the transcriptional coactivator peroxisome proliferator - activated receptor- co - activator 1 ( pgc-1 ) containing 797 amino acids ( full - length ) was first determined to be a cold inducible factor in a differentiated brown fat cell line .
subsequent studies have shown that pgc-1 plays important roles in mitochondrial biogenesis via coactivating several transcription factors such as nuclear respiratory factors and peroxisome proliferator - activated receptors [ 13 ] .
pgc-1 has also been found to mediate fast - to - slow skeletal muscle fiber - type transformation through coactivation of mef2 [ 2 , 4 ] .
exercise / muscle contraction has been found to increase pgc-1 mrna and protein levels [ 5 , 6 ] .
both the activity and expression of pgc-1 appear to be regulated by p38 mapk , which phosphorylates pgc-1 on thr-262 , ser-265 , and thr-298 and increases its stability [ 79 ] .
in addition , pgc-1 expression is also modulated by ampk - dependent phosphorylation at residues thr-177 and ser-570 [ 10 , 11 ] .   recently , a novel biologically active 270-aa isoform of pgc-1 ( nt - pgc-1 ) was cloned by zhang and his colleagues .
nt - pgc-1 is produced by alternative splicing of the full - length pgc-1 through a premature stop condon between exons 6 and 7 , generating a truncated n - terminal form of pgc-1 containing the first 267 amino acids of full - length pgc-1 and 3 additional amino acids from the insertion .
two of the three p38 mapk phosphorylation sites ( thr-262 , ser-265 ) and one of the two ampk phosphorylation sites ( thr-177 ) of full - length pgc-1 are present in nt - pgc-1. although lacking the nuclear localization signals present in the full - length pgc-1 , nt - pgc-1 has a predicted size only about 30  kda which should allow it to be freely diffusible between nucleus and cytosol via the nuclear pores .
however , recent studies found that nt - pgc-1 is predominantly located in cytoplasm [ 1214 ] and that the subcellular localization of nt - pgc-1 is dynamically regulated by protein kinase a ( pka ) , which phosphorylates nt - pgc-1 at residues ser-194 , ser-241 , and thr-256 . in adipocytes and in chinese hamster ovary k1 ( cho - k1 ) cells ,
inhibition of crm1 increased the nuclear content of nt - pgc-1 [ 12 , 13 ] .  
although nt - pgc-1 is found to be expressed in many tissues where pgc-1 plays important roles , including the skeletal muscle , no reports of the subcellular distribution and regulation of nt - pgc-1 in skeletal muscle have been published .
therefore , in the present study , we use flag - tagged nt - pgc-1 to examine the subcellular distribution of nt - pgc-1 in skeletal muscle fibers under control conditions and to demonstrate that the subcellular localization of nt - pgc-1 in muscle fibers is dynamically regulated by pka and by crm1-dependent nuclear export , but not by p38 mapk , or ampk .
dbcamp ( n , 2-o - dibutyryladenosine 3  :  5-cyclic monophosphate sodium ) and monoclonal anti - flag antibody were from sigma ( saint louis , mo ) .
aicar ( 5-aminoimidazole-4-carboxamide-1--d - riboside ) , anti - p38 mapk , anti - phospho - p38 mapk ( thr180/tyr182 ) , anti - ampk , and anti - phospho - ampk ( thr172 ) antibodies were purchased from cell signaling technology ( boston , ma ) .
the expression plasmid of gfp - pgc-1 cdna was obtained from addgene ( cambridge , ma ) .
experiments were carried out on skeletal muscle fibers obtained from 46-week - old cd-1 mice .
experimental protocols were approved by the university of maryland institutional animal care and use committee .
mice were euthanized and single muscle fibers were enzymatically dissociated from isolated flexor digitorum brevis ( fdb ) muscles .
fdb muscle fibers were plated on laminin - coated glass coverslip floors glued over 1  cm holes in 35  mm plastic petri dishes and cultured in mem containing 10% fetal bovine serum and 25  g / ml gentamicin sulfate in 5% co2 at 37c as previously described .
the recombinant adenovirus containing the flag - tagged nt - pgc-1 ( flag - nt - pgc-1 )   was kindly provided by dr .
the constructions of recombinant adenoviruses of nfatc1-gfp and nuclear targeted flag - nfatc1 were described in detail previously .
infection of muscle fiber cultures by recombinant adenoviruses was carried out about 20  h after the fibers were plated as previously described .
after 48 hours of viral infection , muscle fibers expressing flag - nt - pgc-1 were either electrically stimulated or treated pharmacologically .
fiber electrical stimulation in culture was accomplished by mounting stainless steel electrodes in the floor of the culture dish .
28  v , 1  ms pulses produce visible fiber twitches and relaxation between stimuli without fiber detachment from the glass coverslip .
after electrical stimulation or pharmacological treatment , fdb muscle fibers were immediately fixed with 4% paraformaldehyde for 10  min and permeabilized with 0.5% triton - x-100 in pbs for 20  min .
immunostaining for flag - tagged nt - pgc-1 was performed using monoclonal anti - flag antibody ( 1  :  1000 ) followed by incubation with the fluorescent protein - conjugated secondary antibody .
the fluorescence of the stained fibers was visualized using a confocal laser scanning imaging system ( olympus fluoview 500 ) using constant settings of laser intensity and photomultiplier gain . in each fiber image
the mean pixel background fluorescence ( off the fiber ) was determined and subtracted from each pixel throughout the image .
the average pixel fluorescence within user specified areas of interest ( aoi ) over the whole nucleus or the cytoplasm in each image was quantified by software custom - written in the idl programming language ( research systems , boulder , co ) , similar as we used in our previous study , and the ratio of nuclear to cytoplasmic mean pixel fluorescence was then calculated for each nucleus , which should correct for fiber to fiber differences in level of expression of flag - nt - pgc-1 or differences in overall staining level .
cultured fdb muscle fibers were lysed with mammalian protein extraction reagent ( thermo scientific , rockford , il ) plus tablets of tissue protease inhibitor cocktail ( roche diagnostics , indianapolis , in ) and passed through a 21 gauge syringe 10 times .
the lysates were subjected to centrifugation at 10,000  g for 10  min at 4c .
protein concentration was determined by the bradford assay ( bio - rad laboratories , inc . , hercules , ca )
. 30  g protein from each sample was fractionated by nupage 38% tris - acetate gel ( invitrogen , carlsbad , ca ) and transferred to pvdf membranes .
blots were probed with the appropriate primary and secondary antibodies and then visualized by pierce ecl western blotting substrate ( thermo scientific , rockford , il ) and exposed to x ray film ( amersham hyperfilm , ge healthcare , piscataway , nj ) .
film was scanned and band intensity was quantified by image j software ( national institutes of health , bethesda , md ) after subtracting neighboring background intensity .
student 's t - tests were used for comparisons of data obtained from two experimental conditions , and differences were considered significant if  p < 0.05 .
using flag - tagged nt - pgc-1 expressed by adenoviral infection in cultured adult fdb skeletal muscle fibers , we found that flag - nt - pgc-1 is predominantly located in the myoplasm ( figure 1(a ) ) , consistent with the recently described cytoplasmic localization of flag - nt - pgc-1 in adipocytes and cho - k1 cells [ 12 , 13 ] .
quantification of the mean pixel fluorescence in nuclear and cytoplasmic aois of 120 randomly selected resting fibers stained with anti - flag antibody showed that the average value of nuclear / cytoplasmic mean pixel fluorescence ratio ( n / c fluorescence ratio ) was 0.63  0.01 .
this is in contrast to the distribution pattern of expressed full - length gfp - pgc-1 fusion protein , which is predominantly located in the muscle fiber nuclei ( figure 1(b ) ; n / c fluorescence ratio : 9.53  2.86 ) as previously seen in cos cells . as negative controls , fibers either not exposed to flag - nt - pgc-1 viral infection but stained with anti - flag antibody or fibers with flag - nt - pgc-1 viral infection but without incubation with anti - flag antibody
show no evident fluorescence ( data not shown ) , indicating the specificity of the anti - flag antibody used . finally , as a positive control for anti - flag antibody accessibility to muscle fiber nuclei , we transfected muscle fibers with nuclear targeted flag - tagged nfatc1 and found strong nuclear staining with anti - flag antibody ( figure 1(c ) ) , thus establishing nuclear penetration of the anti - flag antibody .
recent studies in adipocytes and cho - k1 cells have shown that activation of pka can significantly increase the nuclear content of nt - pgc-1 [ 12 , 13 ] . to test whether this regulation mechanism also exists in skeletal muscle fibers , we treated muscle fibers with 1  mm dbcamp for 1  h in culture medium in the tissue culture incubator , as previously used to activate pka in cho - k1 cells .
our results show that the flag - nt - pgc-1 fluorescent staining was stronger in nuclei of fibers exposed to dbcamp ( figure 2(a ) , bdcamp ) than in control fibers ( figure 2(a ) , control ) .
the flag - nt - pgc-1 n / c fluorescence ratio was significantly increased from 0.71  0.03 in control to 0.98  0.07 in dbcamp - treated fibers ( figure 2(b ) ; p < 0.05 ) , a 1.4-fold increase .
the nuclear increase of flag - nt - pgc-1 after 1  h treatment of dbcamp found here in skeletal muscle fibers is very close to what has been previously obtained in cho - k1 cells ( about 1.4 fold ) and in differentiated adipocytes ( about 1.5 fold ) .
together , these results support the notion that pka regulates the subcellular distribution of nt - pgc-1 and is thereby involved in modulation of the nt - pgc-1-dependent signaling pathway in muscle fibers . in brown adipocytes , it has been shown that pka - dependent regulation of nuclear content of nt - pgc-1 plays a role in the transcriptional activation of ucp1 and cidea , but the downstream signaling pathway of nt - pgc-1 in skeletal muscle remains to be determined . in previous studies from our laboratory
, we found that repetitive muscle fiber activity elicited by electric field stimulation of isolated fdb fibers is effective for activating nuclear - cytoplasmic redistribution of expressed gfp fusion proteins of the transcriptional regulators nfatc1 ( nfatc1-gfp ) and hdac4 ( hdac4-gfp ) and that both of these effects are mediated by elevated ca , nfatc1-gfp via ca dependent activation of calcineurin [ 16 , 18 ] , and hdac4-gfp via ca dependent activation of cam kinase .
exercise / muscle contraction has also been found to increase the nuclear protein content of full - length pgc-1 [ 5 , 6 ] . since
nuclear nt - pgc-1 has been shown to co - activate ppar receptors as does the full - length pgc-1 , it is of interest to determine whether stimulation of muscle fibers can also induce the nuclear accumulation of nt - pgc-1 in fdb fibers .
our results show that the nuclear flag - nt - pgc-1 fluorescence of fibers stimulated continuously for 4  h at 10  hz ( figure 3(a ) , stimulation ) was essentially the same as in fibers from a parallel culture kept under control conditions without stimulation ( figure 3(a ) , control ) . the mean flag - nt - pgc-1 n / c fluorescence ratio was 0.62  0.03 in control and 0.59  0.03 in stimulated fibers ( figure 3(b ) ; p > 0.05 ) .
thus , flag - nt - pgc-1 does not translocate into the fiber nuclei in response to 4  h of continuous 10  hz stimulation . in order to verify the effectiveness of 10  hz continuous stimulation on isolated fdb fibers , in separate experiments we infected fibers with adenovirus encoding nfatc1-gfp .
two days later , fibers were stimulated continuously for 4  h at 10  hz , the same stimulation pattern used for the flag - nt - pgc-1 expressing fibers above . as previously reported [ 16 , 18 ] , nfatc1-gfp fusion protein is present predominantly at the sarcomeric z line in the cytoplasm of nonstimulated cultured adult fdb fibers ( figure 3(c ) , control ) .
4  h of 10  hz continuous stimulation markedly increased the nuclear nfatc1-gfp fluorescence ( figure 3(c ) , stimulation ) , so fdb fibers clearly respond to this stimulus pattern .
thus , 10  hz muscle fiber activity can cause nuclear accumulation of the transcription factor nfatc1-gfp , but not of flag - nt - pgc-1. however , it remains to be determined whether repetitive muscle activity results in modification of the expression level of endogenous nt - pgc-1.   it was previously reported that activation of p38 mapk , which phosphorylates pgc-1 on thr-262 , ser-265 , and thr-298 , increases the activity and nuclear protein level of full length pgc-1 in various tissues , including skeletal muscle [ 79 ] .
exercise / muscle contraction results in increased phosphorylation of p38 mapk . using western blot analysis
, we found that 4  h of continuous stimulation can significantly increase the level of p38 mapk phosphorylation in cultured fdb muscle fibers ( figure 3(d ) ) .
however , despite this increase in mapk activation , there was no evident nuclear accumulation of flag - nt - pgc-1 as discussed above , even though nt - pgc-1 contains two of the three p38 mapk phosphorylation sites ( thr-262 , ser-265 ) .
these results suggest that p38 mapk is not involved in the regulation of the subcellular distribution of nt - pgc-1 in muscle fibers . whether p38 mapk plays a role or not in regulation of the protein stability and activity of endogenous nt - pgc-1 as it does for the full - length pgc-1 in muscle fibers will be an interesting question for future study .
it was previously reported that ampk can directly phosphorylate full - length pgc-1 at residues thr-177 and ser-570   and activate pgc-1-dependent signaling pathways in skeletal muscle [ 10 , 11 ] .
as nt - pgc-1 contains one of the ampk - phosphorylation sites , we next tested whether activation of ampk can increase the nuclear accumulation of nt - pgc-1 in skeletal muscle fibers .
cultured fdb muscle fibers expressing flag - nt - pgc-1 were treated for 5  h with 2  mm of aicar , a powerful cell permeable amp mimic for activating ampk .
after 5  h of incubation with or without aicar , fibers were fixed and stained with anti - flag antibody .
fibers treated with aicar exhibited similar nuclear flag - nt - pgc-1 protein fluorescence as the control group . the mean flag - nt - pgc-1
n / c fluorescence ratio was 0.61  0.03 in control and 0.55  0.03 in aicar - treated fibers ( figure 4(a ) ; p > 0.05 ) .
figure 4(b ) shows that 5  h of aicar treatment did significantly increase the phosphorylation of ampk .
these results demonstrate that activation of ampk has no effect on the subcellular distribution of flag - nt - pgc-1. one hour treatment with aicar , or one hour of continuous electrical field stimulation at 10  hz , caused no change in the nuclear / cytoplasmic distribution of flag - nt - pgc-1 ( supplementary figure  1 available online at doi : 10.1155/2012/989263 ) .
nt - pgc-1 has a predicted size about 30  kda which should allow it to be freely diffusible between nucleus and cytosol via the nuclear pores , a mechanism which is independent of crm1 .
however , previous studies showed that treatment with the crm1 nuclear export inhibitor leptomycin b ( lmb ) increased the nuclear content of nt - pgc-1 in adipocytes and cho - k1 cells [ 12 , 13 ] , suggesting that nt - pgc-1 shuttles from the nucleus to the cytoplasm in these cells at least partially via a crm1-dependent efflux pathway . whether nucleo - cytoplasmic shuttling of nt - pgc-1 proteins via a crm1-dependent pathway also occurs in skeletal muscle
is not known . to address this issue , we treated muscle fibers expressing flag - nt - pgc-1 with 40  nm leptomycin b. our results show that the flag - nt - pgc-1 fluorescent staining was enhanced in nuclei of fibers exposed to leptomycin b ( figure 5(a ) , lmb ) compared to control fibers ( figure 5(a ) , control ) .
the flag - nt - pgc-1 n / c fluorescence ratio was significantly increased from 0.61  0.02 in control to 1.23  0.06 in fibers treated with leptomycin b for 20  h ( figure 5(b ) ; p < 0.05 ) .
however , the extent of nuclear accumulation of flag - nt - pgc-1 in resting muscle fibers with blocked crm1-dependent nuclear export is much lower than that previously observed for other transcriptional regulators such as nfatc1 and hdac [ 18 , 19 ] , which are too large to pass passively through the nuclear pores .
the relatively low nuclear accumulation of flag - nt - pgc-1 in the presence of leptomycin b ( figure 5 ) compared to other transcriptional regulators such as nfatc1   or hdac4   in adult muscle fibers indicates that either the unidirectional rate of nuclear influx of flag - nt - pgc-1 is extremely low so that there was little entry of flag - nt - pgc-1 into the muscle fiber nuclei during the 20  h exposure to leptomycin b or , more likely , that there is a crm1-independent pathway   for nuclear efflux of flag - nt - pgc-1. in this case , assuming that efflux is proportional to nuclear concentration and that the nuclear volume is negligible compared to the cytoplasm , the approximate doubling of n / c fluorescence ratio in exposure to leptomycin b indicates that nuclear efflux of flag - nt - pgc-1 via the leptomycin b insensitive pathway would have doubled in the presence of leptomycin b , so about half of the nuclear efflux in resting fibers in the absence of leptomycin b would be via the crm1-independent pathway . if the crm1-independent pathway is passive efflux via nuclear pores , which is likely since flag - nt - pgc-1 is below the cut off size for passive movement through the nuclear pores , flag - nt - pgc-1 would be distributed passively between nucleus and cytoplasm in the steady state in the case of full blockade of the crm1-dependent pathway by leptomycin b. in that case the free concentration of flag - nt - pgc-1 would be the same in the nucleus and cytoplasm .
the slightly higher nuclear than cytoplasmic concentration of total ( i.e. , free plus bound ) flag - nt - pgc-1 ( 1.23 fold ; see above ) would indicate that fraction of bound flag - nt - pgc-1 would be slightly higher in the nucleus than in the cytoplasm .
whether the flag tag on flag - nt - pgc-1 might influence its nuclear / cytoplasmic distribution in muscle fibers remains to be determined .
the possibility of rapid intranuclear ubiquitination followed by proteasomal degradation   of flag - nt - pgc-1 within the nucleus as a mechanism for lowering nuclear flag - nt - pgc-1 is unlikely since ubiquitination occurs at the n - terminal residue , which is flag tagged in our flag - nt - pgc-1 construct .
overexpression of full length pgc-1 , which is predominantly nuclear , gives rise to an increase in mitochondrial number and oxidative capacity in skeletal muscle [ 2 , 3 ] .
it will be interesting to determine in future studies whether overexpression of nt - pgc-1 , which is only partially nuclear , would also produce such effects on mitochondrial number and oxidative capacity . in conclusion
, our results suggest that in adult skeletal muscle fibers the nuclear - cytoplasmic distribution and regulation of nt - pgc-1 is very different from that of the full - length pgc-1 , which is exclusively nuclear .
 the transcriptional coactivator peroxisome proliferator - activated receptor  coactivator 1 ( pgc-1 ) regulates expression of genes for metabolism and muscle fiber type . 
 recently , a novel splice variant of pgc-1 ( nt - pgc-1 , amino acids 1270 ) was cloned and found to be expressed in muscle . 
 here we use flag - tagged nt - pgc-1 to examine the subcellular localization and regulation of nt - pgc-1 in skeletal muscle fibers . 
 flag - nt - pgc-1 is located predominantly in the myoplasm . 
 nuclear nt - pgc-1 can be increased by activation of protein kinase a. activation of p38 mapk by muscle activity or of ampk had no effect on the subcellular distribution of nt - pgc-1. inhibition of crm1-mediated export only caused relatively slow nuclear accumulation of nt - pgc-1 , indicating that nuclear export of nt - pgc-1 may be mediated by both crm1-dependent and -independent pathways . 
 together these results suggest that the regulation of nt - pgc-1 in muscle fibers may be very different from that of the full - length pgc-1 , which is exclusively nuclear . 
1. Introduction
2. Materials and Methods
3. Results and Discussion