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J. Biol. Chem., Vol. 277, Issue 47, 45323-45330, November 22, 2002

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Intracellular Calcium and Myosin Isoform Transitions

CALCINEURIN AND CALCIUM-CALMODULIN KINASE PATHWAYS REGULATE PREFERENTIAL ACTIVATION OF THE IIa MYOSIN HEAVY CHAIN PROMOTER^*

David L. Allen and Leslie A. Leinwand

From the Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, Colorado 80309

Received for publication, August 13, 2002, and in revised form, September 9, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Intracellular calcium levels can have profound effects on muscle biology via alterations in gene expression. In particular, intracellular calcium levels increase during muscle activation and are thought to underlie fast-to-slow shifts in muscle gene expression. In the present work, we determined that increased intracellular calcium has a significant effect on the activity of the adult fast myosin heavy chain (MyHC) promoters in the order of MyHC IIa IId/x > IIb. We have identified the pathways by which the calcium signal mediates increased activation of the MyHC IIa promoter. Inhibition of calcineurin or calcium-calmodulin kinase greatly attenuates ionophore-induced activation of the MyHC IIa promoter, whereas protein kinase C inhibitors have no effect. Inhibition and overexpression studies with members of the mitogen-activated protein kinase family reveal roles for MEK1/MEK2 and MEKK1, but not p38 or phosphatidylinositol 3-kinase. Downstream mediators of these effects are the activities of the MEF-2 and NFAT transcription factors, whose binding sites in the MyHC IIa promoter are required for calcium-induced activation of the MyHC IIa promoter.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Mammalian skeletal muscle consists of a mosaic of different fiber types. Four distinct fiber types, one slow (type I) and three fast (IIa, IId/x, and IIb), are found in adult rodent skeletal muscle, and these differ with respect to their fatigability and their strength and speed of contraction. The functional, biochemical, and morphological differences between muscle fiber types arise as a result of different programs of fiber-specific gene expression that result in specific contractile, metabolic, cytoskeletal, and regulatory proteomes associated with each fiber type.

Skeletal muscle fibers demonstrate remarkable plasticity, and shifts in fiber type can occur in response to a number of physiological and pathological conditions, including muscle adaptation, aging, and muscle disease (1). An increase in muscle activity such as endurance exercise training induces muscle fiber adaptations through qualitative and quantitative changes in fiber-specific contractile and metabolic gene expression that result in slower contracting, more oxidative muscle fibers (2). The stimuli that induce these shifts in fiber-specific gene expression are currently not well defined. Likely candidates include many of the consequences of increased or prolonged muscle activation, including changes in the amount or pattern of muscle mechanical and electrical activity; changes in intracellular metabolites such as glycogen/glucose levels, hydrogen ions, or reactive oxygen species; and increased secretion of autocrine/paracrine factors.

The amount and pattern of electrical activity induced in muscle fibers by the motoneuron have been postulated to play a prominent role in defining muscle gene expression and fiber type (3). One of the most critical alterations in response to electrical activation of cells is an increase in intracellular calcium levels. Depolarization-induced increases in intracellular calcium and its effects on gene expression have been studied in a number of electrically active cell types, including cardiac myocytes, neurons, and pancreatic islet cells (4-8). These studies have also identified several signaling molecules involved in transducing the calcium signal, including serum response factor (SRF),¹ cyclic AMP-response element-binding protein, protein kinase C, and various members of the MAP kinase signaling family.

Not surprisingly, several skeletal muscle genes are sensitive to electrical activity and/or intracellular calcium levels. Electrical stimulation of myotubes in culture up-regulates glycogen phosphorylase (9) but represses expression of the acetylcholine receptor (10) and fast sarcoplasmic ATPase genes (11). Increases in intracellular calcium up-regulate several skeletal muscle genes, including the myogenic regulatory factor Myf-5, the inward rectifying potassium channel, and the metabolic enzymes hexokinase II and cytochrome c (12-15). With the exception of the K⁺ channel gene, this up-regulation appears to be induced primarily at the level of transcription (12-15).

In addition, recent evidence has implicated calcium signaling pathways in slow versus fast fiber gene expression. These studies have demonstrated that prolonged low amplitude increases in intracellular calcium, such as those produced by slow motoneurons, result in maximal activation of calcineurin, which in turn dephosphorylates members of the NFAT family of transcription factors, allowing them to activate the promoters of such slow-specific muscle genes as myoglobin and slow troponin I (16). Transgenic mouse studies have also implicated calcineurin as well as CaM kinase and the MEF-2 family of transcription factors in the specification of slow/oxidative gene expression (17-19), although studies using plasmid DNA injection failed to find a role for calcineurin in the specification of slow or fast muscle gene expression in vivo (20).

Calcium-induced activation of calcineurin is also thought to underlie the fast-to-slow transitions in myosin heavy chain (MyHC) isoform expression, both in vitro and in vivo (21-26). What is less clear is whether calcium signaling is involved in the transition between fast fiber subtypes. MyHC isoform transitions typically occur in a stereotypic pattern during muscle adaptation, moving sequentially from IIb to IId/x to IIa to type I (1). Is calcineurin involved only in the transition from fast to slow (i.e. from IIa to type I MyHC expression), or is calcineurin signaling required to move from IIb to IId/x and/or IId/x to IIa? The IIa MyHC isoform is expressed in fibers with a high oxidative enzyme profile more similar to that of type I MyHC-expressing fibers than IIb or IId/x MyHC-expressing fibers, making it attractive to hypothesize that IIa MyHC expression may share common elements with type I MyHC. We recently demonstrated that the upstream promoter region of the IIa MyHC gene was activated to a much greater extent by a constitutively active calcineurin construct than the IId/x or IIb MyHC promoters, suggesting that calcineurin signaling may play a role in transitions between fast subtypes and that IIa MyHC regulation may share common elements with type I MyHC expression (27). However, it is unclear whether expression of the fast MyHC genes is also differentially sensitive to intracellular calcium or whether other signaling pathways are involved in transducing calcium-related signals into shifts in fast MyHC expression.

We examined the responsiveness of isolated segments of the adult mouse skeletal MyHC promoters to calcium signaling in order to better understand the role of intracellular calcium in shifts in fast fiber-specific gene expression. We show that the adult skeletal MyHC upstream promoter regions are differentially sensitive to increased intracellular calcium, with IIa IId/x > IIb. We used overexpression constructs and pharmacological inhibitors to identify specific signaling pathways involved in calcium sensitivity of the IIa MyHC promoter. Our data suggest that changes in intracellular calcium may play a role in the shifts in fast MyHC expression occurring during increased muscle activation and identify several key intracellular pathways that may be involved in this augmentation in vivo.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Promoter and Overexpression Plasmids-- Generation of luciferase reporter constructs containing ~1 kb of the mouse IIa, IId/x, and IIb MyHC promoter sequences was described previously (27). Briefly, ~1 kb of the upstream promoter region for the IIa, IId/x, and IIb MyHC genes was subcloned into the firefly luciferase plasmid VR1255 (Vical). Site-directed mutagenesis of the MEF-2-binding AT-rich region 1, CArG-like box, and proximal NFAT site in the IIa MyHC promoter was accomplished using a mutagenesis kit (Stratagene). "Sensor" constructs containing multimerized copies of the MEF-2 and NFAT binding sites were kindly provided by Dr. Eric Olson, whereas the multimerized SRE construct was obtained from Stratagene. Overexpression constructs for MEF-2C, NFAT, SRF, HDAC4, HDAC5, and MEF-2-interacting transcription repressor (MITR) were also provided by Dr. Eric Olson, whereas the CMV-MEKK1 construct was obtained from Stratagene. A differentiation-sensitive MEKK1 construct was created by Dr. Ulrika Widegren by placing the cDNA for constitutively active MEKK1 under the control of the IId/x MyHC promoter.

Cell Culture Transfections-- Mouse C₂C₁₂ myoblasts (American Type Culture Collection) were grown on gelatin-coated dishes in Dulbecco's modified Eagle's medium (Invitrogen) and 10% fetal bovine serum (Hyclone). Cells were transfected at 70-80% confluence using LipofectAMINE transfection reagent according to the manufacturer's protocol (Invitrogen). In all experiments, the thymidine kinase-Renilla luciferase construct pRLTK (Promega) was co-transfected to correct for transfection efficiency. Myoblasts were switched to differentiation medium consisting of Dulbecco's modified Eagle's medium plus 1% horse serum 1-2 days post-transfection. After 2 days of differentiation, myotubes were either treated with Me₂SO vehicle alone or 0.4 µM of calcium ionophore A23187 (Sigma) for 1-2 days.

Luciferase Assays-- A commercially available dual luciferase assay system was used (Promega). Briefly, cells were lysed in 1× passive lysis buffer and 10 µl of the cell lysate was assayed for both firefly (MyHC promoter constructs and positive controls) and Renilla (internal control) luciferase activity using a standard luminometer. Values are reported as firefly luciferase divided by Renilla luciferase levels.

Western Blotting-- C₂C₁₂ myotubes were treated with either vehicle or A23187 for 1, 3, 6, 12, and 24 h and then scraped in lysis buffer stored at 70 °C until use. Protein concentration was determined using the Bradford assay (Bio-Rad) and was adjusted to 1 mg/ml. Equal protein concentrations were separated by gel electrophoresis using standard techniques. Proteins were transferred to polyvinylidene difluoride membrane using a miniblot transfer apparatus (Bio-Rad) and blocked for 1 h at room temperature in 5% nonfat dry milk, 1% normal goat serum, and 1% bovine serum albumin. Blots were incubated with a monoclonal antibody to the autophosphorylated form of CaM kinase II (Promega) for 1 h at room temperature, rinsed several times in phosphate-buffered saline containing 1% Tween 20 (PBS-T), and then incubated for 2 h with an alkaline phosphatase-conjugated goat anti-mouse secondary antibody (Promega). Binding was visualized using a 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium kit (Vector Laboratories). Bands were densitometrically scanned using NIH Image.

Mobility Shift Assays-- Nuclear extracts were isolated from differentiated control and A23187-treated C₂C₁₂ myotubes using standard techniques (28). Briefly, C₂C₁₂ myoblasts were grown to confluence and allowed to differentiate for 2 days. The medium was removed, and cells were rinsed in phosphate-buffered saline and then scraped into buffer A (10 mM HEPES, pH 7.9, 1.5 mM MgCl₂, 10 mM KCl, and EDTA-free protease inhibitor mixture) and centrifuged at 1400 × g for 10 min. The cells were resuspended in 5 volumes of buffer A, respun, and resuspended in 2 volumes of buffer A. Cells were lysed using a Dounce homogenizer A and centrifuged at 500 × g for 10 min. The pellet was centrifuged at 5000 × g for 20 min and resuspended in buffer C (20 mM HEPES, pH 7.9, 25% glycerol, 0.42 M NaCl, 1.5 mM MgCl₂, 0.2 mM EDTA, and protease inhibitor mixture) and homogenized with 3-5 strokes with a Dounce homogenizer type B. The resulting homogenate was stirred on ice for 30 min and then microcentrifuged at 12,000 rpm for 30 min. The supernatant was dialyzed against Buffer D (20 mM HEPES, pH 7.9, 20% glycerol, 0.1 M KCl, 0.2 mM EDTA, protease inhibitor mixture) for 5 h. The protein concentration was determined using the Bradford method (Bio-Rad). The resulting nuclear extract was stored at 70 °C until use. Oligonucleotides containing the AT-rich-1 region or the proximal NFAT region of the IIa promoter were labeled with [-³²P]dCTP and used for mobility shift reactions. Briefly, 100,000 cpm of labeled probe was added to 10 µg of nuclear extract in a reaction containing 1 µg of dI-dC and binding buffer (100 mM Tris, pH 7.5, 500 mM NaCl, 10 mM EDTA, 10 mM dithiothreitol, 12.5% Ficoll) for 30 min at room temperature. The reaction solution was then run on a 4% acrylamide gel at 300 V. The gel was then dried and placed on a PhosphorImager screen overnight.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Responsiveness of the MyHC Promoters to Intracellular Calcium-- We tested the responsiveness of the three adult fast MyHC promoters to increased intracellular calcium in C₂C₁₂ myotubes. Ionophore treatment for 36 h induced much greater activation of both human and mouse IIa MyHC promoters (~10-20-fold, respectively; Fig. 1, A and B) than the mouse IId/x and IIb MyHC promoters (Fig. 1C). Thus, the activity of the IIa MyHC promoter, which is associated with more oxidative fast fibers, was augmented to a greater extent by intracellular calcium than the IId/x and IIb MyHC promoters.

View larger version (23K): [in this window] [in a new window]   Fig. 1.   Activation of fast MyHC promoters by calcium ionophore. A, relative luciferase levels for 1000 bp of the human type IIa MyHC promoter and 670 bp of the mouse IIa MyHC promoter in control and A23187 ionophore-treated C2C12 myotubes. A23187 treatment resulted in a significant increase in the activity of both mouse and human promoters. All values are normalized to Renilla luciferase levels. B, -fold change in promoter activity following ionophore treatment. The human and mouse IIa promoters were induced 8-13-fold over untreated. C, activity of the mouse IIa, IId/x, and IIb MyHC promoters is increased by ionophore treatment. The mouse adult skeletal fast MyHC promoters were differentially activated by A23187 treatment, with IIa IId/x > IIb. D, shorter durations (1 or 4 h) of ionophore treatment also stimulate IIa MyHC promoter activity. All bars are the mean ± S.E. of 3-5 experiments. *, significantly different from unstimulated control, p < 0.05; , significantly different from IIb, p < 0.05.

IIa MyHC Promoter Activity Is Increased by Shorter Durations of Ionophore Treatment-- We wished to examine whether IIa MyHC promoter activity could be increased in response to shorter periods of ionophore stimulation compared with the 36-h treatment used in Fig. 1. We used 1 and 4 h of ionophore stimulation and examined IIa MyHC promoter activity at several time points poststimulation. Ionophore stimulation of C₂C₁₂ myotubes for these shorter periods resulted in a dose- and time-dependent increase in IIa MyHC promoter activity (Fig. 1D). With 1 h of ionophore stimulation, IIa promoter activity was not significantly activated at 0 or 1 h poststimulation but was significantly increased at 3 and 6 h poststimulation, increasing to about 2-fold over the unstimulated control (Fig. 1D). By 12 h, IIa MyHC promoter activation was decreasing, and by 24 h post-stimulation IIa MyHC promoter activity had returned to base-line values (Fig. 1D). With 4 h of ionophore stimulation, IIa MyHC promoter activity was significantly increased immediately upon cessation of ionophore stimulation and increased thereafter, reaching a peak of ~4-fold stimulation over control at 6 h poststimulation (Fig. 1D). After 12 h poststimulation, IIa MyHC promoter activity was decreasing but was still ~2-fold elevated over control even at 24 h poststimulation. Thus, stimulation for 4 h produced a more rapid and higher peaking response compared with 1 h of ionophore stimulation and did not fully return to base line even 24 h after stimulation.

Calcineurin and CaM Kinase II Mediate Ionophore-induced IIa MyHC Promoter Activation-- Increased intracellular calcium activates at least three major signaling pathways: the calcineurin, CaM kinase, and protein kinase C pathways. We used pharmacological inhibitors for each of these pathways to determine their role in ionophore-induced activation of the IIa MyHC promoter. In addition, we tested the effects of calcineurin on endogenous MyHC IIa expression. Treatment with cyclosporin A, an inhibitor of calcineurin, significantly attenuated ionophore activation of the IIa MyHC promoter in C₂C₁₂ myotubes (Fig. 2A), whereas overexpression of a constitutively activated form of calcineurin increased endogenous IIa MyHC protein expression (Fig. 2, B-D). Treatment of myotubes with KN62, an inhibitor of CaM kinase II, also significantly attenuated ionophore-induced IIa MyHC promoter activity (Fig. 3A). Following activation of CaM kinase II by calcium and calmodulin, the enzyme autophosphorylates, allowing it to remain active independent of calcium levels. Ionophore treatment also resulted in a moderate increase in the autophosphorylation of CaM kinase II at 1-3 h of ionophore treatment, but autophosphorylation was decreased at longer treatment durations (Fig. 3B). In contrast, inhibitors of protein kinase C activity had no significant effect on ionophore-induced activation of the IIa MyHC promoter (Fig. 4). Staurosporine treatment tended to decrease ionophore-induced IIa MyHC promoter activation, but the effect was highly variable and was not statistically significant; the largest effect was seen at the highest concentrations of staurosporine, which is less specific at high concentrations (Fig. 4A). We therefore examined the effect of a more specific protein kinase C inhibitor, chelerythrine, on ionophore-induced IIa MyHC promoter activation. Treatment with chelerythrine had no effect on ionophore-induced IIa MyHC promoter activity (Fig. 4B), confirming that protein kinase C is not involved in increasing IIa MyHC promoter activity in response to increased intracellular calcium. Together these data support a role for calcineurin and CaM kinase II but not protein kinase C in calcium-induced activation of the IIa MyHC promoter.

View larger version (34K): [in this window] [in a new window]   Fig. 2.   Role of calcineurin in IIa MyHC gene expression. A, C2C12 myotubes transfected with the IIa MyHC promoter construct were treated with ionophore and with and different concentrations of the calcineurin inhibitor cyclosporin A. Data are expressed as a percentage of the maximum response of the IIa MyHC promoter to A23187 without cyclosporin A. Cyclosporin significantly inhibited ionophore induction of IIa MyHC promoter activity even at the lowest concentration tested (125 nM). *, significantly different from ionophore treated alone, p < 0.05. B, overexpression of a constitutively active calcineurin gene significantly augments endogenous IIa MyHC expression. The number of IIa-positive myotubes was assessed using immunohistochemistry in five different regions of four wells each in three experiments. Other wells were stained with antibodies to all sarcomeric MyHC isoforms (F59), and the total number of MyHC-positive myotubes was used to determine the percentage of IIa-positive myotubes of the total. Ca-CN, constitutively active calcineurin. , significantly different from -galactosidase-transfected, p < 0.05. C, immunohistochemical staining for IIa MyHC of -galactosidase-transfected well. D, immunohistochemical staining of constitutively active calcineurin field, demonstrating a higher percentage of IIa-positive myotubes.

View larger version (10K): [in this window] [in a new window]   Fig. 3.   CaM kinase pathway and ionophore induction of IIa MyHC promoter activity. A, C2C12 myotubes transfected with the IIa MyHC promoter construct were treated with A23187 plus different concentrations of KN62, an inhibitor of CaM kinase II. Data are expressed as a percentage of the response due to treatment with A23187 alone without KN62 co-treatment. KN62 inhibited A23187 induction of the IIa promoter in a dose-dependent manner. *, significantly different from ionophore treated alone, p < 0.05. B, Western blotting for the autophosphorylated form of CaM kinase II. The top shows a representative Western blot. Bottom, quantification of densitometric scanning of each band.

View larger version (12K): [in this window] [in a new window]   Fig. 4.   Protein kinase C and IIa MyHC promoter calcium sensitivity. A, C2C12 myotubes transfected with the IIa MyHC promoter construct and treated with A23187 plus different concentrations of staurosporine, an inhibitor of protein kinase C. Data are reported as a percentage of the response due to treatment with A23187 alone without staurosporine co-treatment. Staurosporine treatment tended to decrease IIa activation in response to calcium ionophore A23187, but the decrease was variable and not significant. *, significantly different from ionophore treated alone, p < 0.05. B, effect of treatment with a more specific inhibitor of protein kinase C, chelerythrine, at 1 µM. Chelerythrine had no effect on ionophore induction of the IIa MyHC promoter.

MAP Kinase Pathways and Ionophore-induced IIa Promoter Activity-- We also investigated the role of the MAP kinase pathways in ionophore-induced IIa MyHC promoter activation. Treatment of C₂C₁₂ myotubes with UO126 or PD098059, inhibitors of the extracellular signal-regulated kinase pathway activators MEK1 and MEK1/2, respectively, significantly attenuated ionophore stimulation of IIa MyHC promoter activity, whereas SB230580, an inhibitor of p38, and wortmannin, a phosphatidylinositol 3-kinase inhibitor, did not (Fig. 5A). We also examined the effects of overexpression of MEKK1, an activator of the c-Jun N-terminal kinase pathway, on ionophore-induced IIa MyHC activation using both a viral (CMV) and a differentiation-specific (IId/x MyHC) promoter to drive MEKK1 expression. Co-transfection with CMV-MEKK1 almost completely abolished ionophore-induced IIa MyHC promoter activation, whereas co-transfection with IId/x-MEKK1 augmented ionophore-induced IIa MyHC promoter activity (Fig. 5B). Transfection of C₂C₁₂ myoblasts with the CMV-MEKK1 also resulted in a dramatic attenuation of myoblast differentiation into myotubes (Fig. 5D), whereas IId/X-MEKK1 had no effect on differentiation (Fig. 5E). Thus, the effect of MEKK1 on ionophore-induced promoter activity appeared to be differentiation-dependent; CMV-MEKK1 attenuated ionophore-induced IIa MyHC promoter activity indirectly by suppressing differentiation, whereas IId/x-MEKK1 did not affect myoblast differentiation and augmented IIa promoter activity, most likely through differentiation-independent mechanisms.

View larger version (37K): [in this window] [in a new window]   Fig. 5.   MAP kinase pathways and ionophore-induced IIa MyHC promoter activity. A, treatment with specific MAP kinase pathway inhibitors and ionophore-induced IIa MyHC promoter activity. C2C12 myotubes transfected with the IIa MyHC promoter construct were treated with A23187 plus various pharmacological inhibitors. Treatment with PD095059 (50 µM) or UO126 (10 mM), which inhibit MEK1 and MEK1 and MEK2, respectively, significantly attenuated ionophore-induced IIa MyHC promoter activation, whereas SB203580 (20 µM) and wortmannin (1 mM), inhibitors of p38 and phosphatidylinositol 3-kinase, had no effect. *, significantly different from ionophore treated alone, p < 0.05. B, effects of MEKK1 on ionophore-induced IIa MyHC promoter activation. C2C12 myotubes were transfected with the IIa MyHC promoter construct and either a CMV-B-gal construct, a CMV-MEKK1 construct, or a IId/x-MEKK1 construct and treated with A23187. Co-transfection with either CMV-MEKK1 or IId/x-MEKK1 had minimal effects on IIa MyHC promoter activity when treated with vehicle alone. In contrast, CMV-MEKK1 co-transfection completely abolished IIa responsiveness to A23187 treatment, whereas IId/x-MEKK1 co-transfection augmented A23187 activation of the IIa MyHC promoter. *, significantly different from unstimulated control, p < 0.05; , significantly different from CMV-MEKK1, p < 0.05; , significantly different from -galactosidase-transfected, p < 0.05. C-E, immunofluorescence of C2C12 myotubes transfected with either CMV--galactosidase (C), CMV-MEKK1 (D), or IId/x-MEKK1 (E). Myotubes were stained with F59, an antibody that recognizes all sarcomeric isoforms of MyHC, and counterstained with 4',6-diamidino-2-phenylindole. Transfection with CMV-MEKK1 results in a significant attenuation of myotube differentiation, whereas IId/x-MEKK1 has no effect on differentiation.

MEF and NFAT but Not SRF Augment Ionophore-induced IIa Promoter Activity-- We next examined the role of three transcription factor families implicated in calcium signaling pathways (MEF-2, NFAT, and SRF) on IIa MyHC promoter activation by ionophore treatment. Putative binding sites for all three factors are present in the IIa upstream promoter region (27). Co-transfection of MEF-2C or NFAT3 without ionophore treatment resulted in a small but consistent augmentation of IIa MyHC promoter activity (Fig. 6, A and B), as reported previously (27), whereas co-transfection with SRF without ionophore treatment resulted in a 3-fold increase in IIa MyHC promoter activity (Fig. 6C). When combined with ionophore treatment, co-transfection of MEF-2C or NFAT3 resulted in a significant enhancement compared with ionophore treatment alone (Fig 6, A and B). In contrast, co-transfection with SRF did not alter ionophore-induced IIa MyHC promoter activity when compared with treatment with ionophore alone (Fig. 6C).

View larger version (20K): [in this window] [in a new window]   Fig. 6.   Transcription factor co-transfection and ionophore-induced IIa MyHC promoter activation. C2C12 myotubes were co-transfected with the IIa promoter construct and CMV promoter-driven expression plasmids for MEF-2C (A), NFAT3 (B), or SRF (C). -Galactosidase co-transfection was used as a control. Co-transfection with MEF-2C or NFAT3 without ionophore treatment resulted in a modest (50-90%) increase in IIa promoter activity, whereas SRF increased IIa MyHC promoter activity by ~3-4-fold (C). Co-transfection with MEF-2C or NFAT3 significantly potentiated IIa MyHC promoter activation in response to calcium ionophore, whereas co-transfection with SRF did not affect ionophore-induced IIa MyHC promoter activity. D, co-transfection with HDAC4, HDAC5, or MITR all attenuated A23187 activation of the IIa MyHC promoter. *, significantly different from unstimulated control, p < 0.05; , significantly different from -galactosidase-transfected plus ionophore treatment, p < 0.05.

Activation of MEF-2 transcription factors by CaM kinase is thought to be mediated through phosphorylation and removal of class II HDACs from the nucleus (29). We therefore examined the role of HDAC co-transfection on ionophore activation of the IIa MyHC promoter. Co-transfection with HDAC4, HDAC5, or MITR resulted in a significant attenuation of ionophore-induced IIa MyHC activation (Fig. 6D). Together with the MEF-2 co-transfection data, these results support a role for MEF-2 as well as NFAT in ionophore-induced IIa MyHC promoter activation.

MEF-2 and NFAT Binding Sites Are Necessary for Ionophore-induced IIa Promoter Activation-- We examined whether the effect of MEF-2 and/or NFAT was a direct one due to binding of these transcription factors to the IIa MyHC promoter or was an indirect consequence of MEF-2 and/or NFAT activation of other genes. To answer this question, we used "sensor" constructs containing multimerized consensus binding sites for MEF-2, NFAT, and SRF. Consistent with the co-transfection data, activity of the MEF-2 and NFAT sensor constructs was significantly increased by ionophore treatment, but SRF sensor activity was not (Fig. 7A). Together, these data strongly support a role for MEF-2 and NFAT but not SRF in ionophore-induced activation of the IIa MyHC promoter.

View larger version (25K): [in this window] [in a new window]   Fig. 7.   MEF-2 and NFAT binding are critical to ionophore-induced IIa MyHC promoter activation. A, activation of sensor constructs in response to ionophore treatment. Multimerized copies of the MEF-2, NFAT, or SRF consensus binding sites linked to -galactosidase or luciferase reporter genes were used to assess the effects of calcium ionophore A23187 on signaling through these sites. Ionophore treatment increased MEF-2 and NFAT sensor construct activation but not SRF sensor construct activity. B, role of specific binding sites in the IIa MyHC promoter on ionophore-induced IIa promoter activity. The schematic at the left shows the three binding sites within the proximal 200 bp of the IIa MyHC promoter. All elements were mutated in the context of the 670-bp IIa MyHC promoter. Mutation or deletion of the MEF-2 binding site or the NFAT binding site attenuated IIa MyHC promoter activation by ~50%, whereas mutation of the CArG-like element had no effect. * (in both A and B), significantly different from unstimulated control, p < 0.05.

We used mutagenesis to alter the putative binding sites for these factors within the 677-bp IIa MyHC promoter and transfected C₂C₁₂ myoblasts with these constructs to determine their role in ionophore-induced activation. Mutation or internal deletion of the MEF-2-binding AT-rich site or mutation of the proximal NFAT site resulted in a significant attenuation of ionophore-induced IIa MyHC promoter activation (Fig. 7B). Deletion of the MEF-2 site and mutation of the NFAT site in the same construct did not further attenuate ionophore-induced IIa MyHC promoter activity (Fig. 7B), suggesting that other sites contribute to this activation. Mutation of the CArG-like site had no effect on ionophore-induced activation of the IIa MyHC promoter (Fig. 7B).

We examined whether ionophore treatment resulted in changes in the binding activity of MEF-2 and/or NFAT proteins to their respective binding sites on the IId promoter. Mobility shift assays using oligonucleotides containing the MEF-2-binding AT-rich-1 region of the IIa MyHC promoter demonstrated increased formation of three complexes with nuclear extract proteins isolated from ionophore-treated C₂C₁₂ myotubes compared with extract from control C₂C₁₂ myotubes (Fig. 8A). Based on previous research (30),² the upper binding activity is actually a doublet likely to represent binding of MEF-2 family members in the uppermost band, whereas the slightly lower band represents Oct-1 binding. The addition of a polyclonal antibody against MEF-2 results in a supershift of the uppermost band in the complex (Fig. 8A, lane 4, Iono Ab). The third, lower complex probably represents a palindrome-binding protein (Pal-BP) previously identified by Lakich et al. (30). Binding of all three protein complexes is greater in ionophore-treated myotube nuclear extract compared with untreated myotube nuclear extracts (Fig. 8A). Mobility shift assays using oligonucleotides containing the proximal NFAT binding region showed much lower binding activity than the MEF-2 oligonucleotides; Fig. 8B is taken at a much higher exposure than Fig. 8A, suggesting that NFAT binding is much lower than MEF-2 binding in both control and ionophore-treated myotubes. Based on previously published studies on NFAT binding, it is likely that the uppermost binding activity is NFAT, whereas the lower band represents nonspecific binding activity (31). The binding activity of NFAT is increased in ionophore-treated myotube nuclear extract compared with untreated myotube nuclear extract, but the difference is not as great as that for MEF-2 (Fig. 8B). Finally, we used an oligonucleotide containing a consensus binding site for the ubiquitously expressed transcription factor Sp1 to examine the specificity this response. In contrast to the results using the AT-rich region or proximal NFAT site, binding of Sp1 was not increased by ionophore treatment and, in fact, was slightly higher for the control myotube extract (Fig. 8C).

View larger version (56K): [in this window] [in a new window]   Fig. 8.   Mobility shift assays for the MEF-2 and NFAT binding sites. A, mobility shift assays using an oligonucleotide containing the AT-rich-1 MEF-2 binding site in the IIa MyHC promoter. Lane P, probe alone; lane C, unstimulated myotube nuclear extract; Iono, ionophore-treated myotube nuclear extract; Iono Ab, ionophore-treated myotube extract plus polyclonal antibody to either MEF-2 or NFAT. Ionophore treatment results in an increase in MEF-2 binding activity. B, mobility shift assays using an oligonucleotide containing the proximal NFAT binding site in the IIa MyHC promoter. Lanes are the same as in A.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

One of the most prominent signals believed to be involved in regulating fiber-specific gene expression is intracellular calcium levels. In the present work, we determined whether expression of the adult fast skeletal MyHC genes is sensitive to increases in intracellular calcium. Of the three adult fast MyHC promoters, IIa showed the greatest response, increasing by up to 20-fold, followed by IId/x and then IIb (Fig. 1C). The increase in IId/x MyHC promoter activity is in contrast to work by Meisner et al. (22), who demonstrated a decrease in IId/x MyHC RNA and protein in response to prolonged ionophore treatment of myotubes in vitro. In this earlier study, myotubes were stimulated with ionophore for extended periods of time (2 weeks), whereas in the present study we used much shorter durations of ionophore treatment (~36 h). Thus, at least part of the difference between the results of Meisner et al. (22) and the present work may be the difference in treatment duration; the IId/x MyHC gene may be activated during the early phase of adaptation to increased intracellular calcium levels but may eventually be down-regulated as myotubes shift toward a more oxidative phenotype.

Previous studies have demonstrated that overexpression of calcineurin in cultured myotubes increases slow MyHC protein levels and either does not change or decreases fast MyHC expression (23, 24). However, the three different fast MyHC subtypes were not distinguished in those studies, so it cannot be determined whether there were differential shifts within the fast fiber subtypes. Type IIb and/or IId/x MyHC could have decreased while type IIa MyHC was increasing in these studies. Previously, we demonstrated that constitutively active calcineurin preferentially activated the IIa MyHC promoter, increasing it by 50-200-fold compared with 5-10-fold for the IId/x and IIb promoters (27). Here we demonstrate that calcineurin overexpression also increases expression of endogenous IIa MyHC protein (Fig. 2, B-D). Furthermore, the IIa MyHC promoter is preferentially activated to a greater extent than the other two fast MyHC promoters by intracellular calcium levels in a calcineurin-dependent manner (Fig. 2A). These data suggest that calcium-calcineurin signaling may be involved in the shift from IId/x and IIb MyHC toward isoforms of MyHC associated with more oxidative fibers (i.e. I and IIa) that are better suited to meet the work demands involved in prolonged muscle activation.

Studies using transgenic mice have suggested that the calcium-activated calcineurin-NFAT-MEF-2 and CaM kinase-MEF-2-HDAC signaling pathways are involved in fast-to-slow fiber type transitions (16-19). However, it was unclear from these studies whether these factors activated type I expression or were involved in transitions between fast subtypes. Work in vivo using calcineurin inhibitors has suggested that inhibition of calcineurin causes a shift from type I to type IIa MyHC expression only (25), but this was done in the rat soleus muscle, which is normally 80-100% type I, and may not possess the full range of adaptability across all fast fiber types. The present work demonstrates that intracellular calcium and calcineurin are involved in up-regulating IIa MyHC expression and thus may be involved in the transition from IIb or IId/x fibers to IIa. Consistent with this hypothesis, calcineurin-MEF-2 signaling was most active in the fastest, least oxidative muscle fibers in vivo (i.e. IId/x and IIb) during adaptation to increased usage (32), whereas treatment of rat soleus with the endogenous calcineurin inhibitor Cain shifted MyHC expression from type I to type IId/x and IIb (26).

We examined the role of two additional pathways activated by increased intracellular calcium levels on MyHC promoter activity: CaM kinase and protein kinase C. Our results show that CaM kinase II plays an early role in IIa MyHC promoter activation (Fig. 3). In contrast, neither staurosporine nor chelerythrine, two different protein kinase C inhibitors, had a significant effect on IIa MyHC promoter induction by ionophore treatment (Fig. 4). Protein kinase C has previously been shown to inhibit slow MyHC expression in cultured chick myotubes in a muscle-specific manner but did not appear to affect fast MyHC expression (33), consistent with the present results.

We also showed that MEK1 and MEK2 play a role in IIa MyHC promoter activation in response to ionophore treatment. The MAP kinase signaling pathways play critical roles in many cell functions but are particularly prominent in cell proliferation. However, since >80-90% of the cells were in myotubes prior to treatment with ionophore or MEK inhibitors, it is unlikely that the results were due to effects of the MEK1/2 inhibitors on cell proliferation. These data therefore suggest that the MEK1/2-extracellular signal-regulated kinase pathway may regulate calcium activation of the IIa MyHC gene within differentiated muscle cells. However, the extracellular signal-regulated kinase kinases are highly active in both proliferating myoblasts and in fully differentiated myotubes, consistent with a role for this pathway in postdifferentiation muscle gene expression (34). In addition, Ras and Raf, which act upstream of MEK1 and 2, induce slow MyHC expression in regenerating soleus muscle in vivo (35).

MEKK1 is an activator of the c-Jun N-terminal kinase pathway. Inhibition of differentiation by CMV-MEKK1 resulted in the loss in calcium sensitivity of the IIa promoter, which in turn suggests that molecules specific to differentiated myotubes/myofibers are necessary for ionophore induction of the IIa MyHC gene. When the effects of MEKK1 were separated from its effects on cell proliferation using the differentiation-activated IId/x MyHC promoter to drive MEKK1 expression, MEKK1 actually potentiated ionophore-induced IIa activity, suggesting that the c-Jun N-terminal kinase pathway may be involved in IIa MyHC activation in differentiated myotubes.

In contrast, neither p38 nor phosphatidylinositol 3-kinase appears to play a role in the activation of the IIa MyHC promoter. The lack of a role for p38 was somewhat surprising, given that p38 stimulates transactivation activity of MEF-2 family members (36). Our results are also at odds with those of Delling et al. (23), who showed that transfection of C₂C₁₂ myotubes with a MKK6 construct, an activator of p38, results in up-regulation of fast MyHC protein expression. Several isoforms of p38 are expressed in muscle, and there has been some suggestion that SB203580 may not inactivate all p38 isoforms (36), which may explain the effects observed in the present work.

We present several pieces of data suggesting that the MEF-2 and NFAT families of transcription factors are involved in activating the IIa MyHC promoter in response to ionophore treatment. Together with the data on the role of the calcineurin-NFAT-MEF-2 pathway on slow fiber gene expression (16-18), these data are consistent with the hypothesis that MEF-2 and NFAT transcription factors are involved in the activation of genes associated with a more oxidative phenotype such as type I and type IIa MyHC. In contrast, we found no evidence to suggest that SRF is associated with ionophore-induced IIa MyHC promoter activation. The SRF transcription factor has been implicated in calcium-induced shifts in neuronal and myocardial gene transcription (4, 6, 7). SRF is also expressed in a loading-dependent manner in skeletal muscle (37, 38) and activates several muscle-specific genes, including dystrophin (39) and -skeletal actin (40). In addition, SRF is phosphorylated by CaM kinase II at several sites (41). Nonetheless, SRF does not appear to be critical for IIa MyHC promoter activation in response to ionophore treatment.

It has been proposed that changes in the expression of activity-sensitive muscle genes may result from the cumulative effect of repeated exercise bouts on signaling pathways leading to transient increases in gene expression; when these bouts are repeated at regular intervals as during a training regimen, these transient increases in RNA levels reach a threshold beyond which changes in protein levels occur that confer phenotypic changes upon the muscle (42). To date, this has been demonstrated most effectively for metabolic genes such as the GLUT-4 glucose transporter and various mitochondrial genes (42, 43). Endurance exercise has been shown to activate calcineurin-MEF-2 signaling in vivo (44), whereas marathon running in humans also transiently induces the c-Jun N-terminal kinase and extracellular signal-regulated kinase signaling pathways (45, 46). In the studies above, we stimulated C₂C₁₂ myotubes with calcium ionophore for 36 h in order to examine the maximal response to increased intracellular calcium. However, to determine whether durations of increased intracellular calcium that might more closely parallel bouts of exercise could also stimulate IIa MyHC promoter activity, we treated myotubes for either 1 or 4 h and found that this resulted in a transient increase in IIa MyHC promoter activity that was dose- and time-dependent (Fig. 1D). Treatment with ionophore for 1 h resulted in a transient 2-fold increase in IIa MyHC promoter activity that returned to normal by 24 h poststimulation. Treatment for 4 h resulted in a much greater increase in IIa MyHC promoter activity, and activity remained elevated even 24 h poststimulation. These data show that shorter stimulation durations also result in up-regulation of IIa MyHC expression and are consistent with the hypothesis that transient changes in gene expression are a component of the adaptation of skeletal muscle to increased usage.

	ACKNOWLEDGEMENTS

We thank Dr. Eric Olson for the generous gift of various expression constructs, Priya Kumar for assistance with the calcineurin transfection experiments, and Dr. Ulrika Widegren for assistance in cloning the IId/x-MEKK1 construct.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant RO1-GM29090 (to L. A. L.) and an MDA research fellowship grant (to D. L. A.).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.

To whom correspondence should be addressed: Dept. of Molecular, Cellular, and Developmental Biology, University of Colorado, Campus Box 347, Boulder, CO 80309-0347. Tel.: 303-492-8371; Fax: 303-492-8907; E-mail: allendl@stripe.colorado.edu.

Published, JBC Papers in Press, September 15, 2002, DOI 10.1074/jbc.M208302200

² D. L. Allen and L. A. Leinwand, unpublished results.

	ABBREVIATIONS

The abbreviations used are: SRF, serum response factor; MyHC, myosin heavy chain; MEF-2, myocyte-specific enhancer factor 2; NFAT, nuclear factor/activator of T cells; CMV, cytomegalovirus; CaM kinase, calcium-calmodulin kinase; HDAC, histone deacetylase; MAP, mitogen activated protein; MEKK1, mitogen-extracellular kinase kinase-1.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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