Advanced Methodology for Rapid Isolation of Single Myofibers from Flexor Digitorum Brevis Muscle

Abstract

Isolated individual myofibers are valuable experimental models that can be used in various conditions to understand skeletal muscle physiology and pathophysiology at the tissue and cellular level. This report details a time- and cost-effective method for isolation of single myofibers from the flexor digitorum brevis (FDB) muscle in both young and aged mice. The FDB muscle was chosen for its documented history in single myofiber experiments. By modifying published methods for FDB myofiber isolation, we have optimized the protocol by first separating FDB muscle into individual bundles before the digestion, followed by optimizing the subsequent digestion medium conditions to ensure reproducibility. Morphological and functional assessments demonstrate a high yield of isolated FDB myofibers with sarcolemma integrity achieved in a shorter time frame than previous published procedures. This method could be also adapted to other types of skeletal muscle. Additionally, this highly reproducible method can greatly reduce the number of animals needed to yield adequate numbers of myofibers for experiments. Thus, this advanced method for myofiber isolation has the potential to accelerate research in skeletal muscle physiology and screening potential therapeutics "ex vivo" for muscle diseases and regeneration.

Keywords: FDB; flexor digitorum brevis; isolation; muscle physiology; myofiber.

FIG. 1.

Experimental design shows the stages of FDB myofibers isolation. FDB, flexor digitorum brevis.

FIG. 2.

Example of securing the hind paw, extracting FDB, and separation of muscle bundles. Color images are available online.

FIG. 3.

Detailed visualization of the FDB muscle dissection and myofibers isolation. (A) Securing the paw with pins on the dissection board. (B) Removing the skin and exposing the FDB muscle. (C) Clear visualization of the FDB muscle. (D, E) Cutting the proximal tendon to release the FDB muscle. (F) Two intact FDB muscles. (G) Four separated FDB bundles. (H) Bundles transferred into 1 well of 12-well plate with 500 μL digestion media. (I) Microscopic image of the bundles after 15 min of incubation with digestion media. (J) Microscopic image of the bundles from young mice after 35–45 min of incubation with digestion media, indicating dissociation of fibers as seen by the hair-like view. (K) Isolated myofibers in glass bottom dish. Color images are available online.

FIG. 4.

Quantification of viable myofibers isolated from FDB of young mice. Fibers are stained with Calcein AM green stain indicating live viable fibers after 1 h of dissociation. (A) Total number of isolated live myofibers from 2 FDB muscles seeded in 3 dishes versus 10 dishes (B, C) Box plot presenting the total count of viable myofibers after 1 h of seeding. Color images are available online.

FIG. 5.

Quantification of viable myofibers isolated from FDB of aged mice. Fibers are stained with Calcein AM green stain indicating live viable fibers, and Ethidium homodimer-1 red stain indicating dead cells after 1 h of dissociation. Total number of isolated live myofibers from 2 FDB muscles seeded in 10 dishes as labeled from dish 1 to dish 10. At dish 10, zoom out image indicating green-stained fibers (Live) and red-stained fiber (Dead). Box plot presenting the total count of viable myofibers after 1 h of seeding. Color images are available online.

FIG. 6.

MHC and DAPI staining. Myofibers were seeded on laminin-coated glass bottom dishes. Fibers were stained using MHC antibody (A) and counterstained using DAPI (B, C) In the overlay shows a sealed sarcolemma and multinucleation on the perimeter of the myofiber. DAPI, 4′,6-diamidino-2-phenylindole dihydrochloride; MHC, myosin heavy chain. Color images are available online.

FIG. 7.

Desmin staining: fibers were stained using Desmin Rabbit Monoclonal Antibody and DAPI and then fixed with Prolong^® Gold Antifade Reagent according to the manufacturer's protocol. Color images are available online.

FIG. 8.

Membrane integrity of isolated FDB fibers. (A) Green fluorescent dye (Fluo-4) stains intact myofibers and fibers still intact after 1.5 h. (B) Show intact myofibers at 0 h, whereas red dye (FM 1–43) only permeates the damaged membrane as fast as saponin punctures the membrane allowing the influx of FM 1–43 right after 10 min. The compromised myofibers totally damaged and detached after 1.5 h. Scale bar 200 μm is shown on each image. Color images are available online.

FIG. 9.

Calcium transients. The fibers were loaded with Fura-2 AM fluorescent dye. (A) Myofiber during stimulation of 200 mM KCl compared with myofiber at rest (B) The intracellular calcium was calculated from the ratio unit measured from the intensity of the excitation at 340/380 nm and emission wavelength at 510 nm. Color images are available online.