PHYSIOLOGY, ENDOCRINOLOGY, AND REPRODUCTION
Analysis of Myosin Isoform Transitions During Growth
and Development in Diverse Chicken Genotypes
J. M. Reddish, M. Wick, N. R. St-Pierre, and M. S. Lilburn1
Department of Animal Sciences, 2029 Fyffe Road, The Ohio State University, Columbus, Ohio 43210
respectively. The relative concentration of MyHC isoforms was evaluated by semiquantitative ELISA with 3
monoclonal antibodies specific for chicken skeletal fast
embryonic and adult (eMyHC, aMyHC; EB165), neonatal
(nMyHC; 2E9), and adult (aMyHC; AB8) myosin, respectively. The overall temporal expression of the myosin
isoforms, eMyHC, nMyHC, and aMyHC, was similar in
all lines. With eMyHC, at 19 d of incubation, line B had
lower expression than lines A, C, and D. Expression of
nMyHC, in lines C and D was similar with expression
being highest at 7 d and lower at 14 d and 21 d. In lines
A and B, however, nMyHC expression was higher at
hatch than lines C and D. In line D, aMyHC was expressed
at 14 d and increased through 21 d, whereas in lines A,
B, and C, aMyHC isoform was expressed and was higher
at 7 d and increased through 21 d. The results of this
experiment support our hypothesis that commercial broilers have different temporal expression patterns of the
developmental chicken fast MyHC isoforms.
(Key words: pectoralis major muscle, myosin, muscle, broiler)
2005 Poultry Science 84:1729–1734
The ability of poultry breeding companies to select for
heavier BW and higher breast muscle yield due to the high
heritability of BW and breast muscle yield has significantly
decreased the number of days required to reach a given
BW and significantly increased the breast muscle mass of
commercial broilers (Havenstein et al., 2003a,b). Although
primary breeders have made excellent genetic progress
over the years, there are still genetic and phenotypic differences among commercial broiler strains due to company
specific genetic selection practices (Emmerson, 1997).
Within the broiler industry, there is also the recognition
that breast muscle quality issues are becoming more commonplace. Barbut (1997) estimated that the incidence of
pale, soft, exudative (PSE) broiler breast meat could be as
high as 28%. Characteristics noted with PSE syndrome in
broiler breast meat are lower water'holding capacity,
lighter color, and higher shear force (Barbut et al., 2005).
Recent reports in the literature have attempted to address
the variability in broiler breast muscle color, pH changes,
and muscle quality factors related to PSE (Fletcher, 1999;
Bianchi et al., 2005). Overall, the specific muscle proteins
that are involved in or causative of PSE-like syndrome in
broiler breast meat are yet to be identified.
Because broilers that develop PSE breast meat are genetically selected to exhibit increased breast muscle growth, it
is reasonable to speculate that the temporal expression of
the myosin heavy chain (MyHC) isoforms may be correlated with the temporal expression of other muscle specific
proteins that do play mechanistic roles in the development
of PSE-like breast meat. During myogenesis of fast muscle
tissue in the chicken, the developmental MyHC isoforms
are expressed in a temporal and tissue-specific manner
2005 Poultry Science Association, Inc.
Received for publication January 12, 2005.
Accepted for publication July 20, 2005.
1
To whom correspondence should be addressed: [email protected].
Abbreviation Key: aMyHC = adult myosin heavy chain; eMyHC =
embryonic myosin heavy chain; mAb = monoclonal antibody; MyHC =
myosin heavy chain; nMyHC = neonatal myosin heavy chain; SCWL =
Single Comb White Leghorn.
INTRODUCTION
1729
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ABSTRACT The temporal expression of chicken skeletal fast myosin heavy chain (MyHC) isoforms in pectoralis
major muscle was characterized in 3 commercial broiler
lines at embryonic d 19 and at 7, 14, and 21 d posthatch.
Lines A and B have been selected for breast yield, and
line C is a fast'growing commercial line with limited selection for carcass traits. The isoform transitions in breast
muscle samples were compared with samples from Single
Comb White Leghorns (line D) using a semiquantitative
immunoassay. The hypothesis was that selection for
growth and carcass development in broilers would be
accompanied by changes in the temporal expression of
one or more of the chicken fast MyHC isoforms. Embryos
from all lines were sampled at 19 d of incubation, and
chicks were randomly sampled at 7, 14, and 21 d posthatch. Myosin was extracted from pectoralis major muscle
and assayed for purity and total protein concentration
by SDS-PAGE and bincinchoninic acid protein analyses,
1730
REDDISH ET AL.
MATERIALS AND METHODS
Birds
Broiler breeder eggs were obtained from commercial
broiler hatcheries for lines A, B, and C, and Leghorn eggs
were obtained from a commercial layer breeder flock
(American Selected Products, Milton, PA). Eggs from all
4 lines were incubated and hatched at the OARDC (Poultry
Research Center, Wooster, OH). Lines A and B are commercially used for the production of heavy broilers with increased breast yield to serve the boneless breast meat market. Line C is a commercial broiler line that does not show
the extreme breast yield evident in lines A and B (Reddish
and Lilburn, 2003). The Single Comb White Leghorn
(SCWL; line D) served as a comparative control, because
the pattern of developmental fast myosin isoform expression has been previously described in this line. Embryos
were sampled at 19 d of incubation to avoid the variability
associated with different hatching times, and chicks were
subsequently sampled at 7, 14, and 21 d posthatch. These
ages of sampling correspond to the transition times previously reported by Bandman and Bennett (1988) and Tidyman et al. (1997). After hatching, chicks from lines A, B,
and C were reared in floor pens, whereas the Leghorn
chicks were reared in battery brooders for ease of handling.
A commercial starter diet (CP = 21.0%, ME = 3,121 kcal/
kg) and water were provided ad libitum throughout the
experiment. Three birds from each line were sampled
weekly for BW and total pectoralis major muscle weight.
Samples from the pectoralis major breast muscle were
taken from embryos at 19 d of incubation and from chicks
at 7, 14, and 21 d posthatch and stored at −20°C until
further analysis.
Sample Preparation
At each age, pectoralis major samples from 3 chicks per
line were used for myosin extraction. Myosin was extracted
by solubilization in a high-salt buffer and precipitated in
a low-salt buffer as described previously (Rosser, et al.,
1998; Wick et al., 2003) with the following modifications.
Myosin was solubilized in a high-salt buffer solution (0.04
M Na pyrophosphate, 0.001 M MgCl2, and 0.002 M EDTA,
pH 9.5) and precipitated by dialysis in low-salt buffer (0.02
M KCl, 0.002 M KH2PO4, and 0.001 M EDTA, pH 6.8). The
myosin was resuspended in an equal volume of 0.04 M
sodium pyrophosphate, 0.002 M MgCl2, and 0.002 M
EDTA, pH 9.5, and 50% glycerol and was stored at −20°C.
Myosin purity was evaluated by electrophoresis on 10%
SDS polyacrylamide gels (SDS-PAGE) by comparison with
a chicken whole muscle standard that had not been extracted. The protein concentration of individual muscle
extracts was determined by bincinchoninic acid assay according to the manufacturer’s protocol (Pierce Endogen,
Rockford, IL). Gels were loaded with 50 ␮g of protein
per lane for each sample. After electrophoresis, gels were
stained with Coomassie Brilliant Blue G250 and subsequently destained with 10% acetic acid. Gels were analyzed
using Phoretix 1D (Nonlinear Dynamics, Ltd., Durham,
NC) software (Figure 1) to determine relative concentration
of individual peptide bands.
Column purified myosin standards were prepared as
described by Margossian and Lowey, (1982). Standard
curves were generated independently for eMyHC,
nMyHC, and aMyHC developmental fast myosin isoforms
from purified myosin. Serial dilutions of myosin standards
were run on ELISA plates as described below. Linear equations were developed based on the relationship between
myosin isoform concentration and spectrophotometric absorbance values for the serial dilutions. The absorbance
value of each sample was subsequently transformed, and
all data are reported in nanogram concentraqtions of
MyHC per volume.
ELISA
The relative MyHC isoform concentration in each myosin extract was determined by ELISA as described previously (Wick et al., 2003). Briefly, each sample (1,000 ng
of protein) was plated in triplicate onto a 96-well EIA/RIA
plate (Costar Corp., Cambridge, MA) and incubated for
30 min at 37°C. Plates were subsequently blocked with 5%
nonfat dry milk in PBS and incubated with the following
monoclonal antibodies (mAb; isoform specificity): EB165
(eMyHC + aMyHC), 2E9 (nMyHC), AB8 (aMyHC), and
NA4 (all sarcomeric myosin) as a positive control. The
epitopes of the mAb were characterized (Table 1) in SCWL
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(Tidyman et al., 1997; Wick et al., 2003; Reddish et al.,
2005). The sequence of myosin developmental isoform expression in the pectoralis major of the chicken is the appearance of ventricular (Cvent), embryonic (Cemb1, Cemb2,
Cemb3, or eMyHC), neonatal (Cneo or nMyHC), and adult
(Cadult or aMyHC) myosin isoforms (Tidyman et al., 1997;
Rushbrook et al., 1998; Bandman et al., 2000). During posthatch breast muscle development, the sequential appearance of eMyHC, nMyHC, and aMyHC isoforms in the
chicken pectoralis major muscle have been previously reported in vivo with SDS PAGE (Bandman et al., 1982),
by using myosin isoforms-specific monoclonal antibodies
(Bandman, 1985a; Cerny and Bandman, 1987; Rosser et
al., 1998), and Northern analysis (Tidyman et al., 1997;
Rushbrook et al., 1998). The functional diversity of the
MyHC isoforms has yet to be determined.
This study is intended to develop a model for determining the biochemical strategy used by the developing
chicken to comply with the demands for increased breast
muscle yield. Our model is based on the hypothesis that,
during myogenesis, MyHC isoform expression is correlated with other, as yet unidentified, muscle protein isoforms that actively participate in producing PSE in broiler
breast meat. The objective of our experiment was to document the temporal expression of the developmental fast
MyHC isoforms to test the hypothesis that genetic differences in the posthatch growth of the pectoralis major is
accompanied by changes in the temporal transitions of the
developmental fast MyHC isoforms.
ANALYSIS OF MUSCLE GROWTH AND DEVELOPMENT IN POULTRY
1731
(KPL, Inc., Gaithersburg, MD) was added to each well to
determine the spectrophotometric absorbance of the horseradish peroxidase-labeled secondary antibody. The absorbance of each well was read on a Laboratory systems
Multiskan EX at 405nm (version 1.0, Labsystems, Vantaa,
Finland).
Statistical Analyses
The experimental design was a randomized block, splitplot design. Data were analyzed using the mixed procedure of SAS software (SAS Institute, 2002) according to
the following model:
Yijklm = ␮ + Li + Dj + LDij + bk:ij + Pl + Am + LAim
+ DAjm + LDAijm + eijklm
chicks (Moore et al., 1992) at the following dilutions: 2E9,
1:2,500; EB165 and AB8, 1:5,000; and NA4 1:10,000. Antibodies were incubated for 30 min at 37°C. Plates were
subsequently washed with PBS-0.1% Tween 20 (Fisher Biotechnology, Fair Lawn, NJ). Bound mAb was detected with
horseradish peroxidase-conjugated goat antimouse IgG (H
+ L; Pierce-Endogen, Rockford, IL) at a dilution of 1:2,500
in 5% nonfat dry milk in PBS and incubated for 30 min at
37°C. Plates were washed with PBS-0.1% Tween 20, and
100 ␮L of ABTS microwell peroxidase substrate solution
RESULTS
Body weights are summarized in Figure 2. Body weights
at 7 and 14 d posthatch were not different among lines A,
B, and C, but line D (SCWL) BW was less (P < 0.05) than
those of the other lines. At 21 d posthatch, BW of lines A
and C were not different from each other, and both lines
were heavier than lines B and D (P < 0.05); and line B BW
were heavier than those of line D (P < 0.05).
The weight of the pectoralis major muscle, expressed as
a percentage of BW, is represented graphically in Figure
Table 1. Specificity of monoclonal antibodies for chicken myosin heavy chain (MyHC) isoforms
MyHC isoforms
Antibody1
EB165
2E9
AB8
NA4
Embryonic2
Neonatal
Adult
Apparent specificity
+
−
−
+
−
+
−
+
+
−
+
+
Embryonic and adult fast
Neonatal fast
Adult fast
Pan Sarcomeric MyHC
1
The preparation and specificities of these monoclonal antibodies against chicken myosin heavy chains have
been detailed elsewhere (Cerny and Bandman 1987; Bandman and Bennett, 1988; Moore et al., 1992).
2
Embryonic fast refers to multiple embryonic isoforms: CE1, CE2, and CE3 (Moore et al., 1992).
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Figure 1. This SDS-PAGE gel is representative of the relative purity of
myosin extractions of all line × day combinations that were subsequently
used in the ELISA procedures. The lanes of the gel are from muscle
extractions of pectoralis major muscle prepared from all lines (A, lane
2; B, lane 3; C, lane 4; and D, lane 5) at 14 d of age and show relative
purity of extractions in comparison to lane 1, which is a chicken muscle
standard at 14 d of age prepared without extraction, and lane 6 is a
high molecular weight marker (Bio-Rad Inc., Hercules, CA).
where Yijklm is the dependent variable, ␮ is the overall
mean, Li is the fixed effect of the ith line (i = 1,. . .,4), Dj is
the fixed effect of the jth day (k = 1,...,4), bk:ij is the fixed
effect of the kth bird within the ith line on the jth day, Pl
is the random effect of the lth plate (l = 1,. . .12), Am is the
fixed effect of the mth antibody (m = 1,. . .,4), and eijklm is
the random residual error ∼N (o, σ2lm).
Residual errors were not homogeneous according to the
likelihood ratio test (Milliken and Johnson, 1992). Thus,
individual subclass error variances were estimated for each
sample day by antigen subclass. Least square means across
subclasses were compared statistically using Fisher’s protected least significant difference (Snedecor and Cochran,
1980). That is, tests of the differences between pairs of
least squares means were done only when the F'test was
significant. Data are presented as the mean ± standard
error of the mean for each line, and effects are considered
statistically significant at P < 0.05.
1732
REDDISH ET AL.
3. At 7, 14, and 21 d posthatch, lines A and B were not
significantly different from each other, and their relative
breast muscle weights were greater than those in lines C
and D (P < 0.05). The relative breast muscle weight t in
line C was greater than in line D at all sample dates (P
< 0.05).
As shown in Figure 1, the relative purity of the myosin
isoforms for 14 d for lines A, B, C, and D is represented
on the SDS-PAGE gel. Individual band volume in each
lane was calculated using Phoretix 1D software. After extraction, the MyHC band percentage in each lane for extracted myosin samples was 75% or above, and the re-
Figure 3. Weight of the pectoralis major muscle expressed as a percentage of total BW in diverse genotypes of chicken (lines A, B, C and
D) at weekly intervals from 7 to 21 d. Maternal lines A and B are
commercially used for the production of heavy broilers with increased
breast yield. Line C is a commercial broiler used for live market and
has less breast yield than in lines A and B. The Single Comb White
Leghorn line (line D) served as a comparative control. a–cColumns within
day having different letters are significantly different (P < 0.05).
Figure 4. Analysis of the temporal transitions of the developmental
fast embryonic (eMyHC) myosin isoform in samples of the pectoralis
major muscle derived from diverse genotypes of chickens (lines A, B,
C, and D) at 19 d of incubation and 7 d of age. Maternal lines A and
B are commercially used for the production of heavy broilers with
increased breast yield. Line C is a commercial broiler used for live
market and has less breast yield than in lines A and B. Line D is a
Single Comb White Leghorn line, which served as a comparative control.
a,b
Columns within an age group having different letters are significantly
different (P < 0.05).
maining peptide bands in each lane representing other
constituent proteins were less than 25% of the total lane,
which demonstrates relative purification of the myosin
samples. Myosin extraction samples from all other line ×
day combinations were similar (data not shown).
The expression of the developmental fast eMyHC at
embryonic d 19 and 7 d posthatch is graphically represented in Figure 4. The concentration of eMyHC was similar at embryonic d 19 in lines A, C, and D and lower in
line B (P < 0.05). The concentration of eMyHC was lower
in line C and D than lines A and B at 7 d (P < 0.05). In all
4 lines, the concentration of eMyHC was lower at 7 d than
at embryonic d 19 (P < 0.05).
In Figure 5, the differences in nMyHC concentration
within and among lines A, B, C, and D at 7, 14, and 21 d
posthatch are shown. There were no significant differences
in the concentration of nMyHC among the lines at 19 d
of embryonic development (data not shown) or at 7 d
posthatch. The concentration of the nMyHC isoform in
lines A, C and D were similar at 14 d post hatch. The
concentration of nMyHC in Line B was similar to line A
but lower in line B than in lines C and D at this age (P <
0.05). At 21 d posthatch, there was a decrease in nMyHC
concentration in all lines compared with 7 and 14 d (P <
0.05). The concentration of nMyHC was the highest in line
D at 21 d, although the differences between lines A and
D were not significant (P < 0.05). The concentration of
nMyHC in lines A and B were not significantly different
nor were there any significant differences between lines B
Downloaded from http://ps.oxfordjournals.org/ at Pennsylvania State University on May 11, 2016
Figure 2. A comparison of BW in diverse genotypes of chicken (lines
A, B, C, and D), weekly, from 7 to 21 d. Maternal lines A and B are
commercially used for the production of heavy broilers with increased
breast yield. Line C is a commercial broiler used for live market, and
has less breast yield than in lines A and B. The Single Comb White
Leghorn line (line D) served as a comparative control. a–cColumns within
day having different letters are significantly different (P < 0.05).
ANALYSIS OF MUSCLE GROWTH AND DEVELOPMENT IN POULTRY
1733
aMyHC concentration in lines C and D at 7 d posthatch.
At 14 d posthatch, lines A and B had higher concentrations
of aMyHC than lines C and D (P < 0.05), and expression
of aMyHC in line C was higher than in line D. At 21 d
posthatch, expression of aMyHC was higher in line A than
in the other 3 lines (P < 0.05).
DISCUSSION
and C. The concentration in line A, however, was higher
than line C (P < 0.05).
The concentration of aMyHC within and between lines
A, B, C, and D at 7, 14, and 21 d posthatch is shown in
Figure 6. At 19 d of embryonic development, there were
no significant line differences in aMyHC concentrations
(data not shown). There were detectable levels of aMyHC
in all 4 lines at 7 d posthatch, and the concentrations were
significantly higher in lines A and B when compared with
lines C and D (P < 0.05). There were no differences in
Figure 6. Analysis of the temporal transitions of the developmental
fast adult myosin isoforms (aMyHC) in samples of the pectoralis major
muscle derived from diverse genotypes of chickens (lines A, B, C, and
D) at weekly intervals from 7 to 21 d of age. Maternal lines A and B are
commercially used for the production of heavy broilers with increased
breast yield. Line C is a commercial broiler used for live market and
has less breast yield than in lines A and B. Line D is a Single Comb
White Leghorn line, which served as a comparative control. a–cColumns
within a day having different letters are significantly different (P < 0.05).
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Figure 5. Analysis of the temporal transitions of the developmental
fast neonatal myosin isoforms (nMyHC) in samples of the pectoralis
major muscle derived from diverse genotypes of chickens (lines A, B,
C, and D) at weekly intervals from 7 to 21 d of age. Maternal lines A
and B are commercially used for the production of heavy broilers with
increased breast yield. Line C is a commercial broiler used for live
market and has less breast yield than lines A and B. Line D is a Single
Comb White Leghorn line, which served as a comparative control. a–
c
Columns within a day having different letters are significantly different
(P < 0.05).
The temporal expression of the developmental fast
MyHC isoforms in breast muscle were studied in 2 highyielding broiler lines (A and B), a commercial broiler line
with less breast yield than lines A and B (C), and SCWL
chicks (D). Line D (SCWL) is the genotype that has been
used for most of the published studies on poultry skeletal
MyHC isoform expression (Cerny and Bandman, 1987;
Bandman and Bennett, 1988; Moore et al., 1992; Tidyman et
al., 1997). This study is the first comparison of the temporal
expression of the developmental skeletal fast MyHC isoforms in fast-growing commercial broiler lines. The temporal expression of the skeletal fast MyHC isoforms in lines
A, B, and C suggests that these genotypes may serve as
useful models for understanding the mechanisms underlying MyHC transitions in avian species. These lines may also
be useful with respect to understanding the importance of
temporal changes in other muscle specific proteins such
as actin, troponin, or collagen.
The sequential appearance of the MyHC isoforms (eMyHC, nMyHC, and aMyHC) in the 3 broiler lines and the
SCWL chicks was similar and consistent with other reports
in the literature (Bandman et al., 1982; Maruyama and
Kanemaki, 1991; Maruyama et al., 1993; Tidyman et al.,
1997; Rosser et al., 1998). There are multiple mechanisms
underlying the expression of specific MyHC isoforms in
vertebrate muscle (i.e., innervation, muscle activity, thyroid
hormones; Bandman, 1985a,b; Schiaffino and Reggiani,
1994; Bandman, 1999), yet the exact mechanisms controlling the temporal expression of the developmental skeletal
fast MyHC isoforms are not clear.
Unique to the current study is the observation of earlier
expression of the aMyHC isoform in all 3 commercial
broiler lines compared with the Leghorn chicks, which was
concomitant with the observed increase in percentage of
breast muscle weight in the broiler lines. In previous work,
Wick et al. (2003) compared isoform transitions in a fastgrowing broiler line (B/B) and a slower-growing broiler
Leghorn cross (B/L). In that study, broiler hens were mated
with either broiler or Leghorn males, and there was accelerated aMyHC expression in the broiler-broiler chicks. The
results reported herein are consistent with the B/B vs.
B/L data reported by Wick et al. (2003). The increased
expression of aMyHC in lines A and B compared with
line C at 7 d suggests that within fast-growing broiler
genotypes, selection for extremes in conformation (i.e.,
breast yield) may result in altered expression of specific
proteins associated with muscle functionality (product
quality)
The changes in the transition of the myosin isoforms
associated with genetic differences in growth and breast
1734
REDDISH ET AL.
muscle development may allow for some insight into muscle functionality questions that relate to meat quality. This
refers to those components of muscle physiology both in
vivo and postharvest, which influence muscle protein solubility, protein extractability, and the formation of thermally
set meat gels, which is the process of turning muscle into
further-processed meat products. It is important to continually explore the physiological changes underlying the relationships between genetic selection and product quality.
These results support the hypothesis that genetic selection
for increased BW and breast yield has accelerated the temporal expression of developmental fast skeletal MyHC isoforms and that this acceleration of developmental fast skeletal MyHC is necessary for muscle specific growth, regardless of whole muscle growth.
Salaries and research support were provided by state
and federal funds appropriated to the Ohio Agricultural
Research and Development Center, The Ohio State University.
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