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

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.

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.

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 ....

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 .......

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 .......

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.......

This work was supported by grants from the Fonds National de la Recherche Scientifique (FNRS-Crédit de Recherche J.0022.20) and the Société Francophone du Diabète (SFD-Roche Diabetes Care).C.M.S. is the recipient of a Ph.D. fellowship from the FRIA (FNRS). M.A.D.-L.d.C. received a fellowship from the Wallonie-Bruxelles International Excellence Program.

The authors thank Alice Monnier for her contribution to the development of this protocol and Caroline Bouzin for her expertise and technical help in the image acquisition process. We also thank the 2IP-IREC imaging platform for access to the cryostat and ....