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Method for cardiac myosin heavy chain separation by sodium dodecyl sulfate gel electrophoresis
ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 320 (2003) 149–151 www.elsevier.com/locate/yabio
Notes & Tips
Method for cardiac myosin heavy chain separation by sodium dodecyl sulfate gel electrophoresis Chad M. Warren and Marion L. Greaser* Muscle Biology Laboratory, University of Wisconsin-Madison, Madison, WI 53706, USA Received 28 March 2003
Separation of cardiac myosin heavy chain (MHC)1 isoforms using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) is technically difficult. Gradient gels (4–9%) have been used in the past with adequate resolution; however, such gels can be difficult to pour and they often undergo physical distortion upon drying and handling [1]. Nongradient gels, however, give incomplete separation of the alpha and beta MHC isoforms [2,3]. Adequate separation has been achieved only using run times of 30–48 h [4]. A recently described pulse field system does an excellent job of separating a variety of myosin heavy chain species, but it requires specialized equipment and 24- to 64-h run times [5]. It was the aim of this report to describe a SDS–PAGE system that separates the cardiac MHC isoforms easily and reliably.
Materials and methods Sample buffer. The sample buffer contained 8 M urea, 2 M thiourea, 3% SDS (w/v), 75 mM DTT, 0.03% bromophenol blue, and 0.05 M Tris–Cl, pH 6.8 (modified from [6]). Sample buffer (100 ml) is prepared by adding solid urea and thiourea to a small volume of heated water (do not exceed 40 °C due to rapid formation of cyanate) until dissolved completely. Then about 10 g of mixed bed resin (TMD-8, Sigma M-8157) was added and the mixture was stirred for 15 min to deionize the urea. The slurry was then filtered through Whatman No. 1 filter paper to remove the resin. Tris-base, DTT, SDS, and bromophenol blue were then added and the * Corresponding author. Fax: +1-608-265-3110. E-mail address: mgreaser@facstaff.wisc.edu (M.L. Greaser). 1 Abbreviations used: MHC, myosin heavy chain; DTT, dithiothreitol.
pH was carefully adjusted to 6.8 using HCl (use special care near the end since the buffer capacity is very low); if you overshoot, use Tris-base to readjust. Sample buffer may be aliquoted and stored at )20 °C. Sample preparation. Cardiac tissue was dissected from Sprague–Dawley rats and Black Swiss mice and flash frozen in liquid nitrogen. The frozen tissue was pulverized and placed in preweighed 2-ml Dounce homogenizers. The weight of the tissue was determined and sample buffer was added (between 80:1 and 200:1 buffer to tissue v/w ratio). The tissue was dispersed using several strokes in the homogenizer. Samples were then vortexed thoroughly and heated at 100 °C for 3 min. The samples were again vortexed and subsequently centrifuged for 5 min at 13,200g. The supernatant was removed, transferred to clean tubes, and either immediately loaded on the gels or stored at )80 °C prior to electrophoresis. Gel preparation. Glass plates were 16 18 cm, cleaned with soap and water, rinsed with 100% ethanol, and allowed to dry. After the glass plates were dried they were coated with Rain X or Sigmacote (Sigma) to allow for easier removal of the gels from the plates [DATD gels are stickier and more difficult to remove than biscross-linked gels]. A SE600 Hoefer gel system (Pharmacia) was used with 0.75-mm gel spacers. It is important to note that this large gel format is needed to adequately separate the isoforms. The following recipe is sufficient for one 16 18-cm gel. To make 17 ml of a 6%T 2.6%C resolving gel, add 6.56 ml water, 3.4 ml 50% glycerol (v/v), 4.25 ml 1.5 M Tris, pH 8.8, 2.55 ml 40% acrylamide (37.5:1 cross-linked with DATD (Bio-Rad) [7]), 170 ll 10% sodium dodecyl sulfate (w/v), 50 ll 10% ammonium persulfate (w/v), and 20 ll TEMED. The resolving gel was poured to allow a stacking gel with 1-cm deep wells and at least 1-cm length between the bottom of the wells and the resolving gel. Water was added to the top of the resolving gel to form a flat
0003-2697/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0003-2697(03)00350-6
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Notes & Tips / Analytical Biochemistry 320 (2003) 149–151
interface, and the gel was stored in the cold room after polymerization overnight. The stacking gel consisted of 5.085 ml of a 2.95%T 15%C acrylamide. Mix 1.5 ml 10% acrylamide (5.6:1 cross-linked with DATD (Bio-Rad) [7]), 1.3 ml 0.5 M Tris, pH 6.8, 1.15 ml water, 1 ml 50% glycerol (v/v), 50 ll 10% sodium dodecyl sulfate (w/v), 30 ll 10% ammonium persulfate (w/v), 30 ll stacking gel dye [0.1% (w/v) bromophenol blue, 1% (w/v) SDS, and 3% (v/v) glycerol], and 25 ll TEMED [8,9]. The inclusion of stacking dye in the stacking gel does not interfere with the gel polymerization or alter the run characteristics of the gels. However, it allows better visualization of the loading wells and the careful deposit of sample in the base of the well. The water was drained off the resolving gel and the stacking gel was poured at room temperature with a 20-lane comb inserted. Electrophoresis conditions. The upper buffer reservoir had 600 ml of 0.05 M Tris-base, 0.384 M glycine, 0.1% (w/v) SDS, and 10 mM 2-mercaptoethanol, no pH adjustment (modified from [9]). The lower buffer chamber had 4 liters of the same buffer except without 2mercaptoethanol. Usually only one gel was run at a time using 16 mA constant current for 4.5 h with constant cooling to 8 °C using a circulating water bath. After the electrophoresis was complete the gels were stained using Coomassie blue R-250 as previously described [10].
Results and discussion The myosin heavy chains move approximately halfway down in this gel system. Examples of the band resolution of rat and mouse cardiac myosins are shown in Fig. 1. There are clearly separation differences between rat and mouse MHC isoforms (Fig. 1). The beta chains have similar mobilities, but the mouse alpha chain (lane 2) is retarded significantly compared to the rat alpha (upper band, lane 3). The rat aMHC has 1938 residues, a charge of )46, and a molecular mass of 223,506 Da based on the cDNA sequence (GenBank Accession No. NP_058935). The mouse aMHC also has 1938 residues and a charge of –47, and a molecular mass of 223,563 Da based on the cDNA sequence (GenBank
Accession No. NP_034986). Thus the residue numbers are the same; there is a charge difference of only 1 and a molecular mass difference of only 57 Da between the rat and the mouse aMHC. The rat and mouse bMHC has the same charge and residue number; however, that of the rat has a molecular mass that is 30 Da smaller than that of the mouse based on the cDNA sequence (GenBank Accession Nos. X15939 and NP_000248, respectively). Why the MHC isoforms of the mouse and rat migrate so differently is unclear. However, there is some evidence for differential posttranslational modification of myosin heavy chains during development [11]. Posttranslational modifications may, therefore, explain differences in the migration in myosin heavy chains in rat and mouse cardiac tissue. The myosin heavy chain isoforms have been shown to switch in rodents from mainly bMHC in early development to mainly aMHC later in life (lanes 1–4, Fig. 1) [12–14]. In Fig. 1, lane 5 an adult rat ventricle has about 33% bMHC along with two separately migrating bands at the position of aMHC. This particular rat sample is uncharacteristic in that it has more beta and two closely spaced bands at the position of aMHC. It is possible that these two bands are due to a posttranscriptional event where the inclusion or exclusion of a single glutamine at position 1931 causes alternate splicing of the transcript [15,16]. Two rat cardiac aMHC bands have been observed previously using the pulse electrophoresis system [5]. The use of DATD as a cross-linker has been shown to increase the mechanical stability in highly cross-linked gels [7]. This cross-linker results in gels with larger pore sizes than those produced using bis-acrylamide and these gels are thus less restrictive for the movement of high-molecular-weight proteins [7]. In this case the myosin heavy chains move a greater distance in a more rigid gel with excellent resolution of the isoforms. In conclusion, the electrophoresis system described here is simple and reproducible and allows shorter run times than those described previously. It should be a useful tool for investigating myosin heavy chain isoform transitions in health and disease. Acknowledgments This work was supported by the College of Agricultural and Life Sciences, University of Wisconsin-Madison and by grants from the National Institutes of Health (HL47053; HL62466).
Fig. 1. SDS–PAGE gel of cardiac muscle. Only the myosin heavy chain area is shown. Lane 1, 18 day postconception fetal mouse; lane 2, adult cardiac mouse ventricle; lane 3, day 1 neonatal rat cardiac ventricle; lane 4, adult rat cardiac ventricle; lane 5, adult rat cardiac ventricle; lane 6, day 1 neonatal rat cardiac ventricle.
References [1] K.A. Esser, M.O. Boluyt, T.P. White, Separation of cardiac myosin heavy chains by gradient SDS–PAGE, Am. J. Physiol. 255 (1988) H659–H663.
Notes & Tips / Analytical Biochemistry 320 (2003) 149–151 [2] N.K. Sweitzer, R.L. Moss, Determinants of loaded shortening velocity in single cardiac myocytes permeabilized with a-hemolysin, Circ. Res. 73 (1993) 1150–1162. [3] K.L. Ball, J.R. Solaro, Discoordinate regulation of contractile protein gene expression in the senescent rat myocardium, J. Mol. Cell. Cardiol. 26 (1994) 519–525. [4] P.J. Reiser, W.O. Kline, Electrophoretic separation and quantitation of cardiac myosin heavy chain isoforms in eight mammalian species, Am. J. Physiol. 274 (1998) H1048–H1053. [5] J.A.A. SantÕAna Pereira, M. Greaser, R.L. Moss, Pulse electrophoresis of muscle myosin heavy chains in sodium dodecyl sulfatepolyacrylamide gels, Anal. Biochem. 291 (2001) 229–236. [6] L.D. Yates, M.L. Greaser, Quantitative determination of myosin and actin in rabbit skeletal muscle, J. Mol. Biol. 168 (1983) 123–141. [7] G. Baumann, A. Chrambach, A highly crosslinked, transparent polyacrylamide gel with improved mechanical stability for use in isoelectric focusing and isotachophoresis, Anal. Biochem. 70 (1976) 32–38. [8] J.D. Fritz, D.R. Swartz, M.L. Greaser, Factors affecting polyacrylamide gel electrophoresis and electroblotting of high-molecular-weight myofibrillar proteins, Anal. Biochem. 180 (1989) 205–210. [9] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227 (1970) 680–685.
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[10] J. Sambrook, E.F. Fritsch, T. Maniatis, in: Molecular Cloning. A Laboratory Manual, second ed., Cold Spring Harbor Press, Cold Spring Harbor, NY, 1989, pp. 18–55. [11] A.M. Maggs, P. Taylor-Harris, M. Peckham, S.M. Hughes, Evidence for differential post-translational modifications of slow myosin heavy chain during murine skeletal muscle development, J. Muscle Res. Cell Motil. 21 (2000) 101–113. [12] A.M. Lompre, J.J. Mercadier, C. Wisnewsky, P. Bouveret, C. Pantaloni, A. DÕAlbis, K. Schwartz, Species- and age dependent changes in the relative amounts of cardiac myosin isoenzymes in mammals, Dev. Biol. 84 (1981) 286–290. [13] A.M. Lompre, B. Nadal-Ginard, V. Mahdavi, Expression of the cardiac ventricular a- and b-myosin heavy chain genes is developmentally and hormonally regulated, J. Biol. Chem. 259 (1984) 6437–6446. [14] X. Bian, M.M. Briggs, F.H. Schachat, F.J. Seidler, T.A. Slotkin, Glucocorticoids accelerate the ontogenetic transition of cardiac ventricular myosin heavy-chain isoform expression in the rat: promotion by prenatal exposure to a low dose of dexamethasone, J. Dev. Physiol. 18 (1992) 35–42. [15] B.K. Quinn-Laquer, L.E. Kennedy, S.J. Wei, K.W. Beisel, Characterization of the allelic differences in the mouse cardiac amyosin heavy chain coding sequence, Genomics 13 (1992) 176–188. [16] R. Sindhwani, F. Ismail-Beigi, L.A. Leinwand, Post-transcriptional regulation of rat a cardiac myosin heavy chain gene expression, J. Biol. Chem. 269 (1994) 3272–3276.
Notes & Tips
Method for cardiac myosin heavy chain separation by sodium dodecyl sulfate gel electrophoresis Chad M. Warren and Marion L. Greaser* Muscle Biology Laboratory, University of Wisconsin-Madison, Madison, WI 53706, USA Received 28 March 2003
Separation of cardiac myosin heavy chain (MHC)1 isoforms using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) is technically difficult. Gradient gels (4–9%) have been used in the past with adequate resolution; however, such gels can be difficult to pour and they often undergo physical distortion upon drying and handling [1]. Nongradient gels, however, give incomplete separation of the alpha and beta MHC isoforms [2,3]. Adequate separation has been achieved only using run times of 30–48 h [4]. A recently described pulse field system does an excellent job of separating a variety of myosin heavy chain species, but it requires specialized equipment and 24- to 64-h run times [5]. It was the aim of this report to describe a SDS–PAGE system that separates the cardiac MHC isoforms easily and reliably.
Materials and methods Sample buffer. The sample buffer contained 8 M urea, 2 M thiourea, 3% SDS (w/v), 75 mM DTT, 0.03% bromophenol blue, and 0.05 M Tris–Cl, pH 6.8 (modified from [6]). Sample buffer (100 ml) is prepared by adding solid urea and thiourea to a small volume of heated water (do not exceed 40 °C due to rapid formation of cyanate) until dissolved completely. Then about 10 g of mixed bed resin (TMD-8, Sigma M-8157) was added and the mixture was stirred for 15 min to deionize the urea. The slurry was then filtered through Whatman No. 1 filter paper to remove the resin. Tris-base, DTT, SDS, and bromophenol blue were then added and the * Corresponding author. Fax: +1-608-265-3110. E-mail address: mgreaser@facstaff.wisc.edu (M.L. Greaser). 1 Abbreviations used: MHC, myosin heavy chain; DTT, dithiothreitol.
pH was carefully adjusted to 6.8 using HCl (use special care near the end since the buffer capacity is very low); if you overshoot, use Tris-base to readjust. Sample buffer may be aliquoted and stored at )20 °C. Sample preparation. Cardiac tissue was dissected from Sprague–Dawley rats and Black Swiss mice and flash frozen in liquid nitrogen. The frozen tissue was pulverized and placed in preweighed 2-ml Dounce homogenizers. The weight of the tissue was determined and sample buffer was added (between 80:1 and 200:1 buffer to tissue v/w ratio). The tissue was dispersed using several strokes in the homogenizer. Samples were then vortexed thoroughly and heated at 100 °C for 3 min. The samples were again vortexed and subsequently centrifuged for 5 min at 13,200g. The supernatant was removed, transferred to clean tubes, and either immediately loaded on the gels or stored at )80 °C prior to electrophoresis. Gel preparation. Glass plates were 16 18 cm, cleaned with soap and water, rinsed with 100% ethanol, and allowed to dry. After the glass plates were dried they were coated with Rain X or Sigmacote (Sigma) to allow for easier removal of the gels from the plates [DATD gels are stickier and more difficult to remove than biscross-linked gels]. A SE600 Hoefer gel system (Pharmacia) was used with 0.75-mm gel spacers. It is important to note that this large gel format is needed to adequately separate the isoforms. The following recipe is sufficient for one 16 18-cm gel. To make 17 ml of a 6%T 2.6%C resolving gel, add 6.56 ml water, 3.4 ml 50% glycerol (v/v), 4.25 ml 1.5 M Tris, pH 8.8, 2.55 ml 40% acrylamide (37.5:1 cross-linked with DATD (Bio-Rad) [7]), 170 ll 10% sodium dodecyl sulfate (w/v), 50 ll 10% ammonium persulfate (w/v), and 20 ll TEMED. The resolving gel was poured to allow a stacking gel with 1-cm deep wells and at least 1-cm length between the bottom of the wells and the resolving gel. Water was added to the top of the resolving gel to form a flat
0003-2697/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0003-2697(03)00350-6
150
Notes & Tips / Analytical Biochemistry 320 (2003) 149–151
interface, and the gel was stored in the cold room after polymerization overnight. The stacking gel consisted of 5.085 ml of a 2.95%T 15%C acrylamide. Mix 1.5 ml 10% acrylamide (5.6:1 cross-linked with DATD (Bio-Rad) [7]), 1.3 ml 0.5 M Tris, pH 6.8, 1.15 ml water, 1 ml 50% glycerol (v/v), 50 ll 10% sodium dodecyl sulfate (w/v), 30 ll 10% ammonium persulfate (w/v), 30 ll stacking gel dye [0.1% (w/v) bromophenol blue, 1% (w/v) SDS, and 3% (v/v) glycerol], and 25 ll TEMED [8,9]. The inclusion of stacking dye in the stacking gel does not interfere with the gel polymerization or alter the run characteristics of the gels. However, it allows better visualization of the loading wells and the careful deposit of sample in the base of the well. The water was drained off the resolving gel and the stacking gel was poured at room temperature with a 20-lane comb inserted. Electrophoresis conditions. The upper buffer reservoir had 600 ml of 0.05 M Tris-base, 0.384 M glycine, 0.1% (w/v) SDS, and 10 mM 2-mercaptoethanol, no pH adjustment (modified from [9]). The lower buffer chamber had 4 liters of the same buffer except without 2mercaptoethanol. Usually only one gel was run at a time using 16 mA constant current for 4.5 h with constant cooling to 8 °C using a circulating water bath. After the electrophoresis was complete the gels were stained using Coomassie blue R-250 as previously described [10].
Results and discussion The myosin heavy chains move approximately halfway down in this gel system. Examples of the band resolution of rat and mouse cardiac myosins are shown in Fig. 1. There are clearly separation differences between rat and mouse MHC isoforms (Fig. 1). The beta chains have similar mobilities, but the mouse alpha chain (lane 2) is retarded significantly compared to the rat alpha (upper band, lane 3). The rat aMHC has 1938 residues, a charge of )46, and a molecular mass of 223,506 Da based on the cDNA sequence (GenBank Accession No. NP_058935). The mouse aMHC also has 1938 residues and a charge of –47, and a molecular mass of 223,563 Da based on the cDNA sequence (GenBank
Accession No. NP_034986). Thus the residue numbers are the same; there is a charge difference of only 1 and a molecular mass difference of only 57 Da between the rat and the mouse aMHC. The rat and mouse bMHC has the same charge and residue number; however, that of the rat has a molecular mass that is 30 Da smaller than that of the mouse based on the cDNA sequence (GenBank Accession Nos. X15939 and NP_000248, respectively). Why the MHC isoforms of the mouse and rat migrate so differently is unclear. However, there is some evidence for differential posttranslational modification of myosin heavy chains during development [11]. Posttranslational modifications may, therefore, explain differences in the migration in myosin heavy chains in rat and mouse cardiac tissue. The myosin heavy chain isoforms have been shown to switch in rodents from mainly bMHC in early development to mainly aMHC later in life (lanes 1–4, Fig. 1) [12–14]. In Fig. 1, lane 5 an adult rat ventricle has about 33% bMHC along with two separately migrating bands at the position of aMHC. This particular rat sample is uncharacteristic in that it has more beta and two closely spaced bands at the position of aMHC. It is possible that these two bands are due to a posttranscriptional event where the inclusion or exclusion of a single glutamine at position 1931 causes alternate splicing of the transcript [15,16]. Two rat cardiac aMHC bands have been observed previously using the pulse electrophoresis system [5]. The use of DATD as a cross-linker has been shown to increase the mechanical stability in highly cross-linked gels [7]. This cross-linker results in gels with larger pore sizes than those produced using bis-acrylamide and these gels are thus less restrictive for the movement of high-molecular-weight proteins [7]. In this case the myosin heavy chains move a greater distance in a more rigid gel with excellent resolution of the isoforms. In conclusion, the electrophoresis system described here is simple and reproducible and allows shorter run times than those described previously. It should be a useful tool for investigating myosin heavy chain isoform transitions in health and disease. Acknowledgments This work was supported by the College of Agricultural and Life Sciences, University of Wisconsin-Madison and by grants from the National Institutes of Health (HL47053; HL62466).
Fig. 1. SDS–PAGE gel of cardiac muscle. Only the myosin heavy chain area is shown. Lane 1, 18 day postconception fetal mouse; lane 2, adult cardiac mouse ventricle; lane 3, day 1 neonatal rat cardiac ventricle; lane 4, adult rat cardiac ventricle; lane 5, adult rat cardiac ventricle; lane 6, day 1 neonatal rat cardiac ventricle.
References [1] K.A. Esser, M.O. Boluyt, T.P. White, Separation of cardiac myosin heavy chains by gradient SDS–PAGE, Am. J. Physiol. 255 (1988) H659–H663.
Notes & Tips / Analytical Biochemistry 320 (2003) 149–151 [2] N.K. Sweitzer, R.L. Moss, Determinants of loaded shortening velocity in single cardiac myocytes permeabilized with a-hemolysin, Circ. Res. 73 (1993) 1150–1162. [3] K.L. Ball, J.R. Solaro, Discoordinate regulation of contractile protein gene expression in the senescent rat myocardium, J. Mol. Cell. Cardiol. 26 (1994) 519–525. [4] P.J. Reiser, W.O. Kline, Electrophoretic separation and quantitation of cardiac myosin heavy chain isoforms in eight mammalian species, Am. J. Physiol. 274 (1998) H1048–H1053. [5] J.A.A. SantÕAna Pereira, M. Greaser, R.L. Moss, Pulse electrophoresis of muscle myosin heavy chains in sodium dodecyl sulfatepolyacrylamide gels, Anal. Biochem. 291 (2001) 229–236. [6] L.D. Yates, M.L. Greaser, Quantitative determination of myosin and actin in rabbit skeletal muscle, J. Mol. Biol. 168 (1983) 123–141. [7] G. Baumann, A. Chrambach, A highly crosslinked, transparent polyacrylamide gel with improved mechanical stability for use in isoelectric focusing and isotachophoresis, Anal. Biochem. 70 (1976) 32–38. [8] J.D. Fritz, D.R. Swartz, M.L. Greaser, Factors affecting polyacrylamide gel electrophoresis and electroblotting of high-molecular-weight myofibrillar proteins, Anal. Biochem. 180 (1989) 205–210. [9] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227 (1970) 680–685.
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[10] J. Sambrook, E.F. Fritsch, T. Maniatis, in: Molecular Cloning. A Laboratory Manual, second ed., Cold Spring Harbor Press, Cold Spring Harbor, NY, 1989, pp. 18–55. [11] A.M. Maggs, P. Taylor-Harris, M. Peckham, S.M. Hughes, Evidence for differential post-translational modifications of slow myosin heavy chain during murine skeletal muscle development, J. Muscle Res. Cell Motil. 21 (2000) 101–113. [12] A.M. Lompre, J.J. Mercadier, C. Wisnewsky, P. Bouveret, C. Pantaloni, A. DÕAlbis, K. Schwartz, Species- and age dependent changes in the relative amounts of cardiac myosin isoenzymes in mammals, Dev. Biol. 84 (1981) 286–290. [13] A.M. Lompre, B. Nadal-Ginard, V. Mahdavi, Expression of the cardiac ventricular a- and b-myosin heavy chain genes is developmentally and hormonally regulated, J. Biol. Chem. 259 (1984) 6437–6446. [14] X. Bian, M.M. Briggs, F.H. Schachat, F.J. Seidler, T.A. Slotkin, Glucocorticoids accelerate the ontogenetic transition of cardiac ventricular myosin heavy-chain isoform expression in the rat: promotion by prenatal exposure to a low dose of dexamethasone, J. Dev. Physiol. 18 (1992) 35–42. [15] B.K. Quinn-Laquer, L.E. Kennedy, S.J. Wei, K.W. Beisel, Characterization of the allelic differences in the mouse cardiac amyosin heavy chain coding sequence, Genomics 13 (1992) 176–188. [16] R. Sindhwani, F. Ismail-Beigi, L.A. Leinwand, Post-transcriptional regulation of rat a cardiac myosin heavy chain gene expression, J. Biol. Chem. 269 (1994) 3272–3276.