Published December 5, 2014
Myosin heavy chain isoform content and energy metabolism
can be uncoupled in pig skeletal muscle
S. K. Park, A. M. Gunawan, T. L. Scheffler, A. L. Grant, and D. E. Gerrard1
Department of Animal Sciences, Purdue University, West Lafayette, IN 47907
ABSTRACT: Genetic selection for improved growth
and overall meatiness has resulted in the occurrence
of 2 major mutations in pigs, the Rendement Napole
(RN) and Halothane (Hal) gene mutations. At the tissue level, these mutations influence energy metabolism
in skeletal muscle and muscle fiber type composition,
yet also influence total body composition. The RN mutation affects the adenosine monophosphate-activated
protein kinase γ subunit and results in increased glycogen deposition in the muscle, whereas the Hal mutation
alters sarcoplasmic calcium release mechanisms and results in altered energy metabolism. From a meat quality standpoint, these mutations independently influence the extent and rate of muscle energy metabolism
postmortem, respectively. Even though these mutations
alter overall muscle energy metabolism and histochemically derived muscle fiber type independently, their effects have not been yet fully elucidated in respect to
myosin heavy chain (MyHC) isoform content and those
enzymes responsible for defining energetics of the tissue. Therefore, the objective of this study was to determine the collective effects of the RN and Hal genes
on genes and gene products associated with different
muscle fiber types in pig skeletal muscle. To overcome
potential pitfalls associated with traditional muscle
fiber typing, real-time PCR, gel electrophoresis, and
Western blotting were used to evaluate MyHC composition and several energy-related gene expressions in
muscles from wild-type, RN, Hal, and Hal-RN mutant
pigs. The MyHC mRNA levels displayed sequential
transitions from IIb to IIx and IIa in pigs bearing the
RN mutation. In addition, our results showed MyHC
protein isoform abundance is correlated with mRNA
level supporting the hypothesis that MyHC genes are
transcriptionally controlled. However, transcript abundance of genes involved in energy metabolism, including lactate dehydrogenase, citrate synthase, glycogen
synthase, and peroxisome proliferator-activated receptor α, was not different between genotypes. These data
show that the RN and Hal gene mutations alter muscle
fiber type composition and suggest that muscle fiber
energy metabolism and speed of contraction, the 2 determinants of muscle fiber type, can be uncoupled.
Key words: halothane, myosin heavy chain, porcine, Rendement Napole, skeletal muscle
©2009 American Society of Animal Science. All rights reserved.
INTRODUCTION
J. Anim. Sci. 2009. 87:522–531
doi:10.2527/jas.2008-1269
an energy sensor that plays an important role in glucose uptake, glycogen synthesis, and fat oxidation in
skeletal muscle (Kahn et al., 2005). Adenosine monophosphate-activated protein kinase γ3 is predominantly
expressed in type II fast-contracting myofibers (Mahlapuu et al., 2004). Thus, the increase in muscle glycogen content in RN mutant pigs is observed primarily in fast-twitch muscle fibers (Marinova et al., 1992).
Yet, white muscles of RN mutant pigs exhibit greater
oxidative capacities and decreased proportions of the
fastest-contracting MyHC (IIb) as assessed by classical histological examination (Lebret et al., 1999). On
the other hand, Hal-positive pigs (nn) possess mutated
skeletal muscle calcium release channels, which allow
for enhanced calcium release (Mickelson and Louis,
1996). Greater resting cytosolic calcium concentrations
in Hal lead to altered energy metabolism but in a distinctly different manner than the RN mutation. Using
The Rendement Napole (RN) and Halothane (Hal)
gene mutations alter myosin heavy chain (MyHC)
composition, a determinant of muscle fiber type. These
genes independently influence energy metabolism and
meat quality development. The RN mutation leads to
an increase in muscle glycogen and inferior pork quality, characterized by a low ultimate muscle pH. Rendement Napole mutant pigs have a point mutation in
the adenosine monophosphate (AMP)-activated protein kinase (AMPK) γ3 subunit (Milan et al., 2000).
Adenosine monophosphate-activated protein kinase is
1
Corresponding author: [email protected]
Received July 1, 2008.
Accepted September 24, 2008.
522
Myosin and energy metabolism
antibodies against various MyHC, Fiedler et al. (1999)
and Depreux et al. (2000) showed that Hal mutant pigs
have increased IIb fibers at the expense of type I fibers,
indicating the Hal mutation drives fiber-type transition
to a faster, more glycolytic entity.
Indeed, both mutations influence muscle energy metabolism, but their effect on MyHC composition is unclear. Therefore, the purpose of this study was to determine the effects of the RN and Hal genes on porcine
skeletal muscle MyHC profiles and establish whether
energy metabolism and contractile speed is capable of
being uncoupled.
MATERIALS AND METHODS
Experimental procedures with these animals were approved by the Purdue University Animal Care and Use
Committee.
Animals
Animals for this study were maintained at the Purdue University Swine Research Unit. Only those pigs
homozygous at both loci (NN/rn+rn+, normal; NN/
RN−RN−, RN; nn/rn+rn+, Hal; nn/RN−RN−, Hal-RN;
n = 10 per treatment) were used for this experiment.
Genotypes were determined using the PCR-RFLP technique. Halothane and RN PCR products were digested
with appropriate restriction enzymes and separated on
agarose gel following the procedures of O’Brien et al.
(1993) and Meadus et al. (2002), respectively. At approximately 120 kg of BW, pigs were killed and then
exsanguinated following standard industry procedures.
About 6 g of muscle was isolated from LM (area of 10th
and 11th costae), chopped, and snap-frozen in liquid
nitrogen.
RNA Isolation, cDNA Synthesis,
and Real-Time PCR
Total RNA from LM was extracted using the singlestep RNA isolation method (Chomczynski and Sacci,
1987) with modifications. Isolated RNA was treated
with 4 U of DNase (Ambion, Indianapolis, IN) in 2
μL of a 10× DNase buffer containing 100 mM TrisHCl (pH 7.5), 25 mM MgCl2, 5 mM CaCl2, and 3 μL
of nuclease-free water to eliminate genomic DNA contamination. After 30 min of incubation, 5 μL of DNase
inactivation reagent (Ambion) was added to the reaction. Ribonucleic acid was loaded into 0.22-μm SpinX
centrifuge tube filters (VWR International, West Chester, PA) and centrifuged at 10,000 × g for 1 min at
room temperature. One microliter of purified RNA (0.8
μg/μL) was then diluted in TE buffer (249 μL; 10 mM
Tris-Cl, 1 mM EDTA, pH 8.0). Twenty-five microliters
of diluted RNA, 75 μL of TE buffer, and 100 μL of
Ribogreen (Molecular Probes, Carlsbad, CA) reagent
were combined in a 96-well microplate, and fluores-
523
cence signal was measured at 480 (excitation) and 520
(emission) nm by GENios Pro fluoremeter (Tecan, Durham, NC). Complementary DNA was made using the
random primed cDNA synthesis method. Total RNA
(0.5 µg) in 5 μL nuclease-free water was added to 0.5
μL of 100 ng/μL random hexamers and 0.5 μL of 100
µM deoxynucleoside triphosphate. Samples were denatured at 65°C for 5 min after which 1 μL of 10 mM
dithiothreitol (Invitrogen, Carlsbad, CA), 0.5 μL of 5
U Moloney murine leukemia virus reverse transcriptase
(Invitrogen), 0.5 μL of 0.5 U SUPERase-In (Ambion),
and 2 μL of 5× First Strand Buffer (Invitrogen) was
added in a total volume of 10 μL. The cDNA mixture was incubated in the thermal cycler at 25°C for
10 min, 37°C for 50 min, and 70°C for 10 min; it was
then chilled to 4°C. Complementary DNA samples were
then diluted to 10 μg/mL in nuclease-free water. Realtime quantification was performed in duplicate 15-µL
reactions containing 5 μL of cDNA and 10 μL of the
gene-specific master mix. The PCR master mix consisted of 10 μM gene-specific primers and iQ SYBR
Green Supermix (Bio-Rad, Richmond, CA) containing
100 mM KCl, 40 mM Tris-HCL (pH 8.4), 0.4 mM of
each deoxynucleoside triphosphate, 50 U of iTaq DNA
polymerase/mL, 6 mM MgCl2, 7.5 μL of SYBR Green
I, 20 nM fluorescein stabilizers, and nuclease-free water.
Primer sequences for β-actin and MyHC isoforms (type
I, IIa, IIx, and IIb), citrate synthase, peroxisome proliferator activated receptor α, lactate dehydrogenase, and
glycogen synthase are shown in Table 1. β-Actin was
used as an internal control. Real-time quantification
was performed for 40 cycles using the iCycler real-time
PCR detection system (Bio-Rad) according to optimized gene-specific protocols shown in Table 2. Amplified real-time PCR products were sequenced to validate
primer specificity. Samples were amplified in separate
tubes, and the increase in fluorescence was measured
in real time. The first cycle in the log-linear region of
amplification in which a significant increase in fluorescence was detected above background was designated
the threshold cycle (Ct). Therefore, with a greater initial amount of cDNA, the accumulated product was
detected earlier in the PCR process, and a decreased Ct
value was recorded. Gene expression data are presented
as Ct values. Water was used as negative control, and
no PCR product was detected.
SDS-Glycerol Gel Electrophoresis of MyHC
Myosin heavy chain isoforms were separated with
SDS glycerol gel electrophoresis according to the method of Talmadge and Roy (1993) with modifications.
Longissimus dorsi muscles (100 mg) were powdered in
liquid nitrogen, and myosin was extracted with a high
ionic strength buffer (pH 6.5) containing 0.3 M KCl,
0.1 M KH2PO4, 50 mM K2HPO4, 10 mM EDTA, and
50% glycerol. Samples were then centrifuged at 10,000
× g for 20 min at 4°C. Supernatants were diluted 1:1
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Park et al.
Table 1. Nucleotide sequences of the primers used for quantitative reverse transcription-PCR
Gene1
Sequence (5′-3′)
2
MyHC I
MyHC IIa2
MyHC IIx2
MyHC IIb2
β-Actin
Citrate synthase
PPARα
LDH
Glycogen synthase
Forward
Reverse
Forward
Reverse
Forward
Reverse
Forward
Reverse
Forward
Reverse
Forward
Reverse
Forward
Reverse
Forward
Reverse
Forward
Reverse
Accession no.
GGC CCC TTC CAG CTT GA
TGG CTG CGC CTT GGT TT
TTA AAA AGC TCC AAG AAC TGT TTC A
CCA TTT CCT GGT CGG AAC TC
TGC TTC AAG TTC TGC CCC ACT
GGC TGC GGG TTA TTG ATG G
CAC TTT AAG TAG TTG TCT GCC TTG
AG GGC AGC AGG GCA CTA GAT GT
GGC ATC CAC GAG ACC ACC TTC AA
CAG ACA GCA CCG TGT TGG CGT AGA
TGC CAG TGC TTC TTC CAC GAA CTT
GTT GCC GTG TTG CTG CCT GAA G
CAA GGG CTT CTT TCG GAG AAC CAT
GCG CCC AAA TCG AAT TGC ATT AT
GTG CAT CCG ATT TCC ACC ATG ATT
CCA TTC TGT CCC AAG ATG CAA GGA
CCC AGT GGG AGG AGG CAG TCT TG
GAA CCG CCG GTC CAG AAT GTA CA
L10129
U11772
U90719
U90720
U07786
NM_214276
AF175309
U07178
AJ507152
1
MyHC = myosin heavy chain; PPARα = peroxisome proliferator-activated receptor α; LDH = lactate dehydrogenase.
da Costa et al., 2002.
2
(vol/vol) with glycerol. Concentration of the isolated
protein was determined using a BCA Protein Assay kit
(Pierce, Rockford, IL) according to the protocol of the
manufacturer with BSA as a standard. Samples were
then suspended in Laemmli buffer (pH 6.8) containing 10% SDS, 10% β-mercaptoethanol, and 20% glycerol in 0.5 M Tris and boiled for 5 min. Myosin heavy
chain isoforms were separated onto 8% polyacrylamide
gels. Stacking gels were composed of 30% glycerol, 4%
acrylamide:N,N-methylene-bis-acrylamide (50:1), 0.5 M
Tris (pH 6.8), 4 mM EDTA, and 0.4% SDS. The resolving gel consisted of 30% glycerol, 8% acrylamide:N,Nmethylene-bis-acrylamide (50:1), 0.4 M Tris (pH 8.8),
0.1 M glycine, and 4% SDS. Equal amounts of protein
(5 μg per lane) were then loaded onto gels set on a
Mini-Protein II Dual Slab Cell electrophoretic system
(Bio-Rad) and run for 30 h at 70 V at 4°C. After migration, the gels were silver- or Coomassie Blue-stained,
and relative amounts of different MyHC isoforms were
quantified using Kodak 1D Image Analysis Software
(Kodak, New Haven, CT).
Western Blotting
Proteins (5 μg per lane) were separated electrophoretically as described above. Separated proteins were
then transferred to polyvinylidene fluoride membranes
at 200 mA for 2 h, and membranes were stained with
Ponceau S (Sigma, St. Louis, MO) to ensure equal loading and transfer of proteins. Membranes were blocked
for 1 h at room temperature in blocking solution (5%
nonfat dry milk in Tris-buffered saline with 0.1%
Tween-20). Membranes were incubated 1 h at room
temperature with antibodies specific for MyHC type
I (A4.840; Hughes and Blau, 1992), IIa (6B8; Depreux
et al. 2000), IIb (BF-F3; Schiaffino et al. 1989), and
fast all (My-32; Sigma) diluted 1:1,000 in the blocking
solution. Secondary antibodies conjugated with horseradish peroxidase (Jackson ImmunoResearch, West
Grove, PA), diluted 1:1,000 in the blocking solution,
were applied for 1 h. Bands were visualized using enhanced chemiluminescence (ECL; Amersham, Arlington Heights, IL). Several exposures of x-ray films were
conducted. Densitometry (ImageJ software, National
Table 2. Gene-specific protocols for real-time amplification conditions
Gene1
Hot start
Melting
MyHC I
MyHC IIa
MyHC IIx
MyHC IIb
β-Actin
Citrate cynthase
PPARα
LDH
Glycogen synthase
95°C
95°C
95°C
95°C
95°C
95°C
95°C
95°C
95°C
79°C
78°C
79°C
78°C
80°C
78°C
80°C
76°C
83°C
15
15
15
15
15
15
15
15
15
s
s
s
s
s
s
s
s
s
15
15
15
15
15
15
15
15
15
s
s
s
s
s
s
s
s
s
Annealing
Synthesis
72°C
72°C
72°C
72°C
72°C
72°C
72°C
72°C
72°C
55°C
52°C
56°C
56°C
59°C
59°C
54°C
56°C
59°C
30
30
30
30
30
30
30
30
30
s
s
s
s
s
s
s
s
s
15
15
15
15
15
15
15
15
15
s
s
s
s
s
s
s
s
s
1
MyHC = myosin heavy chain; PPARα = peroxisome proliferator-activated receptor α; LDH = lactate
dehydrogenase.
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Myosin and energy metabolism
Figure 1. Body weights of normal, Rendement Napole (RN), Halothane (Hal), and Hal-RN double mutant pigs.
Institutes of Health, Bethesda, MD) was used to quantify bands that resulted from exposure times less than
film saturation. All other chemicals were obtained from
Sigma.
Statistics
Data are represented as the mean ± SE. Differences
between genotypes were assessed utilizing individual Ct
or band intensities with the GLM procedure (SAS Inst.
Inc., Cary, NC). Values were analyzed as a factorial
design with main effects of each Hal and RN gene to
analyze the main effects and their interaction (Hal ×
RN). Least squares means were generated using LSMEANS, and Tukey’s adjustment was used for multiple
comparisons. Statistical significance was established at
P < 0.05.
RESULTS
The BW of the normal, RN, Hal, and Hal-RN genotypes were similar (Figure 1; P = 0.12). β-Actin gene
expression, the control to test for experimental variation, did not differ between muscles from normal and
mutant pigs (Figure 2A). Expression of slow-twitch type
I MyHC was not different between genotypes (Figure
2B). There was an RN (P < 0.05) and Hal (P < 0.05)
gene effect for MyHC IIa mRNA expression but no Hal
× RN interaction (P = 0.34). Thus, the main effects of
the RN (−/rn+rn+ vs. −/RN−RN−) and Hal (NN/−vs.
nn/−) genes are presented separately in Figure 2C.
Myosin heavy chain type IIa mRNA abundance was
1.5-fold greater in RN and 1.4-fold less in Hal compared
with normal, respectively (Figure 2C). The RN mutation increased (P < 0.01) IIx mRNA expression (Figure
2D), and there was tendency for a Hal × RN interaction (P = 0.07) for type IIx MyHC mRNA. There was
no Hal gene effect or Hal × RN interaction for MyHC
IIb MyHC expression (Figure 2E). Myosin heavy chain
IIb expression was decreased (P < 0.05) in pigs bearing
the mutant RN genotype, and this level was approximately 4-fold less than that of normal pigs.
Separation of MyHC isoforms by SDS-PAGE showed
MyHC composition similar to real-time PCR. Although
we could not separate all 4 adult MyHC, 3 bands, including type IIa/IIx, type IIb, and type I, were clearly
discernable using the protocol of Talmadge and Roy
(1993). Scans of these gels (Figure 3) showed type I
MyHC protein abundance did not vary across the genotypes. Expression of MyHC IIa/IIx proteins was greater
in the Hal-RN genotype (P < 0.01). Type IIb protein
expression was decreased in Hal-RN (P < 0.01), which
is consistent with expression patterns of mRNA.
Real-time PCR and SDS-PAGE data were confirmed
by Western blot analysis (Figure 4). No difference in
MyHC type I protein content was detected between
normal, RN, Hal, and Hal-RN genotypes. The fast antibody reacts with all type II fibers and did not reveal
any differences in type II total protein content between
genotypes. In accord with real-time PCR and SDSPAGE data, Western blot analysis revealed the RN mutation increased MyHC IIa and decreased IIb protein
expression (P < 0.01). There was a Hal × RN interaction for MyHC IIb protein expression (P < 0.05). Myosin heavy chain IIb protein expression was decreased
by 28% in the RN genotype and nearly 50% in the
Hal-RN genotype compared with normal pigs. The RN
and Hal-RN genotypes possess a greater proportion of
oxidative MyHC, as evidenced by a decrease in IIb and
an increase in IIa MyHC isoform protein expression
(Figure 4).
Levels of mRNA expression of MyHC I (Figure 5A; P
< 0.01) and IIb (Figure 5B; P < 0.01) were correlated
with protein abundance determined with SDS-PAGE
(R2 = 0.52 and 0.57, respectively). No relationship (P
> 0.3) was found between mRNA and protein abundance for IIa/IIx MyHC (data not shown).
No difference between genotypes was found in mRNA
expression level of citrate synthase, lactate dehydrogenase, peroxisome proliferator-activated receptor α, or
glycogen synthase (Figure 6).
DISCUSSION
Skeletal muscle fiber type diversity is represented,
in part, by differences in MyHC isoforms. Four adult
MyHC isoforms are expressed in the skeletal muscle of
pigs: type I, IIa, IIx, and IIb (Lefaucheur et al., 2002).
Slow-twitch type I myofibers consisting predominately
of type I MyHC receive sustained, tonic contractile
motor activity, whereas type II fast-contracting fibers
containing type II MyHC are recruited by acute motor
neuron activity (Hennig and Lomo, 1985). The type
and abundance of cellular metabolic enzymes generally
match the energetic demands of each fiber-based functional capacity. Type I fibers have greater oxidative capacity to support sustained contraction, whereas type
IIb fibers are predominantly glycolytic and rapidly use
glycogen for short bursts of activity. The IIa and IIx
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Park et al.
Figure 2. Real-time determination of β-actin (A), myosin heavy chain (MyHC) type I (B), IIa (C), IIx (D), and IIb (E) mRNA isoforms of LM
from normal, Rendement Napole (RN), Halothane (Hal), and Hal-RN double mutant pigs. *P < 0.05 vs. control. a–cMeans with different letters
differ (P < 0.05; n = 10 per group for A, B, and D and n = 20 per group for C and E). Ct = threshold cycle.
fibers are intermediate to type I and IIb fibers (Chang
et al., 2003).
Skeletal muscle is a dynamic tissue and adapts to
external cues by changing its metabolism and contractile ability. Transition of MyHC follows a sequential,
yet reversible, pathway: I ↔ IIa ↔ IIx ↔ IIb (Pette
and Staron, 2000). Interestingly, genetic selection for
growth performance pushes fiber type to the right of
this equation (Solomon and West, 1985; Weiler et al.,
1995). Traditionally, muscle fiber type has been characterized using ATPase and enzymatic staining; however,
type IIx fibers cannot be distinguished from IIb fibers.
Enzymatic staining is also problematic, because metabolic activity is a continuum across muscle fibers (Pette
and Staron, 2000). Antibodies against the various isoforms of MyHC have improved muscle fiber-typing protocols, yet reliable IIb and IIx MyHC antibodies are
lacking. To overcome this, expression of each MyHC
isoform (type I, IIa, IIx, and IIb), their respective protein abundance, and various enzymes can be used to
study muscle fiber type (Gunawan et al., 2007).
The combined use of immunohistochemistry with
specific antibodies and quantitative real-time PCR has
been used for more accurate classification of pig MyHC
isoforms (Lefaucheur et al., 2004; Toniolo et al., 2004).
Here, we determined MyHC composition of muscles
from RN and Hal mutant pigs at the mRNA and protein levels using real-time PCR and Western blotting
techniques, respectively. There were limitations to quantifying MyHC protein in pig skeletal muscle because IIa
and IIx MyHC could not be separated by SDS-PAGE,
and a specific type IIx MyHC antibody is not yet available. Nonetheless, significant correlations were found in
MyHC type I and IIb expression between mRNA and
protein levels, which indicates, at least in type I and
IIb MyHC, that MyHC genes are transcriptionally con-
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Myosin and energy metabolism
Figure 3. Myosin heavy chain (MyHC) isoform contents in normal, Rendement Napole (RN), Halothane (Hal), and Hal-RN double mutant
pigs as determined by SDS-PAGE analysis. Bottom, middle, and top bands contain type I, IIb, and IIa/IIx MyHC, respectively. *P < 0.05 vs.
other genotypes within a MyHC isoform (n = 3 per treatment).
trolled. The positive correlation between MyHC protein expression and its corresponding mRNA level has
also been shown by others (Cox and Buckingham, 1992;
Schiaffino and Reggiani, 1996; McKoy et al., 1998; Gunawan et al., 2007). However, we found no relationship
between mRNA and protein level for type IIa and IIx
MyHC, likely due to the absence of a specific antibody
to detect type IIx MyHC.
Real-time PCR data showed RN mutant pigs had a
greater proportion of IIa/IIx MyHC. Gel electrophoresis and Western blots confirmed that patterns of MyHC
protein expression were similar to mRNA expression.
These results support the observations that RN pigs
possess increased IIa isoform and greater relative area
of IIa fibers at the expense of IIb fibers (Lebret et al.,
1999). Fiber type shift from IIb to IIa in RN mutant
Figure 4. Detection of myosin heavy chain (MyHC) isoforms by Western blot analysis using anti-MyHC type I, IIa, and IIb.
different letters differ within a MyHC isoform (P < 0.05; n = 3 in each sample). RN = Rendement Napole; Hal = Halothane.
a–c
Means with
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Park et al.
pigs is consistent with the function of AMPK in skeletal muscle; AMPK is activated by muscle contraction
by increased AMP:ATP ratio (Fujii et al., 2000). Once
activated, AMPK increases glucose uptake, fatty acid
oxidation, and mitochondrial biogenesis (Hayashi et al.,
1998; Zong et al., 2002; Koistinen et al., 2003). Barnes
et al. (2004, 2005) showed that the R225Q mutation is
a loss-of-function mutation that abolishes AMP dependence, resulting in greater basal AMPK activity. They
also demonstrated that increased AMPK activity in
R225Q mutants resulted in enhanced glycogen content
in skeletal muscle by shifting metabolism toward fatty
acid oxidation, thereby sparing glucose and glycogen.
Muscles from Hal mutant pigs have been shown to
contain a greater proportion of glycolytic fibers and
type IIb MyHC isoforms (Fiedler et al., 1999; Depreux
et al., 2000). We also found decreased IIa mRNA and increased MyHC IIx mRNA, indicating the Hal mutation
Figure 5. Relationship between mRNA expression (threshold cycle, Ct) and relative protein abundance (%) of type I (A) and IIb (B) in LM
from normal, Rendement Napole (RN), Halothane (Hal), and Hal-RN double mutant pigs. Each data point represents 1 sample from 1 pig.
Myosin and energy metabolism
529
Figure 6. Real-time PCR analysis for mRNA expression level of citrate synthase (A), lactate dehydrogenase (LDH; B), peroxisome proliferator-activated receptor α (PPARα; C), and glycogen synthase (D) of LM from normal, Rendement Napole (RN), Halothane (Hal), and Hal-RN
double mutant pigs. n = 10 in each group. Ct = threshold cycle.
increases the expression of glycolytic fibers. Increased
sarcoplasmic calcium concentrations in Hal mutant
muscle may stimulate hypercontraction of myofibrils
and glycolysis, thereby increasing the population of glycolytic fibers. Major signaling molecules downstream of
calcium include calcium/calmodulin-dependent kinases
(Fluck et al., 2000), calcium-dependent protein kinase
(Freyssenet et al., 1999), and calcineurin (Chin et al.,
1998). These signaling molecules are known to be selectively activated by calcium in a frequency-dependent
manner and play an important role in activating mitochondrial biogenesis, myosin isoform expression, and
muscle hypertrophy. Therefore, it is possible that specific calcium frequency elicited by Hal mutant muscle
activates genes for glycolytic muscle fiber through these
calcium signals. However, Lopez et al. (1986) found
that resting sarcoplasmic calcium concentration in Hal
mutant muscle is greater than in normal pigs, but is
not sufficient to cause contraction. If this is the case,
subcontraction concentrations of calcium in Hal mutant pigs may directly regulate signaling molecules or
gene expression without causing changes in contractile
activity or energy status. Therefore, more work is necessary to understand the mechanism by which calcium
frequencies and signaling pathways affect muscle adaptation in Hal mutant pigs.
We found fiber type transition from IIb to IIx in
muscles from Hal-RN double mutant pigs. These data
imply that the RN gene predominantly regulates fiber
type transition by promoting a more oxidative phenotype. However, we could not detect a significant increase
in IIa mRNA in Hal-RN double mutant pigs. This indicates that the RN gene may push fibers toward slower
phenotypes, whereas the Hal gene may induce the opposite machinery and increase the proportion of fasttwitch fibers. Therefore, it is possible that RN and Hal
pig mutations may interact with each other in regulation of MyHC gene expression.
Transcript abundance of genes related to energy metabolism may be used to define fast- and slow-twitch
fiber types (Peter et al., 1972). Lactate dehydrogenase,
citrate synthase, and glycogen synthase were measured
to characterize glycolysis, fatty acid oxidation, and glycolytic capacity, respectively. Neither Hal nor RN genotype affected expression of these genes. Collectively,
these data demonstrate that RN and Hal genotypes may
directly regulate MyHC gene expression without causing changes in transcription of genes related to energy
metabolism. In other words, contractile profiles are not
coordinately regulated with gene expression of enzymes
in energy metabolism and are, therefore, uncoupled in
these pigs. Enzyme activity may be more dependent on
substrate availability or posttranslational modifications
rather than transcriptional regulation (Mandarino et
al., 1995). Further study is needed to address the activities of metabolic enzymes, because enzymatic activities could be different between genotypes.
In conclusion, we evaluated muscle fiber type-specific
genes and gene products and showed that the RN and
Hal mutations alter muscle fiber type transition. Specifically, the RN mutation decreases IIb mRNA and
increases IIa and IIx mRNA expression, which corresponds to associated changes in protein expression. This
supports the notion that MyHC are transcriptionally
regulated in porcine skeletal muscle. Also, these data
suggest that metabolism and MyHC are independently
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Park et al.
regulated and capable of being uncoupled. Further studies are needed to determine the mechanisms by which
calcium-mediated signaling events and AMPK activity
regulate enzyme activity, energy metabolism, and the
speed of contraction.
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