Skeletal Muscle Regeneration Research Articles

4. Skeletal Muscle in Inbred Mice Exhibits Divergent Hypoxia and Angiogenesis Transcriptome Expression as Compared to Humans in Aging

Hypoxia signaling has been previously demonstrated to be a critical regulator in age-dependent skeletal muscle regeneration. While animal models provide a crucial scientific substrate for medical innovation and translation, discordant findings have been commonly described in existing literature between animals and humans. The nature and extent of this divergence in the context of aging and its effect on skeletal muscle regeneration remains incompletely understood. Quadriceps muscles from young (Y, 12 weeks, n=3) and old (O, 25 months, n=3) C57BL/6 mice were harvested and analyzed via RNA bulk-sequencing. These results were compared to healthy human vastus lateralis muscles (Y, 22-30 years, n=8; O, 80-83 years, n=5) from the GESTALT study (NIA/NIH) analyses as found in the Gene Expression Omnibus (NIH) database. Gross examination of the most differentially expressed genes demonstrated low variance between samples within each mouse cohort (Disp = 0.04467, BCV = 0.2114) relative to young/old human (Disp = 0.61261, BCV = 0.7827) specimens. Enrichment analyses were agnostically performed on filtered differentially expressed human genes via edgeR (Bioconductor) resulting in significant overlap with the HIF-1 signaling pathway KEGG term (Eno1, Tfrc, Ldha, Eno3, and Pdha1) with significant upregulation in young human muscles. However, these same genes demonstrated no significant differences within mice. Interestingly, when examining angiogenesis gene lists determined a priori, mice demonstrated several significantly upregulated genes in the young cohort (Anxa2, Lox, Mmp9, Angptl4, Adora2b, and Pgf) that thematically correspond to modulators of the extracellular matrix in the process of neoangiogenesis. Conversely, none of these genes were found to be significantly different between old and young humans. To further compare the muscle transcriptomes between humans and mice in the context of aging, genes with the largest magnitude fold changes and most significant p-values were identified. The most significant differentially expressed genes in mice demonstrated robust upregulation of Mss51, Igkv3-2, Ighg2c, Rasd2, Igkc, Eda2r, and Gdf11 in old mice. These same genes in human specimens demonstrated no comparable segregation. Similarly, the most upregulated genes in old human specimens (Gda, Mtcybp32, Fgf7, Fam83b, Lyve1, Plag1, Eda2r, Dkk2, and Col21a1) showed no similar segregations in mice. The most differentially expressed genes for hypoxia and angiogenesis in both mice and humans were not found to be concordantly upregulated when cross-examined across the other species. Thus, skeletal muscle in inbred mice exhibits differential transcriptome expression and decreased variance as compared to humans in aging. Given these findings, great care should be taken in making inferences between findings between mice and humans. As NextGen sequencing becomes routine, incorporation of genetically diverse mice to better approximate human genetic variation may provide further insights in translation from bench to bedside.

E-Selectin/AAV Gene Therapy Promotes Myogenesis and Skeletal Muscle Recovery in a Mouse Hindlimb Ischemia Model.

The response to ischemia in peripheral artery disease (PAD) depends on compensatory neovascularization and coordination of tissue regeneration. Identifying novel mechanisms regulating these processes is critical to the development of nonsurgical treatments for PAD. E-selectin is an adhesion molecule that mediates cell recruitment during neovascularization. Therapeutic priming of ischemic limb tissues with intramuscular E-selectin gene therapy promotes angiogenesis and reduces tissue loss in a murine hindlimb gangrene model. In this study, we evaluated the effects of E-selectin gene therapy on skeletal muscle recovery, specifically focusing on exercise performance and myofiber regeneration. C57BL/6J mice were treated with intramuscular E-selectin/adeno-associated virus serotype 2/2 gene therapy (E-sel/AAV) or LacZ/AAV2/2 (LacZ/AAV) as control and then subjected to femoral artery coagulation. Recovery of hindlimb perfusion was assessed by laser Doppler perfusion imaging and muscle function by treadmill exhaustion and grip strength testing. After three postoperative weeks, hindlimb muscle was harvested for immunofluorescence analysis. At all postoperative time points, mice treated with E-sel/AAV had improved hindlimb perfusion and exercise capacity. E-sel/AAV gene therapy also increased the coexpression of MyoD and Ki-67 in skeletal muscle progenitors and the proportion of Myh7+ myofibers. Altogether, our findings demonstrate that in addition to improving reperfusion, intramuscular E-sel/AAV gene therapy enhances the regeneration of ischemic skeletal muscle with a corresponding benefit on exercise performance. These results suggest a potential role for E-sel/AAV gene therapy as a nonsurgical adjunct in patients with life-limiting PAD.

A concise in vitro model for evaluating interactions between macrophage and skeletal muscle cells during muscle regeneration.

Skeletal muscle has a highly regenerative capacity, but the detailed process is not fully understood. Several in vitro skeletal muscle regeneration models have been developed to elucidate this, all of which rely on specialized culture conditions that limit the accessibility and their application to many general experiments. Here, we established a concise in vitro skeletal muscle regeneration model using mouse primary cells. This model allows evaluation of skeletal muscle regeneration in two-dimensional culture system similar to a typical cell culture, showing a macrophage-dependent regenerative capacity, which is an important process in skeletal muscle regeneration. Based on the concept that this model could assess the contribution of macrophages of various phenotypes to skeletal muscle regeneration, we evaluated the effect of endotoxin pre-stimulation for inducing various changes in gene expression on macrophages and found that the contribution to skeletal muscle regeneration was significantly reduced. The gene expression patterns differed from those of naive macrophages, especially immediately after skeletal muscle injury, suggesting that the difference in responsiveness contributed to the difference in regenerative efficiency. Our findings provide a concise in vitro model that enables the evaluation of the contribution of individual cell types, such as macrophages and muscle stem cells, on skeletal muscle regeneration.

Genomic and Epigenetic Changes Drive Aberrant Skeletal Muscle Differentiation in Rhabdomyosarcoma

Rhabdomyosarcoma (RMS), the most common soft-tissue sarcoma in children and adolescents, represents an aberrant form of skeletal muscle differentiation. Both skeletal muscle development, as well as regeneration of adult skeletal muscle are governed by members of the myogenic family of regulatory transcription factors (MRFs), which are deployed in a highly controlled, multi-step, bidirectional process. Many aspects of this complex process are deregulated in RMS and contribute to tumorigenesis. Interconnected loops of super-enhancers, called core regulatory circuitries (CRCs), define aberrant muscle differentiation in RMS cells. The transcriptional regulation of MRF expression/activity takes a central role in the CRCs active in skeletal muscle and RMS. In PAX3::FOXO1 fusion-positive (PF+) RMS, CRCs maintain expression of the disease-driving fusion oncogene. Recent single-cell studies have revealed hierarchically organized subsets of cells within the RMS cell pool, which recapitulate developmental myogenesis and appear to drive malignancy. There is a large interest in exploiting the causes of aberrant muscle development in RMS to allow for terminal differentiation as a therapeutic strategy, for example, by interrupting MEK/ERK signaling or by interfering with the epigenetic machinery controlling CRCs. In this review, we provide an overview of the genetic and epigenetic framework of abnormal muscle differentiation in RMS, as it provides insights into fundamental mechanisms of RMS malignancy, its remarkable phenotypic diversity and, ultimately, opportunities for therapeutic intervention.

Molecular clocks, satellite cells, and skeletal muscle regeneration.

Skeletal muscle comprises approximately 50% of individual body mass and plays vital roles in locomotion, heat production, and whole body metabolic homeostasis. This tissue exhibits a robust diurnal rhythm that is under control of the suprachiasmatic nucleus (SCN) region of the hypothalamus. The SCN acts as a "central" coordinator of circadian rhythms, while cell-autonomous "peripheral" clocks are located within almost all other tissues/organs in the body. Synchronization of the peripheral clocks in muscles (and other tissues) together with the central clock is crucial to ensure temporally coordinated physiology across all organ systems. By virtue of its mass, human skeletal muscle contains the largest collection of peripheral clocks, but within muscle resides a local stem cell population, satellite cells (SCs), which have their own functional molecular clock, independent of the numerous muscle clocks. Skeletal muscle has a daily turnover rate of 1%-2%, so the regenerative capacity of this tissue is important for whole body homeostasis/repair and depends on successful SC myogenic progression (i.e., proliferation, differentiation, and fusion). Emerging evidence suggests that SC-mediated muscle regeneration may, in part, be regulated by molecular clocks involved in SC-specific diurnal transcription. Here we provide insights on molecular clock regulation of muscle regeneration/repair and provide a novel perspective on the interplay between SC-specific molecular clocks, myogenic programs, and cell cycle kinetics that underpin myogenic progression.

Secreted ADAMTS-like 2 promotes myoblast differentiation by potentiating WNT signaling

Myogenesis is the process that generates multinucleated contractile myofibers from muscle stem cells during skeletal muscle development and regeneration. Myogenesis is governed by myogenic regulatory transcription factors, including MYOD1. Here, we identified the secreted matricellular protein ADAMTS-like 2 (ADAMTSL2) as part of a Wnt-dependent positive feedback loop, which augmented or sustained MYOD1 expression and thus promoted myoblast differentiation. ADAMTSL2 depletion resulted in severe retardation of myoblast differentiation in vitro and its ablation in myogenic precursor cells resulted in aberrant skeletal muscle architecture. Mechanistically, ADAMTSL2 potentiated WNT signaling by binding to WNT ligands and WNT receptors. We identified the WNT-binding ADAMTSL2 peptide, which was sufficient to promote myogenesis in vitro. Since ADAMTSL2 was previously described as a negative regulator of TGFβ signaling in fibroblasts, ADAMTSL2 now emerges as a signaling hub that could integrate WNT, TGFβ and potentially other signaling pathways within the dynamic microenvironment of differentiating myoblasts during skeletal muscle development and regeneration.

NEDD4-1 deficiency impairs satellite cell function during skeletal muscle regeneration

BackgroundSatellite cells are tissue-specific stem cells primarily responsible for the regenerative capacity of skeletal muscle. Satellite cell function and maintenance are regulated by extrinsic and intrinsic mechanisms, including the ubiquitin–proteasome system, which is key for maintaining protein homeostasis. In this context, it has been shown that ubiquitin-ligase NEDD4-1 targets the transcription factor PAX7 for proteasome-dependent degradation, promoting muscle differentiation in vitro. Nonetheless, whether NEDD4-1 is required for satellite cell function in regenerating muscle remains to be determined.ResultsUsing conditional gene ablation, we show that NEDD4-1 loss, specifically in the satellite cell population, impairs muscle regeneration resulting in a significant reduction of whole-muscle size. At the cellular level, NEDD4-1-null muscle progenitors exhibit a significant decrease in the ability to proliferate and differentiate, contributing to the formation of myofibers with reduced diameter.ConclusionsThese results indicate that NEDD4-1 expression is critical for proper muscle regeneration in vivo and suggest that it may control satellite cell function at multiple levels.

Muscle Injuries Induce a Prostacyclin-PPARγ/PGC1a-FAO Spike That Boosts Regeneration.

It is well-known that muscle regeneration declines with aging, and aged muscles undergo degenerative atrophy or sarcopenia. While exercise and acute injury are both known to induce muscle regeneration, the molecular signals that help trigger muscle regeneration have remained unclear. Here, mass spectrometry imaging (MSI) is used to show that injured muscles induce a specific subset of prostanoids during regeneration, including PGG1, PGD2, and the prostacyclin PGI2. The spike in prostacyclin promotes skeletal muscle regeneration via myoblasts, and declines with aging. Mechanistically, the prostacyclin spike promotes a spike in PPARγ/PGC1a signaling, which induces a spike in fatty acid oxidation (FAO) to control myogenesis. LC-MS/MS and MSI further confirm that an early FAO spike is associated with normal regeneration, but muscle FAO became dysregulated during aging. Functional experiments demonstrate that the prostacyclin-PPARγ/PGC1a-FAO spike is necessary and sufficient to promote both young and aged muscle regeneration, and that prostacyclin can synergize with PPARγ/PGC1a-FAO signaling to restore aged muscles' regeneration and physical function. Given that the post-injury prostacyclin-PPARγ-FAO spike can be modulated pharmacologically and via post-exercise nutrition, this work has implications for how prostacyclin-PPARγ-FAO might be fine-tuned to promote regeneration and treat muscle diseases of aging.

Skeletal muscle regeneration at different phases of the menstrual cycle

INTRODUCTION: The effect of estrogen on muscle function and recovery from damage has been studied, but the effects of estrogen fluctuations during the menstrual cycle on muscle regeneration remain inconclusive. PURPOSE: The purpose of this study was to examine the effects of menstrual cycle phases on muscle regeneration after electrically stimulated muscle damage. We hypothesized that muscle damage during the late follicular (LF) phase results in better muscle regeneration as compared to muscle damage during the early follicular (EF) phase. METHODS: 23 premenopausal females (21.3±2 yrs) were recruited and split into two groups, EF and LF. After menstrual cycle tracking and phase confirmation, subjects underwent 200 electrically stimulated eccentric muscle contractions one week after providing a muscle biopsy. 7 days after inducing muscle damage, the subjects reported to give a final biopsy. Sections from muscle samples were stained for markers of laminin, dystrophin, satellite cells (SCs; Pax7), nuclei, total macrophages (CD68), and anti-inflammatory macrophages (CD206/CD68). Data for SCs, and %CD206+CD68+ cells are presented as mean±SD, and damaged fibers and CD206+CD68+ cells/fiber are presented as median±interquartile range. RESULTS: Electrical stimulation caused muscle fiber necrosis (0.005±0.018 damaged fiber/fiber, p=0.0002) as measured by the laminin-containing and dystrophin-deficient fibers. There was no difference between EF and LF groups for the number of damaged fibers (p=0.055) Muscle damage resulted in ~200% increase in the total number of CD68+ macrophages (p=0.008), and ~ 40% increase in the number of SCs (p=0.001) in both groups. However, there was no significant difference between EF and LF groups for the change in total macrophages per fiber (p=0.07) or SCs/fiber (p=0.25) in response to electrically stimulated muscle damage. Anti-inflammatory macrophages increased by about 104%±127 in EF and 240%±390 in LF group, but the difference between groups was not significant (p=0.39). A comparison of the proportion of anti-inflammatory macrophages per total macrophages before and one week after muscle damage did not show any time effect (p=0.25), which could indicate the regenerative and anti-inflammatory state of skeletal muscle one week after damage. CONCLUSION: Contrary to our hypothesis, our data indicate that skeletal muscle regeneration is not affected by different phases of the menstrual cycle. However, this finding is limited to only some of the markers of regeneration one week after muscle damage. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 604: Distinct Fibroadipogenic Progenitor Populations And Trajectories Characterize Regenerative And Non-regenerative Responses In The Ischemic Limb

Introduction: Skeletal muscle possesses the capacity for self-repair. This ability is impaired in chronic limb threatening ischemia (CLTI) patients. Myopathy in CLTI patients is associated with adverse clinical outcomes. Skeletal muscle regeneration requires multicellular interactions between muscle progenitor cells and other cell types. Fibroadipogenic progentiors (FAPs) represent a support cell type in the skeletal muscle niche; they modulate inflammatory and myogenic responses via the secretion of a paracrine factors. Hence, FAPs are an attractive therapeutic target for myopathies. We sought to characterize the temporal dynamics and transcriptional heterogeneity of FAPs in failed and successful skeletal muscle regeneration in the ischemic limb. Methods: We analyzed the FAP regenerative response in BALB/c (non-regenerative) and C57BL/6 (regenerative) mice undergoing hindlimb ischemia using single-cell RNA sequencing spanning 4 distinct time points. We also performed RNA velocity analysis to determine the cellular dynamics of FAP sub-populations during the repair process. Results: We identified 10 FAP populations in the ischemic limb, including specific regenerative and non-regenerative FAP sub-clusters. Gene ontology (GO) enrichment analysis demonstrated distinct transcriptional programs between regenerative and non-regenerative FAPs. Regenerative FAPs were enriched in extracellular matrix and angiogenesis pathways, while non-regenerative FAPs were enriched in chemotactic pathways. RNA velocity analysis demonstrated divergent FAP cellular trajectories. Conclusions: Our study maps the dynamic FAP response in the ischemic limb at single-cell resolution. We identify distinct transcriptional programs and cellular trajectories that are associated with failed and successful skeletal muscle repair. This provides a resource to investigate FAP mediated mechanisms of skeletal muscle regeneration in response to limb ischemia.

The Muscle Stem Cell Mediates Remodeling of Skeletal Muscle Mitochondrial Networks

Skeletal muscle regeneration is an energy-demanding biological process that relies on mitochondria to generate ATP. While interfibrillar mitochondrial networks in healthy skeletal muscle fibers exhibit an organized architecture segregated into columns along the Z-line of the myofiber, regenerating fibers demonstrate altered mitochondrial networks that span several sarcomeres. Although it’s been established that skeletal muscle regeneration parallels altered mitochondrial dynamics and biogenesis, the cells that mediate the remodeling of mitochondrial networks have not been thoroughly investigated. Because muscle stem cells (MuSCs) play a critical role in muscle regeneration, we explored the relationship between MuSCs and mitochondria in regenerating muscle fibers. In order to induce muscle regeneration, we employed the hindlimb ischemia mouse model of peripheral artery disease (PAD) and the mdx mouse model of Duchenne’s muscular dystrophy (DMD). Furthermore, transgenic reporter mice such as Pax7-tdTomato, H2B-EGFP, and mitoDendra2 were used mark MuSCs, track nuclei, and label mitochondria, respectively. Fluorescently-labelled MuSCs were also transplanted into BaCl2-injured muscle to elucidate their behavior and interactions throughout muscle regeneration. Immunofluorescence, single fiber staining, and biochemical assays from harvested muscles were performed for data analysis. Following injury, there was a dramatic increase in the number of endogenous MuSCs 7 days post-injury compared to contralateral control while there was a significant increase in fusion of the accreted MuSCs into the myofiber at days 14 and 28. Interestingly, all fused myofibers at days 14 and 28 were regenerating, demarcated by central nucleation, suggesting that fusion of the MuSC is critical for myofiber regeneration. To determine the origin of the centrally located myonuclei, primary MuSCs from H2B-EGFP reporter mice were isolated and transplanted into injured muscle. For myofibers in which the transplanted MuSCs fused into, centrally located myonuclei were GFP+, implying that these myonuclei were derived from MuSCs. Surprisingly, regenerating muscle fibers isolated from the mitoDendra2 mouse model exhibited high densities of mitochondrial content adjacent to the centrally located myonuclei. In order to test whether MuSCs are responsible for producing these central myonuclei near mitochondria, primary MuSCs isolated from mitoDendra2 reporter mice were transplanted into mdx mice and fused myofibers indeed exhibited high Dendra2+ mitochondrial density between central nuclei as well as a restoration of the mitochondrial network. Finally, we also observed a change in myogenic potential and mitochondrial respiration after transplanting MuSCs that have been preconditioned to enhance mitochondrial biogenesis. Overall, these data indicate that following skeletal muscle injury, MuSC-derived myonuclei synthesize new mitochondria to generat This work was supported by the National Institute of Health under award number (R01AG072309, R21AR072287), Department of Defense under award number W81XWH-20-1-0336, and Regenerative Engineering and Medicine Grant. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Glutathione Levels are Critical for Satellite Cells Response and Skeletal Muscle Regeneration Following Aseptic Myotrauma in Humans

Glutathione Levels are Critical for Satellite Cells Response and Skeletal Muscle Regeneration Following Aseptic Myotrauma in Humans

Inducible deletion of endothelial cell EfnB2 attenuates neuromuscular regeneration in mouse skeletal muscle

Background: Acute injury of skeletal muscle disrupts myofibers, microvessels and neuromuscular junctions (NMJs). While myofiber regeneration is well-characterized, little is known of concurrent events in microvessels and motor nerves following injury. Endothelial cell [EC] ephrin B2 [EfnB2] is required for sprouting angiogenesis during embryonic development, for promoting vascularization of tumors in the adult, and for microvascular-dependent guidance of axon regeneration following nerve resection. Hypothesis: We hypothesized that following acute injury to skeletal muscle 1) EC EfnB2 is required for restoring microvascular volume; and 2) loss of EC EfnB2 would impair recovery of neuromuscular structure and function. Methods: Mice (male and female, age 3-5 months) were bred for EC-specific conditional knockout [CKO] of EfnB2 following tamoxifen injection; noninjected CKO mice served as “wildtype” controls [WT]. Following injury by local injection of the myotoxin BaCl2, the gluteus maximus [GM] muscle was studied for microvascular analysis or the tibialis anterior [TA] muscle was evaluated for recovery of neuromuscular function. Mice were studied (n=4-6/group) at 3 timepoints for vascular studies (0, 5, and 10 days post injury [dpi]) and 2 timepoints for neuromuscular structure and function (0 and 10 dpi). Results: Intravascular staining with fluorescent wheat germ agglutinin revealed a diminished microvascular area in the GM of CKO mice at 5 dpi vs. 10 dpi (means ± SE; CKO 5 dpi 35.5% ± 2.5 vs. CKO 10 dpi 49.8% ± 3.9, P<0.01). No difference in vascular area was recorded in WT groups at 5 vs. 10 dpi (WT 5 dpi 45.5% ± 2.6 vs. WT 10 dpi 46.7% ± 2.2, P=0.99). Direct electrical stimulation of the TA resolved a reduction in maximal force at 10 dpi when compared to 0 dpi for both WT and CKO (WT 0 dpi 128.6 g ± 4.1 vs. WT 10 dpi 64.7 g ± 3.0, P<0.0001) (CKO 0 dpi 112.7 g ± 5.0 vs. CKO 10 dpi 65.9 g ± 5.0, P<0.0001). Indirect stimulation (sciatic nerve) resolved initial neuromuscular transmission failure (INMTF) reduced submaximal force in CKO but not WT (CKO 0 dpi 2.0 ± 4.3% vs. CKO 10 dpi -30.0 ± 6.5 %, P<0.001), which correlated with perturbed recovery of NMJ innervation; (R2=0.328, P=0.003), endplate area; (R2=0.479, P=0.001), and nicotinic receptor clusters; (R2=0.207, P=0.025). Conclusion: Following acute injury of skeletal muscle in the adult mouse, loss of EfnB2 in ECs delays microvessel regeneration in the GM and disrupts the recovery of NMJ structure and function in the TA. Support: NIH grant F32 HL-152558 (NLJ), APS Postdoctoral Fellowship (ABM), NIH LRP (ABM), Margaret Proctor Mulligan Fellowship, MU (NLJ, SSS), NIH R01 AR078045 (DDC), School of Medicine Development Award, MU (SSS), NIH MERIT Award R37 HL-041026 (SSS). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Adult progenitor rejuvenation with embryonic factors

During ageing, adult stem cells' regenerative properties decline, as they undergo replicative senescence and lose both their proliferative and differentiation capacities. In contrast, embryonic and foetal progenitors typically possess heightened proliferative capacities and manifest a more robust regenerative response upon injury and transplantation, despite undergoing many rounds of mitosis. How embryonic and foetal progenitors delay senescence and maintain their proliferative and differentiation capacities after numerous rounds of mitosis, remains unknown. It is also unclear if defined embryonic factors can rejuvenate adult progenitors to confer extended proliferative and differentiation capacities, without reprogramming their lineage-specific fates or inducing oncogenic transformation. Here, we report that a minimal combination of LIN28A, TERT, and sh-p53 (LTS), all of which are tightly regulated and play important roles during embryonic development, can delay senescence in adult muscle progenitors. LTS muscle progenitors showed an extended proliferative capacity, maintained a normal karyotype, underwent myogenesis normally, and did not manifest tumorigenesis nor aberrations in lineage differentiation, even in late passages. LTS treatment promoted self-renewal and rescued the pro-senescence phenotype of aged cachexia patients' muscle progenitors, and promoted their engraftment for skeletal muscle regeneration in vivo. When we examined the mechanistic basis for LIN28A's role in the LTS minimum combo, let-7 microRNA suppression could not fully explain how LIN28A promoted muscle progenitor self-renewal. Instead, LIN28A was promoting the translation of oxidative phosphorylation mRNAs in adult muscle progenitors to optimize mitochondrial reactive oxygen species (mtROS) and mitohormetic signalling. Optimized mtROS induced a variety of mitohormetic stress responses, including the hypoxic response for metabolic damage, the unfolded protein response for protein damage, and the p53 response for DNA damage. Perturbation of mtROS levels specifically abrogated the LIN28A-driven hypoxic response in Hypoxia Inducible Factor-1α (HIF1α) and glycolysis, and thus LTS progenitor self-renewal, without affecting normal or TS progenitors. Our findings connect embryonically regulated factors to mitohormesis and progenitor rejuvenation, with implications for ageing-related muscle degeneration.

Abstract 134: A Single-cell Atlas Of Human Skeletal Muscle Tissue In Chronic Limb-threatening Ischemia

Introduction: Chronic limb-threatening ischemia (CLTI) represents end-stage peripheral arterial disease (PAD) and is a significant cause of limb amputation and mortality. Evidence supports the importance of skeletal muscle responses in the pathobiology of CLTI. Skeletal muscle regeneration depends on the coordinated activity of muscle stem cells (MuSCs) and an array of non-myogenic cells, including immune cells and fibroadipogenic progenitors (FAPs). The mechanisms underlying failed muscle regeneration in the CLTI limb are unknown. Single-cell RNA sequencing (scRNA-seq) facilitates the deconvolution of multicellular tissues in homeostatic and pathologic conditions. Herein, we profiled the transcriptomes of 135,203 cells from lower extremity skeletal muscle of PAD patients to construct a cellular atlas of CLTI. Methods: We recruited 12 PAD patients undergoing major limb amputation for CLTI. We obtained proximal (non-ischemic) and distal (ischemic) muscle samples from each patient and used scRNA-seq to profile the transcriptomes of 1,700 – 20,000 cells per sample. Results: We observed 10 distinct cellular populations in the CLTI limb. The predominant cell types were MuSCs, FAPs, and immune cells. There was significant transcriptional heterogeneity in each population; we defined a range of MuSC, FAP, and macrophage sub-populations and identified specific FAPs and macrophages associated with tissue ischemia. Using CellChat analysis, we found differential cell-cell signaling probabilities in ischemic vs. non-ischemic muscle. In ischemic muscle, FAPs are the primary source of outgoing signals, while macrophages are the primary receiver. Conclusions: Our single-cell atlas highlights the transcriptional heterogeneity of cellular responses in the CLTI limb. This atlas will serve as a resource for future studies of the molecular underpinnings of CLTI, advance our understanding of its pathogenesis, and support the development of novel therapies.

IL-4 Signaling Promotes Myoblast Differentiation and Fusion by Enhancing the Expression of MyoD, Myogenin, and Myomerger.

Myoblast fusion is essential for skeletal muscle development, growth, and regeneration. However, the molecular mechanisms underlying myoblast fusion and differentiation are not fully understood. Previously, we reported that interleukin-4 (IL-4) promotes myoblast fusion; therefore, we hypothesized that IL-4 signaling might regulate the expression of the molecules involved in myoblast fusion. In this study, we showed that in addition to fusion, IL-4 promoted the differentiation of C2C12 myoblast cells by inducing myoblast determination protein 1 (MyoD) and myogenin, both of which regulate the expression of myomerger and myomaker, the membrane proteins essential for myoblast fusion. Unexpectedly, IL-4 treatment increased the expression of myomerger, but not myomaker, in C2C12 cells. Knockdown of IL-4 receptor alpha (IL-4Rα) in C2C12 cells by small interfering RNA impaired myoblast fusion and differentiation. We also demonstrated a reduction in the expression of MyoD, myogenin, and myomerger by knockdown of IL-4Rα in C2C12 cells, while the expression level of myomaker remained unchanged. Finally, cell mixing assays and the restoration of myomerger expression partially rescued the impaired fusion in the IL-4Rα-knockdown C2C12 cells. Collectively, these results suggest that the IL-4/IL-4Rα axis promotes myoblast fusion and differentiation via the induction of myogenic regulatory factors, MyoD and myogenin, and myomerger.

Clinical-pathological features and muscle imaging findings in 36 Chinese patients with rimmed vacuolar myopathies: case series study and review of literature.

Rimmed vacuolar myopathies (RVMs) are a group of genetically heterogeneous diseases that share histopathological characteristics on muscle biopsy, including the aberrant accumulation of autophagic vacuoles. However, the presence of non-coding sequences and structural mutations, some of which remain undetectable, confound the identification of pathogenic mutations responsible for RVMs. Therefore, we assessed the clinical profiles and muscle magnetic resonance imaging (MRI) changes in 36 Chinese patients with RVMs, emphasizing the role of muscle MRI in disease identification and differential diagnosis to propose a comprehensive literature-based imaging pattern to facilitate improved diagnostic workup. All patients presented with rimmed vacuoles with varying degrees of muscular dystrophic changes and underwent a comprehensive evaluation using clinical, morphological, muscle MRI and molecular genetic analysis. We assessed muscle changes in the Chinese RVMs and provided an overview of the RVMs, focusing on the patterns of muscle involvement on MRI. A total of 36 patients, including 24 with confirmed distal myopathy and 12 with limb-girdle phenotype, had autophagic vacuoles with RVMs. Hierarchical clustering of patients according to the predominant effect of the distal or proximal lower limbs revealed that most patients with RVMs could be distinguished. GNE myopathy was the most prevalent form of RVMs observed in this study. Moreover, MRI helped identify the causative genes in some diseases (e.g., desminopathy and hereditary myopathy with early respiratory failure) and confirmed the pathogenicity of a novel mutation (e.g., adult-onset proximal rimmed vacuolar titinopathy) detected using next-generation sequencing. Collectively, our findings expand our knowledge of the genetic spectrum of RVMs in China and suggest that muscle imaging should be an integral part of assisting genetic testing and avoiding misdiagnosis in the diagnostic workup of RVM.

Clinical and Electrophysiological Presentation of a Patient with Multisystem Proteinopathy Associated with Valosin-Containing Protein Mutation (P5-8.005)

<h3>Objective:</h3> Present a rare case of multisystem proteinopathy (MSP) of apparently unrelated multisystem conditions caused by mutation in valosin-containing protein (VCP). <h3>Background:</h3> Multisystem proteinopathy (MSP) is a dominantly inherited disorder causing bone, muscle, central and peripheral nervous systems disease. Mutations within the gene encoding valosin-containing protein (VCP) cause MSP. Such mutations present with a vast phenotypical variability and are associated with inclusion body myopathy, Paget’s disease of bone, frontotemporal dementia, and amyotrophic lateral sclerosis. We present a case of MSP caused by c.464G&gt;A, p. arg155his mutation in VCP. <h3>Design/Methods:</h3> N/A <h3>Results:</h3> A 65-year-old woman presented with proximal muscle weakness and unsteady gait. The symptoms started four years ago when she experienced progressive weakness of the upper and lower extremities leading to recurrent falls. She has no family history of similar symptoms or genetic disease. Examination revealed dropped head, scapular winging, inability to raise the arms above her shoulders, hand weakness, and bilateral foot drop. Her gait was unsteady with a mix of waddling and steppage. Creatine kinase and alkaline phosphatase levels were normal. MRI of spine revealed mild degenerative disc disease from C4–C7 and disc bulges at L3–L4 through L5–S1. The nerve conduction studies showed mixed myopathic and neurogenic changes with ongoing denervation in cervical and lumbar segments suggestive of motor neuronopathy. Genetic testing was remarkable for a heterozygous pathogenic mutation in VCP (c.464G&gt;A, p. arg155his). <h3>Conclusions:</h3> This case highlights the need to consider MSP in patients with a complex neurological presentation and combined myopathic and neurogenic electrodiagnostic findings. The absence of MSP-related symptoms in the patient’s family may indicate the variable severity associated with the same VCP mutation, interaction with environmental factors as well as presence of genetic modifiers.. <b>Disclosure:</b> Mr. Halber has nothing to disclose. Sabrina Bulancea has nothing to disclose. Dr. Treidler has nothing to disclose.

Multisystem Proteinopathies (MSP) and MSP-like Disorders: Clinical-Pathological-Molecular Spectrum and Long-term Follow Up (P5-8.004)

<h3>Objective:</h3> To define phenotypic and genotypic spectrum of multisystem proteinopathies (MSP) and MSP-like disorders. <h3>Background:</h3> MSPs are genetically heterogenous disorders featured by neurodegeneration including amyotrophic lateral sclerosis (ALS)/frontotemporal dementia (FTD), inclusion body myopathy (IBM), and Paget disease of bone (PDB), sharing similar pathological findings of protein aggregation. Additional genes encoding RNA-binding proteins or proteins involved in quality-control pathways also lead to myopathy or neurodegeneration, although not necessarily within the same family, causing similar pathological abnormalities (MSP-like disorders). <h3>Design/Methods:</h3> We searched Mayo Clinic database (1/1/2010–6/30/2022) to identify patients with mutations in genes causative of MSP and MSP-like disorders. Clinical and laboratory data were reviewed. <h3>Results:</h3> Thirty-one individuals (11 female) from 27 unrelated families were identified. Pathogenic mutations were in: <i>VCP</i> (17 patients/14 families), <i>SQSTM1+TIA1</i> (5 patients/5 families), <i>TIA1</i> (5 patients/4 families), <i>MATR3, HNRNPA1</i>, <i>HSPB8</i>, and <i>TFG</i> (1 patient each). Myopathy occurred in all but 2 <i>VCP</i>-MSP patients and was the initial presentation at median age of 52. Patterns of weakness were limb-girdle in 12/15 <i>VCP</i>-MSP and distal-predominant in the remaining patients. Twenty/24 muscle biopsies showed myopathy with rimmed vacuoles. MND and FTD occurred in 5 (4 <i>VCP</i>, 1 <i>TFG</i>) and 4 (3 <i>VCP</i>, <i>1 SQSTM1+TIA1</i>) patients, respectively. PDB was present in 4 <i>VCP</i>-MSP. Twenty-one patients had last echocardiogram 8 years (median; range 3–22 years) after disease onset showing diastolic dysfunction in 2 <i>VCP</i>-MSP patients. Six patients demonstrated restrictive ventilatory impairment 15 years after disease-onset. At median follow-up of 11.5 years, 15 patients were ambulating without gait-aids; loss of ambulation (n=5) and death (n=3) were recorded only in <i>VCP</i>-MSP patients. <h3>Conclusions:</h3> Among MSP and MSP-related disorders, <i>VCP</i> was the most common causative gene, followed by <i>SQSTM1+TIA1</i> and <i>TIA1</i>; myopathy with rimmed vacuoles was the most frequent manifestation; distal-predominant weakness occurred frequently in non-<i>VCP</i>MSP; loss of ambulation was observed only in <i>VCP</i>-MSP at 11.5-year follow-up. <b>Disclosure:</b> Dr. Chompoopong has nothing to disclose. Dr. Oskarsson has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Amylyx. The institution of Dr. Oskarsson has received personal compensation in the range of $5,000-$9,999 for serving as a Consultant for Biogen. Dr. Oskarsson has received personal compensation in the range of $500-$4,999 for serving as a Consultant for AnnJi. The institution of Dr. Oskarsson has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Mitsubishi. Dr. Oskarsson has received personal compensation in the range of $5,000-$9,999 for serving as a Consultant for Tsumura. The institution of Dr. Oskarsson has received personal compensation in the range of $500-$4,999 for serving as a Consultant for MediciNova. The institution of Dr. Oskarsson has received research support from Biogen. The institution of Dr. Oskarsson has received research support from Medicinova. The institution of Dr. Oskarsson has received research support from Cytokinetics. The institution of Dr. Oskarsson has received research support from Calico. The institution of Dr. Oskarsson has received research support from Mitsubishi. The institution of Dr. Oskarsson has received research support from Tsumura. The institution of Dr. Oskarsson has received research support from Sanofi. The institution of Dr. Oskarsson has received research support from AZTherapeutics. The institution of Dr. Oskarsson has received research support from Orion. The institution of Dr. Oskarsson has received research support from Esaii. Dr. Madigan has nothing to disclose. Dr. Mirman has nothing to disclose. Dr. Martinez-Thompson has nothing to disclose. Dr. Liewluck has nothing to disclose. Dr. Milone has received personal compensation in the range of $500-$4,999 for serving on a Scientific Advisory or Data Safety Monitoring board for Argenx. Dr. Milone has received personal compensation in the range of $500-$4,999 for serving as an Editor, Associate Editor, or Editorial Advisory Board Member for Neurology Genetics, AAN. The institution of Dr. Milone has received research support from Mayo Clinic, CCaTS-CBD. The institution of Dr. Milone has received research support from Mayo Clinic, SGP Award. The institution of Dr. Milone has received research support from MDA for Care Center grant. The institution of Dr. Milone has received research support from Regenerative medicine Minnesota.

Arntl deficiency in myeloid cells reduces neutrophil recruitment and delays skeletal muscle repair

After a muscle injury, a process comprising inflammation, repair, and regeneration must occur in a time-sensitive manner for skeletal muscle to be adequately repaired and regenerated. This complex process is assumed to be controlled by various myeloid cell types, including monocytes and macrophages, though the mechanism is not fully understood. Aryl hydrocarbon receptor nuclear translocator-like (Arntl or Bmal1) is a transcription factor that controls the circadian rhythm and has been implicated in regulating myeloid cell functions. In the present study, we generated myeloid cell-specific Arntl conditional knockout (cKO) mice to assess the role of Arntl expressed in myeloid cell populations during the repair process after muscle injury. Myeloid cell-specific Arntl deletion impaired muscle regeneration after cardiotoxin injection. Flow cytometric analyses revealed that, in cKO mice, the numbers of infiltrating neutrophils and Ly6Chi monocytes within the injured site were reduced on days 1 and 2, respectively, after muscle injury. Moreover, neutrophil migration and the numbers of circulating monocytes were significantly reduced in cKO mice, which suggests these effects may account, at least in part, for the impaired regeneration. These findings suggest that Arntl, expressed in the myeloid lineage regulates neutrophil and monocyte recruitment and is therefore required for skeletal muscle regeneration.