Proteogenomic single cell analysis of skeletal muscle myocytes

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.

Download Full-text

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

Skeletal Muscle ◽

10.1186/s13395-021-00269-2 ◽

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.

Download Full-text

Treadmill Running Changes Endothelial Lipase Expression: Insights from Gene and Protein Analysis in Various Striated Muscle Tissues and Serum

Biomolecules ◽

10.3390/biom11060906 ◽

2021 ◽

Vol 11 (6) ◽

pp. 906

Author(s):

Agnieszka Mikłosz ◽

Bartłomiej Łukaszuk ◽

Adrian Chabowski ◽

Jan Górski

Keyword(s):

Protein Expression ◽

Density Lipoprotein ◽

Treadmill Running ◽

Endothelial Lipase ◽

Type I ◽

Fiber Types ◽

Gene And Protein Expression ◽

Single Bout ◽

The Impact ◽

White Gastrocnemius

Endothelial lipase (EL) is an enzyme capable of HDL phospholipids hydrolysis. Its action leads to a reduction in the serum high-density lipoprotein concentration, and thus, it exerts a pro-atherogenic effect. This study examines the impact of a single bout exercise on the gene and protein expression of the EL in skeletal muscles composed of different fiber types (the soleus—mainly type I, the red gastrocnemius—mostly IIA, and the white gastrocnemius—predominantly IIX fibers), as well as the diaphragm, and the heart. Wistar rats were subjected to a treadmill run: 1) t = 30 [min], V = 18 [m/min]; 2) t = 30 [min], V = 28 [m/min]; 3) t = 120 [min], V = 18 [m/min] (designated: M30, F30, and M120, respectively). We established EL expression in the total muscle homogenates in sedentary animals. Resting values could be ordered with the decreasing EL protein expression as follows: endothelium of left ventricle > diaphragm > red gastrocnemius > right ventricle > soleus > white gastrocnemius. Furthermore, we observed that even a single bout of exercise was capable of inducing changes in the mRNA and protein level of EL, with a clearer pattern observed for the former. After 30 min of running at either exercise intensity, the expression of EL transcript in all the cardiovascular components of muscles tested, except the soleus, was reduced in comparison to the respective sedentary control. The protein content of EL varied with the intensity and/or duration of the run in the studied whole tissue homogenates. The observed differences between EL expression in vascular beds of muscles may indicate the muscle-specific role of the lipase.

Download Full-text

How to Get Started with Single Cell RNA Sequencing Data Analysis

Journal of the American Society of Nephrology ◽

10.1681/asn.2020121742 ◽

2021 ◽

pp. ASN.2020121742 ◽

Cited By ~ 1

Author(s):

Michael S. Balzer ◽

Ziyuan Ma ◽

Jianfu Zhou ◽

Amin Abedini ◽

Katalin Susztak

Keyword(s):

Single Cell ◽

Single Cell Analysis ◽

Cell Analysis ◽

Epigenetic Changes ◽

Sequencing Data ◽

Single Experiment ◽

Gene And Protein Expression ◽

Single Cell Rna Sequencing ◽

Analytical Tools ◽

Sequencing Data Analysis

Over the last 5 years, single cell methods have enabled the monitoring of gene and protein expression, genetic, and epigenetic changes in thousands of individual cells in a single experiment. With the improved measurement and the decreasing cost of the reactions and sequencing, the size of these datasets is increasing rapidly. The critical bottleneck remains the analysis of the wealth of information generated by single cell experiments. In this review, we give a simplified overview of the analysis pipelines, as they are typically used in the field today. We aim to enable researchers starting out in single cell analysis to gain an overview of challenges and the most commonly used analytical tools. In addition, we hope to empower others to gain an understanding of how typical readouts from single cell datasets are presented in the published literature.

Download Full-text

Shift from slow- to fast-twitch muscle fibres in skeletal muscle of newborn heterozygous and homozygous myostatin-knockout piglets

Reproduction Fertility and Development ◽

10.1071/rd19103 ◽

2019 ◽

Vol 31 (10) ◽

pp. 1628 ◽

Cited By ~ 2

Author(s):

Mei-Fu Xuan ◽

Zhao-Bo Luo ◽

Jun-Xia Wang ◽

Qing Guo ◽

Sheng-Zhong Han ◽

...

Keyword(s):

Skeletal Muscle ◽

Protein Expression ◽

Muscle Development ◽

Transforming Growth Factor ◽

Myogenic Differentiation ◽

Muscle Fibres ◽

Skeletal Muscle Development ◽

Cross Sectional ◽

Gene And Protein Expression ◽

Fast Twitch

Myostatin (MSTN) is a member of the transforming growth factor-β superfamily that negatively regulates skeletal muscle development. A lack of MSTN induces muscle hypertrophy and increases formation of fast-twitch (Type II) muscle fibres. This study investigated muscle development in newborn heterozygous (MSTN+/−) and homozygous (MSTN−/−) MSTN-knockout piglets. Detailed morphological and gene and protein expression analyses were performed of the biceps femoris, semitendinosus and diaphragm of MSTN+/−, MSTN−/− and wild-type (WT) piglets. Haematoxylin–eosin staining revealed that the cross-sectional area of muscle fibres was significantly larger in MSTN-knockout than WT piglets. ATPase staining demonstrated that the percentage of Type IIb and IIa muscle fibres was significantly higher in MSTN−/− and MSTN+/− piglets respectively than in WT piglets. Western blotting showed that protein expression of myosin heavy chain-I was reduced in muscles of MSTN-knockout piglets. Quantitative reverse transcription–polymerase chain reaction revealed that, compared with WT piglets, myogenic differentiation factor (MyoD) mRNA expression in muscles was 1.3- to 2-fold higher in MSTN+/− piglets and 1.8- to 3.5-fold higher MSTN−/− piglets (P<0.05 and P<0.01 respectively). However, expression of myocyte enhancer factor 2C (MEF2C) mRNA in muscles was significantly lower in MSTN+/− than WT piglets (P<0.05). MSTN plays an important role in skeletal muscle development and regulates muscle fibre type by modulating the gene expression of MyoD and MEF2C in newborn piglets.

Download Full-text

Immobilization Decreases FOXO3a Phosphorylation and Increases Autophagy-Related Gene and Protein Expression in Human Skeletal Muscle

Frontiers in Physiology ◽

10.3389/fphys.2019.00736 ◽

2019 ◽

Vol 10 ◽

Cited By ~ 6

Author(s):

Andreas Buch Møller ◽

Mikkel Holm Vendelbo ◽

Peter Schjerling ◽

Christian Couppé ◽

Niels Møller ◽

...

Keyword(s):

Skeletal Muscle ◽

Protein Expression ◽

Human Skeletal Muscle ◽

Related Gene ◽

Gene And Protein Expression

Download Full-text

Single‐Cell Analysis of Cytokine mRNA and Protein Expression by Flow Cytometry

Current Protocols in Cytometry ◽

10.1002/cpcy.69 ◽

2020 ◽

Vol 92 (1) ◽

Author(s):

Rubina Pal ◽

Jayne Schaubhut ◽

Darcey Clark ◽

Lynette Brown ◽

Jennifer J. Stewart

Keyword(s):

Flow Cytometry ◽

Protein Expression ◽

Single Cell ◽

Single Cell Analysis ◽

Cell Analysis ◽

Cytokine Mrna ◽

Mrna And Protein Expression

Download Full-text

The tissue differentiation and cancer manifolds in gene and protein expression spaces

10.1101/2021.08.20.457160 ◽

2021 ◽

Author(s):

J Nieves ◽

A Gonzalez

Keyword(s):

Protein Expression ◽

Cancer Progression ◽

Tissue Differentiation ◽

The Cancer Genome Atlas ◽

Normal Tissues ◽

Gene And Protein Expression ◽

Human Protein Atlas ◽

Cancer Genome Atlas ◽

Tissue Of Origin ◽

Using Data

AbstractIt is well known that, for a particular tissue, the homeostatic and cancer attractors are well apart both in gene expression and in protein expression spaces. By using data for 15 tissues and the corresponding tumors from The Cancer Genome Atlas, and for 49 normal tissues and 20 tumors from The Human Protein Atlas, we show that the set of normal attractors are also well separated from the set of tumors. Roughly speaking, one may say that there is a cancer progression axis orthogonal to the normal tissue differentiation and cancer manifolds. This separation suggests that therapies targeting common genes, which define the cancer axis, may be effective, irrespective of the tissue of origin.

Download Full-text

Single-Cell Analysis of the Muscle Stem Cell Hierarchy Identifies Heterotypic Communication Signals Involved in Skeletal Muscle Regeneration

Cell Reports ◽

10.1016/j.celrep.2020.02.067 ◽

2020 ◽

Vol 30 (10) ◽

pp. 3583-3595.e5 ◽

Cited By ~ 23

Author(s):

Andrea J. De Micheli ◽

Emily J. Laurilliard ◽

Charles L. Heinke ◽

Hiranmayi Ravichandran ◽

Paula Fraczek ◽

...

Keyword(s):

Skeletal Muscle ◽

Stem Cell ◽

Single Cell ◽

Muscle Regeneration ◽

Single Cell Analysis ◽

Skeletal Muscle Regeneration ◽

Cell Analysis ◽

Muscle Stem Cell ◽

Communication Signals ◽

Stem Cell Hierarchy

Download Full-text

TWEAK-Fn14 pathway activation after exercise in human skeletal muscle: insights from two exercise modes and a time course investigation

Journal of Applied Physiology ◽

10.1152/japplphysiol.00759.2014 ◽

2015 ◽

Vol 118 (5) ◽

pp. 569-578 ◽

Cited By ~ 18

Author(s):

Ulrika Raue ◽

Bozena Jemiolo ◽

Yifan Yang ◽

Scott Trappe

Keyword(s):

Skeletal Muscle ◽

Protein Expression ◽

Time Course ◽

Human Skeletal Muscle ◽

Vastus Lateralis ◽

Cell Surface Receptor ◽

Vastus Lateralis Muscle ◽

Exercise Mode ◽

Gene And Protein Expression ◽

Surface Receptor

The cell surface receptor Fn14/TWEAKR was recently reported by our laboratory to be a prominent marker in the resistance exercise (RE) induced Transcriptome. The purpose of the present study was to extend our Transcriptome findings and investigate the gene and protein expression time course of markers in the TWEAK-Fn14 pathway following RE or run exercise (RUN). Vastus lateralis muscle biopsies were obtained from 6 RE subjects [25 ± 4 yr, 1-repetition maximum (RM): 99 ± 27 kg] pre- and 0, 1, 2, 4, 8, 12, and 24 h post RE (3 × 10 at 70% 1-RM). Lateral gastrocnemius biopsies were obtained from 6 RUN subjects [25 ± 4 yr, maximum oxygen uptake (V̇o2max): 63 ± 8 ml·kg−1·min−1] pre- and 0, 1, 2, 4, 8, 12, and 24 h after a 30-min RUN (75% V̇o2max). After RE, Fn14 gene and protein expression were induced ( P < 0.05) and peaked at 8 and 12 h, respectively. Downstream markers analyzed showed evidence of TWEAK-Fn14 signaling through the alternative NF-κB pathway after RE. After RUN, Fn14 gene expression was induced ( P < 0.05) to a much lesser extent and peaked at 24 h. Fn14 protein expression was only measurable on a sporadic basis, and there was weak evidence of alternative NF-κB pathway signaling after RUN. TWEAK gene and protein expression were not influenced by either exercise mode. These are the first human data to show a transient activation of the TWEAK-Fn14 axis in the recovery from exercise, and our data suggest the level of activation is exercise mode dependent. Furthermore, our collective data support a myogenic role for TWEAK-Fn14 through the alternative NF-κB pathway in human skeletal muscle.

Download Full-text

A maternal high-fat, high-sucrose diet alters insulin sensitivity and expression of insulin signalling and lipid metabolism genes and proteins in male rat offspring: effect of folic acid supplementation

British Journal Of Nutrition ◽

10.1017/s0007114517002501 ◽

2017 ◽

Vol 118 (8) ◽

pp. 580-588 ◽

Cited By ~ 5

Author(s):

Candace E. Cuthbert ◽

Jerome E. Foster ◽

D. Dan Ramdath

Keyword(s):

Skeletal Muscle ◽

Folic Acid ◽

Protein Expression ◽

Insulin Signalling ◽

Folic Acid Supplementation ◽

Male Rat ◽

High Fat ◽

Gene And Protein Expression ◽

High Sucrose ◽

Viral Oncogene Homolog

AbstractA maternal high-fat, high-sucrose (HFS) diet alters offspring glucose and lipid homoeostasis through unknown mechanisms and may be modulated by folic acid. We investigated the effect of a maternal HFS diet on glucose homoeostasis, expression of genes and proteins associated with insulin signalling and lipid metabolism and the effect of prenatal folic acid supplementation (HFS/F) in male rat offspring. Pregnant Sprague–Dawley rats were randomly fed control (CON), HFS or HFS/F diets. Offspring were weaned on CON; at postnatal day 70, fasting plasma insulin and glucose and liver and skeletal muscle gene and protein expression were measured. Treatment effects were assessed by one-way ANOVA. Maternal HFS diet induced higher fasting glucose in offspringv. HFS/F (P=0·027) and down-regulation (P<0·05) of genes coding for v-Akt murine thymoma viral oncogene homolog 2, resistin and v-Raf-1 murine leukaemia viral oncogene homolog 1 (Raf1) in offspring skeletal muscle and acetyl-CoA carboxylase (Acaca), fatty acid synthase and phosphatidylinositol-4,5-biphosphate 3-kinase, catalytic subunitβin offspring liver. Skeletal muscle neuropeptide Y and hepatic Kruppel-like factor 10 were up-regulated in HFSv. CON offspring (P<0·05). Compared with CON,AcacaandRaf1protein expression levels were significantly lower in HFS offspring. Maternal HFS induced higher homoeostasis model of assessment index of insulin resistancev. CON (P=0·030) and HFS/F was associated with higher insulin (P=0·016) and lower glucose (P=0·025). Maternal HFS diet alters offspring insulin sensitivity andde novohepatic lipogenesis via altered gene and protein expression, which appears to be potentiated by folate supplementation.

Download Full-text