Fiber Type and Subcellular-Specific Analysis of Lipid Droplet Content in Skeletal Muscle

Summary

Increasing evidence indicates that excessive infiltration of lipids inside skeletal muscle results in lipotoxicity and diabetes. Here, we present a complete protocol, including tissue processing, staining with Bodipy, image acquisition, and analysis, to quantify the size, density, and subcellular distribution of lipid droplets in a fiber-type specific manner.

Abstract

Skeletal muscle lipid infiltration, known as myosteatosis, increases with obesity and ageing. Myosteatosis has also recently been discovered as a negative prognostic factor for several other disorders such as cardiovascular disease and cancer. Excessive lipid infiltration decreases muscle mass and strength. It also results in lipotoxicity and insulin resistance depending on total intramyocellular lipid content, lipid droplet (LD) morphology, and subcellular distribution. Fiber type (oxidative vs glycolytic) is also important, since oxidative fibers have a greater capacity to utilize lipids. Because of their crucial implications in pathophysiology, in-depth studies on LD dynamics and function in a fiber type-specific manner are warranted.

Herein, a complete protocol is presented for the quantification of intramyocellular lipid content and analysis of LD morphology and subcellular distribution in a fiber type-specific manner. To this end, serial muscle cryosections were stained with the fluorescent dye Bodipy and antibodies against myosin heavy chain isoforms. This protocol enables the simultaneous processing of different muscles, saving time and avoiding possible artifacts and, thanks to a personalized macro created in Fiji, the automatization of LD analysis is also possible.

Introduction

Skeletal muscle lipid infiltration, known as myosteatosis, increases with obesity and ageing. Myosteatosis is negatively correlated with muscle mass and strength and with insulin sensitivity¹. Moreover, recent studies indicate that the degree of myosteatosis could be used as a prognostic factor for other conditions such as cardiovascular disease², non-alcoholic fatty liver disease³, or cancer⁴. Lipids can accumulate in skeletal muscle between muscle fibers as extramyocellular lipids or within the fibers, as intramyocellular lipids (IMCLs). IMCLs are predominantly stored as ....

Protocol

All procedures conducted on mice were approved by the Ethical Committee for Animal Experimentation from the Medical Sector at Université Catholique de Louvain (2019/UCL/MD/013).

1. Dissection and preparation of the samples for freezing

1. Label a 3 mm thick piece of cork for each pair of muscles.
2. Through a small incision made with a blade on the center of the cork, insert perpendicularly a rectangular piece of rigid plastic (0.5 cm W, 1 cm H) that will serve as support (Figure 2B).
   NOTE: The size of the rectangular plastic piece will depend on the size of the musc....

Results

The protocol described herein provides an efficient method to easily quantify LDs in a fiber type and subcellular-specific manner. It shows how, by freezing together two muscles of similar size, such as the EDL and the soleus, the time and resources spent on the following steps are reduced by half.

A complete protocol is provided for immunostaining, image acquisition, and analysis of the different MyHC isoforms expressed in adult mouse muscles. This protocol is based on the one first designed .......

Discussion

The protocol detailed here describes an efficient method to quantify LDs tagged with Bodipy on a fiber-type- and subcellular-specific basis. In recent years, classical lipid dyes, such as ORO or Sudan Black B, have been substituted with a new array of cell-permeable, lipophilic, fluorescent dyes that bind to neutral lipids (e.g., Bodipy). Available as different conjugates, Bodipy has been proven very effective at tagging LDs to study their morphology, dynamics, and interaction with other organelles, not only in different.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Equipment
AxioCam 506 mono 6 Mpix camera	Zeiss
AxioCam MRm 1.4MPix CCD camera	Zeiss
Chemical hood	Potteau Labo	EN-14175
Confocal microscope	Zeiss	LSM800
Cork discs (ø 20 mm, 3 mm thick)	Electron Microscopy Sciences	63305
Cryo-Gloves	Tempshield	16072252
Cryostat	Thermo Scientific	Microm Cryo Star HM 560
Dissecting Stereo Microscope SMZ745	Nikon
Dry Ice
Dumont Forceps	F.S.T	11295-10
Epifluorescence microscope	Zeiss	AxioImage-Apotome Z1
Extra Fine Bonn Scissors	F.S.T	14084-08
FisherBrand Disposable Base Molds (0.7 x 0.7 cm)	ThermoFisher	22-363-552	Used to cut a piece to hold the muscle on the cork for freezing
Glass petri dish (H 25 mm, ø 150 mm)	BRAND Petri dish, MERK	BR455751	Used to place the muscles on ice during dissection
ImmEdge Hydrophobic barrier PAP Pen	Vector Labs	H-4000	Used to create an hidrophobic barrier around the muscle sections
Incubator	MMM Medcenter	Incucell 707
Microscope Cover Glasses (24x50 mm)	Assistent	40990151
Microscope Slide Boxes	Kartell	278	Used as humid chambers for immunohistochemistry
Neck holder	Linie zwo	SB-035X-02	Used as strap to hold the stainless steel tumbler
No 15 Sterile Carbon Steel Scalpel Blade	Swann-Morton	0205
Paint brushes	Van Bleiswijck	Amazon B07W7KJQ2X	Used to handle cryosections
Permanent Marker Pen Black	Klinipath/VWR	98307-R	Used to label slides
Pierce Fixation Forceps	F.S.T	18155-13
Polystyrene Box			H 12 cm x L 25 cm x W 18 cm, used as a liquid nitrogen container and to transport the samples to the cryostat
Scalpel Handle, 125 mm (5"), No. 3	Aesculap	BB073R
Stainless Steel Cup 10oz	Eboxer	B07GFCBPFH	Tumbler to fill with isopentene for muscle freezing
Superfrost Ultra Plus slides	ThermoFisher	J1800AMNZ
Surgical tweezers 1/2 teeth	Medische Vakhandel	1303152	Also called "Rat teeth tweezers"
Vannas Spring Scissors - 3 mm Cutting Edge	F.S.T	15000-00
Weighing boats	VWR international	611-2249
Whole-Slide Scanner for Fluorescence	Zeiss	Axio Scan.Z1
Reagents
Alexa Fluor 405 Goat Anti-Mouse IgG2b	Sigma-Aldrich	SAB4600477	Used at a final concentration of 1:500
Alexa Fluor 488 Goat Anti-Mouse IgG1	ThermoFisher	A-21121	Used at a final concentration of 1:500
Alexa Fluor 568 Goat Anti-Mouse IgM	Abcam	ab175702	Used at a final concentration of 1:1,000
Alexa Fluor 647 goat anti rat-IgG (H+L) secondary antibody	ThermoFisher	A-21247	Used at a final concentration of 1:500
BODIPY-493/503 (4,4-difluoro-1,3,5,7,8-pentametil-4-bora-3a,4a-diaza-s-indaceno)	ThermoFisher	D3922	Used at a final concentration of 1 µg/mL
BODIPY-558/568 C12 (4,4-Difluoro-5-(2-Thienyl)-4-Bora-3a,4a-Diaza-s-Indacene-3-Dodecanoic Acid)	ThermoFisher	D3835	Used at a final concentration of 1 µg/mL
DAPI (4',6-diamidino-2-phenylindole)	ThermoFisher	D1306	Used at a final concentration of 0.5 µg/mL
Dimethyl Sulfoxide (DMSO)	Sigma-Aldrich	D-8418	Used to solve Bodipy for the 1 mg/mL stock solution. CAUTION: Toxic and flammable. Vapors may cause irritation. Manipulate in a fume hood. Avoid direct contact with skin. Wear rubber gloves, protective eye goggles.
Formaldehyde solution 4%, buffered, pH 6.9	Sigma-Aldrich	1004969011	CAUTION: May cause an allergic skin reaction. Suspected of causing genetic defects. May cause cancer. Manipulate in a fume hood. Avoid direct contact with skin. Wear rubber gloves, protective eye goggles.
Isopentane GPR RectaPur	VWR international	24872.298	CAUTION: Extremely flammable liquid and vapor. May be fatal if swallowed and enters airways. May cause drowsiness or dizziness. Repeated exposure may cause skin dryness or cracking. Wear protective gloves/protective clothing/eye protection/face protection.
Liquid Nitrogen			CAUTION:  Extremely cold. Wear gloves. Handle slowly to minimize boiling and splashing and in well ventilated areas. Use containers designed for low-temperature liquids.
Mouse on mouse Blocking Reagent	Vector Labs	MKB-2213-1	Used at concentration of 1:30
Myosin heavy chain Type I (BA-D5-s Primary Antibody) Gene: MYH7, monoclonal bovine anti mouse IgG2b	DSHB University of Iowa	BA-D5-supernatant	Used at a final concentration of 1:10
Myosin heavy chain Type IIA (SC-71-s Primary Antibody) Gene:  MYH2, Monoclonal bovine anti mouse IgG1	DSHB University of Iowa	SC-71-supernatant	Used at a final concentration of 1:10
Myosin heavy chain Type IIX (6H1-s Primary Antibody), Gene:  MYH1, Monoclonal rabbit anti mouse IgM	Developmental Studies Hybridoma Bank, University of Iowa	6H1-supernatant	Used at a final concentration of 1:5
Normal Goat Serum (NGS)	Vector Labs	S-1000
PBS 0.1 M			Commonly used on histology laboratories
ProLong Gold Antifade Mountant	Invitrogen	P36930
Rat anti-Laminin-2 (α-2 Chain) primary antibody (monoclonal)	Sigma-Aldrich	L0663	Used at a final concentration of 1:1,000
Tissue-Tek O.C.T compound	Sakura	4583
Software
Adobe Illustrator CC	Adobe Inc.		Used to design the figures
Adobe Photoshop	Adobe Inc.		Confocal software
BioRender	https://biorender.com/		Used to design the figures
Fiji/ImageJ	https://imagej.net/software/fiji/		Used to analyse the acquired images
Microsoft PowerPoint	Microsoft		Used to reconstruct the histology of the whole muscle after scanning the fiber types
Zen Blue 2.6	Zeiss		Used to reconstruct the histology of the whole muscle after scanning the fiber types