ROBERT J. TALMADGE. AND ROLAND. R. ROY. Department ..... ase histochemical technique of Brooke and Kaiser (7). Subsequently, a third type II MHC was ...
Electrophoretic separation of rat skeletal myosin heavy-chain isoforms ROBERT J. TALMADGE AND ROLAND R. ROY Department of Physiological Science and Brain Research Institute, California 90024- 1527 TALMADGE, ROBERT J., AND ROLAND R. ROY. Electrophoretie separation of rat skeletal muscle myosin heavy-chain isoforms. J. Appl. Physiol. 75(5): 2337-2340, 1993.-A new technique for the sodium dodecyl sulfate-polyacrylamide gel electrophoretic separation of rat skeletal muscle myosin heavy-chain (MHC) isoforms is presented. This technique allows for the separation of the four identified MHC isoforms known to be present in adult rat skeletal muscle. These types of MHC are commonly called I, IIa, 11x or IId, and IIb. The procedure can be performed using minigel electrophoresis systems and does not involve preparation of gradient-separating gels or the use of special cooling devices. The procedure accommodates both silver and Coomasie Blue staining. Thus the procedure is simple to perform and highly repeatable, providing high-resolution separation of MHC protein isoforms. The percent composition of the four adult MHCs in rat soleus, medial gastrocnemius, diaphragm, and levator ani muscles by use of this procedure and Coomasie Blue staining is similar to that previously reported. This new technique provides a novel and easy-to-perform method for the separation of rat skeletal muscle MHC isoforms. isomyosins; myosin isoforms; polyacrylamide gel electrophoresis; sodium dodecyl sulfate-polyacrylamide gel electrophoresis
HEAVY CHAIN (MHC) is the primary component of the thick filament and is the predominant protein in skeletal muscle. MHC is encoded by a multigene family consisting of several members (22), which are expressed in a tissue-specific and developmentally regulated manner (22). To date, four MHC proteins have been identified in the limb, jaw, and respiratory muscles of adult rats (1, 29,33). These are identified as types I, IIa, 11x or IId, and IIb. The unique expression of any one of these MHC protein isoforms in a single muscle fiber is primarily responsible for the distinct fiber types observed in skeletal muscle after staining for qualitative myofibrillar adenosinetriphosphatase (ATPase) after acid and alkaline preincubations (33). The coexpression of multiple MHC isoforms within a given muscle fiber can occur (3, 4, 16, 28, 30), but this coexpression is thought to occur only during MHC transitions. The MHC protein expressed in a single muscle fiber is correlated to its maximum contraction velocity (5, 28, MYOSIN
0161-7567193
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muscle
University of California, Los Angeles,
32), which is related to the maximum myosin ATPase activity of a muscle (2). The myofibrillar and myosin ATPase activities of a muscle are related to the isoforms of myosin expressed in the muscle (34). Ranatunga and Thomas (27) demonstrated that the MHC-based histochemical fiber type composition of a muscle can be used to predict the contractile properties of the whole muscle. Thus the MHC expressed in a single muscle fiber, to a large degree, determines the histochemical type, myosin and myofibrillar ATPase activities, and contractile properties of the muscle fiber. One of the responses of skeletal muscle to chronic changes in the amount and/or pattern of neuromuscular activity is an adaptation at the level of the MHC (26). Similarly, changes in the MHC expressed by a muscle are observed during development (8,19) and aging (17,21) in response to endogenous and exogenous endocrine factors (9, 13, 15), with changes in innervation status (23, 24), and in response to neuromuscular diseases (14). Therefore electrophoretic techniques that allow for the separation of MHC isoforms are critical to the analysis of skeletal muscle adaptation to 1) alterations in neuromuscular activity, 2) neuromuscular disease, 3) alterations in circulating levels of endogenous and exogenous hormones, and 4) signals related to development and aging. In an attempt to obtain complete electrophoretic resolution of MHCs, the method of Doucet and Trifaro (12) was modified by 1) reducing the acrylamide content of the separating gel to 8%, 2) increasing the glycerol content of separating and stacking gels to 30%, 3) decreasing the gel thickness to 0.75 mm, 4) increasing the running time to 24 h, 5) decreasing the applied current to 6 mA/ gel for a minigel system and 12 mA/gel for a large-gel system, and 6) omitting urea from the sample buffer. Thus a new technique is described for the electrophoretic separation of adult rat muscle MHCs. This technique has distinct advantages over previously published protocols (1, 9, 10, 20). MATERIALS
AND METHODS
Muscles from adult male and female Sprague-Dawley rats were removed while the animal was anesthetized with pentobarbital sodium. Soleus, medial gastrocne-
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2338 TABLE
MYOSIN
HEAVY-CHAIN
ELECTROPHORESIS
1. SDS-PAGE gel mixtures and stock solutions Stock Solution
100% Glycerol 30% Acrylamide-bis (5O:l) Tris 1.5 M (pH 8.8) 0.5 M (nH 6.7) 1 M glycine 100 mM EDTA (pH 7.0) 10% SDS Distilled H,O TEMED 10% Ammonium persulfate
Seuaratine 3.000 2.667
Gel
Stackine
Gel
3.000 1.333
1.333 1.400 1.000 0.400 1.495 0.005 0.100
0.400 0.400 3.362 0.005 0.100
Values are in ml. Gel mixtures are degassed before initiation of polymerization. All stock solutions are stored at 4”C, except 10% SDS, which is stored at room temperature. All stock chemicals are of electrophoresispurity. Acrylamide-bis, acrylamide-N,N’-methylene-bis-acrylamide; TEMED, N,N,N’,N’-tetramethylethylenediamine; PAGE, polyacrylamide gel electrophoresis.
mius, diaphragm, and levator ani were chosen for study because of their varied MHC percent compositions (1, 33). The muscles were weighed, frozen in liquid nitrogen, and stored at -70°C. Frozen muscles were minced with scissors in 9 vol of ice-cold homogenization buffer [250 mM sucrose, 100 mM KCl, 5 mM EDTA, and 20 mM tris(hydroxymethyl)aminomethane (Tris), pH 6.81. The muscle minces were subsequently homogenized by hand in glass tissue grinders. The homogenates were used for the preparation of washed myofibrils (34). The washed myofibrils were boiled in sample buffer (18) for 2 min at a final protein concentration of 0.125 mg/ml. Total protein was assayed according to the method of Bradford (6). The stacking gels were composed of 30% glycerol, 4% acrylamide-NJ/‘-methylene-bis-acrylamide (his) (50:1), 70 mM Tris (pH 6.7), 4 mM EDTA, and 0.4% sodium dodecyl sulfate (SDS). The separating gels were composed of 30% glycerol, 8% acrylamide-bis (50:1), 0.2 M Tris (pH 8.8), 0.1 M glycine, and 0.4% SDS. The gel constituents (12) were prepared from stock solutions, and polymerization was initiated with 0.05% N,N,N’,N’-tetramethylethylenediamine and 0.1% ammonium persulfate. The pH values of the stacking and separating gels were not adjusted after the stock solutions were mixed. All chemicals were of electrophoresis grade. Table 1 lists the stock concentrations and volumes used for the preparation of minigels in the Biorad Mini-Protean II Dual Slab Cell electrophoretic system utilizing a Biorad lOOO/ 200 power supply. The gel volumes listed provide for two 0.75-mm-thick gels. For a large-gel apparatus (CBS Scientific SG-200), doubling the volumes would be appropriate. A lo-space comb was used for the minigel system, and a 15-space comb was used for the large gel. Separate upper and lower running buffers were used. The upper running buffer consisted of 0.1 M Tris (base), 150 mM glycine, and 0.1% SDS. The lower running buffer consisted of 50 mM Tris (base), 75 mM glycine, and 0.05% SDS. The pH values of the running buffers were not adjusted. Upper and lower buffers were cooled to 4°C in a refrigerator before use, and the entire gel unit was placed in a Styrofoam box containing cooling packs and/or ice to maintain the temperature below 10°C for the duration of the electrophoretic run. The running conditions for the
minigel were 70 V (constant voltage) for 24 h. For the large-gel apparatus, the running time was 24 h at 275 V (constant voltage). In the minigel system the blue tracking dye ran off the gel, but in the large-gel system it ran to the bottom of the gel (20 cm total length of gel). The amount of protein run on the gel was varied between 1.0 and 0.5 hg of total protein per lane. The gels were stained with Coomasie Blue if the protein per lane was 1.0 pg or with silver (Biorad silver stain plus kit) if the total protein per lane was 0.5 pg. The stained gels were scanned with a Pharmacia LKB Ultrascan scanning densitometer and photographed. RESULTS
Typical MHC isoform separations of rat muscles with use of the minigel system and Coomasie Blue staining are shown in Fig. 1. Identification of the region of the gel containing MHC was accomplished with use of rabbit muscle myosin as a standard. The separation of the MHC bands is distinct. The success rate of this protocol for yielding four well-differentiated MHC bands was 100%. Identification of the various MHC bands with this protocol was based on the previously published (1,33) MHC compositions and migration patterns of soleus, diaphragm, and levator ani (Fig. 1). Soleus contained only types I and IIa MHC, with a predominance of type I MHC. Type I MHC was the fastest-migrating band (lower band), and type IIa MHC was the slowest-migrating band (upper band). The diaphragm contained type 11x MHC in addition to types I and IIa MHC. The type 11x MHC migrated as a distinct band immediately below the type IIa band. The levator ani contained almost exclusively type IIb MHC, which migrated as a distinct band between types I and 11x MHC. The medial gastrocnemius contained all four MHC types but predominantly types 11x and IIb MHC. The overall order of migration by MHC type was I > IIb > 11x > IIa. This migration order was identical to that in previously published protocols (20, 31, 33). The separation of the MHC isoforms was similar when the large-gel apparatus was used; however, the distance between the bands was slightly greater (data not shown). Figure 2 shows a gel similar to that in Fig. 1, displaying the four MHC bands in rat fast- and slow-
2
3
4
FIG. 1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of isolated myofibrils as outlined in MATERIALS AND METHODS on minigel system and stained with Coomasie Blue. Lane 1, soleus lmvosin heavy-chain (MHC) types I and IIaJ; lane 2, midcostal diaphragm (MHC types I, IIa, and 11x); lane 3, levator ani (MHC type IIb); he 4, medial gastrocnemius (MHC types I, IIa, 11x, and IIb). All 4 MHC bands are clearly separated using minigel system. a, Type IIa MHC; b, type IIb MHC; x, type 11x MHC; I, type I MHC. Dual arrowheads in 2une 2 show separation of types IIa and 11x MHC.
MYOSIN
HEAVY-CHAIN
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ELECTROPHORESIS
2. MHC percent composition of various rat skeletal muscles
TABLE
FIG. 2. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of isolated myofibrils as in Fig. 1, but silver stained. Muscle samples are different from those in Fig. 1. Lane 1, soleus; lane 2, diaphragm; lane 3, media1 gastrocnemius.
twitch muscles, but is silver stained. A densitometric tracing of the MHC region of rat medial gastrocnemius (lane 3, Fig. 2) is shown in Fig. 3. The four MHC bands (types I, IIa, 11x or IId, and IIb) of rat medial gastrocnemius are clearly separated and observed as separate peaks on the densitometric tracing. The type IIa and 11x or IId bands are separated into two clearly resolvable peaks with the minigel system. The percent compositions of the four MHC isoforms in the various muscles are shown in Table 2 and are similar to those in previously published reports (1, 31, 33). DISCUSSION
The separation of mammalian MHC isoforms was initially accomplished by Carraro and Catani (10). This procedure only allowed for the separation of two MHC bands designated as slow (type I) and fast (type II). Later, adaptations of this procedure distinguished two isoforms of fast MHC designated IIa and IIb (9, 11, 25), which appeared to correlate with the myofibrillar ATPase histochemical technique of Brooke and Kaiser (7). Subsequently, a third type II MHC was independently identified by two groups and called type 11x (29) or type IId (1,33). However, the electrophoretic techniques that have been used to separate the four adult rat MHCs require labor-intensive silver staining and utilize either gradient gels (1, 33), which are difficult to reproduce, or special cooling devices, which are not readily available to
Migration
Direction
e
FIG. 3. Densitometric tracing of MHC region of rat media1 gastrocnemius muscle (lane 3, Fig. 2). Note 4 distinct peaks that correspond to 4 MHC isoforms (types I, IIa, 11x or IId, and IIb) found in rat limb muscle. Peaks are labeled with corresponding MHC isoform.
Muscle
n
TYPO I
Type IIa
Type 11x
Type IIb
Soleus Medial gastrocnemius Diaphragm (costal) Levator ani
6 4 4 4
89t3 7+1 28+6 0
11+3 lo+1 24+3 0
0 25*2 43+4 2*1
0 58?2 523 98&l
Values are means rt SE. MHC, myosin heavy chain.
all laboratories (20). Therefore the need for a consistent and high-resolution SDS-polyacrylamide gel electrophoresis technique for the separation of rat MHCs that could be used in conjunction with Coomasie Blue staining was apparent. The electrophoretic method described here can differentiate the three type II rat MHC isoforms, making it useful in the analysis of muscle MHC content. The technique has advantages over previously published protocols; for example, the technique 1) allows for the identification of the four identified MHC isoforms found in adult rat skeletal muscle, 2) does not require the laborious and difficult procedure of preparing a gradient-separating gel, 3) does not require the use of a special cooling apparatus, 4) can be run on a minigel system, and 5) accommodates Coomasie Blue or silver staining of protein bands. Finally, because the method described here makes use of a single percent separating gel, diffusional gradients that may be encountered when silver staining gradient gels are not present. We thank Dr. V. R. Edgerton for critical review of the manuscript and Dr. K. M. Baldwin and R. E. Herrick for helpful discussions. This work was supported by National Institutes of Health National Research Service Award DE-07212 to R. J. Talmadge and Grant NS16333 to R. R. Roy. Address for reprint requests: R. J. Talmadge, Dept. of Physiological Science, University of California at Los Angeles, 405 Hilgard Ave., 1804 Life Science Bldg., Los Angeles, CA 90024-1527. Received 7 July 1993; accepted in final form 16 August 1993. REFERENCES 1. BAR, A., AND D. PETTE. Three fast myosin heavy chains in adult rat skeletal muscle. FEBS Lett. 235: 153-155, 1988. 2. BARANY, M. ATPase activity of myosin correlated with speed of muscle shortening. J. Gen. Physiol. 50: 197-218, 1967. 3. BILLETER, R., H. WEBER, H. LUTZ, H. HOWALD, H. M. EPPENBERGER, AND E. JENNY. Myosin types in human muscle fibers. Histochemistry 65: 249-259, 1980. 4. BIRAL, D., R. BETTO, D. DANIELLI-BETTO, AND G. SALVIATTI. Myosin heavy chain composition of single fibres from normal human muscle. Biochem. J. 250: 307-308, 1988. 5. BOTTINELLI, R., S. SCHIAFFINO, AND C. REGGIANI. Force-velocity relations and myosin heavy chain isoform compositions of skinned fibres from rat skeletal muscle. J. Phvsiol. Lond. 437: 655-673, 1991. 6. BRADFORD, M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the m-inciule of protein-dye binding. Anal. Biockem. 72: 248-252, 1976. 7. BROOKE, M. H., AND K. K. KAISER. Three “myosin adenosine triphosphatase” systems: the nature of their pH lability and sulfhydry1 dependence. J. Histochen. Cytochem. 18: 670-672, 1970. 8. BUTLER-BROWNE, G. S., AND R. G. WHALEN. Myosin isozyme transitions occuring during the postnatal development of the rat soleus muscle. Deu. Biol. 102: 324-334, 1984. 9. CAIOZZO,V. J., R. E. HERRICK, AND K. BALDWIN. Influence of hyperthyroidism on maximal shortening velocity and myosin isoform
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