A calcineurin-dependent transcriptional pathway controls skeletal muscle fiber type

Abstract

Slow- and fast-twitch myofibers of adult skeletal muscles express unique sets of muscle-specific genes, and these distinctive programs of gene expression are controlled by variations in motor neuron activity. It is well established that, as a consequence of more frequent neural stimulation, slow fibers maintain higher levels of intracellular free calcium than fast fibers, but the mechanisms by which calcium may function as a messenger linking nerve activity to changes in gene expression in skeletal muscle have been unknown. Here, fiber-type-specific gene expression in skeletal muscles is shown to be controlled by a signaling pathway that involves calcineurin, a cyclosporin-sensitive, calcium-regulated serine/threonine phosphatase. Activation of calcineurin in skeletal myocytes selectively up-regulates slow-fiber-specific gene promoters. Conversely, inhibition of calcineurin activity by administration of cyclosporin A to intact animals promotes slow-to-fast fiber transformation. Transcriptional activation of slow-fiber-specific transcription appears to be mediated by a combinatorial mechanism involving proteins of the NFAT and MEF2 families. These results identify a molecular mechanism by which different patterns of motor nerve activity promote selective changes in gene expression to establish the specialized characteristics of slow and fast myofibers.

Figure 1

Response of different promoters to forced expression of a constitutively active form of calcineurin (O’Keefe et al. 1992) in cultured C2C12 myotubes or NIH-3T3 fibroblasts. Promoter–reporter plasmids were constructed to link the indicated promoters: (CMV) cytomegalovirus; (TATA) a minimal promoter consisting of the TATA element from the human hsp70 gene; (MCK) a 4.8-kb 5′ flanking region from the murine muscle creatine kinase gene; (TnI slow) a 4.2-kb 5′ flanking region from the human slow-fiber-specific troponin I gene; (myoglobin) a 2-kb 5′ flanking region from the human myoglobin gene to a firefly luciferase reporter gene. The response to activated calcineurin was calculated as the fold change in luciferase activity induced by activated calcineurin above that measured following transfection of the empty vector alone, corrected for transfection efficiency (β-galactosidase activity). (Open bars) NIH-3T3 cells; (solid bars) C2C12 cells; (hatched bars) C2C12 + CsA. Cyclosporin A (CsA) was added to the culture medium at the indicated final concentrations. Histograms depict mean values (±s.e.) from 4–8 independent transfections in each cell background.

Figure 2

Role of NFAT proteins in calcineurin-dependent transactivation. Activity of wild-type and mutated myoglobin (A) or troponin I slow (B) gene promoters in differentiated C2C12 cells as a function of increasing doses of the calcineurin-expression plasmid. Consensus NFAT recognition motifs at the indicated positions relative to the transcriptional start sites (see Fig. 6) were altered (ΔNFAT) by site-directed mutagenesis, and transfections were performed as described in Figure 1. Data points represent mean values of luciferase activity, corrected for transfection efficiency (β-galactosidase activity), from duplicate transfections in a representative experiment, and expressed as a percentage of native promoter activity after transfection with the indicated amounts of activated calcineurin expression plasmid (CMV–CnA*). (A) (○) 2 kb of myoglobin; (•) ΔNFAT myoglobin (−690 and −232). (B) (○) 4.2 kb TnIs; (•) ΔNFAT TnIs (−743 and −683). (C) Activated calcineurin promotes nuclear translocation of NFAT proteins. C2C12 cells were transfected with plasmids expressing native GFP (upper left), a truncated variant of NFATc fused to GFP (ΔNFATc–GFP) that removes the amino-terminal regulatory domain controlled by calcineurin (upper right), or full-length NFATc fused to GFP (NFATc–GFP) in the absence (lower left) or presence (lower right) of activated calcineurin.

Figure 2

Role of NFAT proteins in calcineurin-dependent transactivation. Activity of wild-type and mutated myoglobin (A) or troponin I slow (B) gene promoters in differentiated C2C12 cells as a function of increasing doses of the calcineurin-expression plasmid. Consensus NFAT recognition motifs at the indicated positions relative to the transcriptional start sites (see Fig. 6) were altered (ΔNFAT) by site-directed mutagenesis, and transfections were performed as described in Figure 1. Data points represent mean values of luciferase activity, corrected for transfection efficiency (β-galactosidase activity), from duplicate transfections in a representative experiment, and expressed as a percentage of native promoter activity after transfection with the indicated amounts of activated calcineurin expression plasmid (CMV–CnA*). (A) (○) 2 kb of myoglobin; (•) ΔNFAT myoglobin (−690 and −232). (B) (○) 4.2 kb TnIs; (•) ΔNFAT TnIs (−743 and −683). (C) Activated calcineurin promotes nuclear translocation of NFAT proteins. C2C12 cells were transfected with plasmids expressing native GFP (upper left), a truncated variant of NFATc fused to GFP (ΔNFATc–GFP) that removes the amino-terminal regulatory domain controlled by calcineurin (upper right), or full-length NFATc fused to GFP (NFATc–GFP) in the absence (lower left) or presence (lower right) of activated calcineurin.

Figure 2

Role of NFAT proteins in calcineurin-dependent transactivation. Activity of wild-type and mutated myoglobin (A) or troponin I slow (B) gene promoters in differentiated C2C12 cells as a function of increasing doses of the calcineurin-expression plasmid. Consensus NFAT recognition motifs at the indicated positions relative to the transcriptional start sites (see Fig. 6) were altered (ΔNFAT) by site-directed mutagenesis, and transfections were performed as described in Figure 1. Data points represent mean values of luciferase activity, corrected for transfection efficiency (β-galactosidase activity), from duplicate transfections in a representative experiment, and expressed as a percentage of native promoter activity after transfection with the indicated amounts of activated calcineurin expression plasmid (CMV–CnA*). (A) (○) 2 kb of myoglobin; (•) ΔNFAT myoglobin (−690 and −232). (B) (○) 4.2 kb TnIs; (•) ΔNFAT TnIs (−743 and −683). (C) Activated calcineurin promotes nuclear translocation of NFAT proteins. C2C12 cells were transfected with plasmids expressing native GFP (upper left), a truncated variant of NFATc fused to GFP (ΔNFATc–GFP) that removes the amino-terminal regulatory domain controlled by calcineurin (upper right), or full-length NFATc fused to GFP (NFATc–GFP) in the absence (lower left) or presence (lower right) of activated calcineurin.

Figure 3

Upstream regulatory elements of the myoglobin gene participating in calcineurin-dependent transactivation. Data are presented as reporter–gene expression (mean ± s.e.m. of six independent transfections) normalized to activity of a cotranfected CMV–lacZ plasmid [luminometer units (×10⁵)/well (1.9 × 10⁵ cells)]. (A) Responses of native (Mb380) or mutated variants of a truncated segment (−373 to +7) of the human myoglobin gene promoter to activated calcineurin. Nucleotide substitutions were introduced into each of two upstream regulatory elements shown previously to be essential for muscle-specific promoter activity (Devlin et al. 1989; Bassel-Duby et al. 1993; Grayson et al. 1995, 1998). These mutated promoters (MbΔA/T and MbΔCCAC) are likewise defective for calcineurin-stimulated transactivation. (Stippled box) −CnA*; (solid bars) +CnA*. (B) Responses to activated calcineurin of synthetic promoters constructed with various combinations of multimerized oligonucleotide cassettes representing protein-binding motifs (CCAC) Sp1 binding site; (A/T) MEF2 binding site; (NRE) putative NFAT binding site; (TATA) TBP binding site and core promoter from the myoglobin promoter.

Figure 3

Upstream regulatory elements of the myoglobin gene participating in calcineurin-dependent transactivation. Data are presented as reporter–gene expression (mean ± s.e.m. of six independent transfections) normalized to activity of a cotranfected CMV–lacZ plasmid [luminometer units (×10⁵)/well (1.9 × 10⁵ cells)]. (A) Responses of native (Mb380) or mutated variants of a truncated segment (−373 to +7) of the human myoglobin gene promoter to activated calcineurin. Nucleotide substitutions were introduced into each of two upstream regulatory elements shown previously to be essential for muscle-specific promoter activity (Devlin et al. 1989; Bassel-Duby et al. 1993; Grayson et al. 1995, 1998). These mutated promoters (MbΔA/T and MbΔCCAC) are likewise defective for calcineurin-stimulated transactivation. (Stippled box) −CnA*; (solid bars) +CnA*. (B) Responses to activated calcineurin of synthetic promoters constructed with various combinations of multimerized oligonucleotide cassettes representing protein-binding motifs (CCAC) Sp1 binding site; (A/T) MEF2 binding site; (NRE) putative NFAT binding site; (TATA) TBP binding site and core promoter from the myoglobin promoter.

Figure 4

Fiber composition of soleus muscles from intact rats treated with cyclosporin A. Myosin ATPase activity determined by pH-dependent histochemistry distinguishes slow (darkly stained) and fast (unstained) fibers in sections of soleus muscle from vehicle-treated (A) and cyclosporin A-treated (B) rats. Immunohistochemistry using an antibody raised against fast myosin heavy chain identifies fibers expressing fast myosin (red) in sections of soleus muscle from vehicle-treated (C) and cyclosporin A-treated (D) rats. Nuclei are stained blue. (Bar, 200 μm). (E) Circles represent individual animals. (○) Vehicle treated; (•) cyclosporin A treated and mean values in each group (± s.e.) are shown as horizontal lines. The difference in group means was highly significant (P < 0.001 by unpaired Student’s t-test).

Figure 4

Fiber composition of soleus muscles from intact rats treated with cyclosporin A. Myosin ATPase activity determined by pH-dependent histochemistry distinguishes slow (darkly stained) and fast (unstained) fibers in sections of soleus muscle from vehicle-treated (A) and cyclosporin A-treated (B) rats. Immunohistochemistry using an antibody raised against fast myosin heavy chain identifies fibers expressing fast myosin (red) in sections of soleus muscle from vehicle-treated (C) and cyclosporin A-treated (D) rats. Nuclei are stained blue. (Bar, 200 μm). (E) Circles represent individual animals. (○) Vehicle treated; (•) cyclosporin A treated and mean values in each group (± s.e.) are shown as horizontal lines. The difference in group means was highly significant (P < 0.001 by unpaired Student’s t-test).

Figure 5

Model for a calcineurin-dependent pathway linking specific patterns of motor nerve activity to distinct programs of gene expression that establish phenotypic differences between slow and fast myofibers. MEF2 is shown to represent the requirement for collaboration between activated NFAT proteins and muscle-restricted transcription factors in slow-fiber-specific gene transcription, but other proteins (not shown) also are likely to participate.

Figure 6

NFAT consensus binding sequences are present within transcriptional control regions shown previously to direct transcription selectively in slow-oxidative myofibers (Parsons et al. 1993; Levitt et al. 1995; Qin et al. 1997). (A) Consensus NFAT binding motifs in myoglobin, troponin I slow (TnI slow), and sarcomeric mitochondrial creatine kinase (sMtCK) promoters. (B) Conserved sequence blocks (CAGG, CCAC, MEF2, and E box) common to a SURE from the rat troponin I slow gene and a FIRE from the quail troponin I fast gene (Nakayama et al. 1996). A predicted NFAT response element (darkly shaded) overlapping the E-box is a unique feature of the SURE element.

Figure 6

NFAT consensus binding sequences are present within transcriptional control regions shown previously to direct transcription selectively in slow-oxidative myofibers (Parsons et al. 1993; Levitt et al. 1995; Qin et al. 1997). (A) Consensus NFAT binding motifs in myoglobin, troponin I slow (TnI slow), and sarcomeric mitochondrial creatine kinase (sMtCK) promoters. (B) Conserved sequence blocks (CAGG, CCAC, MEF2, and E box) common to a SURE from the rat troponin I slow gene and a FIRE from the quail troponin I fast gene (Nakayama et al. 1996). A predicted NFAT response element (darkly shaded) overlapping the E-box is a unique feature of the SURE element.