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

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 another^1,2,3. 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 diseases^3,4,5,6,7,8.

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 20^th 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 relaxation^{9,10,11,12,13,14,15,16}. 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 characterization^17,18,19. 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 studies^{5,21,22,23,24,25,26,27,28,29,30,31,32}. 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 muscles^{24,33,34,35,36,37,38,39,40}, 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.

All procedures were approved by the Committee for Ethics in Experiments with animals of the University of Antioquia (UdeA) (minutes 104 of June 21^st, 2016, and 005 of April 15^th, 2021), according to Law 84 of 1989 and Resolution 8430 of 1993 issued by the Colombian Government and were performed and reported in compliance with the Animal Research: Reporting In Vivo Experiments (ARRIVE) guidelines⁴¹. All results presented here come from healthy, 7-13 weeks old, 20-26 g, C57BL/6 male mice. Figure 1 shows the general design of this study and the order of the procedures. All reagents, materials, and equipment details are listed in the Table of Materials.

1. Animals

1. House a maximum of six mice per acrylic, transparent, rectangular cage, with wood-derived bedding, under conditions of controlled temperature (21 ± 2 °C) and light:darkness (12:12 h) cycles.
2. Give the animals free access to food and tap water in specific pathogen-free animal facilities with no environmental enrichment.

2. Dissection

1. Solutions, materials, and reagents
   1. Prepare and filter (0.22 µm) the working solutions with the following composition (all concentrations in mM):
      1. Tyrode: 5.4 KCl, 1 MgCl₂, 140 NaCl, 0.33 NaH₂PO₄, 2 CaCl₂, 10 glucose, 10 HEPES, pH 7.3
      2. Dissociation: 2.7 KCl, 1.2 KH₂PO₄, 0.5 MgCl₂, 138 NaCl, 0.1 Na₂HPO₄, 1 CaCl₂, pH 7.4
      3. Phosphate-buffered saline (PBS): 137 NaCl, 8.6 Na₂HPO₄, 2.8 KH₂PO₄, pH 7.34
   2. Prepare two dissection chambers; stereoscope; operating scissors; fine scissors; fine forceps; and clean, transparent, non-conic, 1-1.5 cm wide, glass vials of 3-4 mL total volume with caps. Arrange a system for electrically stimulating the muscles in the dissection chambers.
   3. Prepare fire-polished Pasteur glass pipettes of different width tips: 5, 4, 3, 2, and 1 mm.
   4. Set the water bath to 37 °C. Weigh aliquots of 3 mg of collagenase type 2.
2. Procedure
   1. Sacrifice the mouse using methods approved by the local Ethics Committee. Cervical dislocation is recommended because it is rapid, less stressful, and avoids exposure to drugs, which may affect the muscle tissue (such as CO₂ or some anesthetics). Start dissection immediately to obtain better results.
   2. Place the mouse on a foam surface and tape or pin the forelimbs. Cut both hindlimbs over the knees with the operating scissors, transfer each of them to a separate dissection chamber, and add cold (10-20 °C) Tyrode to cover the tissue.
      NOTE: Each hindlimb will give six different muscles in the following order: FDB, soleus, EDL, EHL, PL, and PDQA. Detailed anatomical references to dissect the six muscles intact from tendon to tendon are given in Figure 2 and elsewhere⁴².
   3. Pin the first hindlimb to the dissection chamber in a position in which the posterior face of the legs is visible. Remove the skin under magnification; then expose and remove the FDB (Figure 2). Store it in one labeled glass vial with 1 mL of Tyrode solution.
      NOTE: Appropriate magnification and previous training are required to avoid any undesired cut in the muscle tissue.
   4. Expose, remove, and store the soleus in a separate vial with 1 mL of Tyrode. Use fine scissors to first separate the gastrocnemius and then to remove the soleus, as indicated in Figure 2.
   5. Expose the anterior face of the leg, remove the skin, and identify the distal tendons of the tibialis anterior and the EDL muscles in the ankle. Remove and discard the tibialis; then cut the distal tendons of the EDL (Figure 2). Continue dissection until removing the EDL and place it in a separate glass vial with 1 mL of Tyrode.
   6. Remove the EHL muscle, which lies just posterior and medial to the EDL. Start dissection by identifying and following the tendon to the 1^st digit, as indicated in the corresponding panel of Figure 2. Keep the muscle in a separate glass vial with 1 mL of Tyrode.
   7. Identify and follow the most external tendon of the peronei to cut it and remove the PL muscle (Figure 2). Place the muscle in a separate glass vial with 1 mL of Tyrode.
   8. Identify and follow the tendon to the 4^th digit; cut it and remove the PDQA muscle (Figure 2). Place it in a separate glass vial with 1 mL of Tyrode.
   9. Repeat the procedure with the second hindlimb.
   10. Gather both muscles of the same type in one labeled glass vial or a small Petri dish with Tyrode solution.
       ​NOTE: If more than two pairs of muscles are planned to be dissected during a working session, recruit two researchers for the dissection procedure.

3. Muscle fiber isolation protocol

1. Renew the Tyrode solution in the dissection chambers to remove debris and mouse fur. Pour the FDB muscles into one dissection chamber, verify their integrity, and transfer them to a new glass vial with 1 mL of dissociation solution. Repeat this procedure with the EHL, PL, and PDQA muscles.
   NOTE: If a muscle looks hypercontracted, cut, or is unresponsive to the electrical stimulation, do not continue to the next protocol step. Instead, optimize the dissection protocol by verifying the quality of the solutions (pH, contamination, osmolarity) and gaining more dissection skills (Figure 1C and Supplemental Video S1).
2. Perform longitudinal or diagonal cuts to the soleus and EDL muscles, following the orientation of the fibers (Figure 2). For the soleus, follow the central tendon, cutting ~80% of its length. For EDL, just follow one or two tendons and cut about the same length as for soleus. Put each pair of muscles in glass vials with 1 mL of dissociation solution.
   NOTE: This procedure makes the EDL and soleus smaller and allows for the collagenase to better enter the tissue. Sufficient magnification (40-50x), as well as fine scissors and forceps, are mandatory. Always check sample integrity by visual inspection and electrical stimulation before continuing to the next step of the dissociation protocol.
3. Add 3 mg of collagenase type 2 (with an activity of 250-300 U/mg) to each vial containing 1 mL of dissociation solution and a pair of muscles. Standardize the exact amount of collagenase by considering the activity of the enzyme batch used.
4. Incubate the pairs of muscles in the water bath for 65-90 min at 36.8-37 °C, with gentle shaking.
   NOTE: Be rigorous with the temperature control. Standardize the procedure so that the muscles do not remain in collagenase for more than 100 min to avoid damage.
5. Check the vials under stereoscope magnification every 5 min after the 65^th min of incubation. When the muscles look slightly rippled, ragged, and loose, gently shake the vial and verify if some fibers start detaching readily. If this is the case, wash the muscles with Tyrode at room temperature to inactivate and remove the collagenase.
   NOTE: Washing must be done carefully, without touching the muscles with the pipettes. Start by adding 0.8 mL of Tyrode and then remove 0.8 mL of the solution. Repeat this procedure 4-5x and verify that the solution becomes fully transparent.
6. Separate more fibers from the bulk of the muscles with very gentle trituration in Tyrode with the help of the set of fire-polished Pasteur pipettes. Start by agitating the solution around the muscle with the widest pipette (5 mm tip) and then gently pull the muscles up into and out of the pipette 3-4x. When the muscle starts releasing fibers and becomes thinner, repeat the procedure with the next pipette (4 mm tip).
   ​NOTE: Fibers rendered via this procedure remain excitable and contract briskly for more than 24 h, as exemplified using PL, EDL, EHL, and soleus fibers in Supplemental Video S2, Supplemental Video S3, Supplemental Video S4, and Supplemental Video S5.

4. Experimental procedures

NOTE: Isolated fibers were used for sarcoplasmic Ca²⁺ concentration estimations, morphometric measurements, and myosin heavy chain (MHC) expression studies.

1. Measurement of the sarcoplasmic Ca²⁺ concentration during a twitch
   1. Mount a clean, glass slide on the experimental bath chamber. Coat the slide with 2-3 µL of laminin and allow it to dry for 30 s before pouring ~400 µL of the fiber suspension onto the slide. Allow the fibers to adhere to the laminin for 10-15 min at room temperature.
   2. Mount the experimental chamber onto the stage of an inverted microscope equipped for epifluorescence (Figure 3A).
   3. Evoke single twitches to verify the viability of the fibers by applying rectangular current pulses (0.8-1.2 ms) through the two platinum electrodes placed along either side of the experimental chamber. Even when attached to laminin, the contraction of the fibers is still visible mainly at the extremes.
   4. Load the fibers with 3.5-4.5 µM of the fast Ca²⁺ dye Mag-Fluo-4, AM for 4-5 min in Tyrode solution. After this time, gently wash with Tyrode to remove the extracellular dye. Allow the intracellular dye to be de-esterified for ~15-20 min under dark conditions. Always keep the temperature below 22 °C to avoid the dye compartmentalization.
      NOTE: Prepare a stock of Mag-Fluo-4, AM in dimethyl sulfoxide (DMSO) only, so that the final concentration of DMSO in the loading Tyrode solution is less than 0.5%.
   5. Illuminate the fiber with a white light-emitting diode (LED) and a filter set with the following wavelengths for excitation/dichroic/emission: 450-490/510/515 nm (Figure 3A).
      NOTE: Alternative sources of excitation include mercury and xenon fluorescence lamps. Use the lowest possible intensity and size of the excitation spot to avoid photobleaching of the dye and damage to the cell.
   6. Evoke the fiber´s Ca²⁺ response (sarcoplasmic Ca²⁺ transients) by applying rectangular current pulses (0.8-1.2 ms) through the two platinum electrodes placed along either side of the experimental chamber at 20-22 °C.
   7. Collect and save the light signals with an oil immersion 40x long-distance objective suitable for fluorescence and a photomultiplier tube (PMT) connected to a digitizer (Figure 3A and Supplemental Video S6). Ensure a scale in the acquisition software of 0-200 arbitrary units (AU) and set the resting fluorescence (F_rest) of the experiment to 10 AU on that scale by modulating the size of the excitation spot and the gain of the PMT. Once the procedure is standardized, keep the gain unmodified from one experiment to the other and set the scale only through minor adjustments in the spot size.
      NOTE: If movement artifacts arise, use 20-30 µM N-benzyl-p-toluene sulphonamide (BTS) in the Tyrode solution.
   8. Analyze and calibrate the signals as follows:
      1. Lowpass filter the whole trace at 1 kHz.
      2. Calculate the F_rest in 1 s of the trace, adjust the F_rest to 0, and measure the peak sarcoplasmic Ca²⁺ transients ' amplitude (F_peak). Present the amplitude as in equation (1):
            (1)
      3. Calculate the peak Ca²⁺ concentration ([Ca²⁺], µM) using equation (2)²⁶ and the following parameters: in situ dissociation constant (Kd) = 1.65 × 10⁵ µM², maximum fluorescence (F_max) of 150.9 AU, minimum fluorescence (F_min) of 0.14 AU, Mag-Fluo-4 concentration [D]_T of 229.1 µM²⁶. F_peak was already obtained in step 4.1.8.2.
            (2)
      4. Measure the rise time from 10% to 90% of the amplitude (RT, ms), the duration at half maximum (HW, ms), and the decay time from 90% to 10% of the amplitude (DT, ms). Then, estimate the decay kinetics according to a fit with the biexponential function (equation 3):
            (3)
      5. Save the values of the time constants of decay τ₁ and τ₂ (ms) and amplitudes A₁ and A₂²⁶.
2. Morphometric measurements
   1. Mount a clean, glass slide on the experimental bath chamber. Coat the slide with 2-3 µL of laminin and allow it to dry for 30 s before pouring ~400 µL of the fiber suspension onto the slide. Allow the fibers to adhere to the laminin for 10-15 min at room temperature.
   2. Evoke single twitches to verify the viability of the fibers by applying rectangular current pulses (0.8-1.2 ms) through the two platinum electrodes placed along either side of the experimental chamber. Even when attached to laminin, the contraction of the fibers is still visible mainly at the extremes.
   3. Acquire images of the alive fibers using 10x and 20x objectives and a camera of at least 5 megapixels mounted on an inverted fluorescence microscope. Store the images in .TIFF format for offline analyses.
      NOTE: A set of ~2-6 images may be needed to completely capture a long fiber.
   4. Image a microscope micrometer calibration ruler under the same magnification. Store the images in .TIFF format for offline analyses.
   5. Measure the lengths and diameters of the fibers using the calibration tool of free software for image analyses as follows:
      1. Establish a relation between pixels and the known distance (µm) in the images with the help of the microscope micrometer calibration ruler by using the Analyze/Set scale tool as shown in Supplemental Figure S1.
      2. Measure lengths once from one tip to the other of the fiber and diameters in 2-6 different places along the fiber (1-2 measurements per image, depending on its length), as in Supplemental Figure S1.
      3. Report the value of length (µm or mm) and the average of all diameters (µm) measured per fiber.
3. Myosin heavy chain expression studies
   NOTE: For details about the determination of MHC by immunofluorescence⁴³ and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)^33,44,45,46 in whole muscles, please see Supplemental File 1. The protocol for fiber typing by immunofluorescence determination of MHC in the suspension of FDB-isolated fibers is as follows:
   1. Coat each of five clean, glass slides with 2-3 µL of laminin and allow it to dry for 30 s before pouring ~300 µL of the fiber suspension onto each slide. Allow the fibers to adhere to the laminin for 4 h at room temperature.
   2. Fix the preparations with freezer-cooled acetone for 30 min at room temperature.
   3. Wash gently 3x with PBS.
   4. Permeabilize the cell membranes with PBS supplemented with 0.7% Triton X-100 for 15 min at room temperature.
   5. Wash gently 3x with PBS supplemented with 0.2% bovine serum albumin (BSA) and 0.04% Triton X-100, and subsequently block with PBS with 2% BSA, 2% goat serum, and 0.4% Triton X-100 for 30 min at room temperature.
   6. Wash gently 3x with PBS supplemented with 0.2% BSA and 0.04% Triton X-100 and incubate with the primary antibodies as follows:
      1. Dilute each anti-MHC primary antibody in a separate vial in PBS with 1% BSA and 0.04% Triton X-100: anti-I (1:1,500), anti-II (1:600), anti-IIA (use entire conditioned media from the hybridoma), and anti-IIB (1:500).
      2. Incubate each slide with one antibody and the remaining slide with PBS as a control for 12-16 h at 4 °C.
         NOTE: In this protocol, fibers type IIX remained unlabeled in all samples.
   7. Wash gently 3x with PBS and incubate all slides with the secondary antibody (1:800) coupled to a fluorescent green molecule for 1-2 h at room temperature.
   8. Stain nuclei with 1 µg/mL Hoechst for 15 min.
   9. Wash gently 3x with PBS, carefully add 20-40 µL of mounting medium, and place a coverslip.
      NOTE: Gentle solution exchanges and washing ensure that dozens of fibers remain attached to the slide, making the experiment statistically sound.
   10. Visualize each slide using a 10x objective suitable for fluorescence and a filter set with the following wavelengths for excitation/dichroic/emission: 450-490/510/515 nm and count all positive and negative fibers. Alternatively, acquire fluorescence images using the same technical conditions and a camera of at least 5 megapixels mounted on an inverted fluorescence microscope and store them in .TIFF format for offline analyses.
   11. Record the positive and negative fibers of each slide in a database and calculate the percentages of positive I, IIA, IIB, and total II fibers based on the total number of fibers present in the corresponding slide. Calculate the percentage of IIX fibers by subtracting the sum of IIA+IIB from the percentage of total II fibers. Estimate the percentage of hybrid I/IIA fibers by subtracting the sum of I+II from a value of 100%. Finally, subtract the percentage of hybrid cells from the total of I and II to have the pure type I and II fibers.
       NOTE: In MHC composition studies, fiber types are designated by a capital letter while isoforms are designated by a lowercase letter⁴⁶.
4. Hematoxylin and eosin staining
   1. Coat a clean, glass slide with 2-3 µL of laminin and allow it to dry for 30 s before pouring ~300 µL of the fiber suspension onto the slide. Allow the fibers to adhere to the laminin for 4 h at room temperature.
   2. Fix the preparation with Carnoy´s solution (60% absolute ethanol, 30% chloroform, 10% acetic acid) for 5 min at room temperature.
   3. Incubate with hematoxylin for 90 s.
   4. Wash gently 3x with tap water.
   5. Incubate with 1% eosin Y prepared in 70% ethanol for 30 s.
   6. Wash gently 3x with tap water.
   7. Immerse 3x into absolute ethanol.
   8. Incubate in xylol for 60 s.
   9. Add 20-40 µL of mounting medium and visualize with a conventional microscope. Acquire images at the desired magnification using a color camera of at least 5 megapixels.

5. Statistical analyses and graphing

NOTE: The experimental unit is a muscle fiber.

1. Express results as mean ± standard deviation and calculate confidence intervals 95% (CI95%) for some analyses.
2. To compare length, diameter, and Ca²⁺ transients´ kinetics among groups, perform analysis of variance (ANOVA) and post-hoc tests with the correction of Bonferroni.
3. Assess normality and variance equality using the Shapiro-Wilk and Levene's tests, respectively.
4. Consider the differences significant when p < 0.05.

Sarcoplasmic Ca²⁺ concentration during a twitch
To demonstrate the feasibility of physiological experiments in the set of dissociated fibers and to extend our previous findings on excitation-contraction coupling (ECC) and fiber types, Ca²⁺ transients were acquired in fibers from all muscles. First, FDB (n = 5) and EDL (n = 7) showed Ca²⁺ kinetics known as morphology type II (MT-II). These are fast, spiky signals, whose RT lasts ~1 ms; its decay phase can be fitted with a biexponential function with the first component (A₁) larger than 30% of the whole amplitude, and its peak [Ca²⁺] is between 15 and 30 µM^33,47 (Figure 3B). Their results are pooled (n = 12) in the first column of Table 1, while the results of the soleus´ Ca²⁺ transients (n = 6) are presented in the second column of Table 1. The soleus´ signals were classified as morphology type I (MT-I, Figure 3B) -wider, with an RT over 1.2 ms, an A₁ lower than 30%, and a peak [Ca²⁺] between 7 and 13 µM^33,47. These two columns were regarded as references for comparing the Ca²⁺ transients of the new muscles. Since all fibers from PDQA (n = 4), PL (n = 6), and EHL (n = 4) shared the MT-II (Figure 3B), their data are pooled (n = 14) in the third column of Table 1. These signals showed an average RT of ~1 ms, an A₁ of ~45%, a peak [Ca²⁺] over 15 µM, and compare very well with the results presented in the first column but clearly differ from those shown in the second column, as confirmed by the statistical analysis (Table 1). The fastest signal of the whole sample came from an EHL fiber, with a ΔF/F of 0.66, [Ca²⁺] of 16.99 µM, RT of 0.85 ms, HW of 2.42 ms, and DT of 10.56 ms. τ₁ and τ₂ were 1.63 and 7.21 ms, respectively, while A₁ and A₂ values were 56.60% and 43.40%. The slowest signal came from a soleus fiber, with a ΔF/F of 0.41, [Ca²⁺] of 9.76 µM, RT of 1.56 ms, HW of 9.43 ms, and DT of 31.88 ms. τ₁ and τ₂ were 2.81 and 96.42 ms, respectively, while A₁ and A₂ values were 19.55% and 80.45%.

A battery of short, intermediate, and long fibers
The observation of clear differences among the fibers according to their muscle source enabled a more complete morphometric characterization. The histograms of Figure 4 show striking variations in the lengths of the fibers across all muscles. This is highlighted when comparing the shortest fiber from FDB (227.06 µm) with the longest one from soleus (5.69 mm). The mean length values are summarized in Table 2. There were significant statistical differences among groups (p < 0.01). These results allow for the classification of FDB fibers as short (<1 mm); PDQA and PL as intermediate (1 to 3 mm); and EDL, EHL, and soleus as long (>3 mm) (Supplemental Figure S2).

Conversely, minor differences were observed in the average diameters of the fibers of all muscles (Table 2) and the distribution of the values (Figure 4). Still, there were significant statistical differences among groups (p < 0.01). When evaluated in detail, there were differences between FDB and each other muscle and between FDB (FDB 38.40 ± 9.40 µm, n = 370) and the pool of intermediate and long fibers (45.07 ± 9.99 µm, n = 422, p < 0.05). The thinnest cell of the whole sample measured 18.42 µm (FDB) and the thickest reached 82.79 µm (PDQA).

Fiber types used in physiological experiments
First, fiber types present in each whole muscle were determined by immunofluorescence. Except for the soleus, the muscles showed a predominance of over 76% of type II fibers (Supplemental Figure S3, Table 3, and Table 4). EHL, PL, and PDQA are particularly fast muscles, with over 90% of fast fibers and up to 58.8% of type IIB fibers, as found in PDQA. EHL and PDQA were virtually devoid of type I fibers. Hybrid I/IIA fibers were present in all muscles in low percentages. As expected, type I and IIA fibers accounted for over 82% of the total fibers of the soleus. This muscle is almost devoid of the fastest IIB fibers. According to the profile of fiber types, the soleus is the slowest and the PDQA is the fastest muscle of the six analyzed.

Thereafter, and because it is more relevant for single-fiber physiological experiments, the question of which types of fibers appear in the cell suspension derived from the dissociated FDB was addressed. It was found that the profile of fibers in the cell suspension reflects the composition of fibers present in the cryosections: three out of four dissociated fibers were type II (Figure 5 and Table 3).

Finally, the composition of the muscles was confirmed by separating their MHC isoforms through SDS-PAGE. The results were consistent with the general pattern observed in the immunofluorescence assays. The soleus was enriched in MHC IIa and I, while the EDL, EHL, and peronei were enriched in MHC IIx and IIb but were devoid of I (Supplemental Figure S4).

Figure 1: Design of the study. (A) Equipment and solutions to be set up before starting the dissection procedure. 1. Stereoscope number one. 2. From bottom to top: a set of five fire-polished pipettes, dissection tools, and collagenase vials inside a portable cooler (yellow case). 3. Filter case, dissection chamber, labeled glass vials with cap, rack with filtered solutions. 4. Dissection tools. 5. Stereoscope with dissection chamber numbers two coupled to a camera and an electric stimulator. (B) The dissection procedure of the set of six hindlimb muscles of mice as further explained in Figure 2. (C) After dissection, muscle contraction and integrity must be verified before continuing to the next protocol step. The left muscle in the left panel shows a wavy, hypercontracted, unresponsive PL muscle (blue arrow), which shall not be used to obtain dissociated fibers. Instead, the dissection protocol needs to be optimized and started again. The well-dissected PL counterpart (right muscle in the left panel) displays an elongated appearance and visibly contracts (Supplemental Video S1). The panel on the right shows two EDL (blue arrow) and two FDB muscles within the collagenase solution with the correct, straight appearance. (D) Once the muscles are dissociated, pour the isolated fibers into the experimental chamber and check their contraction and integrity. On the left panel, live (blue arrow) and dead PL fibers. On the right panel, live (blue arrow) and dead EDL fibers. (E-G) Three sets of experiments were performed with the isolated fibers: (E) Sarcoplasmic Ca²⁺ concentration measurements during a twitch (results in Figure 3). (F) Morphometric analyses (results presented in Figure 4). This example shows a PL fiber. (G) Immunofluorescence for myosin heavy chain expression studies (results illustrated in Figure 5). This example shows an isolated FDB fiber. Scale bars = 5 mm (B,C), 100 µm (D,F,G). Abbreviations: FDB = flexor digitorum brevis; PL = peroneus longus; EDL = extensor digitorum longus. Please click here to view a larger version of this figure.

Figure 2: Anatomical references and general procedure to dissect the set of six hindlimb muscles of mouse. Rows, from top to bottom, present the muscles according to the best order of dissection: FDB, soleus, EDL, EHL, PL, and PDQA. Columns, from left to right, present the milestones during dissection. The first column shows the muscles or their distal tendons exposed so the dissection can start. For instance, blue arrows point to the FDB and soleus in situ and to the distal tendons of the EDL. The following two columns illustrate the dissection itself and the muscles exposed after the distal tendon(s) is/are cut, as labeled, for example, with the blue arrow in the EDL row. It is recommended that the branch of the FDB directed to the fifth digit is removed. The rightmost column presents the muscles once completely removed, with the proximal tendons oriented to the upper part of the images. Blue, discontinuous lines (extreme right column) illustrate the longitudinal or diagonal cuts that need to be performed in the soleus and EDL muscles to ensure that the collagenase enters their bulk. Scale bars = 5 mm. Abbreviations: FDB = flexor digitorum brevis; PL = peroneus longus; EDL = extensor digitorum longus; EHL = extensor hallucis longus; PDQA = peroneus digiti quarti. Please click here to view a larger version of this figure.

Figure 3: Ca²⁺ transients recorded in fibers obtained from a set of six hindlimb muscles of mouse. (A) Setup used for the acquisition of Ca²⁺ transients under dimmed illumination. Left panel: 1. PMT connected to an inverted fluorescence microscope (2). 3. Camera coupled to the microscope. 4. Electric stimulator coupled to the experimental chamber. 5. The micromanipulation system can be used when electrophysiology and injection assays are planned to complement the Ca²⁺ transients acquisition. Middle panel: the experimental chamber (insert) with the loaded cells is mounted onto the stage of the microscope and illuminated with blue light (450-490 nm, blue arrow) to excite the dye. In this setup, items 1 to 5 are placed over an antivibration table and inside a Faraday´s cage. Right panel: Once the quality of contraction and dye loading is verified with the camera (6), the light emitted is directed to the PMT, the electrical stimulation starts, and the signal is fed to the digitizer (7) and to the acquisition software (8) to record the Ca²⁺ transient. The integrated function of these elements during a live experiment can be seen in Supplemental Video S6. (B) Representative, calibrated Ca²⁺ transients of FDB, PDQA, PL, EDL, EHL, and soleus fibers of mouse. The similar, rapid, narrow signals of FDB, PDQA, PL, EDL, and EHL confirm that they have MT-II already known to derive from IIX and IIB fibers, which are the most abundant in these muscles. In contrast, the soleus transient is slower and smaller, typical of MT-I, originally described in I and IIA fibers. The red curve over the EDL and soleus signals demonstrates that the decay phase of MT-I and MT-II can be fitted with a biexponential decay function. Calibration bars apply to all panels. Vertical bar = 2.5 µM of [Ca²⁺], horizontal bar = 10 ms. The color key at the bottom helps identify the muscles. Abbreviations: FDB = flexor digitorum brevis; PDQA = peroneus digiti quarti; PL = peroneus longus; EDL = extensor digitorum longus; EHL = extensor hallucis longus; PMT = photomultiplier tube; MT-I = morphology type I; MT-II = morphology type II. Please click here to view a larger version of this figure.

Figure 4: Morphometric measurements of the short, intermediate, and long fibers obtained by enzymatic dissociation of the set of six hindlimb muscles of mouse. Intact, healthy, representative fibers from each muscle are depicted in the leftmost column panels. Images were edited only to reduce background noise and enhance contrast, with no direct manipulation of the muscle fibers. Scale bars = 100 µm (all panels). The measurement protocol, as well as the appearance and quality of the complete long fibers, can be further viewed in Supplemental Figure S1. Middle column shows the distributions of lengths, ordered from the muscle that rendered the shortest fibers (FDB), to the one with the longest fibers (soleus). There is a continuum of lengths so that there is some overlap between muscles, spanning a total of ~5.5 mm. Diameter distributions are presented in the rightmost column panels. Most fibers are between 30 and 60 µm, with small differences among muscles. Test-retest analyses (n = 78 fibers, equivalent to 9.85% of the whole sample of n = 792) of the morphometric measurements showed very high reproducibility (mean coefficient of variation of 1.77%─confidence interval 95%, CI95%, 1.26-2.27%─mean coefficient of correlation of 0.99 (CI95% 0.98-0.99)), highlighting the good reliability of the results. Gaussian distribution curves were added with graphing licensed software. Supplemental Figure S2 shows bar plots of the mean values of length and diameter and the merges of length and diameter distributions of all muscles for easier comparison. Color key at the bottom helps identify the muscles. Abbreviations: FDB = flexor digitorum brevis; PDQA = peroneus digiti quarti; PL = peroneus longus; EDL = extensor digitorum longus; EHL, = extensor hallucis longus. Please click here to view a larger version of this figure.

Figure 5: Immunofluorescence assays for fiber types in the cell suspension in dissociated FDB muscles. Images of different fields show intact, dissociated FDB fibers adhered to the bottom of the slides. (A) An antimyosin II-positive fiber (green fluorescence, diagonal light blue arrow) clearly contrasts with a negative one (light blue arrowhead), demonstrating the discrimination power of the assay. The presence of both fibers in the image can be confirmed because of the labeling of the nuclei (blue fluorescence). (B) A different field shows another antimyosin II-positive fiber. (C) The correct identification of the myosin heavy chain in the A bands (green fluorescence) by the antibodies was confirmed using confocal microscopy. The most notorious transversal black striations correspond to the I bands, enriched in actin, and thus are expected not to be recognized by the antibodies. (D) Hematoxylin labels in purple the typical peripheral, abundant nuclei, and eosin labels the sarcoplasm of the fiber, showing an intact, mature cell. Scale bars = 50 µm (A,B,D); 10 µm (C). Abbreviation: FDB = flexor digitorum brevis. Please click here to view a larger version of this figure.

Table 1: Ca²⁺ transients' kinetics in enzymatically dissociated fibers obtained from a set of six hindlimb muscles of mouse. Values are mean ± standard deviation. p values correspond to the Analysis of variance test. ^*Significantly different from the FDB and EDL groups. ^†Significantly different from the PDQA, PL, and EHL groups. Abbreviations: F = fluorescence; [Ca²⁺] = cytosolic peak Ca²⁺ concentration; RT = rise time; HW = duration at half maximum; DT = decay time; τ₁ and τ₂ = time constants of decay; A₁ and A₂ = amplitudes of decay; FDB = flexor digitorum brevis; PDQA = peroneus digiti quarti; PL = peroneus longus; EDL = extensor digitorum longus; EHL = extensor hallucis longus; MT-I = morphology type I; MT-II = morphology type II.

Table 2: Morphometric measurements of the short, intermediate, and long fibers obtained by enzymatic dissociation of the set of six hindlimb muscles of mouse. Values are mean ± standard deviation. p values correspond to the Analysis of variance test. ^*Statistically different from PDQA, PL, EDL, EHL, and soleus. ^**Significantly different from PL, EDL, EHL, and soleus. ^†Significantly different from EDL, EHL, and soleus. ^††Significantly different from EHL and soleus. ^‡Significantly different from soleus. ^¶Significantly different from EHL. Abbreviations: FDB = flexor digitorum brevis; PDQA = peroneus digiti quarti; PL = peroneus longus; EDL = extensor digitorum longus; EHL = extensor hallucis longus.

Table 3: Fiber types in cryosections and in the cell suspension in dissociated FDB muscles of mouse. Values are mean ± standard deviation. N refers to the number of animals, while n refers to the total number of fibers analyzed in the experiments. FDB row presents data of the whole muscle as determined in cryosections, while *FDB row corresponds to isolated fibers in the cell suspension after dissociating the muscle. The "Total type II" column reflects pure fibers type IIA, IIX, and IIB. Abbreviation: FDB = flexor digitorum brevis.

Table 4: Fiber types in cryosections of the muscles which give intermediate and long fibers of mouse. Values are mean ± standard deviation. N refers to the number of animals, while n refers to the total number of fibers analyzed in the experiments. All rows present data of the whole muscle as determined in cryosections. Type I, IIA, IIX, and IIB columns reflect pure fibers, while the I/IIA column refers to hybrid fibers. The "Total type II" column is the summation of the type IIA, IIX, and IIB columns. Abbreviations: PDQA = peroneus digiti quarti; PL = peroneus longus; EDL = extensor digitorum longus; EHL = extensor hallucis longus.

Supplemental File 1: Myosin heavy chain expression studies in whole muscles. The protocols present details for the determination of myosin heavy chain isoforms by immunofluorescence in cryosections and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in homogenates of the whole muscles. Please click here to download this File.

Supplemental Figure S1: Details of the morphology of the fibers obtained by enzymatic dissociation of the set of six hindlimb muscles of mouse. (A) The microscope micrometer calibration ruler open to set the scale in the image analyses software is shown on the left. The right panel shows how the diameter was repeatedly measured as indicated by the blue lines perpendicular to the long axis of the fiber (blue arrows), while the length was measured once from tip to tip as illustrated by the blue line along the major axis of the fiber. (B) Several images were assembled with minor editing to demonstrate the long and healthy appearance of PDQA, PL, EDL, EHL, and soleus fibers (small blue, green, yellow, orange, and pink inserts). The assembled images were further edited to give the fibers a better look (large, black, and white images). (C) DIC images of FDB, PDQA, PL, and soleus fibers highlighting the normal irregularity of their tip and their well-known transversal striations. DIC appearance of the remaining EDL and EHL fibers can be seen in Supplemental Video S3 and Supplemental Video S4. Scale bars = 100 µm. The color key at the bottom helps identify the muscles. Abbreviations: FDB, flexor digitorum brevis; PDQA, peroneus digiti quarti; PL, peroneus longus; EDL, extensor digitorum longus; EHL, extensor hallucis longus; DIC = Differential interference contrast. Please click here to download this File.

Supplemental Figure S2: Bar plots and merged length and diameter distributions of the fibers obtained by enzymatic dissociation of the set of six hindlimb muscles of mouse. (A) Bar plots (mean ± standard deviations) and (B) merged histograms confirm the disparity of the lengths but the similarity of the diameters across all groups. ^*Significantly different from PDQA, PL, EDL, EHL, and soleus. ^**Significantly different from PL, EDL, EHL, and soleus. ^†Significantly different from EDL, EHL, and soleus. ^††Significantly different from EHL and soleus. ^‡Significantly different from soleus. ^¶Significantly different from EHL. The color key at the bottom helps identify the muscles. Abbreviations: FDB = flexor digitorum brevis; PDQA = peroneus digiti quarti; PL = peroneus longus; EDL = extensor digitorum longus; EHL = extensor hallucis longus. Please click here to download this File.

Supplemental Figure S3: Immunofluorescence studies for fiber type composition of the set of six hindlimb muscles of mouse. Representative antibody-labeled cryosections used for the analyses demonstrate the good quality and intactness of the samples. There is a clear predominance of fiber type II in all muscles, except for the soleus in which the amount of fiber type I is sizeable. Scale bars = 100 µm. The color key at the bottom helps identify the muscles. Abbreviations: FDB = flexor digitorum brevis; PDQA = peroneus digiti quarti; PL = peroneus longus; EDL = extensor digitorum longus; EHL = extensor hallucis longus. Please click here to download this File.

Supplemental Figure S4: Electrophoretic separation of MHC isoforms in the set of six hindlimb muscles of mouse. (A) Two representative complete gels run on different days with different samples demonstrate the quality, cleanness, and reproducibility of the separation and migration distance of the MHC isoforms when stained with Coomassie blue. Although these two images were slightly edited to enhance contrast and clarity for the reader, the analyses were performed in unedited gels. (B) Examples of the analyses of the bands for each of the six muscles. After an ROI is selected in the lanes of the gel, a plot profile is generated and a Gaussian fit is used to estimate the proportion of MHC isoforms in the set of six muscles. The MHC composition found was: FDB: I 9.0 ± 5.0%, IIa + IIx 91.0 ± 5.0% (n = 3); PDQA: IIa + IIx 25.9 ± 2.4%, IIb 74.1 ± 2.4% (n = 3); PL: IIa + IIx 24.9 ± 2.2%, IIb 75.1 ± 2.2% (n = 3); EDL: IIa + IIx 22.0 ± 2.3%, IIb 78.0 ± 2.3% (n = 4); EHL: IIa + IIx 27.5 ± 1.0%, IIb 72.5 ± 1.0% (n = 3); soleus: I 35.8 ± 2.5%, IIa (+IIx + IIb when present) 64.2 ± 2.5% (n = 4). The color key at the bottom helps identify the muscles. The gray rectangle below the rightmost gel in A refers to the molecular weight marker indicating the migration of the IIa to ~ 200 KDa. Abbreviations: FDB = flexor digitorum brevis; PDQA = peroneus digiti quarti; PL = peroneus longus; EDL = extensor digitorum longus; EHL = extensor hallucis longus; MHC = myosin heavy chain; ROI = region of interest. Please click here to download this File.

Supplemental Video S1: The damaged, wavy, peroneus longus (PL, left) does not contract upon stimulation, while the intact PL (right) contracts well, even when it is farther from the electrodes. 0.8x magnification. The stimulation protocol releases rectangular current pulses of 1.2 ms, 0.7 Hz, and 50 V through two electrodes. Please click here to download this Video.

Supplemental Video S2: Contracting peroneus longus fiber. 20x magnification. Differential interference contrast mode. The stimulation protocol releases rectangular current pulses of 1.2 ms, 0.5 Hz, and 50 V through two platinum electrodes. Please click here to download this Video.

Supplemental Video S3: Contracting extensor digitorum longus fiber. 20x magnification. Differential interference contrast mode. The stimulation protocol releases rectangular current pulses of 1.2 ms, 0.5 Hz, and 50 V through two platinum electrodes. Please click here to download this Video.

Supplemental Video S4: Contracting extensor hallucis longus fiber. 20x magnification. Differential interference contrast mode. The stimulation protocol releases rectangular current pulses of 1.2 ms, 0.5 Hz, and 50 V through two platinum electrodes. Please click here to download this Video.

Supplemental Video S5. Contracting soleus fiber. 20x magnification. Differential interference contrast mode. The stimulation protocol releases rectangular current pulses of 1.2 ms, 0.5 Hz, and 50 V, through two platinum electrodes. Please click here to download this Video.

Supplemental Video S6: Live recording of a Ca²⁺ transient from an extensor digitorum longus fiber loaded with Mag-Fluo-4, AM as described in step 4.1.4. The stimulation protocol releases rectangular current pulses of 1.2 ms, 0.5 Hz, and 50 V through two platinum electrodes. Please click here to download this Video.

To complement the models available for studying mature skeletal muscle biology, here we demonstrate the successful enzymatic dissociation of a range of mouse muscles with short, intermediate, and long fibers. These fibers allow for the demonstration of the generalizability of the MT-II kinetics of the Ca²⁺ transients in skeletal muscle. Further, the fiber types in the intact, whole muscles were classified. Given that the FDB is the most used muscle for physiological experiments, the types of fibers present in the cell suspension after dissociation were assessed.

A battery of short, intermediate, and long intact fibers for muscle biology studies
The dissection technique, the duration of the enzymatic treatment, the type of enzyme used, and the trituration procedure are critical steps within the protocol to obtain intact enzymatically dissociated fibers. The dissection must be done as fast as possible to reduce the time under hypoxia while avoiding unnecessary stretching. The latter point is particularly relevant for the soleus, which must not be removed by pulling up the gastrocnemius-soleus complex. Moreover, an extensive enzymatic treatment (~3 h)^20,48 and poor trituration procedures (e.g., harsh, low experience of the researcher) damage the cells, as confirmed empirically in several laboratories. Collagenase type 2 is recommended over other collagenase types. The formation of bubbles during trituration must be avoided and either the muscles or the fibers must be manipulated only with glass pipettes instead of plastic pipette tips. The use of glass vials is preferred during the whole procedure over plastic, conic vials, because the first give more visibility, can be placed upwards on the stereoscope for a top-view observation of the muscle when in the solutions, give more space for the trituration of the muscles longer and bulkier than the FDB, and are resistant to scratches caused by the glass Pasteur pipettes. Since age, weight, and metabolic condition (e.g., healthy vs. high-fat obesity) affect the efficiency of the method, several parameters could need optimization when working with animals out of the range used in this manuscript. Importantly, a mild exposure of a muscle to certain enzymes such as those used here and a correct dissociation procedure do not functionally affect the fibers, as shown by evidence gathered during decades by multiple groups, some of which was summarized a few years ago⁴⁹. This is further supported by the fact that the peak [Ca²⁺] measured with fast Ca²⁺ dyes during a twitch obtained in manually dissected bundles and enzymatically dissociated fibers of mice can be considered comparable (reviewed in ⁴⁷).

Although there are other models for muscle biology studies, such as C2C12 myotubes, normal and mutated human myoblast cell lines, or pluripotent stem cell (iPSC)-derived fibers^{31,39,50,51,52,53}, they are immature and are not discussed here. Permeabilized or skinned fibers are also informative, well-characterized models^54,55, but they are not intact. The model of manually isolated fibers offers several advantages as presented elsewhere²⁰ but has low efficiency and requires high training. These facts highlight that the model of interest here-fibers obtained by enzymatic dissociation-offers the possibility of obtaining intact, abundant, mature skeletal muscle fibers of different types. One limitation of the model, though, is that the lack of tendons limits the direct assessment of contractile properties in the isolated fibers.

Given that the length of the fibers is not the same as the length of the muscles^14,42 and that the fibers are the experimentation unit, it is more relevant to classify the length of the fibers, not of the muscles. Given our results and their potential experimental implications, the FDB fibers can be classified as short (<1 mm); PDQA and PL as intermediate (1 to 3 mm); and EDL, EHL, and soleus as long (>3 mm). Short fibers are the easiest and most abundantly obtained (~400-500 fibers per mouse) and are suitable for fluorescence microscopy experiments in which the attachment of the fiber to the bottom chamber is key, or when movement artifacts must be avoided (e.g., Ca²⁺ transients). Long fibers are the most difficult to obtain, have the lowest rendering, and are better for protein expression using separation techniques or single-cell molecular biology studies. Intermediate fibers can be obtained in a good quantity (~50 fibers per mouse) after moderate training and are suitable for many experimental approaches, as shown for instance with the Ca²⁺ transients' experiments. Acknowledging that FDB, EDL, and soleus have been used before, this is the first work to successfully isolate fibers from the PDQA, PL, and EHL muscles, thus, increasing the availability of models for mature skeletal muscle studies. Moreover, obtaining fibers from different muscles ensures that more information is obtained from the same animal, reducing experimental variability, increasing statistical power and biological soundness of the conclusions, and reducing the number of animals used in experimentation.

Generalizability of Ca²⁺ transients´ kinetics
When analyzing the kinetics of the Ca²⁺ transients in mammalian mature skeletal muscle fibers, we have previously shown that fibers type I and IIA share the so-called morphology type I (MT-I), while fibers IIX and IIB share the MT-II morphology. Typically, MT-II signals have an RT of less than 1.2 ms, DT of less than 25 ms, A₁ higher than 30%, [Ca²⁺] over 15 µM, and are easily found in FDB and EDL fibers^33,47. After comparing the results of the novel Ca²⁺ transients of PDQA, PL, and EHL with those found here and those previously published for FDB and EDL, it is straightforward to conclude that they are all MT-II as well. A further interesting observation is that increasing the availability of new muscles enriched in fibers type IIX and IIB as models to obtain dissociated fibers allows one to confirm that the kinetics of the Ca²⁺ transients can be generalized-type IIX and IIB fibers have the MT-II regardless of their source (i.e., flexors (FDB), extensors (EDL and EHL), or peronei (PDQA and PL)). This strengthens the idea of an established gene cellular program or profile of the ECC machinery, which is rather similar within all fibers of the same type, and that differences in the molecular machinery (i.e., isoforms and protein quantity) that underly the generation of Ca²⁺ transients explain the differences in morphologies reported among fibers³³. Lastly, a practical implication is that by recording with Mag-Fluo-4 the Ca²⁺ transient of a fiber, it is possible to reliably know its type.

MHC expression in muscles and isolated fibers: implications for types of fibers used in physiological experiments
Knowing the type of fiber increases the reliability of muscle research. For instance, in knockout mice of any protein differentially expressed according to fiber types, using fibers of the FDB without any typing may lead to misleading results. Our data confirm that the FDB is a mixed muscle with a predominance of type IIX fibers, in agreement with previous papers ^25,31,56. More importantly, there was a high concordance between the proportion of fiber types present in the whole muscle and that obtained after dissociation. Since this information is novel and is not usually discussed in papers using FDB fibers, it can be speculated that, by chance, a lot of results gained in dissociated FDB fibers may truly reflect mixed information of ~20-25% coming from fibers type I and 75-80% from type II. Future studies using FDB muscle ought to present data and their corresponding discussion, relative to fiber types.

Since soleus is highly enriched in oxidative type I, IIA, and I/IIA fibers^57,58,59, these are the fibers found in the suspension after dissociation³³. EDL, another well-known muscle, is almost devoid of type I fibers^57,58,59, therefore rendering only fast fibers after dissociation³³. Following the same logic and according to the typing results showing enrichment of type II fibers in the three novel muscles, further supported by the fact that the few type I fibers found in peronei muscles of mouse are central⁶⁰ and not expected to be reached during a mild dissociation, we hypothesize that the probability of employing a fast IIX or IIB fiber in an experiment with dissociated fibers of PDQA, PL, or EHL can be as high as 80-90%. This seems to be true according to the Ca²⁺ transients' data in which no MT-I signals were found. Given their size, these fibers offer the advantage of being suitable for typing after finishing a functional experiment. Further, BTS´s specificity for MHC II helps discriminate among fiber types while removing movement artifacts. Finally, while this fiber typing method was more laborious than recently described optimized ones^61,62, it did not affect the results of the present work.

Conclusions
The presented methodological details may foster the use of a variety of enzymatically dissociated muscle fibers in the study of muscle biology. The versatility of the model is supported by the possibility of getting fibers within a large range of lengths and types and their use across several experimental applications. Besides FDB, EDL, and soleus whose dissociation was reported years ago, we demonstrated the feasibility of obtaining intermediate and long fibers from other muscles such as PDQA, PL, and EHL. Given the ease with which the dissection can be performed and the number of fibers that can be obtained, as well as the homogeneity and size of the fibers, we propose PL as a routine, suitable model for mature skeletal muscle studies. This is supported by our findings that the kinetics of the Ca²⁺ transients in skeletal muscle can be generalized regardless of their source.