Expression of utrophin A mRNA correlates with the oxidative capacity of skeletal muscle fiber types and is regulated by calcineurin/NFAT signaling.
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.
Chakkalakal
Joe V
JV
Department of Cellular and Molecular Medicine and Centre for Neuromuscular Disease, Faculty of Medicine, University of Ottawa, Ottawa, ON, Canada K1H 8M5.
Stocksley
Mark A
MA
Harrison
Mary-Ann
MA
Angus
Lindsay M
LM
Deschenes-Furry
Julie
J
St-Pierre
Simon
S
Megeney
Lynn A
LA
Chin
Eva R
ER
Michel
Robin N
RN
Jasmin
Bernard J
BJ
D000818:Animals; D015153:Blotting, Western; D019703:Calcineurin / Q000378:metabolism*; D003598:Cytoskeletal Proteins / Q000096:biosynthesis / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism*; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D015151:Immunoblotting; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D008856:Microscopy, Fluorescence; D018485:Muscle Fibers, Skeletal / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000378:metabolism*; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D010100:Oxygen / Q000378:metabolism*; D010641:Phenotype; D011401:Promoter Regions, Genetic; D020033:Protein Isoforms; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D014157:Transcription Factors / Q000378:metabolism*; D014162:Transfection; D049411:Utrophin
D003598:Cytoskeletal Proteins; D004268:DNA-Binding Proteins; D008565:Membrane Proteins; D050778:NFATC Transcription Factors; C494005:Nfatc1 protein, mouse; D009687:Nuclear Proteins; D020033:Protein Isoforms; D012333:RNA, Messenger; D014157:Transcription Factors; C483600:Utrn protein, mouse; D049411:Utrophin; D012313:RNA; D019703:Calcineurin; D010100:Oxygen