Fiber Type Identification of Human Skeletal Muscle

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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 suction^11,12. 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.......

Discussion

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.......

Materials

Name	Company	Catalog Number	Comments
1x Denaturing buffer	Make according to recipe	Constituents 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 buffer	Make according to recipe	Constituents can be sourced from Sigma-Aldrich or other chemical distributors	3x 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% Ethanol	N/A	100% ethanol can be sourced from any company	Diluted to 95% with ultra-pure H2O.
Actin rabbit polyclonal antibody	Sigma-Aldrich	A2066	Dilute 1 in 1,000 with BSA buffer.
Analytical scales	Mettler Toledo	Model number: MSZ042101
Antibody enhancer	Thermo Fischer Scientific	32110	Product name is Miser Antibody Extender Solution NC.
Beaker (100 mL)	N/A	N/A
Benchtop centrifuge	Eppendorf	5452	Model name: Mini Spin.
Blocking buffer: 5% Skim milk in Wash buffer.	Diploma	Store bought
BSA buffer: 1 % BSA/PBST, 0.02 % NaN3	BSA: Sigma-Aldrich              PBS: Bio-Rad Laboratories. NaN3 : Sigma-Aldrich	BSA: A6003-25G                         10x PBS: 1610780                       NaN3: S2002	Bovine serum albumin (BSA), Phosphate-buffered saline (PBS), and Sodium azide (NaN3). Store at 4 °C.
Cassette opening lever	Bio-Rad Laboratories	4560000	Used to open the precast gel cassettes.
Chemidoc MP Imager	Bio-Rad Laboratories	Model number: Universal hood III	Any imaging system with Stain-Free gel imaging capabilities.
Criterion blotter	Bio-Rad Laboratories	1704070	Includes ice pack, transfer tray, roller, 2 cassette holders, filter paper, foam pads and lid with cables.
Criterion Cell (Bio-Rad)	Bio-Rad Laboratories	1656001
ECL (enhanced chemiluminescence)	Bio-Rad Laboratories	1705062	Product name: Clarity Max Western ECL Substrate.
Electrophoresis buffer 1x Tris Glycine SDS (TGS)	Bio-Rad Laboratories	1610772	Dilute 10x TGS 1 in 10 with ultra-pure H2O.
Filter paper, 0.34 mm thick	Whatmann	3030917	Bulk size 3 MM, pack 100 sheets, 315 x 335 mm.
Fine tissue dissecting forceps	Dumont	F6521-1EA	Jeweller’s forceps no. 5.
Flat plastic tray/lid	N/A	N/A	Large enough to place the membrane on. Ensure the surface is completely flat.
Freeze-drying System	Labconco	7750030	Freezone 4.5 L with glass chamber sample stage.
Freezer -80 oC	N/A	N/A	Any freezer with a constant temperature of -80 °C is suitable.
Gel releasers	1653320	Bio-Rad	Slide under the membrane to gather or move the membrane.
Grey lead pencil	N/A	N/A
Image lab software	Bio-Rad Laboratories	N/A	Figures refers to software version 5.2.1 but other versions can used.
Incubator	Bio-Rad Laboratories	1660521	Any incubator that can be set to 37 °C would suffice.
Lamp	N/A	N/A
Magnetic stirrer with flea	N/A	N/A
Membrane roller	Bio-Rad Laboratories	1651279	Can 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/A	N/A
Mouse IgG HRP secondary	Thermo Fisher Scientific	31430	Goat anti-Mouse IgG (H+L), RRID AB_228341. Dilute at 1 in 20,000 in blocking buffer.
Mouse IgM HRP secondary	Abcam	ab97230	Goat Anti-Mouse IgM mu chain. Use at the same dilution as mouse IgG.
Myosin Heavy Chain I (MHCI) primary antibody	DSHB	A4.840	Dilution range: 1 in 200 to 1 in 500 in BSA buffer.
Myosin Heavy Chain IIa (MHCIIa) primary antibody	DSHB	A4.74	Dilution range: 1 in 200 to 1 in 500 in BSA buffer.
Myosin Heavy Chain IIx (MHCIIx) primary antibody	DSHB	6H1	Dilution range: 1 in 200 to 1 in 500 in BSA buffer.
Nitrocellulose Membrane 0.45 µm	Bio-Rad Laboratories	1620115	For Western blotting.
Petri dish lid	N/A	N/A
Plastic tweezers	N/A	N/A
Power Pack	Bio-Rad Laboratories	164-5050	Product name: Basic power supply.
Protein ladder	Thermo Fisher Scientific	26616	PageRuler Prestained Protein Ladder, 10 to 180 kDa.
PVDF Membrane 0.2 µm	Bio-Rad Laboratories	1620177
Rabbit HRP secondary	Thermo Fisher Scientific	31460	Goat anti-Rabbit IgG (H+L), RRID AB_228341. Dilution same as mouse secondary antibodies.
Rocker	N/A	N/A
Ruler	N/A	N/A
Scissors	N/A	N/A
Stereomicroscope	Motic	SMZ-168
Stripping buffer	Thermo Fisher Scientific	21059	Product name: Restore Western Blot Stripping Buffer.
Tissue (lint free)	Kimberly-Clark professional	34120	Product name: Kimwipe.
Transfer buffer (1x Tris Glycine buffer (TG), 20% Methanol)	TG: Bio-Rad Laboratories   Methanol: Merck	TG buffer: 1610771           Methanol: 1.06018	dilute 10x TG buffer with ultra-pure H2O to 1x. Add 100% Methanol to a final concentration of 20% Methanol. Store at 4 °C.
Transfer tray	Bio-Rad Laboratories	1704089
UV-activation precast gel	Bio-Rad Laboratories	5678085	Gel type: 4–15% Criterion TGX Stain-Free Protein Gel, 26 well, 15 µL.
Vortex	N/A	N/A
Wash buffer (1x TBST)	10x TBS: Astral Scientific  Tween 20: Sigma	BIOA0027-4L	1x 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 containers	Sistema	Store bought	Any tupperware container, that suits the approximate dimensions of the membrane would suffice.