mouse hindlimb muscle dissection and subsequent muscle fiber isolation using collagenase digestion for calcium transient studies

Enzymatic Isolation and Characterization of Variable Length Mouse Muscle Fibers

The mature skeletal muscle of mammals is a multifunctional tissue. It heavily regulates metabolism, is the main source of heat production, and its dynamical properties confer upon it a key role in respiration, movement of body segments, or displacement from one point to another1,2,3. Skeletal muscle is also relevant for the pathophysiology of many illnesses, including inherited and chronic conditions, such as myopathies, dystrophies, or sarcopenia, as well as many non-muscle chronic conditions, such as cardiometabolic diseases3,4,5,6,7,8.
The ex vivo study of the structural and functional properties of mature skeletal muscle in the context of health and disease has been possible mainly through two experimental models: whole muscle and isolated fibers. In the 20th century, researchers exploited the properties of the whole, intact extensor digitorum longus (EDL), soleus, tibialis anterior, and gastrocnemius muscles of different small species as pivotal models to learn about motor units, fiber types, and dynamic properties such as force and kinetics of contraction and relaxation9,10,11,12,13,14,15,16. However, the advent of more refined cell biology studies moved the area toward the study of single muscle fibers. Pioneering work then enabled the isolation of intact flexor digitorum brevis (FDB) fibers of rats by enzymatic dissociation for subsequent characterization17,18,19. Although FDB fibers can also be obtained by manual dissection20, the ease and high throughput of enzymatic dissociation of murine muscles, in addition to their suitability for a variety of experimental approaches, have made the latter model widely used during the last two decades.
The short FDB fibers are suitable for electrophysiological and other biophysical studies, biochemical, metabolic, and pharmacological analyses, electron and fluorescence microscopy experiments, transfection for cell biology approaches, or as a source of stem cells in myogenesis studies5,21,22,23,24,25,26,27,28,29,30,31,32. However, using only FDB fibers in muscle experiments narrows the scope of research dealing with fiber types and limits the amount of biological material available for some methodological techniques or for gaining more information from one animal. These limitations hinder a clear correlation of cellular physiological phenomena with previous biochemical and dynamical studies performed in different whole, intact, muscles (e.g., EDL, soleus, peronei).
Overcoming these limitations, some groups succeeded in dissociating the longer EDL and soleus muscles24,33,34,35,36,37,38,39,40, opening the door to further extend the method to other relevant muscles. However, the use of EDL and soleus fibers is still scarce, likely due to the lack of methodological details for getting them as intact fibers. Here, we describe in detail how to isolate fibers of different lengths and types from six muscles: three of them already described (FDB, EDL, and soleus) and three of them successfully dissociated for the first time (extensor hallucis longus [EHL], peroneus longus [PL], and peroneus digiti quarti [PDQA]). The results of the present work confirm that the model of enzymatically dissociated fibers is apt for a wide range of studies and future correlations with previously published data, thus increasing the availability of models for mature skeletal muscle studies.

Author Spotlight: Isolation of Long Muscle Fibers from Mouse Hindlimb Muscles for Studying Excitation-Contraction Coupling Across Fiber Types

Intact Short, Intermediate, and Long Skeletal Muscle Fibers Obtained by Enzymatic Dissociation of Six Hindlimb Muscles of Mice: Beyond Flexor Digitorum Brevis

In this study, we describe a method to isolate fibers of different lengths and types from six hindlimb muscles from mice. Also, we tested the suitability of the fibers for physiological experiments dealing with the study of the excitation-contraction coupling mechanism. For years, obtaining long muscle fibers was challenge, limiting the scope of many studies in the field.Now we show that it is possible to obtain lean fibers with mouse flexors, extensors, and evertors with lengths of up to six millimeters. As an example, obtaining fibers from six different muscles has allowed us to show that the calcium transient kinetics known as morphology type II, can be generalized to type IIB and IIX regardless of the location or function of their muscle source. We believe that the model of enzymatically isolated fibers is suitable for a variety of experimental approaches with different types of technologies that allow us to answer mechanistic questions within the range of biochemistry, physiology, biophysics, cell biology, and molecular biology.

Mouse Hindlimb Muscle Dissection and Subsequent Muscle Fiber Isolation Using Collagenase Digestion for Calcium Transient Studies

After sacrificing the mouse, place it on a foam surface and secure the forelimbs with tape or pins. Use operating scissors to cut both hind limbs over the knees. Transfer each hind limb to a separate dissection chamber, and add cold Tyrode's solution to cover the tissue.Pin the first hind limb to the dissection chamber, ensuring the posterior face of the legs is visible. Under magnification, remove the skin, then expose and excise the flexor digitorum brevis or FDB muscle. Store the FDB muscle in a labeled glass vial containing one milliliter of Tyrode's solution.Using fine scissors, separate the gastrocnemius muscle, then excise the soleus muscle. Remove the skin from the anterior face of the leg. Then, identify the distal tendons of the tibialis anterior and the extensor digitorum longus or EDL muscles in the ankle region.Remove and discard the tibialis. Then, cut the distal tendons of the EDL and continue the dissection to completely remove the EDL muscle. After identifying the extensor hallucis longus or EHL muscle, begin the dissection following the tendon to the first digit.Next, identify and follow the most external tendon of the peronei to sever it and remove the peroneus longus or PL muscle. Identify and trace the tendon to the fourth digit. Then, cut and remove the peroneus digiti quarti or PDQA muscle.After dissecting the second hind limb, collect muscles of the same type in a labeled glass vial containing Tyrode's solution. Replace the Tyrode's solution in the dissection chamber to remove debris and mouse fur. Pour the PL muscles into the dissection chamber and verify their integrity and contraction.Perform longitudinal or diagonal cuts on the soleus muscle following the central tendon, cutting approximately 80%of its length. For the EDL muscle, follow one or two tendons and cut about the same length as the soleus. Transfer each pair of muscles to a new glass vial containing three milligrams of collagenase type 2 in dissociation solution, and incubate them in a 37 degree Celsius water bath for 65 to 90 minutes with gentle shaking.After 65 minutes of incubation, check the vial under stereoscope every five minutes. Once the muscle appears rippled and loose, and fibers begin to detach, rinse the muscles with Tyrode's at room temperature to deactivate and remove the collagenase. Using a set of fire-polished Pasteur pipettes, agitate the solution around the muscle with a five millimeter tip pipette.Then, gently pull the muscles in and out of the tip three to four times. As the muscles starts to release fibers and becomes thinner, using a four millimeter tip, repeat the procedure to separate more fibers.

Research

JoVE Journal

Biology

Skeletal muscle fibers obtained by enzymatic dissociation of mouse muscles are a useful model for physiological experiments. However, most papers deal ...