scholarly journals Single cell RNA-seq analysis of the flexor digitorum brevis mouse myofibers

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Rohan X. Verma ◽  
Suraj Kannan ◽  
Brian L. Lin ◽  
Katherine M. Fomchenko ◽  
Tim O. Nieuwenhuis ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Single Cell ◽  
Fiber Type ◽  
Expression Patterns ◽  
Transcript Abundance ◽  
Rna Seq ◽  
Whole Cell ◽  
Gene And Protein Expression ◽  
Flexor Digitorum Brevis ◽  
Flexor Digitorum

Abstract Background Skeletal muscle myofibers can be separated into functionally distinct cell types that differ in gene and protein expression. Current single cell expression data is generally based upon single nucleus RNA, rather than whole myofiber material. We examined if a whole-cell flow sorting approach could be applied to perform single cell RNA-seq (scRNA-seq) in a single muscle type. Methods We performed deep, whole cell, scRNA-seq on intact and fragmented skeletal myofibers from the mouse fast-twitch flexor digitorum brevis muscle utilizing a flow-gated method of large cell isolation. We performed deep sequencing of 763 intact and fragmented myofibers. Results Quality control metrics across the different gates indicated only 171 of these cells were optimal, with a median read count of 239,252 and an average of 12,098 transcripts per cell. scRNA-seq identified three clusters of myofibers (a slow/fast 2A cluster and two fast 2X clusters). Comparison to a public skeletal nuclear RNA-seq dataset demonstrated a diversity in transcript abundance by method. RISH validated multiple genes across fast and slow twitch skeletal muscle types. Conclusion This study introduces and validates a method to isolate intact skeletal muscle myofibers to generate deep expression patterns and expands the known repertoire of fiber-type-specific genes.

Disruption of microtubules in rat skeletal muscle does not inhibit insulin- or contraction-stimulated glucose transport

2003 ◽  
Vol 285 (4) ◽  
pp. E836-E844 ◽  
Author(s):  
Hua Ai ◽  
Evelyn Ralston ◽  
Hans P. M. M. Lauritzen ◽  
Henrik Galbo ◽  
Thorkil Ploug
Keyword(s):  
Skeletal Muscle ◽  
Glucose Transport ◽  
Immunofluorescence Microscopy ◽  
Fiber Type ◽  
Force Production ◽  
Rat Skeletal Muscle ◽  
Microtubule Network ◽  
Type Composition ◽  
Flexor Digitorum Brevis ◽  
Flexor Digitorum

Insulin and muscle contractions stimulate glucose transport in skeletal muscle through a translocation of intracellular GLUT4 glucose transporters to the cell surface. Judged by immunofluorescence microscopy, part of the GLUT4 storage sites is associated with the extensive microtubule cytoskeleton found in all muscle fibers. Here, we test whether microtubules are required mediators of the effect of insulin and contractions. In three different incubated rat muscles with distinct fiber type composition, depolymerization of microtubules with colchicine for ≤8 h did not inhibit insulin- or contraction-stimulated 2-deoxyglucose transport or force production. On the contrary, colchicine at least partially prevented the ∼30% decrease in insulin-stimulated transport that specifically developed during 8 h of incubation in soleus muscle but not in flexor digitorum brevis or epitrochlearis muscles. In contrast, nocodazole, another microtubule-disrupting drug, rapidly and dose dependently blocked insulin- and contraction-stimulated glucose transport. A similar discrepancy between colchicine and nocodazole was also found in their ability to block glucose transport in muscle giant “ghost” vesicles. This suggests that the ability of insulin and contractions to stimulate glucose transport in muscle does not require an intact microtubule network and that nocodazole inhibits glucose transport independently of its microtubule-disrupting effect.

scholarly journals Proteogenomic single cell analysis of skeletal muscle myocytes

2020 ◽  
Author(s):  
Katherine M. Fomchenko ◽  
Rohan X. Verma ◽  
Suraj Kannan ◽  
Brian L. Lin ◽  
Xiaoping Yang ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Protein Expression ◽  
Single Cell ◽  
Single Cell Analysis ◽  
Fiber Types ◽  
Genomic Expression ◽  
Variable Expression ◽  
Gene And Protein Expression ◽  
Human Protein Atlas ◽  
Flexor Digitorum Brevis

AbstractSkeletal muscle myocytes have evolved into slow and fast-twitch types. These types are functionally distinct as a result of differential gene and protein expression. However, an understanding of the complexity of gene and protein variation between myofibers is unknown. We performed deep, whole cell, single cell RNA-seq on intact and fragments of skeletal myocytes from the mouse flexor digitorum brevis muscle. We compared the genomic expression data of 171 of these cells with two human proteomic datasets. The first was a spatial proteomics survey of mosaic patterns of protein expression utilizing the Human Protein Atlas (HPA) and the HPASubC tool. The second was a mass-spectrometry (MS) derived proteomic dataset of single human muscle fibers. Immunohistochemistry and RNA-ISH were used to understand variable expression. scRNA-seq identified three distinct clusters of myocytes (a slow/fast 2A cluster and two fast 2X clusters). Utilizing 1,605 mosaic patterned proteins from visual proteomics, and 596 differentially expressed proteins by MS methods, we explore this fast 2X division. Only 36 genes/proteins had variable expression across all three studies, of which nine are newly described as variable between fast/slow twitch myofibers. An additional 414 genes/proteins were identified as variable by two methods. Immunohistochemistry and RNA-ISH generally validated variable expression across methods presumably due to species-related differences. In this first integrated proteogenomic analysis of mature skeletal muscle myocytes we confirm the main fiber types and greatly expand the known repertoire of twitch-type specific genes/proteins. We also demonstrate the importance of integrating genomic and proteomic datasets.

scholarly journals Clustering Single-Cell RNA-Seq Data with Regularized Gaussian Graphical Model

Genes ◽  
2021 ◽  
Vol 12 (2) ◽  
pp. 311
Author(s):  
Zhenqiu Liu
Keyword(s):  
Single Cell ◽  
Free Parameter ◽  
Graphical Model ◽  
Expression Patterns ◽  
Information Criterion ◽  
Log P ◽  
Rna Seq ◽  
Clustering Methods ◽  
Wide Range ◽  
Free Parameters

Single-cell RNA-seq (scRNA-seq) is a powerful tool to measure the expression patterns of individual cells and discover heterogeneity and functional diversity among cell populations. Due to variability, it is challenging to analyze such data efficiently. Many clustering methods have been developed using at least one free parameter. Different choices for free parameters may lead to substantially different visualizations and clusters. Tuning free parameters is also time consuming. Thus there is need for a simple, robust, and efficient clustering method. In this paper, we propose a new regularized Gaussian graphical clustering (RGGC) method for scRNA-seq data. RGGC is based on high-order (partial) correlations and subspace learning, and is robust over a wide-range of a regularized parameter λ. Therefore, we can simply set λ=2 or λ=log(p) for AIC (Akaike information criterion) or BIC (Bayesian information criterion) without cross-validation. Cell subpopulations are discovered by the Louvain community detection algorithm that determines the number of clusters automatically. There is no free parameter to be tuned with RGGC. When evaluated with simulated and benchmark scRNA-seq data sets against widely used methods, RGGC is computationally efficient and one of the top performers. It can detect inter-sample cell heterogeneity, when applied to glioblastoma scRNA-seq data.

scholarly journals Consequences of SUR2[A478V] Mutation in Skeletal Muscle of Murine Model of Cantu Syndrome

Cells ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1791
Author(s):  
Rosa Scala ◽  
Fatima Maqoud ◽  
Nicola Zizzo ◽  
Giuseppe Passantino ◽  
Antonietta Mele ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Katp Channel ◽  
Ex Vivo ◽  
Channel Modulation ◽  
Flexor Digitorum Brevis ◽  
Inflammatory Edema ◽  
Flexor Digitorum ◽  
Channel Currents

(1) Background: Cantu syndrome (CS) arises from gain-of-function (GOF) mutations in the ABCC9 and KCNJ8 genes, which encode ATP-sensitive K+ (KATP) channel subunits SUR2 and Kir6.1, respectively. Most CS patients have mutations in SUR2, the major component of skeletal muscle KATP, but the consequences of SUR2 GOF in skeletal muscle are unknown. (2) Methods: We performed in vivo and ex vivo characterization of skeletal muscle in heterozygous SUR2[A478V] (SUR2wt/AV) and homozygous SUR2[A478V] (SUR2AV/AV) CS mice. (3) Results: In SUR2wt/AV and SUR2AV/AV mice, forelimb strength and diaphragm amplitude movement were reduced; muscle echodensity was enhanced. KATP channel currents recorded in Flexor digitorum brevis fibers showed reduced MgATP-sensitivity in SUR2wt/AV, dramatically so in SUR2AV/AV mice; IC50 for MgATP inhibition of KATP currents were 1.9 ± 0.5 × 10−5 M in SUR2wt/AV and 8.6 ± 0.4 × 10−6 M in WT mice and was not measurable in SUR2AV/AV. A slight rightward shift of sensitivity to inhibition by glibenclamide was detected in SUR2AV/AV mice. Histopathological and qPCR analysis revealed atrophy of soleus and tibialis anterior muscles and up-regulation of atrogin-1 and MuRF1 mRNA in CS mice. (4) Conclusions: SUR2[A478V] “knock-in” mutation in mice impairs KATP channel modulation by MgATP, markedly so in SUR2AV/AV, with atrophy and non-inflammatory edema in different skeletal muscle phenotypes.

scholarly journals Differential impact of mitochondrial positioning on mitochondrial Ca2+ uptake and Ca2+ spark suppression in skeletal muscle

2011 ◽  
Vol 301 (5) ◽  
pp. C1128-C1139 ◽  
Author(s):  
Ann E. Rossi ◽  
Simona Boncompagni ◽  
Lan Wei ◽  
Feliciano Protasi ◽  
Robert T. Dirksen
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Osmotic Shock ◽  
Progressive Increase ◽  
Tetanic Stimulation ◽  
Differential Impact ◽  
Flexor Digitorum Brevis ◽  
Flexor Digitorum ◽  
Activity Dependent ◽  
Old Mice

Muscle contraction requires ATP and Ca2+ and, thus, is under direct control of mitochondria and the sarcoplasmic reticulum. During postnatal skeletal muscle maturation, the mitochondrial network exhibits a shift from a longitudinal (“longitudinal mitochondria”) to a mostly transversal orientation as a result of a progressive increase in mitochondrial association with Ca2+ release units (CRUs) or triads (“triadic mitochondria”). To determine the physiological implications of this shift in mitochondrial disposition, we used confocal microscopy to monitor activity-dependent changes in myoplasmic (fluo 4) and mitochondrial (rhod 2) Ca2+ in single flexor digitorum brevis (FDB) fibers from 1- to 4-mo-old mice. A robust and sustained Ca2+ accumulation in triadic mitochondria was triggered by repetitive tetanic stimulation (500 ms, 100 Hz, every 2.5 s) in FDB fibers from 4-mo-old mice. Specifically, mitochondrial rhod 2 fluorescence increased 272 ± 39% after a single tetanus and 412 ± 45% after five tetani and decayed slowly over 10 min following the final tetanus. Similar results were observed in fibers expressing mitochondrial pericam, a mitochondrial-targeted ratiometric Ca2+ indicator. Interestingly, sustained mitochondrial Ca2+ uptake following repetitive tetanic stimulation was similar for triadic and longitudinal mitochondria in FDB fibers from 1-mo-old mice, and both mitochondrial populations were found by electron microscopy to be continuous and structurally tethered to the sarcoplasmic reticulum. Conversely, the frequency of osmotic shock-induced Ca2+ sparks per CRU density decreased threefold (from 3.6 ± 0.2 to 1.2 ± 0.1 events·CRU−1·min−1·100 μm−2) during postnatal development in direct linear correspondence ( r2 = 0.95) to an increase in mitochondrion-CRU pairing. Together, these results indicate that mitochondrion-CRU association promotes Ca2+ spark suppression but does not significantly impact mitochondrial Ca2+ uptake.

Effect of electrical stimulation on intracellular triacylglycerol in isolated skeletal muscle

1990 ◽  
Vol 68 (1) ◽  
pp. 348-354 ◽  
Author(s):  
J. F. Hopp ◽  
W. K. Palmer
Keyword(s):  
Skeletal Muscle ◽  
Electrical Stimulation ◽  
Glucose Control ◽  
Muscle Preparation ◽  
Bicarbonate Buffer ◽  
Intermittent Stimulation ◽  
Flexor Digitorum Brevis ◽  
Muscle Groups ◽  
Flexor Digitorum

The contribution of intracellular triacylglycerol (TG) as a substrate for skeletal muscle during electrical stimulation is equivocal. Therefore, the purpose of this study was to investigate the effect of electrical stimulation on the TG content in the isolated intact rat flexor digitorum brevis skeletal muscle preparation by use of two different stimulation protocols. Muscles were electrically stimulated for 1 h either continuously at 1 Hz or intermittently (30 s on, 60 s off) at 5 Hz while incubated in 21 degrees C Krebs bicarbonate buffer (pH 7.4) that contained 11 mM glucose. Control muscles were either frozen immediately after excision or incubated for 1 h. TG content was significantly decreased (P less than 0.05) compared with control concentrations in both stimulated muscle groups, with the greatest reduction (60%) occurring after 5-Hz intermittent stimulation. These data indicate that intramuscular TG is hydrolyzed in response to electrical stimulation in the isolated flexor digitorum brevis muscle preparation. In addition, the type of stimulation (higher frequency intermittent vs. lower frequency continuous) employed influences the amount of intracellular TG hydrolyzed.

scholarly journals Effects of myofiber isolation technique on sarcolemma biomechanics

BioTechniques ◽  
2020 ◽  
Vol 69 (5) ◽  
pp. 388-391
Author(s):  
Karla P Garcia-Pelagio ◽  
Stephen JP Pratt ◽  
Richard M Lovering
Keyword(s):  
Skeletal Muscle ◽  
Extensor Digitorum Longus ◽  
Sarcomere Length ◽  
Biomechanical Properties ◽  
Isolation Technique ◽  
Enzymatic Dissociation ◽  
Flexor Digitorum Brevis ◽  
Flexor Digitorum ◽  
Biomechanical Measurements

Isolated myofibers are commonly used to understand the function of skeletal muscle in vivo. This can involve single isolated myofibers obtained from dissection or from enzymatic dissociation. Isolation via dissection allows control of sarcomere length and preserves tendon attachment but is labor-intensive, time-consuming and yields few viable myofibers. In contrast, enzymatic dissociation is fast and facile, produces hundreds of myofibers, and more importantly reduces the number of muscles/animals needed for studies. Biomechanical properties of the sarcolemma have been studied using myofibers from the extensor digitorum longus, but this has been limited to dissected myofibers, making data collection slow and difficult. We have modified this tool to perform biomechanical measurements of the sarcolemma in dissociated myofibers from the flexor digitorum brevis.

scholarly journals Finding cell-specific expression patterns in the early Ciona embryo with single-cell RNA-seq

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Garth R. Ilsley ◽  
Ritsuko Suyama ◽  
Takeshi Noda ◽  
Nori Satoh ◽  
Nicholas M. Luscombe
Keyword(s):  
Single Cell ◽  
Expression Patterns ◽  
Rna Seq ◽  
Specific Expression
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