Vol. 181, No. 3, 1991 December 31, 1991
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1164-l 172
DYSTROPHIN: A SENSITIVE IMMUNOCHEMICAL
AND RELIABLE ASSAY
IN TISSUE AND
CELL CULTURE HOMOGENATES
My-Anh
Ho-Rim, Anny BCdard, Michel and Peter A. Rogers*
Vincent
Lava1 University Hospital Research Center, 2705 Blvd Laurier, Ste-Foy, Quebec, Canada GlV 4G2 Received
October
25,
1991
A modified polyacrylamide gel electrophoresis system is described which provides excellent resolution of very high molecular weight proteins. This system has been successfully applied to the immunochemical detection of dystrophin in mouse and rat skeletal muscle, mouse myotubes in cell culture, and in human muscle-biopsy specimens. The mass of total homogenate protein (3-12 pg) and the relative quantity of dystrophin detected immunologically were found to be strongly correlated (r = 0.970 - 0.995). The method described here requires minute quantities of tissue or cells to accurately evaluate the relative amount of dystrophin present. The entire procedure for the detection of dystrophin is simple, rapid and cost efficient compared to other available techniques. 0 1991*cademuPress, Inc.
Dystrophin is the protein product of the Duchenne and Becker muscular dystrophy (DMD/BMD) gene locus (reviewed in ref. 1). Since the initial identification of the gene (2) there has been a significant accumulation of important information regarding its organization, the multiplicity of mutations resulting in DMD and the less severe form BMD, and our understanding of certain aspects of the molecular nature of dystrophin (3‘4). For example, the organization of the primary structure of this protein into four distinct domains has led to suggestion that dytrophin assembles as antiparallel dimers which then are potentially capable of forming a honeycomb-like network which, it is proposed, offers a degree of resilience to the muscle cell membrane (5). The rapid advancement in our understanding of the dystrophin molecule provides an appropriate basis for the formulation of testable hypotheses concerning the cellular function of this protein as well as how the absence of this protein results
1To whom correspondence
should be addressed .
0006-291X/91 $1.50 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
1164
Vol.
181, No. 3, 1991
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
in the characteristic muscle fiber degeneration which is observed in DMD (6). The isolation of dystrophin (7,8), the myoblast transfer technique (9,101, and the successful transfer of the human dystrophin gene into the myofibers of the mdx mouse (11) collectively illustrate the extent of experimental possibilities that are now available to approach the question of the function of dystrophin. An essential and critical element in such experiments involving high molecular weight polypeptides In the present communication we is that of accurate detection and quantification. describe a modified polyacrylamide gel electrophoresis system which provides a rapid, reliable and quantitative immunochemical method to detect dystrophin. MATERIALS
AND METHODS
Muscle tissues and cell cultures. The soleus and superficial portion of the vastus lateralis muscles were dissected from C57 BL 105 +/+ female mice and Spraguespecimens (vastus lateralis and gluteus Dawley female rats. Human muscle-biopsy maximus muscles) were obtained by informed consent from two patients undergoing hip surgery at Lava1 University Hospital. The muscle tissues were either processed immediately or snap frozen in liquid nitrogen and stored at -8O’C until use. The tissue specimens were weighed, finely minced with scissors, and placed in 10 volumes of homogenizing buffer (10 mM Tris, pH 8.0, 1.0 mM EDTA, 3.3% sodium dodecyl sulfate [SDS], 10% glycerol and 40 mM dithiothreitol [D’IT]) supplemented with a protease inhibitor combination (17 pg/ml phenylmethylsulfonyl fluoride [PMSF] and 5 pg/ml each of leupeptin, antipain and pepstatin). The homogenization was carried out using glass Dual1 tissue grinders (Kontes Glass Co., N.J., U.S.A.). The SDS sample buffer was preheated to w 9O“C and the tissue homogenate was intermittently placed in a boiling water bath during the homogenization. The total heating time of the homogenate should not exceed 2 min. When no visible pieces of tissue could be seen, the resulting homogenate was centrifuged at 10,000 g for 20 min at room temperature. The supernatant was divided into aliquots and stored at -8O’C. Before electrophoresis, the samples were diluted to the desired concentration of protein with SDS sample buffer, heated for 1 min and centrifuged at 10,000 g for 2 min. Primary myoblast cultures were propagated from mouse satellite cells and prepared as follows. Small pieces of muscle were treated with 0.2% collagenase (Sigma Chemical Co., St-Louis, MO) solution in Hank’s Buffered salt solution (HBSS) for a period of 60 min. The tissue was subsequently treated for 30 n-tin with a 0.1% trypsin (Gibco/BRL, Bethesda, MD) solution prepared in HBSS. Satellite cells were collected by low speed centrifugation and plated at a density of 600 x 103 cells per 35 mm gelatin coated culture dishes. Myoblasts were allowed to proliferate for three days in medium 199 supplemented with 15% horse serum, 1% penicillin, 1% streptomycin and 1% L-glutamine. Myoblast fusion and transition to the myotube stage were initiated by reduction of the serum concentration to 2%. Cross-striated myotubes were collected by centrifugation and homogenized exactly as described for rodent and human muscle samples. The mass of total protein in the homogenates was determined by the filter paper dye-binding technique (12). Electrophoretic procedures. Muscle tissue and cell culture proteins were resolved on SDS polyacrylamide gels according to the technique of Laemmli (13) with the following modifications. The resolving gel consisted of a 7-15% linear gradient over which a 6% stacking gel was poured. The ratio of acrylamide to bisacrylamide was 1165
Vol. 181, No. 3, 1991
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
30:0.15 (w/w) in our stock solution instead of 30.0:0.8 as used in the original method. A minigel apparatus (BioRad Inc., Missisauga, ON, Canada) with 1 mm thick spacers was used. Electrophoresis was performed at 150 to 200 volts and terminated when the bromophenol blue marker entered the lower reservoir. The gels were soaked for 15 to 30 min in transfer buffer (25 mM Tris, 192 mM glycine, 0.03% SDS and 20% (v/v) methanol), pH 8.3, then placed in a BioRad minitransblot electrophoretic transfer cell. Fractionated proteins were transferred to polyvinylidene fluoride membranes (Millipore Corp., Missisauga, ON, Canada) for 1 h at 100 volts. All gels were stained with Coomassie blue. detection of dystrophin. Following the electrophoretic transfer, the membranes were stained with Ponceau S solution to evaluate the efficiency of transfer, then rinsed in distilled water. Nonspecific binding sites were saturated by incubating the membranes in 5% (w/v) non fat milk in TBST (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 0.05% (v/v) Tween-20) at room temperature for 1 h or at 4’C overnight. The membranes were first incubated with a monoclonal antibody (NCLDys 2, Novocastra Laboratories Ltd, Newcastle upon Tyne, England) diluted 1:lOO in TBST, then with an affinity-purified goat antimouse IgG conjugated to alkaline phosphatase (BIO/CAN Scientific, Mississauga, ON, Canada) diluted l:lO,OOO in TBST. Following each incubation period, which lasted for 1 h at room temperature, the membranes were washed 3 x 5 min in TBST. The antigen-antibody complex was revealed by chromogenic development using 5-bromo-4 chloro-3 indodyl phosphate and nitroblue tetrazolium (BCIP/NBT). Parallel gels were stained with Coomassie blue to evaluate the degree of resolution of the high molecular weight proteins. Immunochemical
Quantification
of
immunoreaction
products
and
stained
protein
bands.
Immunoblots and stained polyacrylamide gels were photographed and the resulting images digitized using the Loats Associates Research Analysis System (Amersham Corp., Mississauga, ON, Canada). The digitized images were quantitatively analysed using the GL 1000 program. Values are expressed as integrated optical density units (IOD) and are corrected for background. RESULTS
AND DISCUSSION
In order to study the high molecular weight (> 200 kd) structural proteins of mammalian skeletal muscle, a polyacrylamide gel electrophoresis (PAGE) system exhibiting high protein resolution, good mechanical A series of experiments was therefore strength, and cost efficiency was needed. performed in which the acrylamide:bisacrylamide ratio was systematically varied in the resolving gel system; the optimal ratio was found to be 30:0.15 (w/w>. The use of the minigel system, which is widely available commercially, resulted in gels in which protein banding patterns were consistently reproducible and also significantly reduced time, labor and reagent costs. Polyacrylamide
Detection
subsequently muscle (Fig. (600-800 kd) protein was
gel electrophoresis.
The PAGE system was employed to analyse the protein composition of the mouse soleus lB-c). These experiments revealed that both titin (2,600 kd) and nebulin could be readily visualized when as little as 3.0 pg of total homogenate applied to the gel. Four other proteins, whose apparent high molecular
of dystrophin
in mouse skeletal
1166
muscle.
Vol.
181, No. 3, 1991
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
B a
-
DYS
-MHC -T -N 20-Ml-C
s-
- 20 pg) significantly reduced the degree of correlation between the two variables. The intensity of residual myosin heavy chain (MHC), quantitated by densitometry, in 1167
Vol. 181, No. 3, 1991
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
each lane of the post-transfer Coomassie blue stained gels (Fig. lB-b) may also be used as a sensitive indicator of the amount of muscle protein loaded (r = 0.999). The same experimental identical
approach was applied to rat soleus muscle with
results (data not shown).
comparing
slow- and fast-twitch
However,
it is important
rat skeletal muscles, which
to note that in contain
a high
proportion of type I and type II fibers respectively, the relative content of dystrophin (per ug of total protein) can vary up to two-fold (manuscript in preparation). This observation
is important
transplantation
for the evaluation
of the efficiency
technique should it be applied to different
of the myoblast
skeletal muscle of the
mdx mouse which vary with respect to fiber composition. Detection of dystrophin therapy
technique
in mouse muscle cell culture.
(9,lO) has led to investigations
accumulation and distribution
of dystrophin
The advent of the myoblast concerning
the synthesis,
in hybrid myotubes obtained from the
fusion of normal myoblasts and those from DMD patients.
It was therefore of
interest to apply the methodology described here to muscle cell cultures. The results of a typical experiment, shown in Fig. 2, indicated that dystrophin could be reliably detected in myotube cultures. Further, this series of experiments served to assessthe limits of sensitivity of the immunochemical system. Dystrophin is developmentally regulated in both cardiac and skeletal muscle although it is not certain if the cellular accumulation
of this protein parallels that of other muscle
specific proteins (14). The intensity of the immunochemical staining of dystrophin from cell cultures shown in Fig. 2 (inset) was of relatively lesser intensity compared to that obtained from an equivalent amount (8 pg) of total protein from mouse skeletal muscle homogenate. actively
synthesizing
However, it should be noted that these myotubes are
and accumulating
contractile
proteins
and have not yet
attained a steady state level of protein turnover. Therefore, the relative quantity of dystrophin would be more accurately evaluated with respect to the MHC subunit content in developing
myotubes.
The relative quantity
of dystrophin
observed in
this sample may therefore be adjusted accordingly since the MHC subunit content is reduced when compared to developmentally stable muscle cells. Recently the cloning and expression of a full-length dystrophin cDNA in nonmuscle cells has been reported (11). The evaluation of the cellular content of dystrophin
in experiments
of this type is essential for the assessment of the
transfection process and the efficiency of the expression of the transfected gene. The sensitivity of the technique described in this paper constitutes a clear advantage for experiments involving dystrophin gene transfection. Detection of dystrophin
in human muscle-biopsy
specimens. A separate series of
experiments was carried out with human vastus-biopsy specimens using the same protocol described for animal tissues and cell cultures. The results of the 1168
BIOCHEMICAL
Vol. 181, No. 3, 1991
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
-0ystrophin
16 -A 14 -
A
6
12 y-6.3641
/
+1.6619x
r=l.OOO
Figure 2. Immunochemical detection of dystrophin in mouse myotubes in cell
The relative quantity of dystrophin as measured by immunoblotting is highly correlated (r = 1.000) with the mass of cell culture homogenate protein applied to the polyacrylamide gel. The equation describing the relation between the two variables is provided for reference. The stained polyacrylamide gel loaded with 8.0 pg of protein and the corresponding dystrophin immunoblot are shown as insets A and B respectively. Abbreviations are the sameas those indicated in Figure 1.
culture.
experiments are summarized in Fig. 3. As shown in Fig. 3A, the mass of vastus muscle protein applied to the gel and the relative quantity of dystrophin detected were found to be strongly correlated (r = 0.970). A similar degree of correlation (r = 0.990) was also obtained between the residual mass of MHC of the post-transfer Coomassie blue stained gel and the amount of total protein loaded (Fig. 3B-b). A definite and clear identification of dystrophin was also observed with 3.0 pg of total protein (Fig. 3B-a). Both titin and nebulin were easily visualized on the stained gel (Fig. 3B-c). Similar results were obtained with the human gluteus muscle (data not shown). The resolving capacity of this gel system for very high molecular mass proteins should therefore be of value in determining whether titin and nebulin are degraded in skeletal muscle of DMD patients as conflicting reports exist regarding this aspect of the pathophysiology of DMD (15,16). A notable advantage of the electrophoresis and immunoblotting system described in this paper is the small mass of protein required, which consequently reduces the amount of the MHC in the gel. We have noted that when quantities of more than 30 pg of protein were applied to the gel, the 1169
Vol. 181, No. 3, 1991
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
50 1
40.
30. : 20.
10.
, : 0
970
04 0
3
6
9
3
12
PROTEIN
6
9
12
(/&J)
Fimre 3. Dystrophin immunoblotting in human skeletal muscle-biopsy sample. Increasing massesof homogenate protein (3-12 pg) of the vastus lateralis muscle are strongly correlated (r = 0.970) to the relative intensity of the dystrophin immunoreaction product (panel B-a) evaluated by image analysis. The myosin heavy chain subunit (MHC) is shown in the stained post-transfer gel (panel B-b). Note the MHC isoform separation in the gel lane corresponding to 3.0 pg of homogenate protein. A stained gel identical to that used in the immunoblotting experiments is illustrated in panel B-c for reference. Abbreviations used are the sameas identified in Figure 1.
large amount of MHC rendered the banding pattern and identification of high molecular mass proteins difficult and variable from experiment to experiment. The present study defines a set of optimal conditions for the resolution and subsequent immunochemical detection of dystrophin in human and animal tissue homogenates as well as in muscle cell culture homogenates. By reducing the amount of bisacrylamide in the polyacrylamide gel and employing a gradient in the resolving gel, we achieved a high degree of resolution of high molecular weight proteins, including titin and nebulin, two major proteins of the myofibrillar cytoskeleton (20). The low bisacrylamide content also enhanced the electrophoretic transfer of these proteins from the gel. The high degree of sensitivity of dystrophin detection was also partially attributed to this modification. This method should hence be generally applicable to immunochemical detection and quantification of other proteins from a wide variety of tissues and cell types. There are numerous of reports in the literature which describe modifications of various parameters of the polyacrylamide gel electrophoresis technique and the 1170
Vol. 181, No. 3, 1991
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
subsequent electrotransfer of the proteins to solid supports (17,18,19,20). The method described in the present report offers several advantages over currently employed techniques. The low bisacrylamide content facilitates the transfer of protein to the membrane support and in the case of dystrophin, the transfer is quantitative. The use of an acrylamide gradient of 7-15 % produces sharp and distinct stained protein bands ranging from titin to actin. Moreover, this particular gradient results in gels which are very easily handled and no particular precautions or additional steps are required for the electrotransfer procedure (20). The entire method described herein for the immunodetection of dystrophin can be applied to several high molecular mass protein including titin and nebulin (data not shown). However, it should be noted that the exact conditions which are ideal for one protein may not apply to another. For example, in the case of titin the duration of the electrotransfer is extended to 2 hours. Interestingly, we have observed that titin from slow-twitch skeletal muscle can be electrotransferred with a significantly greater degree efficiency than titin from fast-twitch skeletal muscle (unpublished observations). Therefore it is suggested that conditions for the immunodetection of a particular protein be fully explored prior to comparative analyses, especially those involving comparisons between fast- and slow-twitch skeletal muscles. As the number of site specifc antibodies and interest in the physiological function of dystrophin continues to grow there will be a need for a standard protocol to quantitatively evaluate the mass of dystrophin as well as provide accurate information concerning the status of other small and large polypeptides of the muscle cell. It is suggested that the methodology described here adequately fulfils these requirements. ACKNOWLEDGMENTS This work was supported by the Muscular Dystrophy Association of Canada. Dr. M. Vincent was supported by the Fonds de la recherche en Sante du Quebec. We would like to thank Dr. J. Tremblay and Mr. J. Huard for providing the muscle cell cultures and constructive discussion, Dr. J. Gagnon for the human muscle-biopsy specimens, Mr. R. Maheux for the densitometric quantification of dystrophin on the immunoblots and stained gels, and Ms. L. Turcotte for typing the manuscript. REFERENCES 1. 2. 3. 4. 5.
Beggs, A.H., and Kunkel, L.M. (1990) J. Clin. Monaco, A.P., Neve, R.L., Colletti-Feener, Kunkel, L.M. (1986) Nature 323,646-650. Hoffman, E.P., Brown, R.H. Jr., and Kunkel, Koenig, M., Monaco, A.P., and Kunkel, L.M. Koenig, M., and Ktmkel, L.M. (1990) J. Biol. 1171
Invest. 85,613-619. C., Bertelson, C.J., Kumit, L.M. (1987) Cell 51,919-928. (1988) Cell 53,219-228. Chem. 265,4560-4566.
D.M., and
Vol. 181, No. 3, 1991
6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Moser, H. (1984) Hum. Genet. 66,17-40. Ports, F., Augier, N., Heilig, R., Leger, J., Mornet, D., and Leger, J.J. (1990) Proc. Natl. Acad. Sci USA 87,7851-7855. Ervasti, J.M., Kahl, SD., and Campbell, K.P. (1991) J. Biol. Chem. 266,9161-9165. Partridge, T.A., Morgan, J.E., Coulton, G.R., Hoffman, E.P., and Kunkel, L.M. (1989) Nature 337, 176-179. Karpati, G., Pouliot, Y., Zubrzycha-Gaarn, E., Carpenter, S., Ray, P.N., Warton, R.G., and Holland, P. (1989) Am. J. Pathol. 135,27-32. Acsadi, G., Dickson, G., Love, D.R., Jani, A., Walsh, F.S., Gurusinghe, A., Wolff, J.A., and Davies, K.E. (1991) Nature 352,815-818. Ninamide, L.S., and Bamburg, J.R. (1990) Anal. Biochem. 190, 66-70. Laemmli, U.K. (1970) Nature 227,680-685. Tanaka, H., and Ozawa, E. (1990) Histochem. 94,449-453. Wood, D.S., Zeviani, M., Prelle, A., Bonilla, E., Salviati, G., Miranda, A.F., DiMauro, S., and Rowland, L.P. (1987) N. Engl. J. Med. 316,107-108. Fiirst, D., Nave, R., Osborn, M., Weber, K., Bardosi, A., Archidiacono, N., Ferro, M., Romano, V., and Romeo, G. (1987) FEBS Lettr. 224,49-53. Doucet, J.-P., and Trifaro, J.-M. (1988) Anal. Biochem. 168, 265-271. Doucet, J.-P., Murphy, B.J., and Tuana, B.S. (1990) Anal. Biochem. 190,209-211. Fritz, J.D., Swartz, D.R., and Greaser, M.L. (1989) Anal. Biochem. 180,205-210. Wang, K., Fanger, B.O., Guyer, C.A., and Staros, T.V. (1989) Meth. Enzymol. 172, 687-696.
1172
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1164-l 172
DYSTROPHIN: A SENSITIVE IMMUNOCHEMICAL
AND RELIABLE ASSAY
IN TISSUE AND
CELL CULTURE HOMOGENATES
My-Anh
Ho-Rim, Anny BCdard, Michel and Peter A. Rogers*
Vincent
Lava1 University Hospital Research Center, 2705 Blvd Laurier, Ste-Foy, Quebec, Canada GlV 4G2 Received
October
25,
1991
A modified polyacrylamide gel electrophoresis system is described which provides excellent resolution of very high molecular weight proteins. This system has been successfully applied to the immunochemical detection of dystrophin in mouse and rat skeletal muscle, mouse myotubes in cell culture, and in human muscle-biopsy specimens. The mass of total homogenate protein (3-12 pg) and the relative quantity of dystrophin detected immunologically were found to be strongly correlated (r = 0.970 - 0.995). The method described here requires minute quantities of tissue or cells to accurately evaluate the relative amount of dystrophin present. The entire procedure for the detection of dystrophin is simple, rapid and cost efficient compared to other available techniques. 0 1991*cademuPress, Inc.
Dystrophin is the protein product of the Duchenne and Becker muscular dystrophy (DMD/BMD) gene locus (reviewed in ref. 1). Since the initial identification of the gene (2) there has been a significant accumulation of important information regarding its organization, the multiplicity of mutations resulting in DMD and the less severe form BMD, and our understanding of certain aspects of the molecular nature of dystrophin (3‘4). For example, the organization of the primary structure of this protein into four distinct domains has led to suggestion that dytrophin assembles as antiparallel dimers which then are potentially capable of forming a honeycomb-like network which, it is proposed, offers a degree of resilience to the muscle cell membrane (5). The rapid advancement in our understanding of the dystrophin molecule provides an appropriate basis for the formulation of testable hypotheses concerning the cellular function of this protein as well as how the absence of this protein results
1To whom correspondence
should be addressed .
0006-291X/91 $1.50 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
1164
Vol.
181, No. 3, 1991
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
in the characteristic muscle fiber degeneration which is observed in DMD (6). The isolation of dystrophin (7,8), the myoblast transfer technique (9,101, and the successful transfer of the human dystrophin gene into the myofibers of the mdx mouse (11) collectively illustrate the extent of experimental possibilities that are now available to approach the question of the function of dystrophin. An essential and critical element in such experiments involving high molecular weight polypeptides In the present communication we is that of accurate detection and quantification. describe a modified polyacrylamide gel electrophoresis system which provides a rapid, reliable and quantitative immunochemical method to detect dystrophin. MATERIALS
AND METHODS
Muscle tissues and cell cultures. The soleus and superficial portion of the vastus lateralis muscles were dissected from C57 BL 105 +/+ female mice and Spraguespecimens (vastus lateralis and gluteus Dawley female rats. Human muscle-biopsy maximus muscles) were obtained by informed consent from two patients undergoing hip surgery at Lava1 University Hospital. The muscle tissues were either processed immediately or snap frozen in liquid nitrogen and stored at -8O’C until use. The tissue specimens were weighed, finely minced with scissors, and placed in 10 volumes of homogenizing buffer (10 mM Tris, pH 8.0, 1.0 mM EDTA, 3.3% sodium dodecyl sulfate [SDS], 10% glycerol and 40 mM dithiothreitol [D’IT]) supplemented with a protease inhibitor combination (17 pg/ml phenylmethylsulfonyl fluoride [PMSF] and 5 pg/ml each of leupeptin, antipain and pepstatin). The homogenization was carried out using glass Dual1 tissue grinders (Kontes Glass Co., N.J., U.S.A.). The SDS sample buffer was preheated to w 9O“C and the tissue homogenate was intermittently placed in a boiling water bath during the homogenization. The total heating time of the homogenate should not exceed 2 min. When no visible pieces of tissue could be seen, the resulting homogenate was centrifuged at 10,000 g for 20 min at room temperature. The supernatant was divided into aliquots and stored at -8O’C. Before electrophoresis, the samples were diluted to the desired concentration of protein with SDS sample buffer, heated for 1 min and centrifuged at 10,000 g for 2 min. Primary myoblast cultures were propagated from mouse satellite cells and prepared as follows. Small pieces of muscle were treated with 0.2% collagenase (Sigma Chemical Co., St-Louis, MO) solution in Hank’s Buffered salt solution (HBSS) for a period of 60 min. The tissue was subsequently treated for 30 n-tin with a 0.1% trypsin (Gibco/BRL, Bethesda, MD) solution prepared in HBSS. Satellite cells were collected by low speed centrifugation and plated at a density of 600 x 103 cells per 35 mm gelatin coated culture dishes. Myoblasts were allowed to proliferate for three days in medium 199 supplemented with 15% horse serum, 1% penicillin, 1% streptomycin and 1% L-glutamine. Myoblast fusion and transition to the myotube stage were initiated by reduction of the serum concentration to 2%. Cross-striated myotubes were collected by centrifugation and homogenized exactly as described for rodent and human muscle samples. The mass of total protein in the homogenates was determined by the filter paper dye-binding technique (12). Electrophoretic procedures. Muscle tissue and cell culture proteins were resolved on SDS polyacrylamide gels according to the technique of Laemmli (13) with the following modifications. The resolving gel consisted of a 7-15% linear gradient over which a 6% stacking gel was poured. The ratio of acrylamide to bisacrylamide was 1165
Vol. 181, No. 3, 1991
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
30:0.15 (w/w) in our stock solution instead of 30.0:0.8 as used in the original method. A minigel apparatus (BioRad Inc., Missisauga, ON, Canada) with 1 mm thick spacers was used. Electrophoresis was performed at 150 to 200 volts and terminated when the bromophenol blue marker entered the lower reservoir. The gels were soaked for 15 to 30 min in transfer buffer (25 mM Tris, 192 mM glycine, 0.03% SDS and 20% (v/v) methanol), pH 8.3, then placed in a BioRad minitransblot electrophoretic transfer cell. Fractionated proteins were transferred to polyvinylidene fluoride membranes (Millipore Corp., Missisauga, ON, Canada) for 1 h at 100 volts. All gels were stained with Coomassie blue. detection of dystrophin. Following the electrophoretic transfer, the membranes were stained with Ponceau S solution to evaluate the efficiency of transfer, then rinsed in distilled water. Nonspecific binding sites were saturated by incubating the membranes in 5% (w/v) non fat milk in TBST (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 0.05% (v/v) Tween-20) at room temperature for 1 h or at 4’C overnight. The membranes were first incubated with a monoclonal antibody (NCLDys 2, Novocastra Laboratories Ltd, Newcastle upon Tyne, England) diluted 1:lOO in TBST, then with an affinity-purified goat antimouse IgG conjugated to alkaline phosphatase (BIO/CAN Scientific, Mississauga, ON, Canada) diluted l:lO,OOO in TBST. Following each incubation period, which lasted for 1 h at room temperature, the membranes were washed 3 x 5 min in TBST. The antigen-antibody complex was revealed by chromogenic development using 5-bromo-4 chloro-3 indodyl phosphate and nitroblue tetrazolium (BCIP/NBT). Parallel gels were stained with Coomassie blue to evaluate the degree of resolution of the high molecular weight proteins. Immunochemical
Quantification
of
immunoreaction
products
and
stained
protein
bands.
Immunoblots and stained polyacrylamide gels were photographed and the resulting images digitized using the Loats Associates Research Analysis System (Amersham Corp., Mississauga, ON, Canada). The digitized images were quantitatively analysed using the GL 1000 program. Values are expressed as integrated optical density units (IOD) and are corrected for background. RESULTS
AND DISCUSSION
In order to study the high molecular weight (> 200 kd) structural proteins of mammalian skeletal muscle, a polyacrylamide gel electrophoresis (PAGE) system exhibiting high protein resolution, good mechanical A series of experiments was therefore strength, and cost efficiency was needed. performed in which the acrylamide:bisacrylamide ratio was systematically varied in the resolving gel system; the optimal ratio was found to be 30:0.15 (w/w>. The use of the minigel system, which is widely available commercially, resulted in gels in which protein banding patterns were consistently reproducible and also significantly reduced time, labor and reagent costs. Polyacrylamide
Detection
subsequently muscle (Fig. (600-800 kd) protein was
gel electrophoresis.
The PAGE system was employed to analyse the protein composition of the mouse soleus lB-c). These experiments revealed that both titin (2,600 kd) and nebulin could be readily visualized when as little as 3.0 pg of total homogenate applied to the gel. Four other proteins, whose apparent high molecular
of dystrophin
in mouse skeletal
1166
muscle.
Vol.
181, No. 3, 1991
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
B a
-
DYS
-MHC -T -N 20-Ml-C
s-
- 20 pg) significantly reduced the degree of correlation between the two variables. The intensity of residual myosin heavy chain (MHC), quantitated by densitometry, in 1167
Vol. 181, No. 3, 1991
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
each lane of the post-transfer Coomassie blue stained gels (Fig. lB-b) may also be used as a sensitive indicator of the amount of muscle protein loaded (r = 0.999). The same experimental identical
approach was applied to rat soleus muscle with
results (data not shown).
comparing
slow- and fast-twitch
However,
it is important
rat skeletal muscles, which
to note that in contain
a high
proportion of type I and type II fibers respectively, the relative content of dystrophin (per ug of total protein) can vary up to two-fold (manuscript in preparation). This observation
is important
transplantation
for the evaluation
of the efficiency
technique should it be applied to different
of the myoblast
skeletal muscle of the
mdx mouse which vary with respect to fiber composition. Detection of dystrophin therapy
technique
in mouse muscle cell culture.
(9,lO) has led to investigations
accumulation and distribution
of dystrophin
The advent of the myoblast concerning
the synthesis,
in hybrid myotubes obtained from the
fusion of normal myoblasts and those from DMD patients.
It was therefore of
interest to apply the methodology described here to muscle cell cultures. The results of a typical experiment, shown in Fig. 2, indicated that dystrophin could be reliably detected in myotube cultures. Further, this series of experiments served to assessthe limits of sensitivity of the immunochemical system. Dystrophin is developmentally regulated in both cardiac and skeletal muscle although it is not certain if the cellular accumulation
of this protein parallels that of other muscle
specific proteins (14). The intensity of the immunochemical staining of dystrophin from cell cultures shown in Fig. 2 (inset) was of relatively lesser intensity compared to that obtained from an equivalent amount (8 pg) of total protein from mouse skeletal muscle homogenate. actively
synthesizing
However, it should be noted that these myotubes are
and accumulating
contractile
proteins
and have not yet
attained a steady state level of protein turnover. Therefore, the relative quantity of dystrophin would be more accurately evaluated with respect to the MHC subunit content in developing
myotubes.
The relative quantity
of dystrophin
observed in
this sample may therefore be adjusted accordingly since the MHC subunit content is reduced when compared to developmentally stable muscle cells. Recently the cloning and expression of a full-length dystrophin cDNA in nonmuscle cells has been reported (11). The evaluation of the cellular content of dystrophin
in experiments
of this type is essential for the assessment of the
transfection process and the efficiency of the expression of the transfected gene. The sensitivity of the technique described in this paper constitutes a clear advantage for experiments involving dystrophin gene transfection. Detection of dystrophin
in human muscle-biopsy
specimens. A separate series of
experiments was carried out with human vastus-biopsy specimens using the same protocol described for animal tissues and cell cultures. The results of the 1168
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-0ystrophin
16 -A 14 -
A
6
12 y-6.3641
/
+1.6619x
r=l.OOO
Figure 2. Immunochemical detection of dystrophin in mouse myotubes in cell
The relative quantity of dystrophin as measured by immunoblotting is highly correlated (r = 1.000) with the mass of cell culture homogenate protein applied to the polyacrylamide gel. The equation describing the relation between the two variables is provided for reference. The stained polyacrylamide gel loaded with 8.0 pg of protein and the corresponding dystrophin immunoblot are shown as insets A and B respectively. Abbreviations are the sameas those indicated in Figure 1.
culture.
experiments are summarized in Fig. 3. As shown in Fig. 3A, the mass of vastus muscle protein applied to the gel and the relative quantity of dystrophin detected were found to be strongly correlated (r = 0.970). A similar degree of correlation (r = 0.990) was also obtained between the residual mass of MHC of the post-transfer Coomassie blue stained gel and the amount of total protein loaded (Fig. 3B-b). A definite and clear identification of dystrophin was also observed with 3.0 pg of total protein (Fig. 3B-a). Both titin and nebulin were easily visualized on the stained gel (Fig. 3B-c). Similar results were obtained with the human gluteus muscle (data not shown). The resolving capacity of this gel system for very high molecular mass proteins should therefore be of value in determining whether titin and nebulin are degraded in skeletal muscle of DMD patients as conflicting reports exist regarding this aspect of the pathophysiology of DMD (15,16). A notable advantage of the electrophoresis and immunoblotting system described in this paper is the small mass of protein required, which consequently reduces the amount of the MHC in the gel. We have noted that when quantities of more than 30 pg of protein were applied to the gel, the 1169
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50 1
40.
30. : 20.
10.
, : 0
970
04 0
3
6
9
3
12
PROTEIN
6
9
12
(/&J)
Fimre 3. Dystrophin immunoblotting in human skeletal muscle-biopsy sample. Increasing massesof homogenate protein (3-12 pg) of the vastus lateralis muscle are strongly correlated (r = 0.970) to the relative intensity of the dystrophin immunoreaction product (panel B-a) evaluated by image analysis. The myosin heavy chain subunit (MHC) is shown in the stained post-transfer gel (panel B-b). Note the MHC isoform separation in the gel lane corresponding to 3.0 pg of homogenate protein. A stained gel identical to that used in the immunoblotting experiments is illustrated in panel B-c for reference. Abbreviations used are the sameas identified in Figure 1.
large amount of MHC rendered the banding pattern and identification of high molecular mass proteins difficult and variable from experiment to experiment. The present study defines a set of optimal conditions for the resolution and subsequent immunochemical detection of dystrophin in human and animal tissue homogenates as well as in muscle cell culture homogenates. By reducing the amount of bisacrylamide in the polyacrylamide gel and employing a gradient in the resolving gel, we achieved a high degree of resolution of high molecular weight proteins, including titin and nebulin, two major proteins of the myofibrillar cytoskeleton (20). The low bisacrylamide content also enhanced the electrophoretic transfer of these proteins from the gel. The high degree of sensitivity of dystrophin detection was also partially attributed to this modification. This method should hence be generally applicable to immunochemical detection and quantification of other proteins from a wide variety of tissues and cell types. There are numerous of reports in the literature which describe modifications of various parameters of the polyacrylamide gel electrophoresis technique and the 1170
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subsequent electrotransfer of the proteins to solid supports (17,18,19,20). The method described in the present report offers several advantages over currently employed techniques. The low bisacrylamide content facilitates the transfer of protein to the membrane support and in the case of dystrophin, the transfer is quantitative. The use of an acrylamide gradient of 7-15 % produces sharp and distinct stained protein bands ranging from titin to actin. Moreover, this particular gradient results in gels which are very easily handled and no particular precautions or additional steps are required for the electrotransfer procedure (20). The entire method described herein for the immunodetection of dystrophin can be applied to several high molecular mass protein including titin and nebulin (data not shown). However, it should be noted that the exact conditions which are ideal for one protein may not apply to another. For example, in the case of titin the duration of the electrotransfer is extended to 2 hours. Interestingly, we have observed that titin from slow-twitch skeletal muscle can be electrotransferred with a significantly greater degree efficiency than titin from fast-twitch skeletal muscle (unpublished observations). Therefore it is suggested that conditions for the immunodetection of a particular protein be fully explored prior to comparative analyses, especially those involving comparisons between fast- and slow-twitch skeletal muscles. As the number of site specifc antibodies and interest in the physiological function of dystrophin continues to grow there will be a need for a standard protocol to quantitatively evaluate the mass of dystrophin as well as provide accurate information concerning the status of other small and large polypeptides of the muscle cell. It is suggested that the methodology described here adequately fulfils these requirements. ACKNOWLEDGMENTS This work was supported by the Muscular Dystrophy Association of Canada. Dr. M. Vincent was supported by the Fonds de la recherche en Sante du Quebec. We would like to thank Dr. J. Tremblay and Mr. J. Huard for providing the muscle cell cultures and constructive discussion, Dr. J. Gagnon for the human muscle-biopsy specimens, Mr. R. Maheux for the densitometric quantification of dystrophin on the immunoblots and stained gels, and Ms. L. Turcotte for typing the manuscript. REFERENCES 1. 2. 3. 4. 5.
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