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Department of Oral Biology, The Ohio State University, Columbus, Ohio 43210-1241
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ABSTRACT |
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Top
Abstract
Introduction
Materials
Results
Discussion
References
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A protocol for
sample preparation and gel electrophoresis is described that reliably
results in the separation of the 
- and 
-isoforms of cardiac
myosin heavy chain (MHC- and MHC-) in eight mammalian species.
The protocol is based on a simple, nongradient denaturing gel. The
magnitude of separation of MHC- and MHC- 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- and MHC- in myocardial samples from different species. The
effect of sample load on MHC- and MHC- 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.
isoenzymes; sodium dodecyl sulfate-polyacrylamide gel electrophoresis; contractile proteins; myocardium; heart
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INTRODUCTION |
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Top
Abstract
Introduction
Materials
Results
Discussion
References
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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- and MHC-. MHC- predominates
in atrial tissue of most mammals, whereas MHC- predominates in the
ventricles of many, especially larger, mammals with lower resting heart
rates (13). Cardiac tissues that contain relatively more MHC- than MHC- contract faster than those in which the ratio of MHC- to MHC- (MHC-/MHC-) is low (2, 20). This difference in
contraction rate can be related directly to the relative amounts of
MHC- and MHC- 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- and MHC- isoforms, respectively (17).
MHC-/MHC- (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-/MHC- 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- and MHC- isoforms have been relatively difficult to
electrophoretically separate because of their large and similar size.
For example, the molecular masses of rat MHC- and MHC- 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- from MHC- isoforms in
small samples of myocardial tissue from eight mammalian species. The
procedures for sample preparation and gel staining are also described.
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METHODS AND MATERIALS |
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Top
Abstract
Introduction
Materials
Results
Discussion
References
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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 (see
RESULTS) 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- and MHC- 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 in
RESULTS. 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 in
RESULTS). 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.
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RESULTS |
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Top
Abstract
Introduction
Materials
Results
Discussion
References
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Consistent electrophoretic separation of MHC- and MHC- 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 and
2). The predominant isoform in the atrial
samples was identified as MHC- in all of these species. MHC-
predominated in the ventricles of each species studied, except in the
neonatal mouse ventricles, in which only a relatively small amount of
MHC-, along with the predominating MHC-, was observed. MHC-
migrated farther than MHC- 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- from MHC- 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- isoforms in the different species were more
similar to each other than were those of the MHC- isoforms on the
separating gels consisting of 8% total acrylamide. Therefore,
differences in the mobilities of the MHC- isoform on these gels were
primarily responsible for the species differences in the magnitude of
the separation of MHC- from MHC-. Greater species differences
were observed in the migrations of both MHC- and MHC- 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.
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It was not possible to consistently separate rabbit MHC- and MHC-
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- and MHC- 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-
and MHC- 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- and MHC- 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- and MHC- 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- and MHC- 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- from MHC- 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.
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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- and MHC-
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 illustrates
1) 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, and
3) 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- and MHC- 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.
4B); however, this same band
migrates slightly ahead of at least one of the bands in the psoas
sample in gels with 5% glycerol (Fig.
4A). 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.
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Comparison of the results from the different species that were included
in this study indicate that rabbit MHC- and MHC- 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.
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DISCUSSION |
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Top
Abstract
Introduction
Materials
Results
Discussion
References
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The results of this study illustrate the utility of a simple,
nongradient denaturing gel electrophoresis system that reliably yields
sufficient separation of MHC- from MHC- 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- and
MHC- 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- and
MHC- in rat cardiac tissues that was sufficient for quantitative
determination of the ratio, MHC-/MHC-. 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- and MHC-, 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- and MHC-
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.
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ACKNOWLEDGEMENTS |
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The technical assistance of Bi Zhou is gratefully acknowledged.
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FOOTNOTES |
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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.
Address for reprint requests: P. J. Reiser, Oral Biology Box 192, The Ohio State Univ., 305 W. 12th Ave., Columbus, OH 43210-1241.
Received 16 June 1997; accepted in final form 21 November 1997.
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Top
Abstract
Introduction
Materials
Results
Discussion
References
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 M. Bergrin, S. Bicer, C. A. Lucas, and P. J. Reiser Three-dimensional compartmentalization of myosin heavy chain and myosin light chain isoforms in dog thyroarytenoid muscle Am J Physiol Cell Physiol, May 1, 2006; 290(5): C1446 - C1458. [Abstract] [Full Text] [PDF]
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B. C. Blunt, Y. Chen, J. D. Potter, and P. A. Hofmann Modest actomyosin energy conservation increases myocardial postischemic function Am J Physiol Heart Circ Physiol, March 1, 2005; 288(3): H1088 - H1096. [Abstract] [Full Text] [PDF]
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V. L. M. Rundell, V. Manaves, A. F. Martin, and P. P. de Tombe Impact of {beta}-myosin heavy chain isoform expression on cross-bridge cycling kinetics Am J Physiol Heart Circ Physiol, February 1, 2005; 288(2): H896 - H903. [Abstract] [Full Text] [PDF]
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 T. Hashimoto, N. Kambara, R. Nohara, M. Yazawa, and S. Taguchi Expression of MHC-{beta} and MCT1 in cardiac muscle after exercise training in myocardial-infarcted rats J Appl Physiol, September 1, 2004; 97(3): 843 - 851. [Abstract] [Full Text] [PDF]
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C. A. Carnes, T. P. Geisbuhler, and P. J. Reiser Age-dependent changes in contraction and regional myocardial myosin heavy chain isoform expression in rats J Appl Physiol, July 1, 2004; 97(1): 446 - 453. [Abstract] [Full Text] [PDF]
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M. Iemitsu, T. Miyauchi, S. Maeda, T. Tanabe, M. Takanashi, M. Matsuda, and I. Yamaguchi Exercise training improves cardiac function-related gene levels through thyroid hormone receptor signaling in aged rats Am J Physiol Heart Circ Physiol, May 1, 2004; 286(5): H1696 - H1705. [Abstract] [Full Text] [PDF]
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Y. Zhong, P. J Reiser, and M. A. Matlib Gender differences in myosin heavy chain-{beta} and phosphorylated phospholamban in diabetic rat hearts Am J Physiol Heart Circ Physiol, December 1, 2003; 285(6): H2688 - H2693. [Abstract] [Full Text] [PDF]
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N. R. Alpert, C. Brosseau, A. Federico, M. Krenz, J. Robbins, and D. M. Warshaw Molecular mechanics of mouse cardiac myosin isoforms Am J Physiol Heart Circ Physiol, October 1, 2002; 283(4): H1446 - H1454. [Abstract] [Full Text] [PDF]
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 M. J. Mihm, F. Yu, C. A. Carnes, P. J. Reiser, P. M. McCarthy, D. R. Van Wagoner, and J. A. Bauer Impaired Myofibrillar Energetics and Oxidative Injury During Human Atrial Fibrillation Circulation, July 10, 2001; 104(2): 174 - 180. [Abstract] [Full Text] [PDF]
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T. L. Clanton, V. P. Wright, P. J. Reiser, P. F. Klawitter, and N. R. Prabhakar Physiological and Genomic Consequences of Intermittent Hypoxia: Selected Contribution: Improved anoxic tolerance in rat diaphragm following intermittent hypoxia J Appl Physiol, June 1, 2001; 90(6): 2508 - 2513. [Abstract] [Full Text] [PDF]
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P. J. Reiser, M. A. Portman, X.-H. Ning, and C. S. Moravec Human cardiac myosin heavy chain isoforms in fetal and failing adult atria and ventricles Am J Physiol Heart Circ Physiol, April 1, 2001; 280(4): H1814 - H1820. [Abstract] [Full Text] [PDF]
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J. Wattanapermpool and P. J. Reiser Differential effects of ovariectomy on calcium activation of cardiac and soleus myofilaments Am J Physiol Heart Circ Physiol, August 1, 1999; 277(2): H467 - H473. [Abstract] [Full Text] [PDF]
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H.-J. Wang, Y.-C. Zhu, and T. Yao Effects of all-trans retinoic acid on angiotensin II-induced myocyte hypertrophy J Appl Physiol, May 1, 2002; 92(5): 2162 - 2168. [Abstract] [Full Text] [PDF]
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B) of gel is
indicated. Length of arrow corresponds to 2.5 mm of separation on
gels.
