Expression of utrophin A mRNA correlates with the oxidative capacity of skeletal muscle fiber types and is regulated by calcineurin/NFAT signaling

Abstract

Utrophin levels have recently been shown to be more abundant in slow vs. fast muscles, but the nature of the molecular events underlying this difference remains to be fully elucidated. Here, we determined whether this difference is due to the expression of utrophin A or B, and examined whether transcriptional regulatory mechanisms are also involved. Immunofluorescence experiments revealed that slower fibers contain significantly more utrophin A in extrasynaptic regions as compared with fast fibers. Single-fiber RT-PCR analysis demonstrated that expression of utrophin A transcripts correlates with the oxidative capacity of muscle fibers, with cells expressing myosin heavy chain I and IIa demonstrating the highest levels. Functional muscle overload, which stimulates expression of a slower, more oxidative phenotype, induced a significant increase in utrophin A mRNA levels. Because calcineurin has been implicated in controlling this slower, high oxidative myofiber program, we examined expression of utrophin A transcripts in muscles having altered calcineurin activity. Calcineurin inhibition resulted in an 80% decrease in utrophin A mRNA levels. Conversely, muscles from transgenic mice expressing an active form of calcineurin displayed higher levels of utrophin A transcripts. Electrophoretic mobility shift and supershift assays revealed the presence of a nuclear factor of activated T cells (NFAT) binding site in the utrophin A promoter. Transfection and direct gene transfer studies showed that active forms of calcineurin or nuclear NFATc1 transactivate the utrophin A promoter. Together, these results indicate that expression of utrophin A is related to the oxidative capacity of muscle fibers, and implicate calcineurin and its effector NFAT in this mechanism.

Fig. 1.

Localization of utrophin in extrasynaptic compartments of type IIb-negative fibers. Shown are representative examples of photomicrographs of serial sections processed to detect utrophin by using the DRP2 antibody (A and C) and MyHC IIb (B and D) by immunofluorescence. Note the lack (or low level) of utrophin staining in extrasynaptic regions of IIb fibers from both normal (A and B) and mdx (C and D) mice.*, The same fibers are identified on these serial sections. Images for utrophin were taken with increased exposure times to show the difference in the staining pattern between fiber types.

Fig. 2.

Extrasynaptic utrophin is the A isoform. (A) A polyclonal antibody raised against utrophin A recognizes a single high molecular mass band in Western blots by using muscle proteins. (B and C) Shown is the presence of AChR and utrophin A in cryostat sections of muscle fibers, respectively. Note the accumulation of utrophin A at the neuromuscular junctions. Preincubation of the rabbit serum with the utrophin A peptide, used to raise the antibody, completely abolished the utrophin A labeling (E) at AChR-rich regions (D). This labeling was also absent at AChR-rich regions from utrophin-deficient mice (F and G). In both the peptide block experiments (H) and in sections from utrophin-deficient mice (I), no labeling could be observed using this utrophin A antibody in extrasynaptic compartment of muscle fibers. Double immunofluorescence experiments showed that utrophin A (J) is expressed at the sarcolemma of MyHC IIb-negative fibers (K) and is thus not confined to junctional regions.*, The same fibers are identified in these serial sections.

Fig. 3.

Expression of utrophin and utrophin A mRNAs in single fibers. (A) Examples of ethidium bromide-stained agarose gels showing PCR products obtained from two single fibers (1 and 2) for total utrophin, utrophin A, MyHC I, and MyHC IIb. Note the presence of strong utrophin signals in the fiber expressing MyHC I. Quantitation of the RT-PCR data shows that MyHC I and IIa fibers contained considerably more total utrophin (B) and utrophin A (C) transcripts.*, Denoted are significant differences from type I (P ≤ 0.05). A total of 60 fibers were analyzed from three different mice. Mean ± SEM are shown.

Fig. 4.

Expression of utrophin transcripts is increased in overloaded plantaris muscle. (A) Examples of ethidium bromide-stained agarose gels showing PCR products obtained from control (CTL) and overloaded (OV) muscles for total utrophin, utrophin A, and MyHC IIb transcripts. Note the increased utrophin and utrophin A mRNAs concurrent with a decrease in MyHC IIb transcripts in OV muscles (B).*, Denoted are significant differences from control (P ≤ 0.05). n = 3 for CTL and n = 6 for OV. Means ± SEM are shown.

Fig. 5.

Expression of utrophin A mRNA is regulated by calcineurin. (A and C) Examples of ethidium bromide-stained agarose gels showing PCR products for utrophin A mRNA in control (CTL) and cyclosporine (CsA)-treated muscles, and in WT and transgenic (CnA*) muscles, respectively. Quantitation showed that, in comparison with vehicle-treated CTL muscles, muscles from CsA-treated mice contained significantly fewer utrophin A transcripts (B). By contrast, muscles from CnA* transgenic mice contained significantly more utrophin A transcripts than WT mice (D).*, Denoted are significant differences (P < 0.05). Muscles from three to six mice were analyzed for each group. Means ± SEM are shown.

Fig. 6.

Increase in utrophin A expression in muscle fibers from CnA* mice. Immunofluorescence experiments were done using the utrophin A antibody on soleus muscle sections from control (A and C) and CnA* (B and D). Note that muscles from CnA* mice express high levels of utrophin A at the sarcolemma of each individual fiber.

Fig. 7.

Activity of the utrophin A promoter is modulated by calcineurin and NFAT. (A) Schematic representation of a putative NFAT site in the utrophin A promoter. (B) EMSAs using protein extracts (lane “+ Ext”) incubated with ³²P-labeled double-stranded oligonucleotides corresponding to the NFAT motif (black arrow). Note the specific binding activity that is competed by a 200× molar excess of unlabeled probe (lane “+ C”). This binding can be supershifted by adding NFATc1 antibodies in the reaction mixture (white arrow; lane “+ Ab”). (C) C2C12 muscle cells transfected with plasmids containing the utrophin A promoter-LacZ reporter gene alone or with expression vectors containing CnA* or nNFATc1. (D) Mouse soleus muscles were transduced with plasmids containing the utrophin A promoter-reporter gene alone or with an expression vector containing CnA*. Note that, in these two sets of experiments, expression of active CnA* or nuclear NFATc1 increased (P < 0.05) the activity of the utrophin A promoter as measured by the relative abundance of LacZ transcripts. For the transfection experiments, n = 2 to 4 in duplicate. For the injection, n = 4 mice. Means ± SEM are shown.