Biochemical Isolation of Myonuclei from Mouse Skeletal Muscle Tissue

Cutler, Alecia; Corbett, Anita H.; Pavlath, Grace K.

doi:10.21769/bioprotoc.2654

Cited by 10 publications

(12 citation statements)

References 8 publications

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“…Soleus muscles were excised immediately following euthanasia and the protocol developed by Cutler et al was followed for isolation of myonuclei. 48 Briefly, muscles were minced with scissors in homogenization buffer (500 µL HEPES [1 M] 3 mL KCl [1 M] 250 µL spermidine [100 mM] 750 µL spermine tetrahydrochloride [10 mM] 10 mL EDTA [10 mM] 250 µL EGTA [100 mM] 2.5 mL MgCl [100 mM] 5.13 g sucrose), dounced on ice in homogenization buffer and passed through a 40 µm filter into sorting buffer. DAPI was added to label nuclei and fluorescently-labeled nuclei were purified via fluorescence-activated cell sorting (FACS) and collected in reverse transcription (RT) buffer.…”

Section: Methodsmentioning

confidence: 99%

Satellite Cell Depletion Disrupts Transcriptional Coordination and Muscle Adaptation to Exercise

et al. 2020

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Satellite cells are required for post-natal development, skeletal muscle regeneration across the lifespan and skeletal muscle hypertrophy prior to maturity. Our group has aimed to address whether satellite cells are required for hypertrophic growth in mature skeletal muscle. Here, we generated a comprehensive characterization and transcriptome-wide profiling of skeletal muscle during adaptation to exercise in the presence or absence of satellite cells in order to identify distinct phenotypes and gene networks influenced by satellite cell content. We administered vehicle or tamoxifen to adult Pax7-DTA mice and subjected them to progressive weighted wheel running (PoWeR). We then performed immunohistochemical analysis and whole-muscle RNA-seq of vehicle (SC+) and tamoxifen-treated (SC-) mice. Further, we performed single myonuclear RNA-seq to provide detailed information on how satellite cell fusion affects myonuclear transcription. We show that while skeletal muscle can mount a robust hypertrophic response to PoWeR in the absence of satellite cells, growth and adaptation are ultimately blunted. Transcriptional profiling reveals several gene networks key to muscle adaptation are altered in the absence of satellite cells.

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Section: Methodsmentioning

confidence: 99%

Satellite Cell Depletion Disrupts Transcriptional Coordination and Muscle Adaptation to Exercise

et al. 2020

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“…TDP-43 are low in mature muscle but are increased in isolated primary myoblasts (42), which can be differentiated into myotubes in vitro (43). Thus, we examined TDP-43 in both primary myoblasts and myotubes prepared from control WT Ala-10/ Ala-10 PABPN1 mice and the OPMD model Ala-17/Ala-10 PABPN1 mice.…”

Section: Tdp-43 Is Present In High-molecular-weight Complexes That Comentioning

confidence: 99%

Proteomic analysis reveals that wildtype and alanine-expanded nuclear poly(A)-binding protein exhibit differential interactions in skeletal muscle

Banerjee

Phillips

Deng

et al. 2019

Journal of Biological Chemistry

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Oculopharyngeal muscular dystrophy (OPMD) is a late-onset, primarily autosomal dominant disease caused by a short GCN expansion in the PABPN1 (polyadenylate-binding protein nuclear 1) gene that results in an alanine expansion at the N terminus of the PABPN1 protein. Expression of alanine-expanded PABPN1 is linked to the formation of nuclear aggregates in tissues from individuals with OPMD. However, as with other nuclear aggregate-associated diseases, controversy exists over whether these aggregates are the direct cause of pathology. An emerging hypothesis is that a loss of PABPN1 function and/or aberrant protein interactions contribute to pathology in OPMD. Here, we present the first global proteomic analysis of the protein interactions of WT and alanine-expanded PABPN1 in skeletal muscle tissue. These data provide both insight into the function of PABPN1 in muscle and evidence that the alanine expansion alters the protein-protein interactions of PABPN1. We extended this analysis to demonstrate altered complex formation with and loss of function of TDP-43 (TAR DNA-binding protein 43), which we show interacts with alanine-expanded but not WT PABPN1. The results from our study support a model where altered protein interactions with alanine-expanded PABPN1 that lead to loss or gain of function could contribute to pathology in OPMD.

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“…MuSCs and myonuclei are discriminated by Tmem38a expression, a gene uniquely expressed by myonuclei (Fig. 1G) (27). Among myonuclei, distinct subsets were observed expressing NMJ-associated transcripts and MTJassociated transcripts, as well as Myh1, Myh2, and Myh4 isoforms, representative of type-IIx, type IIa, or type IIb myofibers, respectively (Fig.…”

Section: Resultsmentioning

confidence: 94%

“…1B-C). TA muscle nuclei were enriched for myonuclei to increase coverage of low-abundant myogenic populations present during regeneration (27). To assess the coverage of the nuclear isolation and snRNA-seq experiments, the nuclear transcriptomes from each experiment were computationally aggregated to assess the proportions of nuclei derived from mononuclear cells and multinucleated myofibers (8,28,29).…”

Section: Resultsmentioning

confidence: 99%

“…Nuclei comprising the myogenic cluster express either Pax7 identifying quiescent MuSCs, Myod1 or Myog identifying myogenic progenitors, or Ckm or Mylk2 identifying myonuclei (31)(32)(33)(34)(35). The majority of nuclei in the myogenic cluster are myonuclei from myofibers as they express Tmem38a, Ttn, Ckm, and Neb (27,33,36) (Fig. 1G; SI Appendix, Fig.…”

Section: Resultsmentioning

confidence: 99%

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Myonuclear maturation dynamics in aged and adult regenerating mouse skeletal muscle

Pawlikowski

et al. 2021

Preprint

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Skeletal muscle cells are multinucleated syncytial cells arising from cell fusion, yet despite sharing a common cytoplasm individual myonuclei express distinct transcriptional programs. Whether individual myonuclei acquire heterogenous transcriptional states via differences in their progenitors, during differentiation, or once their anatomical position is acquired, is not known. We performed transcriptome and pseudotime analysis of single myogenic nuclei from uninjured and post-injury murine skeletal muscle to assess when myonuclear heterogeneity is acquired. Two distinct progenitors contribute to myonuclei, one a non-myogenic fibroblast subtype, and skeletal muscle stem cells the other. Both progenitors enter a single pseudotime trajectory that bifurcates as myonuclei mature into two branches segregated by myosin isoform expression and metabolic profiles, suggesting transcriptional heterogeneity is acquired as myonuclei mature. In aged skeletal muscle myogenic progenitor expansion is perturbed and nuclei from aged muscle display distinct pseudotemporal kinetics compared to nuclei from young mice. In aged mice, the inferred myogenic differentiation trajectory is delayed, altering the distribution of myogenic nuclei in pseudotime, suggesting that altered transcriptional dynamics in nuclei in aged mice may drive age-associated muscle deficits and bias myonuclei towards acquiring oxidative metabolic profiles.

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Biochemical Isolation of Myonuclei from Mouse Skeletal Muscle Tissue

Cited by 10 publications

References 8 publications

Satellite Cell Depletion Disrupts Transcriptional Coordination and Muscle Adaptation to Exercise

Satellite Cell Depletion Disrupts Transcriptional Coordination and Muscle Adaptation to Exercise

Proteomic analysis reveals that wildtype and alanine-expanded nuclear poly(A)-binding protein exhibit differential interactions in skeletal muscle

Myonuclear maturation dynamics in aged and adult regenerating mouse skeletal muscle

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