skNAC, a Smyd1-interacting transcription factor, is
involved in cardiac development and skeletal muscle
growth and regeneration
Chong Yon Parka,b,c, Stephanie A. Pierced, Morgan von Drehlea,b,c, Kathryn N. Iveya,b,c, Jayson A. Morgane,
Helen M. Blaue, and Deepak Srivastavaa,b,c,1
aGladstone Institute of Cardiovascular Disease and Departments ofbPediatrics andcBiochemistry and Biophysics, University of California, San Francisco, CA
94158;dDepartment of Pediatrics and Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390; andeBaxter Laboratory for Stem
Cell Biology, Stanford University, Stanford, CA 94350
Edited by Eric N. Olson, University of Texas Southwestern, Dallas, TX, and approved October 12, 2010 (received for review September 13, 2010)
Cardiac and skeletal muscle development and maintenance require
complex interactions between DNA-binding proteins and chroma-
tin remodeling factors. We previously reported that Smyd1,
a muscle-restricted histone methyltransferase, is essential for
cardiogenesis and functions with a network of cardiac regulatory
proteins. Here we show that the muscle-specific transcription
factor skNAC is the major binding partner for Smyd1 in the
developing heart. Targeted deletion of skNAC in mice resulted in
partial embryonic lethality by embryonic day 12.5, with ventricular
hypoplasia and decreased cardiomyocyte proliferation that were
similar but less severe than in Smyd1 mutants. Expression of Irx4,
a ventricle-specific transcription factor down-regulated in hearts
lacking Smyd1, also depended on the presence of skNAC. Viable
skNAC−/−adult mice had reduced postnatal skeletal muscle
growth and impaired regenerative capacity after cardiotoxin-in-
duced injury. Satellite cells isolated from skNAC−/−mice had im-
paired survival compared with wild-type littermate satellite cells.
Our results indicate that skNAC plays a critical role in ventricular
cardiomyocyte expansion and regulates postnatal skeletal muscle
growth and regeneration in mice.
fashion by numerous signaling, transcriptional, and trans-
lational networks (1, 2). Epigenetic events, including post-
translational modification of histones, modulate the structure of
chromatin and the accessibility of regulatory sequences to tran-
scriptional activators and repressors. Acetylation of conserved
lysine residues in histone tails by histone acetyltransferases stim-
ulates chromatin relaxation and transcription, whereas deacety-
lation by histone deacetylases represses transcription (3).
Methylation of lysine and arginine residues in histones affects
chromatin conformation and either facilitates or inhibits tran-
Smyd1 is a muscle-restricted member of a family of chromatin
remodeling proteins that contains both MYND and SET domains
to recruit a corepressor complex, and the SET domain commonly
functions as a methyltransferase for histones or other proteins (6,
7). Targeted deletion of Smyd1 in mice results in a failure of
similar to cardiac phenotypes in embryos lacking Mef2c or Hand2
and regulates the expression of Hand2 and Irx4, all of which
function in a transcriptional network to control ventricular car-
embryonic lethality of Smyd1 mutant mice, its role in skeletal
muscle development remains unclear in mice. Knockdown of
SmyD1a and SmyD1b expression in zebrafish embryos by mor-
(11). Although Smyd1 may not bind DNA, it is likely recruited to
muscle-specific target genes through physical interactions with
unknown DNA-binding partners.
ardiogenesis is regulated in a temporally and spatially precise
Here, we show that the muscle-restricted isoform of the DNA-
is a major interacting partner of Smyd1 in the developing heart
by yeast two-hybrid screening, similar to skeletal muscle (12), and
that disruption of skNAC in mice results in partial embryonic le-
thality between embryonic day (E) 9.5 and E12.5 due to ventric-
survived to adulthood had reduced postnatal skeletal muscle
growth. In addition, we found that skNAC-null skeletal muscle
had markedly impaired regenerative capacity in response to in-
jury. Thus, our findings demonstrate that skNAC is involved in
early cardiac development and postnatal skeletal muscle growth
skNAC Is a Muscle-Specific Partner of Smyd1 During Cardiogenesis.
To understand the molecular mechanism by which Smyd1 reg-
ulates embryonic heart development, we constructed a yeast two-
hybrid library from mouse embryonic heart (E11.5) and screened
for interacting partners with full-length Smyd1 as bait. Half of
the candidates isolated from the screen (35 of 70) encoded
skNAC, a muscle-specific transcription factor that interacts with
Smyd1 in skeletal muscle (12) (Fig. 1A). Expression of skNAC
and Smyd1 is induced during skeletal myogenesis in culture, and
the interaction domains in both Smyd1 and skNAC have been
mapped previously (12). Several other proteins were isolated,
and their interactions were confirmed by reversing prey and bait.
These included a cytoplasmic protein with a flavoprotein mon-
ooxygenase domain, MICAL (4 of 70), involved in myofilament
organization and actin dynamics (13); a unique protein with
a helicase domain (4 of 70); and FK506 binding protein 8 (4 of
70), which regulates BCL2 (14). Each of these interactions was
confirmed by coimmunoprecipitation assays (Fig. 1B).
We focused our further studies on skNAC because of its
strong interaction with Smyd1 and its muscle-specific expression,
although Smyd1 interaction with the other proteins may be im-
portant as well. skNAC is generated by splicing-in of a 6-kb
second exon (Fig. 1C) and encodes a transcriptional activator
of the myoglobin promoter (15). The ubiquitous αNAC does
not contain exon 2 but can function as a transcriptional co-
activator of osteocalcin through an interaction with Jun and
Author contributions: C.Y.P., H.M.B., and D.S. designed research; C.Y.P., S.A.P., M.v.D.,
K.N.I., and J.A.M. performed research; C.Y.P., H.M.B., and D.S., analyzed data; and C.Y.P.
and D.S. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Freely available online through the PNAS open access option.
Data deposition: Microarray data were deposited to the Gene Expression Omnibus data-
base (accession no. GSE5841).
1To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| November 30, 2010
| vol. 107
| no. 48www.pnas.org/cgi/doi/10.1073/pnas.1013493107
also acts as a positive regulator of human erythroid cell dif-
ferentiation (16, 17).
Whole-mount in situ hybridization revealed high levels of
skNAC expression in all four chambers of the looping heart at
E9.5 (Fig. 1 D and E), but lower expression before E9.0. Section
in situ hybridization showed weak expression in the heart at E8–
E8.5. At E10.5 and E12.5, expression was restricted to the
myocardial layers, including the compact zone, interventricular
septum, and trabeculae (Fig. 1 F–I). At E12.5, high levels of
expression were also observed in skeletal muscle precursors
(Fig. 1I). Transcripts in the heart and skeletal muscle persisted
throughout subsequent development and adulthood (Fig. 1K).
The temporal and spatial expression patterns of skNAC were al-
most identical to those of Smyd1 (5), consistent with their
skNAC−/−Embryos Exhibit Ventricular Hypoplasia. To investigate the
in vivo function of skNAC, we deleted skNAC in mice. Because
Northern analysis demonstrated ubiquitous αNAC expression
from E7 onward, we designed a targeting vector to replace only
the skNAC-specific exon 2 with the neomycin-resistance gene
cassette (Neo), flanked by Flp recombinase (FRT) sites (Fig.
2A). This approach was designed to disrupt skNAC expression,
while maintaining αNAC expression. Targeted ES cells were
identified by Southern analysis (Fig. 2B), and chimeras were
generated from two independent lines. Heterozygous mice lack-
ing skNAC were generated either on a mixed or isogenic back-
ground and were normal. The offspring from intercrosses of
heterozygous skNAC mice were genotyped before weaning. As
summarized in Table S1, 17% of pups from the mixed 129S6/
C57BL/6 background and 8% from the 129S6 isogenic back-
specific expression of skNAC. (A) Frequency of Smyd1-inter-
acting partners isolated by yeast two-hybrid screening of an
embryonic heart cDNA library. (B) Immunoprecipitation (IP)
of Smyd1 with anti-MYC antibodies and immunoblot (IB)
with anti-FLAG antibodies are shown along with Western
blots of input protein used. (C) Genomic organization of the
NAC gene with splice forms of skNAC and αNAC indicated. (D
and E) Whole-mount in situ hybridizations with exon 2-spe-
cific probe at E9.5 in lateral (D) and frontal views (E). (F–K)
Section in situ hybridizations at indicated stages with dark-
field and bright-field images. h, head; ht, heart; oft, outflow
tract; ra, right atrium; la, left atrium; rv, right ventricle; lv, left
ventricle; ao, aorta; sk, skeletal muscle precursor.
Smyd1-interacting partners in the heart and muscle-
exon 2 in mice. (A) Organization of the skNAC wild-type allele
(skNACWT) and targeting vector (skNACTV) and replacement of
exon 2 by homologous recombination (skNACNull). (B) South-
ern analysis of genomic DNA of targeted ES cells after di-
gestion with HindIII and HpaI and after hybridization with 5′
and 3′ probes, respectively. (C) RT-PCR analysis showing the
absence of skNAC transcripts and the maintenance of αNAC
transcripts in skNAC−/−embryonic heart. GAPDH is a loading
control. (D) qPCR showing expression of αNAC transcripts in
skNAC−/−hearts. Data are presented as mean with SE (n = 6,
*P < 0.01).
Targeting strategy for deletion of the skNAC-specific
Park et al.PNAS
| November 30, 2010
| vol. 107
| no. 48
ground were homozygous, instead of the expected Mendelian
ratio of 25%. This partial embryonic lethality occurred even
upon deletion of Neo on the null allele by intercrossing with
ubiquitous ACTb:FLPe mice (18). The absence of skNAC but
maintenance of αNAC expression in homozygous mutant mice
was confirmed by RT-PCR (Fig. 2C). By real-time quantitative
PCR (qPCR), we found that αNAC expression was not reduced
in the skNAC mutants but rather slightly up-regulated (Fig. 2D).
To investigate the timing and cause of prenatal death upon loss
of skNAC, we examined embryos from timed pregnancies of
skNAC heterozygous intercrosses. Most of the deaths occurred
skNAC−/−embryos were identical on the mixed and isogenic
backgrounds, we used 129S6 isogenic mice for all subsequent
experiments. The development of skNAC−/−embryos was slightly
delayed, but most embryos were generally normal until E10.5.
Approximately half of the embryos beyond E10.5 displayed peri-
cardial edema and some developmental delay, typical of many
mouse mutants with cardiac dysfunction (Fig. 3 Aa and Ab). From
the muscle-specific expression of skNAC and maintenance of
αNAC expression, this phenotype is likely secondary to hemody-
namic insufficiency from cardiac dysfunction. At E11.5, pericar-
dial hemorrhage was also frequently noted on gross inspection,
suggesting breach of the myocardial wall (Fig. 3 Ac and Ad).
Histological analysis of affected skNAC-null embryos con-
firmed the pericardial effusion and revealed blood cells in the
pericardial chamber (Fig. 3B). In skNAC−/−embryos, the myo-
cardial wall was thinner than in wild-type littermates, the in-
terventricular septum was poorly formed, and trabeculae were
decreased and poorly organized. In agreement with the gross
appearance, section in situ hybridization analysis with cardiac
chamber-specific markers, such as Hand2, Hand1, and Tbx5, and
markers of valve-forming regions, such as Tbx2 and TGFβ2, re-
vealed appropriate specification of subdomains within the heart.
Although skNAC expression was restricted to myocardial layers
of the heart, we performed extensive histological analyses for
potential vascular or placental defects in the mutant embryos,
because these can also cause hemodynamic insufficiency. We did
not find any abnormalities outside the heart of skNAC-null em-
bryos. Vascular patterning appeared normal as marked by an
antibody against the endothelial-specific protein PECAM.
Ventricular Hypoplasia Is Caused by Proliferation Defects. We in-
vestigated whether the thin ventricular myocardial layer in
skNAC−/−embryos was caused by a proliferation defect or by
abnormal apoptosis. We examined hearts at multiple times, fo-
cusing on hearts before obvious pathologic abnormalities to
avoid secondary effects. At E10.0, before evidence of pericardial
edema or developmental delay, we found that the myocardium
of skNAC−/−hearts had a lower percentage of phosphohistone
H3 (pH3)-positive cells than wild-type littermates by counting
pH3 and MF20 double-positive myocytes (Fig. 3C). Quantitative
analyses of the proliferation defect showed 30% fewer pro-
liferating myocytes in skNAC−/−than in wild-type hearts (Fig.
3D; P < 0.01). The proliferation rates in the pericardium and
other parts of the embryo were similar in wild-type and mutant
mice at E10.0 (Fig. 3D). TUNEL assays revealed no apoptotic
cells in the hearts of either wild-type or skNAC−/−embryos.
Altered Gene Expression in the Hearts of skNAC Mutants. To identify
gene expression changes associated with the myocardial defects
in skNAC−/−hearts, we performed mRNA microarray analysis of
skNAC−/−and wild-type hearts at E10.5. We excluded any hearts
that showed signs of slow contraction, pericardial edema, or
hemorrhage to avoid gene expression changes secondary to
cardiac dysfunction. Three independent biologic samples for
each genotype were analyzed. Surprisingly, only 23 genes were
differentially expressed in all skNAC−/−hearts, 17 of which are
currently annotated genes.
and frontal views focused on the heart show hemorrhage within the pericardium. h, head; ht, heart; oft, outflow tract; rv, right ventricle; lv, left ventricle. (B)
Transverse sections of wild type and mutant (×2) E11.5 embryos stained with H&E. Note blood cells in the pericardial sac of skNAC mutants (arrow). Areas for
ventricular septum and left ventricle wall are shown at higher magnification. Thin myocardial wall in skNAC−/−hearts isindicated (black bar) (vi and ix). ra, right
atrium; la, left atrium; p, pericardium; ivs, interventricular septum; t, trabeculae. (Scale bars, 100 μm.) (C) Histological sections of wild type and skNAC−/−E10.0
hearts stained with either H&E, MF20 antibody, anti-pH3 antibody, or DAPI. Merged image of boxed area is shown at higher magnification. (D) Percentage
proliferating myocytes was determined as the number of the pH3-positive, MF20-positive cells divided by the total number of DAPI/MF20-stained nuclei per
levels of dysregulated genes in hearts of skNAC mutants at E10.5. Data are presented as mean of each genotype (n = 3) with SE (*P < 0.01).
| www.pnas.org/cgi/doi/10.1073/pnas.1013493107Park et al.
We validated candidates from the microarray by qPCR (Fig.
3E). Myoglobin, a muscle-specific hemoprotein (19), is a repor-
ted direct target of skNAC (15) and was one of the most down-
regulated genes in skNAC mutant hearts. Interestingly, two of
the other down-regulated genes, Periostin and Cxcl12 (Sdf-1),
positively regulate cardiomyocyte or endothelial proliferation in
the developing heart, consistent with the proliferation defect in
skNAC mutant hearts (20, 21). We reported that expression of
Irx4, a ventricular-specific member of the family of transcription
factors that contains an Iroquois box (22), was decreased in
Smyd1 mutants (5). The observation that Irx4 was also down-
regulated in the skNAC-null heart (Fig. 3E) suggests that some
targets may be commonly regulated, although the Smyd1-de-
pendent gene Hand2 (9) was unaffected in skNAC mutants,
implying that it is independently regulated. Finally, we found
that some genes normally expressed at low levels in differenti-
ated working myocardium were up-regulated. These genes in-
cluded the epithelial marker Krt2.8 (23) and Shox2, a trans-
cription factor required for normal cardiac conduction system
development (24), possibly indicating incomplete myocyte dif-
ferentiation in skNAC mutant hearts. Many of these dysregu-
lated genes are critical for normal cardiac physiology and could
individually or collectively contribute to the cardiac dysfunction
in embryos lacking skNAC.
skNAC Is Important for Postnatal Skeletal Muscle Growth and
Regulates Fiber Type Specification. In the subset of viable
skNAC−/−mice that survived postnatally, growth retardation was
evident as early as the end of the first week after birth, although at
the time of birth there was no obvious difference among litter-
4A). skNAC−/−mice were smaller than gender-matched litter-
mates at 3 mo of age, and the weight difference was statistically
significant in males (P < 0.01) (Fig. 4B). skNAC−/−mice were
physically active and fertile. Cardiac function was normal
Histological comparison showed less muscle mass in the calf
muscles of skNAC−/−than of wild-type mice, although the sizes of
individual myofibers were similar in both (Fig. 4C). Quantitative
inthe cross-sectional areaofmyofibers(Fig.4D) orthenumberof
myofibers per unit area (Fig. 4E). The combined results suggest
that the reduced muscle mass was more likely due to fewer myo-
fibers rather than to less growth of individual myofibers.
Because myoglobin and skNAC both are highly expressed in
slow oxidative skeletal muscle and myoglobin is a skNAC target
(15), we determined whether loss of skNAC affected the specifi-
cation of oxidative vs. glycolytic fibers of the muscle. Skeletal
muscle fibers can be classified with respect to myoglobin content.
Slow oxidative (type I) and fast oxidative (type IIa) fibers have
largeamounts ofmyoglobin and manymitochondria, whereas fast
glycolytic (type IIb) fibers have low myoglobin content and large
amounts of glycogen. Myoglobin-null mice have myofiber type
switching from oxidative to glycolytic fibers (25). More of the type
muscle, and less of oxidative fiber markers, such as Myh7 (type I)
and Myh2 (type IIA) (Fig. 4F). We confirmed the lower oxidative
potential of skNAC−/−muscle by staining for succinate dehy-
growth in skNAC−/−mice. (A) Growth re-
tardation of skNAC−/−mice at 1 wk (P7) and 5
mo of age. (B) Body weight of skNAC−/−mice
and gender-matched littermates at 3 mo of
age (male,n=6;female, n =11).The averageis
marked with a black line (*P < 0.01). (C) Re-
comparison of H&E-stained sections of wild-
type and skNAC−/−calf muscles shows the
smaller size of skNAC−/−muscle at 3 mo of age.
(iii and iv) At higher magnification, cross-sec-
tional area of individual myofibers looks simi-
lar in wild-type and skNAC−/−soleus. (Scale
bars, 100 μm.) (D and E) Quantitative analysis
of myofibers in skNAC−/−or wild-type muscle.
The number of myofibers in 480,000 μm2was
counted, and the cross-sectional area of in-
dividual fibers was measured by using ImageJ
software. Data are presented as mean with SE
(n = 3). (F) Muscle fiber type markers in soleus
of skNAC−/−mice. qPCR revealed that expres-
sion of Myh4 (glycolytic, type IIb) was signifi-
cantly increased in skNAC−/−muscle, whereas
Myh7 (oxidative, type I) and Myh2 (oxidative,
type IIa) markers were reduced. Data are pre-
sentedas meanwithSEs(n= 3,*P<0.05, **P<
0.01). (G) Histochemical assay for SDH activity
(blue stain) to measure oxidative potential in
skNAC−/−soleus muscle. The skNAC−/−soleus
demonstrated less oxidative potential than
wild-type soleus. Representative samples are
shown (n = 3).
Defects in postnatal skeletal muscle
Park et al. PNAS
| November 30, 2010
| vol. 107
| no. 48
drogenase (SDH) in frozen sections. SDH is located in the inner
membrane of mitochondria and is responsible for oxidizing suc-
cinate to fumarate in the citric acid cycle. skNAC−/−sections
showed lower oxidative potential as marked by SDH activity (Fig.
4G). These findings suggest that loss of skNAC causes myofiber
type switching from oxidative to glycolytic fibers.
skNAC Is Involved in Postnatal Muscle Regeneration. The reduced
muscle mass of skNAC−/−mice was apparent early in life (Fig.
4A). Postnatal muscle growth is mainly achieved by addition of
myoblasts derived from satellite cells to existing myofibers (26).
Satellite cells are a population of muscle precursor cells that exist
in postnatal muscle tissues. During periods of muscle growth and
regeneration after injury, satellite cells reenter the cell cycle and
fuse with myofibers (27, 28). Thus, we studied muscle growth
defects in skNAC−/−mice during regeneration after injury.
Necrotic injury was induced in the calf muscles (gastrocnemius
and soleus) of 2- to 3-mo-old skNAC−/−and wild-type mice by
injection of cardiotoxin. The injuries were histologically analyzed
12 d after the injury (Fig. 5A). Wild-type muscle showed small
myofibers containing centrally located nuclei, indicating normal
muscle regeneration, and the overall muscle architecture was
almost restored. However, regeneration of skNAC−/−muscle was
significantly impaired, as evidenced by myonecrosis and in-
filtration of mononuclear cells. In addition, skNAC−/−muscle
exhibited abnormal fat accumulation, marked by Oil Red O
staining. One month after injury, regenerated wild-type muscle
was indistinguishable from uninjured muscle, whereas injured
skNAC−/−muscle still had many myofibers with centrally located
nuclei and infiltrated mononuclear cells and adipocytes (Fig.
5B). Twelve days after the injury, numbers of cells with central
nuclei were similar in skNAC−/−and wild-type muscle (Fig. 5C).
Quantitative analysis of the cross-sectional area of myofibers
1 mo after the injury showed significantly smaller myofiber size in
skNAC−/−muscle (Fig. 5D). These findings suggest that skNAC−/−
satellite cells may be able to initiate the regeneration process but
may not be able to complete the regeneration program.
To determine whether skNAC plays a role in satellite cell
activation and proliferation, we isolated satellite cells from the
tibialis anterior muscle by FACS and monitored proliferation of
individual cells for 96 h on a microwell coated with hydrogel. We
found no significant difference in the proliferation profiles of
control (skNAC+/−) or skNAC−/−satellite cells (Fig. 5E). How-
ever, skNAC−/−satellite cells had more cell death than control
satellite cells over a 96-h period (Fig. 5F). These results suggest
that skNAC is likely involved in survival and possibly differen-
tiation of satellite cell-driven myoblasts, rather than satellite
cardiomyocyte expansion in asubset ofembryos.Dysregulation of
numerous genes involved in cardiac proliferation and differenti-
ation was observed in skNAC mutant hearts, many overlapping
with genes altered in Smyd1 mutants. skNAC-null mice that sur-
vived to adulthood revealed a role for skNAC during postnatal
injury. Thus, in skeletal muscle, and possibly developing cardiac
muscle,skNACmayregulate progressive additionorexpansion of
muscle cells from the pool of progenitors.
The physical interaction of skNAC and Smyd1 in the embry-
onic heart and the coincident expression of these two genes are
consistent with their cooperative regulation of downstream
events. Interestingly, Irx4 was down-regulated in skNAC−/−em-
bryonic hearts, similar to the previous observation in Smyd1−/−
mice (5). Irx4 is a member of the Iroquois homeobox tran-
scription factor family that is expressed specifically in the ven-
tricular chambers of the looped heart, where it activates
ventricle-specific genes and represses atrial genes (22). Similarly,
cle in skNAC−/−mice. (A) H&E staining of skel-
etal muscle 12 d after cardiotoxin-induced
injury. Note normalizing architecture in wild-
type muscle, with regenerating myofibers con-
taining centrally located nuclei (arrowhead).
skNAC-deficient muscle had signs of myonec-
rosis, with infiltrating mononuclear cells (ar-
row) and fat deposits (asterisk). Accumulation
of fat in regenerating myofibers in skNAC−/−
mice was marked by Oil Red O staining. (Scale
bars, 100 μm.) (B) skNAC−/−muscle still had
many myofibers with central nuclei (arrow-
head), mononuclear infiltrating cells (arrow),
and adipocytes (asterisk) 1 mo after injury;
wild-type muscle was almost indistinguishable
from preinjury muscle. Oil Red O staining indi-
cates fat replacement. (Scale bars, 100 μm.) (C)
Quantitative analysis of regenerating myo-
fibers with central nuclei 12 d after the injury (n
= 3). (D) Quantitative analysis of cross-sectional
area of regenerated myofibers 1 mo after the
injury. Myofibers of skNAC−/−mice had smaller
cross-sectional area than wild-type myofibers
(*P < 0.0001). (E and F) Proliferation and cell
death profiles of satellite cells isolated from
skNAC−/−mice. Single satellite cells isolated from
the tibialis anterior muscle of skNAC−/−or control
microwell array and monitored for 96 h by time-
lapse microscopy (34). A representative graph
from the three experimental replicates is dis-
played. Data are shown as mean with SE (*P <
a higher cell death percentage than those from control skNAC+/−littermates over the 96-h period.
Regeneration defects of skeletal mus-
| www.pnas.org/cgi/doi/10.1073/pnas.1013493107Park et al.
periostin, which promotes reentry of adult cardiomyocytes into Download full-text
the cell cycle by activating phosphoinositol-3 kinase, was down-
regulated in skNAC mutants and may contribute to the car-
diomyocyte proliferation defect (20).
skNAC is a Smyd1-interacting protein in cultured skeletal
myoblasts (12) and is markedly up-regulated after skeletal muscle
injury (29). Adult skeletal muscle possesses a remarkable re-
generative capacity because of satellite cells, which serve as
aresident poolofmuscleprogenitors.Inrapidly growingneonatal
muscle, nuclei of satellite cells and newly derived myoblasts from
activated satellite cells account for approximately 30% of myo-
fiber-associated nuclei, but after cessation of muscle growth by 3
wk of age, satellite cells are less than 5% of adult nuclei (30). The
postnatal muscle growth defect we observed in skNAC−/−neo-
nates and the failure of muscle regeneration in adults suggest a
defect in the satellite cells. Inaddition, we also observed extensive
replacement of myofibers by adipocytes during regeneration of
skNAC−/−mice. Recent studies showed that brown fat cells arise
in vivo from the Myf5-positive myoblast lineage by the action of a
PRDM16/EBP-b transcriptional complex (31, 32). Because sat-
culture system, skNAC might have a role in progenitor survival
and differentiation into the muscle lineage rather than activation
of satellite cells. However, the mechanism of action remains elu-
sive. It will be important to determine whether the interaction
with Smyd1, a SET domain-containing partner, is necessary for
skNAC function in muscle growth and reneration.
Materials and Methods
Yeast Two-Hybrid Library Construction and Screening. A cDNA library for yeast
two-hybrid screening was constructed with 200 ng of poly(A) RNA isolated
from E11.5 embryonic hearts with the MATCHMAKER Library Construction
kit, according to the manufacturer’s protocol (Clontech). Approximately 5 ×
106clones were screened with a full-length Smyd1 as bait; colonies that
were positive for β-galactosidase activity within 1 h were selected, and
inserts were amplified by PCR and sequenced.
Targeted Deletion and Generation of skNAC-Deficient Mice. Genomic frag-
ments surrounding exon 2 of skNAC were isolated and subcloned into
a vector containing a PGK-Neo cassette, which was introduced into 129S6 ES
cells as previously described (33). Two correctly targeted ES cell clones were
injected into C57BL/6 blastocysts to ultimately generate skNAC−/−mice.
Cardiotoxin-Induced Muscle Injury. The calf muscle of adult mice (2–3 mo old)
were injected with 100 μL of 10 μM cardiotoxin (Calbiochem), and mice were
sacrificed at specified times. Calf muscles (gastrocnemius and soleus) were
dissected, mounted on 7% tragacanth, snap-frozen in isopentane cooled in
liquid nitrogen, cryosectioned, and stained for histological examination.
Satellite Cell Isolation and Microwell Culture. Isolation of satellite cells and
microwell arrays from skNAC−/−and control animals were performed as
previously described (34, 35). Additional detail is provided in SI Materials
ACKNOWLEDGMENTS. We thank the Genomics Core (C. Barker, L. Ta, and
Y. Hao) and Histology Core (J. Fish and C. Miller) at the Gladstone Institutes
for technical support; R. Yeh for bioinformatics support and array analyses;
B. Taylor for manuscript preparation; S. Ordway and G. Howard for editorial
assistance; Drs. Penney Gilbert and Alessandra Sacco for expert instruction in
satellite cell preparation; Karen Havenstrite for hydrogel microwell fabrica-
tion and timelapse in the H.M.B laboratory; and P. Tucker and members of
our laboratory for discussion and review of the manuscript. J.A.M. was
supported by the Stanford Medical Scholars Program. H.M.B. is supported by
grants from the National Institutes of Health (NIH), Juvenile Diabetes
Research Foundation, Muscular Dystrophy Association, Leukemia and Lym-
phoma Society, California Institute for Regenerative Medicine (CIRM), Stan-
ford BioX Award, and the Baxter Foundation in Stem Cell Biology. D.S. is
supported by the National Heart, Lung, and Blood Institute/NIH, CIRM, and
the Younger Family Foundation. K.N.I. was a postdoctoral scholar of the
CIRM. This work was also supported by NIH/National Center for Research
Resources Grant C06 RR018928 (to the Gladstone Institutes).
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Park et al.PNAS
| November 30, 2010
| vol. 107
| no. 48