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

This study, we describe a method to isolate fibers of different lengths and types from six hindlimb muscles of 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 challenged 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 with length 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 two, can be generalized to type 2B and 2X 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.

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 vile 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 perinea 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 quinti or PDQA muscle After dissecting the second hind limb, collect muscles of the same type in a labeled glass vile 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 sous 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 veil containing three milligrams of collagenase type two 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 vile 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 muscle starts to release fibers and becomes thinner. Using a four millimeter tip, repeat the procedure to separate more fibers. To begin mount a clean glass slide on the experimental bath chamber.

Coat the slide with two to three microliters of laminin and let it dry for 30 seconds. Then pour approximately 400 microliters of the muscle fiber suspension onto the slide and allow the fibers to adhere to the laminin for 10 to 15 minutes. Place the experimental chamber onto the stage of an inverted microscope equipped for epifluorescence to verify the viability of the fibers.

Place two platinum electrodes on either side of the experimental chamber after applying rectangular current pulses for 0.8 to 1.2 milliseconds observe the muscle fiber contractions at the extremes. Load the fibers with 3.5 to 4.5 micro molars of the fast calcium di-mag flow 4AM for four to five minutes in Tyrode's solution. After washing the slide with Tyrode's solution, let the intracellular dye de-esterify for 15 to 20 minutes in the dark.

During the acquisition of calcium transient. Under dimmed illumination, collect and save the light signals with an oil immersion 40 x objective and a photo multiplier tube connected to a digitizer. In the acquisition software ensure a scale of zero to 200 arbitrary units.

Then adjust the size of the excitation spot and the gain of the photo multiplier tube to set the resting fluorescence or F rest to 10 arbitrary units. For analyzing the recordings, set a low-pass filter to the whole trace at one kilohertz. Then adjust the peak to calculate the F rest in one second of the trace.

Adjust the fret to zero and measure the peak sarcoplasmic calcium transients amplitude. Measure the rise time from 10%to 90%of the amplitude, the duration at half maximum, and the decay time from 90%to 10%of the amplitude. Then estimate the decay kinetics according to a fit.

With the bi-exponential function, Calculate the peak calcium concentration in micromolar. Finally, save the values of the time constants of decay tau one and tau two, and amplitudes A one and A two. The FDB and EDL muscles showed calcium kinetics known as morphology type two.

Soleus muscles displayed morphology type one calcium transients Fibers from PDQA, PL and EHL muscles shared the type two morphology. The calcium transient kinetic signals of PDQA, PL and EHL were similar to the signals of FDBEDL muscles, but differed from soleus muscle signals.