1 Department of Biology and 2 Department of Kinesiology and Health Science, York University, Toronto, Ontario, Canada M3J 1P3
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We evaluated contractile activity-induced alterations in cytochrome c transcriptional activation and mRNA stability with unilateral chronic stimulation (10 Hz, 3 h/day) of the rat tibialis anterior (TA) muscle for 1, 2, 3, 4, 5, and 7 days (n = 3-11/group). Transcriptional activation was assessed by direct plasmid DNA injection into the TA with a chloramphenicol acetyltransferase (CAT) reporter gene linked to 326 bp of the cytochrome c promoter. Cytochrome c mRNA in stimulated muscles increased by 1.3- to 1.7-fold above control between 1 and 7 days. Cytochrome c protein was increased after 5 days of stimulation to reach levels that were 1.9-fold higher than control by 7 days. Cytochrome c mRNA stability, determined with an in vitro decay assay, was greater in stimulated TA than in control between 2 and 4 days, likely mediated by the induction of a cytosolic factor. In contrast, cytochrome c transcriptional activation was elevated only after 5 days of stimulation when mRNA stability had returned to control levels. Thus the contractile activity-induced increase in cytochrome c mRNA was due to an early increase in mRNA stability, followed by an elevation in transcriptional activation, leading to an eventual increase in cytochrome c protein levels.
cell-free messenger ribonucleic acid decay; chronic stimulation; direct gene injection; mitochondrial biogenesis
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
IT IS WELL ESTABLISHED that mitochondrial biogenesis can be elicited in skeletal muscle in response to sustained contractile activity (10, 12). Increases in the volume of skeletal muscle mitochondria (9, 20), as well as concomitant elevations in the activity of many mitochondrial enzymes, occur in response to increased muscle activity (8, 13). These changes occurring at the protein level of expression are also accompanied by elevations in mRNAs derived from both the nuclear and the mitochondrial genomes (14, 32). Alterations in gene transcription and/or mRNA stability could potentially explain these increases. However, the relative contribution of these processes in response to contractile activity has not been established.
In the present study, we used the expression of cytochrome c as a representative model of the possible adaptations in mRNA turnover that transpire during chronic stimulation. Cytochrome c is a nuclear-encoded mitochondrial protein involved in electron transport between complexes III and IV of the mitochondrial respiratory chain. The sequence of the cytochrome c gene has been extensively studied, and both the coding region (24) and the upstream regulatory elements have been determined (11). In skeletal muscle, cytochrome c mRNA and protein levels are known to be upregulated in parallel with changes in contractile activity (8, 36). Recently, it has been shown that continuous contractile activity (24 h/day) induced an alteration in RNA-protein interactions at the 3' end of the cytochrome c mRNA, suggesting that increases in skeletal muscle cytochrome c mRNA expression may be at least partially mediated by changes in message stability (36). To further investigate this hypothesis, we evaluated both cytochrome c transcriptional activation and mRNA degradation with a physiologically relevant contraction (3 h/day) and recovery period stimulation paradigm. To measure transcriptional activation, we used direct plasmid DNA injection into skeletal muscle. A number of studies have shown that promoter-reporter chimeric gene constructs are successfully taken up and expressed in skeletal muscle (30, 33). Transcriptional activation can be subsequently determined by measuring the expression of the protein encoded by the reporter gene. To evaluate cytochrome c mRNA degradation, we utilized a cell-free mRNA decay system (21) with extracts derived from stimulated and nonstimulated control skeletal muscle. Thus measurements of transcriptional activation as well as mRNA stability were used to interpret the time course changes in cytochrome c mRNA expression during chronic contractile activity.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Animal care and surgery. Male Sprague-Dawley rats (283 ± 5 g) were housed individually in a temperature-controlled room (21°C) with a 12:12-h light-dark cycle and were allowed food and water ad libitum. A smaller group of animals was used for a separate mRNA stability experiment (352 ± 14 g; n = 6). Animals were anesthetized with an intraperitoneal injection of pentobarbital sodium (65 mg/kg body wt), and platinum electrodes (Med-Wire, Leico Industries, New York, NY) were surgically implanted on both sides of the common peroneal nerve of the left hindlimb as previously described (28). This procedure was used to stimulate both the tibialis anterior (TA) and the extensor digitorum longus (EDL) muscles. The contralateral nonstimulated limb served as an internal control. Animals subject to this protocol are able to locomote freely within the cage and appear to eat and drink in unaffected fashion.
Plasmids. The plasmid constructs
(pRC4CAT/-326 and -66) used to assess cytochrome
c transcriptional activation were
generous gifts of Dr. R. Scarpulla (Northwestern University, Chicago,
IL). The pRC4CAT/-326 construct contains sequences of the
rat cytochrome c promoter, which
include 326 bp upstream of the transcription start site, linked to a
chloramphenicol acetyltransferase (CAT) reporter gene. This construct
has been previously shown to give full cytochrome
c promoter activity in COS-1 cells
(11). The pRC4CAT/-66 construct, which represents the minimal promoter
region (11), was used in initial studies for comparison to the
pRC4CAT/-326 promoter. The plasmid directing the synthesis of
-galactosidase under the control of the Rous sarcoma virus long
terminal repeat (pRSV/
-galactosidase) was also used with the intent
of correcting for DNA transfection efficiency. Plasmid DNA was isolated
with an alkaline lysis technique followed by phenol-chloroform
extractions. DNA was then dissolved in 0.9% sodium chloride, and the
DNA concentration was determined at 600 nm with the diphenylamine assay
(31). The quality of DNA was assessed by restriction endonuclease
digestion and 1% agarose gel electrophoresis.
Direct plasmid injection. One week
postoperatively, animals were anesthetized with an intraperitoneal
injection of pentobarbital sodium (65 mg/kg body wt), and a single
incision was made in the skin covering the right and left TA muscles. A
27-gauge needle was inserted obliquely 0.1- to 0.2-cm deep into the TA
muscle to inject a combination of pRC4CAT/-326 or -66 and
pRSV/-galactosidase (50 µg each) in a final volume of 100 µl.
The incision was then sutured, and animals were allowed to recover for
2 days before the onset of chronic stimulation.
Stimulation protocol and tissue sampling. Two days after the plasmid DNA injections, TA and EDL muscles were stimulated (10 Hz, 0.1-ms duration, 3 h/day) for 1, 2, 3, 4, 5, and 7 days (27, 29). On the day after the indicated stimulation time period (21 h later), animals were anesthetized, and TA muscles were removed from both the stimulated and contralateral limbs, weighed, and frozen. In a separate experiment designed in part to evaluate the effect of continuous contractile activity on cytochrome c mRNA stability alone, animals were stimulated at 10 Hz for 10 days (24 h/day), as done previously (28). Six hours after the cessation of stimulation, bilateral TA muscles were removed, frozen, and stored. All muscles were then powdered and stored in liquid N2 until required for analyses.
RNA analyses. Total RNA was isolated
from frozen muscle powders (50-70 mg) as previously described (6).
RNA concentration and purity were determined by ultraviolet photometry
at 260 and 280 nm, respectively. Total RNA (6 µg) was then separated
on a denaturing formaldehyde-1% agarose gel and transferred overnight to a nylon membrane (Hybond N, Amersham, Mississauga, Canada). Cytochrome c and 18S rRNA radiolabeled
cDNAs were generated by random primer labeling in the presence of
[-32P]dCTP
(Amersham). After the removal of unincorporated nucleotides, label
incorporation was determined by Cerenkov counting. Blots were
prehybridized overnight (42°C), and the membranes were subsequently hybridized overnight at 42°C with the appropriate radiolabeled cDNA
probe (2 × 106 counts/min)
as done previously (6). The blots were rinsed with 2×
saline-sodium citrate (SSC; 1× SSC = 0.15 M NaCl/0.015 M sodium
citrate), 0.1% SDS, and subsequently washed 3 × 10 min at room temperature with 2× SSC, 0.1% SDS, followed by a
15-min wash at 50°C in 0.1× SSC, 0.1% SDS. The blots were
quantified by electronic autoradiography (Instantimager, Packard),
which measures the total radioactivity in the region of the cytochrome c mRNA. In this case, all three bands
corresponding to the cytochrome c mRNA
species were quantified. Blots were then corrected for uneven loading
with subsequent probing with a cDNA encoding 18S rRNA.
Tissue extraction for protein assays and immunoblotting. Powdered tissues (20-25 mg) were diluted 40-fold (wt/vol) in 0.1 M KH2PO4 buffer (pH 7.2) containing 2 mM EDTA and sonicated (8 × 10 s) on ice. Samples were then centrifuged for 6 min in a Microfuge at 4°C. The supernatants were removed, and protein concentration was determined photometrically (2).
SDS gel electrophoresis and immunoblotting. Muscle protein extracts (75 µg) were applied to one-dimensional SDS polyacrylamide gels [12.5% (wt/vol)] and electrophoresed overnight. The separated proteins were then electrotransferred to nitrocellulose membranes (Hybond C, Amersham) and incubated overnight with a rabbit anti-rat cytochrome c polyclonal antibody (1:500; Ref. 25). The secondary antibody was an alkaline phosphatase-conjugated goat anti-rabbit antibody (1:1,000), affording visualization of the antigen by a standard color reaction. The immunoblots were then quantified by laser densitometry (27).
CAT and -galactosidase activities.
Muscle powders (100-125 mg) were diluted threefold (wt/vol) in
0.25 M Tris (pH 7.9), subsequently frozen in an ethanol-dry ice bath,
and thawed at 37°C three times. The resulting homogenates were then
centrifuged in a Microfuge for 5 min at 4°C, and the supernatants
were used as muscle extracts for CAT and
-galactosidase activities.
To assess CAT activity, 10 µl of muscle extract were mixed with 4 µl of 10 mM acetyl-CoA and 8.6 µl (0.215 µCi) of
[14C]chloramphenicol
(55 mCi/mmol; Amersham). The mixture was adjusted to a final volume of
40 µl with 0.25 M Tris (pH 7.9) and was subsequently incubated at
37°C for 3.5 h. Chloramphenicol was then extracted from the
reaction mixture with ethyl acetate, dried in a vacuum dessicator, and
resuspended in 30 µl of ethyl acetate. Samples were applied to TLC
plates, and the acetylated and nonacetylated forms of chloramphenicol
were separated for 30 min at room temperature with chloroform-methanol
[95:5% (vol/vol)] as the mobile phase. Signals were then
visualized and quantified by electronic autoradiography (Instantimager,
Packard). To assess
-galactosidase activity, 20 µl of the muscle
extracts were combined with 130 µl of water and an equal volume (150 µl) of assay buffer, consisting of 120 mM
Na2HPO4,
80 mM
NaH2PO4,
2 mM MgCl2, 100 mM
-mercaptoethanol and 1.18 mM
o-nitrophenyl-
-D-galactopyranoside.
The reaction mixture was then incubated at 37°C for 2.5 h, and
-galactosidase activity was determined photometrically at 420 nm.
Reactions containing extracts derived from muscles that had not been
transfected with the
-galactosidase construct possessed an
endogenous enzyme activity (7) ranging from 0.045 to 0.069 absorbance
U/h. There also appeared to be a modest effect of stimulation on
endogenous
-galactosidase activity, because this activity was
elevated by 25-30% from day 1 to
day 7 (results not shown). Thus, to use
-galactosidase expression to
correct CAT activity for transfection efficiency, we calculated the
actual transfected
-galactosidase activity by subtracting the
endogenous activity from the total activity measured during the time
course of the experiment.
In vitro mRNA decay. Protein extracts
from stimulated and nonstimulated control EDL muscles were prepared
according to previously published protocols (15, 36), with some
modifications. Briefly, skeletal muscle powders (50-100 mg) were
homogenized (Ultra Turrax, 7-mm probe) 3 × 10 s (70% maximum) in
1 ml of homogenization buffer comprised of 25% glycerol, 0.42 M NaCl,
1.5 mM MgCl2, 0.2 mM EDTA, 20 mM
HEPES, 0.5 mM 1,4-dithiothreitol, and 0.5 mM phenylmethylsulfonyl fluoride. These homogenates were then centrifuged for 15 min at 5,000 g (Beckman, Avanti J-25) at 4°C.
The resulting supernatant fractions were then subjected to further
centrifugation at 15,000 g (4°C)
for 15 min, and the resultant postmitochondrial supernatant (S15) was
transferred to a sterile Eppendorf tube. Protein concentrations of the
S15 fractions from stimulated and control muscles were determined
photometrically (2). Total RNA (30 or 60 µg) from stimulated or
control EDL muscles was incubated with 60 or 200 µg of S15 extract
obtained from the same muscles in a 300-µl reaction volume at
37°C. Aliquots were removed at various times as indicated in Fig.
1-5, and the RNA was extracted with a phenol-chloroform-isoamyl alcohol extraction procedure. The RNA was then precipitated at 70°C for 1 h and subsequently pelleted, dried, and
resuspended in 10 µl of sterile
H2O. The RNA was separated on a
1% agarose gel, transferred to a nylon membrane (Hybond-N, Amersham),
and fixed to the membrane with ultraviolet light. These membranes were
probed with
[32P]dCTP-labeled
cDNAs specific for cytochrome c mRNA,
and signals were quantified with electronic autoradiography
(Instantimager, Packard).
Statistical analyses. The effects of chronic stimulation on muscle mass, total protein, total RNA, cytochrome c mRNA, and cytochrome c protein levels were evaluated with paired t-tests. Changes in cytochrome c transcriptional activation and mRNA stability after increased contractile activity were determined with two-way ANOVA, followed by Tukey's post-hoc test. All values are presented as means ± SE, and differences were considered to be statistically significant at the 0.05 level of confidence.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Body mass, TA muscle weights, total protein, and total
RNA concentrations. Chronic contractile activity had no
effect on body mass or TA muscle mass over the 7-day time period (Table
1). Control TA muscle mass averaged 485.9 ± 12.8 mg (n = 43). Total protein
concentration in control muscle averaged 84.7 ± 4.4 mg/g wet wt
(n = 27) and was not influenced by
contractile activity. The total RNA concentration in nonstimulated
control TA muscle was 1,846.2 ± 77.2 µg/g
(n = 34) and was not significantly
affected by the simulation protocol (Table 1).
|
Cytochrome c mRNA and protein
levels. Chronic contractile activity
induced an increase in steady-state cytochrome
c mRNA content in the TA muscle, from
1.3-fold on day
1 to 1.7-fold between
days
5 and
7 (P < 0.05; Fig 1,
A and
C). The 1.5-fold increases apparent
at days
2 and
4 tended toward, but did not attain,
statistical significance (0.05 < P < 0.1). There was no effect of contractile activity on 18S rRNA
levels, as reported previously (27). The time course of the
stimulation-induced alterations in cytochrome
c mRNA was different from that
observed for cytochrome c protein
content (Fig. 1C). No changes in
protein level were evident until 5 days of stimulation (Fig. 1,
B and C). Cytochrome
c protein remained elevated in the
stimulated TA muscle at 7 days of stimulation, attaining levels that
were 1.9-fold above those observed in control muscle (Fig.
1C). These increases in expression
are typical of those observed with this chronic stimulation model (14,
27-29, 36).
|
Transfected -galactosidase activity in control and
stimulated TA muscles. Transfected
-galactosidase activity, used as a correction factor for
transfection efficiency, varied between 0.05 and 0.08 absorbance U/h
over the 7-day experimental period. There was no effect of time or
stimulation on transfected
-galactosidase activity.
Cytochrome c transcriptional activation. Initial studies were performed with control, unstimulated TA muscles to confirm that transcriptional activation of the cytochrome c promoter would occur after injection with the cytochrome c DNA constructs. Transcriptional activation of the pRC4CAT/-66 construct was only marginally above background levels of detectability (n = 8). In contrast, CAT activity driven by the pRC4CAT/-326 construct was much higher than background levels, and it was found to progressively increase over time after injection, even as much as 5 wk later (results not shown). These initial studies confirmed the effectiveness of our injection protocol, verified the stability of the transcriptional activation product (CAT) in our injected muscles, and led us to the use of the pRC4CAT/-326 construct in evaluating the effect of contractile activity on cytochrome c transcriptional activation.
A time-dependent increase in cytochrome
c promoter-driven CAT activity was
evident over the 7-day period in nonstimulated TA muscle. An
approximate fourfold accumulation of corrected CAT activity above that
found at day
1 was evident between days 4 and 7 of the experimental protocol (Fig.
2B;
n = 27). In the stimulated muscle, an
increase in CAT activity was only evident after 5 days of contractile
activity, reaching values that were 2.1-fold higher (P < 0.05) than those measured in
contralateral, nonstimulated TA muscles (Figs. 2,
A and
B; n = 11).
|
Cytochrome c mRNA stability. In
establishing the in vitro mRNA decay assay conditions, we first showed
that mRNA stability was unaffected in the presence of the individual
components of the decay reaction. We also documented that mRNA levels
progressively decreased as a function of incubation time (5, 10, 20, and 40 min) in the presence of cytosol and that decay was linear with the amount of cytosolic fraction added (20, 40, and 66 µg
protein/lane; Connor and Hood, unpublished observations). The assay
conditions chosen (10 and 20 min; 20 µg protein/lane) represent the
outcome of those preliminary studies. We assessed the effect of
contractile activity on cytochrome c
mRNA stability at each time point of the 7-day experimental protocol.
Because similar results were observed in 2-, 3-, and 4-day stimulated
and nonstimulated muscle, only the data obtained from muscle subjected
to 3 days of contractile activity are illustrated (Fig.
3A).
When total RNA was incubated with cytosol isolated from the same
muscle, cytochrome c mRNA derived from
the stimulated muscle (Fig. 3A,
lanes
1-3)
was degraded at a slower rate than cytochrome
c mRNA from control muscle [Fig. 3, A
(lanes
4-6)
and B]. This contractile
activity-mediated increase in cytochrome
c mRNA stability was no longer evident
after 5 days of stimulation, when rates of degradation in the
stimulated muscle were equivalent to the cytosol-induced degradation of
cytochrome c mRNA from control muscle
[Fig. 4,
A
(lanes
1-3
vs. lanes
4-6) and B]. These results at 5 days
were also similar to rates of cytochrome
c mRNA decay at both 1 and 7 days (not
shown).
|
|
In a separate subset of animals, we further evaluated the effect of a
cytosolic fraction in mediating cytochrome
c mRNA stability under conditions of
continuous contractile activity (24 h/day for 10 days), similar to that
used recently (36). This treatment resulted in a 1.9-fold increase in
cytochrome c mRNA in the stimulated muscle. In these decay reactions, cytosolic fractions obtained from
both stimulated and control muscles were incubated with RNA isolated
from control muscle. Cytochrome c mRNA
was more stable in the presence of cytosolic fraction derived from the
muscle subject to continuous chronic contractile activity (Fig.
5). The t1/2 value (26 min) was 88% higher in the presence of the cytosol from stimulated
muscle, compared with the value obtained in the presence of the control
cytosol (14 min). Taken together, these data are consistent with the
idea that a cytosolic component is induced in response to chronic
contractile activity and that it acts to reduce the degradation rate of
cytochrome c mRNA. In addition, this
effect appears to be at least partially mRNA specific, because no
effect of contractile activity (for 2-4 days, 3 h/day) on
-actin mRNA stability was observed (Connor and Hood, unpublished observations).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Contractile activity is a potent stimulus for the induction of mitochondrial biogenesis in skeletal muscle (for reviews, see Refs. 10 and 12) and thus serves as a good experimental model to study the underlying mechanisms involved in organelle synthesis and turnover. In the present study, contractile activity was induced with a chronic low-frequency electrical stimulation protocol (10 Hz, 3 h/day), which has been shown to lead to mitochondrial phenotypic alterations, as well as increases in muscle mitochondrial content (27, 29). To document some of the underlying mechanisms involved in these adaptations, we chose to measure cytochrome c protein and mRNA levels, as well as the attendant processes of gene transcription and mRNA stability. The results indicate that increases in cytochrome c expression brought about by chronic contractile activity are due to time-dependent adaptations, which appear to involve an increase in mRNA stability, followed by a subsequent increase in transcriptional activation.
The application of a cell-free mRNA decay assay (21) to measure mRNA degradation in the presence of a cytosolic fraction permitted the conclusion that the early increases in cytochrome c mRNA expression were due to a change in mRNA stability. The mRNA decay assay employed appears to be sensitive enough to measure subtle alterations in mRNA stability, even when studying mRNAs possessing relatively low rates of turnover, as found in skeletal muscle (6). The cellular events mediating the stabilization of cytochrome c mRNA as a result of contractile activity likely involve the 3' untranslated region (UTR), because it is established that this is an important site of RNA-protein interactions, which confers alterations in mRNA stability (22, 23). Yan et al. (36) recently demonstrated a reduced RNA-protein interaction in the 3'-UTR of cytochrome c mRNA in response to 9 days of stimulation (24 h/day), at a time when cytochrome c mRNA levels had increased over twofold. This suggested that contractile activity induced the activation of an inhibitor of RNA-protein interactions. This important observation is probably related to our findings, because we provide direct evidence for an increase in cytochrome c mRNA stability brought about by a factor endogenous to the cytosol of 10-day, continuously stimulated muscle. We also show that an increase in stability can be a relatively early occurrence as a result of contractile activity, because the effect appeared after only 2 days of treatment when the muscle was allowed a recovery phase. As with the induction of the transcription factors responsible for transcriptional activation (see below), we hypothesize that the recovery phase may have permitted the induction of an mRNA stabilizing factor; however, its characterization and its effect on other nuclear-encoded mRNAs remain to be determined. It is interesting to note that the inhibition of mitochondrial protein synthesis in Hep G2 hepatocytes leads to an increase in the stability of a variety of nuclear-encoded mRNAs (5). In this context, we have recently observed that 5 min of 10-Hz contractile activity markedly reduced mitochondrial protein synthesis in subsarcolemmal mitochondria (M. K. Connor, O. Bezborodova, and D. A. Hood, unpublished observations). This decrease in intramitochondrial translation rate may be transmitted to the nuclear genome via a putative signal (18) leading to the induction of stabilizing proteins (5).
The early mRNA stabilization was followed by an increase in
transcriptional activation, leading to an eventual accumulation of
cytochrome c mRNA, evident before an
increase in protein level (Fig. 1). Our assessment of transcriptional
activation was afforded by the availability of the pRC4CAT/-326 bp
cytochrome c promoter-reporter DNA
construct (11). The technique of direct plasmid DNA injection into
muscle, one which is useful for the investigation of transcriptional events in skeletal muscle during alterations in contractile activity (4, 26), was employed. This technique requires the coinjection of a
nonspecific reporter gene to correct for transfection efficiency. In
the present study, muscles were injected with plasmid DNA containing an
RSV promoter linked to a -galactosidase reporter gene. An endogenous
muscle
-galactosidase activity consistent with reports in the
literature (7) was measured, and we also found that total (endogenous + transfected)
-galactosidase enzyme activity was progressively
elevated in response to 7 days of muscle stimulation (results not
shown). There was no effect of contractile activity on transfected
-galactosidase activity, and this value was used to correct CAT
activity for transfection efficiency. It should be noted, however, that
a variety of stressors have been reported to activate the
cytomegalovirus promoter in other systems (3). Thus the use of RSV or
cytomegalovirus promoter-driven reporter constructs must
be used with caution in skeletal muscle subject to chronic contractile activity.
Recently, the transcriptional activation of cytochrome c in cardiac myocytes subject to electrical pacing in cell culture was reported (34). Both the nuclear respiratory factor (NRF)-1 and cAMP response element sites are important responsive elements within the cytochrome c promoter that mediate transcription under those conditions. The cAMP response element site appears to bind c-Jun, and both NRF-1 and c-Jun mRNA levels increase in response to cardiac pacing (35). In skeletal muscle, it is known that c-Jun mRNA is increased as a result of contractile activity (16). It is noteworthy that the pattern of induction of immediate early genes such as c-fos, c-jun, and egr-1 is particularly pronounced during the 0.5- to 8-h recovery phase after the cessation of contractile activity (1, 17, 19). This is well within the time frame of the 21-h recovery period that we employed between each 3-h bout of contractile activity. As a result, with repeated 3-h bouts of contractile activity followed by recovery, a progressive accumulation of transcription factors (e.g., c-Jun and NRF-1) could have occurred, leading to the transcriptional activation that we observed after 5 days of contractile activity. Thus the recovery period may have allowed for an earlier onset, but not necessarily a greater magnitude, of cytochrome c mRNA induction in comparison with continuous contractile activity (36) with the same absolute workload (i.e., 10 Hz).
In summary, we used the technique of direct plasmid DNA injection to measure transcriptional activation, combined with a sensitive mRNA decay assay to measure mRNA stability. The increase in cytochrome c mRNA in skeletal muscle undergoing contractile activity-induced mitochondrial biogenesis can be explained largely by the existence of time-dependent, rapid increases in mRNA stability, followed by increases in transcriptional activation. These changes precede increases in cytochrome c protein expression. The data suggest the utility of these two complementary experimental approaches for the study of muscle adaptations and identify mRNA stability as a rapidly altered physiological process that deserves greater attention in the area of skeletal muscle gene expression.
![]() |
ACKNOWLEDGEMENTS |
---|
Michael Connor and Damien Freyssenet contributed equally to this study.
![]() |
FOOTNOTES |
---|
We thank Dr. Richard Scarpulla (Northwestern University, Chicago, IL) for the kind donation of the cytochrome c promoter constructs.
This work was supported by the Natural Science and Engineering Research Council of Canada (to D. A. Hood).
Present address of D. Freyssenet: Laboratoire de Physiologie, Faculté de Médicine, 15 rue A. Paré, 42023 Saint-Etienne Cedex 2, France.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: D. A. Hood, Dept. of Biology, York Univ., 4700 Keele St., Toronto, ON, Canada M3J 1P3 (E-mail: dhood{at}yorku.ca).
Received 30 November 1998; accepted in final form 25 March 1999.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Abu-Sakra, S. R.,
A. J. Cole,
and
D. B. Drachman.
Nerve stimulation and denervation induce differential patterns of immediate early gene mRNA expression in skeletal muscle.
Mol. Brain Res.
18:
216-220,
1993[Medline].
2.
Bradford, M. M.
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding.
Anal. Biochem.
72:
255-260,
1976[Medline].
3.
Bruening, W.,
B. Giasson,
W. Mushynski,
and
H. D. Durham.
Activation of stress-activated MAP protein kinases up-regulates expression of transgenes driven by the cytomegalovirus immediate/early promoter.
Nucleic Acids Res.
26:
486-489,
1998
4.
Carson, J. A.,
Z. Yan,
F. W. Booth,
M. E. Coleman,
R. J. Schwartz,
and
C. S. Stump.
Regulation of skeletal -actin promoter in young chickens during hypertrophy caused by stretch overload.
Am. J. Physiol.
268 (Cell Physiol. 37):
C918-C924,
1995
5.
Chrzanowska-Lightowlers, Z. M. A.,
T. Preiss,
and
R. N. Lightowlers.
Inhibition of mitochondrial protein synthesis promotes increased stability of nuclear-encoded respiratory gene transcripts.
J. Biol. Chem.
269:
27322-27328,
1994
6.
Connor, M. K.,
M. Takahashi,
and
D. A. Hood.
Tissue-specific stability of nuclear- and mitochondrially-encoded mRNAs.
Arch. Biochem. Biophys.
333:
103-108,
1996[Medline].
7.
Dhawan, J.,
T. A. Rando,
S. L. Elson,
H. Bujard,
and
H. M. Blau.
Tetracycline-regulated gene expression following direct gene transfer into mouse skeletal muscle.
Somat. Cell Mol. Genet.
21:
233-240,
1995[Medline].
8.
Dudley, G. A.,
W. M. Abraham,
and
R. L. Terjung.
Influence of exercise intensity and duration on biochemical adaptations in skeletal muscle.
J. Appl. Physiol.
53:
844-850,
1982
9.
Eisenberg, B. R.,
and
S. Salmons.
The reorganization of subcellular structure in muscle undergoing fast-to-slow type transformation.
Cell Tissue Res.
220:
449-471,
1981[Medline].
10.
Essig, D. A.
Contractile activity-induced mitochondrial biogenesis in skeletal muscle.
Exerc. Sport Sci. Rev.
24:
289-319,
1996[Medline].
11.
Evans, M. J.,
and
R. C. Scarpulla.
Both upstream and intron sequence elements are required for elevated expression of the rat somatic cytochrome c gene in Cos-1 cells.
Mol. Cell. Biol.
8:
35-41,
1988[Medline].
12.
Freyssenet, D.,
P. Berthon,
and
C. Denis.
Mitochondrial biogenesis in skeletal muscle in response to endurance exercises.
Arch. Physiol. Biochem.
104:
129-141,
1996[Medline].
13.
Holloszy, J. O.
Biochemical adaptations in muscle. Effects of exercise on mitochondrial oxygen uptake and respiratory enzyme activity in skeletal muscle.
J. Biol. Chem.
242:
2278-2282,
1967
14.
Hood, D. A.,
R. Zak,
and
D. Pette.
Chronic stimulation of rat skeletal muscle induces coordinate increases in mitochondrial and nuclear mRNAs of cytochrome-c-oxidase subunits.
Eur. J. Biochem.
179:
275-280,
1989[Abstract].
15.
Locke, M. E.,
E. G. Noble,
R. M. Tanguay,
M. R. Field,
S. E. Ianuzzo,
and
C. D. Ianuzzo.
Activation of heat-shock transcription factor in rat heart after heat shock and exercise.
Am. J. Physiol.
268 (Cell Physiol. 37):
C1387-C1394,
1995
16.
Michel, J. B.,
G. A. Ordway,
J. A. Richardson,
and
R. S. Williams.
Biphasic induction of immediate early gene expression accompanies activity-dependent angiogenesis and myofiber remodelling of rabbit skeletal muscle.
J. Clin. Invest.
94:
277-285,
1994[Medline].
17.
Neufer, P. D,
G. A. Ordway,
and
R. S. Williams.
Transient regulation of c-fos, B-crystallin, and hsp70 in muscle during recovery from contractile activity.
Am. J. Physiol.
274 (Cell Physiol. 43):
C341-C346,
1998
18.
Poyton, R. O.,
and
J. E. McEwen.
Crosstalk between nuclear and mitochondrial genomes.
Annu. Rev. Biochem.
65:
563-607,
1996[Medline].
19.
Puntschart, A.,
E. Wey,
K. Jortarndt,
M. Vogt,
M. Wittwer,
H. R. Widmer,
H. Hoppeler,
and
R. Billeter.
Expression of fos and jun genes in human skeletal muscle after exercise.
Am. J. Physiol.
274 (Cell Physiol. 43):
C129-C137,
1998
20.
Reichmann, H.,
H. Hoppeler,
O. Mathieu-Costello,
F. Von Bergen,
and
D. Pette.
Biochemical and ultrastructural changes of skeletal muscle mitochondria after chronic electrical stimulation in rabbits.
Pflügers Arch.
404:
1-9,
1985[Medline].
21.
Ross, J.
Analysis of messenger RNA turnover in cell-free extracts from mammalian cells.
In: RNA Processing Volume II: A Practical Approach. New York, NY: IRL, 1994, p. 107-133.
22.
Ross, J.
Control of messenger RNA stability in higher eukaryotes.
Trends Genet.
12:
171-175,
1996[Medline].
23.
Russel, J. E.,
J. Morales,
and
S. A. Liebhaber.
The role of mRNA stability in the control of globin gene expression.
Prog. Nucleic Acid Res. Mol. Biol.
57:
249-287,
1997[Medline].
24.
Scarpulla, R. C.,
K. M. Agne,
and
R. Wu.
Isolation and structure of a rat cytochrome c gene.
J. Biol. Chem.
256:
6480-6486,
1981
25.
Stevens, R. J.,
M. L. Nishio,
and
D. A. Hood.
Effect of hypothyroidism on the expression of cytochrome c and cytochrome c oxidase in heart and muscle during development.
Mol. Cell. Biochem.
143:
119-127,
1995[Medline].
26.
Swoap, S. J.
In vivo analysis of the myosin heavy chain IIB promoter region.
Am. J. Physiol.
274 (Cell Physiol. 43):
C681-C687,
1998
27.
Takahashi, M.,
A. Chesley,
D. Freyssenet,
and
D. A. Hood.
Contractile activity-induced alterations in the mitochondrial protein import system.
Am. J. Physiol.
274 (Cell Physiol. 43):
C1380-C1387,
1998
28.
Takahashi, M.,
and
D. A. Hood.
Chronic stimulation-induced changes in mitochondria and performance in rat skeletal muscle.
J. Appl. Physiol.
74:
934-941,
1993[Abstract].
29.
Takahashi, M.,
D. T. M. McCurdy,
D. A. Essig,
and
D. A. Hood.
-Aminolaevulinate synthase expression in muscle after contractions and recovery.
Biochem. J.
291:
219-223,
1993[Medline].
30.
Thomason, D. B.,
and
F. W. Booth.
Stable incorporation of a bacterial gene into adult rat skeletal muscle in vivo.
Am. J. Physiol.
258 (Cell Physiol. 27):
C578-C581,
1990
31.
Waterborg, J. H.,
and
H. R. Matthews.
The Burton assay for DNA.
In: Methods in Molecular Biology Volume II: Nucleic Acids. Clifton, NJ: Humana, 1984, p. 1-3.
32.
Williams, R. S.,
S. Salmons,
E. A. Newsholme,
R. E. Kaufman,
and
J. Mellor.
Regulation of nuclear and mitochondrial gene expression by contractile activity in skeletal muscle.
J. Biol. Chem.
261:
376-380,
1986
33.
Wolff, J. A.,
R. W. Malone,
P. Williams,
W. Chong,
G. Acsadi,
A. Jani,
and
P. L. Felgner.
Direct gene transfer into mouse muscle in vivo.
Science
247:
1465-1468,
1990[Medline].
34.
Xia, Y.,
L. M. Buja,
and
J. B. McMillan.
Activation of the cytochrome c gene by electrical stimulation in neonatal rat cardiac myocytes.
J. Biol. Chem.
273:
12593-12598,
1998
35.
Xia, Y.,
L. M. Buja,
R. C. Scarpulla,
and
J. B. McMillan.
Electrical stimulation of neonatal cardiomyocytes results in the sequential activation of nuclear genes governing mitochondrial proliferation and differentiation.
Proc. Natl. Acad. Sci. USA
94:
11399-11404,
1997
36.
Yan, Z.,
S. Salmons,
Y. L. Dang,
M. T. Hamilton,
and
F. W. Booth.
Increased contractile activity decreases RNA-protein interaction in the 3'-UTR of cytochrome c mRNA.
Am. J. Physiol.
271 (Cell Physiol. 40):
C1157-C1166,
1996