG uptake in myotubes were suppressed by IGF2R knockdown and Compound C, an AMPK inhibitor. These results suggest that CREG1 stimulates glucose uptake in skeletal muscles partially through AMPK activation. Hence, CREG1 plays an essential role in muscle regeneration by affecting glucose metabolism in skeletal muscles.

Long-term evaluation parameters in GNE myopathy: a 5-year observational follow-up natural history study

BackgroundA number of clinical trials targeting GNE myopathy patients have been conducted. However, useful clinical parameters for postmarketing surveillance and long-term clinical observation have not yet been established.ObjectiveWe conducted a...

Auxetic material in biomedical applications: a systematic review

&lt;span lang="EN-US"&gt;This study reviews and analyzes the different auxetic materials that have been developed in recent years. The search for research articles was carried out through one of the largest databases such as ScienceDirect, where 845 articles were collected, of which several filters were carried out to have a base of 386 articles. There are a variety of materials depending on their structure, composition, and industrial application, highlighting biomedical applications from tissue engineering, cell proliferation, skeletal muscle regeneration, transportation, bio-prosthesis to biomaterial. The present paper provides an overview of auxetic materials and its applications, providing a guide for designers and manufacturers of devices and accessories in any industry.&lt;/span&gt;

Not young but still immature: a HIF-1α-mediated maturation checkpoint in regenerating muscle.

Muscle fibers express particular isoforms of contractile proteins, depending on the fiber's function and the organism's developmental stage. In the adult, after a muscle injury, newly generated fibers transition through embryonic and neonatal myosins, prior to selecting their distinctive adult myosin isoform. In this issue of the JCI, Wang et al. discover a checkpoint that regulates the neonatal-to-adult myosin isoform transition. They found that HIF-1α regulated this checkpoint, with elevated HIF-1α levels blocking progression, while HIF-1α knockout accelerated the transition. They further related these findings to centronuclear myopathy, a disease in which HIF-1α is similarly elevated and neonatal myosin expression is maintained. These findings highlight a maturation checkpoint that impacts the skeletal muscle regeneration following ischemic injury, providing a pharmacologically accessible pathway in injury and diseases such as centronuclear myopathy.

Tissue Engineering Applied to Skeletal Muscle: Strategies and Perspectives.

Muscle tissue is formed by elongated and contractile cells with specific morphofunctional characteristics. Thus, it is divided into three basic types: smooth muscle tissue, cardiac striated muscle tissue and skeletal striated muscle tissue. The striated skeletal muscle tissue presents high plasticity, regeneration and growth capacity due to the presence of satellite cells, quiescent myoblasts that are activated in case of injury to the tissue and originate new muscle fibers when they differentiate. In more severe deficiencies or injuries there is a loss of their regenerative capacity, thus compromising the body's functionality at different levels. Tissue engineering studies the development of biomaterials capable of stimulating the recovery of cellular activity in injured body tissues, as well as the activity of cells with muscle differentiation potential in injury repair. However, the need for three-dimensional re-assembly in a complex organization makes it difficult to mimic this tissue and fully regenerate it for the sake of precise and effective movements. Thus, this article aims to provide a narrative review of tissue engineering strategies applied to the regeneration of skeletal muscle, in a critical evaluation of research, whether aimed at injury or atrophies such as spinal muscular atrophy.

Extracellular matrix: Brick and mortar in the skeletal muscle stem cell niche.

The extracellular matrix (ECM) is an interconnected macromolecular scaffold occupying the space between cells. Amongst other functions, the ECM provides structural support to tissues and serves as a microenvironmental niche that conveys regulatory signals to cells. Cell-matrix adhesions, which link the ECM to the cytoskeleton, are dynamic multi-protein complexes containing surface receptors and intracellular effectors that control various downstream pathways. In skeletal muscle, the most abundant tissue of the body, each individual muscle fiber and its associated muscle stem cells (MuSCs) are surrounded by a layer of ECM referred to as the basal lamina. The core scaffold of the basal lamina consists of self-assembling polymeric laminins and a network of collagens that tether proteoglycans, which provide lateral crosslinking, establish collateral associations with cell surface receptors, and serve as a sink and reservoir for growth factors. Skeletal muscle also contains the fibrillar collagenous interstitial ECM that plays an important role in determining tissue elasticity, connects the basal laminae to each other, and contains matrix secreting mesenchymal fibroblast-like cell types and blood vessels. During skeletal muscle regeneration fibroblast-like cell populations expand and contribute to the transitional fibronectin-rich regenerative matrix that instructs angiogenesis and MuSC function. Here, we provide a comprehensive overview of the role of the skeletal muscle ECM in health and disease and outline its role in orchestrating tissue regeneration and MuSC function.

The Efficacy of HGF/VEGF Gene Therapy for Limb Ischemia in Mice with Impaired Glucose Tolerance: Shift from Angiogenesis to Axonal Growth and Oxidative Potential in Skeletal Muscle

Combined non-viral gene therapy (GT) of ischemia and cardiovascular disease is a promising tool for potential clinical translation. In previous studies our group has developed combined gene therapy by vascular endothelial growth factor 165 (VEGF165) + hepatocyte growth factor (HGF). Our recent works have demonstrated that a bicistronic pDNA that carries both human HGF and VEGF165 coding sequences has a potential for clinical application in peripheral artery disease (PAD). The present study aimed to test HGF/VEGF combined plasmid efficacy in ischemic skeletal muscle comorbid with predominant complications of PAD-impaired glucose tolerance and type 2 diabetes mellitus (T2DM). Male C57BL mice were housed on low-fat (LFD) or high-fat diet (HFD) for 10 weeks and metabolic parameters including FBG level, ITT, and GTT were evaluated. Hindlimb ischemia induction and plasmid administration were performed at 10 weeks with 3 weeks for post-surgical follow-up. Limb blood flow was assessed by laser Doppler scanning at 7, 14, and 21 days after ischemia induction. The necrotic area of m.tibialis anterior, macrophage infiltration, angio- and neuritogenesis were evaluated in tissue sections. The mitochondrial status of skeletal muscle (total mitochondria content, ETC proteins content) was assessed by Western blotting of muscle lysates. At 10 weeks, the HFD group demonstrated impaired glucose tolerance in comparison with the LFD group. HGF/VEGF plasmid injection aggravated glucose intolerance in HFD conditions. Blood flow recovery was not changed by HGF/VEGF plasmid injection either in LFD or HFD conditions. GT in LFD, but not in HFD conditions, enlarged the necrotic area and CD68+ cells infiltration. However, HGF/VEGF plasmid enhanced neuritogenesis and enlarged NF200+ area on muscle sections. In HFD conditions, HGF/VEGF plasmid injection significantly increased mitochondria content and ETC proteins content. The current study demonstrated a significant role of dietary conditions in pre-clinical testing of non-viral GT drugs. HGF/VEGF combined plasmid demonstrated a novel aspect of potential participation in ischemic skeletal muscle regeneration, through regulation of innervation and bioenergetics of muscle. The obtained results made HGF/VEGF combined plasmid a very promising tool for PAD therapy in impaired glucose tolerance conditions.