Single-nucleus RNA-seq and FISH identify coordinated transcriptional activity in mammalian myofibers

Santos, Matthieu Dos; Backer, Stéphanie; Saintpierre, Benjamin; Izac, Brigitte; Andrieu, Muriel; Letourneur, Franck; Relaix, Frédéric; Sotiropoulos, Athanassia; Maire, Pascal

doi:10.1038/s41467-020-18789-8

Cited by 169 publications

(265 citation statements)

References 85 publications

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“…This group of enhancers are characterized by spanning several kb and being important for defining the identity of the cell(78)(79)(80). As one of the most enriched area in EDL, it suggests that the super-enhancer also plays a role in the adult glycolytic fiber in vivo and might stabilize fiber types and be an obstacle to transformations.For the regulatory network several of the transcription factors are well known regulators of skeletal muscle phenotype and function, such as NFAT, SOX, SIX and MYOD1 (4, 60, 81).Interestingly our enriched factors are largely in agreement with a recent study, using an alternative approach to investigate the epigenetic regulatory environment in slow and fast muscles at the single nuclei level(82).…”

supporting

confidence: 88%

“…In addition, we found differences in distal enhancers at the loci reflecting different regulatory Other factors we identified, such as MAF BZIP Transcription Factors (MAF) and ZBTB14, is just recently described to have a role in muscle regulation at the gene level (82,83). The latter is identified to be involved in regulation of metabolism, a function also assigned to the factor in our regulatory network.…”

Section: Discussionmentioning

confidence: 61%

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The epigenetic landscape in purified myonuclei from fast and slow muscles

Bengtsen

Eftestøl

et al. 2021

Preprint

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Muscle cells have different phenotypes adapted to different usage and can be grossly divided into fast/glycolytic and slow/oxidative types. While most muscles contain a mixture of such fiber types, we aimed at providing a genome-wide analysis of chromatin environment by ChIP-Seq in two muscle extremes, the almost completely fast/glycolytic extensor digitorum longus (EDL) and slow/oxidative soleus muscles. Muscle is a heterogeneous tissue where less than 60% of the nuclei are inside muscle fibers. Since cellular homogeneity is critical in epigenome-wide association studies we devised a new method for purifying skeletal muscle nuclei from whole tissue based on the nuclear envelope protein Pericentriolar material 1 (PCM1) being a specific marker for myonuclei. Using antibody labeling and a magnetic-assisted sorting approach we were able to sort out myonuclei with 95% purity. The sorting eliminated influence from other cell types in the tissue and improved the myo-specific signal. A genome-wide comparison of the epigenetic landscape in EDL and soleus reflected the functional properties of the two muscles each with a distinct regulatory program involving distal enhancers, including a glycolytic super-enhancer in the EDL. The two muscles are also regulated by different sets of transcription factors; e.g. in soleus binding sites for MEF2C, NFATC2 and PPARA were enriched, while in EDL MYOD1 and SOX1 binding sites were found to be overrepresented. In addition, novel factors for muscle regulation such as MAF, ZFX and ZBTB14 were identified.

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supporting

confidence: 88%

Section: Discussionmentioning

confidence: 61%

The epigenetic landscape in purified myonuclei from fast and slow muscles

Bengtsen

Eftestøl

et al. 2021

Preprint

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“…For immunostaining against Myh4, Myh2, Myh7 and Laminin, mouse hindlimbs (no skin) without fixation were embedded with Tissue-TEK OCT (Sakura Finetek, Netherlands) and directly frozen in cold isopentane pre-cooled in liquid nitrogen as described [40]. Hindlimb cross sections were prepares using a cryostat (Leica 3050s) with a thickness of 10 μm.…”

Section: Materials and Mthodsmentioning

confidence: 99%

Compound- and fiber type-selective requirement of AMPKγ3 for insulin-independent glucose uptake in skeletal muscle

Rhein

Desjardins

Rong

et al. 2021

Preprint

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Objective: The metabolic master-switch AMP-activated protein kinase (AMPK) mediates insulin-independent glucose uptake in muscle and regulates the metabolic activity of brown and beige adipose tissue (BAT). The regulatory AMPKγ3 isoform is uniquely expressed in skeletal muscle and also potentially in BAT. Here, we investigated the role that AMPKγ3 plays in mediating skeletal muscle glucose uptake and whole-body glucose clearance in response to small-molecule activators that act on AMPK via distinct mechanisms. We also assessed if γ3 plays a role in adipose thermogenesis and browning. Methods: Global AMPKγ3 knockout (KO) mice were generated. A systematic whole-body, tissue and molecular phenotyping linked to glucose homeostasis was performed in γ3 KO and wild type (WT) mice. Glucose uptake in glycolytic and oxidative skeletal muscle ex vivo, as well as blood glucose clearance in response to small molecule AMPK activators that target nucleotide-binding domain of γ subunit (AICAR) and allosteric drug and metabolite (ADaM) site located at the interface of the α and β subunit (991, MK-8722) were assessed. Oxygen consumption, thermography, and molecular phenotyping with a β3-adrenergic receptor agonist (CL-316,243) treatment were performed to assess BAT thermogenesis, characteristics and function. Results: Genetic ablation of γ3 did not affect body weight, body composition, physical activity, and parameters associated with glucose homeostasis under chow or high fat diet. γ3 deficiency had no effect on fiber-type composition, mitochondrial content and integrity, or insulin-stimulated glucose uptake in skeletal muscle. Glycolytic muscles in γ3 KO mice showed a partial loss of AMPKα2 activity, which was associated with reduced levels of AMPKα2 and β2 subunit isoforms. Notably, γ3 deficiency resulted in a selective loss of AICAR-, but not MK-8722-induced blood glucose lowering in vivo and glucose uptake specifically in glycolytic muscles ex vivo. We detected γ3 in BAT and found that it preferentially interacts with α2 and β2. We observed no differences in oxygen consumption, thermogenesis, morphology of BAT and inguinal white adipose tissue (iWAT), or markers of BAT activity between WT and γ3 KO mice. Conclusions: These results demonstrate that γ3 plays a key role in mediating AICAR- but not ADaM site binding drug-stimulated blood glucose clearance and glucose uptake specifically in glycolytic skeletal muscle. We also showed that γ3 is dispensable for thermogenesis and browning of iWAT.

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“…The size of the aggregated dataset enabled identification of subtle cell type differences. For example, we were able to distinguish the blood vessel type for endothelial cells 28 , the fiber type for mature myonuclei [9][10][11] , and the differentiation potential for fibro-adipogenic progenitors (FAPs 36 ) ( Figs. 1D and S4).…”

Section: Large-scale Integration Enables a High-resolution View Of Skmentioning

confidence: 99%

Strength in numbers: Large-scale integration of single-cell transcriptomic data reveals rare, transient muscle progenitor cell states in muscle regeneration

McKellar

Walter

Song

et al. 2020

Preprint

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Skeletal muscle repair is driven by the coordinated self-renewal and fusion of myogenic stem and progenitor cells. Single-cell gene expression analyses of myogenesis have been hampered by the poor sampling of rare and transient cell states that are critical for muscle repair, and do not provide spatial information that is needed to understand the context in which myogenic differentiation occurs. Here, we demonstrate how large-scale integration of new and public single-cell and spatial transcriptomic data can overcome these limitations. We created a large-scale single-cell transcriptomic dataset of mouse skeletal muscle by integration, consensus annotation, and analysis of 23 newly collected scRNAseq datasets and 79 public single-cell (scRNAseq) and single-nucleus (snRNAseq) RNA-sequencing datasets. The resulting compendium includes nearly 350,000 cells and spans a wide range of ages, injury, and repair conditions. Combined, these data enabled identification of the predominant cell types in skeletal muscle with robust, consensus gene expression profiles, and resolved cell subtypes, including endothelial subtypes distinguished by vessel-type of origin, fibro/adipogenic progenitors marked by stem potential, and many distinct immune populations. The representation of different experimental conditions and the depth of transcriptome coverage enabled robust profiling of sparsely expressed genes. We built a densely sampled transcriptomic model of myogenesis, from stem-cell quiescence to myofiber maturation and identified rare, short-lived transitional states of progenitor commitment and fusion that are poorly represented in individual datasets. We performed spatial RNA sequencing of mouse muscle at three time points after injury and used the integrated dataset as a reference to achieve a high-resolution, local deconvolution of cell subtypes. This analysis identified the temporal variation in cell subtype colocalized interactions during injury recovery. We will release an interactive public web tool to enable exploration and visualization of this rich single-cell transcriptomic resource. Our work supports the utility of large-scale integration of single-cell transcriptomic data as a tool for biological discovery.

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Single-nucleus RNA-seq and FISH identify coordinated transcriptional activity in mammalian myofibers

Cited by 169 publications

References 85 publications

The epigenetic landscape in purified myonuclei from fast and slow muscles

The epigenetic landscape in purified myonuclei from fast and slow muscles

Compound- and fiber type-selective requirement of AMPKγ3 for insulin-independent glucose uptake in skeletal muscle

Strength in numbers: Large-scale integration of single-cell transcriptomic data reveals rare, transient muscle progenitor cell states in muscle regeneration

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