Activity-dependent repression of muscle genes by NFAT

Abstract

Adult skeletal muscles retain an adaptive capacity to switch between slow- and fast-twitch properties that largely depend on motoneuron activity. The NFAT (nuclear factor of activated T cells) family of calcium-dependent transcription factors has been implicated in the up-regulation of genes encoding slow contractile proteins in response to slow-patterned motoneuron depolarization. Here, we demonstrate an unexpected, novel function of NFATc1 in slow-twitch muscles. Using the troponin I fast (TnIf) intronic regulatory element (FIRE), we identified sequences that down-regulate its function selectively in response to patterns of electrical activity that mimic slow motoneuron firing. A bona fide NFAT binding site in the TnIf FIRE was identified by site-directed mutations and by electrophoretic mobility and supershift assays. The activity-dependent transcriptional repression of FIRE is mediated through this NFAT site and, importantly, its mutation did not alter the up-regulation of TnIf transcription by fast-patterned activity. siRNA-mediated knockdown of NFATc1 in adult muscles resulted in ectopic activation of the FIRE in the slow soleus, without affecting enhancer activity in the fast extensor digitorum longus muscle. These findings demonstrate that NFAT can function as a repressor of fast contractile genes in slow muscles and they exemplify how an activity pattern can increase or decrease the expression of distinct contractile genes in a use-dependent manner as to enhance phenotypic differences among fiber types or induce adaptive changes in adult muscles.

Fig. 1.

The TnI SURE/FIRE chimeric enhancer drives transcription in adult fast muscles. (A) The 130-bp TnI SURE/FIRE chimeric enhancer consists of the pan muscle-specific half of the SURE (red) plus the complementary downstream half of the FIRE, which harbors the two potential NFAT sites S1 and S2 (underlined). The E-box and MEF-2, CACC, and CAGG motifs conserved between SURE and FIRE are shown. (B) Luciferase reporter levels in solei (red) and EDL (blue) muscles of SURE/FIRE-luc transgenic mice harvested at P7, P14, and P28. Measurements represent the mean ± SEM (n = 5 for each bar; *, P < 0.05; Kruskal–Wallis test with Dunn's post test). Assays were normalized for protein.

Fig. 2.

NFAT binds to a cis element in the TnI FIRE. Competition EMSAs and supershift assays indicate that the TnIf site S1, but not S2, binds NFAT. (A) Probe and competitor sequences from the mouse IL-2 enhancer (IL-NFAT) and TnI FIRE are shown. The bona fide IL-2 and putative FIRE NFAT sites are underlined; mutations are shown in red. (B) Competition and supershift EMSAs using the IL-NFAT sequences as the labeled probe are shown. Nuclear extracts (2 μg) were prepared from myotubes expressing ca-NFATc1, and unlabeled competitor ds-oligos were added at 200-fold molar excess where indicated. The migration of the NFAT-bound probe (arrow) and the band supershifted by the NFATc1 antiserum (asterisk) are marked. (C) EMSA using different labeled probes for wild-type or mutant IL-NFAT (lanes 1–3), and wild-type or mutant TnI FIRE (lanes 4–9). Nuclear extracts and supershifts were used as in B.

Fig. 3.

The FIRE is necessary to drive the developmental and fiber-type transcription of the TnIf2100 construct. Diagram of TnIf2100 luciferase reporter constructs with and without the FIRE (TnIf2100ΔFIRE) is shown at the top. (A) Reporter activity in C2C12 myoblasts (Left) and myotubes (Right) transfected with TnIf2100-luc or TnIf2100ΔFIRE-luc is shown. (B) Reporter activity in adult EDL and soleus muscles transfected by electroporation is shown. Firefly luciferase levels were normalized for transfection efficiency by using HSV- and RSV-renilla luciferase in cultured cells and muscle, respectively. Measurements represent the mean ± SEM (n = 6 for each bar; ***, P < 0.001; Kruskal–Wallis test with Dunn's post test).

Fig. 4.

NFAT represses transcription from TnIf2100. Luciferase activity driven by TnIf2100 (lanes 1–3) and TnIf2100mutS1 (lanes 4–6) in C2C12 myotubes cotransfected with an empty expression vector (lanes 1 and 4), or expression vectors for ca-NFATc1 (lanes 2 and 5) or VIVIT (lanes 3 and 6) is shown. The mean luciferase level of myotubes transfected with TnIf2100 and the empty expression vector was set to 100%. HSV-renilla luciferase levels were used to normalize for transfection efficiency. Measurements represent the mean ± SEM (n = 6 each bar; *, P < 0.05; Kruskal–Wallis test with Dunn's post test).

Fig. 5.

Mutation of the NFAT site selectively prevents TnIf FIRE repression by slow-patterned activity. (A and B) Micrographs of soleus myofibers transfected by electroporation with TnIf2100 (A) and TnIf2100mut-S1 (B) EGFP reporter constructs and stimulated with either fast or slow activity patterns. To measure enhancer function, myofibers were imaged 1 week after transfection, at the time of denervation and onset of stimulation (Before). A second image was taken after 12 days of stimulation (After). Numbers identify the same fiber before and after stimulation. (Scale bar: 200 μm.) (C) Bars represent relative changes (%) in EGFP in single soleus fibers stimulated for 12 days with either fast (blue) or slow (red) patterns. Reporter levels were normalized by using fluorescence calibration beads (arrowhead) implanted at the time of surgery. Values represent the mean ± SEM (40–72 myofibers from six muscles per condition; ***, P < 0.001; one-way ANOVA with Bonferroni post test).

Fig. 6.

NFATc1 knockdown prevents the activity-dependent suppression of TnIf2100. (A) EDL (blue) and soleus (red) muscles were cotransfected with a trimerized NFAT-luc reporter construct (3xNFAT) and NFATc1-specific siRNAs. Normalized mean luciferase levels in EDL transfected with 3xNFAT were set as 100%. Luciferase activity was normalized as described in Fig. 3B (n = 4). (B) EDL and soleus muscles were cotransfected with TnIf2100-luc and either a nontargeting control siRNA or NFATc1 siRNA. Normalized mean luciferase levels in EDL transfected with TnIf2100 and no siRNA were set as 100%. Reporter activities were measured 4 days after electroporation (n = 5). (C) Luciferase activity in EDL and soleus muscles from FIRE-SURE transgenic mice transfected with NFATc1 siRNA is shown. Assays were performed 4 days after siRNA transfection. Mean luciferase activity in EDL transfected with control siRNA was set as 100%. Luciferase activity was normalized to protein. Data represent the means ± SEM (n = 6; *, P < 0.05; **, P < 0.01; Kruskal–Wallis test with Dunn's post test).