Uninjured Muscle Research Articles

Mitochondria transplant therapy improves regeneration and restoration of injured skeletal muscle.

Injection of exogenous mitochondria has been shown to improve the ischaemia-damaged myocardium, but the effect of mitochondrial transplant therapy (MTT) to restore skeletal muscle mass and function has not been tested following neuromuscular injury. Therefore, we tested the hypothesis that MTT would improve the restoration of muscle function after injury. BaCl2 was injected into the gastrocnemius muscle of one limb of 8-12-week-old C57BL/6 mice to induce damage without injury to the resident stem cells. The contralateral gastrocnemius muscle was injected with phosphate-buffered saline (PBS) and served as the non-injured intra-animal control. Mitochondria were isolated from donor mice. Donor mitochondria were suspended in PBS or PBS without mitochondria (sham treatment) and injected into the tail vein of BaCl2 injured mice 24h after the initial injury. Muscle repair was examined 7, 14 and 21days after injury. MTT did not increase systemic inflammation in mice. Muscle mass 7days following injury was 21.9±2.1% and 17.4±1.9% lower (P<0.05) in injured as compared with non-injured intra-animal control muscles in phosphate-buffered saline (PBS)- and MTT-treated animals, respectively. Maximal plantar flexor muscle force was significantly lower in injured as compared with uninjured muscles of PBS-treated (-43.4±4.2%, P<0.05) and MTT-treated mice (-47.7±7.3%, P<0.05), but the reduction in force was not different between the experimental groups. The percentage of collagen and other non-contractile tissue in histological muscle cross sections, was significantly greater in injured muscles of PBS-treated mice (33.2±0.2%) compared with MTT-treated mice (26.5±0.2%) 7days after injury. Muscle wet weight and maximal muscle force from injured MTT-treated mice had recovered to control levels by 14days after the injury. However, muscle mass and force had not improved in PBS-treated animals by 14days after injury. The non-contractile composition of the gastrocnemius muscle tissue cross sections was not different between control, repaired PBS-treated and repaired MTT-treated mice 14days after injury. By 21days following injury, PBS-treated mice had fully restored gastrocnemius muscle mass of the injured muscle to that of the uninjured muscle, although maximal plantar flexion force was still 19.4±3.7% (P<0.05) lower in injured/repaired gastrocnemius as compared with uninjured intra-animal control muscles. Our results suggest that systemic mitochondria delivery can enhance the rate of muscle regeneration and restoration of muscle function following injury.

Impact of elevated serum advanced glycation end products and exercise on intact and injured murine tendons

ABSTRACT Overview: Delayed tendon healing is a significant clinical challenge for those with diabetes. We explored the role of advanced glycation end-products (AGEs), a protein modification present at elevated levels in serum of individuals with diabetes, on injured and intact tendons using a mouse model. Cell proliferation following tissue injury is a vital component of healing. Based on our previous work demonstrating that AGEs limit cell proliferation, we proposed that AGEs are responsible for the delayed healing process commonly observed in diabetic patients. Further, in pursuit of interventional strategies, we suggested that moderate treadmill exercise may support a healing environment in the presence of AGEs as exercise has been shown to stimulate cell proliferation in tendon tissue. Materials and Methods: Mice began receiving daily intraperitoneal injections of bovine serum albumin (BSA)-Control or AGE-BSA injections (200μg/ml) at 16-weeks of age. A tendon injury was created in the central third of both patellar tendons. Animals assigned to an exercise group began a moderate treadmill protocol one week following injury. The intact Achilles tendon and soleus muscle were also evaluated to assess the effect of BSA and AGE-BSA on un-injured muscle and tendon. Results: We demonstrate that our injection dosing and schedule lead to an increase in serum AGEs. Our findings imply that AGEs indeed modulate gene expression following a patellar tendon injury and have modest effects on gene expression in intact muscle and tendon. Conclusions: While additional biomechanical analysis is warranted, these data suggest that elevated serum AGEs in persons with diabetes may impact tendon health.

Movement compensation is driven by the deltoid and teres minor muscles following severe rotator cuff tear

BackgroundRotator cuff tears are common in older adults, negatively affecting function. Previous simulation-based studies reported more posterior and superior oriented glenohumeral loading with increased cuff tear severity and task performance, although corresponding muscle compensation strategies are unclear. Our objective is to determine how shoulder muscle forces change with increased rotator cuff tear severity during functional task performance. MethodsEight musculoskeletal models of increasing tear severity were developed to represent no rotator cuff tear to massive three-tendon tears. Simulations were performed using each combination of model and kinematics for five functional tasks. Individual muscle forces were averaged for each task and tear severity, then normalized by the sum of the muscle forces across the shoulder. Forces were compared across tear severity and muscle. FindingsFor muscle force contribution, interactions between tear severity and muscle and a main effect of muscle were seen for all tasks (P < 0.0001). Middle deltoid increased force contribution by >10% in the greatest tear severity model compared to no cuff tear model for all tasks (all P < 0.0001). Teres minor contribution increased by 7.7%, 5.6%, and 11% in the greatest tear severity model compared to the no cuff tear model for forward reach, axilla wash, and upward reach 105° tasks, respectively (all P < 0.0001). InterpretationFunctional tasks elicit compensatory responses from uninjured muscles following severe cuff tears, notably in middle deltoid and teres minor, leading to posterior-superior glenohumeral loading. The muscles are potential targets for strengthening to avoid injury from sustained increased muscle force.

Pharmaceutical Agents for Contractile-Metabolic Dysfunction After Volumetric Muscle Loss.

Volumetric muscle loss (VML) injuries represent a majority of military service member casualties and are common in civilian populations following blunt and/or penetrating traumas. Characterized as a skeletal muscle injury with permanent functional impairments, there is currently no standard for rehabilitation, leading to lifelong disability. Toward developing rehabilitative strategies, previous research demonstrates that the remaining muscle after a VML injury lacks similar levels of plasticity or adaptability as healthy, uninjured skeletal muscle. This may be due, in part, to impaired innervation and vascularization of the remaining muscle, as well as disrupted molecular signaling cascades commonly associated with muscle adaptation. The primary objective of this study was to assess the ability of four pharmacological agents with a strong record of modulating muscle contractile and metabolic function to improve functional deficits in a murine model of VML injury. Male C57BL/6 mice underwent a 15% multimuscle VML injury of the posterior hindlimb and were randomized into drug treatment groups (formoterol [FOR], 5-aminoimidazole-4-carboxamide riboside [AICAR], pioglitazone [PIO], or sildenafil [SIL]) or untreated VML group. At the end of 60 days, the injury model was first validated by comparison to age-matched injury-naive mice. Untreated VML mice had 22% less gastrocnemius muscle mass, 36% less peak-isometric torque, and 27% less maximal mitochondrial oxygen consumption rate compared to uninjured mice (p < 0.01). Experimental drug groups were, then, compared to VML untreated, and there was minimal evidence of efficacy for AICAR, PIO, or SIL in improving contractile and metabolic functional outcomes. However, FOR-treated VML mice had 18% greater peak isometric torque (p < 0.01) and permeabilized muscle fibers had 36% greater State III mitochondrial oxygen consumption rate (p < 0.01) compared to VML untreated mice, suggesting an overall improvement in muscle condition. There was minimal evidence that these benefits came from greater mitochondrial biogenesis and/or mitochondrial complex protein content, but could be due to greater enzyme activity levels for complex I and complex II. These findings suggest that FOR treatment is candidate to pair with a rehabilitative approach to maximize functional improvements in VML-injured muscle. Impact statement Volumetric muscle loss (VML) injuries result in deficiencies in strength and mobility, which have a severe impact on patient quality of life. Despite breakthroughs in tissue engineering, there are currently no treatments available that can restore function to the affected limb. Our data show that treatment of VML injuries with clinically available and FDA-approved formoterol (FOR), a beta-agonist, significantly improves strength and metabolism of VML-injured muscle. FOR is therefore a promising candidate for combined therapeutic approaches (i.e., regenerative rehabilitation) such as pairing FOR with structured rehabilitation or cell-seeded biomaterials as it may provide greater functional improvements than either strategy alone.

QS2. High Frequency Spectral Ultrasound Imaging Detects Early Post Traumatic Muscle Fibrosis in Rodents

Purpose: Muscle fibrosis is a devastating condition that can occur following traumatic injury leading to loss of function and poor outcomes. Limited noninvasive modalities exist to diagnose and characterize the extent of muscle damage. Here we investigate spectral ultrasound imaging (SUSI), a quantitative imaging technique which detects tissue parameters based on intrinsic structure and composition, to identify early muscle fibrosis based on fibrotic changes established in a rat model. We hypothesize that SUSI will detect early traumatic muscle fibrosis in a clinically translational rat muscle injury model. Methods: Adult male Sprague Dawley rats were sedated, prepped, and underwent a soft tissue crush injury that generated 20lbs/in2 of pressure using a digital force gauge. A superficial 1.5 cm incisional wound was then made over the quadriceps and closed. Injured and contralateral uninjured hind limbs were collected and fixed for SUSI imaging. Imaging was performed with a VisualSonics VEVO770 high resolution small animal ultrasound imaging system, using a 30 MHz center frequency single element transducer. Injured muscles were imaged at the crush injury and the corresponding place on the contralateral limb. Analysis of radiofrequency data was done as previously described in literature and SUSI parameters were calculated using the average acoustic concentration (AAC) of the fibrotic region and the corresponding region in the contralateral muscle. AAC values were grouped by time point and tissue type for statistical comparison using unpaired t tests. Results: In our rodent model, SUSI identified muscle fibrosis as early as 7 days post injury. The average AAC of injured fibrotic versus uninjured muscle was significantly increased at post injury day 7 (36.33 vs 18.36 db/mm3, injured versus uninjured, p = 0.003) and day 28 (54.64 vs 28.29 db/mm3, injured versus uninjured, p<0.0001). Though not statistically significant at day 14 (38.69 vs 27.61 db/mm3, injured vs uninjured, p=0.14) the fibrotic muscle did trend toward a higher AAC value. These results indicate that at day 7 and 28, the SUSI measurement AAC can be used to distinguish injured fibrotic muscle from uninjured muscle. Conclusions: SUSI offers a portable, non-invasive, and safe diagnostic modality to detect muscle fibrosis, making it an ideal modality for post traumatic injuries. Here, we show SUSI distinguishes muscle fibrosis from healthy tissue at day 7 and 28 post injury in our model allowing for early treatment. Unlike qualitative grayscale imaging, SUSI determines unique parameters of the tissue based on a calibrated spectrum related to the inherent properties of the tissue, making it less user-dependent and more reproducible.

68. Adipocytes Transition to Pro-fibrotic Fibroblasts and Contribute to Muscle Fibrosis Following Nerve Injury

Purpose: Recent studies have suggested that interconversion between adipocytes and fibroblasts (the cellular mediators of skin scarring) occurs in the wound environment. Muscle fibrosis occurs following aging, muscle injury, and muscular dystrophies causing weakness and pain. The role of adipocytes in muscle fibrosis has not been fully explored. Our study examines whether mature adipocytes participate in muscle fibrosis following nerve injury via conversion into dermal fibroblasts. Methods:AdipoqCre transgenic driver mice were crossed to R26mTmG reporter mice to generate AdipoqCre;ROSA26mTmG mice, in which mature adipocytes (Adipoq/adiponectin-expressing cells) express green fluorescent protein (GFP), and all other cells express a red fluorescent protein (RFP). AdipoqCre;ROSA26mTmG mice underwent sciatic nerve injury per established protocol. Injured tibialis anterior and gluteus maximus muscle was harvested at postoperative day-14 (POD14) and underwent immunofluorescent staining for fibroblast (collagen type IV/col-IV, alpha-smooth muscle actin/a-SMA) and adipocyte (perilipin) cell markers. To achieve local adipocyte ablation, AdipoqCre;ROSA26mTmG;R26tm1(HBEGF)Awai mice were generated and wounded, and diphtheria toxin (DT;400ng/mouse) was injected into the muscle at the nerve injury site. DT- and vehicle control-treated muscle underwent histologic analysis. Results: We identified a significantly greater number of adiponectin-lineage-positive cells (GFP+) within injured muscle at POD14 compared to uninjured muscle (*p<0.05,n=5). The GFP+ cells that infiltrated the muscle injury exhibited upregulation of fibrotic markers including col-IV and α-SMA, and downregulation of adipocyte markers including perilipin, indicating conversion of adipocytes into pro-fibrotic fibroblasts. DT-induced ablation of Adipoq-lineage-positive cells resulted in reduced muscle scarring, with decreased collagen deposition compared to vehicle-control injured muscle (*p<0.05,n=5). Conclusion: Our study demonstrates that adipocytes undergo conversion to pro-fibrotic fibroblasts in response to nerve injury and contribute to muscle fibrosis. Targeted adipocyte ablation resulted in reduced muscle scarring, suggesting that adipocyte-derived fibroblasts are important contributors to muscle fibrosis.

Glutamine supplementation improves contractile function of regenerating soleus muscles from rats.

This study evaluated the effects of glutamine supplementation immediately after freezing injury on morphological and contractile function of regenerating soleus muscles from rats. Young male Wistar rats were subjected to cryolesion of soleus muscles, and immediately after received a daily supplementation of glutamine (1g/kg/day). The muscles were evaluated on post-injury days 3 and 10. Glutamine-supplemented injured muscles had a lower number of CD11b positive immune cells and higher mRNA levels of IL-4 compared to those from the cryolesioned muscles analyzed on post-injury day 3. The mRNA and protein expression levels of the myogenic transcription factor MyoD were also higher in glutamine-supplemented injured muscles than in injured muscles examined on post-cryolesion day 3. In addition, glutamine-supplemented injured muscles had a higher size of their regenerating myofibers, attenuated decline in maximum tetanic strength and improved fatigue resistance compared to those from injured muscles evaluated on post-cryolesion day 10. No effect was observed in uninjured muscles supplemented with glutamine. Our results suggest that glutamine supplementation improves the resolution of inflammation, as well as the size and functional recovery of regenerating myofibers from soleus muscles by accelerating the up-regulation of IL-4 and MyoD expression. Future non-pharmacological rehabilitation studies are warranted to investigate the effect of glutamine supplementation on the outcome of injured skeletal muscles.

A Long Journey before Cycling: Regulation of Quiescence Exit in Adult Muscle Satellite Cells.

Skeletal muscle harbors a pool of stem cells called muscle satellite cells (MuSCs) that are mainly responsible for its robust regenerative capacities. Adult satellite cells are mitotically quiescent in uninjured muscles under homeostasis, but they exit quiescence upon injury to re-enter the cell cycle to proliferate. While most of the expanded satellites cells differentiate and fuse to form new myofibers, some undergo self-renewal to replenish the stem cell pool. Specifically, quiescence exit describes the initial transition of MuSCs from quiescence to the first cell cycle, which takes much longer than the time required for subsequent cell cycles and involves drastic changes in cell size, epigenetic and transcriptomic profiles, and metabolic status. It is, therefore, an essential period indispensable for the success of muscle regeneration. Diverse mechanisms exist in MuSCs to regulate quiescence exit. In this review, we summarize key events that occur during quiescence exit in MuSCs and discuss the molecular regulation of this process with an emphasis on multiple levels of intrinsic regulatory mechanisms. A comprehensive understanding of how quiescence exit is regulated will facilitate satellite cell-based muscle regenerative therapies and advance their applications in various disease and aging conditions.

Is Muscle Architecture Different in Athletes with a Previous Hamstring Strain? A Systematic Review and Meta-Analysis.

Hamstring strains are a frequent injury in sports and are characterized by a high recurrence rate. The aim of this review was to examine the muscle and tendon architecture in individuals with hamstring injury. A systematic literature search in four databases yielded eleven studies on architecture following injury. Differences in the fascicle length (FL), pennation angle (PA) and muscle size measures (volume, thickness and physiological cross-sectional area) at rest were not significantly different between the previously injured limb and the contralateral limb (p > 0.05). There was moderate evidence that biceps femoris long head (BFlh) FL shortening was greater during contraction in the injured compared to the contralateral limb. The BFlh FL was smaller in athletes with a previous injury compared to uninjured individuals (p = 0.0015) but no differences in the FL and PA of other muscles as well as in the aponeurosis/tendon size were observed (p > 0.05). An examination of the FL of both leg muscles in individuals with a previous hamstring strain may be necessary before and after return to sport. Exercises that promote fascicle lengthening of both injured and uninjured leg muscles may be beneficial for athletes who recover from a hamstring injury.

Repairing Volumetric Muscle Loss in the Ovine Peroneus Tertius Following a 6-Month Recovery.

Tissue-engineered skeletal muscle is a promising novel therapy for the treatment of volumetric muscle loss (VML). Our laboratory has developed tissue-engineered skeletal muscle units (SMUs) and engineered neural conduits (ENCs), and modularly scaled them to clinically relevant sizes for the treatment of VML in a large animal (sheep) model. In a previous study, we evaluated the effects of the SMUs and ENCs in treating a 30% VML injury in the ovine peroneus tertius muscle after a 3-month recovery period. The goal of the current study was to expand on our 3-month study and evaluate the SMUs and ENCs in restoring muscle function after a 6-month recovery period. Six months after implantation, we found that the repair groups with the SMU (VML+SMU and VML+SMU+ENC) restored muscle mass to a level that was statistically indistinguishable from the uninjured contralateral muscle. In contrast, the muscle mass in the VML-Only group was significantly less than groups repaired with an SMU. Following the 6-month recovery from VML, the maximum tetanic force was significantly lower for all VML injured groups compared with the uninjured contralateral muscle. However, we did demonstrate the ability of our ENCs to effectively regenerate nerve between the distal stump of the native nerve and the repair site in 14 of the 15 animals studied. Impact Statement Volumetric muscle loss (VML) is a clinically relevant problem for which current treatment options are lacking and for which tissue-engineered skeletal muscle presents a promising novel therapeutic option. However, the fabrication of tissues of clinically relevant sizes is necessary for advancement of the technology to the clinic. This study aimed to evaluate the efficacy of our scaled-up tissue-engineered skeletal muscle to treat VML in a large animal (sheep) model after a 6-month recovery.

Membrane Proteins Increase with the Repeated Bout Effect.

The ability of skeletal muscle to adapt to eccentric (ECC) contraction-induced injury is known as the repeated bout effect (RBE). Despite the RBE being a well-established phenomenon observed in skeletal muscle, cellular and molecular events particularly those at the membranes that contribute to the adaptive potential of muscle have yet to be established. Therefore, the purpose of this study was to examine how membrane-associated proteins respond to the RBE. Anterior crural muscles of C57BL/6 female mice (3-5 months) were subjected to repeated bouts of in vivo ECCs, with isometric torque being measured immediately before and after injury. A total of six bouts were completed with 7 d between each bout. Protein content of dystrophin, β-sarcoglycan, and junctophilin were then assessed via immunoblotting in injured and uninjured muscles. When expressed relative to preinjury isometric torque of bout 1, deficits in postinjury isometric torque during bout 2 (38%) did not differ from bout 1 (36%; P = 0.646) and were attenuated during bouts 3 through 6 (range, 24%-15%; P ≤ 0.014). Contents of dystrophin, β-sarcoglycan, and junctophilin did not change immediately after a single bout of 50 maximal ECCs (P ≥ 0.155); however, as a result of repeated bouts, contents of dystrophin, β-sarcoglycan, and junctophilin all increased compared with muscles that completed one or no bouts of ECC contractions (P ≤ 0.003). The RBE represents a physiological measure of skeletal muscle plasticity. Here, we demonstrate that repeated bouts of ECC contractions increase contents of dystrophin, β-sarcoglycan, and junctophilin and attenuate postinjury torque deficits. Given our results, accumulation of membrane-associated proteins likely contributes to strength adaptations observed after repeated bouts of ECC contractions.

Reliability and discriminative validity of real-time ultrasound elastography in the assessment of tissue stiffness after calf muscle injury

ObjectiveTo investigate the reliability and discriminative validity of real-time ultrasound elastography (RTE) measures of soft-tissue elasticity after calf muscle tear. DesignCross-sectional, intra/inter-examiner reliability and comparative validity study. SettingDepartment of Physical Therapy. ParticipantsTwenty-one recreational athletes were included and examined 6 weeks after sustaining a grade I-II calf musculature tear. Main outcome measuresSoft-tissue elasticity was measured by two experienced assessors using RTE assessments in both the longitudinal and transverse planes of the athletes’ injured and uninjured calf muscles. Elasticity was estimated by using the strain ratio (SR), which was calculated by dividing the strain (displacement) value taken at the medial gastrocnemius-soleus myotendinous junction (reference) by the strain value taken at the centre of the injury (index) as visualized on B-mode sonogram. Intra- and inter-observer reliability was estimated calculating intra-class correlation coefficients (ICCs) and standard error of measurement (SEM). Differences in elasticity between injured and healthy legs were assessed using t-tests or Wilcoxon tests for repeated measures. ResultsAll RTE assessments in both planes showed ICC values ranging from 0.77 to 0.95 and SEM values ranging from 0.72 to 0.99. Additionally, RTE enabled both assessors to determine differences in elastic properties between injured and control legs (p < 0.001). ConclusionRTE measures of calf muscles demonstrated good reliability and were able to differentiate injured from non-injured muscle tissue. RTE may provide a fast and objective measure in sports medicine to improve the detection of risk factors for muscle injury related to alterations of the mechanical behaviour of soft tissues during healing process.

Progressive and Coordinated Mobilization of the Skeletal Muscle Niche throughout Tissue Repair Revealed by Single-Cell Proteomic Analysis.

Background: Skeletal muscle is one of the only mammalian tissues capable of rapid and efficient regeneration after trauma or in pathological conditions. Skeletal muscle regeneration is driven by the muscle satellite cells, the stem cell population in interaction with their niche. Upon injury, muscle fibers undergo necrosis and muscle stem cells activate, proliferate and fuse to form new myofibers. In addition to myogenic cell populations, interaction with other cell types such as inflammatory cells, mesenchymal (fibroadipogenic progenitors—FAPs, pericytes) and vascular (endothelial) lineages are important for efficient muscle repair. While the role of the distinct populations involved in skeletal muscle regeneration is well characterized, the quantitative changes in the muscle stem cell and niche during the regeneration process remain poorly characterized. Methods: We have used mass cytometry to follow the main muscle cell types (muscle stem cells, vascular, mesenchymal and immune cell lineages) during early activation and over the course of muscle regeneration at D0, D2, D5 and D7 compared with uninjured muscles. Results: Early activation induces a number of rapid changes in the proteome of multiple cell types. Following the induction of damage, we observe a drastic loss of myogenic, vascular and mesenchymal cell lineages while immune cells invade the damaged tissue to clear debris and promote muscle repair. Immune cells constitute up to 80% of the mononuclear cells 5 days post-injury. We show that muscle stem cells are quickly activated in order to form new myofibers and reconstitute the quiescent muscle stem cell pool. In addition, our study provides a quantitative analysis of the various myogenic populations during muscle repair. Conclusions: We have developed a mass cytometry panel to investigate the dynamic nature of muscle regeneration at a single-cell level. Using our panel, we have identified early changes in the proteome of stressed satellite and niche cells. We have also quantified changes in the major cell types of skeletal muscle during regeneration and analyzed myogenic transcription factor expression in satellite cells throughout this process. Our results highlight the progressive dynamic shifts in cell populations and the distinct states of muscle stem cells adopted during skeletal muscle regeneration. Our findings give a deeper understanding of the cellular and molecular aspects of muscle regeneration.

Paxbp1 controls a key checkpoint for cell growth and survival during early activation of quiescent muscle satellite cells

Adult mouse muscle satellite cells (MuSCs) are quiescent in uninjured muscles. Upon muscle injury, MuSCs exit quiescence, reenter the cell cycle to proliferate and self-renew, and then differentiate and fuse to drive muscle regeneration. However, it remains poorly understood how MuSCs transition from quiescence to the cycling state. Here, we report that Pax3 and Pax7 binding protein 1 (Paxbp1) controls a key checkpoint during this critical transition. Deletion of Paxbp1 in adult MuSCs prevented them from reentering the cell cycle upon injury, resulting in a total regeneration failure. Mechanistically, we found an abnormal elevation of reactive oxygen species (ROS) in Paxbp1-null MuSCs, which induced p53 activation and impaired mTORC1 signaling, leading to defective cell growth, apoptosis, and failure in S-phase reentry. Deliberate ROS reduction partially rescued the cell-cycle reentry defect in mutant MuSCs. Our study reveals that Paxbp1 regulates a late cell-growth checkpoint essential for quiescent MuSCs to reenter the cell cycle upon activation.

FOS licenses early events in stem cell activation driving skeletal muscle regeneration

SUMMARYMuscle satellite cells (SCs) are a quiescent (non-proliferative) stem cell population in uninjured skeletal muscle. Although SCs have been investigated for nearly 60 years, the molecular drivers that transform quiescent SCs into the rapidly dividing (activated) stem/progenitor cells that mediate muscle repair after injury remain largely unknown. Here we identify a prominent FBJ osteosarcoma oncogene (Fos) mRNA and protein signature in recently activated SCs that is rapidly, heterogeneously, and transiently induced by muscle damage. We further reveal a requirement for FOS to efficiently initiate key stem cell functions, including cell cycle entry, proliferative expansion, and muscle regeneration, via induction of “pro-regenerative” target genes that stimulate cell migration, division, and differentiation. Disruption of one of these Fos/AP-1 targets, NAD(+)-consuming mono-ADP-ribosyl-transferase 1 (Art1), in SCs delays cell cycle entry and impedes progenitor cell expansion and muscle regeneration. This work uncovers an early-activated FOS/ART1/mono-ADP-ribosylation (MARylation) pathway that is essential for stem cell-regenerative responses.

Intramuscular injection of skeletal muscle derived extracellular matrix mitigates denervation atrophy after sciatic nerve transection

Peripheral nerve injury and the associated muscle atrophy has an estimated annual healthcare burden of $150 billion dollars in the United States. When considering the total annual health-related spending of $3.5 trillion, these pathologies alone occupy about 4.3%. The prevalence of these ailments is rooted, at least in part, in the lack of specific preventative therapies that can be administered to muscle while it remains in the denervated state. To address this, skeletal muscle-derived ECM (skECM) was injected directly in denervated muscle with postoperative analysis performed at 20 weeks, including gait analysis, force production, cytokine quantification, and histological analysis. skECM was shown to be superior against non-injected muscle controls showing no difference in contraction force to uninjured muscle at 20 weeks. Cytokines IL-1β, IL-18, and IFNγ appeared to mediate regeneration with statistical regression implicating these cytokines as strong predictors of muscle contraction, showing significant linear correlation.

Satellite cell activation and retention of muscle regenerative potential after long-term denervation.

Irreversible denervation atrophy remains an unsolved clinical problem, and the role of skeletal muscle stem cell (MuSC, satellite cell) depletion in this process is unclear. We investigated the ability of MuSCs to regenerate muscle in the context of denervation. Three to 12 months following sciatic denervation in mice, MuSC number, size, EdU uptake, rate of division, and mitochondrial activity were increased. Following acute myotoxin injury, denervated muscles formed new muscle fibers in situ. MuSCs isolated via flow cytometry from denervated mouse muscle, or from atrophic denervated gluteus maximus muscles of humans with complete spinal cord injuries two decades prior, formed new muscle fibers and reoccupied the anatomic niche after transplantation into uninjured muscle. Our results show unequivocally that, even after prolonged denervation, MuSCs retain intrinsic regenerative potential similar to that of uninjured MuSCs. Treatment of denervation atrophy will require elucidating the non-MuSC environmental changes in muscle that prevent functional regeneration.

CCAAT/enhancer-binding protein beta promotes muscle stem cell quiescence through regulation of quiescence-associated genes.

Regeneration of skeletal muscle depends on resident muscle stem cells called satellite cells that in healthy, uninjured muscle remain quiescent (noncycling). After activation and expansion of satellite cells postinjury, satellite cell numbers return to uninjured levels and return to mitotic quiescence. Here, we show that the transcription factor CCAAT/enhancer-binding protein beta (C/EBPβ) is required to maintain quiescence of satellite cells in uninjured muscle. We show that C/EBPβ is expressed in quiescent satellite cells in vivo and upregulated in noncycling myoblasts in vitro. Loss of C/EBPβ in satellite cells promotes their premature exit from quiescence resulting in spontaneous activation and differentiation of the stem cell pool. Forced expression of C/EBPβ in myoblasts inhibits proliferation by upregulation of 28 quiescence-associated genes. Furthermore, we find that caveolin-1 is a direct transcriptional target of C/EBPβ and is required for cell cycle exit in muscle satellite cells expressing C/EBPβ. The induction of mitotic quiescence is considered necessary for the long-term maintenance of adult stem cell populations with dysregulation driving increased differentiation of progenitors and depletion of the stem cell pool. Our findings place C/EBPβ as an important transcriptional regulator of muscle satellite cell quiescence.

A tissue engineering approach for repairing craniofacial volumetric muscle loss in a sheep following a 2, 4, and 6-month recovery.

Volumetric muscle loss (VML) is the loss of skeletal muscle that results in significant and persistent impairment of function. The unique characteristics of craniofacial muscle compared trunk and limb skeletal muscle, including differences in gene expression, satellite cell phenotype, and regenerative capacity, suggest that VML injuries may affect craniofacial muscle more severely. However, despite these notable differences, there are currently no animal models of craniofacial VML. In a previous sheep hindlimb VML study, we showed that our lab's tissue engineered skeletal muscle units (SMUs) were able to restore muscle force production to a level that was statistically indistinguishable from the uninjured contralateral muscle. Thus, the goals of this study were to: 1) develop a model of craniofacial VML in a large animal model and 2) to evaluate the efficacy of our SMUs in repairing a 30% VML in the ovine zygomaticus major muscle. Overall, there was no significant difference in functional recovery between the SMU-treated group and the unrepaired control. Despite the use of the same injury and repair model used in our previous study, results showed differences in pathophysiology between craniofacial and hindlimb VML. Specifically, the craniofacial model was affected by concomitant denervation and ischemia injuries that were not exhibited in the hindlimb model. While clinically realistic, the additional ischemia and denervation likely created an injury that was too severe for our SMUs to repair. This study highlights the importance of balancing the use of a clinically realistic model while also maintaining control over variables related to the severity of the injury. These variables include the volume of muscle removed, the location of the VML injury, and the geometry of the injury, as these affect both the muscle's ability to self-regenerate as well as the probability of success of the treatment.

A Type 2 Deiodinase-Dependent Increase in Vegfa Mediates Myoblast-Endothelial Cell Crosstalk During Skeletal Muscle Regeneration.

Background: The type 2 deiodinase (DIO2) converts thyroxine to 3,3',5-triiodothyronine (T3), modulating intracellular T3. An increase in DIO2 within muscle stem cells during skeletal muscle regeneration leads to T3-dependent potentiation of differentiation. The muscle stem cell niche comprises numerous cell types, which coordinate the regeneration process. For example, muscle stem cells provide secretory signals stimulating endothelial cell-mediated vascular repair, and, in turn, endothelial cells promote muscle stem differentiation. We hypothesized that Dio2 loss in muscle stem cells directly impairs muscle stem cell-endothelial cell communication, leading to downstream disruption of endothelial cell function. Methods: We assessed the production of proangiogenic factors in differentiated C2C12 cells and in a C2C12 cell line without Dio2 (D2KO C2C12) by real-time quantitative-polymerase chain reaction and enzyme-linked immunosorbent assay. Conditioned medium (CM) was collected daily in parallel to evaluate its effects on human umbilical vein endothelial cell (HUVEC) proliferation, migration and chemotaxis, and vascular network formation. The effects of T3-treatment on vascular endothelial growth factor (Vegfa) mRNA expression in C2C12 cells and mouse muscle were assessed. Chromatin immunoprecipitation (ChIP) identified thyroid hormone receptor (TR) binding to the Vegfa gene. Using mice with a targeted disruption of Dio2 (D2KO mice), we determined endothelial cell number by immunohistochemistry/flow cytometry and evaluated related gene expression in both uninjured and injured skeletal muscle. Results: In differentiated D2KO C2C12 cells, Vegfa expression was 46% of wildtype (WT) C2C12 cells, while secreted VEGF was 45%. D2KO C2C12 CM exhibited significantly less proangiogenic effects on HUVECs. In vitro and in vivo T3 treatment of C2C12 cells and WT mice, and ChIP using antibodies against TRα, indicated that Vegfa is a direct genomic T3 target. In uninjured D2KO soleus muscle, Vegfa expression was decreased by 28% compared with WT mice, while endothelial cell numbers were decreased by 48%. Seven days after skeletal muscle injury, D2KO mice had 36% fewer endothelial cells, coinciding with an 83% decrease in Vegfa expression in fluorescence-activated cell sorting purified muscle stem cells. Conclusion:Dio2 loss in the muscle stem cell impairs muscle stem cell-endothelial cell crosstalk via changes in the T3-responsive gene Vegfa, leading to downstream impairment of endothelial cell function both in vitro and in vivo.