Download Protein expression in pectoral skeletal muscle of chickens as

Protein expression in pectoral skeletal muscle of chickens
as influenced by dietary methionine1
W. Zhai,* L. F. Araujo,* S. C. Burgess,† A. M. Cooksey,† K. Pendarvis,†
Y. Mercier,‡ and A. Corzo*2,3
*Department of Poultry Science, and †Institute for Genomics, Biocomputing and Biotechnology,
Mississippi State University, Mississippi State 39762;
and ‡Adisseo France S.A.S. Commentry, 03 600, Commentry, France
ABSTRACT Effects of dietary methionine (Met) on
pectoralis muscle development and the effect that Met
as a nutritional substrate has on protein expression of
skeletal muscle cells of pectoralis muscle of chickens
were evaluated in this study. Broiler chickens received
a common pretest diet up to 21 d of age and were subsequently fed either a low (LM) or high Met (HM) diet
(0.41 vs. 0.51% of diet) from 21 to 42 d of age. Dietary
deficiency was shown in vivo judging by the depression
in breast meat weight and yield when broilers were fed
the LM diet. Global protein expression was analyzed
by quantitative high-performance liquid chromatography nanospray ionization tandem mass spectrometry.
Up- and downregulated proteins were analyzed via Ingenuity Pathways Analysis to identify the metabolic
pathways affected. Four canonical pathways related
to muscle development were identified as being differentially regulated between LM- and HM-fed chickens.
These pathways included the citrate cycle and calcium,
actin cytoskeleton, and clathrin-mediated endocytosis
signaling. The HM diet may have allowed for increased
muscle growth by an increased availability of nutrients
to muscle cells. Although the Met supplementation was
associated with enhanced breast muscle growth, contraction fiber concentrations in muscles decreased and
were associated with a lower calcium transportation
rate and sensitivity and with a lower energy supply.
It is further suggested that increased muscle protein
deposition, that was induced by Met supplementation,
may have been largely due to sarcoplasmic rather myofibrillar hypertrophy.
Key words: broiler, methionine, pectoral skeletal muscle, protein expression
2012 Poultry Science 91:2548–2555
http://dx.doi.org/10.3382/ps.2012-02213
INTRODUCTION
Methionine (Met) is typically the first limiting amino
acid among common feed ingredients used in poultry
diets. In addition to being a structural component of
nucleic acids and proteins in the chicken, Met is also
recognized as a precursor in the biosynthesis of various
metabolites (sarcosine, betaine, and choline via transmethylation) and as an intermediary in the conversion
to cystine or cysteine (via homocysteine).
The pectoralis (breast) major and minor muscles of
chickens have been subject to intensive genetic selection
for maximal breast meat tissue deposition (Scheuermann et al., 2003). Although among all essential amino
©2012 Poultry Science Association Inc.
Received February 9, 2012.
Accepted June 19, 2012.
1 Approved for publication as Journal article no. J-12103 of the
Mississippi Agricultural and Forestry Experiment Station, Mississippi
State University.
2 Corresponding author: [email protected]
3 Present address: Elanco Animal Health, 2500 Innovation Way, PO
Box 708, Greenfield, IN 46140.
acids, the concentration of Met within these muscles is
the lowest (Murphy, 1994); a dietary deficiency of Met
has been shown to hinder breast muscle development
(Hickling et al., 1990; Schutte and Pack, 1995). Consequently, levels of Met in the diets of broiler chickens has
been extensively evaluated in recent years to further
understand its role in the metabolism and development
of rapidly growing chickens (NRC, 1994; Schutte and
Pack, 1995; Kalinowski et al., 2003).
Recently, Corzo et al. (2006) modeled the expression
of pectoral skeletal muscle protein in chickens in response to dietary Met inadequacy. After Met-adequate
or -deficient diets were fed to broiler chickens, the global
protein expression of their breast muscle was analyzed
using 2-dimensional liquid chromatography (LC) tandem mass spectrometry (MS). At that time, only limited methodologies and experimental techniques were
available for the analysis of differential protein expression in tissues. Therefore, only a small number of proteins were identified that were affected by dietary Met.
However, the more recent availability of LC MS-based
proteomics methods and systems has greatly improved
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DIETARY METHIONINE INFLUENCES PROTEIN EXPRESSION
chromatographic resolution and sensitivity. This has
involved the conversion of micro-flow to nano-flow in
HPLC, an upgrading of the ESI source to nanoESI, and
other improvements in mass spectrophotometers. Many
more mass spectra can be generated for analysis using
these new systems (Sokale et al., 2011). Therefore, the
current study, using updated technology, was designated to replicate the in vivo model provided by Corzo et
al. (2006). Furthermore, differential protein expressions
in pectoral skeletal muscle samples were compared
which elucidate the anabolic mechanisms by which Met
influences breast muscle deposition in chickens.
MATERIALS AND METHODS
Chickens and Diets
In total, 216 one-day-old male broiler chicks (Ross
× Ross 708) were obtained (Aviagen North America,
Huntsville, AL) and placed in 18 floor pens (0.9 × 1.2
m/pen) after equalization of BW across pens (±2 g/
chick). Water and feed were supplied for ad libitum
intake with a 23 h of light and 1 h of dark lighting program. Temperatures were set for the birds to achieve
thermoneutrality throughout the study (Aviagen North
America, 2005). All animal procedures were approved
by the Mississippi State University Institutional Animal Care and Use Committee.
To ensure accurate diet formulation, samples of corn
and soybean meal were analyzed before formulation for
total amino acids (AOAC International, 2006; 985.28,
994.12) and crude protein composition (AOAC International, 2006; 968.06). Upon analyses, the nutrient
matrix of the feed ingredients used was updated and
all the test diets were then formulated using linear programming that solved for energy, amino acids, and mineral and vitamin needs for optimal growth. A common
pretest diet in crumbled form, formulated to satisfy all
nutrient recommendations (NRC, 1994), was fed to all
the broilers in the study from placement until 21 d
of age (Table 1). Subsequently, the test diet was fed
from 21 to 42 d of age (Table 1) and was formulated
to satisfy all nutrient recommendations for the 21- to
42-d period, with the exception of Met (NRC, 1994;
Kalinowski et al., 2003). Because poultry can efficiently
utilize dietary Met in both d- and l-isoforms, dl-methionine (99%) was supplemented at the expense of an
inert filler to achieve a low dietary Met level (LM) and
fed to half of the pens, whereas the other half received
the high Met (HM) test diet (total Met: 0.41 vs. 0.51%
of diet).
Composite samples of both dietary treatments were
later analyzed for amino acid composition by HPLC
(AOAC International, 2006; 985.28, 994.12). To prevent sulfur amino acid degradation during hydrolysis
of the raw feed ingredients (before formulation of the
Table 1. Experimental diet composition (%, as-is)
Item
Ingredient
Corn
Soybean meal
Poultry oil
Dicalcium phosphate
Calcium carbonate
Sodium chloride
dl-Methionine
Premix1
Coccidiostat2
l-Lysine-HCl
Filler3
l-Threonine
Calculated composition
ME (kcal/kg)
CP
Calcium
Available phosphorus
Digestible sulfur amino acids
Digestible methionine
Total methionine4
Digestible lysine
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Common starter
(0–21 d)
Experimental
grower (21–42 d)
52.891
39.558
3.989
1.841
0.619
0.487
0.265
0.250
0.050
0.050
—
—
 
3,100
22.5
0.94
0.47
0.96
0.55
0.60
1.31
65.990
27.421
2.871
1.713
0.715
0.493
0.105
0.250
0.05
—
0.350
0.043
 
3,150
20.2
0.86
0.43
0.69
0.37
0.41
1.10
1The vitamin and mineral premix contained per kilogram of diet: retinyl acetate, 2,654 μg; cholecalciferol,
110 μg; dl-α-tocopherol acetate, 9.9 mg; menadione, 0.9 mg; B12, 0.01 mg; folic acid, 0.6 μg; choline, 379 mg;
d-pantothenic acid, 8.8 mg; riboflavin, 5.0 mg; niacin, 33 mg; thiamin, 1.0 mg; d-biotin, 0.1 mg; pyridoxine, 0.9
mg; ethoxyquin, 28 mg; manganese, 55 mg; zinc, 50 mg; iron, 28 mg; copper, 4 mg; iodine, 0.5 mg; selenium, 0.3
mg.
2Dietary inclusion of coccidiostat provides 60 g of salinomycin sodium per 907.2 kg of feed.
3Inert filler included in the experimental grower diet and used as an inert filler to supplement dl-methionine.
4Analyzed total methionine dietary concentrations were in close agreement with calculated values. Calculated
and analyzed (in parentheses) total methionine values were low methionine: 0.41% (0.42%); high methionine:
0.51% (0.50%).
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Zhai et al.
diets) and the test diets, oxidation was initiated using
performic acid to derive stable products of Met and
cystine. The formed products, methionine sulphone and
cysteic acid, were then determined by chromatography
and calculated back to the Met and cystine content in
the experimental diets in accordance with AOAC International (2006; 985.28, 994.12). Mean bird BW in all
pens was recorded at the initiation (21 d) and termination (42 d) of the experimental phase.
Processing Measurements
At 42 d of age, 10 birds per pen were randomly selected, tagged, and individually weighed and placed in
chicken coops 12 h before processing. Birds were processed at a pilot processing plant. Electrical stunning
was performed by applying 11.5 V (<0.05 mA, AC to
DC current) for 30 s to each broiler. Broiler carcasses
were scalded, picked, and eviscerated automatically using commercial prototype equipment. Birds were then
chilled for 4 h, at which point all birds were manually
deboned and weights were obtained for their pectoralis muscles. Absolute and relative pectoralis muscles
weights were then calculated.
Protein Expression Analyses
One bird from each pen (n = 9 per diet) was randomly selected at 41 d of age and euthanized via cervical dislocation. One cubic centimeter from each bird
was aseptically dissected from the cranial region of the
left pectoralis major postmortem and immediately frozen at −80°C. A 150-mg sample of pectoralis major
from each bird was processed by differential detergent
fractionation and trypsin digested as outlined by McCarthy et al. (2005). Following digestion, samples were
desalted using a peptide macrotrap (TR1/25108/52;
Michrom BioResources, Auburn, CA), followed by a
strong cation exchange (Michrom TR1/25108/53) step
to remove any detergents and other polymers that can
interfere with MS/MS analysis.
Each sample was loaded on a BioBasic C18 reversed
phase column (Thermo 72105–100266) and flushed for
20 min with 5% acetonitrile (ACN) and 0.1% formic
acid to remove salts. Peptide separation was achieved
using a Thermo Surveyor MS pump with a 655-min
nano-HPLC method consisting of a gradient from 5%
to 50% ACN for 620 min, followed by a 20-min wash
with 95% ACN and subsequent equilibration with 5%
ACN for 15 min (all solvents contain 0.1% formic acid
as a proton source). Ionization of peptides was achieved
via nanospray ionization using a Thermo Finnigan
nanospray source type I operated at 1.85 kV with 8
μm internal diameter silica tips (New Objective FS360–
75–8-N-20-C12). High voltage was applied using a tconnector with a gold electrode in contact with the
HPLC solvent. A Thermo LCQ DECA XP Plus ion
trap mass spectrometer was used to collect data over
the 655 min of duration of each HPLC run. Precursor
mass scans were performed using repetitive MS scans,
each immediately followed by 3 MS/MS scans of the
3 most intense MS peaks. Dynamic exclusion was enabled for a duration of 2 min with 2 repeat counts.
Once a mass is measured twice, it is added to a list to
be excluded from further analysis for a predetermined
amount of time, which was 2 min in the current trial.
This allows the MS to collect data on different masses.
In the meantime, the mass will have eluted from the
column. Dynamic exclusion allows for a more efficient,
deeper depth of sample coverage (Zhang et al., 2009b).
Statistical Analyses
Data collected and analyzed for pectoral breast meat
muscle weight and yield were evaluated by one-way
ANOVA in a completely randomized design. Pen was
used as the experimental unit for analysis. Data were
analyzed using the GLM procedure of SAS Institute
(2004). Treatment effects (P ≤ 0.05) were separated
using a t-test. Database searches of mass spectrometry
data for protein expression were performed using the
SEQUEST (Yates et al., 1995) algorithm in Bioworks
3.3. The database for peptide spectral matching was
the Chicken Affymetrix nucleotide database. Decoy
searches were performed using a randomized version of
the target database. Search results were filtered using
a decoy-based, statistical method in which a probability of being a false-positive match is assigned to each
peptide (Pendarvis et al., 2009). Proteins containing
peptides with a probability of 0.05 or less were evaluated for differential expression using Monte Carlo resampling statistics (Pendarvis et al., 2009). Proteins with a
probability of differential expression of 0.05 or less were
selected for further modeling. Expression fold changes
were calculated as described by Old et al. (2005).
Ingenuity Pathways Analysis
Data were analyzed through the use of Ingenuity
Pathways Analysis (Ingenuity Systems, www.ingenuity.
com). Canonical pathways analysis identified the pathways from the Ingenuity Pathways Analysis library of
canonical pathways that were most significant to the
data set. Molecules from the data set that were differentially regulated between LM- and HM-fed chickens
and were associated with a canonical pathway in Ingenuity’s Knowledge Base were considered for the analysis. The significance of the association between the data
set and the canonical pathway was measured by the
following 2 ways: 1) a ratio of the number of molecules
from the data set that map to the pathway divided by
the total number of molecules that map to the canonical pathway displayed; 2) Fisher’s exact test and B-H
multiple testing correction (Benjamini and Hochberg,
multiple testing correction which is for controlling the
false discovery rate) was used to calculate a P-value determining the probability that the association between
the genes in the data set and the canonical pathway is
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DIETARY METHIONINE INFLUENCES PROTEIN EXPRESSION
explained by chance alone. Pathways were considered
statistically significantly associated with the data set if
the P-value for the B-H was 0.05 or less.
RESULTS
Growth and Pectoralis Muscle Accretion
All broiler chicks being raised on a common diet from
placement until 21 d of age grew at an adequate growth
rate (866 ± 27.2 g/bird) before being fed the experimental diets. Calculated and analyzed total Met levels of the experimental diets were in close agreement,
therefore, validating the experimental dietary treatments fed to the broiler chickens (Table 1; footnote 4).
Table 2 shows the effect that feeding the LM diet had
on pectoral muscle development of broilers when compared with HM-fed birds. Feeding the LM diet resulted
in a 10% decrease in pectoralis muscle weight and a
0.72 percentage point drop in breast meat yield (% of
live BW), and both of these parameters were found
to be statistically different from those of chickens fed
HM diets. The drop in breast meat yield was found to
be 1 percentage point when the pectoral muscles were
expressed relative to the carcass weight.
Protein Identification
A total of 5,016 unique proteins was identified from
biological replicates of LM chickens, and 4,296 unique
proteins were identified from biological replicates of
HM chickens. Of these proteins, 1,468 were differentially regulated (593 upregulated; 875 downregulated)
between LM and HM chickens.
Six canonical pathways were differentially regulated
between LM- and HM-fed chickens. Those pathways
were citrate cycle, calcium signaling, actin cytoskeleton signaling, clathrin-mediated endocytosis signaling,
germ cell-sertoli cell junction signaling, and cellular effects of sildenafil (Figure 1). The differentially regulated
proteins of the first 4 pathways (citrate cycle, calcium
signaling, actin cytoskeleton signaling, clathrin-mediated endocytosis signaling) have been listed in Table 3.
The germ cell-sertoli cell junction signaling and cellular
Figure 1. Protein expression difference in pectoralis muscles of
broilers fed a low (LM) or high (HM) dietary methionine diet from 21
to 42 d of age. Color version available in the online PDF.
effects of sildenafil pathways were not presented due to
their indistinct function on breast muscle development
and growth.
DISCUSSION
Methionine is encoded by a single codon (AUG) in
the standard genetic code. The codon AUG is also the
“start” message for a ribosome that signals the initiation of protein translation from mRNA. As a consequence, Met is incorporated into the N-terminal position of all proteins in eukaryotes and archaea during
translation (Berg et al., 2002). In the current study,
dietary treatments imposed to these broiler chickens
resulted in markedly different pectoral muscle development, thus allowing for a valid proteome comparison to
obtain a better understanding of dietary Met-induced
metabolic and physiological differences. Protein expression was affected by Met supplementation, as indicated by higher absolute and relative muscle weights, as
well as changes in 6 canonical pathways. However, 2
pathways (Germ cell-sertoli cell junction signaling and
cellular effects of sildenafil) are important only in the
male reproductive system. Their functions in muscle
development remain obscure and should require further
Table 2. Body weight gain, feed conversion rate, pectoralis muscles weight and yield of broilers fed
a low or high dietary methionine diet from 21 to 42 d of age1
Pectoralis muscles
Treatment
BW gain
(g)
Feed
conversion2
0.41% Methionine
0.51% Methionine
SEM
P-value
1,972
2,013
52.5
0.17
1.92
1.85
0.051
0.32
a,bValues
 
 
 
 
Weight (g)
Yield3
Yield4
552b
606a
11.5
0.002
19.9b
20.6a
0.16
0.0004
29.7b
30.7a
0.22
0.0008
within a column with different superscripts differ significantly.
means are calculated from 10 carcasses from each experimental unit.
2Values represent the feed conversion of birds for the experimental phase (21 to 42 d of age) after being corrected for mortality weight.
3Yield data expressed relative to the live weight.
4Yield data expressed relative to the carcass weight.
1Treatment
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Zhai et al.
Table 3. Protein or peptide that has been differentially regulated in the 4 pathways
Pathway
Symbol
Entrez gene name
Citrate cycle
ACLY
ATP5G3
ATP citrate lyase
ATP synthase, H+ transporting, mitochondrial Fo complex, subunit C3
(subunit 9)
Malate dehydrogenase 2, NAD (mitochondrial)
Phosphoenolpyruvate carboxykinase 1 (soluble)
Aconitase 2, mitochondrial
Isocitrate dehydrogenase 3 (NAD+) α
Succinate dehydrogenase complex, subunit B, iron sulfur (Ip)
Succinate dehydrogenase complex, subunit D, integral membrane protein
Succinate-CoA ligase, α subunit
Succinate-CoA ligase, GDP-forming, β subunit
Glutamate receptor, ionotropic, AMPA 1
Histone deacetylase 4
Myocyte enhancer factor 2A
Troponin C type 1 (slow)
Actin, α 1, skeletal muscle
ATPase, Ca++ transporting, cardiac muscle, slow twitch 2
ATPase, Ca++ transporting, ubiquitous
cAMP responsive element binding protein 1
Histone deacetylase 9
Myosin, light chain 1, alkali; skeletal, fast
Myosin, light chain 3, alkali; ventricular, skeletal, slow
Protein phosphatase 3, regulatory subunit B, α
Troponin C type 2 (fast)
Troponin I type 2 (skeletal, fast)
Tropomyosin 3
ARP2 actin-related protein 2 homolog (yeast)
v-crk sarcoma virus CT10 oncogene homolog (avian)-like
Fibronectin 1
Phosphoinositide-3-kinase, catalytic, α polypeptide
Radixin
Vinculin
Actin, α 1, skeletal muscle
Actin, β
Actin, gamma 1
Cell division cycle 42 (GTP binding protein, 25 kDa)
Myosin, light chain 1, alkali; skeletal, fast
Myosin, light chain 3, alkali; ventricular, skeletal, slow
Neuroblastoma RAS viral (v-ras) oncogene homolog
Protein phosphatase 1, regulatory (inhibitor) subunit 12A
ras-related C3 botulinum toxin substrate 1 (rho family, small GTP
binding protein Rac1)
Slingshot homolog 1 (Drosophila)
WAS protein family, member 2
ARP2 actin-related protein 2 homolog (yeast)
Clathrin, heavy chain (Hc)
Huntingtin interacting protein 1 related
Numb homolog (Drosophila)
Phosphoinositide-3-kinase, catalytic, α polypeptide
Actin, α 1, skeletal muscle
Actin, β
Actin, gamma 1
Amphiphysin
Cell division cycle 42 (GTP binding protein, 25 kDa)
Heat shock 70 kDa protein 8
Protein phosphatase 3, regulatory subunit B, α
RAB5C, member RAS oncogene family
RAB7A, member RAS oncogene family
ras-related C3 botulinum toxin substrate 1 (rho family, small GTP
binding protein Rac1)
Ubiquitin specific peptidase 9, X-linked
Calcium signaling
Actin cytoskeleton signaling
Clathrin-mediated endocytosis
MDH2
PCK1
ACO2
IDH3A
SDHB
SDHD
SUCLG1
SUCLG2
GRIA1
HDAC4
MEF2A (includes EG:4205)
TNNC1
ACTA1
ATP2A2
ATP2A3
CREB1
HDAC9 (includes EG:9734)
MYL1
MYL3
PPP3R1
TNNC2
TNNI2
TPM3
ACTR2
CRKL
FN1
PIK3CA
RDX
VCL
ACTA1
ACTB
ACTG1
CDC42
MYL1
MYL3
NRAS
PPP1R12A
RAC1
SSH1
WASF2
ACTR2
CLTC
HIP1R
NUMB
PIK3CA
ACTA1
ACTB
ACTG1
AMPH
CDC42
HSPA8
PPP3R1
RAB5C
RAB7A
RAC1
USP9X
1Positive
Fold
changes1
5.50
7.97
1.13
4.72
−5.41
−0.57
−3.67
−4.51
−4.54
−6.41
4.57
5.37
5.31
8.19
−0.47
−0.32
−4.62
−3.60
−1.80
−3.56
−0.98
−7.03
−0.49
−1.94
−0.34
6.07
2.66
5.52
5.37
5.69
0.67
−0.47
−4.39
−0.25
−1.03
−3.56
−0.98
−3.68
−4.44
−2.61
−0.78
−2.40
6.07
5.74
1.36
3.41
5.37
−0.47
−4.39
−0.25
−8.35
−1.03
−0.52
−7.03
−5.56
−3.99
−2.61
−4.23
numbers mean upregulated; negative numbers mean downregulated.
investigation. Therefore, only 4 of the 6 canonical pathways that were differentially regulated between LM and
HM chickens are addressed in this manuscript.
Citrate Cycle
The citrate cycle is a series of enzyme-catalyzed
chemical reactions that converts carbohydrates, fats,
and proteins into carbon dioxide and water to generate
usable energy within mitochondria (Berg et al., 2002).
In the current study, supplementary dietary Met decreased the overall expression of several proteins in the
citrate cycle. Adenosine triphosphate (ATP) citrate
lyase expression was upregulated (Figure 2; Table 3).
Adenosine triphosphate citrate lyase catalyzes the for-
DIETARY METHIONINE INFLUENCES PROTEIN EXPRESSION
Figure 2. Simplified citrate cycle that shows the enzyme expression difference in pectoralis muscles of broilers fed a low (LM) or high
(HM) dietary methionine diet from 21 to 42 d of age. Symbols: ↑
means upregulated and ↓ means downregulated, ◊ are enzymes and □
are substrates. Color version available in the online PDF.
mation of acetyl-CoA and oxaloacetate from citrate
and CoA with a concomitant hydrolysis of ATP, which
is in opposition to ATP synthesis that is occurring in
the TCA cycle.
The expression of various enzymes that catalyze
various chemical reactions of the citrate cycle was
downregulated in HM groups (Figure 2). For example,
aconitase (ACO2), isocitrate dehydrogenase (IDH3A),
succinate-CoA ligase (SUCLG1 and SUCLG2), and
succinate dehydrogenase complex (SDHB and SDHD)
were all downregulated (Figure 2; Table 3). Isocitrate
dehydrogenase (IDH3A) catalyzes the oxidative decarboxylation of isocitrate to 2-oxoglutarate and is a ratelimiting step of the citrate cycle. These enzyme level
changes could lead to a reduction in mitochondrial ATP
production and an accumulation of oxaloacetate. The
increase in oxaloacetate may also have been used for
gluconeogenesis, as indicated by the increase of phosphoenolpyruvate carboxykinase (PCK1), a main control point for the regulation of gluconeogenesis. Phosphoenolpyruvate carboxykinase (PCK1) catalyzes the
formation of phosphoenolpyruvate from oxaloacetate,
and various amino acids in the body are metabolized
through the citrate cycle. Thus, a balanced amino acid
composition in the HM diet may have lowered the rate
of amino acid metabolism in the citrate cycle, allowing
more amino acids to be preserved for muscle growth
rather than being entered into the citrate cycle for energy production.
Calcium Signaling
As a second messenger, Ca2+ activates a protein kinase which translocates to the cell nucleus, where it
activates a cAMP responsive element binding protein
2553
(CREB). The CREB in turn acts as a DNA-binding
protein that plays an important role in cellular growth
and development (Lehrmann et al., 2002). In the current study, CREB expression was found to be downregulated in HM-fed chickens. In association with the
decrease in CREB, several proteins of the fast muscle
fiber contraction complex (dominate in broiler breast
muscle) including actin-α, myosin, tropomyosin, and
troponins were downregulated. These findings concur
with a previous report by Corzo et al. (2006), in which
several myosin-related proteins were found downregulated in Met-supplemented groups. In addition, 2 isoforms (ATP2A2 and ATP2A3) of sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA)-2, were
observed to be downregulated. The current results were
similar to a previous report in which SERCA was also
downregulated in HM-fed chickens (Corzo et al., 2006).
Sarcoplasmic/endoplasmic reticulum calcium ATPase
(SERCA) catalyzes the hydrolysis of ATP coupled
with the translocation of calcium from the cytosol into
the sarcoplasmic reticulum lumen and is involved in
the regulation of muscular contraction and relaxation.
Downregulation of the contraction complex and SERCA suggest that HM birds may exhibit a lower muscle
contraction capability. In addition, calcineurin (regulation of Ca2+ sensitivity; Groenendyk et al., 2004) was
downregulated in HM chickens. The downregulation
of calcineurin further suggests that muscle contraction
was lowered in HM chickens.
Actin Cytoskeleton Signaling
The main functions of the actin cytoskeleton are to
mediate cell motility and cell shape, for cytoplasmic
organization, and to generate mechanical forces within the cell (Schmidt and Hall, 1998). Similar to the
changes in the calcium signaling pathway, contraction
fibers (myosin and F-actin) were downregulated in HM
chickens. The expression of 2 myosin isoforms (MYL1
and MYH6) was downregulated. Myosins are a large
family of motor proteins that share common features,
including ATP hydrolysis, actin binding, and kinetic
energy transduction. They are involved in muscle contraction through cyclic interactions with actin-rich
thin filaments, creating a contractile force. Methionine
supplementation downregulated the expression level of
various isoforms of F-actin: ACTA1, which is a major
constituent of the contractile apparatus; ACTB and
ACTG1, which are involved in the regulation of cell
motility. Results of the current study were similar to a
study conducted by Yalçin et al. (2010), in which a high
protein diet decreased myofiber density and increased
sarcoplasmic protein solubility in broiler muscle. The
interaction of myosin and actin is regulated by phosphorylation and intracellular Ca2+ concentrations. Calcium also activates pyruvate dehydrogenase, IDH3A,
and α-ketoglutarate dehydrogenase to increase reaction
rate (Denton et al., 1975). Supplementation of Met decreased IDH3A in this study.
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Zhai et al.
The expression of 2 other proteins (slingshot homolog
and α actinins) involved in the formation of actin was
also downregulated. Slingshot homolog plays a role in
actin dynamics to stabilize actin filaments (Niwa et al.,
2002). In skeletal muscle, α actinins are localized to
the Z-disc and analogous dense bodies, where they help
anchor the myofibrillar actin filaments (Luther, 2009).
Associated with the decrease of contraction fibers, the
expression of Rho-like GTPases, such as RAC1 and
CDC42 was downregulated. Rho-like GTPases are key
regulators in signaling pathways that link extracellular
growth signals or intracellular stimuli to the assembly
and organization of the actin cytoskeleton (Schmidt
and Hall, 1998).
Although the contraction components (proteins that
lead to actin polymerization and actomyosin assembly
contraction) were downregulated, the 2 proteins for anchoring and linking (Radixin and Vinculin) involved in
focal complex assembly and focal adhesion assembly,
were found to be upregulated in HM-fed chickens. Focal
adhesions and adherens junctions are membrane-associated complexes that serve as nucleation sites for actin
filaments and as cross-linkers between the cell exterior,
the plasma membrane, and the actin cytoskeleton (Yamada and Geiger, 1997; Schmidt and Hall, 1998). Additionally, fibronectin (FN1) involved in cell adhesion
and migration processes (Pankov and Yamada, 2002),
was also upregulated in HM chickens.
Clathrin-Mediated Endocytosis Signaling
Clathrin-mediated endocytosis is the major route for
endocytosis in most cells. It is mediated by the molecule
clathrin (CLTC), which plays a major role in the formation of coated vesicles. Clathrin-mediated endocytosis
is required for a large number of essential cell functions,
including nutrient uptake, cell-cell communication, and
modulation of membrane composition (Ramanan et al.,
2011). Clathrin expression was upregulated in HM-fed
chickens. It is hypothesized that increased clathrin expression could increase the number of clathrin-coated
pits, quicken endocytosis, and enhance recycling and
absorption of nutrients from outside of the cell. Because
more nutrients were taken into the cell, the overall endocytosis may have been increased.
Furthermore, the expression of 2 other related proteins (numb and HIP1R, huntingtin interacting protein 1 related) was found to be upregulated in HM-fed
chickens. Numb (an endocytic adaptor) has been shown
to be associated with Eps15, which is involved in cell
growth regulation (Gulino et al., 2010) and plays an
important role in the assembly of clathrin-coated pits
(Naslavsky and Caplan, 2005). Huntingtin interacting
protein (HIP1R) is a component of clathrin-coated pits
and vesicles that may link the endocytic machinery to
the actin cytoskeleton (Boettner et al., 2011). However,
not all endocytosis procedures were upregulated. The
expression of 2 proteins (CDC42 and RAC1) that are
responsible for clathrin-mediated endocytosis of virus-
es and actin polymerization (Li et al., 1998; Van den
Broeke et al., 2010) was downregulated. This may suggest that substrates or nutrients were selectively taken
up by the cell.
Even the overall rate of endocytosis would presumably be upregulated in HM chickens, as indicated by
higher clathrin and HIP1R expression levels. However,
the disassembly of the clathrin-coated vesicles would
not be increased because of the downregulation of the
following 3 proteins: heat shock 70-kDa protein (responsible for disassembly of the vesicle; Eisenberg and
Greene, 2007), RAB5C, and RAB7A (RAS oncogene
family responsible for docking or fusion of vesicle and
vesicular transport; Han et al., 1996; Zhang et al.,
2009a).
Conclusion
Despite the report of an increase in the absolute and
relative weights of pectoralis muscle in HM-fed chickens,
F-actin and myosin (2 main contractile proteins) were
found to be downregulated. There are 2 types of muscle
hypertrophy: myofibrillar hypertrophy and sarcoplasmic hypertrophy. Myofibrillar hypertrophy is characterized by an increase in contractile proteins, such as
actin and myosin, which subsequently increases the
strength of muscle contraction and is associated with
physical activity such as exercise. Sarcoplasmic hypertrophy is characterized by the growth of sarcoplasm
(a semifluid interfibrillar substance lacking contractile
proteins) without contribution to the production of
muscle force. The results of this study would suggest
that sarcoplasmic hypertrophy predominated over myofibrillar hypertrophy. A predominance of sarcoplasmic
hypertrophy would lead to a decrease in muscle fiber
density, whereas muscle fiber cross-sectional area would
increase, without an accompanying increase in muscle
strength (Zatsiorsky and Kraemer, 2006). In the current study, the dietary supplementation of Met to levels
considered to be adequate led to an increase in breast
muscle development. However, the contraction fiber
concentration in the muscle was found to be downregulated, as suggested by the lower calcium transportation
rate, sensitivity, and energy supply. More nutrients may
have been taken up into the muscle cells and were preserved for growth instead of being converted to energy
for contraction. The increase of muscle protein deposition that was induced by Met supplementation might
have been caused mainly by sarcoplasm hypertrophy
rather than by the myofibrillar hypertrophy. Maltin et
al. (1997), Shackelford et al. (1997), and Rehfeldt et
al. (2000) reported that an increase in muscle mass,
largely as a result of myofibrillar hypertrophy, may decrease the tenderness of pig and lamb muscle and thereby affect its meat quality. Sarcoplasm hypertrophy via
Met supplementation in the current study suggests that
further investigations into the effects of dietary Met
supplementation on meat tenderness and meat quality
are needed.
DIETARY METHIONINE INFLUENCES PROTEIN EXPRESSION
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