Contractile Activity-induced Transcriptional Activation of Cytochrome c Involves Sp1 and Is Proportional to Mitochondrial ATP Synthesis in C2C12 Muscle Cells*

Michael K. Connor, Isabella Irrcher, and David A. HoodDagger

From the Departments of Biology and Kinesiology and Health Science, York University, Toronto, Ontario M3J 1P3, Canada

Received for publication, January 11, 2001


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Contractile activity induces adaptations in the expression of genes encoding skeletal muscle mitochondrial proteins; however, the putative signals responsible for these adaptations remain unknown. We used electrical stimulation (5 Hz, 65 V) of C2C12 muscle cells in culture to define some of the mechanisms involved in contractile activity-induced changes in cytochrome c gene expression. Chronic contractile activity (4 days, 3 h/day) augmented cytochrome c mRNA by 1.6-fold above control cells. This was likely mediated by increases in transcriptional activation, because cells transfected with full-length (-726 base pairs) or minimal (-66 base pairs) cytochrome c promoter/chloramphenicol acetyltransferase reporter constructs demonstrated contractile activity-induced 1.5-1.7-fold increases in the absence of contractile activity-induced increases in mRNA stability. Transcriptional activation of the -726 promoter was abolished when muscle contraction was inhibited at various subcellular locations by pretreatment with either the Na+ channel blocker tetrodotoxin, the intracellular Ca2+ chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester, or the myosin ATPase inhibitor 2,3-butanedione monoxime. It was further reduced in unstimulated cells when mitochondrial ATP synthesis was impaired using the uncoupler 2,4-dinitrophenol. Because the contractile activity-induced response was evident within the minimal promoter, electromobility shift assays performed within the first intron (+75 to +104 base pairs) containing Sp1 sites revealed an elevated DNA binding in response to contractile activity. This was paralleled by increases in Sp1 protein levels. Sp1 overexpression studies also led to increases in cytochrome c transactivation and mRNA levels. These data suggest that variations in the rate of mitochondrial ATP synthesis are important in determining cytochrome c gene expression in muscle cells and that this is mediated, in part, by Sp1-induced increases in cytochrome c transcription.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Elevations in skeletal muscle contractile activity are known to induce large and rapid augmentations in the mRNA expression of genes encoding mitochondrial proteins (1-4). These increases result from a disruption in the equilibrium that exists between gene transcription and mRNA stability during nonadaptive steady-state conditions. Elevations in the level of the mRNA encoding the nuclear gene product cytochrome c in response to in vivo contractile activity have been reported (5), and our recent work has established that this adaptation is mediated via sequential, time-dependent elevations in both of these processes (6). This necessitates alterations in the expression of proteins involved in regulating transcriptional activation and/or message stabilization within the cytosol. However, the specific cellular events that occur during muscle contraction to initiate these adaptations remain largely undefined. These events could include membrane depolarization, Ca2+ mobilization, cross-bridge cycling, and alterations in energy metabolism. Each of these are known to initiate intracellular signaling cascades (7-11), which could ultimately alter rates of transcription and/or mRNA stability. It is now established that acute elevations in contractile activity can stimulate a number of kinases involved in signal transduction, including mitogen-activated protein kinase, c-Jun N-terminal kinase, and p38 kinase activities in skeletal muscle (4, 12, 13). However, the temporal relationship between the onset of the putative signaling event (i.e. kinase activity), transcription factor activation, and the up-regulation of nuclear genes encoding mitochondrial proteins in response to contractile activity remains unknown.

We have previously used cytochrome c as a model, nuclear-encoded mitochondrial protein to define some of the adaptations that occur in response to contractile activity and artificially elevated muscle Ca2+ levels (6, 8). The cytochrome c promoter contains multiple GC-rich regions that can serve as binding sites for the transcription factor Sp1 (14-16). However, the zinc finger transcription factor Egr-1 (17) may also interact with these elements, because the consensus binding sequences are similar, and it may be that Sp1 and Egr-1 can compete for similar, nonconsensus sites (14). Furthermore, Sp1-mediated gene transcription is inhibited by Egr-1 displacement of Sp1 from the promoter (18, 19). In addition, Egr-1 is rapidly induced in response to elevations in contractile activity (20, 21) as well as elevated intracellular Ca2+ levels (22). These findings suggest potential roles for both Sp1 and Egr-1 in the regulation of cytochrome c expression during conditions of increased contractile activity. Thus, in the present study we adopted a contracting C2C12 murine skeletal muscle cell model to define some of the events occurring during muscle contraction that initiate the increase in cytochrome c expression and to identify some of the transcription factors that are immediately responsible for this increase.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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Materials-- BDM,1 TTX, DNP, and ACT were purchased from Sigma. [14C]Chloramphenicol, [alpha -32P]dCTP, [gamma -32P]ATP, nitrocellulose, and nylon membranes (Hybond N) were obtained from Amersham Pharmacia Biotech. BAPTA-AM was purchased from Calbiochem (San Diego, CA). Fetal bovine serum and horse serum were obtained from Summit Biotechnologies (Fort Collins, CO) and Life Technologies, Inc., respectively. Polyclonal antibodies directed toward Egr-1, Sp1, and Cyclin D1 were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). The dual luciferase reporter assay system was from Promega (Madison, WI). All other reagents were purchased from Sigma and were of the highest grade available. The oligodeoxynucleotides used for electromobility shift assays were 1) Sp1, 5'-ATTCGATCGGGGCGGGGCGAGC-3' (Dalton Chemicals, Toronto); 2) Egr-1, 5'-GGATCCAGCGGGGGCGAGCGGGGGCGA-3' (Santa Cruz Biotechnology, Santa Cruz); and 3) a sequence composed of +75 to +104 of the first intron of the cytochrome c gene, 5'-GGGGACGCGGGGCGGGAAGAGGGCGAGGAG-3' (Dalton Chemicals, Toronto, Canada).

Cell Culture-- C2C12 murine skeletal muscle cells were maintained at 37 °C in 5% CO2 on 100-mm gelatin-coated plastic dishes containing Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. Upon reaching 90% confluence, myoblast differentiation was induced by switching to a lower serum medium (Dulbecco's modified Eagle's medium supplemented with 5% heat-inactivated horse serum and 1% penicillin/streptomycin). Treatments were routinely carried out when the myotubes occupied 90-100% of the dish (~8-10 days).

Electrical Stimulation-- The design of the electrical stimulation apparatus was derived by modifying existing methods (23-25). Lids from plastic 100-mm culture dishes (Sarstedt, Montreal, Canada) were fitted with two platinum wire electrodes such that 5-cm lengths ran parallel to each other at opposite ends of the dish ~5 cm apart. Electrodes were attached to the plates using miniature banana plugs (Electrosonic, Toronto, Canada). Multiple plates were arranged in parallel and connected to a stimulator unit, which reversed the polarity of the output every second. Cells were stimulated at a frequency of 5 Hz and an intensity of 65 V (1.2 V/cm2). Stimulation was either performed acutely for 5, 15, 30, 60, or 240 min or chronically for 4 days (3 h/day). Cell extracts obtained from the chronic experiments were prepared 21 h after the last stimulation period.

Inhibitors-- To determine the event(s) of muscle contraction responsible for activity-induced alterations in gene expression, sarcomere shortening was inhibited at three different levels 24 h prior to the onset of stimulation: 1) membrane depolarization was inhibited by preventing the opening of voltage-gated Na+ channels by treatment with 10 µM TTX, 2) contraction was disrupted by pretreatment with the membrane-permeable Ca2+ chelator BAPTA-AM (25 µM); this was done to prevent the increase in cytosolic [Ca2+], which occurs subsequent to membrane depolarization; and 3) contraction was inhibited by preventing cross-bridge cycling while allowing membrane depolarization and Ca2+ fluxes to occur by treatment with 1.5 mM BDM. This concentration of BDM has been shown to have no effect on Ca2+ transients in phenylepherine treated cardiac myocytes (26) and appears to act by inhibiting myosin ATPase activity (27). Cells were electrically stimulated for 4 days during each treatment (3 h/day), and results from drug-treated cells were compared with quiescent cells treated with the corresponding vehicle (VEH). Finally, mitochondrial ATP synthesis was inhibited by treatment of unstimulated cells with 200 µM DNP to uncouple mitochondrial respiration. In this case, cells were treated for 9 h on two consecutive days. Cells were harvested immediately after treatment on the second day.

Steady-state mRNA Measurements-- Total RNA was isolated from stimulated and unstimulated control cells as done previously (6) and resuspended in diethyl pyrocarbonate-treated H2O. Determination of the quality and subsequent size separation of total RNA (15 µg for cytochrome c or 30 µg for Egr-1) was achieved by electrophoresis using denaturing formaldehyde-1% agarose gels, which were transferred and subsequently fixed to nylon membranes. These membranes were then probed with radiolabeled cDNA probes encoding cytochrome c, Egr-1, and 18 S rRNA as done previously (28). Stringent washes were performed at 55 °C for 15 min in 0.1× SSC, 0.1% SDS followed by 15 min at 60 °C. Signals were quantified by electronic autoradiography (Instantimager, Packard), and cytochrome c and Egr-1 mRNAs were normalized to 18 S rRNA levels to correct for uneven loading.

Plasmids-- Plasmid constructs containing various lengths of the cytochrome c promoters linked to a reporter gene (pRC4CAT and pRC4LUC) were provided by Dr. R. Scarpulla (Northwestern University, Chicago, IL) and Dr. F. Booth (University of Missouri, Columbia, MO), respectively, and have been previously characterized (16, 29). The pRC4CAT/-726 construct is composed of sequences from the cytochrome c promoter from -726 bp upstream of the transcription start site to position +115 within the first intron, fused to a chloramphenicol acetyltransferase (CAT) reporter gene. Experiments utilizing the minimal cytochrome c promoter (pRC4CAT/-66 or pRC4LUC/-66) were also conducted, because this region has been shown to be sufficient to confer cytochrome c transcriptional activation in response to elevated levels of intracellular Ca2+ (8). Overexpression of wild type Sp1 driven by the cytomegalovirus promoter was achieved with a vector provided by Dr. G. Suske (University of Marburg, Germany). beta -Galactosidase activity, under the control of the Rous sarcoma virus promoter or Renilla luciferase activity driven by the cytomegalovirus promoter was used to correct for transfection efficiency.

DNA Transfection and Expression Assays-- C2C12 myoblasts were transfected with the appropriate cytochrome c promoter/reporter construct (5 µg/100-mm dish) along with pRSV/beta -gal (5 µg/dish) or pCMV/RL (5 ng/dish) when they reached 70% confluence. Where applicable, wild type Sp1 or empty vector were also cotransfected (5 µg/dish) in combination with cytochrome c promoter/reporter constructs. The total amount of DNA added was maintained constant in all transfected cells. Transfections were done using a poly-L-ornithine method followed by a Me2SO shock (30). Cells were then differentiated by switching to a low serum medium. CAT and beta -galactosidase activities in stimulated and quiescent cells were measured as described previously (8). Luciferase activities were measured in a EG & G Berthold (Lumat LB 9507) luminometer using the dual luciferase reporter assay system according to the manufacturer's instructions.

Measurement of mRNA Stability-- Differentiated myotubes were either electrically stimulated for 4 days (3 h/day) or left untreated for a similar time period. Immediately after the final bout of stimulation, cells (stimulated and control) were treated with either 10 µg/ml ACT to inhibit mRNA synthesis or an equivalent volume of methanol, which served as the VEH. Myocytes were incubated with ACT or VEH for 4, 24, or 48 h. At the appropriate time points, cells were harvested, and total RNA was isolated. Equal amounts of total RNA (15 µg) from ACT- and VEH-treated cells were subjected to Northern blotting as described above. Blots were probed for cytochrome c mRNA and uneven loading was corrected using 18 S rRNA. Cytochrome c mRNA degradation was assessed by expressing the mRNA levels found at all time points as a percentage of the t = 0 value.

Electromobility Shift Assays-- Nuclear proteins were isolated from stimulated and control cells by scraping them from culture dishes in ice-cold phosphate-buffered saline followed by centrifugation for 10 s in a microcentrifuge (4 °C). The supernatant was discarded, and the pellet was resuspended in 400 µl of swelling buffer (10 mM Hepes-KOH, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride). Following a 10-min incubation on ice, cells were vortexed and pelleted in a microcentrifuge (4 °C). The supernatant was discarded, and the pellet was resuspended in 100 µl of resuspension buffer (20 mM Hepes-KOH, pH 7.9, 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride). Following a 20-min incubation on ice, cells were pelleted for 2 min in a microcentrifuge (4 °C). The supernatant was removed and used in electromobility shift assays following determination of protein content using a Bradford assay (31). These nuclear extracts (25 µg) were incubated with 20 µg/ml poly(dI-dC), 50 µM pyrophosphate, and 40,000 cpm of a [gamma -32P]ATP end-labeled oligonucleotide (containing the sequence between +75 and +104 bp of the cytochrome c gene) in a binding buffer (20 mM Tris, pH 7.6, 0.1 M MgCl2, 50 mM dithiothreitol, 1 mM spermidine, 1 mM EDTA) at room temperature for 20 min. To determine the specificity of binding, competition assays were conducted by preincubating extracts (20 min) with a 100 molar excess of cold oligonucleotide before the addition of labeled oligonucleotide. The oligonucleotides used in these competition assays were composed of 1) +75 to +104 bp of the first intron of the cytochrome c gene, 2) the consensus Sp1 binding site, and 3) the consensus Egr-1 binding site. Samples were run on a nondenaturing 4% acrylamide gel and were electrophoresed for 3 h (200 V). The gel was subsequently fixed for 15 min in acetic acid/methanol/H2O (10:30:60), dried, and imaged utilizing an Instantimager (Packard).

Immunoblotting-- C2C12 muscle cells were rinsed in ice-cold phosphate-buffered saline and subsequently scraped from culture dishes in 200 µl of Laemmli buffer (62.5 mM Tris, 20% glycerol, 2% SDS, and 5% 2-mercaptoethanol). Equal amounts of nuclear extracts (50 µg) were size-separated by electrophoresis on a 12% SDS-polyacrylamide gel. Proteins were transferred to nitrocellulose membranes and probed with polyclonal antibodies for Egr-1, Sp1, or cyclin D1 (1:500). Blots were then probed with the appropriate horseradish peroxidase-conjugated secondary antibody (1:1000) and visualized with an enhanced chemiluminescence kit (Amersham Pharmacia Biotech).

Cytochrome c Oxidase Enzyme Activity-- Cytochrome c oxidase activity was measured as described previously (2).

Statistical Analyses-- The data presented are the means ± S.E. Student's t test was used to evaluate the effects of contractile activity, Sp1 overexpression, or DNP treatment on mRNA levels and transcriptional activation. One-way analysis of variance was used to evaluate the effects of TTX, BAPTA-AM, and BDM on cytochrome c transcriptional activation in stimulated and unstimulated control cells. In all cases individual differences were determined using Tukey's post-hoc test and differences were considered significant if p < 0.05.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Effectiveness of Stimulation of C2C12 Muscle Cells-- Using our cell culture model, myotubes were observed to contract synchronously at the correct frequency (5 Hz) using voltages as low as 35 V. Cells appeared to reach maximal shortening at 55 V with no further increase in contraction intensity apparent at voltages as high as 110 V. Thus, cells in subsequent experiments were stimulated at an intensity of 65 V (1.2 V/cm2). This voltage is much lower than that previously used to stimulate cardiac myocytes in culture (32, 33) and resulted in minimal myotube detachment following up to 8 h of continuous stimulation.2 To more objectively assess the effectiveness of the treatment, we evaluated the acute effects of stimulation on Egr-1 mRNA levels, because Egr-1 mRNA is known to increase rapidly with contractile activity (20, 21). Increases (p < 0.05) in mRNA were evident as early as 15 min after the onset of stimulation (Fig. 1). This increase was transient in nature, reaching a maximum of 4.0-fold above that in unstimulated control cells following 30 min of contractile activity and declining to 2.0-fold above control after 4 h of stimulation. This effect is similar to that observed previously in vivo (20), and it demonstrates the effectiveness of the myocyte electrical stimulation model in rapidly altering muscle gene expression.


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Fig. 1.   Effect of contractile activity on Egr-1 mRNA levels in C2C12 myocytes. Cells were electrically stimulated (5 Hz, 65 V) for 5, 15, 30, and 60 min and harvested immediately following the cessation of stimulation. Total RNA was extracted, and Northern blot analyses were carried out as described under "Experimental Procedures." A typical Northern blot showing the levels of Egr-1 mRNA in electrically stimulated cells (S) compared with unstimulated control cells (C) is shown (inset), and the results of repeated experiments are depicted graphically. *, p < 0.05 versus control. Egr-1 mRNA levels were corrected for uneven loading with 18 S rRNA and are expressed as percentages of the levels observed in control cells. Values are the means ± S.E.

Electrical Stimulation Increases Cytochrome c mRNA Levels as a Result of Transcriptional Mechanisms-- Cytochrome c was chosen as a representative model of the effect of contractile activity on the expression of nuclear genes encoding mitochondrial proteins. Four days of electrical stimulation (3 h/day) resulted in an elevation in cytochrome c mRNA to levels that were 1.6-fold above those in unstimulated cells (Fig. 2A, p < 0.05). This adaptation occurred prior to changes in mitochondrial enzymes reflected by cytochrome c oxidase activity, which was 157.3 ± 20.1 and 150.0 ± 2.7 nmol/min/mg (n = 3) in control and stimulated cells, respectively. To assess whether electrical stimulation affects cytochrome c transcription, myocytes were transfected with pRC4CAT/-726 plasmid constructs, which contained the full-length cytochrome c promoter linked to a CAT reporter gene. CAT assays revealed a 1.5-fold higher (p < 0.05) cytochrome c transcriptional activation in stimulated cells compared with unstimulated control cells (Fig. 2B), which paralleled the stimulation-induced increase in cytochrome c mRNA. Because it has been shown previously that the factors involved in Ca2+-mediated increases in transcription act within the minimal -66 bp cytochrome c promoter (8), we transfected cells with plasmid constructs containing this region (pRC4CAT/-66). Electrical stimulation of these cells elicited similar increases (1.7-fold higher; p < 0.05) in CAT activity as with the -726 construct, compared with unstimulated control cells (Fig. 2B). This observation indicates the presence of a contractile activity response element within this minimal -66-bp promoter region.


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Fig. 2.   Effects of contractile activity on cytochrome c mRNA, transcriptional activation, and mRNA stability in skeletal muscle cells. A, C2C12 cells were electrically stimulated (5 Hz, 65 V) for 4 days (3 h/day) or remained quiescent for a similar time period. Total RNA extraction and Northern blot analyses were carried out as described under "Experimental Procedures." Autoradiograms demonstrating the levels of cytochrome c mRNA (inset) in control and stimulated cells were quantified by electronic autoradiography. *, p < 0.05 versus control. B, C2C12 myoblasts were transfected with 5 µg of either pRC4CAT/-726 or pRC4CAT/-66 along with 5 µg of pRSV/beta -gal. Cells were then electrically stimulated (5 Hz, 65 V) for 4 days (3 h/day) or remained quiescent for a similar time period. CAT and beta -galactosidase activities were measured as described under "Experimental Procedures." Autoradiograms of CAT assays (inset) from control cells (C) and electrically stimulated cells (S) were quantified by electronic autoradiography. *, p < 0.05 versus control. Values are the means ± S.E. of at least seven independent experiments. C, degradation of cytochrome c mRNA in stimulated and control cells was measured following treatment with 10 µg/ml ACT. RNA was isolated at 4, 24, and 48 h after the addition of ACT, and cytochrome c mRNA levels were measured and expressed as percentages of the t = 0 value. Values are the means ± S.E. of three or four experiments.

To evaluate the possible contribution of contractile activity-induced changes in mRNA stability to the increase in cytochrome c mRNA observed, we measured cytochrome c mRNA decay in stimulated and unstimulated cells. Similar degradation rates of cytochrome c mRNA were observed in stimulated and control cells, which declined by 23.8 ± 4.5 and 30.1 ± 9.7%, respectively, after 48 h of transcriptional inhibition with ACT (Fig. 2C). Cytochrome c mRNAs were very stable in these cells, exhibiting an extrapolated half-life of ~89 h. These data suggest that the contractile activity-induced increase in cytochrome c mRNA was due to increases in gene transcription.

Sp1 Binding within the First Intron of the Cytochrome c Promoter Is Increased by Contractile Activity-- Following 4 days of electrical stimulation (3 h/day), there was a contractile activity-induced 2.4 ± 0.5-fold increase in Sp1 protein levels, whereas no effect of electrical stimulation on Egr-1 protein levels was evident (Fig. 3A). Coincident with this increase in Sp1 protein levels was a 1.8 ± 0.2-fold increase in DNA binding within the first intron (+75 to +104 bp) of the cytochrome c promoter (Fig. 3B, lane 2 versus lane 3). This binding was prevented by incubation with 100-molar excess of a cold oligodeoxynucleotide containing a portion of the first intron of the cytochrome c gene (lane 4). In addition, DNA binding was eliminated by preincubation with a nonradiolabeled consensus Sp1 deoxyribonucleotide (lane 5), suggesting that Sp1 may be responsible for the activity-induced activation of cytochrome c transcription. In contrast, there was no significant effect of preincubation with a cold oligonucleotide containing the consensus Egr-1 binding sequence (lane 6). To further evaluate the effects of contractile activity on Sp1 activation, extracts from stimulated and control cells were incubated with a radiolabeled consensus Sp1 sequence (Fig. 3C, lanes 1-4). These analyses revealed a stimulation-induced elevation in Sp1 DNA binding (lane 2 versus lane 3), which was completely eliminated by the addition of an excess of cold Sp1 consensus oligonucleotide (lane 4). In contrast, there was no effect of contractile activity on binding to an Egr-1 consensus oligonucleotide (lane 6 versus lane 7). As expected, Egr-1 binding was not evident in the presence of an excess of cold Egr-1 consensus oligonucleotides (lane 8). Taken together, these results suggest that electrical stimulation increases Sp1 binding within the first intron of the cytochrome c gene and that Egr-1 does not bind within this region.


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Fig. 3.   The effects of contractile activity on DNA binding within the first intron of the cytochrome c gene. A, C2C12 muscle cells were electrically stimulated (3 h/day for 4 days), and nuclear proteins were isolated as described under "Experimental Procedures." Nuclear extracts (50 µg) from stimulated (S) and control (C) cells were separated by SDS-polyacrylamide gel electrophoresis and probed with polyclonal antibodies for Sp1, Egr-1, or cyclin D1. B, nuclear proteins (25 µg) from control (lane 2) and stimulated cells (lanes 3-6) were incubated with a 32P-labeled oligonucleotide corresponding to +75 to +104 bp of the first intron of the cytochrome c gene. Nuclear extracts were also preincubated with nonradiolabeled oligonucleotides corresponding to this region (-66, lane 4), the Sp1 consensus binding site (Sp, lane 5) or the Egr-1 consensus binding sequence (Egr, lane 6). C, cell extracts (25 µg) from control (lanes 2 and 6) and S (lanes 3, 4, 7, and 8) cells were incubated as in A with radiolabeled Sp1 (lanes 1-4) or Egr-1 (lanes 5-8) consensus oligonucleotides. Competition reactions were conducted with 100 molar excess nonradiolabeled Sp1 (lane 4) or Egr-1 (lane 8) oligonucleotides. FP, free probe.

Overexpression of Sp1 Increases Cytochrome c Transactivation and Cytochrome c mRNA-- To further evaluate the role of Sp1 in cytochrome c transcriptional activation, cells were cotransfected with either empty vector or a vector directing the overexpression of Sp1, along with the pRC4LUC/-66 construct. Luciferase activity controlled by this minimal promoter was ~3-fold higher in Sp1 overexpressing cells (Fig. 4A; p < 0.05). This was accompanied by a modest but significant 25% increase in cytochrome c mRNA levels (Fig. 4B; p < 0.05).


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Fig. 4.   Sp1 overexpression transactivates the cytochrome c promoter and increases cytochrome c mRNA levels. A, C2C12 myoblasts were transfected with 5 µg of CMV-SP1 (Sp1) or an empty vector (EV), 5 µg of pRC4LUC/-66, and 5 ng of pCMV/RL. Firefly and Renilla luciferase activities were measured as described under "Experimental Procedures." Luciferase activities observed in empty vector cells were set at 1.0, and activities measured in Sp1-transfected cells were normalized to this value. Values are the means ± S.E. of 10 separate experiments. *, p < 0.05 versus empty vector. B, C2C12 myoblasts were transfected with 5 µg of CMV-SP1 (Sp1) or an empty vector (EV). Total RNA extraction and Northern blot analyses were performed as described under "Experimental Procedures." Cytochrome c mRNA levels were determined by Northern blot analyses and quantitated by electronic autoradiography. Equal loading was verified by inspection of the ethidium bromide (EtBr) stained gel (bottom panel).

Transcriptional Activation of Cytochrome c Is Proportional to Mitochondrial ATP Synthesis-- To evaluate the event(s) occurring during contractile activity that provide the putative signal responsible for activity-induced cytochrome c transactivation, muscle contraction was inhibited at various subcellular levels. First, cells were transfected with the -726-bp cytochrome c promoter and were treated 24 h prior to the onset of stimulation with 1) TTX (10 µM) to prevent membrane depolarization, 2) BAPTA-AM (25 µM) to prevent the increase in [Ca2+]i associated with Ca2+ release from the sarcoplasmic reticulum, or 3) BDM (1.5 mM) to prevent cross-bridge cycling. Pretreatment of control cells with TTX abolished muscle contraction entirely but had no effect on basal levels of cytochrome c transcriptional activation compared with VEH-treated cells (Fig. 5A). Contractile activity induced a 1.8-fold activation (p < 0.05) of the -726-bp cytochrome c promoter in the presence of VEH. This situation represents the condition in which the mitochondrial ATP synthesis rate is the highest, because myosin ATPase is activated by Ca2+, and the resulting production of free ADP is high and capable of activating mitochondrial ATP regeneration through coupled oxidative phosphorylation. This effect was completely prevented by TTX treatment (Fig. 5A), as expected. BAPTA-AM (25 µM) was used to permit membrane depolarization while preventing stimulation-induced muscle shortening via the chelation of intracellular Ca2+. BAPTA-AM treatment resulted in a 37% decrease (p < 0.05) in cytochrome c transcriptional activation in unstimulated cells compared with VEH-treated cells (Fig. 5B), suggesting the involvement of Ca2+ in cytochrome c transcription, as described recently (8). Myotubes subjected to electrical stimulation and VEH treatment demonstrated a 1.5-fold increase (p < 0.05) in cytochrome c transactivation above that in unstimulated control cells. Stimulated cells pretreated with BAPTA-AM were completely unable to contract and showed no elevation in cytochrome c transcriptional activation above that found in C + VEH cells (Fig. 5B). In this situation, mitochondrial ATP synthesis is low and similar to unstimulated cells, because there is no Ca2+ available to permit cross-bridge cycling and ATP utilization. In addition, because BAPTA-AM prevented the increase in Ca2+ associated with muscle contraction, and this occurred coincident with a normalization of cytochrome c transactivation, these data support a potential role for Ca2+ in mediating the transcriptional response. We then inhibited contractile activity at the level of cross-bridge cycling with BDM to allow membrane depolarization and an increase in [Ca2+]i to occur in response to electrical stimulation, while preventing sarcomere shortening and maintaining ATP turnover at a rate similar to unstimulated cells. BDM had no effect on cytochrome c transcriptional activation in unstimulated cells (Fig. 5C). Electrical stimulation of VEH-treated cell resulted in a 1.4-fold elevation (p < 0.05) in cytochrome c transcriptional activity, similar to the results illustrated in Fig. 5 (A and B). However, coincident with the low ATP turnover and mitochondrial ATP synthesis rate, cytochrome c transcriptional activation was completely prevented in the presence of BDM, despite the high levels of Ca2+ available, expected in view of continued membrane depolarization and Ca2+ release.


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Fig. 5.   The roles of membrane depolarization, intracellular Ca2+ levels, and cross-bridge cycling in activity-induced cytochrome c transcriptional activation. A, C2C12 myoblasts were transfected with 5 µg of pRC4CAT/-726 and 5 µg of pRSV/beta -gal. Cells were treated with 10 µM TTX to prevent opening of voltage-gated Na+ channels or treated with vehicle (V) 24 h prior to the onset of stimulation. Cells either remained quiescent (C) or were electrically stimulated (S) for 4 days (5 Hz, 65 V, 3 h/day). CAT and beta -galactosidase activities were measured as described under "Experimental Procedures." CAT activities observed in control and vehicle (C + V) cells were set at 100%, and all other activities were expressed as percentages of this value. Values are the means ± S.E. of six experiments. B, myoblasts were transfected with 5 µg of pRC4CAT/-726. To prevent the contraction-induced elevation in [Ca2+]i, cells were treated with 25 µM BAPTA-AM or VEH 24 h prior to the onset of stimulation. CAT activities were measured as in A and expressed as percentages of the C + V value. Values are the means ± S.E. of five separate experiments. C, myoblasts were transfected with 5 µg of pRC4CAT/-726 and subsequently treated with 1.5 mM BDM, to prevent the cross-bridge cycling, or VEH 24 h prior to the onset of stimulation. CAT activities were measured as in A and expressed as percentages of the C + V value. Values are the means ± S.E. of five independent experiments. *, p < 0.05 versus C + V.

To reduce the mitochondrial ATP synthesis rate below that found in resting, noncontracting cells, we treated cells with DNP to inhibit oxidative phosphorylation. Mitochondrial uncouplers such as DNP have been used previously as energy stressors (34) that do not affect ATP utilization (i.e. myosin ATPase activity) but that impair ATP synthesis, reduce the mitochondrial transmembrane potential (Delta Psi ), and lead to a reduction in ATP levels by about 50% (11). Coincident with reduced mitochondrial ATP synthesis was a diminished cytochrome c transactivation to ~45-50% of Me2SO-treated cells (Fig. 6). The addition of BDM did not further increase the effect of the uncoupler on cytochrome c transactivation.


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Fig. 6.   Treatment of cells with DNP reduces cytochrome c transcriptional activation in C2C12 myotubes. Myoblasts were transfected with 5 µg of pRC4CAT/-726 and 5 ng of pCMV/RL. Cells were treated with 200 µM DNP, 1.5 mM BDM, alone or in combination, or dimethyl sulfoxide vehicle (VEH) for two successive 9-h periods prior to harvesting myotubes for CAT reporter assays. CAT and Renilla luciferase activities were measured as described under "Experimental Procedures." CAT activities observed in vehicle (DMSO) cells were set at 100%, and all other activities were expressed as percentages of this value. Values are the means ± S.E. of four separate experiments. *, p < 0.05 versus dimethyl sulfoxide.


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Contractile activity is a potent stimulus for the induction of numerous cellular adaptations in skeletal muscle, including mitochondrial biogenesis (1, 4). This complex process involves the coordinated expression of proteins encoded within the nucleus as well as those encoded in mitochondrion, suggesting that a vital intracellular communication exists between these two organelles. It is likely that mitochondrial assembly is regulated mainly by proteins originating from the nucleus, because most of the factors required for the expression of mitochondrial proteins are nuclear-encoded, and the vast majority of the proteins found within the organelle are derived from the nuclear genome. We have adopted the respiratory chain protein cytochrome c as a model for the regulation of nuclear gene expression in response to contractile activity. Recently, we demonstrated that an elevation in contractile activity evokes time-dependent augmentations in cytochrome c transcriptional activation in vivo (6). Increases in cytochrome c mRNA stability were also partly responsible for the contractile activity-induced increase in mRNA observed. Here our goal was to utilize a cell culture model of contractile activity to mimic the in vivo induction of cytochrome c mRNA, while allowing for the definition of some of the intracellular signals and transcription factors responsible for this adaptation. Electrical stimulation of C2C12 cells in culture elevated the contractile activity of differentiated myotubes, which was clearly visible upon inspection under the microscope compared with unstimulated control cells.

Contractile activity imposed for 4 days (3 h/day) induced an elevation in cytochrome c mRNA. This was likely due to transcriptional activation, as indicated by cytochrome c promoter-reporter activity, as well as by the fact that no change in cytochrome c mRNA stability was observed under these conditions. Previous studies have shown that transcriptional activation of cytochrome c can occur in response to elevated intracellular Ca2+. This appears to be mediated by factors that bind to elements within the minimal (-66 to +115 bp) promoter region of the cytochrome c gene (8). Our data indicate that this region is also responsive to contractile activity. Within the first intron of the cytochrome c gene are GC-rich regions that may serve as binding sites for the zinc finger transcription factors Sp1 and Egr-1 (14, 15). Indeed, Egr-1 mRNA levels were elevated very rapidly after the onset of muscle contraction (Fig. 1), although no changes in Egr-1 protein were evident after 4 days of stimulation (Fig. 3A), emphasizing the transient nature of Egr-1 gene expression, as reported previously (20). This lack of increase evident at 4 days was reflected by the results of the DNA binding assay, because no increases in Egr-1 DNA binding were evident at that time.

In contrast to Egr-1, it is known that Sp1 is widely involved in the expression of a variety of mammalian genes involved in oxidative phosphorylation (35, 36). In particular, Sp1 affects cytochrome c expression by binding within the region between +83 and +104 bp of the gene (16). Here we show that contractile activity increases Sp1 protein levels and causes an elevation in DNA binding within this region and to a consensus Sp1 oligonucleotide. In both cases DNA binding is completely eliminated by preincubation with a nonlabeled Sp1 oligonucleotide. Further, overexpression of Sp1 led to an increase in cytochrome c transactivation and mRNA levels. Thus, it appears that contractile activity in skeletal muscle cells induces cytochrome c transcription via mechanisms that involve Sp1, at least with respect to the activation of the -66 bp promoter. However, the effect of contractile activity on the transactivation of the full (-726 bp) promoter is not likely entirely due to Sp1 activity. Using cardiac myocytes, Xia et al. (37) showed that a mutation of the Sp1 site within the first intron did not abolish the transcriptional activation induced by cardiac pacing, although the magnitude of transcriptional activation was reduced. In particular, they showed that the NRF-1 and CRE sites were also important for full contractile activity-induced transcriptional activation of the gene, at least in cardiac cells. Our data compliment those findings and uniquely illustrate the inducibility of Sp1-mediated transcriptional activation in skeletal muscle cells in response to a physiological stimulus.

To define more precisely the intracellular events associated with muscle contraction that mediated this transcriptional response, muscle contraction was systematically disrupted at three different levels. Treatment of cells with TTX completely abolished muscle contraction by preventing membrane depolarization, which should eliminate the subsequent release of Ca2+ and actin-myosin interactions. As expected, this prevented the activity-induced cytochrome c transcriptional activation that was apparent in vehicle-treated cells. Next, the role of Ca2+ in activity-induced cytochrome c expression was evaluated using the membrane-permeable Ca2+ chelator BAPTA-AM. The potential involvement of intracellular Ca2+ in activity-induced cytochrome c expression was suggested from observations that Ca2+ serves as a potent intracellular second messenger for a variety of cellular adaptations (11, 37, 38). In addition, we recently reported that a marked elevation in cytochrome c transcriptional activation occurs following the treatment of rat L6E9 muscle cells with the Ca2+ ionophore A23187 (8), an effect that is reproducible in the mouse C2C12 muscle cells used in the present study.2 Treatment with BAPTA-AM should permit the transmission of membrane depolarization but eliminate sarcoplasmic reticulum-mediated Ca2+ transients, subsequent actin-myosin interactions, and the accelerated ATP turnover. The complete inhibition of contractile activity that we observed during these conditions was also associated with the abolition of the simulation-induced cytochrome c transcriptional activation. This suggests that membrane depolarization alone, leading to voltage-sensitive activation of signaling cascades, which are known to activate the transcription of numerous genes in excitable cells (see Ref. 7 for review), cannot account for the observed increases in cytochrome c transactivation. We then evaluated contractile activity-induced increases in cytochrome c transactivation in the presence and absence of BDM. This agent is known to eliminate cross-bridge cycling and, therefore, the increased ATP turnover associated with contractile activity, but it allows both membrane depolarization and the increase in Ca2+ transients to occur. We hypothesized that if Ca2+ was a primary intracellular signal mediating the increase in muscle cytochrome c transactivation (8), this transcriptional activation should be evident even when muscle contraction was inhibited in electrically stimulated cells by preventing cross-bridge cycling. However, under these conditions no elevation in cytochrome c transcription was evident. These data suggest that the increases in Ca2+ invoked during the contractile activity conditions employed were not a sufficient stimulus (i.e. either in magnitude or duration) to induce the effect, as compared with treatment with a calcium ionophore like A23187 (8). In contrast, our data support the idea that, during skeletal muscle contractile activity, alterations in the rate of mitochondrial ATP synthesis represent a more important signal for the induction mitochondrial biogenesis, as reflected by cytochrome c expression. This is suggested by the three levels of mitochondrial ATP synthesis induced in the present study, using cells subject to contractile activity, unstimulated cells, as well as those in which oxidative phosphorylation is inhibited. Whether this effect is mediated directly via effects on kinase or phosphatase activities or indirectly via putative signaling molecules involved in mitochondrial-to-nuclear interorganellar communication remains to be determined.

    ACKNOWLEDGEMENTS

We thank Dr. R. C. Scarpulla (Department of Cell and Molecular Biology, Northwestern University, Chicago, IL) and Dr. F. Booth (University of Missouri, Columbia, MO) for the donation of the cytochrome c promoter constructs and Dr. G. Suske (University of Marburg, Marburg, Germany) for the Sp1 expression vector.

    FOOTNOTES

* This work was supported by a grant from the Natural Sciences and Engineering Council of Canada.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. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Biology, York University, North York, ON M3J 1P3, Canada. Tel.: 416-736-2100, Ext. 66640; Fax: 416-736-5698; E-mail: dhood@yorku.ca.

Published, JBC Papers in Press, February 20, 2001, DOI 10.1074/jbc.M100272200

2 M. K. Connor and D. A. Hood, unpublished observations.

    ABBREVIATIONS

The abbreviations used are: BDM, 2,3-butanedione monoxime; ACT, actinomycin D; BAPTA-AM, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester; CAT, chloramphenicol acetyltransferase; DNP, 2,4-dinitrophenol; LUC, luciferase; TTX, tetrodotoxin; VEH, vehicle; bp, base pair(s).

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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