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First published online 12 December 2006
doi: 10.1242/jcs.03307

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A combinatorial role for NFAT5 in both myoblast migration and differentiation during skeletal muscle myogenesis

¹ Graduate Program in Molecular and Systems Pharmacology Emory University, Atlanta, GA 30322, USA
² Department of Pharmacology, Emory University, Atlanta, GA 30322, USA
³ Biogen Idec, Inc., San Diego, CA 92122, USA

View larger version (68K): [in this window] [in a new window]   Fig. 1. NFAT5 expression and activity during myogenesis in vivo. (A) Immunostaining of TA sections from uninjured mice with an anti-NFAT5 antibody revealed a diffuse low level of staining throughout the section. No immunostaining was observed in sections following incubation with control IgG. Serial sections were stained with hematoxylin and eosin (H&E). Bar, 50 µm. (B) Immunostaining of TA sections 3 and 4 days following injury with an antibody against NFAT5 indicated bright staining of both mononucleated cells (*) and regenerating myofibers. NFAT5 appeared both nuclear (arrows) and cytoplasmic in newly formed myofibers (day 4). Arrow indicates the same myofiber in both sections. Bar, 50 µm. (C) TA muscles were electroporated with either an NFAT5 or control (C) reporter. Muscles were homogenized after 6 days and luciferase activity was determined. NFAT5 was transcriptionally active in vivo. Data are expressed as fold increase in luminescence relative to the control reporter containing mutated NFAT5 binding sites. Data are mean ± s.e.m. from four mice. (D) TA muscles were electroporated with a construct encoding ß-gal and collected 6 days later. ß-gal activity was confined to regenerating areas. The H&E stained section is a higher magnification view of the area in the indicated rectangle on the ß-gal stained section. Bar, 100 µm.

View larger version (46K): [in this window] [in a new window]   Fig. 2. NFAT5 expression and localization during myogenesis in vitro. (A) Lysates were prepared from primary myoblasts (Mb), nascent myotubes (nMt) and mature myotubes (mMt) as well as from murine kidney (+). Representative immunoblot analyses demonstrated NFAT5 protein was increased in myotubes compared with myoblasts. (B) NFAT5 localization during myogenesis was detected by immunostaining with an antibody against NFAT5. Nuclei were counterstained with the fluorescent dye DAPI. NFAT5 appeared predominantly nuclear in myoblasts, nascent myotubes (nMt) and mature myotubes (mMt). A subset of nuclei in mMT contained less NFAT5 (arrows) than others. No immunostaining was observed with an IgG control (data not shown).

View larger version (31K): [in this window] [in a new window]   Fig. 3. NFAT5 is transcriptionally active in skeletal muscle cells in vitro. (A) Wild-type (+/+) and NFAT5+/- myoblasts containing an NFAT5 reporter plasmid were differentiated for 24 hours and subsequently treated with 100 mM mannitol for 5 hours. Luciferase activity was diminished by 52% in mannitol-treated NFAT5+/- muscle cells compared to the wild type. Data are fold induction over basal levels of luciferase activity in unstimulated cells. (B) Wild-type myoblasts containing either control or NFAT5 reporters were differentiated for 40 hours and subsequently treated with lower concentrations of mannitol (mM) for 5 hours. Data are fold induction over basal levels of luciferase activity in unstimulated cells. (C) Wild-type myoblasts containing an NFAT5 reporter plasmid and VIVIT, a peptide inhibitor of NFATc1-c4 activation, were differentiated for 48 hours and treated with 100 mM glucose. Luciferase activity was similar in the presence or absence of VIVIT. Data are fold induction over basal levels of luciferase activity in unstimulated cells. (D) In parallel experiments, co-transfection of an NFATc1-c4 reporter plasmid and VIVIT led to decreased luciferase activity in cells treated with PMA and ionomycin (*P<0.05). Data are fold induction over basal levels of luciferase activity in unstimulated cells. (E) Wild-type myoblasts containing either control or NFAT5 reporters were differentiated for 24 hours (nMt) or 48 hours (mMt) and analyzed by luciferase assay. NFAT5 was transcriptionally active at all stages of myogenesis. Data are fold increase over levels of luciferase activity in cells containing a reporter with mutated NFAT5 binding sites. (F) NFAT5 transcriptional activity was analyzed as in E, but cells were treated with 100 mM mannitol to induce NFAT5 activity. Maximal NFAT5 activity was observed in myotubes (*P<0.05 for Mb relative to ctrl; **P<0.05 for Mt relative to Mb). Data are fold induction over basal levels of luciferase activity in unstimulated cells. All data in this figure are mean ± s.e.m. from three to four independent experiments.

View larger version (62K): [in this window] [in a new window]   Fig. 4. NFAT5 regulates myofiber formation during muscle regeneration in vivo. (A) Cells were isolated from the TA muscles of wild-type (+/+) and NFAT5+/- mice 3 days following injury and analyzed for Mac-1 expression by flow cytometry. Similar percentages of Mac-1+ macrophages were observed in regenerating muscle from wild-type (+/+) and NFAT5+/- mice. (B) TA sections obtained from wild-type(+/+) and NFAT5+/- mice were stained with hematoxylin and eosin 5 and 14 days following injury. Bar, 50 µm. (C) The number of regenerating TA myofibers per field was reduced in NFAT5+/- mice 5 days (*P<0.05) but not 14 days following injury. (D) The XSA of regenerating TA myofibers was similar in both wild-type (+/+) and NFAT5+/- mice 5 days following injury but decreased in NFAT5+/- mice 14 days following injury. (E) Myofiber number was similar in uninjured soleus muscles of both wild-type (+/+) and NFAT5+/- mice. All data are mean ± s.e.m., n=4-8 for each genotype.

View larger version (28K): [in this window] [in a new window]   Fig. 5. Myoblast differentiation is transiently decreased in muscle cells containing dominant-negative NFAT5. (A) Primary myoblasts containing either control (C) or dominant-negative NFAT5 (DN) plasmids were labeled with BrdU and subsequently immunostained. Nuclei were counterstained with DAPI. Bar, 100 µm. (B) The percentage of BrdU+ cells was similar in both control and DN cultures. (C) Nascent myotubes containing the NFAT5 reporter and either C or DN plasmids were treated with 100 mM mannitol for 5 hours. Luciferase activity was reduced by 74% in DN cells (*P<0.05). Data are fold induction over basal levels of luciferase activity in unstimulated cells. (D) eMyHC immunostaining (arrows) in primary myoblasts containing either C or DN retroviruses. Bar, 100 µm. (E) Ten hours following the induction of differentiation, the percentage of eMyHC+ cells in DN cultures was decreased (*P<0.05). (F) After 24 hours of differentiation, the percentage of eMyHC+ cells in DN cultures was similar to levels in the control. All data are mean ± s.e.m. from three to four independent experiments.

View larger version (27K): [in this window] [in a new window]   Fig. 6. NFAT5+/- muscle cells are less motile. (A) Migration of wild-type (+/+) and NFAT5+/- myoblasts was analyzed using time-lapse microscopy. Individual cell traces from 30 cells of each genotype are shown. NFAT5+/- myoblasts migrated shorter distances than the wild type. Three independent isolates for each genotype were analyzed. (B) Mean velocity was decreased in NFAT5+/- myoblasts (*P<0.05). Data are mean ± s.e.m. of 60 cells (20 cells from each of three independent isolates). (C) Frequency histogram illustrating the distribution of cell velocities for wild-type and NFAT5+/- myoblasts. A population of rapidly moving cells was absent in NFAT5+/- myoblasts. Data are mean ± s.e.m. of 60 cells (20 cells from each of three independent isolates).

View larger version (23K): [in this window] [in a new window]   Fig. 7. Cyr61, an NFAT5 target gene in skeletal muscle, regulates myoblast motility. (A) RNA was isolated from primary myoblasts (Mb), nascent myotubes (nMt) and mature myotubes (mMt) as well as from murine kidney (+). RT-PCR analyses demonstrated that Cyr61 was expressed in skeletal muscle cells at all stages of myogenesis. Equivalent input of cDNA was confirmed by amplification of the ribosomal gene 18S. (B) Immunoblot analyses demonstrated that Cyr61 protein was expressed in both uninjured (C) and regenerating (n=3 animals at each time point except n=2 for day 5) TA muscles at days 1 and 5 after injury. Equivalent protein loading was confirmed by Coomassie Blue staining. (C) Lysates from myoblasts containing either control (C) or dominant-negative NFAT5 (DN) plasmids were immunoblotted for Cyr61. Cyr61 was decreased in DN cells. Tubulin was detected as a loading control. (D) The average velocity of myoblasts expressing DN was decreased relative to the control. (*P<0.05). Average velocity returned to control levels following the addition of 5 µg/ml Cyr61 to these cells (**P<0.05 relative to DN). Data are mean ± s.e.m. of 60 cells (20 cells from each of three independent isolates). (E) Frequency histogram illustrating the distribution of cell velocities for control, DN and DN+Cyr61 cells. The DN curve was shifted to the left relative to control. The addition of 5 µg/ml Cyr61 to DN cells caused a rightward shift in the population. Data are mean ± s.e.m. of 60 cells (20 cells from each of three independent isolates).

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