Myf5 is a member of the muscle regulatory factor family of transcription factors and plays an important role in the determination, development, and differentiation of skeletal muscle. However, factors that regulate the expression and activity of Myf5 itself are not well understood. Recently, a role for the calcium-dependent phosphatase calcineurin was suggested in three distinct pathways in skeletal muscle: differentiation, hypertrophy, and fiber-type determination. We propose that one downstream target of calcineurin and the calcineurin substrate NFAT in skeletal muscle is regulation of Myf5 gene expression. For these studies, we used myotube cultures that contain both multinucleated myotubes and quiescent, mononucleated cells termed ‘reserve’ cells, which share many characteristics with satellite cells. Treatment of such myotube cultures with the calcium ionophore ionomycin results in an approximately 4-fold increase in Myf5 mRNA levels, but similar effects are not observed in proliferating myoblast cultures indicating that Myf5 is regulated by different pathways in different cell populations. The increase in Myf5 mRNA levels in myotube cultures requires the activity of calcineurin and NFAT, and can be specifically enhanced by overexpressing the NFATc isoform. We used immunohistochemical analyses and fractionation of the cell populations to demonstrate that the calcium regulated expression of Myf5 occurs in the mononucleated reserve cells. We conclude that Myf5 gene expression is regulated by a calcineurin- and NFAT-dependent pathway in the reserve cell population of myotube cultures. These results may provide important insights into the molecular mechanisms responsible for satellite cell activation and/or the renewal of the satellite cell pool following activation and proliferation.

Abbott
K. L.
,
Friday
B. B.
,
Thaloor
D.
,
Murphy
T. J.
,
Pavlath
G. K.
(
1998
).
Activation and cellular localization of the cyclosporine A-sensitivetranscription factor NF-AT in skeletal muscle cells.
Mol. Biol. Cell
9
,
2905
2916
Aramburu
J.
,
Yaffe
M. B.
,
Lopez-Rodriguez
C.
,
Cantley
L. C.
,
Hogan
P. G.
,
Rao
A.
(
1999
).
Affinity-driven peptide selection of an NFAT inhibitor more selective than cyclosporin A.
Science
285
,
2129
2133
Arnold
H. H.
,
Braun
T.
(
1996
).
Targeted inactivation of myogenic factor genes reveals their role during mouse myogenesis: a review.
Int. J. Dev. Biol
40
,
345
353
Arnold
H. H.
,
Winter
B.
(
1998
).
Muscle differentiation: more complexity to the network of myogenic regulators.
Curr. Opin. Genet. Dev
8
,
539
544
Aurade
F.
,
Pfarr
C. M.
,
Lindon
C.
,
Garcia
A.
,
Primig
M.
,
Montarras
D.
,
Pinset
C.
(
1997
).
The glucocorticoid receptor and AP-1 are involved in a positive regulation of the muscle regulatory gene myf5 in cultured myoblasts.
J. Cell Sci
110
,
2771
2779
Barth
J. L.
,
Morris
J.
,
Ivarie
R.
(
1998
).
An Oct-like binding factor regulates Myf-5 expression in primary avian cells.
Exp. Cell Res
238
,
430
438
Boss
V.
,
Abbott
K. L.
,
Wang
X. F.
,
Pavlath
G. K.
,
Murphy
T. J.
(
1998
).
The cyclosporin A-sensitive nuclear factor of activated T cells (NFAT) proteins are expressed in vascular smooth muscle cells. Differential localization of NFAT isoforms and induction of NFAT-mediated transcription by phospholipase C-coupled cell surface receptors.
J. Biol. Chem
273
,
19664
19671
Bradford
M. M.
(
1976
).
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.
Anal. Biochem
72
,
248
254
Chin
E. R.
,
Olson
E. N.
,
Richardson
J. A.
,
Yang
Q.
,
Humphries
C.
,
Shelton
J. M.
,
Wu
H.
,
Zhu
W.
,
Bassel-Duby
R.
,
Williams
R. S.
(
1998
).
A calcineurin-dependent transcriptional pathway controls skeletal muscle fiber type.
Genes Dev
12
,
2499
2509
Cooper
R. N.
,
Tajbakhsh
S.
,
Mouly
V.
,
Cossu
G.
,
Buckingham
M.
,
Butler-Browne
G. S.
(
1999
).
In vivo satellite cell activation via Myf5 and MyoD in regenerating mouse skeletal muscle.
J. Cell Sci
112
,
2895
2901
Cornelison
D. D.
,
Wold
B. J.
(
1997
).
Single-cell analysis of regulatory gene expression in quiescent and activated mouse skeletal muscle satellite cells.
Dev. Biol
191
,
270
283
Davis
R. L.
,
Cheng
P. F.
,
Lassar
A. B.
,
Weintraub
H.
(
1990
).
The MyoD DNA binding domain contains a recognition code for muscle-specific gene activation.
Cell
60
,
733
746
Delling
U.
,
Tureckova
J.
,
Lim
H. W.
,
De Windt
L. J.
,
Rotwein
P.
,
Molkentin
J. D.
(
2000
).
A calcineurin-NFATc3-dependent pathway regulates skeletal muscle differentiation and slow myosin heavy-chain expression.
Mol. Cell Biol
20
,
6600
6611
Dunn
S. E.
,
Burns
J. L.
,
Michel
R. N.
(
1999
).
Calcineurin is required for skeletal muscle hypertrophy.
J. Biol. Chem
274
,
21908
21912
Friday
B. B.
,
Horsley
V.
,
Pavlath
G. K.
(
2000
).
Calcineurin activity is required for the initiation of skeletal muscle differentiation.
J. Cell Biol
149
,
657
666
Hasty
P.
,
Bradley
A.
,
Morris
J. H.
,
Edmondson
D. G.
,
Venuti
J. M.
,
Olson
E. N.
,
Klein
W. H.
(
1993
).
Muscle deficiency and neonatal death in mice with a targeted mutation in the myogenin gene.
Nature
364
,
501
506
Hughes
S. M.
,
Taylor
J. M.
,
Tapscott
S. J.
,
Gurley
C. M.
,
Carter
W. J.
,
Peterson
C. A.
(
1993
).
Selective accumulation of MyoD and myogenin mRNAs in fast and slow adult skeletal muscle is controlled by innervation and hormones.
Development
118
,
1137
1147
Hughes
S. M.
,
Koishi
K.
,
Rudnicki
M.
,
Maggs
A. M.
(
1997
).
MyoD protein is differentially accumulated in fast and slow skeletal muscle fibres and required for normal fibre type balance in rodents.
Mech. Dev
61
,
151
163
Hughes
S. M.
,
Chi
M. M.
,
Lowry
O. H.
,
Gundersen
K.
(
1999
).
Myogenin induces a shift of enzyme activity from glycolytic to oxidative metabolism in muscles of transgenic mice.
J. Cell Biol
145
,
633
642
Jacobs-El
J.
,
Zhou
M. Y.
,
Russell
B.
(
1995
).
MRF4, Myf-5, and myogenin mRNAs in the adaptive responses of mature rat muscle.
Am. J. Physiol
268
,
1045
1052
Kitzmann
M.
,
Carnac
G.
,
Vandromme
M.
,
Primig
M.
,
Lamb
N. J.
,
Fernandez
A.
(
1998
).
The muscle regulatory factors MyoD and myf-5 undergo distinct cell cycle-specific expression in muscle cells.
J. Cell Biol
142
,
1447
1459
Kraus
B.
,
Pette
D.
(
1997
).
Quantification of MyoD, myogenin, MRF4 and Id-1 by reverse-transcriptase polymerase chain reaction in ratmuscles—effects of hypothyroidism and chronic low-frequency stimulation.
Eur. J. Biochem
247
,
98
106
Lai
M. M.
,
Burnett
P. E.
,
Wolosker
H.
,
Blackshaw
S.
,
Snyder
S. H.
(
1998
).
Cain, a novel physiologic protein inhibitor of calcineurin.
J. Biol. Chem
273
,
18325
18331
Lindon
C.
,
Montarras
D.
,
Pinset
C.
(
1998
).
Cell cycle-regulated expression of the muscle determination factor Myf5 in proliferating myoblasts.
J. Cell Biol
140
,
111
118
Lo Russo
A.
,
Passaquin
A. C.
,
Andre
P.
,
Skutella
M.
,
Ruegg
U. T.
(
1996
).
Effect of cyclosporin A and analogues on cytosolic calcium and vasoconstriction: possible lack of relationship to immunosuppressive activity.
Br. J. Pharmacol
118
,
885
892
Lo Russo
A.
,
Passaquin
A. C.
,
Cox
C.
,
Ruegg
U. T.
(
1997
).
Cyclosporin A potentiates receptor-activated [Ca2+]c increase.
J. Recept Signal Transduct. Res
17
,
149
161
Lowe
D. A.
,
Alway
S. E.
(
1999
).
Stretch-induced myogenin, MyoD, and MRF4 expression and acute hypertrophy in quail slow-tonic muscle are not dependent upon satellite cell proliferation.
Cell Tissue Res
296
,
531
539
Megeney
L. A.
,
Rudnicki
M. A.
(
1995
).
Determination versus differentiation and the MyoD family of transcription factors.
Biochem. Cell Biol
73
,
723
732
Megeney
L. A.
,
Kablar
B.
,
Garrett
K.
,
Anderson
J. E.
,
Rudnicki
M. A.
(
1996
).
MyoD is required for myogenic stem cell function in adult skeletal muscle.
Genes Dev
10
,
1173
1183
Musaro
A.
,
McCullagh
K. J.
,
Naya
F. J.
,
Olson
E. N.
,
Rosenthal
N.
(
1999
).
IGF-1 induces skeletal myocyte hypertrophy through calcineurin in association with GATA-2 and NF-ATc1.
Nature
400
,
581
585
Naya
F. J.
,
Mercer
B.
,
Shelton
J.
,
Richardson
J. A.
,
Williams
R. S.
,
Olson
E. N.
(
2000
).
Stimulation of slow skeletal muscle fiber gene expression by calcineurin in vivo.
J. Biol. Chem
275
,
4545
4548
Nervi
C.
,
Benedetti
L.
,
Minasi
A.
,
Molinaro
M.
,
Adamo
S.
(
1995
).
Arginine-vasopressin induces differentiation of skeletal myogenic cells and up-regulation of myogenin and Myf-5.
Cell Growth Differ
6
,
81
89
Patapoutian
A.
,
Miner
J. H.
,
Lyons
G. E.
,
Wold
B.
(
1993
).
Isolated sequences from the linked Myf-5 and MRF4 genes drive distinct patterns of muscle-specific expression in transgenic mice.
Development
118
,
61
69
Rando
T. A.
,
Blau
H. M.
(
1994
).
Primary mouse myoblast purification, characterization, and transplantation for cell-mediated gene therapy.
J. Cell Biol
125
,
1275
1287
Rao
A.
,
Luo
C.
,
Hogan
P. G.
(
1997
).
Transcription factors of the NFAT family: regulation and function.
Annu. Rev. Immunol
15
,
707
747
Rudnicki
M. A.
,
Schnegelsberg
P. N.
,
Stead
R. H.
,
Braun
T.
,
Arnold
H. H.
,
Jaenisch
R.
(
1993
).
MyoD or Myf-5 is required for the formation of skeletal muscle.
Cell
75
,
1351
1359
Rudnicki
M. A.
,
Jaenisch
R.
(
1995
).
The MyoD family of transcription factors and skeletal myogenesis.
BioEssays
17
,
203
209
Schultz
E.
,
McCormick
K. M.
(
1994
).
Skeletal muscle satellite cells.
Rev. Physiol. Biochem. Pharmacol
123
,
213
257
Semsarian
C.
,
Wu
M. J.
,
Ju
Y. K.
,
Marciniec
T.
,
Yeoh
T.
,
Allen
D. G.
,
Harvey
R. P.
,
Graham
R. M.
(
1999
).
Skeletal muscle hypertrophy is mediated by a Ca2+-dependent calcineurin signalling pathway.
Nature
400
,
576
581
Teti
A.
,
Naro
F.
,
Molinaro
M.
,
Adamo
S.
(
1993
).
Transduction of arginine vasopressin signal in skeletal myogenic cells.
Am. J. Physiol
265
,
113
121
Wu
H.
,
Naya
F. J.
,
McKinsey
T. A.
,
Mercer
B.
,
Shelton
J. M.
,
Chin
E. R.
,
Simard
A. R.
,
Michel
R. N.
,
Bassel-Duby
R.
,
Olson
E. N.
(
2000
).
MEF2 responds to multiple calcium-regulated signals in the control of skeletal muscle fiber type.
EMBO J
19
,
1963
1973
Yablonka-Reuveni
Z.
(
1995
).
Development and postnatal regulation of adult myoblasts.
Microsc. Res. Tech
30
,
366
380
Yoon
J. K.
,
Olson
E. N.
,
Arnold
H. H.
,
Wold
B. J.
(
1997
).
Different MRF4 knockout alleles differentially disrupt Myf-5 expression: cis-regulatory interactions at the MRF4/Myf-5 locus.
Dev. Biol
188
,
349
362
Yoshida
N.
,
Yoshida
S.
,
Koishi
K.
,
Masuda
K.
,
Nabeshima
Y.
(
1998
).
Cell heterogeneity upon myogenic differentiation: down-regulation of MyoD and Myf-5 generates ‘reserve cells’.
J. Cell Sci
111
,
769
779
Zhang
W.
,
Behringer
R. R.
,
Olson
E. N.
(
1995
).
Inactivation of the myogenic bHLH gene MRF4 results in up-regulation of myogenin and rib anomalies.
Genes Dev
9
,
1388
1399
Zweigerdt
R.
,
Braun
T.
,
Arnold
H. H.
(
1997
).
Faithful expression of the Myf-5 gene during mouse myogenesis requires distant control regions: a transgene approach using yeast artificial chromosomes.
Dev. Biol
192
,
172
180
This content is only available via PDF.