Analytical Biochemistry 482 (2015) 22–24
Contents lists available at ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
Notes & Tips
Protocol for rat single muscle fiber isolation and culture Yusuke Komiya a, Judy E. Anderson b, Mariko Akahoshi a, Mako Nakamura a, Ryuichi Tatsumi a, Yoshihide Ikeuchi a, Wataru Mizunoya a,⇑ a b
Department of Bioresource Sciences, Faculty of Agriculture, Kyushu University, Higashi-ku, Fukuoka 812-8581, Japan Department of Biological Sciences, University of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada
a r t i c l e
i n f o
Article history: Received 1 December 2014 Received in revised form 20 March 2015 Accepted 24 March 2015 Available online 23 April 2015 Keywords: Skeletal muscle Muscle fiber Primary culture Rat
a b s t r a c t To attain a superior in vitro model of mature muscle fibers, we modified the established protocol for isolating single muscle fibers from rat skeletal muscle. Muscle fiber cultures with high viability were obtained using flexor digitorum brevis muscle and lasted for at least 7 days. We compared the expression levels of adult myosin heavy chain (MyHC) isoforms in these single muscle fibers with myotubes formed from myoblasts; isolated fibers contained markedly more abundant adult MyHC isoforms than myotubes. This muscle fiber model, therefore, will be useful for studying the various functions and cellular processes of mature muscles in vitro. Ó 2015 Elsevier Inc. All rights reserved.
Skeletal muscle, the largest organ in the human body, governs our metabolic and locomotive ability. It comprises bundles of muscle fibers, the multinucleated cells formed by the fusion of a large number of differentiated myogenic cells during development. Study of skeletal muscle function is expected to contribute to our understanding of how to alleviate sarcopenia and various metabolic syndromes. Myotubes, differentiated from myogenic cell lines such as L6 (derived from rat) and C2C12 (derived from mouse) or cultures of muscle satellite cells isolated from skeletal muscles of animals, are often used as in vitro models of skeletal muscle fibers. Previously, Ravenscroft and coworkers highlighted the murine isolated single muscle fiber culture system as a more mature culture system for adult skeletal muscle [1]. Although this idea has merit, rat fibers have not yet been tested despite the prevalence of rat tissue use in the skeletal muscle field. Here, we endeavored to modify an established protocol for isolating rat single muscle fibers to achieve long-term culture. Furthermore, we examined the validity of fiber cultures as a model for mature muscle tissue by comparing our model with myotubes differentiated from myoblast cultures. Muscle function is underpinned by a contractile protein called myosin that is composed of two heavy chains and four light-chain subunits. To date, four isoforms of myosin heavy chain (MyHC)1 expressed in adult skeletal muscle tissues have been ⇑ Corresponding author. Fax: +81 92 642 2951. E-mail address: [email protected] (W. Mizunoya). Abbreviations used: MyHC, myosin heavy chain; FDB, flexor digitorum brevis; PRS, physiological rodent saline; FBS, fetal bovine serum; PM, proliferation medium; DMEM, Dulbecco’s modified Eagle’s medium; MM, maintenance medium; EDL, extensor digitorum longus; calcein–AM, calcein–acetoxymethyl ester; mRNA, messenger RNA; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis. 1
http://dx.doi.org/10.1016/j.ab.2015.03.034 0003-2697/Ó 2015 Elsevier Inc. All rights reserved.
classified as MyHC1, -2A, -2X, and -2B (listed from slow-twitch to fast-twitch myosin). In myotubes formed from differentiated C2C12 cells, MyHC1, -2A, -2X, and -2B—the adult isoforms—are much less abundantly expressed than in normal skeletal muscle tissue [2]. Instead, the predominant isoforms are embryonic and neonatal MyHC, which are immature developmental isoforms. This is a critical issue in analyzing muscle fiber type because the adult MyHC isoforms are the common fiber type marker proteins. Primary culture of isolated muscle fibers is the most suitable model for mature muscle fibers. The initial method of rodent fiber isolation was pioneered by Bekoff and Betz [3] and later established by Bischoff [4]. A detailed description of the procedure was published recently [5–7]. This method, involving isolated single muscle fibers, has largely been used to take and examine muscle satellite cells resident on muscle fibers and isolated together with fibers from muscle tissue. These satellite cells, along with fibers, have been examined for gene expression [6,7], myogenesis [6], regenerative capacity [4], and the activation processes [4,6]. It remains uncertain whether isolated fibers are a useful in vitro model for studying mature muscle fiber properties. In this study, single muscle fibers were prepared from flexor digitorum brevis (FDB) muscle in the sole of the rat foot by modification of the method published by Anderson and coworkers [6] and Ravenscroft and coworkers [1] using mice. The major modifications were enzymatic digestion and the method of transferring fibers during medium changes. F344 male rats (3–4 weeks old) were sacrificed by cervical dislocation under inhalation anesthesia with sevoflurane, and muscles were dissected from the hind leg. First, we placed the
Notes & Tips / Anal. Biochem. 482 (2015) 22–24
dissected FDB muscle in physiological rodent saline (PRS: 138 mM NaCl, 2.7 mM KCl, 1.8 mM CaCl2, 1.06 mM MgCl2, 12.4 mM Hepes, and 5.6 mM glucose, pH 7.3) and removed all connective tissues, fat, blood vessels, and other nonmuscle tissues using micro scissors under a dissecting microscope. This was essential for subsequent collagenase digestion because rat FDB is covered with many nonmuscle tissues such as connective tissue and fat. Next, muscles were incubated in collagenase solution (PRS containing 0.2% collagenase type 1 [Worthington, USA], 0.1% elastase [Worthington], 0.0625% protease from Streptomyces griseus [Sigma–Aldrich, USA], 0.033% dispase [Invitrogen, USA], and 10% fetal bovine serum [FBS, Invitrogen]) at 37 °C under 5% CO2 for 90 min to digest any remaining connective tissues. The cocktail of four types of enzyme enabled quick dissociation of muscle fibers with less trituration and a shorter incubation period than the previous protocol, which required 2.5 to 3.0 h [6]. After collagenase digestion, muscles were moved to proliferation medium (PM: high-glucose Dulbecco’s modified Eagle’s medium [DMEM, Life Technologies, USA] containing 10% FBS, 1% antibiotic–antimycotic mix [Life Technologies], and 0.1% gentamycin [Life Technologies]) using a flame-polished wide-bore Pasteur pipette. Muscles were triturated with pipettes of decreasing diameter to dissociate them into single muscle fibers. Fibers were transferred into a glass centrifuge tube containing 10 ml of PM for a gravity sedimentation wash. The gravity sedimentation was repeated twice more. Then, as much PM as possible was removed and fibers were resuspended carefully in 1 ml of maintenance medium (MM: high-glucose DMEM supplemented with 20% Serum Replacement 2 [Sigma–Aldrich]). A 100-ll aliquot of isolated fibers was placed in a 35-mm Petri dish containing 1.9 ml of MM. Fig. 1A shows the typical composition of FDB fibers immediately after isolation. Culture medium was replaced every 2 days during long-term culturing, removing as much old MM as possible, and then fibers were transferred with any remaining MM into a new dish containing 2.0 ml of fresh MM. Using this method, mononuclear cells (including myoblasts and other cells derived from muscle tissue) were left in the old dish and removed in each MM replacement. This potentially allows analysis of gene and protein expression in relatively pure isolated muscle fibers. Fibers were also isolated from soleus and extensor digitorum longus (EDL) muscles in the same manner. All instruments used in this protocol were sterilized. Animal experiments were performed according to the guidelines for animal experiments of Kyushu University and with the ethical approval of the animal care and use committee of Kyushu University (protocol 05-002-01). The viability of cultured fibers was measured after 1, 3, 5, and 7 days using a cell-permeable green fluorescent dye, calcein–AM (acetoxymethyl ester) (Nacalai Tesque, Japan). Fibers were incubated in 2 lM calcein–AM in phosphate-buffered saline (PBS) for 15 min at 37 °C. Viability was defined as the percentage of calcein–AM-positive fibers among the total number of fibers in a dish. Green fluorescence derived from calcein–AM was observed in living fibers (Fig. 1B). More than 90% of muscle fibers isolated from the FDB remained alive at day 7 (Fig. 1C). Conversely, the viability of fibers isolated from the soleus and EDL decreased rapidly, and the overwhelming majority of fibers were dead by day 5. Next, we examined the validity of the single fibers as an in vitro cell culture model for adult skeletal muscles by comparing them with myotubes formed from the L6 rat myoblast cell line. L6 cells were differentiated for 4 days in low-glucose DMEM with 2% horse serum to obtain myotubes. Total RNA was extracted from isolated fibers and myotubes using Trizol reagent (Life Technologies) and reverse transcribed using reverse transcriptase SuperScript III (Invitrogen) and Oligo d(T)16 primer (Applied Biosystems, USA) according to the manufacturers’ protocols. Real-time quantitative polymerase chain reaction (PCR) using a LightCycler 1.5 (Roche
23
Fig.1. Representative images and survival curve of isolated rat single muscle fibers. (A) Phase contrast image of fibers immediately after isolation from rat FDB. (B) Fluorescence image of calcein–AM positive fibers. Green fluorescence indicates living fibers. (C) Survival curve in culture of isolated single fibers originating from three different hind limb muscles: FDB, EDL, and soleus (Sol). Values are means ± standard errors for three dishes at each time point. Each dish contained at least 200 fibers.
Diagnostics, Switzerland) was performed by the intercalator method using specific primers (see Supplementary Table 1 in online supplementary material) and EvaGreen dye (Biotium, USA). Amplicon specificity was verified by melting curve analysis and a preliminary Taqman probe assay. Hypoxanthine–guanine phosphoribosyl transferase (HPRT) was used as an internal standard. As expected, messenger RNA (mRNA) expression of all four adult MyHC isoforms in isolated single muscle fiber cultures from FDB muscle was much higher than in cultured L6 myotubes (Fig. 2A). The levels of MyHC protein isoforms were also quantified using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS– PAGE) and silver staining [8]. Notably, only adult MyHC isoforms were detected in cultures of isolated single fibers, and the MyHC composition in fibers was the same as the composition in whole FDB muscle tissue (Fig. 2B). FDB was composed of 42% MyHC2A, an intermediate isoform close to slow MyHC1, and 58% MyHC2X, an intermediate isoform close to fast MyHC2B. These results also indicated that there was no selection for muscle fiber type during the process of fiber isolation (Fig. 2B). We also examined MyHC expression in myotubes formed from primary cultures of muscle satellite cells isolated from rat skeletal muscle. These myotubes are considered to have higher differentiation potential than L6 cells. In fact, satellite cells used in this study formed myotubes in 2 days, which was faster than observed in cultures of L6 cells. After culturing myoblasts until 70% confluence in Opti-MEM (Life Technologies), cultures were maintained in differentiation medium and sampled every 2 days during differentiation for a total of 8 days according to a previously reported procedure [9]. No MyHC isoforms were detected in satellite cells before differentiation. Clear MyHC bands appeared after 2 days in differentiation medium (Fig. 2C). In cultures of differentiated satellite cells, immature isoforms of neonatal and embryonic MyHC were observed, and adult MyHC isoforms (as indicated by samples of pooled homogenate of EDL and soleus muscle) were not detected even after analysis of enhanced amounts of the day 8 sample (Fig. 2C). In this protocol, the origin of muscle fibers is the critical factor in acquiring vital fibers. We found that fibers derived from FDB could stay viable because isolation, trituration, and transfer every 2 days reduced the level of damage, likely because of the short fiber length. In contrast, the excision of EDL and soleus and subsequent isolation of fibers seemed to cause more severe damage to the fibers. The fibers in EDL and soleus are longer than in FDB, and trituration in a Pasteur pipette seemed to lead to inevitable lethal damage. Rosenblatt and coworkers reported a similar result, where
24
Notes & Tips / Anal. Biochem. 482 (2015) 22–24
Fig.2. MyHC expression, a marker of mature muscle, in isolated single fibers and myotubes differentiated from myogenic cell cultures. (A) Comparison of mRNA expression levels of four adult MyHC isoforms between isolated single muscle fibers and L6 myotubes differentiated for 4 days. (B) Protein composition of MyHC isoforms in isolated single fibers and FDB muscle tissue as revealed by silver-stained SDS–PAGE. Isolated single fibers of FDB had similar MyHC isoform composition to homogenized FDB tissue. (C) MyHC proteins in cultures of rat satellite cells differentiated into myotubes for up to 8 days. Lanes labeled as 1.5 and 2 indicate loading of 1.5- and 2.0-fold more protein than the day 8 sample. A mixed sample of rat EDL and soleus was used as a marker to demonstrate four adult MyHC isoforms—MyHC1, -2A, -2X, and -2B—in one lane. N.D.: not detected.
fibers isolated from muscles with long fibers tended to be tangled and damaged, especially during gravity sedimentation [10]. Modification of tissue handling and trituration procedures is expected to improve the viability of muscle fibers isolated from EDL or soleus, but currently FDB muscles seem to be most practical for fiber culture studies. Muscle fiber formation is almost impossible using the current culture protocol for myoblasts (muscle satellite cells). Extensive myotubes formed from cultured [11] satellite cells during the first 2 or 3 days in differentiation medium. On the 8th day of differentiation, myotube formation seemed to be stable, although it was rare to observe well-differentiated myotubes with sarcomeric striations. The shapes of myotubes were irregular, in contrast to the clear-cut cylindrical shape of isolated fibers, and the diameter of myotubes was smaller than the diameter of isolated muscle fibers. Interestingly, in Western blotting studies using specific antibodies against slow MyHC (clone NOQ7.5.4D, Sigma–Aldrich) and pan-MyHC (clone MF20, R&D Systems, USA), slow adult isoforms were faintly detected in myotubes differentiated from rat satellite cells [11]; this finding suggested that adult isoforms were present in myotubes but at a very low level compared with the levels of immature neonatal and embryonic MyHC isoforms. Therefore, we conclude that single muscle fibers have a great advantage over myotubes as an in vitro model of skeletal muscle because the pattern of MyHC isoforms is significantly closer to that in mature adult muscle tissue than the pattern observed in myotube cultures from established cell lines or primary satellite cells. In conclusion, we have modified well-established protocols for isolating rat single muscle fibers and enabled long-term culture of skeletal muscle fibers. Isolated fibers contained abundant adult MyHC isoforms compared with myotubes. A high purity of fibers was attained by eliminating mononuclear cells that might have migrated off fibers onto culture dishes, leading to subsequent spurious analysis of satellite cells and connective tissue remnants rather than muscle fibers. Acknowledgments This research was supported by funding from Japan Society for the Promotion of Science (JSPS) KAKENHI (22580136 and
24658228) and Kyushu University Interdisciplinary Programs in Education and Projects in Research Development (P&P A-type, 25005). The publication was supported in part by a Research Grant for Young Investigators of Faculty of Agriculture, Kyushu University. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ab.2015.03.034. References [1] G. Ravenscroft, K.J. Nowak, C. Jackaman, S. Clement, M.A. Lyons, S. Gallagher, A.J. Bakker, N.G. Laing, Dissociated flexor digitorum brevis myofiber culture system: a more mature muscle culture system, Cell Motil. Cytoskeleton 64 (2007) 727–738. [2] W.A. LaFramboise, R.D. Guthrie, D. Scalise, V. Elborne, K.L. Bombach, C.S. Armanious, J.A. Magovern, Effect of muscle origin and phenotype on satellite cell muscle-specific gene expression, J. Mol. Cell. Cardiol. 35 (2003) 1307– 1318. [3] A. Bekoff, W. Betz, Properties of isolated adult rat muscle fibres maintained in tissue culture, J. Physiol. 271 (1977) 537–547. [4] R. Bischoff, Proliferation of muscle satellite cells on intact myofibers in culture, Dev. Biol. 115 (1986) 129–139. [5] A.C. Wozniak, J.E. Anderson, Single-fiber isolation and maintenance of satellite cell quiescence, Biochem. Cell Biol. 83 (2005) 674–676. [6] J.E. Anderson, A.C. Wozniak, W. Mizunoya, Single muscle-fiber isolation and culture for cellular, molecular, pharmacological, and evolutionary studies, Methods Mol. Biol. 798 (2012) 85–102. [7] A. Pasut, A.E. Jones, M.A. Rudnicki, Isolation and culture of individual myofibers and their satellite cells from adult skeletal muscle, J. Vis. Exp. (2013) e50074. [8] W. Mizunoya, J. Wakamatsu, R. Tatsumi, Y. Ikeuchi, Protocol for highresolution separation of rodent myosin heavy chain isoforms in a mini-gel electrophoresis system, Anal. Biochem. 377 (2008) 111–113. [9] T. Suzuki, M.K. Do, Y. Sato, K. Ojima, M. Hara, W. Mizunoya, M. Nakamura, M. Furuse, Y. Ikeuchi, J.E. Anderson, R. Tatsumi, Comparative analysis of semaphorin 3A in soleus and EDL muscle satellite cells in vitro toward understanding its role in modulating myogenin expression, Int. J. Biochem. Cell Biol. 45 (2013) 476–482. [10] J.D. Rosenblatt, A.I. Lunt, D.J. Parry, T.A. Partridge, Culturing satellite cells from living single muscle fiber explants, Vitro Cell. Dev. Biol. Anim. 31 (1995) 773– 779. [11] Y. Komiya, J.E. Anderson, M. Akahoshi, M. Nakamura, R. Tatsumi, Y. Ikeuchi, W. Mizunoya, Data in support of protocol for rat single muscle fiber isolation and culture, Data in Brief, (submitted for publication).
Contents lists available at ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
Notes & Tips
Protocol for rat single muscle fiber isolation and culture Yusuke Komiya a, Judy E. Anderson b, Mariko Akahoshi a, Mako Nakamura a, Ryuichi Tatsumi a, Yoshihide Ikeuchi a, Wataru Mizunoya a,⇑ a b
Department of Bioresource Sciences, Faculty of Agriculture, Kyushu University, Higashi-ku, Fukuoka 812-8581, Japan Department of Biological Sciences, University of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada
a r t i c l e
i n f o
Article history: Received 1 December 2014 Received in revised form 20 March 2015 Accepted 24 March 2015 Available online 23 April 2015 Keywords: Skeletal muscle Muscle fiber Primary culture Rat
a b s t r a c t To attain a superior in vitro model of mature muscle fibers, we modified the established protocol for isolating single muscle fibers from rat skeletal muscle. Muscle fiber cultures with high viability were obtained using flexor digitorum brevis muscle and lasted for at least 7 days. We compared the expression levels of adult myosin heavy chain (MyHC) isoforms in these single muscle fibers with myotubes formed from myoblasts; isolated fibers contained markedly more abundant adult MyHC isoforms than myotubes. This muscle fiber model, therefore, will be useful for studying the various functions and cellular processes of mature muscles in vitro. Ó 2015 Elsevier Inc. All rights reserved.
Skeletal muscle, the largest organ in the human body, governs our metabolic and locomotive ability. It comprises bundles of muscle fibers, the multinucleated cells formed by the fusion of a large number of differentiated myogenic cells during development. Study of skeletal muscle function is expected to contribute to our understanding of how to alleviate sarcopenia and various metabolic syndromes. Myotubes, differentiated from myogenic cell lines such as L6 (derived from rat) and C2C12 (derived from mouse) or cultures of muscle satellite cells isolated from skeletal muscles of animals, are often used as in vitro models of skeletal muscle fibers. Previously, Ravenscroft and coworkers highlighted the murine isolated single muscle fiber culture system as a more mature culture system for adult skeletal muscle [1]. Although this idea has merit, rat fibers have not yet been tested despite the prevalence of rat tissue use in the skeletal muscle field. Here, we endeavored to modify an established protocol for isolating rat single muscle fibers to achieve long-term culture. Furthermore, we examined the validity of fiber cultures as a model for mature muscle tissue by comparing our model with myotubes differentiated from myoblast cultures. Muscle function is underpinned by a contractile protein called myosin that is composed of two heavy chains and four light-chain subunits. To date, four isoforms of myosin heavy chain (MyHC)1 expressed in adult skeletal muscle tissues have been ⇑ Corresponding author. Fax: +81 92 642 2951. E-mail address: [email protected] (W. Mizunoya). Abbreviations used: MyHC, myosin heavy chain; FDB, flexor digitorum brevis; PRS, physiological rodent saline; FBS, fetal bovine serum; PM, proliferation medium; DMEM, Dulbecco’s modified Eagle’s medium; MM, maintenance medium; EDL, extensor digitorum longus; calcein–AM, calcein–acetoxymethyl ester; mRNA, messenger RNA; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis. 1
http://dx.doi.org/10.1016/j.ab.2015.03.034 0003-2697/Ó 2015 Elsevier Inc. All rights reserved.
classified as MyHC1, -2A, -2X, and -2B (listed from slow-twitch to fast-twitch myosin). In myotubes formed from differentiated C2C12 cells, MyHC1, -2A, -2X, and -2B—the adult isoforms—are much less abundantly expressed than in normal skeletal muscle tissue [2]. Instead, the predominant isoforms are embryonic and neonatal MyHC, which are immature developmental isoforms. This is a critical issue in analyzing muscle fiber type because the adult MyHC isoforms are the common fiber type marker proteins. Primary culture of isolated muscle fibers is the most suitable model for mature muscle fibers. The initial method of rodent fiber isolation was pioneered by Bekoff and Betz [3] and later established by Bischoff [4]. A detailed description of the procedure was published recently [5–7]. This method, involving isolated single muscle fibers, has largely been used to take and examine muscle satellite cells resident on muscle fibers and isolated together with fibers from muscle tissue. These satellite cells, along with fibers, have been examined for gene expression [6,7], myogenesis [6], regenerative capacity [4], and the activation processes [4,6]. It remains uncertain whether isolated fibers are a useful in vitro model for studying mature muscle fiber properties. In this study, single muscle fibers were prepared from flexor digitorum brevis (FDB) muscle in the sole of the rat foot by modification of the method published by Anderson and coworkers [6] and Ravenscroft and coworkers [1] using mice. The major modifications were enzymatic digestion and the method of transferring fibers during medium changes. F344 male rats (3–4 weeks old) were sacrificed by cervical dislocation under inhalation anesthesia with sevoflurane, and muscles were dissected from the hind leg. First, we placed the
Notes & Tips / Anal. Biochem. 482 (2015) 22–24
dissected FDB muscle in physiological rodent saline (PRS: 138 mM NaCl, 2.7 mM KCl, 1.8 mM CaCl2, 1.06 mM MgCl2, 12.4 mM Hepes, and 5.6 mM glucose, pH 7.3) and removed all connective tissues, fat, blood vessels, and other nonmuscle tissues using micro scissors under a dissecting microscope. This was essential for subsequent collagenase digestion because rat FDB is covered with many nonmuscle tissues such as connective tissue and fat. Next, muscles were incubated in collagenase solution (PRS containing 0.2% collagenase type 1 [Worthington, USA], 0.1% elastase [Worthington], 0.0625% protease from Streptomyces griseus [Sigma–Aldrich, USA], 0.033% dispase [Invitrogen, USA], and 10% fetal bovine serum [FBS, Invitrogen]) at 37 °C under 5% CO2 for 90 min to digest any remaining connective tissues. The cocktail of four types of enzyme enabled quick dissociation of muscle fibers with less trituration and a shorter incubation period than the previous protocol, which required 2.5 to 3.0 h [6]. After collagenase digestion, muscles were moved to proliferation medium (PM: high-glucose Dulbecco’s modified Eagle’s medium [DMEM, Life Technologies, USA] containing 10% FBS, 1% antibiotic–antimycotic mix [Life Technologies], and 0.1% gentamycin [Life Technologies]) using a flame-polished wide-bore Pasteur pipette. Muscles were triturated with pipettes of decreasing diameter to dissociate them into single muscle fibers. Fibers were transferred into a glass centrifuge tube containing 10 ml of PM for a gravity sedimentation wash. The gravity sedimentation was repeated twice more. Then, as much PM as possible was removed and fibers were resuspended carefully in 1 ml of maintenance medium (MM: high-glucose DMEM supplemented with 20% Serum Replacement 2 [Sigma–Aldrich]). A 100-ll aliquot of isolated fibers was placed in a 35-mm Petri dish containing 1.9 ml of MM. Fig. 1A shows the typical composition of FDB fibers immediately after isolation. Culture medium was replaced every 2 days during long-term culturing, removing as much old MM as possible, and then fibers were transferred with any remaining MM into a new dish containing 2.0 ml of fresh MM. Using this method, mononuclear cells (including myoblasts and other cells derived from muscle tissue) were left in the old dish and removed in each MM replacement. This potentially allows analysis of gene and protein expression in relatively pure isolated muscle fibers. Fibers were also isolated from soleus and extensor digitorum longus (EDL) muscles in the same manner. All instruments used in this protocol were sterilized. Animal experiments were performed according to the guidelines for animal experiments of Kyushu University and with the ethical approval of the animal care and use committee of Kyushu University (protocol 05-002-01). The viability of cultured fibers was measured after 1, 3, 5, and 7 days using a cell-permeable green fluorescent dye, calcein–AM (acetoxymethyl ester) (Nacalai Tesque, Japan). Fibers were incubated in 2 lM calcein–AM in phosphate-buffered saline (PBS) for 15 min at 37 °C. Viability was defined as the percentage of calcein–AM-positive fibers among the total number of fibers in a dish. Green fluorescence derived from calcein–AM was observed in living fibers (Fig. 1B). More than 90% of muscle fibers isolated from the FDB remained alive at day 7 (Fig. 1C). Conversely, the viability of fibers isolated from the soleus and EDL decreased rapidly, and the overwhelming majority of fibers were dead by day 5. Next, we examined the validity of the single fibers as an in vitro cell culture model for adult skeletal muscles by comparing them with myotubes formed from the L6 rat myoblast cell line. L6 cells were differentiated for 4 days in low-glucose DMEM with 2% horse serum to obtain myotubes. Total RNA was extracted from isolated fibers and myotubes using Trizol reagent (Life Technologies) and reverse transcribed using reverse transcriptase SuperScript III (Invitrogen) and Oligo d(T)16 primer (Applied Biosystems, USA) according to the manufacturers’ protocols. Real-time quantitative polymerase chain reaction (PCR) using a LightCycler 1.5 (Roche
23
Fig.1. Representative images and survival curve of isolated rat single muscle fibers. (A) Phase contrast image of fibers immediately after isolation from rat FDB. (B) Fluorescence image of calcein–AM positive fibers. Green fluorescence indicates living fibers. (C) Survival curve in culture of isolated single fibers originating from three different hind limb muscles: FDB, EDL, and soleus (Sol). Values are means ± standard errors for three dishes at each time point. Each dish contained at least 200 fibers.
Diagnostics, Switzerland) was performed by the intercalator method using specific primers (see Supplementary Table 1 in online supplementary material) and EvaGreen dye (Biotium, USA). Amplicon specificity was verified by melting curve analysis and a preliminary Taqman probe assay. Hypoxanthine–guanine phosphoribosyl transferase (HPRT) was used as an internal standard. As expected, messenger RNA (mRNA) expression of all four adult MyHC isoforms in isolated single muscle fiber cultures from FDB muscle was much higher than in cultured L6 myotubes (Fig. 2A). The levels of MyHC protein isoforms were also quantified using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS– PAGE) and silver staining [8]. Notably, only adult MyHC isoforms were detected in cultures of isolated single fibers, and the MyHC composition in fibers was the same as the composition in whole FDB muscle tissue (Fig. 2B). FDB was composed of 42% MyHC2A, an intermediate isoform close to slow MyHC1, and 58% MyHC2X, an intermediate isoform close to fast MyHC2B. These results also indicated that there was no selection for muscle fiber type during the process of fiber isolation (Fig. 2B). We also examined MyHC expression in myotubes formed from primary cultures of muscle satellite cells isolated from rat skeletal muscle. These myotubes are considered to have higher differentiation potential than L6 cells. In fact, satellite cells used in this study formed myotubes in 2 days, which was faster than observed in cultures of L6 cells. After culturing myoblasts until 70% confluence in Opti-MEM (Life Technologies), cultures were maintained in differentiation medium and sampled every 2 days during differentiation for a total of 8 days according to a previously reported procedure [9]. No MyHC isoforms were detected in satellite cells before differentiation. Clear MyHC bands appeared after 2 days in differentiation medium (Fig. 2C). In cultures of differentiated satellite cells, immature isoforms of neonatal and embryonic MyHC were observed, and adult MyHC isoforms (as indicated by samples of pooled homogenate of EDL and soleus muscle) were not detected even after analysis of enhanced amounts of the day 8 sample (Fig. 2C). In this protocol, the origin of muscle fibers is the critical factor in acquiring vital fibers. We found that fibers derived from FDB could stay viable because isolation, trituration, and transfer every 2 days reduced the level of damage, likely because of the short fiber length. In contrast, the excision of EDL and soleus and subsequent isolation of fibers seemed to cause more severe damage to the fibers. The fibers in EDL and soleus are longer than in FDB, and trituration in a Pasteur pipette seemed to lead to inevitable lethal damage. Rosenblatt and coworkers reported a similar result, where
24
Notes & Tips / Anal. Biochem. 482 (2015) 22–24
Fig.2. MyHC expression, a marker of mature muscle, in isolated single fibers and myotubes differentiated from myogenic cell cultures. (A) Comparison of mRNA expression levels of four adult MyHC isoforms between isolated single muscle fibers and L6 myotubes differentiated for 4 days. (B) Protein composition of MyHC isoforms in isolated single fibers and FDB muscle tissue as revealed by silver-stained SDS–PAGE. Isolated single fibers of FDB had similar MyHC isoform composition to homogenized FDB tissue. (C) MyHC proteins in cultures of rat satellite cells differentiated into myotubes for up to 8 days. Lanes labeled as 1.5 and 2 indicate loading of 1.5- and 2.0-fold more protein than the day 8 sample. A mixed sample of rat EDL and soleus was used as a marker to demonstrate four adult MyHC isoforms—MyHC1, -2A, -2X, and -2B—in one lane. N.D.: not detected.
fibers isolated from muscles with long fibers tended to be tangled and damaged, especially during gravity sedimentation [10]. Modification of tissue handling and trituration procedures is expected to improve the viability of muscle fibers isolated from EDL or soleus, but currently FDB muscles seem to be most practical for fiber culture studies. Muscle fiber formation is almost impossible using the current culture protocol for myoblasts (muscle satellite cells). Extensive myotubes formed from cultured [11] satellite cells during the first 2 or 3 days in differentiation medium. On the 8th day of differentiation, myotube formation seemed to be stable, although it was rare to observe well-differentiated myotubes with sarcomeric striations. The shapes of myotubes were irregular, in contrast to the clear-cut cylindrical shape of isolated fibers, and the diameter of myotubes was smaller than the diameter of isolated muscle fibers. Interestingly, in Western blotting studies using specific antibodies against slow MyHC (clone NOQ7.5.4D, Sigma–Aldrich) and pan-MyHC (clone MF20, R&D Systems, USA), slow adult isoforms were faintly detected in myotubes differentiated from rat satellite cells [11]; this finding suggested that adult isoforms were present in myotubes but at a very low level compared with the levels of immature neonatal and embryonic MyHC isoforms. Therefore, we conclude that single muscle fibers have a great advantage over myotubes as an in vitro model of skeletal muscle because the pattern of MyHC isoforms is significantly closer to that in mature adult muscle tissue than the pattern observed in myotube cultures from established cell lines or primary satellite cells. In conclusion, we have modified well-established protocols for isolating rat single muscle fibers and enabled long-term culture of skeletal muscle fibers. Isolated fibers contained abundant adult MyHC isoforms compared with myotubes. A high purity of fibers was attained by eliminating mononuclear cells that might have migrated off fibers onto culture dishes, leading to subsequent spurious analysis of satellite cells and connective tissue remnants rather than muscle fibers. Acknowledgments This research was supported by funding from Japan Society for the Promotion of Science (JSPS) KAKENHI (22580136 and
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