Periostin regulates atrioventricular valve maturation
Russell A. Norrisa,1, Ricardo A. Moreno-Rodrigueza,1, Yukiko Sugia, Stanley Hoffmana,
Jenny Amosb, Mary M. Harta, Jay D. Pottsb, Richard L. Goodwinb, Roger R. Markwalda,⁎
aDepartment of Cell Biology and Anatomy, Medical University of South Carolina, BSB Suite 601, 173 Ashley Avenue, Charleston, SC 29425, USA
bDepartment of Cell and Developmental Biology and Anatomy, University of South Carolina, Columbia, SC, USA
Received for publication 27 September 2007; revised 20 December 2007; accepted 3 January 2008
Available online 17 January 2008
Cardiac valve leaflets develop from rudimentary structures termed endocardial cushions. These pre-valve tissues arise from a complex
interplay of signals between the myocardium and endocardium whereby secreted cues induce the endothelial cells to transform into migratory
mesenchyme through an endothelial to mesenchymal transformation (EMT). Even though much is currently known regarding the initial EMT
process, the mechanisms by which these undifferentiated cushion mesenchymal tissues are remodeled “post-EMT” into mature fibrous valve
leaflets remains one of the major, unsolved questions in heart development. Expression analyses, presented in this report, demonstrate that
periostin, a component of the extracellular matrix, is predominantly expressed in post-EMT valve tissues and their supporting apparatus from
embryonic to adult life. Analyses of periostin gene targeted mice demonstrate that it is within these regions that significant defects are observed.
Periostin null mice exhibit atrial septal defects, structural abnormalities of the AV valves and their supporting tensile apparatus, and aberrant
differentiation of AV cushion mesenchyme. Rescue experiments further demonstrate that periostin functions as a hierarchical molecular switch that
can promote the differentiation of mesenchymal cells into a fibroblastic lineage while repressing their transformation into other mesodermal cell
lineages (e.g. myocytes). This is the first report of an extracellular matrix protein directly regulating post-EMTAV valve differentiation, a process
foundational and indispensable for the morphogenesis of a cushion into a leaflet.
Published by Elsevier Inc.
Keywords: Periostin; Fasciclin; Valvulogenesis; Heart; Collagen; Differentiation; Fibroblast; Myocyte; Knock-out; Atomic force microscopy; Gene deletion
Valvulogenesis commences with an endothelial to mesench-
ymal transition (EMT) resulting in a swellings of undiffer-
entiated mesenchymal tissue, known as endocardial cushions
(ECs) (de la Cruz and Markwlad, 1998; de la Cruz et al., 1977;
Eisenberg and Markwald, 1995; Kinsella and Fitzharris, 1980,
1982; Krug et al., 1985; Manasek, 1970; Manasek et al., 1973;
Markwald et al., 1975, 1977; Markwald and Smith, 1972;
Person et al., 2005). As blood circulates through the heart, these
EC swellings function as primitive valves prohibiting retrograde
blood flow between the atria and ventricle (atrioventricular
canal) and the ventricle and aortic sac (outflow tract). Over the
past 30 years much effort has been put forth to determine the
inductive signals essential for initiating and sustaining EMT. To
date, more than 100+ genes have been linked to this important
morphogenetic event (Camenisch et al., 2002; Holifield et al.,
2004; Lakkis and Epstein, 1998; Ma et al., 2005; Mjaatvedt and
Markwald, 1989; Mjaatvedt et al., 1998; Savagner, 2001;
Schroeder et al., 2003; Yamamura et al., 1997; Sugi et al.,
2004). However, the EMT event leading to the formation of
prevalvular cushions is just the initial phase in valvuloseptal
morphogenesis. The molecular and cellular events that occur
after this initial EMT event are quite complex and poorly
understood. This “post-EMT” process, which will yield a
mature valve leaflet or cusp and associated tension supporting
apparatus involves the migration and proliferation of mesench-
ymal cells to distend the forming cushions into the lumen,
followed by further elongation of the tissue and its gradual at-
tenuation (sculpting/thinning) and maturation. This remodeling
Available online at www.sciencedirect.com
Developmental Biology 316 (2008) 200–213
⁎Corresponding author. Fax: +1 843 792 0664.
E-mail address: email@example.com (R.R. Markwald).
1Authors contributed equally to this work.
0012-1606/$ - see front matter. Published by Elsevier Inc.
of cushions into valves is directed and proceeds through the
differentiation of cushion mesenchyme into fibroblastic inter-
stitial cells (Aikawa et al., 2006; Goldsmith et al., 2004; Hinton
et al., 2006; Kruithof et al., 2007; Lincoln et al., 2004, 2006;
Schoen, 1999; Weber, 1989). Based on this fundamental change
in cell phenotype, the matrix surrounding these fibroblasts
matures into highly organized and compacted lamellar arrays of
collagen rich fibrous tissue that confers the structural stability to
the growing valve necessary for handling the increasing
hemodynamic stress and strain of the beating heart (Icardo
and Colvee, 1995a,b; Kruithof et al., 2007). The process by
which the valve leaflet remodels and matures during later stages
of valvulogenesis is poorly understood. However, the spatio-
temporal pattern of periostin expression invokes a critical role
for this gene in post-EMT valvulogenesis (Kern et al., 2005;
Kruzynska-Frejtag et al., 2001; Lindsley et al., 2005; Norris et
al., 2004, 2005). Periostin is a secreted, matricellular protein
which is evolutionarily conserved and contains 4-repeated
domains related to the Drosophila midline fasciclin-1 gene
(Horiuchi et al., 1999; Litvin et al., 2005; Takeshita et al.,
1993). The mammalian fasciclin gene family comprises 4
members: periostin, βIG-H3, stabilin-1, and stabilin-2. These
genes have been shown to play important roles in cellular
processes such as adhesion, migration, and differentiation
(Diamond et al., 1993; Elkins et al., 1990; Falkowski et al.,
2003; Gillan et al., 2002; Hortsch and Goodman, 1990; Kim et
al., 2000, 2002; Nakamoto et al., 2002; Oshima et al., 2002;
Park et al., 2004; Snow et al., 1989; Takeshita et al., 1993; Yan
and Shao, 2006). In this report, we demonstrate that periostin
protein is highly expressed in the post-EMT murine cushions as
they undergo remodeling and maturation. Expression is
maintained throughout valvulogenesis; being expressed in the
mature fibrous AV and OT leaflets, the tendinous chords of the
supporting apparatus and the fibrous anchoring annulus into
post-natal and adult life.
Although gene-targeted periostin mice have previously been
described, a detailed characterization of a heart valve phenotype
was not reported (Kii et al., 2006; Norris et al., 2007; Oka et al.,
2007; Rios et al., 2005). As a starting point, we sought to
determine if periostin was required for proper maturation of AV
valve leaflets where we have found structural defects in a subset
abnormallypositive formyosinheavychain weredetected inthe
superior and inferior AV cushions of null animals. This ectopic
myocardial staining persisted throughout development and was
maintained into adulthood. AV valves in the adult periostin null
animals were hypoplastic, failed to completely delaminate, and
contained significant presence of myocardial tissue. Addition-
ally, these valves had a significant reduction in collagen
expression and organization which resulted in a reduction of
biomechanical properties. The chordae tendineae, an area of
intense periostin expression, exhibited significant defects in the
numberofbranchesand thickness intheirstemsinthenull mice.
Rescue experiments demonstrated that periostin not only
promoted post-EMT AV cushion migration, but was also
essential for the synthesis of collagen while repressing cardiac
for periostin in the differentiation and remodeling pathways of
cushion mesenchymal cells into mature valve fibroblasts which
is indispensable for proper leaflet formation and function.
Materials and methods
Immunohistochemical localization of periostin
Preparation and characterization of affinity purified anti-mouse periostin
antibodies were described previously (Kruzynska-Frejtag et al., 2004).
Immunohistochemical procedures for mouse embryos and adult hearts were
described previously (Sugi et al., 2004).Briefly, mouse embryos from ED9.0–
ED13.5, hearts from mouse fetuses at ED16 and adults were collected in Earl's-
buffered saline solution (EBSS, Invitrogen) and fixed with 100% methanol at
−20 °C overnight. Fixed samples were processed through descending methanol
series at 4 °C and embedded in Paraplast X-TRA. Serial 6 μm sections were cut
and deparaffinized in xylene. Sections were blocked with 10% normal goat
serum (MP Biomedicals) in 1% bovine serum albumin (BSA, Sigma)/phosphate
buffered saline (PBS, pH.7.4) and processed for double immunohistochemistry.
Sections were incubated with anti-mouse periostin antibodies (3 μg/ml)
followed by treatment with FITC-labeled goat anti-rabbit IgG (MP Biomedi-
cals). Normal rabbit IgG was used as a negative control for anti-mouse periostin
antibodies. The sections were then rinsed and incubated with MF20
(Developmental Studies Hybridoma Bank) followed by RITC-labeled goat
anti-mouse IgG (MP Biomedicals). Immunostained sections were examined
under a Leica BMLB fluorescent microscope.
Periostin null mice analysis
The generation of the periostin null mouse has previously been described
(Kii et al., 2006; Norris et al., 2007; Oka et al., 2007; Rios et al., 2005). For
analysis of embryonic time points, matings were set up between heterozygote
parents. Following caesarian section, embryo's at E12.5 were harvested (n=50),
genotyped by PCR and analyzed by either immunohistochemistry, or Masson's
trichrome staining (DakoCytomation) per standard procedures. Adult wild-type
(n=15), heterozygote (n=20) and periostin null (n=28) mice were analyzed for
3-D hanging drop assays
AV cushions were dissected free of myocardium from E12.5 wild-type and
periostin null mice. Individual AV cushions were placed in 25 μl hanging drops
(OptiMEM supplemented with 1% heat inactivated fetal bovine serum, 5 μg/ml
insulin, 5 μg/ml transferring, 5 ng/ml selenium, 100 units/ml Penicillin, 100 mg/
ml Streptomycin). 16 cushions were isolated from periostin null mice of which 8
were randomly chosen for the rescue experiments. For rescue experiments,
10 μg/ml of full-length purified mouse periostin (R&D systems) was added to
the hanging drops. 8 AV cushions were isolated from wild-type mice and placed
in culture as described above.Cushion explants were grown in culture for 7 days
after which total protein lysate was taken and processed for Western analysis.
The entire procedure was repeated 3 times resulting in the analysis of 24 null, 24
wild-type, and 24 rescued AV cushions.
Western blot analysis
AV cushions were lysed in 1× RIPA cocktail which included protease
inhibitors (Sigma). Lysate was mixed with a 2× protein loading dye and loaded
onto 4–15% SDS-PAGE protein gel (BIO-RAD). Gels were blotted onto
nitrocellulose and probedfor various markerantibodies:(MHC-abcam(1:1000),
MLC-abcam (1:1000), Periostin-abcam (1:10,000), collagen I-abcam (1:1000),
actin-chemicon (1:5000). Secondary antibodies used were goat anti-mouse
were detected with Visualizer (UpState Biologicals). Western analyses were
performed on each of the triplicate set of experiments. Densitometric analyses
was performed using Adobe Photoshop CS3 and results are presented
graphically. Actin is used as a normalization control.
201R.A. Norris et al. / Developmental Biology 316 (2008) 200–213
Atomic force microscopy measurements
Chordae tendineae (dissected from the right AV tricuspid valve apparatus)
and mural tricuspid valve leaflets were isolated from adult periostin null and
wild-type mice. The isolated mouse chordae and leaflets were placed on
individual glass slides. The tissues were rinsed of any residual salts in double
distilled water and maintained in a moist condition within the isolation
chamber. An Asylum MFP-3D Series AFM instrument (Asylum Research)
was used to scan the sample. The scanning head used to make the
measurements has a 100 μm scanning area. The cantilever used was silicon
nitride coated with a diving-board tip at 0.01 N/m constant force (Asylum
Research). These tips have a radius of curvature of less than 20 nm. To obtain
stiffness, the AFM was used in the “indentation mode”. First, the sample was
imaged to ensure that the tip was on the sample. The tip was then brought
toward the surface until it contacted the sample then was retracted a few
nanometers. A force distance curve was taken at this point by allowing the tip
to approach the surface and retract, in quick pulses; this then relayed the static
deflection of the tip on the surface. An average of 6 independent spots had a
force distance curve performed on them. These were then averaged and
normalized against the glass slide surface from which the force distance curve
was generated. The result was a plot with two averaged curves, one for the
approach and one for the retraction demonstrating pico-Newton forces along
the nanometer scale of approach and retraction. In order to corroborate that the
data was reliable, the curves generated were analyzed for similar slopes on
both the approach and retraction. Stiffness, by definition, is calculated as the
force/distance (F/D) traveled which is the slope of the curve obtained from the
force distance curve.
Periostin is predominantly expressed during post-EMT
The expression of periostin RNA during cardiac develop-
ment has been previously documented in the chick and the
mouse (Kern et al., 2005; Kruzynska-Frejtag et al., 2001;
Norris et al., 2004, 2005). However, the expression and
localization of periostin protein has not been rigorously
addressed, especially in post-EMT development of the mouse
heart. Therefore, specific anti-mouse periostin antibodies were
developed and utilized in immunohistochemical analyses of
Fig. 1. Periostin protein localization during mouse embryonic cardiac development. (A) Sagittal section of an ED 9.5 mouse embryo shows onset of periostin
expression (green) in the atrioventricular (AV) canal (arrows). Note the strong periostin expression within umbilical vessels (UV). MF20 staining is confined to the
myocardium (red). (B) Higher magnification view of the AV canal in panel A. Arrows indicate subendocardial expression of periostin. Periostin expression is also
detected in the cytoplasm of forming cushion mesenchymal cells (arrows). (C) Sagittal section of an ED11 mouse embryo shows periostin expression in the AVand
outflow tract (OT) cushion mesenchyme. (D) Higher magnification view of the cushion mesenchyme in panel C. Both AVand OTcushion mesenchyme show intense
periostinexpression.(E) Frontal section of an ED 12.5mouseembryo shows intenseperiostin expressioninthe OTcushion.Note, periostin expressionextendsbeyond
the border of the OTcovered by the myocardium and is also expressed in the epicardium (arrow heads). (F) Frontal section of an ED 12.5 mouse embryo shows intense
periostin expression in the AV cushion. Examination by serial sections did not reveal any MF20 staining in the AV cushion mesenchyme at ED12.5. Also note,
expression within the epicardium (arrow heads) and endothelial lining of the ventricular trabeculae (arrows). A, atrium; DA — Dorsal Aorta; 1st, first branchial arch;
IC, inferior cushion; SC, superior cushion; V, ventricle. Scale Bars: A, C–F=100 μm; B=50 μm.
202 R.A. Norris et al. / Developmental Biology 316 (2008) 200–213
Fig. 2. Periostin protein localization during mouse fetal cardiac development and within the adult heart. (A) Frontal section of an ED 13.5 heart, showing intense
periostin immunostaining (green) in the developing aortic valves (AoV) and aorta (Ao). Periostin expression is also detected within the inter ventricular septum (IVS,
arrows). (B) Frontal section of an ED 13.5 heart, showing intense periostin immunostaining in the developing tricuspid (TV) and mitral valves (MV) and the
mesenchymal cap of the dorsal mesenchymal protrusion (DMP) (arrow). (C) Higher magnification view of the TV, MV, and DMP in B reveals fibrous immunostaining
of periostin. (D) Periostin expression is detected within the forming epicardium (Ep) covering the ventricle and atrium and is also detected in the subendocardial space
of the trabeculae (Tb). (E) Frontal section of an ED16 heart, showing intense periostin immunostaining in the forming pulmonary valves (PV), aorta (Ao), pulmonary
trunk(PT),andformingfibrousannulus(arrow heads).(F) Frontalsectionof an ED16heart, showingintenseperiostin immunostaining withinthe developingtricuspid
valves (TV), epicardium (Ep), fibrous annulus (arrow head) and AV sulcus (asterisk). (G) Frontal section of an ED16 heart, showing intense periostin staining in the
developing mitral valves (MV). Periostin staining is also detected near the luminal edge of the inter ventricular septum (arrows), and the fibrous annulus (arrow head).
(H) Highermagnification view of the epicardiumat ED16, showingrobustepicardialand EPDCstaining(arrows).(I) Image of an adultheart showingintenseperiostin
immunostaining within the aortic valves (AoV) and aorta (Ao). Note, the fibrous staining of periostin in the aortic wall. (J) Intense immunostaining is detected in the
tendinous chord (TC), attached to the papillary muscle (PM). (K, L) Intense periostin immunostaining is detected in the adult tricuspid (TV) and mitral valves (MV).
LA, left atrium; LV, left ventricle; RA right atrium; RV, right ventricle. Scale bars: A, C–I, K, L=100 μm, B, J=500 μm.
203 R.A. Norris et al. / Developmental Biology 316 (2008) 200–213
various timepoints during cardiovascular development. Peri-
ostin protein is first detected within the atrioventricular (AV)
canal during the EMT process at embryonic day (ED) 9.5
(Figs. 1A, B). At this stage, cytoplasmic and extracellular
expression/secretion of periostin is seen within transformed
mesenchymal cells (Fig. 1B). Periostin expression is not
detected in the myocardium of the AV canal. Intense periostin
immunostaining is also detected in the umbilical vessels in
ED9.5 embryos (Fig. 1A). As EMT progresses, periostin
expression becomes predominantly extracellular with intense
staining in the enlarging AVand outflow tract (OT) cushions at
ED 11 and ED12.5 (Figs. 1C–F). By E12.5, the endothelium
lining the ventricular trabeculae and the epicardial epithelium
express periostin protein (arrow heads and arrows, respectively
in Figs. 1E, F).
After the initial EMT process has been completed, strong
expression is detected within the developing aortic and
pulmonary valve leaflets at E13.5 and E16 (Figs. 2A, E).
Expression within the aortic valve appears to be widespread
whereas the pulmonary valve exhibits more intense expression
on the ventricular aspect of the leaflet. Weak expression is also
evident within the aorta and the anterior aspect of the
interventricular septum, representing the ventral mesenchyme
of the fused AV cushions (arrows in Fig. 2A). Additionally,
intense expression is seen in the mesenchymal cap of the atrial
septum that attaches to the fused AV cushions (Figs. 2 B, C —
arrow head). The developing mitral and tricuspid AV valves
also exhibit intense periostin staining at E13.5 and E16 (Figs.
2A–C, E–G). Within these AV valves, expression is
predominantly localized to the atrial aspect of each valve.
Additionally, periostin is expressed in the endothelial lining of
the ventricular trabeculae and epicardium at E13.5 (Fig. 2D)
and can also be seen in epicardial derived cells in the
myocardial wall as well as the AV sulcus at E16 (arrows in Fig.
2H and asterisk in F, respectively). At E16, on either side of
the AV septum, strands of periostin expression are seen
associated with fibrous components of the developing
conduction system (Fig. 2G — arrows). Periostin expression
is more intense where the future annulus will develop that
serves to anchor the base of the free AV valve leaflets to the
myocardial wall (asterisks in Figs. 2E–G). In the adult mouse,
periostin expression is also intense within the developing
tendinous cords of the valve supporting apparatus (Figs. 2I–
L). Within the AV valves (tricuspid and mitral), expression is
more pronounced on the ventricular aspect of the leaflets.
Periostin expression is diminished within the epicardium
epithelium after ED16 and absent in the adult heart (data not
Periostin null mice exhibit embryonic lethality coincident with
AV cushion anomalies
The periostin gene targeted mouse and over-expressor
mouse have been previously described by us and others (Kii et
al., 2006; Norris et al., 2007; Oka et al., 2007; Rios et al.,
2005). Using these mouse models, results on tooth develop-
ment and adult myocardial remodeling in the ventricles have
been published. However, to date, a thorough investigation of
potential cardiac valve defects have not been reported. An
analysis of mice generated from het×het matings (n=136)
have indicated that the expected Mendelian numbers of
offspring were not statistically different between genotypes
(Fig. 3). However, it was noted that het×het matings generated
less offspring than the wild-type matings. This suggested that a
sub-population of the null animals were probably dying during
development. Fig. 3 shows that the viability of the embryos is
significantly reduced when both of the periostin alleles are
removed; lethality being most apparent after the E10.5
timepoint. Based on the intense expression of periostin during
post-EMT cushion morphogenesis we hypothesized that
knocking out this gene may have resulted in valve defects
that were not compatible with survival. At E10.5, no
morphological defects were observed (data not shown).
However, by E12.5, periostin null mice exhibited small islands
of MF20 positive cells within both the superior and inferior
cushions (Fig. 4). MF20 is a well-established marker for
contractile cardiac sarcomeric myocytes and is not normally
expressed within either the superior or inferior AV cushions of
wild-type mice. This suggested that periostin may function to
repress aberrant myocardial differentiation within the AV
cushion mesenchyme. To further test this possibility, AV
cushions from periostin null and wild-type mice were dissected
and placed in hanging drop cultures. Consistent with in vivo
findings, periostin null AV cushions were positive for the
Fig. 3. Embryonic lethality of periostin deficient mice. Numbers of viable neonates at P1 were counted from heterozygote matings (n=136) and compared to the
theoretical Mendelian numbers. No statistically significant differences were observed between each of the genotypes. However, a comparison between litter size of
Het×Het and WT×WT matings demonstrated some degree of lethality. This was further substantiated in the null×null matings which have less than one-half the litter
size of the wild-type mice. An analysis of embryo numbers at E10.5 indicated that it was after this timepoint when the embryos began to die.
204R.A. Norris et al. / Developmental Biology 316 (2008) 200–213
cardiac muscle markers myosin heavy chain (MHC) and
myosin light chain (MLC) (Fig. 5).
Purified periostin was administered to half of the null tissues
in culture and after 7 days the tissues were analyzed by Western
blotting for MHC, MLC and collagen I expression (Fig. 5).
Both myocardial markers, MHC and MLC, were decreased
whereas the expression of the fibroblast markers, collagen Iα1
and collagen Iα2, were increased in null AV cushion
mesenchyme that received purified periostin. This addition of
exogenous periostin significantly repressed the muscle markers
in the nulls while stimulating fibroblast markers, indicating
during normal development periostin in undifferentiated
cushion mesenchyme correlated with expression of fibroblast
markers, whereas its absence correlated with expression of
Interatrial septal defects observed in the periostin null mice
AV cushion tissue isalsocrucial for properformation of septal
septation requires the fusion of the dorsal mesenchymal
protrusion (DMP), or spinal vestibuli, and the mesenchymal
cap on the leading edge of the primary atrial septum with the
mesenchyme of the AV septum (formed by the fusion of the
superior and inferior AV cushions). The formation of this AV
mesenchymal complex anchors the atrial septum and progres-
sively closes the primary atrial foramen (Snarr et al., 2007). Due
to the intense expression of periostin in the AV cushions and in
were examined for defects in the closure of the primary atrial
foramen. 100% of the periostin null mice exhibited large primary
Fig. 4. Periostin null mice exhibit aberrant differentiation of AV cushion mesenchyme. (A) Sagittal sections through E12.5 wild-type mouse hearts were analyzed for
MF20 (green) and periostin (red) co-expression. (B) MF20 expression is absent within the AV cushions of wild-type mice. (C) Periostin null mice exhibit ectopic, de
novo, sarcomeric myosin expression indicating aberrant differentiation of AV cushion mesenchyme into a cardiac myocyte lineage.
Fig. 5. Western analysis of myocyte and fibroblast markers on periostin null, wild-type, and “rescued” AV cushion mesenchyme. AV cushions from periostin null mice
were placed in hanging drop cultures and incubated with either purified periostin protein (10 μg/ml) or PBS. AV cushions from wild-type mice were used as control
tissues. After 7 days tissues were harvested and analyzed for differentiation markers by immunoblotting. (A) Periostin null cushions exhibit high levels of the
myocardial markers: myosin heavy and light chains (MHC, MLC) and low levels of fibroblastic markers: collagen 1α1 and 1α2. Addition of periostin to the culture
medium induces expression of collagen 1α1 and 1α2 while repressing MHC and MLC (arrow heads). (B) Graphical representation of Western analyses presented in
panel A. Densitometric analyses were performed as described in Materials and methods. Values obtained were compared against wild-type values (baseline) and
represented as relative percent change.
205 R.A. Norris et al. / Developmental Biology 316 (2008) 200–213
planes and regions of wild-type mice and no defects were seen.
Due to the change in overall shape of the periostin null hearts
(being smaller and rounder), it was not possible to get the exact
plane.However, througha whole mount view, itispossibletosee
the extent of the ASDs which were “probe patent” (Fig. 6C).
Periostin is essential for the proper differentiation and
maturation of the AV valves
As the mitral and tricuspid valves elongate, attenuate and
maturate during fetal and post-natal development, the
mesenchymal cells differentiate into fibroblasts and secrete
collagen (Kruithof et al., 2007). Periostin is most intensely
expressed during these post-EMT stages of valvular modeling.
Thus, adult periostin null mice were examined for defects in
post-EMT cardiac valve maturation. The periostin null mice
did exhibit significant defects in the mitral and tricuspid valve
architecture and cellular content. In wild-type mice, these
valves stained exclusively blue with Masson's trichrome
stain, which indicates a primarily fibrous, collagen composi-
tion. However, the periostin null mice have a significant
reduction in blue staining, indicating a lack of organized
fibrillar collagen containing matrix (Fig. 7). Quantitative
Western analyses indicated that there is a ~70% reduction in
collagen 1α1 and 1α2 protein in the heterozygote and null
leaflets. Additionally, the tricuspid septal leaflet of the
periostin null mouse had extensive myocardial tissue (red)
associated with the ventricular aspect of the leaflet. This
finding was also present on the ventricular surface of the left
and right mural AV valve leaflets indicating a potential defect
in delamination. The mural leaflets normally develop upon a
template/substrate of myocardial tissue that is later remodeled
to remove or replace myocardial cells during delamination of
the leaflet from the ventricular wall, ultimately leaving a
purely fibrous leaflet as shown in the wildtype heart (Gaussin
et al., 2005). Periostin is most intensely expressed in the
boundary between AV cushion tissue and associated myo-
cardial tissue, suggesting that periostin may play a role in the
remodeling and/or separation (delamination) of developing
AV mural valves from myocardium. Finally, the leaflets of the
periostin null mouse had mesenchymal-like swellings contain-
ing abundant ECM and cells resemebling post-EMT cushion
mesenchyme. The cell type(s) within these swellings could
not be specifically identified. But due to their cell shape
(stellate), size (small) and lack of Masson's staining, these
cells did not appear to be either myocytes or fibroblasts.
However, we cannot rule out that other cell types may
contribute to these swellings (Fig. 7).
Periostin is required for chordae tendineae formation and
Another area of intense periostin expression is the tendinous
chords, or chordae tendineae (CT). The chords are fibrous
structures that ramify through several generations of branching
that connects the mural leaflets of the mitral and tricuspid valves
to papillary muscles (Morse et al., 1984). Periostin is expressed
throughout the chordae tendineae with expression being most
intense at the myotendinous junction where the stem or trunk of
tree-like chords inserts into the papillary muscle. The periostin
null mice exhibit striking defects in chordal development as
depicted in Fig. 8. The chordal trunks of the tricuspid valve in
periostin nulls have few ifany branches compared to neonatal or
adult wild-type mice. In wild-type development, the trunks of
the murine tricuspid valve apparatus have 2–3 generations of
branching at birth. As the mouse ages, the tendinous chords
continue to divide and branch, giving rise to ~10–15 mature
fibrous chords (Figs. 8A, D). In null hearts, the persisting
chordal trunks were shortened and abnormally thickened in
diameter (compared to wild-type chordae tendineae). This
defect was seen in nearly 40% of all neonates and 20% of all
adult mice (data not shown). In addition, a gene dosage affect
was seen in the percentage of chordal trunks that were not
branched, demonstrating a correlation between both periostin
alleles and the chordal branching process.
Fig. 6. Periostin null mice exhibit interatrial septal defects. Frontal sections of adult wild-type (A) and periostin null (B) animals stained for Masson's trichrome.
Periostin null mice have a failure of fusion between the dorsal mesenchymal protrusion and the extending primary atrial septum resulting in atrial septal defects. The
hole formed by this failed fusion results in an interatrial communication between the right and left atrium evidenced by an insect needle on gross examination (C).
RA — right atrium, RV — right ventricle, LA — left atrium, IVS — interventricular septum, IAS — interatrial septum, IAC — interatrial communication.
206 R.A. Norris et al. / Developmental Biology 316 (2008) 200–213
Biomechanical properties of tricuspid leaflets and chordae
tendineae are altered in periostin null mice
In vivo and in vitro analyses demonstrated that periostin is
necessary for fibroblast differentiation, collagen synthesis,
attenuation and compaction of cushions into leaflets and the
formation of their suspensory tension apparatus. How periostin
ultimately affected the integrity and strength of these fibrous
tissues was further evaluated. The small size of valve tissue
made utilization of a conventional materials testing system
(MTS) not possible. However, the biomechanical properties for
mouse valves could be attained using atomic force microscopy
(AFM). AFM is a technique that provides high-resolution
scanning and cantilever measurements of micromechanical
properties along a tissue surface. This technique has been
previously used to generate topographical and biomechanical
profiles ofcardiaccellsandtissues, including human and bovine
heart valves (Brody et al., 2006; Jastrzebska et al., 2006, 2008;
Mathur et al., 2001; Merryman et al., 2007). We compared both
the surface structure and the mechanical properties of freshly
isolated wild-type and periostin null tricuspid valve leaflets and
associated tendinous chords. Results demonstrated that the
surface of the tricuspid valve leaflet and tendinous chords of the
periostin null animals were significantly smoother than that of
their wild-type counterparts. Organized collagen bundles were
only observed within the wild-type tendinous chords and valve
leaflets and never within the periostin null tissues (Figs. 9A–E).
Importantly, the cantilever force indentation data collected from
50–100 randomized locations within the wild-type and null
tricuspid leaflets and tendinous chords indicated that these
tissues were mechanically less rigid (i.e. reduced tensile
properties) in the periostin null mice (Fig. 9F). Thus, periostin
is not only essential for regulating collagen synthesis and valve
properties of mature cardiac valves.
The identification of genes related to normal and abnormal
valvuloseptal development remains largely unknown due to the
Fig. 7. Periostin null mice exhibit defects in delamination, fibroblastic differentiation, and remodeling of associated myocardium. (A–E) Frontal section of an adult
periostin null mouse heart stained with Masson's trichrome. A significant reduction of collagen matrix (blue staining) is seen in the tricuspid mural and septal leaflets
(B–E) compared to wild-type mice (F). Failure of complete delamination of the mural tricuspid leaflet is evident in the periostin null mouse as are large foci, or
swellings (B–D). In the septal leaflet of the tricuspid leaflet there is failure of normal remodeling/removal and/or transdifferentiation of associated myocardium (B, E).
Collagen 1α1 and 1α2 are reduced by ∼70% in the periostin heterozygote and null mice as shown by Western analysis. Actin is used as a loading control.
207 R.A. Norris et al. / Developmental Biology 316 (2008) 200–213
lack of appropriate model systems to study. Most genes
important for the EMT phase of valvulogenesis results in an
early embryonic lethality and shed little light on how post-EMT
mesenchymalized cushions become mature cusps of leaflets.
Post-EMT valvulogenesis, as defined in this study, involves
three fundamental processes: (i) the differentiation of cushion
mesenchymal cells into valvular interstitial fibroblasts, (ii)
modeling and attenuation of the valve leaflet, and (iii) formation
of valve-associated fibrous structures such as the annulus
fibrosae and chordae tendineae. It is these processes that act in
concert with fluid dynamics to ultimately sculpt the rudimentary
cushions into a mature valve apparatus (Butcher and Markwald,
2006; Butcher et al., 2007a). The lack of functional data and
appropriate gene knock-out animals have made the post-EMT
process difficult to understand. In this report, we describe the
detailed expression and null phenotype of periostin, a candidate,
post-EMT valvulogenic protein expressed during intrauterine
and postnatal life. Periostin null mice described herein display
defects in the three fundamental processes (as described above)
required for the normal maturation and development of the
cardiac valve apparatus. Importantly, a large subset of the
periostin null mice is viable and thus may provide a powerful
tool to begin understanding mechanisms underlying post-EMT
Fig. 8. Periostin is required for proper formation of the chordae tendineae. (A) AVapparatus of an adult wild-type mouse showing (1) anterior tricuspid leaflet, (2)
tendinous chords, and (3) papillary muscles. (B and C) Comparison of adult wild-type (Pn+/+) and periostin null (Pn−/−) tendinous chordal structure (asterisks). The
periostin null mice exhibit thickened, non-branched, severely shortened tendinous chords. (D and E) Graphical representation of defects in chordal structure in the
periostin null mouse. (D) The number of chordae tendineae normally doubles from neonate to adult. This doubling of the chordae tendineae is not evident in the
periostin null mice. (E) The chordae tendineae of the adult periostin null tricuspid valve apparatus has a significantly thicker diameter than that of the wild-type mouse.
This defect appears to be nearly as prevalent in the heterozygote as in the null further suggesting the importance of having both intact periostin alleles.
208 R.A. Norris et al. / Developmental Biology 316 (2008) 200–213
Periostin is an evolutionary conserved extracellular matrix
(ECM) protein that has significant similarity with its other
family members: βIG-H3, stabilin-1, and stabilin-2. The
expression of the stabilin and βIG-H3 genes have been
evaluatedthroughoutdevelopment (Lindsley etal.,2005; Norris
et al., 2005). Whereas expression of the stabilin genes do not
overlap with periostin, there is partial overlap of periostin with
βIG-H3 which might confer sufficient compensation to permit
survival. A periostin/βIG-H3 double knock-out mouse would
help address this possibility. Periostin has been demonstrated to
play an important role in cellular processes such as adhesion,
migration/cell sorting, and differentiation.
It is well known that periostin expression is significantly
increased upon TGFβ or BMP stimulation in a variety of cell
types, but is also known to be transcriptionally regulated during
development by the basic helix–loop–helix gene, twist1
(Horiuchi et al., 1999; Li et al., 2006; Lindner et al., 2005;
Afanador et al., 2005; Connerney et al., 2006; Oshima et al.,
Fig. 9. Atomic force microscopy (AFM) on mouse chordae tendineae and valves. Chordae tendineae and valve leaflets were removed from wild-type (A, C) and
periostin null mice (B, D) and processed for AFM. (A) A surface scan at high resolution was performed on a wild-type mouse chordae tendineae. Fibrillar structures
(arrows) are apparent at the surface of the chordae. (B) A similar scan of a chordae from a periostin null mouse shows a smoother surface with less distinct fibril-like
structures. (C) A control valve leaflet was scanned at high resolution with numerous pore-like depressions observed (arrows). In comparison, periostin null leaflets
were again smooth with minimal surface depressions. The scan sizes were 4 μm2in panels A and B and 1 μm2in panel C and 2.5 μm2in panel D. (E) Demonstrates the
view prior to scanning the tissues taken from the AFM. The cantilever is shown as the triangular structure with the tip placed on the center of the 40 chordae tendineae.
For reference the cantilever is 160 μm in size. (F) Represents the force measurements from the tissues illustrating that there is a significant decrease in stiffness of
chordae tendineae(red and green lines) as comparedto the leaflets (blueand gold lines). In addition, there is a dramatic decrease in stiffness in the periostin-null leaflet.
The control leaflet (gold line) is more than twice as stiff as the periostin-null valve leaflet (blue line). Relative stiffness numbers are represented in μN.
209 R.A. Norris et al. / Developmental Biology 316 (2008) 200–213
2002). Periostin can interact in vivo with other ECM scaffold
proteins, such as collagens, fibronectin, and tenascin as well as
integrin receptors at the cell surface including: αV/β3, αV/β5,
and β1 (Butcher et al., 2007b; Gillan et al., 2002; Norris et al.,
2007; Takayama et al., 2006). Recently, we have found that
periostin binding to integrins αV/β3 and β1 on cardiac
mesenchymal progenitor cells can initiate signaling related
to migration and collagen crosslinking or compaction trans-
duced through Rho and PI3 kinases (Butcher et al., 2007b;
Norris et al., 2007). Thus, there is an integrin-based receptor
mechanism for mediating the potential effects of periostin on
Although RNA in situ expression of periostin has been well
documented in mouse and chick, a detailed analysis of the
protein expression in the mouse has yet to be reported. Our
analysis has revealed temporal–spatial patterning of periostin
not previously appreciated. For example, while expression of
periostin commences at E9.5, which coincides with the ongoing
EMT process, the majority of expression at this time remains
cytoplasmic suggesting that even though the protein is
produced, it is not secreted or fully functional. By E10.5,
periostin is expressed and secreted by the cushion mesenchyme
of the AVand OFT regions as seen by ECM staining. As post-
EMT valvuloseptal morphogenesis commences, periostin
expression intensifies in areas that are undergoing active
morphogenetic remodeling such as the fibrous annulus, the
AV and OFT valve leaflets, the chordae tendineae, the
mesenchymal cap of the septum primum, and the epicardium.
These areas express high levels of periostin are fated to
normally become fibrous tissue. Thus, it is our working
hypothesis that periostin expression is required for fibrogenesis.
In fact, our recent data have demonstrated that periostin
regulates collagen fibrillogenesis and the biomechanical proper-
ties of connective tissues (Norris et al., 2007). Consistent with
this observations, periostin has been shown to be integral to the
formation of a fibrous scar following a myocardial infarct (MI)
(Iekushi et al., 2007; Oka et al., 2007). Inducing an MI in the
periostin null mice gave precisely the opposite affect: a decrease
in fibrous tissue and formation and/or survival of cardiomyo-
cytes (Oka et al., 2007; Norris et al., in press). However, it
should be pointed out that these data conflict with what has
recently been described by Kuhn et al. who reported that
periostin promoted cardiac regeneration and reduction in scar
tissue following the addition of “purified periostin” to the heart
after a myocardial infarction (Kuhn et al., 2007). The periostin
used in these studies was a carboxyl-truncated form of periostin
lacking 163 amino acids of the carboxyl terminus. It has been
shown that the carboxyl terminus encodes a functional domain
that is important for migration, invasion, and adhesion (Kim
et al., 2005). Thus, one alternative interpretation is that
absence of this domain may act as a dominant-negative protein
when applied in vivo. Our data, which includes deleting
expression of the entire protein, strongly suggests that periostin
is profibrogenic and important for the differentiation of
cushion mesenchyme into fibroblastic tissues.
By E12.5, the periostin null mouse displays defects in AV
cushion mesenchymal differentiation as pockets of cells that are
positive for the myocardial marker, MF20, within the AV
cushions. In the case of the proximal (conal) cushions of the
outflow tract, this process happens naturally whereby the
cushion mesenchyme normally undergoes muscularization
(termed “myocardialization”) (van den Hoff et al., 2001).
Periostin expression decreased in the conus mesenchyme prior
with the observation that mesenchyme in the periostin null AV
cushions can differentiate into myocardial tissue suggested to us
that this matrix factor is functioning to promote fibrogenesis of
the AV cushions, and/or block aberrant differentiation into a
non-fibroblastic lineage (e.g. myocardial). Furthermore, in
culture, periostin null AV mesenchymal cells expressed
cardiomyocyte markers, whereas expression of fibroblasts
markers was low or absent. The addition of exogenous periostin
to null cushion mesenchyme significantly reduced myocardial
marker expression while enhancing fibroblast differentiation
markers. To date, this is the first example of a secreted,
extracellular protein that can directly, or indirectly, alter the
differentiation pathway of AV cushion mesenchyme. We
interpret these findings to indicate that periostin may function
as a “binary or hierarchical switch” promoting firbogenesis (if
present) vs. cardiomyogenesis (if absent). What is clear is that
these altered patterns of differentiation seen during intrauterine,
post-EMT valve development in periostin null animals is
sustained into postnatal and adult life. Thus, periostin is a
the normal differentiation of cushion mesenchyme.
In wild-type hearts, by ED 16, periostin is expressed
predominantly within the mural leaflets at sites where the
developing tricuspid AV valve is separated from the ventricular
myocardium. Conversely, in the adult periostin null mouse, the
delamination or separation of the leaflet from myocardial tissue
appears incomplete and the valve retains primitive character-
istics (swollen foci of ECM containing undifferentiated
mesenchyme). To date, the mechanism(s) by which the
myocardium is removed from the tricuspid leaflet is not
known. However, there is intense expression of periostin at
sites where cushion prevalvular mesenchyme directly contacts
myocytes (e.g. ventricular trabeculae and the AV junctional
myocardium) which correlates with the normal removal or
“disappearance” of the associated myocardial tissue (Gaussin et
al., 2005). This suggests to us a possible role for periostin as an
anti-myocardial factoraffectingthefate orsurvival of myocytes.
A more speculative alternative would be that specific myocytes
in direct contact with periostin secreting fibroblasts are capable
of “transdifferentiating” into myofibroblasts or fibroblasts. Such
a mechanism has been proposed for myocytes during patholo-
gical remodeling in adult life (d'Amati et al., 2000). This theory
is further supported by recent work by Kolditz et al. who find
that periostin is expressed around the myocardial accessory
pathways of the conduction system during quail development.
By adulthood, the expression of periostin within these
myocardial accessory pathways eventually result in inhibition
of the myocardial phenotype and transdifferentiation of the
myocytes into fibrous tissue (Kolditz et al., 2007). Additionally,
this theory is consistent with data presented by Katsuragi et al.
210 R.A. Norris et al. / Developmental Biology 316 (2008) 200–213
whereby transfection of periostin into adult rat hearts not only
induced ventricular fibrosis but also resulted in a loss of
ventricular cardiomyocytes (Katsuragi et al., 2004) and recent
work involving the Alk3/Bmpr1A knockout mouse also
implicated periostin as a putative mediator in regulating
myocyte–fibroblast interactions during tricuspid leaflet delami-
nation and maturation (Gaussin et al., 2005). The tricuspid
leaflets of the Alk3/cGATA6-Cre conditional knockout (CKO)
mouse exhibit a reduced level of periostin expression. In this
mouse, Alk3 was specifically deleted in myocytes, a cell type
that does not secrete periostin, suggesting that there is dynamic
signaling and communication between the myocytes and
fibroblasts that could affect myocyte survival, adhesion, and/or
migration (Gaussin et al., 2005). Whether this interaction is
mediated by periostin remains to be determined.
Congenital heart defects represent the most common form of
birth defects in humans, with those attributable to post-EMT
valvulogenesis among the most prevalent (Hinton et al., 2006;
Hoffman, 1990). These include structural (anatomical) defects
such as Ebstein's anomaly, mitral valve stenosis, mitral valve
syndrome, Ehlers–Danlos syndrome, and polyvalvular disease
(Anderson et al., 1979; Bonnet et al., 1997; Castaneda et al.,
1969; Chesler and Gornick, 1991; Gittenberger-de Groot et al.,
2003; Oosthoek et al., 1997; Ruckman and Van Praagh, 1978;
Zuberbuhler et al., 1979). It is important to note that defects in
post-EMT valvulogenesis (except severe mitral regurgitation)
are compatible with embryonic and post-natal life and may not
be immediately recognized at birth but over time cause
morphological or functional changes. Thus genes that encode
ECM proteins, like periostin, may not produce an immediate
phenotype. It is their regulation, organization, alignment, and
turn-over which, over time, accumulate to adversely affect valve
function, resulting in progression of prolapsed valves, ectopic
fibrosis, formation of myxomatous lesions, and calcifications.
Also, hemodynamic responses to aberrant valvular ECM
composition can have secondary consequences such as
myocardial hypertrophy, dilated cardiomyopathies, ectopic
cardiac fibrosis, and cardiac failure. Collectively, structural
and ECM based functional defects contribute to 100,000+ valve
replacement surgeries per year in the United States. Thus, a
greater understanding of how developmental process, such as
post-EMT valve maturation contribute to adult diseases is
necessary which will provide the foundation for the generation
of better therapeutics to combat these diseases. Here, we
have described the function of periostin during post-EMT
valve maturation. To date, this is the first known candidate
valvulogenic protein capable of controlling cell fate, either
directly or indirectly, within the maturing valve apparatus,
and as such has provided us with a potential novel tool for
understanding mechanisms underlying cardiac valvular diseases.
of Health: RO1 HL33756 (RRM), COBRE P20RR016434-07
(RRM), RO1 HL072958 (JDP), HL086856-01 (RLG), SC
INBRE 5MO1RR001070-28 (RAN); American Heart Associa-
tion: 0755525U (YS), 0765280U (RAN); and National Science
Foundation: FIBRE EF0526854 (RRM and RAN).
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