A protocol for sample preparation and gel electrophoresis is described that reliably results in the separation of the α- and β-isoforms of cardiac myosin heavy chain (MHC-α and MHC-β) in eight mammalian species. The protocol is based on a simple, nongradient denaturing gel. The magnitude of separation of MHC-α and MHC-β achieved with this protocol is sufficient for quantitative determination of the relative amounts of these two isoforms in mouse, rat, guinea pig, rabbit, canine, pig, baboon, and human myocardial samples. The sensitivity of the protocol is sufficient for the detection of MHC isoforms in samples at least as small as 1 μg. The glycerol concentration in the separating gel is an important factor for successfully separating MHC-α and MHC-β in myocardial samples from different species. The effect of sample load on MHC-α and MHC-β band resolution is illustrated. The results also indicate that inclusion of a homogenization step during sample preparation increases the amount of MHC detected on the gel for cardiac samples to a much greater extent than for skeletal muscle samples. Although the protocol described in this study is excellent for analyzing cardiac samples, it should be noted that the same protocol is not optimal for separating MHC isoforms expressed in skeletal muscle, as is illustrated.
- sodium dodecyl sulfate-polyacrylamide gel electrophoresis
- contractile proteins
myosin is a hexameric enzyme in muscle that hydrolyzes ATP, the energy source for contraction. The two myosin heavy chain (MHC) subunits of each myosin molecule are relatively large, each with a molecular mass of ∼200 kDa. At least 12 MHC isoforms are expressed in mammalian smooth, skeletal, and cardiac muscles (recently reviewed in Refs. 27 and 31). Mammalian cardiac muscle expresses two MHC isoforms, MHC-α and MHC-β. MHC-α predominates in atrial tissue of most mammals, whereas MHC-β predominates in the ventricles of many, especially larger, mammals with lower resting heart rates (13). Cardiac tissues that contain relatively more MHC-α than MHC-β contract faster than those in which the ratio of MHC-α to MHC-β (MHC-α/MHC-β) is low (2, 20). This difference in contraction rate can be related directly to the relative amounts of MHC-α and MHC-β in a given myocardial preparation, on the basis of recent reports of a difference in the velocity of actin filament sliding by isolated cardiac myosin isoenzymes, V1 and V3, in vitro (5, 14, 30), and given that V1 and V3 isoenzymes contain homodimers of MHC-α and MHC-β isoforms, respectively (17).
MHC-α/MHC-β (or the ratio V1/V3) decreases during aging (6, 22) and during the development of several cardiovascular abnormalities (e.g., Ref. 15). Quantitation of MHC-α/MHC-β in myocardial samples is one method for documenting and assessing changes that occur in cardiac MHC expression during normal development and aging and in association with cardiovascular disorders. Determination of this ratio could also contribute to a more complete understanding of the effects of experimentally induced perturbations, such as coronary artery ligation (19), and could, thereby, provide a mechanistic interpretation for more macroscopic observations, as reported by Li et al. (18).
MHC-α and MHC-β isoforms have been relatively difficult to electrophoretically separate because of their large and similar size. For example, the molecular masses of rat MHC-α and MHC-β are both ∼223 kDa and differ from each other by <0.2% (21). This study provides a description of a simple, nongradient denaturing gel protocol that reliably yields separation of MHC-α from MHC-β isoforms in small samples of myocardial tissue from eight mammalian species. The procedures for sample preparation and gel staining are also described.
METHODS AND MATERIALS
The care and use of all of the animals from which samples were obtained for this study were in accordance with institutionally approved protocols. All of the animals were adults, except the mice, which were 3–4 days postnatal. The animals were euthanized with either a combination of stunning and rapid cardiectomy (guinea pig), an intravenous injection of an overdose of pentobarbital sodium (rabbit, canine, pig, and baboon), inhalation of CO2 (rat), or cervical dislocation (mice). Atrial and ventricular tissues were immediately removed and either freshly processed or frozen at −85°C and thawed before processing. All of the ventricular samples were prepared from the left ventricle unless noted otherwise. Human atrial and left ventricular tissues were obtained through the Cooperative Human Tissue Network from a 38-yr-old male who died from sudden cardiac arrest. These samples were obtained from noninfarcted areas of the heart. Samples from all of the species were prepared as described in Blough et al. (3) with the following modifications. Some samples (seeresults) were homogenized (model PRO200 homogenizer, PRO Scientific, Monroe, CT) for 5–10 s after adding the sample buffer (3), and the originally prepared samples were diluted 1:100, unless noted otherwise, with sample buffer before the gels were loaded. All gel sample volumes were 3 μl, except for mouse samples, which were 6 μl. Samples were also prepared from mouse, rat, and rabbit skeletal muscles, as described in Blough et al. (3).
The preparation and composition of the gels were modifications of those described by Talmadge and Roy (29). Except where indicated otherwise, the stacking and separating gels (0.75 mm thick) consisted of 4 and 8% acrylamide (wt/vol), respectively, with acrylamide:N,N′-methylene-bis-acrylamide in the ratio of 50:1. Gel polymerization initiators were the same as in Talmadge and Roy (29). All of the stacking gels included 5% (vol/vol) glycerol. Glycerol was also included in the separating gels, and the concentration was varied between 5 and 45% (vol/vol). The electrophoretic separation between MHC-α and MHC-β among the species studied was dependent on the glycerol concentration in the separating gel, as well as the total run time, which increased with the glycerol concentration, as described inresults. The gel and electrode buffers were identical to those in Talmadge and Roy (29), except that 2-mercaptoethanol was added to the upper electrode buffer at a final concentration of 10 mM (3, 10). The gels were run in a Hoefer SE600 unit (Hoefer Scientific, San Francisco, CA) at 8°C. Most of the gels were run at a constant voltage of 200 V (exceptions are stated inresults). The gels were fixed and silver-stained as in Blough et al. (3), with the following modifications. The gels were soaked in the fixing solutions in 9 × 9 in. glass trays so that the gels were always lying flat while they were continuously agitated at a low rotational speed. This resulted in an increase in the uniformity of staining across the gels. Also, the gels were rinsed briefly in water five times between the staining and developing steps. Each rinse lasted ∼30 s with manual rocking. This latter modification resulted in very low background staining. The gels were dried before scanning as described in Giulian et al. (11). A GS 300 scanning densitometer (Hoefer Scientific) was utilized to scan the stained gels. The water utilized for all of the procedures was distilled and deionized.
Consistent electrophoretic separation of MHC-α and MHC-β isoforms from mouse, rat, guinea pig, dog, pig, baboon, and human hearts was achieved with 5% glycerol in the separating gel and a run time of 30 h (Figs. 1 and2). The predominant isoform in the atrial samples was identified as MHC-α in all of these species. MHC-β predominated in the ventricles of each species studied, except in the neonatal mouse ventricles, in which only a relatively small amount of MHC-β, along with the predominating MHC-α, was observed. MHC-β migrated farther than MHC-α in all of the species and at every concentration of glycerol in the separating gel that yielded separation of these two isoforms. There were consistent differences in the amount of separation of MHC-α from MHC-β between species when the same samples were run on replicate gels. The amount of separation between these two isoforms was the least for rat and guinea pig compared with that for mouse, pig, dog, baboon, and human, whereas the separations for all of the latter were similar to each other. The migrations of MHC-β isoforms in the different species were more similar to each other than were those of the MHC-α isoforms on the separating gels consisting of 8% total acrylamide. Therefore, differences in the mobilities of the MHC-α isoform on these gels were primarily responsible for the species differences in the magnitude of the separation of MHC-α from MHC-β. Greater species differences were observed in the migrations of both MHC-α and MHC-β with separating gels consisting of 6 or 7% total acrylamide (run for 20 or 26 h, respectively) with all of the other parameters held constant (not shown). The separation of MHC bands on these lower percentage acrylamide gels was slightly greater (the greatest increases were observed with guinea pig and rat samples), but the bands were generally less well resolved.
It was not possible to consistently separate rabbit MHC-α and MHC-β from each other with the gel system described above, that is, with 5% glycerol in separating gels consisting of 8% total acrylamide, even with gel runs much longer than 30 h. A small increase in the separation of rabbit MHC-α and MHC-β was achieved by lowering the total acrylamide concentration in the separating gel to 6%, keeping all of the other gel parameters constant and using a constant voltage of 200 V for 20 h. However, the magnitude of the separation of rabbit MHC-α and MHC-β on these lower percentage acrylamide gels was still insufficient for reliable quantitative determination of their relative amounts (not shown). Therefore, rabbit cardiac samples, as well as those from several other species, were run on separating gels that included 30, 40, or 45% (vol/vol) glycerol. The best separation of rabbit MHC-α and MHC-β was achieved with a separating gel containing 45% glycerol (Fig. 1) that was run for 45–48 h at a constant voltage of 275 V. Thirty hours was an insufficient run time for this type of gel to separate the two rabbit cardiac MHC isoforms. All of the other gel parameters were the same as described above for all the other species tested. A comparison of the results obtained from the separating gels that contained either 5 or 30–45% glycerol also indicates that the glycerol concentration in the separating gel has a profound effect on the relative mobilities of cardiac MHC isoforms in different species. Human MHC-α and MHC-β had mobilities that were very similar to each other at these high glycerol concentrations and essentially did not separate when atrial and ventricular samples were electrophoresed in the same lane. The separation between MHC-α and MHC-β isoforms from baboon heart was less at these high glycerol concentrations also compared with the results obtained with separating gels that contained 5% glycerol, but these two isoforms still separated from each other when atrial and ventricular samples were coelectrophoresed.
Densitometric scans of single gel lanes that contained both isoforms of cardiac MHC for each of the species studied are shown in Fig.3. Three microliters of both atrial and ventricular samples (except for rat and mouse samples, which were ventricular only), diluted 1:100, were coelectrophoresed in the scanned lanes. The scan records indicate that the electrophoretic separation of MHC-α from MHC-β for each species is sufficient in magnitude and that the stain procedure is sufficiently sensitive for reliable determination of the relative amounts of these two MHC isoforms in a tissue sample with a mass at least as small as 1 μg.
The linearity of silver staining was tested by scanning gels on which several loads, ranging from 1 to 6 μl, of rat left ventricle (diluted 1:100), a mixture of guinea pig right and left ventricles (both diluted 1:100), and neonatal mouse right ventricle (diluted 1:10) were run. The mean linear correlation coefficients (±SD) for MHC-α and MHC-β were 0.970 ± 0.024 and 0.976 ± 0.033, respectively. The mean coefficient of variation for the determination of the relative amounts of the two MHC isoforms was 9.9% over the same loads for these samples. Therefore, the described staining and densitometric scanning procedures are reliable over the sample loads that were tested.
Figure 4 illustrates1) the effect of the inclusion of a homogenization step during sample preparation on the amount of MHC extracted during sample preparation,2) the effect of the magnitude of sample load on band resolution, and3) the effect of the glycerol concentration in the separating gel on the magnitude of MHC band separation and relative migration of different MHC isoforms. One homogenized sample and one nonhomogenized sample were prepared from rat left ventricle, psoas, and soleus muscles. Inclusion of the homogenization step in the preparation of rat ventricle resulted in nearly 120% more MHC on the stained gel, as determined by scanning densitometry, compared with the nonhomogenized sample. The effect of homogenization in the preparation of psoas and soleus samples was much less, with ∼10% more MHC being detected by densitometry for both muscles. The gels shown in Fig. 4 also illustrate that lower sample loads result in a marked increase in band resolution. However, relatively minor bands are detected only with greater sample loads (e.g., compare the lanes loaded with 3 μl of 1:10 or 1:100 diluted psoas samples on the gel with 30% glycerol shown in Fig. 4). It is important to emphasize that the gel described in this study results in excellent separation of MHC-α and MHC-β in cardiac samples from eight mammalian species but is not suitable for separation of skeletal muscle MHC isoforms. This point is also illustrated in Fig. 4. Note that, although all four of the adult skeletal muscle MHC isoforms are clearly separated on the gel containing 30% glycerol, this is not the case with gels containing 5% glycerol, which yield better separation of the cardiac MHC isoforms. In addition, the relative order of migration of some skeletal muscle MHC isoforms is reversed between gels containing 5 and 30% glycerol. For instance, a minor band (presumably, MHC-IIa) that is visible in the 1:10 diluted soleus sample migrates as the slowest of all skeletal muscle MHC bands (visible in the psoas sample in the adjacent lane) on gels consisting of 30% glycerol (Fig.4 B); however, this same band migrates slightly ahead of at least one of the bands in the psoas sample in gels with 5% glycerol (Fig.4 A). Therefore, sample homogenization, sample load, and the glycerol concentration of the separating gel can affect the detection, resolution, and overall separation of protein bands, as well as the relative order of isoform migration.
Comparison of the results from the different species that were included in this study indicate that rabbit MHC-α and MHC-β were the least separated in gels with 5% glycerol. We therefore examined the relative separation between rabbit skeletal muscle MHC isoforms compared with that of two other species (mouse and rat). The separating gel contained 30% glycerol, and the gel was run at a constant voltage of 275 V for 24 h, consistent with the conditions utilized in another recent study (24) in this laboratory on rabbit skeletal muscle MHC isoforms. The results indicate that, as is the case for cardiac MHC isoforms, the overall separation between rabbit skeletal muscle MHC isoforms (i.e., the distance between the fastest and slowest migrating isoforms) is the least among the three species tested (Fig.5). However, the species differences in the overall separation of skeletal muscle MHC isoforms were less than the species differences in the separation of cardiac MHC isoforms.
The results of this study illustrate the utility of a simple, nongradient denaturing gel electrophoresis system that reliably yields sufficient separation of MHC-α from MHC-β for the quantitation of the relative amounts of these two isoforms in small samples of myocardium from eight mammalian species. The entire procedure, from sample preparation to gel scanning, requires ∼4 days but involves <8 h of actual “hands-on” time. It might be possible to reduce the total time required to <4 days (e.g, by utilizing a minigel format, by changing the gel composition or running conditions, and/or by employing Coomassie blue stain), but this was not extensively tested.
Other electrophoretic protocols that yield separation of MHC-α and MHC-β in individual species have been described (e.g., Refs. 7, 16, and 26). Esser et al. (8) reported more than a decade ago the utility of a gradient gel that results in excellent separation of MHC-α and MHC-β in rat cardiac tissues that was sufficient for quantitative determination of the ratio, MHC-α/MHC-β. The protocol described in the present study is simpler in that it is based on a nongradient gel format that is easier to prepare consistently. Furthermore, the protocol is applicable to at least eight mammalian species with changes in the separating gel glycerol concentration, the voltage setting, and the duration of electrophoresis. It is likely that the same or a similar protocol can be extended to other species. Variations in glycerol concentration, the ratio of acrylamide to bis-acrylamide, pH, the tris(hydroxymethyl)aminomethane concentration in the separating and/or stacking gel(s), and other factors have been employed by others to successfully separate cardiac and skeletal muscle isoforms by gel electrophoresis. These and other parameters that can have profound effects on the separation and/or resolution of proteins during electrophoresis have been discussed by Hames (12).
This protocol provides a method for quantitation of the relative amounts of cardiac MHC isoforms in a sample. It is assumed that the two isoforms are extracted in proportion to their relative composition in intact samples and that staining of the two cardiac MHC isoforms is stoichiometric. Silver staining procedures and potential associated problems have been reviewed by Syrovy and Hodny (28) and Wirth and Romano (32). Quantitation of absolute amounts of MHC in muscle samples can be accomplished by other techniques, such as radioimmunoassay or isotope dilution (4, 9).
The basis for the relatively lower amount of separation between rabbit MHC-α and MHC-β, as well as skeletal muscle MHC isoforms to a lesser extent, compared with the other species tested is not understood. Also, the mechanism of the effect of glycerol concentration in the separating gel on the relative migrations of MHC-α and MHC-β between species and the relative order of MHC-IIa and MHC-IId in skeletal muscle samples is unclear. An effect of the separating gel glycerol concentration on the relative order of skeletal muscle MHC isoforms was reported previously (3). Evidence for an alteration of the free mobility of proteins during electrophoresis by glycerol (possibly due to displacement of sodium dodecyl sulfate from specific proteins) has been presented elsewhere (25). Clearly, the effect of glycerol on MHC migration and possible interactions with other gel constituents that may enhance isoform separation and band resolution deserve further study. These results illustrate the need for careful consideration of gel conditions and the interpretation of the results obtained with the application of the gel system described in this study, as well as other systems, when MHC isoforms are examined in additional species.
The technical assistance of Bi Zhou is gratefully acknowledged.
Address for reprint requests: P. J. Reiser, Oral Biology Box 192, The Ohio State Univ., 305 W. 12th Ave., Columbus, OH 43210-1241.
This study was supported by a grant-in-aid from the American Heart Association. Human tissue samples were provided by the Cooperative Human Tissue Network, which is funded by the National Cancer Institute.
- Copyright © 1998 the American Physiological Society