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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Biochemistry

Fiber Type Identification of Human Skeletal Muscle

Published: September 22nd, 2023

DOI:

10.3791/65750

1Department of Biochemistry and Chemistry, La Trobe Institute for Molecular Science, School of Agriculture, Biomedicine and Environment, La Trobe University
* These authors contributed equally

This protocol demonstrates single-fiber isolation from freeze-dried human skeletal muscle and fiber-type classification according to Myosin heavy chain (MHC) isoform using the dot blotting technique. Identified MHC I and II fiber samples can then be further analyzed for fiber type-specific differences in protein expression using western blotting.

The technique described here can be used to identify specific myosin heavy chain (MHC) isoforms in segments of individual muscle fibers using dot blotting, hereafter referred to as Myosin heavy chain detection by Dot Blotting for IDentification of muscle fiber type (MyDoBID). This protocol describes the process of freeze-drying human skeletal muscle and isolating segments of single muscle fibers. Using MyDoBID, type I and II fibers are classified with MHCI- and IIa-specific antibodies, respectively. Classified fibers are then combined into fiber type-specific samples for each biopsy.

The total protein in each sample is determined by Sodium Dodecyl-Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) and UV-activated gel technology. The fiber type of samples is validated using western blotting. The importance of performing protein loading normalization to enhance target protein detection across multiple western blots is also described. The benefits of consolidating classified fibers into fiber type-specific samples compared to single-fiber western blots, include sample versatility, increased sample throughput, shorter time investment, and cost-saving measures, all while retaining valuable fiber type-specific information that is frequently overlooked using homogenized muscle samples. The purpose of the protocol is to achieve accurate and efficient identification of type I and type II fibers isolated from freeze-dried human skeletal muscle samples.

These individual fibers are subsequently combined to create type I and type II fiber type-specific samples. Furthermore, the protocol is extended to include the identification of type IIx fibers, using Actin as a marker for fibers that were negative for MHCI and MHCIIa, which are confirmed as IIx fibers by western blotting. Each fiber type-specific sample is then used to quantify the expression of various target proteins using western blotting techniques.

Skeletal muscle is a heterogeneous tissue, with distinct cellular metabolic and contractile properties that are dependent on whether the cell (fiber) is slow twitch (type I) or fast twitch (type II). The fiber type can be identified by examining the myosin heavy chain (MHC) isoforms, which differ from each other in several ways, including contraction time, speed of shortening, and fatigue resistance1. Major MHC isoforms include type I, type IIa, type IIb, and type IIx and their metabolic profiles are either oxidative (type I and IIa) or glycolytic (IIx, IIb)1. The proportion of these fiber types varies in muscle type and....

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Human muscle samples were obtained from the vastus lateralis from n = 3 (2 males, 1 female), aged 70-74 years old under sterile conditions using local anesthesia (Xylocaine) and a Bergstrom needle modified for manual suction11,12. Samples were a subset of a previous study approved by the Victoria University Human Research Ethics Committee (HRETH11/221) and conducted in accordance with the Declaration of Helsinki13. Participants pr.......

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Identification of individual MHCI, MHCIIa, and MHCIIx muscle fibers using dot blotting
A feature of MyDoBID is the categorization of the varying MHC and Actin signal intensity strength in a given fiber (Figure 4A). Fiber type was identified by the presence or absence of MHCI and IIa isoforms (Figure 4B). Six fibers showed no MHC or actin detection, indicating that there was no fiber collected. The results of this specific dot blot were the.......

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Fiber collection
Based on several years of experience, most researchers can master this technique; however, practice leads to faster and more efficient fiber collection for downstream analyses. To be able to isolate 30 single fiber segments of a quality for pooling, it is recommended that 50 fiber segments are collected per sample. Studying the fiber collection video carefully and after performing two practice sessions (~50 fibers per session) is recommended to achieve a reasonable standard. All fi.......

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The antibodies against MHC I (A4.840) and MHCIIa (A4.74) used in this study were developed by Dr. H. M. Blau and the antibody against MHCIIx (6H1) was developed by Dr. C. A. Lucas and obtained from the Developmental Studies Hybridoma Bank (DSHB), thanks to the auspices of the National Institute of Child Health and Human Development and maintained by the University of Iowa, Department of Biological Sciences (Iowa City, IA). We thank Victoria L. Wyckelsma for providing the human muscle samples for this study. The majority of the images in Figure 1 was sourced from BioRender.com.

Funding:
This study received no external ....

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NameCompanyCatalog NumberComments
1x Denaturing bufferMake according to recipeConstituents can be sourced from Sigma-Aldrich or other chemical distributing companies 1x denaturing buffer is made by diluting 3x denaturing buffer 1 in 3 v/v with 1X Tris-HCl (pH 6.8). Store at -20 °C.
3x Denaturing bufferMake according to recipeConstituents can be sourced from Sigma-Aldrich or other chemical distributors3x denaturing buffer contains: 0.125M Tris-HCI, 10% glycerol, 4% SDS, 4 M urea, 10% 2-mercaptoethanol, and 0.001% bromophenol blue, pH 6.8.  Store at -20 °C.
95% EthanolN/A100% ethanol can be sourced from any companyDiluted to 95% with ultra-pure H2O.
Actin rabbit polyclonal antibodySigma-Aldrich  A2066Dilute 1 in 1,000 with BSA buffer.
Analytical scalesMettler ToledoModel number: MSZ042101
Antibody enhancerThermo Fischer Scientific32110Product name is Miser Antibody Extender Solution NC.
Beaker (100 mL)N/AN/A
Benchtop centrifugeEppendorf5452Model name: Mini Spin.
Blocking buffer: 5% Skim milk in Wash buffer.DiplomaStore bought
BSA buffer: 1 % BSA/PBST, 0.02 % NaN3BSA: Sigma-Aldrich              PBS: Bio-Rad Laboratories. NaN3 : Sigma-Aldrich BSA: A6003-25G                         10x PBS: 1610780                       NaN3: S2002Bovine serum albumin (BSA), Phosphate-buffered saline (PBS), and Sodium azide (NaN3). Store at 4 °C.
Cassette opening lever Bio-Rad Laboratories4560000Used to open the precast gel cassettes.
Chemidoc MP Imager Bio-Rad LaboratoriesModel number: Universal hood IIIAny imaging system with Stain-Free gel imaging capabilities.
Criterion blotterBio-Rad Laboratories1704070Includes ice pack, transfer tray, roller, 2 cassette holders, filter paper, foam pads and lid with cables.
Criterion Cell (Bio-Rad)Bio-Rad Laboratories1656001
ECL (enhanced chemiluminescence)Bio-Rad Laboratories1705062Product name: Clarity Max Western ECL Substrate.
Electrophoresis buffer 1x Tris Glycine SDS (TGS)Bio-Rad Laboratories1610772Dilute 10x TGS 1 in 10 with ultra-pure H2O.
Filter paper, 0.34 mm thickWhatmann3030917Bulk size 3 MM, pack 100 sheets, 315 x 335 mm.
Fine tissue dissecting forcepsDumontF6521-1EAJeweller’s forceps no. 5.
Flat plastic tray/lid  N/AN/ALarge enough to place the membrane on. Ensure the surface is completely flat.
Freeze-drying SystemLabconco7750030Freezone 4.5 L with glass chamber sample stage.
Freezer -80 oN/AN/AAny freezer with a constant temperature of -80 °C is suitable.
Gel releasers1653320Bio-RadSlide under the membrane to gather or move the membrane.
Grey lead pencilN/AN/A
 Image lab software Bio-Rad LaboratoriesN/AFigures refers to software version 5.2.1 but other versions can used.
IncubatorBio-Rad Laboratories1660521Any incubator that can be set to 37 °C would suffice.
LampN/AN/A
Magnetic stirrer with fleaN/AN/A
Membrane roller Bio-Rad Laboratories1651279Can be purchased in the Transfer bundle pack. However, if this product is not available, any smooth surface cylindrical tube long enough to roll over the membrane would suffice. 
Microcentrifuge tubes  (0.6 mL)N/AN/A
Mouse IgG HRP secondaryThermo Fisher Scientific31430Goat anti-Mouse IgG (H+L), RRID AB_228341. Dilute at 1 in 20,000 in blocking buffer.
Mouse IgM HRP secondaryAbcamab97230Goat Anti-Mouse IgM mu chain. Use at the same dilution as mouse IgG.
Myosin Heavy Chain I (MHCI) primary antibodyDSHBA4.840 Dilution range: 1 in 200 to 1 in 500 in BSA buffer.
Myosin Heavy Chain IIa (MHCIIa) primary antibodyDSHBA4.74 Dilution range: 1 in 200 to 1 in 500 in BSA buffer.
Myosin Heavy Chain IIx (MHCIIx) primary antibodyDSHB6H1Dilution range: 1 in 200 to 1 in 500 in BSA buffer.
Nitrocellulose Membrane 0.45 µm Bio-Rad Laboratories1620115For Western blotting.
Petri dish lidN/AN/A
Plastic tweezersN/AN/A
Power Pack Bio-Rad Laboratories164-5050Product name: Basic power supply.
Protein ladderThermo Fisher Scientific26616PageRuler Prestained Protein Ladder, 10 to 180 kDa.
PVDF Membrane 0.2 µmBio-Rad Laboratories1620177
Rabbit HRP secondaryThermo Fisher Scientific31460Goat anti-Rabbit IgG (H+L), RRID AB_228341. Dilution same as mouse secondary antibodies.
RockerN/AN/A
RulerN/AN/A
ScissorsN/AN/A
StereomicroscopeMoticSMZ-168
Stripping bufferThermo Fisher Scientific21059Product name: Restore Western Blot Stripping Buffer.
Tissue (lint free)Kimberly-Clark professional34120Product name: Kimwipe.
Transfer buffer (1x Tris Glycine buffer (TG), 20% Methanol)TG: Bio-Rad Laboratories   Methanol: MerckTG buffer: 1610771           Methanol: 1.06018dilute 10x TG buffer with ultra-pure H2O to 1x. Add 100% Methanol to a final concentration of 20% Methanol. Store at 4 °C.
Transfer trayBio-Rad Laboratories1704089
 UV-activation precast gelBio-Rad Laboratories5678085Gel type: 4–15% Criterion TGX Stain-Free Protein Gel, 26 well, 15 µL.
VortexN/AN/A
Wash buffer (1x TBST)10x TBS: Astral Scientific  Tween 20: Sigma BIOA0027-4L1x TBST recipe: 10x Tris-buffered saline (TBS) is diluted down to 1x with ultra-pure H2O, Tween 20 is added to a final concentration of 0.1%. Store buffer at 4 °C.
Wash containersSistemaStore boughtAny tupperware container, that suits the approximate dimensions of the membrane would suffice.

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