We describe a protocol to obtain enzymatically dissociated fibers of different lengths and types from six muscles of adult mice: three of them already described (flexor digitorum brevis, extensor digitorum longus, soleus) and three of them successfully dissociated for the first time (extensor hallucis longus, peroneus longus, peroneus digiti quarti).
Skeletal muscle fibers obtained by enzymatic dissociation of mouse muscles are a useful model for physiological experiments. However, most papers deal with the short fibers of the flexor digitorum brevis (FDB), which restrains the scope of results dealing with fiber types, limits the amount of biological material available, and impedes a clear connection between cellular physiological phenomena and previous biochemical and dynamical knowledge obtained in other muscles.
This paper describes how to obtain intact fibers from six muscles with different fiber type profiles and lengths. Using C57BL/6 adult mice, we show the muscle dissection and fiber isolation protocol and demonstrate the suitability of the fibers for Ca2+ transient studies and their morphometric characterization. The fiber type composition of the muscles is also presented. When dissociated, all muscles rendered intact, living fibers that contract briskly for more than 24 h. FDB gave short (<1 mm), peroneus digiti quarti (PDQA) and peroneus longus (PL) gave intermediate (1-3 mm), while extensor digitorum longus (EDL), extensor hallucis longus (EHL), and soleus muscles released long (3-6 mm) fibers.
When recorded with the fast dye Mag-Fluo-4, Ca2+ transients of PDQA, PL, and EHL fibers showed the fast, narrow kinetics reminiscent of the morphology type II (MT-II), known to correspond to type IIX and IIB fibers. This is consistent with the fact that these muscles have over 90% of type II fibers compared with FDB (~80%) and soleus (~65%). Moving beyond FDB, we demonstrate for the first time the dissociation of several muscles, which render fibers spanning a range of lengths between 1 and 6 mm. These fibers are viable and give fast Ca2+ transients, indicating that the MT-II can be generalized to IIX and IIB fast fibers, regardless of their muscle source. These results increase the availability of models for mature skeletal muscle studies.
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.
All procedures were approved by the Committee for Ethics in Experiments with animals of the University of Antioquia (UdeA) (minutes 104 of June 21st, 2016, and 005 of April 15th, 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
2. Dissection
3. Muscle fiber isolation protocol
4. Experimental procedures
NOTE: Isolated fibers were used for sarcoplasmic Ca2+ concentration estimations, morphometric measurements, and myosin heavy chain (MHC) expression studies.
5. Statistical analyses and graphing
NOTE: The experimental unit is a muscle fiber.
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 µM33,47 (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 µM33,47. 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.
Fibers | FDB and EDL | Soleus | PDQA, PL and EHL | p |
n | 12 | 6 | 14 | |
ΔF/F | 0.68±0.15 | 0.46±0.06*,† | 0.66±0.12 | <0.01 |
[Ca2+] (µM) | 17.46±5.18 | 11.14±1.86*,† | 16.82±3.29 | <0.01 |
RT (ms) | 0.95±0.10 | 1.26±0.20*,† | 0.95±0.09 | <0.01 |
Rise slope (F/ms) | 5.97±1.41 | 3.12±0.79*,† | 5.83±0.97 | <0.01 |
HW (ms) | 3.59±0.85 | 8.17±3.00*,† | 3.19±0.54 | <0.01 |
DT (ms) | 16.98±7.37 | 27.26±7.13*,† | 11.32±2.06* | <0.01 |
Decay slope (F/ms) | -0.24±0.07 | -0.10±0.03*,† | -0.35±0.08* | <0.01 |
τ1 (ms) | 1.86±0.37 | 2.07±0.66† | 1.57±0.18 | <0.05 |
τ2 (ms) | 11.50±3.93 | 46.38±27.14*,† | 8.29±2.14 | <0.01 |
A1 (%) | 48.14±7.27 | 25.94±8.98*,† | 44.59±8.29 | <0.01 |
A2 (%) | 51.86±7.27 | 74.06±8.98*,† | 55.41±8.29 | <0.01 |
Morphology | MT-II | MT-I | MT-II |
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.
Fibers | FDB | PDQA | PL | EDL | EHL | Soleus | p |
n | 370 | 142 | 80 | 70 | 87 | 43 | |
Length (mm) | 0.42±0.05* | 2.20±0.26** | 2.69±0.26† | 3.51±0.53†† | 3.83±0.44‡ | 4.62±0.64 | <0.01 |
Diameter (µm) | 38.40±9.40* | 46.39±9.50¶ | 45.98±11.18 | 44.43±7.62 | 41.96±9.16 | 46.36±12.85 | <0.01 |
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.
Muscle/Fibers | N | n | Total type I + I/IIA (%) | Total type II (%) |
FDB | 5 | 747 | 21.94±11.47 | 78.06±11.47 |
*FDB | 8 | 1483 | 23.46±2.51 | 76.54±2.51 |
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.
Muscle | N | n | Type I (%) | Type I/IIA (%) | Type IIA (%) | Type IIX (%) | Type IIB (%) | Total type II (%) |
PDQA | 4 | 597 | 0.00±0.00 | 10.15±2.62 | 19.33±6.19 | 11.68±7.57 | 58.84±7.63 | 89.85±2.62 |
PL | 4 | 576 | 3.44±3.94 | 2.04±2.48 | 23.01±7.03 | 35.85±5.66 | 35.66±9.36 | 94.52±3.90 |
EDL | 4 | 826 | 0.00±0.00 | 10.44±8.33 | 5.67±5.07 | 24.75±10.93 | 59.15±11.36 | 89.56±8.33 |
EHL | 5 | 618 | 0.24±0.76 | 5.29±3.31 | 19.04±6.83 | 21.18±10.91 | 54.25±11.73 | 94.47±3.05 |
Soleus | 4 | 729 | 32.18±4.48 | 1.74±1.30 | 50.37±7.46 | 14.58±6.94 | 1.12±1.93 | 66.08±5.59 |
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 fibers31,39,50,51,52,53, they are immature and are not discussed here. Permeabilized or skinned fibers are also informative, well-characterized models54,55, 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 muscles14,42 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 fibers33,47. 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 fibers57,58,59, these are the fibers found in the suspension after dissociation33. EDL, another well-known muscle, is almost devoid of type I fibers57,58,59, 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 ones61,62, 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.
The authors have nothing to disclose.
The authors express their gratitude to Professor Robinson Ramírez from UdeA for help with animals and some photos and to Carolina Palacios for technical support. Johan Pineda from Kaika helped us to set up the color and fluorescence cameras. Shyuan Ngo, from the University of Queensland, kindly proofread the manuscript. This study was funded by the CODI-UdeA (2020-34909 from February 22nd, 2021, and 2021-40170 from March 31st, 2022, SIU), and Planning Office-UdeA (E01708-K and ES03180101), Medellín, Colombia, to JCC. Funders did not participate in data collection and analysis, manuscript writing or submission.
Reagents | |||
Absolute ethanol | Sigma Aldrich | 32221 | |
Acetone | Merck | 179124 | |
Acrylamide | Gibco BRL | 15512-015 | |
Ammonium persulfate | Panreac | 141138.1610 | |
Anti myosin I antibody | Sigma Aldrich | M4276 | Primary antibody |
Anti myosin II antibody | Sigma Aldrich | M8421 | Primary antibody |
Anti myosin IIA antibody | American Type Culture Collection | SC-71 | Primary antibody. Derived from HB-277 hybridoma |
Anti myosin IIB antibody | Developmental Studies Hybridoma Bank | BF-F3-c | Primary antibody |
Bis-acrylamide | AMRESCO | 0172 | |
Bovine serum albumin | Thermo Scientific | B14 | |
Bradford reagent | Merck | 1.10306.0500 | |
Bromophenol blue | Carlo Erba | 428658 | |
Calcium carbonate | Merck | 102066 | |
Calcium dichloride (CaCl2) | Merck | 2389 | |
Chloroform | Sigma Aldrich | 319988 | |
Collagenase type 2 | Worthington | CLS-2/LS004176 | |
Consul-Mount | Thermo Scientific | 9990440 | |
Coomassie Brilliant blue R 250 | Merck | 112553 | |
Dimethyl sulfoxide (DMSO) | Sigma Aldrich | D2650 | |
Dithiothreitol (DTT) | AMRESCO | 0281 | |
Edetic acid (EDTA | AMRESCO | 0322 | |
Eosin Y | Sigma Aldrich | E4009 | |
Glycerol | Panreac | 1423291211 | |
Glycine | Panreac | 151340.1067 | |
Goat serum | Sigma Aldrich | G9023 | |
Hematoxylin | Thermo Scientific | 6765015 | |
HEPES | AMRESCO | 0511 | |
Hoechst 33258 | Sigma Aldrich | 861405 | |
Imidazole | AMRESCO | M136 | |
Isopentane | Sigma Aldrich | M32631 | |
Laminin | Sigma Aldrich | L2020 | |
Mag-Fluo-4, AM | Invitrogen | M14206 | Prepared only in DMSO. Pluronic acid is not required and should not be used to avoid fiber deterioration. |
Mercaptoethanol | Applichem | A11080100 | |
Methanol | Protokimica | MP10043 | |
Mice | Several | Several | For this manuscript, we only used C57BL/6 mice. However, some preliminary results have shown that the protocol works well for Swiss Webster mice of the same age and weight. |
Mowiol 4-88 | Sigma Aldrich | 81381 | |
N,N,N',N'-tetramethylethane-1,2-diamine (TEMED) | Promega | V3161 | |
N-benzyl-p-toluene sulphonamide (BTS) | Tocris | 1870 | |
Optimal cutting compound (OCT) | Thermo Scientific | 6769006 | |
Secondary antibody | Thermo Scientific | A-11001 | Goat anti-mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 |
Sodium dodecil sulfate | Panreac | 1323631209 | |
TRIS 0.5 M, pH 6.8 | AMRESCO | J832 | |
Tris(Hydroxymethyl)aminomethane | AMRESCO | M151 | |
Triton X-100 | AMRESCO | M143 | |
Materials | |||
Dissection chamber | Custom-made | ||
Charged slides | Erie Scientific | 5951PLUS | |
Experimental bath chamber | Warner Instruments | RC-27NE2 | Narrow Bath Chamber with Field Stimulation, ensembled on a heated platform PH-6 |
Fine forceps | World Precision Instruments | 500338, 500230 | |
Fine scissors | World Precision Instruments | Vannas Scissors 501778 | |
Glass Pasteur pipettes | Several | Fire-polished tips | |
Glass vials with cap | Several | 2-3 mL volumen | |
Operating scissors | World Precision Instruments | 501223-G | |
Equipment | |||
Centrifuge | Thermo Scientific | SL 8R | |
Confocal microscope | Olympus | FV1000 | |
Cryostat | Leica | CM1850 | |
Digital camera | Zeiss | Erc 5s and Axio 305 | Axio 305, coupled to the Stemi 508 stereoscope, was used to take pictures during dissection; while Erc 5s or Axio 208, coupled to the Axio Observer A1 microscope, were used to take images of the isolated fibers and the immunofluorescence assays |
Digitizer | Molecular Devices | 1550A Digidata | |
Electrophoresis chamber | Bio Rad | Mini-Protean IV | |
Inverted microscope coupled to fluorescence | Zeiss | Axio Observer A1 | Coupled to an appropriate light source, filters and objectives for fluorescence |
Photomultiplier | Horiba | R928 tube, Hamamatsu, in a D104 photometer, Horiba | Coupled to the lateral port of the fluorescence microscope |
Stereoscope | Zeiss | Stemi 508 | |
Stimulator | Grass Instruments | S6 | |
Water bath | Memmert | WNE-22 | |
Xilol | Sigma Aldrich | 808691 | |
Software | |||
Free software for electrophoreses analyses | University of Kentucky | GelBandFitter v1.7 | http://www.gelbandfitter.org |
Free software for image analysis and morphometry | National Institutes of Health | ImageJ v1.54 | https://imagej.nih.gov/ij/index.html |
Licensed software for Ca2+ signals acquisition and analyses | Molecular Devices | pCLAMP v10.05 | https://www.moleculardevices.com |
Licensed software for statistical analyses and graphing | OriginLab | OriginPro 2019 | https://www.originlab.com/ |