Calcium-regulated changes in mitochondrial phenotype in skeletal muscle cells

Damien Freyssenet,1 Isabella Irrcher,1 Michael K. Connor,1 Martino Di Carlo,2 and David A. Hood1,2

1Department of Biology and 2Department of Kinesiology and Health Science, York University, Toronto, Ontario, Canada M3J 1P3

Submitted 30 September 2003 ; accepted in final form 23 December 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cytochrome c expression and mitochondrial biogenesis can be invoked by elevated intracellular Ca2+ in muscle cells. To characterize the potential role of Ca2+ as a messenger involved in mitochondrial biogenesis in muscle, we determined the effects of the Ca2+ ionophore A-23187 on the expression of nuclear- and mitochondrially encoded genes. Treatment of myotubes with 1 µM A-23187 for 48–96 h increased nuclear-encoded {beta}-subunit F1ATPase and malate dehydrogenase (MDH) mRNA levels by 50–100% (P < 0.05) but decreased mRNA levels of glutamate dehydrogenase (GDH) by 19% (P < 0.05). mRNA levels of the cytochrome c oxidase (COX) nuclear-encoded subunits IV, Vb, and VIc were unchanged, whereas the mitochondrially encoded subunits COX II and COX III were decreased by 30 and 70%, respectively (P < 0.05). This was paralleled by a 20% decrease (P < 0.05) in COX activity. These data suggest that cytoplasmic Ca2+ differentially regulates the mRNA level of nuclear and mitochondrial genes. The decline in COX II and III mRNA may be mediated by Tfam, because A-23187 modestly reduced Tfam levels by 48 h. A-23187 induced time-dependent increases in Egr-1 mRNA, along with the activation of ERK1/2 and AMP-activated protein kinase. MEK inhibition with PD-98059 attenuated the increase in Egr-1 mRNA. A-23187 also increased Egr-1, serum response factor, and Sp1 protein expression, transcription factors implicated in mitochondrial biogenesis. Egr-1 overexpression increased nuclear-encoded cytochrome c transcriptional activation by 1.5-fold (P < 0.05) and reduced GDH mRNA by 37% (P < 0.05) but had no effect on MDH or {beta}-subunit F1ATPase mRNA. These results indicate that changes in intracellular Ca2+ can modify mitochondrial phenotype, in part via the involvement of Egr-1.

mitochondrial biogenesis; malate dehydrogenase; cytochrome c oxidase mitochondrial transcription factor-A; early growth response gene-1; glutamate dehydrogenase


THE MAMMALIAN MITOCHONDRION contains its own genome encoding two ribosomal (r)RNAs, 22 transfer RNAs, and 13 mRNAs that participate in the formation of important components of the mitochondrial respiratory chain (5). Other mitochondrial proteins are derived from the nuclear genome, translated in the cytosol, and subsequently imported into the mitochondria. The dual genomic organization required for the synthesis and assembly of nascent and functional multisubunit holoenzyme complexes within mitochondria suggests the involvement of a coordinated mechanism. Earlier studies (23, 41) have shown that an increase in the expression of nuclear- and mitochondrial-encoded mRNAs occurs in rat skeletal muscle in response to chronic electrical stimulation. It has also been shown that these increases can be dissociated to some degree by the manipulation of thyroid status (21, 40), depletion of mitochondrial (mt)DNA (3, 28, 33), or by the inhibition of mitochondrial translation (9).

We have reported that cytochrome c gene expression was induced in skeletal muscle cells in response to treatment with the Ca2+ ionophore A-23187, providing the potential identification of a contractile activity-induced intracellular signal, which could induce mitochondrial biogenesis in skeletal muscle (15). Subsequently, it has been shown that the intermittent exposure of muscle cells to the Ca2+ ionophore ionomycin or to caffeine (34, 35), which stimulates the release of Ca2+ from the sarcoplasmic reticulum, induces the expression of genes involved in mitochondrial biogenesis. The orchestration of mitochondrial biogenesis in muscle via the Ca2+-induced activation of intracellular signals has also been shown in transgenic mice selectively expressing a constitutively active form of Ca2+/calmodulin-dependent protein kinase IV in muscle (42). Thus changes in intracellular Ca2+ have emerged as important signals for the synthesis of nuclear proteins leading to mitochondrial biogenesis. However, for Ca2+ to be viewed as a dominant signal for the synthesis of mitochondria in muscle, the generalizability of this response to multiple nuclear genes encoding mitochondrial proteins, as well to their potential regulating transcription factors, should be evident. Thus in this study, we extended our analysis of the involvement of calcium-mediated regulation of gene expression in skeletal muscle mitochondrial biogenesis by determining the effect of A-23187 on the expression of the number of nuclear genes encoding mitochondrial proteins, including important nuclearencoded transcription factors, such as the early growth response gene-1 (Egr-1), specificity protein-1 (Sp1), serum response factor (SRF), and mitochondrial transcription factor A (Tfam), in addition to the expression of mitochondrial-encoded cytochrome c oxidase (COX) subunits II and III. Because a marked enhancement of Egr-1 mRNA was noted in response to A-23187, we subsequently examined the effect of Egr-1 overexpression on the expression of nuclear genes encoding mitochondrial proteins. Finally, because the activation of AMP-activated protein kinase (AMPK) has also been shown to increase mitochondrial biogenesis (43), we investigated the possibility of an interaction between Ca2+ levels and AMPK activity.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture. L6E9 cells were cultured on 100 mm gelatin-coated tissue culture plates containing DMEM supplemented with 10% FBS and 1% penicillin/streptomycin at 37°C and 5% CO2 in air. All cell culture materials were purchased from Sigma (St. Louis, MO). At ~60% confluence, cells were switched to differentiation medium (DMEM supplemented with 5% heat-inactivated horse serum and 1% penicillin/streptomycin). Alternatively, cells were transfected (see below) before differentiation. All treatments were carried out when myotubes reached 80–90% confluence (15).

Drugs and treatments. All experiments were done with differentiated cells (myotubes) and were matched with vehicle-treated control myotubes. A-23187 and ruthenium red were purchased from Sigma. They were prepared as stock solutions of 0.25 mM in Me2SO (Sigma) and 7 mM in distilled water, respectively. PD-98059 was obtained from Calbiochem (San Diego, CA) and prepared as 25 mM solution in Me2SO. For most experiments, A-23187 was used at a concentration of 1 µM for 48 h. The dose-dependent effect of A-23187 on malate dehydrogenase mRNA level was evaluated with concentrations ranging from 0.25 to 1.50 µM. The effect of time was evaluated by incubating myotubes with 1 µM A-23187 for 8, 24, 48, 72, or 96 h. The effect of ruthenium red was evaluated by preincubating myotubes for 24 h with 10 µM ruthenium red before the treatment with 1 µM A-23187 was started for 72 h. The effect of the MEK inhibitor was evaluated by preincubating myotubes for 30 min with 10 µM PD-98059 before starting the treatment with 1 µM A-23187 for 2, 4, and 8 h.

RNA isolation, Northern blotting, and radiolabeled probes. Total RNA was extracted as previously described (15). The concentration and purity were determined by ultraviolet spectrophotometry at 260 and 280 nm, respectively. Total RNA (10 µg) was separated on denaturing formaldehyde-1% agarose gel and transferred overnight to nylon membranes (Hybond N, Amersham; Arlington Heights, IL) (10). Radiolabeled probes were generated by random primer labeling in the presence of [{alpha}-32P]dCTP (Amersham-Pharmacia Biotech, Mississauga, Ontario, Canada), as done previously (15). Membranes were prehybridized overnight at 42°C. Blots were then incubated overnight at 42°C after the addition of the appropriate radiolabeled probe (2 x 106 cpm) encoding COX subunits II, III, IV, Vb, VIc, {alpha}-actin, {beta}-subunit of F1ATPase, malate dehydrogenase (MDH), glutamate dehydrogenase (GDH), or Egr-1. The blots were then rinsed and washed 3 x 10 min at room temperature with 2x SSC (0.15 M NaCl/0.030 M sodium citrate), 0.1% SDS, followed by 1 x 15 min at 55°C and 1 x 15 min at 65°C. The final wash for membranes hybridized with cytochrome oxidase subunit II or the {beta}-subunit of F1ATPase was 60°C for 15 min. Signals were corrected for loading using hybridization signals produced by 18S rRNA and were quantified by electronic autoradiography.

Western blot analysis. Total protein extracts from cultured L6E9 myotubes treated were treated for varying times up to 48 h with 1 µM A-23187 were harvested and electrophoresed through SDS-polyacrylamide gels, and electroblotted onto nitrocellulose membranes (Amersham, Baie D'Urfé, Québec, Canada). Overnight incubations with antibodies diluted in blocking buffer directed toward phospho-AMPK-{alpha} (1:400; New England Biolabs, Mississauga, Ontario, Canada) or total AMPK-{alpha} (1:750; New England Biolabs), cytochrome c (1:750) (34), Egr-1 (1:500; Santa Cruz Biotechnology, Santa Cruz, CA), phospho-extracellular regulated kinases-1 and -2 (ERK1 and ERK2; 1:400; New England Biolabs) or total ERK1/2 (1:1,000; New England Biolabs), Sp1 (1:500; Santa Cruz Biotechnology), SRF (1:1,000; Santa Cruz Biotechnology), and Tfam (1:1,000) (15) were followed by incubations at room temperature with the appropriate secondary antibody conjugated to horseradish peroxidase, visualized with enhanced chemiluminescence and quantified using SigmaGel (Jandel, San Rafael, CA).

Cell transfection. L6E9 myoblasts were transfected with either the -66 or the -726 bp cytochrome c promoter/luciferase construct (2.5 µg/100 mm dish) when they reached 70% confluence. Cells were cotransfected with p-cytomegalovirus/Renilla luciferase (5 ng/dish) to correct for differences in transfection efficiency. Where applicable, wild-type Egr-1, or empty vector expression vectors were transfected (2.5 µg/dish) in combination with cytochrome c promoter/luciferase constructs. The wild-type Egr-1 expression vector contains the full-length Egr-1 cDNA under the transcriptional control of the cytomegalovirus promoter. The mutant construct is expressed at similar levels but lacks DNA binding activity and has been previously described (15). The total amount of DNA added was maintained constant in all transfected cells. Transfections were done with the use of the poly-L-ornithine method, followed by a Me2SO shock (14) or with the use of Lipofectamine 2000 (Invitrogen, Mississauga, Ontario) following the manufacturer's recommendations. Cells were then differentiated by switching to a low-serum medium and subsequently scraped for either total RNA isolation or for measurement of luciferase activities. Firefly and renilla luciferase activities were measured using luminometer (Lumat 9507, Berthold) and a Promega (Madison, WI) Dual Luciferase Assay kit, according to the manufacturer's instructions.

Cell extracts and enzyme activities. Cells were harvested after 72 h of treatment and centrifuged at 4°C for 5 min. The cell pellet was then resuspended with 100 µl of 0.1 M KH2PO4/Na2PO4 buffer (pH 7.2) containing 2 mM EDTA. The cells were then sonicated (4 x 10 s) on ice and centrifuged at 4°C for 5 min. Protein concentrations of the resulting supernates were obtained by determining absorbance at 280 nm. COX enzyme activity was measured by the rate of oxidation of fully reduced cytochrome c (22). MDH activity was measured as the rate of reduction of NAD (20).

ATP measurements. Control and 48-h-treated L6E9 myotubes were trypsinized and centrifuged for 4 min at 2,000 g. The pellet was deproteinized with 6 M perchloric acid, 80 µl of cold PBS, and centrifuged for 5 min at 14,000 g. The supernatant was neutralized with 2 M KOH and centrifuged for 5 min at 14,000 g. The resulting supernatant fraction was used to determine ATP production using a Berthold Luminometer. All measurements were normalized to total protein content.

Statistical analysis. Values were expressed as means ± SE. One-way analyses of variance, followed by Scheffé's post hoc test (P < 0.05), were used for multiple data comparisons (Figs. 1, 2A, and 4B). Student's t-test was used for comparing two groups of data (slopes in Fig. 2A, COX II mRNA data), as appropriate.



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Fig. 1. Effect of A-23187 on the mRNA levels of nuclear genes encoding mitochondrial proteins. A: quantification of Northern blot analyses of malate dehydrogenase (MDH) mRNA levels and MDH enzyme activities as a function of time post-A-23187 treatment (1 µM; *P < 0.05 vs. control), and (B) as a function of A-23187 concentration, incubated for 48 h (*P < 0.05 vs. 0 µM A-23187). The effect of preincubation (5 h) with EGTA is also shown. C: effect of 1 µM A-23187 (48 h) on mRNA levels encoding cytochrome c (Cyt c; Ref. 15), {beta}-F1 ATPase, cytochrome c oxidase (COX) IV, COX Vb, COXVIc, glutamate dehydrogenase (GDH), and {alpha}-actin [*P < 0.05 vs. (-) A-23187; n = 3–5 experiments/condition].

 


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Fig. 2. Effect of A-23187 on the expression of mitochondrially encoded COX subunits. A: representative Northern blot showing the effect of A-23187 on COXII and III mRNA expression after 48 h of A-23187 (or vehicle) treatment. COXIII mRNA expression was also evaluated in a subset of A-23187-treated cells pretreated with ruthenium red (10 µM). The effects of A-23187 and ruthenium red were quantified and graphically represented (*P < 0.05 vs. 0 µM A-23187; n = 3 to 4 experiments/condition). B: effect of A-23187 on COX activity in cells treated for 48 h with 1 µM A-23187 (*P < 0.05 vs. 0 h; n = 4 experiments).

 


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Fig. 4. Effect of A-23187 treatment on transcription factor protein expression. A: representative Western blots (50–100 µg/lane) showing the effect of A-23187 on Egr-1, specificity protein-1 (Sp1), serum response factor (SRF), and mitochondrial transcription factor A (Tfam) after 8 and 48 h of treatment. Data obtained at the two time points were collected in separate experiments each compared with appropriate controls (time 0), resulting in variable signal intensities when compared between 8 and 48 h. B: effect of 8 and 48 h of A-23187 treatment was quantified, and a summary of repeated experiments (n = 3) is shown. *P < 0.05 vs. 0 h.

 


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Effect of A-23187 treatment on MDH expression in L6E9 cells. We initially characterized the response of the nuclear-encoded mitochondrial matrix enzyme MDH to A-23187 treatment, similar to our previous work with cytochrome c (15). The treatment of myotubes with the Ca2+ ionophore A-23187 for 48 h increased MDH mRNA level in a time-dependent fashion, such that an ~50–100% increase (P < 0.05) was evident between 48–96 h of treatment (Fig. 1A). This effect was also reflected at the protein level as MDH enzyme activity was significantly increased by 80% (P < 0.05; n = 4) in response to A-23187 treatment for 48 h (Fig. 1A). The effect of A-23187 on MDH mRNA expression was also concentration-dependent up to 1.5 µM and was abolished when myotubes were preincubated with the extracellular chelating agent EGTA (Fig. 1B). This strongly suggests that the effect of A-23187 was mediated through Ca2+ mobilization from the extracellular pool.

A-23187 differentially regulates nuclear genes encoding mitochondrial proteins. A-23187 treatment of myotubes for 48 h elicited a similar response when Northern blot analyses were performed with a probe encoding the {beta}-subunit of the F1ATPase (Fig. 1C). Quantification of this effect revealed a 90% increase (P < 0.05) in mRNA expression level. In contrast to the response observed for cytochrome c (15), MDH and the {beta}-subunit of F1ATPase, no changes in the COX IV, COX Vb, and COX VIc mRNA levels were observed in response to 48 h of A-23187 treatment (Fig. 1C), whereas glutamate dehydrogenase (GDH) mRNA expression was reduced by 19% (P < 0.05). In addition, {alpha}-actin mRNA, used to assess the specificity of the effect of A-23187 on nuclear genes encoding mitochondrial proteins, was expressed at similar levels both in control and A-23187-treated cells (Fig. 1C).

Effect of A-23187 on mitochondrially encoded COXII and III mRNA expression, and COX enzyme activity. The lack of response among nuclear-encoded COX subunit mRNA expression to A-23187 prompted us to investigate the expression of the mitochondrially encoded COX subunits II and III. Surprisingly, COX II mRNA expression declined by ~30% (P < 0.05), whereas COX III mRNA level was more markedly affected, exhibiting an ~70% decrease (P < 0.05) when myotubes were incubated with 1 µM A-23187 for 48 h (Fig. 2A). This effect does not appear to depend on Ca2+ entry into the organelle via the mitochondrial Ca2+ uniporter, the major route of Ca2+ entry into the organelle (18, 19, 29) because preincubation of myotubes for 24 h with 10 µM ruthenium red did not change the response elicited by 48 h of A-23187 treatment on COX III mRNA (Fig. 2A). Furthermore, these data also suggest that the A-23187-mediated effect is initiated outside the mitochondrion, possibly via changes in the expression of nuclear-encoded transcription factors (see below). The relevance of the decrease in COX II and III mRNA expression was also reflected at the protein level, because A-23187 induced a 20% decrease in COX activity (n = 4; P < 0.05; Fig. 2B). Taken together, these data suggest that an increase in intracellular cytosolic Ca2+ concentration mediated by A-23187 differentially regulates the expression of genes encoding mitochondrial proteins.

Effect of A-23187 on Egr-1 expression. We investigated the effect of A-23187 on the mRNA level of Egr-1, a transcription factor that has putative binding sites within the upstream promoter regions of several nuclear genes encoding mitochondrial proteins (32), including GDH (12). A sequence similar to, but not identical to the consensus site, is also found in the cytochrome c promoter. A rapid 100–150% increase (P < 0.05, n = 3 experiments) in Egr-1 mRNA was observed as early as 1 h posttreatment, was maintained at 2 h, and declined by 4 h (Fig. 3A). This early response is consistent with the induction of Egr-1 as an upstream event in a cascade of reactions leading to changes in gene expression in response to A-23187. Interestingly, the increase in Egr-1 mRNA expression by 2 h was abolished by preincubation of the cells for 30 min with 12.5 µM PD-98059, a MEK inhibitor (Fig. 3A). At 4 h, when the effect of A-23187 was reduced to ~30% (n = 3), the effect of MEK inhibition was more pronounced. This finding is consistent with other results, in which MEK inhibition with PD-98059 led to a dose-dependent reduction in baseline Egr-1 mRNA levels (Lowe S and Hood DA, unpublished observations). We then evaluated whether the down-stream targets of MEK, the extracellular regulated kinases-1 and 2 (ERK1/2), could be activated via A-23187. ERK 2 activation was evident by 2 h, was significantly increased ~2.5-fold (P < 0.05) by 4 h, and returned to control levels by 8 h of treatment. The kinetics of these responses, along with the inhibition data, suggest that the induction of Egr-1 mRNA levels by A-23187 is mediated by both ERK1/2-dependent and -independent effects.



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Fig. 3. Effect of A-23187 on early growth-response gene-1 (Egr-1) mRNA expression and mitogen-activated protein (MAP) kinase activation. A: Egr-1 mRNA expression as a function of time posttreatment with A-23187 (+) or vehicle (-). Preincubation (30 min) with the MEK inhibitor PD-98059 (PD; 10 µM) is also shown and is representative of three experiments. 18S rRNA is also shown to verify gel loading. B: representative Western blot (75 µg/lane) showing the effect of A-23187 on ERK 1 and 2 MAP kinase activation, and total ERK2 protein levels as a function of time posttreatment. ERK2 kinase activation was quantified, and a summary of repeated experiments (n = 3) is shown. *P < 0.05 vs. 0 h.

 

To investigate whether the early A-23187-mediated increase in Egr-1 mRNA could lead to subsequent changes in Egr-1 protein expression, we examined Egr-1 protein expression following treatment with A-23187 for 8 and 48 h. A-23187 treatment for 8 h increased Egr-1 protein 3.5-fold (P < 0.05), a response that was diminished by 48 h (Fig. 4, A and B).

Time-dependent changes in Sp1, SRF, and Tfam protein expression after A-23187 treatment. We investigated the possibility that the effect of A-23187 on COX II and III mRNA levels (Fig. 2A) was mediated via Ca2+-induced changes in the levels of Tfam, a nuclear-encoded transcription factor protein that is imported into mitochondria and binds to the light and heavy-strand promoter of mtDNA (36). We therefore evaluated Tfam protein expression after 8 and 48 h of exposure to A-23187. No change in Tfam protein was observed after 8 h of treatment. This is consistent with our observations of a lack of change in Tfam mRNA expression for up to 12 h of treatment with A-23187 (data not shown). However, a 32% decrease (P < 0.05) by 48 h was observed, in parallel with the overall decrease in the mitochondrially encoded COX II and III subunits, and the overall decrease in COX activity observed at that time.

We extended our investigation beyond Egr-1 and Tfam, to identify additional calcium-regulated transcription factors that may be responsible for mediating changes in both nuclear and mitochondrial gene expression. We chose to investigate Sp1 because almost all nuclear genes encoding mitochondrial proteins, including Tfam and cytochrome c, contain Sp1 binding sites within their promoter regions (7, 10, 14, 20, 32, 39). We also investigated the effect of A-23187 on serum response factor (SRF) protein expression, as it has been implicated in regulating the transcription of Egr-1 (8), and it has a binding site within the F1ATPase promoter (32). A-23187 treatment for 8 h increased (P < 0.05) Sp1 and SRF protein levels by 4.9- and 2.4-fold, respectively (Fig. 4, A and B). The effect of A-23187 on SRF and Sp1 expression was completely dissipated by 48 h (Fig. 4, A and B).

Effect of Egr-1 overexpression on mRNA levels, cytochrome c transcriptional activation, and protein expression. We hypothesized that the A-23187-mediated changes in Egr-1 mRNA and protein expression could lead to an increase in the expression of nuclear genes encoding mitochondrial proteins. To evaluate this, we overexpressed Egr-1 and measured MDH, {beta}-F1ATPase, and GDH mRNA levels. Although no changes in the levels of MDH or {beta}-F1ATPase mRNA were observed (Fig. 5A), Egr-1 overexpression decreased GDH mRNA expression by 37% (P < 0.05; Fig. 5, A and B).



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Fig. 5. Effect of Egr-1 overexpression on mRNA expression and cytochrome c transcriptional activation. A: MDH, {beta}-F1 ATPase, and GDH mRNA expression in the presence of wild-type (wt) or mutant (mt) Egr-1 overexpression, or with empty vector (ev). B: quantification of GDH mRNA obtained from multiple Northern blots as shown in A (*P < 0.05 vs. ev; n = 5 experiments). C: cytochrome c transcriptional activation of the full (-726 bp) and minimal (-66 bp) promoters in the presence of wild-type Egr-1 overexpression or empty vector. {dagger}P < 0.05 -726Cytc vs. -66Cytc; *P < 0.05 wt vs. ev.

 

Because the cytochrome c promoter contains potential binding sites for Egr-1 based on sequence similarities with Sp1, we also sought to determine the effect of Egr-1 overexpression on cytochrome c transcriptional activation. To verify this, both the minimal (-66 bp) and the full (-726 bp) length cytochrome c promoter/luciferase constructs were cotransfected with Egr-1 into L6E9 cells. Egr-1 overexpression had no effect on cytochrome c transcriptional activation of the minimal promoter, but increased the full-length cytochrome c promoter activity 1.5-fold over the empty vector-matched control (Fig. 5C).

Effect of A-23187 treatment on AMPK-{alpha} activation. The intracellular signals involved in stimulating mitochondrial biogenesis in skeletal muscle likely involve crosstalk between multiple signaling pathways. This was most recently demonstrated in mice expressing a muscle-specific dominant negative AMPK transgene, where the expression of the calcium-regulated CamKIV appeared to be dependent on AMPK-{alpha} (43). We therefore explored the possibility that AMPK-{alpha} activation may also be calcium dependent. Forty-eight hours of A-23187 treatment dramatically increased (P < 0.05) AMPK-{alpha} activation by 10-fold (Fig. 6A). To confirm that chronic elevations in intracellular Ca2+ did not comprise cellular energy status, ATP levels were measured in cells treated with A-23187 for 48 h. No significant change in ATP concentrations were observed, suggesting that A-23187 had no effect in compromising the energy status of the cells (Fig. 6B).



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Fig. 6. A: representative Western blots showing the effect of 48 h of A-23187 on AMP-activated protein kinase (AMPK-{alpha}) activation and total AMPK-{alpha} protein levels. Kinase activation, measured as a fold change of the phosphorylated protein over the total protein, was quantified and a summary of repeated experiments (n = 3) is shown; *P < 0.05 vs. DMSO. B: immediately after A-23187 treatment, cells were harvested and ATP levels were measured as described in MATERIALS AND METHODS. A summary of two experiments, each measurement repeated in triplicate, is shown.

 


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Our recent finding that elevations in intracellular Ca2+ concentration within skeletal muscle cells increased cytochrome c transcriptional activation and mRNA levels (15) led us to examine whether this Ca2+ effect was specific to cytochrome c, or common to other nuclear genes encoding mitochondrial proteins. Our results show that, similar to cytochrome c, the nuclear-encoded MDH and F1{beta}-ATPase respond to elevated intracellular Ca2+ levels by increasing their mRNA concentrations, whereas GDH mRNA expression decreased in response to the same treatment. We also show that the mRNAs encoding subunits of the COX holoenzyme are differentially regulated by changes in intracellular Ca2+ concentrations. In particular, we show that A-23187 treatment decreases the mRNA level of mitochondrially encoded genes COX II and COX III of cytochrome oxidase, whereas the nuclear-encoded subunits IV, Vb, and VIc of COX remained unaffected. This pattern of change in mRNA level was meaningful because it was reflected at the protein level by elevated MDH and reduced COX enzyme activities. Thus our findings uniquely show that elevating intracellular Ca2+ differentially regulates mRNA levels of genes encoding mitochondrial proteins and therefore suggest that Ca2+ does not constitute a signal that can lead to stoichiometric increases in all mitochondrial proteins, resulting in mitochondrial biogenesis.

Although a continuously elevated cytosolic Ca2+ concentration does not compare with the transient physiological oscillations in Ca2+ observed in contracting muscle cells, continuously elevated levels have been observed in specific pathophysiological states and under conditions of muscle adaptation. For example, muscle cells that have been depleted of mtDNA, and fibroblast cells from mitochondrial encephalopathy and lactic acid syndrome patients, which harbor a mtDNA tRNAleu mutation, both exhibit chronically elevated levels of cytosolic Ca2+. These cells exhibit significant changes in mitochondrial phenotype (3, 31). Furthermore, cytosolic Ca2+ levels are also markedly elevated in skeletal muscle subject to chronic electrical stimulation, and it is well known that one of the consequences of chronic stimulation is mitochondrial biogenesis (6, 37). Thus the A-23187-mediated elevation in cytosolic Ca2+ concentrations, which can be sustained for up to 16 days after treatment (26), models cellular environments in which changes in mitochondrial phenotype can be produced.

In skeletal muscle cells, increases in cytosolic Ca2+ concentration elicited by muscle-specific agents such as nicotinic agonists, KCl depolarization in Ca2+-free medium, or caffeine are amplified four- to sixfold within the mitochondrial matrix (4). This raises the important question as to whether Ca2+ is a direct regulator of mitochondrial gene expression via actions within the organelle, on the mitochondrial transcription machinery, or whether it acts outside the mitochondrion (i.e., in the nucleus or cytosol) to modify the expression or activity of molecules that are subsequently imported to alter mitochondrial gene expression. Under physiological conditions, mitochondrial Ca2+ influx is mediated by a uniporter that facilitates the diffusion of Ca2+ down its electrochemical gradient (18, 19, 29). Ruthenium red, a potent noncompetitive inhibitor of the Ca2+ uniporter, was used here to determine whether Ca2+ entry via this route could account for the effect of A-23187 on COX II and III mRNA levels. Our results demonstrate that 10 µM ruthenium red did not abolish the effect mediated by A-23187 on COX III mRNA levels. Because Ca2+ influx into mitochondria via the uniporter can be attenuated and completely blocked at concentrations much less than the dose used in this study (19), we conclude that Ca2+ entry via this pathway is not responsible for the effect observed, and that this effect likely originates external to the organelle.

The nuclear-encoded transcription factor Tfam is an important protein involved in the transcription of the mitochondrial genome (36). In the present study, Tfam mRNA and protein levels were determined to assess whether changes in Tfam expression could explain the decrease in mitochondrial-encoded COX II and III mRNA levels. Although early time points revealed no change in Tfam mRNA or protein levels, Tfam protein expression was reduced following a longer treatment period. This decrease coincided precisely with decreases in COX II and III mRNA expression, as well as with the decrease in COX activity reported here. Thus these data support our hypothesis that the A-23187-mediated effect on mitochondrial gene transcription originates outside the mitochondrion via a decrease in Tfam protein expression. The use of a number of different experimental conditions has shown that a positive relationship exists between Tfam protein expression and the regulation of mtDNA-derived transcripts of the COX holoenzyme. For example, chronic stimulation of skeletal muscle in vivo has been shown to increase Tfam protein expression. This corresponded to an increase in Tfam import into mitochondria, an increase in Tfam/DNA binding to the D-loop of mtDNA, and corresponding increases in mitochondrial-encoded COXIII mRNA expression (17). In addition, elevated Tfam expression in cancer cells was matched by parallel increases in mitochondrial-encoded COXI and COXII mRNA expression (13). Conversely, when Tfam expression was experimentally decreased (24), the expression of mitochondrially encoded COX subunits was also reduced. Our findings in the present study using A-23187 to influence changes in gene expression also support this relationship.

Egr-1 is an immediate-early gene, which has been previously shown to increase in response to contractile activity (2, 10, 30) and also to increased intracellular Ca2+ (1). Importantly, putative binding sites for Egr-1 in the upstream region of GDH (12) and possibly cytochrome c exist, suggesting that Egr-1 may be involved in the Ca2+-mediated signal leading to mitochondrial biogenesis. In the present study, the response of Egr-1 mRNA and protein expression to elevated internal Ca2+ was large and rapid, and it occurred before any changes in the mRNA for cytochrome c (15), MDH, GDH, or F1{beta}-ATPase, consistent with our hypothesis. Thus, to investigate a direct role for Egr-1, we evaluated the effect of Egr-1 overexpression on cytochrome c transcriptional activation and the mRNA expression of GDH, examples of nuclear-encoded gene products, which increase and decrease, respectively, in response to A-23187. Here we show that Egr-1 overexpression transcriptionally activated the full-length cytochrome c promoter and also led to a marked decrease in GDH mRNA levels. These data support a role for this transcription factor in the regulation of nuclear genes encoding mitochondrial proteins, leading to an altered organelle phenotype. Although putative Egr-1 binding sites within the cytochrome c promoter are thought to exist, multiple Sp1 sites have been identified in the cytochrome c promoter, and it is well known that Egr-1 and Sp1 can compete for similar GC-rich promoter elements (25). Thus, increases in Egr-1, via initial elevations in intracellular Ca2+, represent a possible mechanism by which cytochrome c is transcriptionally transcriptional activated. It is also known that the GDH promoter contains overlapping Egr-1 and Sp1 sites. Under these circumstances, an increase in Egr-1 expression can facilitate the displacement of Sp1 from its binding site, resulting in a reduction in mRNA expression (24), thus demonstrating that Egr-1 can act as a transcriptional repressor. The decrease in GDH mRNA expression via A-23187, and also by Egr-1 overexpression, is supportive of a Ca2+-initiated, Egr-1-mediated pathway (Fig. 7).



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Fig. 7. Summary of Ca2+-regulated changes in gene expression leading to alterations in mitochondrial phenotype in skeletal muscle cells. A-23187 activates both MAP kinase and AMPK and increases SRF expression, leading to increases in Egr-1 mRNA and protein (7, and present study). Egr-1 upregulates cytochrome c transcription and downregulates GDH mRNA expression. The A-23187-induced increase in Sp1 can also affect cytochrome c levels (14), whereas the decrease in Tfam will result in a reduction of mitochondrial (mt)DNA transcript levels. These effects lead to an alteration in muscle mitochondrial phenotype.

 

To establish whether additional Ca2+-regulated factors may be involved in regulating the expression of genes encoding mitochondrial proteins, we also evaluated the effect of A-23187 on Sp1 and SRF in addition to Egr-1 and Tfam because putative binding sites for these factors exist within the promoters of many nuclear genes encoding mitochondrial proteins, including COX enzyme subunits (27, 32) and Tfam (7). On the basis of Western blot analyses (Fig. 4), our results suggest that the early increase in SRF and Sp1 protein, in addition to Egr-1, may serve as additional factors that aid in the regulation of mitochondrial gene expression (Fig. 7).

The transduction of intracellular signals into changes in gene expression often occurs via the activation of signaling kinases. Several Ca2+-regulated signaling pathways have been identified, including the MAP kinases. With respect to Egr-1, we have shown that the increase in Egr-1 mRNA can be markedly attenuated in the presence of the MEK inhibitor PD-98059. Our previous work (15) has demonstrated that the cytochrome c transactivation mediated by A-23187 was reduced by ~40% in the presence of PD-98059 and that MAP kinases (ERK1 and ERK2) were rapidly and transiently activated by A-23187 (Fig. 3B). The fact that the increase in Egr-1 mRNA level was also blunted by the MEK inhibitor is consistent with the involvement of MAP kinase as an early signaling event in the activation of Egr-1 and, subsequently, cytochrome c expression (Fig. 7).

Finally, an interesting result of the current study is the A-23187-mediated activation of AMPK. Only one investigation to date has documented a link between Ca2+/calmodulin-dependent protein kinase IV and AMPK (43). However, in that study it was reported that the regulation of Ca2+/calmodulin-dependent protein kinase IV expression occurs downstream of AMPK activation. Our results suggest that AMPK activation occurs directly as a result of Ca2+ signaling, independent of changes in cellular energy status as reflected by ATP concentration, since decreases in ATP levels, and presumably AMP levels in A-23187-treated cells were not observed. More studies are warranted to investigate the direct or indirect role of Ca2+-evoked changes in AMPK activation, and other proteins involved in mediating mitochondrial biogenesis.


    ACKNOWLEDGMENTS
 
We thank Ponni Kumar for the measurement of COX Vb mRNA. We are grateful to Dr. V. Sukhatme (Harvard University, Cambridge, MA), Dr. F. Booth (University of Missouri, Columbia, MO), Dr. N. Avadhani (University of Pennsylvania, Philadelphia, PA), Dr. A. Das (University of Amsterdam, Amsterdam, The Netherlands), and Dr. R. Scarpulla (Northwestern University,Chicago, IL) for the provision of the Egr-1, actin, COX Vb, GDH, and cytochrome c DNA constructs, respectively.

GRANTS

This work was supported by a grant from the Natural Science and Engineering Research Council of Canada (NSERC) and Canadian Institutes of Health Research. D. Freyssenet was the recipient of a postdoctoral fellowship from the Région Rhônes-Alpes (France). I. Irrcher is the recipient of a NSERC postgraduate scholarship. D. A. Hood is the holder of a Canada Research Chair in Cell Physiology.

Present address of D. Freyssenet: Laboratoire de Physiologie, Physiologie et Physiopathologie de l'Exercise et Handicap, GIP-E2S, Faculté de Médicine, Université Jean Monnet, 15 rue A. Paré, 42023 Saint-Etienne Cedex 2, France.


    FOOTNOTES
 

Address for reprint requests and other correspondence: D. A. Hood, Dept. of Biology, York University, Toronto, Ontario, M3J 1P3 Canada (E-mail: dhood{at}yorku.ca).

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.


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