Zhen Yan and Frank W. Booth
J Appl Physiol 85:973-978, 1998.
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Myotubes originating from single fast and slow satellite cells display similar patterns of
AChE expression
C. Boudreau-Lariviere, D. J. Parry and B. J. Jasmin
Am J Physiol Regulatory Integrative Comp Physiol, January 1, 2000; 278 (1): R140-R148.
[Abstract] [Full Text] [PDF]
Cytochrome c promoter activity in soleus and white vastus
lateralis muscles in rats
ZHEN YAN AND FRANK W. BOOTH
Department of Integrative Biology, Pharmacology, and Physiology,
University of Texas Medical School, Houston, Texas, 77030
mitochondria; direct plasmid injections; muscle fiber types;
oxidative capacity
SKELETAL MUSCLE FIBER diversity may arise from the
commitment of distinct myoblast lineages during myogenesis (27). However, the phenotypes of adult muscles
in living animals are very plastic because the quantity
of muscle contraction regulates the expression of most
contractile protein isoforms and metabolic enzymes
(22, 34). Pette and Staron (22) have commented that
future research should be directed at improving our
understanding of the molecular basis for the functional
specialization of phenotypic expression in skeletal
muscle fibers. One approach to understanding the
molecular mechanisms that control muscle diversification and plasticity is to identify the DNA-regulatory
sequences that confer fiber type-specific expression of
contractile proteins and metabolic enzymes. Extensive
studies of troponin slow and fast genes have indicated
that several core sequences in the promoters, such as
E-box, CCAC-box, and MEF-2-like sequence, bind their
cognate factors and these transcriptional factor-DNA
interactions determine fiber type diversity (18). Others
have found the E box and MEF-2 sequences in fiber
type elements of troponin I slow (4) and myosin heavy
chain IIB promoters (29). A more recent study showed
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that both the nuclear respiratory factor 1 (NRF-1)binding site and cAMP-response element (CRE) in the
2326 cytochrome c promoter are necessary and sufficient for the induction of its promoter activity in
electrically stimulated cardiomyocytes in serum-free
media (36). Because the same treatment (electrical
stimulation) does not increase mitochondrial density in
cultured myotubes (15, 25), it seemed necessary to
learn whether the same promoter’s elements confer the
fiber type-specific difference in cytochrome c gene expression in adult skeletal muscle. Moreover, because
muscle cultures do not manifest the specific properties
of muscles in living animals (9), the identification of the
cis-acting sequences in the cytochrome c promoter that
confer fiber specificity needs to be addressed by using a
living animal.
In 1969, Gauthier (10) published electron micrographs that showed that red muscle fibers were richer
in mitochondria than were white fibers in animals. The
concentration of cytochrome c protein, a mitochondrial
protein in the respiratory chain, has been used to
determine the mitochondrial density and/or oxidative
capacity in the skeletal muscle due to its parallel
changes with other mitochondrial oxidative enzymes in
animals (35) and its possible limiting role for electron
transport (12). Winder et al. (35) reported that cytochrome c protein concentration was 3 and 12 nmol/g
wet weight for the white vastus lateralis (predominantly fast-twitch glycolytic) and soleus (predominantly slow-twitch oxidative) muscles, respectively, in
sedentary rats. The soleus and white vastus lateralis
muscles are recruited ,7 h/day and ,2 min/day, respectively, in sedentary rats according to Hennig and Lomo
(14). These data suggested that the cytochrome c level
in skeletal muscle could be regulated, in part, by
differences in the quantity of contractile activities
among various fiber types in the animal. Mechanistically, the fourfold higher concentration of cytochrome c
protein in slow-twitch oxidative muscle than in fasttwitch glycolytic muscles (17) may be contributed, in
part, by the twofold difference in cytochrome c mRNA
level. Therefore, it is very important to know whether
the cytochrome c promoter’s activity is higher in the
slow-twitch oxidative than in the fast-twitch glycolytic
muscles because this information would be the basis for
future experiments to generate transgenic mice lacking
the fiber type-specific element in the cytochrome c
promoter. A recent advance in gene delivery, the direct
plasmid injection technique, has allowed the relatively
rapid estimation of promoter activities in striated
muscles of living animals (2, 4, 13, 16, 20, 21, 24,
29–31), and such results provide a more rational basis
for future transgenic animal models (4). The present
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973
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Yan, Zhen, and Frank W. Booth. Cytochrome c promoter
activity in soleus and white vastus lateralis muscles in rats.
J. Appl. Physiol. 85(3): 973–978, 1998.—Cytochrome c protein and mRNA are 300 and 100% higher, respectively, in the
soleus muscle (predominantly slow-twitch oxidative) than the
white vastus lateralis (predominately fast-twitch glycolytic)
muscle (W. W. Winder, K. M. Baldwin, and J. O. Holloszy. Eur. J.
Biochem. 47: 461–467, 1974; M. M. Lai and F. W. Booth. J.
Appl. Physiol. 69: 843–848, 1990). However, the mechanisms
controlling these differences in cytochrome c mRNA are
largely unknown. The present study employed direct plasmid
injection techniques to determine whether the proximal
promoter (2726 to 1610) of the rat somatic cytochrome c gene
was more active in the soleus than in white vastus lateralis
muscles in rats. No difference between the soleus and white
vastus lateralis muscles for the activities of the 2726, 2631,
2489, 2326, 2215, 2159 and 2149 cytochrome c promoters
was noted. The results of this study suggest that additional
elements (outside of 2726 to 1610) in the cytochrome c gene
may be required, or posttranscriptional regulation may account, for the higher cytochrome c mRNA in the slow-twitch
oxidative muscle.
974
CYTOCHROME
C
PROMOTER IN SKELETAL MUSCLE
study was undertaken to determine whether the 2726
promoter of the rat somatic cytochrome c gene was
more active in the soleus muscle than in the white
vastus lateralis muscle in the living animal (6, 22).
METHODS
Fig. 1. Direct correlation between constitutive reporter gene activities and amount of DNA injected. Chloramphenicol acetyltransferase
(CAT; A) and luciferase (Luc; B) activities from RSV and SV40
promoters, respectively, were measured 4 days after rat skeletal
muscles were injected with 0, 50, 100, or 200 µg of each of pSV40-Luc
and pRSV-CAT in 100 µl of normal saline. Values are means 6 SE of 4
injections at each dose. %Conv., %conversions; RLU, relative luciferase light units.
min at 15,000 g, the pellet fraction was discarded and the
supernatant was used for luciferase and CAT assays. Assays
for either CAT or luciferase were performed at the same time
for all muscle samples
Luciferase assays. Luciferase activity was assayed immediately after muscle homogenates were obtained by using a
previously described procedure (32) with modification (2).
Briefly, the reaction was started by addition of 20 µl of the
homogenate to 100 µl luciferase reagent containing (in mM)
20 tricine (pH 7.8), 1.07 (MgCO3 )4Mg(OH)2b · 5H2O, 2.67
MgSO4, 0.1 EDTA, 0.53 ATP, 0.27 CoA, and 33.3 1,4dithiothreitol as well as 0.47 µM luciferin. Seventy seconds
after the initiation of the reaction, light production becomes
stable and it was measured by using a Monolight model 2010
luminometer (Analytical Luminescence Laboratory) and integrated for the next 10 s. Total luciferase activity was calculated in relative light units after correction for the background, which was the value from the uninjected control
muscles.
CAT assays. Each muscle homogenate was assayed for CAT
activity as previously described (11) with modification (1).
Briefly, the reaction was initiated by adding 25 µl homogenate
to 65 µl of reaction cocktail containing (in mM) 192.3 Tris · HCl
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Animals. Female Sprague-Dawley rats, weighing 100–149
g, were obtained from Harlan (Houston) and housed under
temperature-controlled (21°C) conditions with a 12:12-h lightdark cycle. Rat chow and water were provided ad libitum.
Protocols were approved by the University of Texas Health
Science Center Institutional Animal Welfare Committee.
Plasmids. Rat somatic cytochrome c promoter (pRC)luciferase plasmids were constructed from pRC4-chloramphenicol acetyltransferase (CAT) (8) and pGL2-Basic (Promega). Briefly, the Xho I-Hind III fragments containing the
rat cytochrome c 58-regions were inserted by T4 ligase into
the 5.6-kb Xho I-Hind III fragment of pGL2-Basic. pRCluciferase chimeric genes were constructed as follows: various
deletion constructs of the 58-rat somatic cytochrome c promoter (from either 2726, 2631, 2491, 2326, 2215, 2159, or
2146 to 1610) (8), the luciferase-coding region, and the SV40
polyadenylation site and splice signal at the 38-end in pGL2
were ligated (Promega) (Fig. 1). Plasmid DNAs were transformed in JM109 bacteria (Promega). The bacteria were
grown for 12 h in Luria-Bertani medium with moderate
shaking at 37°C. Plasmid DNAs were isolated and purified by
using alkaline lysis with differential polyethylene glycol
precipitation and subsequent phenol extractions (26).
Plasmid injections. Rats were anesthetized with a mixture
of ketamine (54 mg/ml), xylazine (2.2 mg/ml), and acepromazine (3.5 mg/ml) injected intramuscularly into the right
gluteus muscle (1.4 ml/kg). All DNA deletional constructs
were injected on the same day. Two-centimeter incisions were
made lateral to the soleus muscle and to the white vastus
lateralis muscles so that these muscles could be directly
visualized. We selected these two muscles because they have
a twofold difference in cytochrome c mRNA (17) and are
visually accessible to verify the injections. To compare cytochrome c chimeric gene expression between the soleus and
white vastus lateralis muscles, 20 µl of double-distilled water
containing plasmid DNA cocktail [50 µg of chimeric reporter
gene and 50 µg of constitutive reporter gene Rous sarcoma
virus promoter driving CAT (pRSV-CAT)] were injected for
each muscle. In preliminary experiments, luciferase and CAT
activities were measured 4 days after injection of various
amounts of DNA (0, 50, 100, or 200 µg for each construct; n 5
4 for each dose) to determine linearity of injected plasmid. For
the fiber type experiments, 200 µg of each plasmid were
injected. Either 4 days (linearity experiments) or 7 days (fiber
type experiments) after these injections, rats were again
anesthetized. The entire soleus and white vastus lateralis
muscles were dissected, weighed, quickly cut into seventeen
,3-mm3 pieces, fast-frozen with a pair of Wollenberger tongs
prechilled in liquid nitrogen, and held at 280°C until homogenized.
Tissue homogenization. Each sample was placed on ice and
5 ml/g (1 ml minimum) of ice-cold homogenization buffer [(in
mM) 25 Tris-phosphate, pH 7.8, 2 trans-1,2-diaminocyclohexane-N, N8,N8-tetraacetic acid, 10% glycerol, 2 1,4-dithiothreitol, 1 phenylmethylsulfonyl fluoride, 1 EDTA, and 1 benzamidine as well as 10 µg/ml leupeptin, 10 µg/ml pepstatin,
1 µg/ml aprotinin] were added to each sample in a 15-ml
polyethylene tube. The samples were homogenized at 4°C
with a Polytron (Brinkman) at 70% of its maximal intensity
with three 10-s intervals. After centrifugation at 4°C for 15
CYTOCHROME
C
PROMOTER IN SKELETAL MUSCLE
975
(pH 7.8), 1.85 acetyl-CoA, and 3.77 14C-labeled chloramphenicol (53 µCi/mmol). The promoter activity in each muscle
sample was calculated as relative luciferase activity, i.e., the
ratio of luciferase to CAT activity. For each animal, the mean
value was taken from the left and right hindlimbs for the
soleus and white vastus lateralis muscles.
Statistics. To determine the linear correlation between the
constitutive reporter gene activities and the amount of DNA
injected, regression with replication analysis was performed
and the P value was calculated by analysis of variance. P ,
0.05 was interpreted as a significant linear relationship
between DNA-injection dose and the reporter gene activity.
Cytochrome c promoter activity values are expressed as
means 6 SE. A paired Student’s t-test was used to determine
whether differences between means from the soleus and white
vastus lateralis muscles were significant at a P , 0.05 level.
RESULTS
Fig. 2. 58-Deletional analysis of rat somatic cytochrome c-Luc chimeric gene in soleus and white vastus lateralis muscles of rats.
Histogram shows relative Luc activities (Luc driven by cytochrome c
promoter/RSV-CAT activity) for 2 muscles. Values are means 6 SE;
n 5 8 rats/group. No significant differences (P , 0.05) existed for any
deletion length between soleus and white vastus lateralis muscles.
Open bar, white vastus muscle; solid bar, soleus muscle; downward
arrows, relative position of deletion to identified potential regulatory
elements in cytochrome c promoter (schematic diagram at bottom);
x-axis, bp of 58-cytochrome c promoter for each deletion construct;
WV, white vastus lateralis muscle; SO, soleus muscle; Mt, sequence
elements that bind proteins in human cytochrome c1 (28); Sp1,
GC-rich regions (7); CRE, cAMP-response element; NRF-1, nuclear
respiratory factor 1; CCAAT, enhancer binding proteins (C/EBP) (7).
Inset: mean of relative Luc values for both soleus and white vastus
lateralis muscles for each deletion length.
Deletion to 266 decreased promoter activity to the
background of muscles injected with promoterless constructs (data not shown). The major difference with
these data from a previous deletion study in cultured
kidney cells was that deletion of the regions containing
the 58-CRE site (2326/2215) in the cytochrome c
promoter region decreased the promoter activity in
skeletal muscle of the living rat but not in cultured
monkey kidney CV-1 cells (8). This difference might
indicate that the regions containing the 58-CRE site
and region I are required for normal cytochrome c gene
expression in adult skeletal muscles, not in cultured
kidney cells.
DISCUSSION
No difference in the activities of the 2726-bp cytochrome c promoter or of its deletion constructs has been
found between rat skeletal muscles with greater and
lesser cytochrome c mRNA concentrations. The following options could explain these negative results. The
first possibility is that higher cytochrome c mRNA in
the more active muscle is not a consequence of more
promoter activity for the cytochrome c gene but is a
result of greater mRNA stability. However, Connor et
al. (3) were unable to detect a half-life for either
cytochrome c oxidase subunit III or VIc mRNA in
skeletal muscle of living rats, so determination of
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Coinjection of pSV40-luciferase and pRSV-CAT into
rat skeletal muscles was performed to determine the
linearity of the injection procedure. Expression of luciferase driven by either the SV40 promoter or the RSV
promoter was linear up to 200 µg of plasmid DNA
injected into the tibialis anterior, lateral gastrocnemius, or quadriceps muscles (r 5 0.74, P , 0.01 for
luciferase and r 5 0.65, P , 0.05 for CAT) (Fig. 1).
To determine the difference in the cytochrome c
promoter activities among muscles with different inherent contractile activities, pRSV-CAT or plasmids containing various lengths of the cytochrome c promoter
driving the luciferase reporter gene were coinjected
into the more active soleus (predominantly slow-twitch
oxidative fibers) and less active white vastus lateralis
(predominantly fast-twitch glycolytic fibers) muscles.
The CAT enzyme activities from the control plasmid,
pRSV-CAT, were not significantly different between
soleus and white vastus lateralis muscles (19.0 6 2.3
vs. 14.9 6 1.7% conversion/min, respectively; P . 0.05;
n 5 140). This result indicates that both the soleus and
white vastus lateralis muscles have similar efficiency
in taking up the pRSV-CAT plasmid DNA and expressing it, which, in addition to the above result of direct
correlation between DNA dose and reporter gene activities, validated the use of pRSV-CAT as a control for
DNA injection. There were variations in reporter gene
activities from sample to sample, probably due to
variations in plasmid DNA uptake by the muscle fibers
(5). However, this error is diminished by correcting
luciferase activity against its CAT activity from a
constitutive promoter of a coinjected plasmid in the
same muscle homogenate.
No differences in the 2726, 2631, 2489, 2326, 2215,
2159 and 2149 cytochrome c promoter activities, expressed as relative luciferase activities, were noted
between the soleus and white vastus lateralis muscles
(Fig. 2). Because there were no significant differences
in activity between the two muscles, we pooled the data
from the soleus and white vastus lateralis muscles to
examine the deletion effect on the promoter activity
(Fig. 2, inset). Deletion from 2726 to 2489 did not
markedly alter cytochrome c activity. Deletion from
2489 to 2159 progressively reduced promoter activity.
976
CYTOCHROME
C
PROMOTER IN SKELETAL MUSCLE
The second methodological possibility for the lack of
a difference in cytochrome c promoter activity between
muscles with different cytochrome c mRNA levels is
that the sensitivity of the technique employed is not
suitable for detecting the difference in transcriptional
rate. However, the standard error of the mean for
promoter activity in the various cytochrome c deletion
constructs in our present deletion experiments was
15 6 2 relative luciferase light units (mean of 14
groups; 7 deletions tested in 2 muscles). Thus the
methods employed appear to be sensitive enough to
detect a difference between soleus and white vastus
lateralis muscles when means differed by 35%, which is
less than the twofold difference in cytochrome c mRNA.
Moreover, others (2, 4, 13, 16, 20, 21, 24, 29–31) have
been able to detect differences in promoter activities by
using the direct injection of plasmids into striated
muscles (Table 1).
Nevertheless, one limitation of the plasmid injection
technique is that the expression of the coinjected
reporter gene may be altered by the treatment independent of the reporter sequence. For example, if the
activity in coinjected control plasmid increases with
Table 1. Studies that have employed plasmid
injections into striated muscles
Promoter (Ref. No.)
2613- to 132-bp
a-Myosin heavy
chain (16)
2613- to 1421-bp
a-Myosin heavy
chain (20)
23,542-bp b-Myosin
heavy chain (13)
Species/Tissue
Rat heart
100% Increase after
thyroid treatment
Rat heart
300% Increase after
thyroid treatment
Rat heart
200% Increase in
hypertrophy;
mutation of
GATA4 eliminated
increase with
hypertrophy
2667-bp Greater
activity than
2354- or 2215-bp
promoters
Fiber type expression element is
within the
2295-bp promoter
and doubles in
hindlimb
unloading
Deletion of 2124- to
279-bp abolished
promoter activity
2700 and 2179 bps
Required for
activity in heart
and quadriceps,
respectively
157-bp Enhancer
conferred slowmuscle phenotype
100% Increase in
hypertrophy; 291
to 281 bp required
for hypertrophy
2667-bp b-Myosin
heavy chain (31)
Dog heart
22,600-bp Myosin
heavy chain IIB
(29)
Rat tibialis anterior
and soleus
muscles
2156-bp Cardiac
troponin C (21)
Rat heart
2700-bp M-isozyme
of creatine kinase
(30)
Rat heart and quadriceps muscle
24,200-bp Troponin
Islow promoter (4)
Rat extensor digitorum longus and
soleus muscles
Rooster anterior
latissimus dorsi
muscle
22,090- to 1318-bp
Skeletal a-actin
(1, 2)
Change Observed
Downloaded from jap.physiology.org on May 19, 2009
cytochrome c mRNA half-life in muscles of living rats
also seemed unfeasible. Furthermore, it is technically
difficult to employ nuclear run-on (a more direct measurement of transcription rate) for cytochrome c gene
expression in the skeletal muscles of living rats. For
example, Neufer and Dohm (19) found that the measured transcription rates of the cytochrome c gene in
skeletal muscle of control and treadmill-run rats were
too close to background level to report. In the same
muscles, GLUT-4 transcription rate was not only detectable but shown to be elevated transiently postexercise
(19). Thus nuclear run-on assays for cytochrome c
transcription rate in rat skeletal muscle were also
deemed not feasible in the present study. If promoter
activities are deemed as an index of transcription rate,
observations from the present report imply that cytochrome c transcription is not different between the
soleus and white vastus lateralis muscles.
The second possibility for the lack of a difference in
cytochrome c promoter activity between muscles with
different cytochrome c mRNA levels is that the methods
employed in the present study failed to detect a difference in promoter activities. However, we believe this
option is unlikely for the following reasons. Others
have detected electrically responsive regulatory elements within 2726 to 1610 of this promoter in cultured cardiomyocytes. Two elements [the NRF-1 motif
and CRE, which are located within 2326 to 1610 bp of
the cytochrome c promoter] were shown to be necessary
and sufficient for the induction of the cytochrome c
promoter in electrically stimulated cardiomyocytes that
had been cultured in serum-free media (36). In contrast, our study showed that reporter genes bearing
these same elements were not activated by the inherently higher contractile activity of the soleus muscle
(compared with the white vastus lateralis muscle) in
living rats. One potential interpretation is that this
discrepancy could be the result of differences in gene
regulation pathways between the cardiac myocytes and
skeletal muscle cells. If this is true, then a signaling
pathway of electrical stimulation to the activation of
the cytochrome c promoter via NRF-1 and CRE sites in
embryonic cardiomyocytes is missing in the skeletal
muscle of young rats. Another potential interpretation
for the differences between the previous and present
study is that cytochrome c promoter regulation differs
between cultured cells and living animals. Precedence
for this possibility exists. For example, electrical stimulation of skeletal myotubes in tissue culture (15, 25) did
not result in an increase in mitochondrial enzyme
activities, whereas the indirect electrical stimulation of
skeletal muscle in the living animal resulted in very
large increases in mitochondrial proteins (33). Similar
observations have been made in developmental studies;
Firulli and Olson (9) wrote that it is becoming clear that
significant discordances in gene regulation often exist
between cell culture and whole animals. A final potential interpretation is that the fiber type and electrical
activity elements for the cytochrome c gene differ.
CYTOCHROME
C
PROMOTER IN SKELETAL MUSCLE
treatment, the corrected value of the reporter gene of
interest will be artificially lowered as a result. In
earlier studies an effect of the stretch-induced hypertrophy (2) on the expression of the reporter gene was
noted. This limitation is not applicable to the present
study because we did not find a significant difference
with a large sample size (n 5 140) in the constitutive
promoter activities between the soleus and white vastus lateralis muscles.
In summary, regulatory regions responsible for the
fiber type differences in cytochrome c mRNA in rat
skeletal muscle may reside outside of the 2726 to 1610
region of the gene, or it is posttranscriptional control
that mediates the upregulation of the cytochrome c
gene in the soleus, compared with the white vastus
lateralis, muscle. Modulation of cytochrome c gene
expression in various fiber types remains a complex
problem in exercising live animals.
Received 12 January 1998; accepted in final form 7 May 1998.
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The authors thank Drs. Marc Hamilton, Martin Flück, Scott
Gordon, Warren McClure, Chris Carlson, Brian Tseng, Ron Gomes,
and Brian Pavey for discussions and thank Dr. Richard Scarpulla for
pRC4CATs.
This research was supported by National Institute of Arthritis and
Musculoskeletal and Skin Diseases Grant AR-44208.
Present address of Z. Yan: Molecular Cardiology Laboratories, Div.
of Cardiology, Dept. of Internal Medicine, 5323 Harry Hines Blvd.,
Univ. of Texas Southwestern Medical Center, Dallas, TX 75235-8573.
Address for reprint requests: F. W. Booth, Dept. of Integrative
Biology and Pharmacology, Univ. of Texas Medical School, 6431
Fannin St., Houston, TX 77030.
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