|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online 28 November 2007
doi: 10.1242/dev.010363
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Department of Cell Biology, Duke University Medical Center, Durham, NC 27710, USA.
* Author for correspondence (e-mail: k.poss{at}cellbio.duke.edu)
Accepted 1 October 2007
 |
SUMMARY |
|---|
 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Key words: Cardiomyocyte, Epicardium, Heart, Regeneration, Tissue homeostasis, Zebrafish
|
INTRODUCTION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Potter and Xu,
2001; Taub, 2004).
Understanding the cellular and molecular mechanisms of organ homeostasis is a
major research goal, with potential to shed light on therapies for trauma,
degenerative disease and aging.
Although cellular and molecular mechanisms of homeostatic regulation in
most organs are incompletely understood, cardiac homeostasis is particularly
mysterious. For many years, it was believed that the adult mammalian heart is
a post-mitotic organ, and that all postnatal cardiac growth is achieved
through hypertrophy of a fixed number of cardiomyocytes (CMs). Indeed,
analyses of DNA synthesis in the adult murine heart estimated a very low
percentage (a maximum of 0.0005%) of ventricular CMs entering the cell cycle
each day (Soonpaa and Field,
1997). Similar analyses during postnatal rodent growth indicated
that CMs undergo a transition shortly after birth from hyperplastic to
hypertrophic growth, associated with the production of large, multinucleated
CMs through karyokinesis rather than with new, mononucleated CMs through
cytokinesis (Li et al., 1996;
Soonpaa et al., 1996).
Furthermore, there is little evidence of consequential regeneration after
cardiac injuries such as ischemic infarction, and the resulting hypertrophy of
existing muscle that occurs concomitantly with scar formation is detrimental
to cardiac function. The notion that the major structural cells within such a
vigorous and vital organ survive 70 to 100 years in humans without support
from new CMs is contested, especially given recent identification of cell
populations that may possess progenitor activity in the postnatal mammalian
heart (Beltrami et al., 2003;
Laugwitz et al., 2005;
Martin et al., 2004;
Oh et al., 2003). However, a
natural in vivo contribution of these progenitor cells to adult tissue
homeostasis has not been experimentally demonstrated.
The mechanisms by which non-myocardial cardiac cell types are maintained
and replaced are also poorly understood. Interestingly, recent studies have
found that cells of the adult mammalian epicardium, a thin epithelial tissue
surrounding the myocardium proper, can be experimentally stimulated to
differentiate into smooth muscle and endothelial cells in vitro
(Smart et al., 2007;
van Tuyn et al., 2007). In
this way, the in vitro activity of the adult epicardium appears to mimic the
capacity of the embryonic epicardium, a structure that serves as the primary
source of coronary vasculature during heart development
(Reese et al., 2002). Thus, it
is possible that adult epicardial cells also function as a progenitor tissue
to maintain the vasculature or other cardiac cell populations.
Certain non-mammalian vertebrates such as amphibians and fish may have
greater potential for hyperplastic cardiac homeostasis. By contrast with
mammals, many of these species display indeterminate growth, and can rapidly
increase adult mass in response to changes in population density and nutrition
(Jordan, 1905), an increase
that is typically accompanied by organ augmentation. Moreover, some of these
species have demonstrated various degrees of regeneration after mechanical
injury of the cardiac ventricle, a response that may be perceived as a
homeostatic method to restore cardiac mass
(Flink, 2002;
Oberpriller and Oberpriller,
1974; Poss et al.,
2002). In particular, the zebrafish, which displays indeterminate
growth (Tsai et al., 2007),
has a strong cardiac regenerative response, and is amenable to molecular
genetic approaches, represents a unique model system to visualize and dissect
cardiac homeostasis.
Here, we show that adult zebrafish display dramatic, hyperplastic cardiac growth in response to aquarium conditions that stimulate rapid animal growth, whereas animals maintaining cardiac size show distinct but rare addition of new CMs. Additionally, we find that rapid growth conditions induce epicardial expression of embryonic markers such as raldh2 (also known as aldh1a2 - Zfin) and tbx18, and that the epicardium regularly contributes cells to the adult ventricular wall even in the absence of cardiac growth or myocardial injury. Inhibition of Fgf signaling, a pathway necessary for normal heart regeneration, disrupts epicardial cell supplementation and causes spontaneous ventricular scarring in uninjured adult fish. Our study exposes dynamic myocardial and epicardial mechanisms that mediate cardiac homeostasis.
|
MATERIALS AND METHODS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
). All
experiments on animals were performed in accordance with institutional
guidelines and regulations.
Histological techniques
In situ hybridization on cryosections of paraformaldehyde (PFA)-fixed
hearts was performed using digoxigenin-labeled cRNA probes as described
previously (Poss et al.,
2002). Acid fuchsin-Orange G staining was performed as described
previously (Poss et al.,
2002). Immunofluorescence was performed
(Poss et al., 2002) using
antibodies against BrdU (rat; Accurate), Mef2 (rabbit; Santa Cruz
Biotechnology), PCNA (mouse; Sigma) and DsRed (rabbit; Clontech). Terminal
deoxynucleotidyl transferase biotin-dUTP nick end-labeling (TUNEL) reactions
were performed on 10 µm cryosections of PFA-fixed hearts with reagents from
Invitrogen, and visualized with peroxidase substrate reagents from Vector
Laboratories.
Ventricular size, CM BrdU incorporation, CM density, %RFPcyto cells and EPDC density
To calculate ventricular section surface area, hearts were sectioned
longitudinally, stained with TRITC-phalloidin, and the three largest sections
of the ventricle were imaged and measured for calculation of the area using
Openlab software. The measurements were averaged to give one value for each
heart.
For BrdU incorporation experiments, animals were injected once daily with 2.5 mg/ml BrdU for 3 days prior to collection. Hearts from RG (injected 7, 8 and 9 days after introducing density conditions), SG, and MS animals were cryosectioned and immunostained for Mef2 and BrdU. Images of the middle of the lateral ventricle wall (opposite the atrium) from the three largest sections of each ventricle were selected for analysis. Nuclei labeled with Mef2, a marker of CMs, BrdU, or both were counted by hand using Adobe Photoshop images. The region analyzed included the compact muscular wall and a region approximately six cells thick of trabecular muscle, so that both trabecular and compact muscle would be included. For these experiments, 150-300 Mef2pos nuclei were counted per section, or 500-900 per animal.
To calculate CM density, Mef2-stained ventricular sections were used to
produce similar images. Then, the area of the region was calculated using
Openlab software, and the number of CM nuclei within the traced region counted
by hand. About 100-300 nuclei were counted per section, or 400-900 per animal.
To assess CM nucleation, ventricles were isolated from cmlc2:nRFP RG,
SG and MS fish (see Results for description of transgenic zebrafish), and
cells were dissociated as described previously
(Warren et al., 2001).
Isolated cells were collected in L-15 medium and live cells imaged to
determine the number of nuclei.
For analysis of RFPcyto cells (see Results for RFPcyto description), cryosections from cmlc2:nRFP ventricles were stained with anti-DsRed antibody and the three largest sections from each heart were used for imaging. For these experiments, images of the apex were selected for analysis. RFPnuc and RFPcyto cells were counted by hand from regions including the ventricular wall and approximately four cell layers of trabecular muscle. About 150-300 CMs per section were counted, or 600-900 per animal.
For analysis of epicardial-derived cells (EPDCs) within the ventricular wall, in situ hybridizations for tbx18 were performed on hearts from wild-type or hsp70:dn-fgfr1 animals under various growth conditions. Images from the middle of the lateral ventricle wall (opposite the atrium) were taken from the three largest sections of each heart. For each section, only tbx18-positive cells clearly separated from the epicardium and within the compact myocardial wall were counted (see Figs 5 and 6).
RNA isolation and real-time Q-PCR analysis
RNA was collected from 12-15 MS, RG, SG and regenerating [7 days
post-amputation (dpa)] ventricles by extracting the heart in PBS on ice and
mechanically removing the atrium and outflow tracts. Ventricles were
transferred to TRI reagent (Sigma) and the RNA was isolated according to the
manufacturer's instructions, before purification using Qiagen RNAeasy columns.
RNA integrity was assessed by gel electrophoresis, and concentration
determined by spectroscopy. cDNA was made from 200 ng RNA using oligo(dT)
primer and SuperScript III reverse transcriptase (Invitrogen). Quantitative
PCR (Q-PCR) was run using LightCycler FastStart DNA MasterPLUS SYBR
Green I (Roche) on a LightCycler 2.0 machine (software version 4.05x).
Dilutions of cDNA generated from pooled RNA of 20 hours and 56 hours
post-fertilization embryos was used to determine the reaction efficiency (E)
for each primer pair, and 
CT for each group was calculated
using 7 dpa regenerating hearts as a standard. hand2 expression was
normalized to gapdh for each sample using the following equation:
X=((E)gapdh)-CTgapdh)/((E)hand2)-CThand2).
All samples were run in triplicate, and the averages of two independent
experiments are shown.
Pericardial manipulations
Adult zebrafish 
6 months of age were placed into 1.5 l tanks (3
fish/l) to maintain a constant density during the experiment. Hearts from
uninjured control animals, as well as manipulated animals, were fixed 3 days
after manipulations. In one group of animals (Open), a single incision was
made in the pericardial sac using iridectomy scissors. In saline-treated
animals, 5 µl Hank's buffered saline was injected into the pericardial
cavity from a 30 gauge needle until expansion of the sac was visible. For an
additional group, a needle was similarly inserted into the pericardial sac,
but no fluid was injected (Pierce). Only animals that showed no bleeding after
any of these treatments were used for this study. As a control, iridectomy
scissors were used to make a small incision on the dorsal side of the animal
(Dorsal). For each group 9-11 animals were used in two experimental
trials.
DiI labeling of the adult epicardium
DiI labeling was performed by pipetting 0.5 µl of 1 mg/ml Cell
Tracker CM-DiI (Molecular Probes) into the pericardial cavity of
6-month-old zebrafish through a small incision in the pericardial sac.
Smaller animals used for RG and SG conditions did not survive this procedure.
Hearts were extracted and fixed at 1 hour, 3 days, 7 days and 14 days
post-labeling. Labeled hearts were sectioned and imaged without coverslips, as
mounting with a coverslip commonly caused diffusion of the dye. At least 15
animals were used for 1 hour, 3 day, and 7 day timepoints in three
experimental trials.
Fgfr inhibition
For RG experiments, heterozygous hsp70:dn-fgfr1 transgenic and
wild-type clutchmates (weighing 70-100 mg each) were selected at 8-10 weeks of
age. Fish received a daily, transient increase in temperature from 26°C to
38°C as described previously (Lee et
al., 2005). Animals remained at 38°C for 30 minutes each
day. We found that this temperature increase could not be achieved in 10 l
tanks using our system, so we instead placed one fish each in a 3 l tank, and
performed analyses at 14 days. For adult maintenance experiments, uninjured 4-
to 6-month-old hsp70:dn-fgfr1 and wild-type adult clutchmates
received a daily temperature increase for 60-70 days.
|
|
RESULTS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
208% increase from day
0). Clutchmates maintained at SG displayed only a 13% increase in mass
over this period. Fully mature, 6-month-old fish, considerably larger at the
onset of experiments than RG and SG fish, maintained mass for at least 28 days
when kept at standard aquarium density conditions
(Fig. 1B). In general we found
that starting size was a greater determinant of animal growth rate than age,
consistent with other published studies
(Tsai et al., 2007).
In these experiments, the large increases in body mass seen in RG fish were
accompanied by dramatic increases in the size of both heart chambers
(Fig. 1A). To quantify changes
in ventricular size, we measured the surface area of longitudinal ventricular
sections using digital imaging software. These analyses revealed that
ventricular size more than tripled after only 14 days in RG conditions (a
221% increase from day 0), a rate of cardiac growth approximately
equivalent to that of animal growth under these conditions. Clutchmates in SG
conditions exhibited a more modest increase in ventricular size over this
period (48%). Because this increase was greater than the increase in
animal size during this period, it is possible that the rate of cardiac growth
may slightly diverge from the rate of animal growth in SG conditions. MS
animals experienced no significant change in ventricular size over 14 days
(Fig. 1A,C). Thus, by varying
aquarium density and starting size of the animal, we closely controlled the
rate of cardiac homeostasis. In particular, low aquarium density conditions
induced remarkably rapid cardiogenesis in adult zebrafish.
New cardiomyocyte creation accompanies cardiac growth and maintenance
To determine whether density-dependent control of ventricular size resulted
from hyperplasia or hypertrophy, we administered three daily injections of
BrdU prior to harvesting tissue after 9 days in RG, SG or MS conditions. CMs
were specifically assayed for BrdU incorporation by costaining with an
antibody against Mef2, a transcription factor that regulates myocardial
differentiation (Molkentin and Markham,
1993). We observed a high rate of BrdU incorporation in CMs within
both trabecular and compact muscle compartments of RG fish, with
10.1±1.8% of CMs labeled by BrdU (mean ± s.e.m.;
Fig. 2A,B). This labeling index
was almost eight times that of SG animals (1.3±0.3%), and 25 times that
of MS animals sustaining constant body mass and ventricular size
(0.4±0.1%; Fig. 2A,B).
We obtained similar results when identifying proliferating cells with an
antibody against PCNA (see Fig. S1 in the supplementary material). We suspect
that rare CM proliferation in MS animals serves to replace dying cells, as we
observed occasional CM apoptosis events by TUNEL staining of ventricular
sections (see Fig. S2 in the supplementary material).
To assess possible contributions of hypertrophy to growth, we measured the density of cells positive for the nuclear marker Mef2 in ventricular sections. There were no differences in CM nuclear density per myocardial area in RG, SG and MS fish, indicating a lack of CM hypertrophy (Fig. 2C). In addition, we dissociated ventricles and assessed the extent of binucleation, a consequence of karyokinesis without cytokinesis that is common in adult mammalian CMs. We found that the vast majority of ventricular CMs in rapidly growing fish were mononucleate (95.6%), as were CMs from fish kept at SG (97.9%) and MS conditions (95.1%), indicating derivation through cytokinesis (Fig. 2D,E). Thus, homeostatic cardiac growth and maintenance are primarily the result of bona fide CM hyperplasia, with the vigor of this hyperplasia dependent on the rate of animal growth.
Evidence that myocardial homeostasis is aided by progenitor cells
Homeostatic CM generation must occur through the division of mature CMs, or
the maturation of myocardial progenitor cells, or both. During regeneration,
cells expressing markers of embryonic cardiac progenitor cells such as
hand2 (Yelon et al.,
2000) are established at the apical edge of the wounded muscle
within 3-4 days post injury, and are maintained at that edge as regeneration
progresses (Lepilina et al.,
2006). The origin of these cells is unknown; they might exist as
resident non-myocardial cells activated upon injury, or be created through
reduction in contractile function of existing CMs, known as
de-differentiation. Strong, discrete hand2 in situ hybridization
signals analogous to that seen during myocardial regeneration were common in
RG animals particularly within the compact myocardium, sparingly present in SG
animals, and largely absent in MS adults
(Fig. 3A). Real-time
quantitative PCR experiments indicated that hand2 expression in RG
ventricles was 1.4 fold that of SG clutchmates, and 2.5 fold that of MS
ventricles (Fig. 3C).
|
). Mature CMs in these transgenic fish show nuclear-localized
DsRed fluorescence. However, an anti-DsRed antibody detects unfolded,
cytosolic DsRed fluorescence in the new CMs of the 24-hour post-fertilization
embryo and the regenerating edge of the injured adult ventricle, apparently
before nuclear localization occurs and natural fluorescence is manifest. Thus,
this marker (RFPcyto) appears to serve as an indicator of
developmental transition from undifferentiated progenitor cells to
differentiated CMs (Lepilina et al.,
2006). We found that 10.7±2.1% of CMs throughout the
ventricle of RG animals expressed the RFPcyto marker, a percentage
over three times that of SG clutchmates (3.1±0.3%) and 36 times that of
MS fish (0.3±0.1%; Fig.
3B,D). In ventricles of cmlc2:nRFP RG animals
that had been injected with BrdU, both RFPcyto and
RFPnuc CMs showed BrdU-labeled nuclei, suggesting that both the
most recently maturing CMs and earlier-maturing CMs undergo proliferation
(data not shown). In total, these results suggest that myocardial progenitor
cell populations contribute at least in part to the vigorous generation of new
CMs stimulated by rapid animal growth, and in rare events during homeostatic
maintenance of heart size.
Homeostatic developmental activation of the adult epicardium
During zebrafish heart regeneration, local injury strongly activates
expression of the embryonic epicardial markers raldh2 and
tbx18 throughout the entire ventricular and atrial epicardium. This
activation is accompanied by epicardial cell proliferation, expanding the
epithelial cover of the ventricle to eventually cover the injury
(Lepilina et al., 2006).
raldh2 and tbx18 were expressed at low levels in a small
number of epicardial cells in both SG and MS ventricles, indicating moderate
activation. By contrast, these markers were robustly induced in contiguous
stretches of RG atrial and ventricular epicardium
(Fig. 4A,
Fig. 5B). In addition,
endocardial cells surrounding cardiac myofibers near the injury site have been
shown to induce raldh2 during regeneration (R.J.M. and K.D.P.,
unpublished) (Lepilina et al.,
2006). We found that RG animals, but not SG or MS animals,
displayed strong raldh2 expression in endocardial cells throughout
the ventricle (arrows in Fig.
4A; A.A.W., J.E.H. and K.D.P., unpublished). Thus, embryonic gene
expression within both the epicardium and endocardium is activated by rapid
animal growth.
|
; Shabetai et al.,
1985; Spodick,
1997). To test whether these procedures affect epicardial gene
expression, we examined raldh2 expression in several groups of
experimentally manipulated 6-month-old MS animals. We found that ventricular
raldh2 expression was induced to roughly the same level as in RG
animals (but at levels lower than after partial ventricular resection) in
experiments in which (1) the pericardial sac was surgically opened, or (2)
saline was carefully injected into the pericardial cavity
(Fig. 4B). Different
manipulations that resulted in no effect on epicardial raldh2
expression included: (1) piercing the pericardial sac without saline
injection, (2) lesions to the dorsal region of the animal and (3) fin
amputation. These results show that the epicardium is a dynamic tissue
responsive to changes in the milieu surrounding the heart, and suggest that
the effects of rapid animal growth on epicardial gene expression are due in
part to an ability of the epicardial tissue to sense changes in resistance
within the extra-cardiac space.
Epicardial-derived cells recurrently supplement the ventricular wall
During embryonic heart development, a subset of epicardial cells undergoes
epithelial-mesenchymal transition (EMT) and migrates into the underlying
subepicardial space and myocardial wall as epicardial-derived cells (EPDCs).
There, EPDCs contribute fibroblasts as well as smooth muscle and/or
endothelial cells for building coronary vasculature
(Olivey et al., 2004;
Reese et al., 2002). To
determine whether the adult zebrafish epicardium actively transfers cells
subepicardially to the ventricular wall, we performed a pulse-chase
experiment. We labeled the epicardium by filling the pericardial sac of MS
zebrafish with the fluorescent lipophilic dye DiI, and then harvested hearts
at different timepoints post-injection. Dye was limited to the epicardium at 1
hour post-injection; by contrast, we noticed a small number of labeled cells
separated from the epicardium by 3 days post-injection. Occasionally, these
labeled cells had a tubular morphology reminiscent of vascular tissue (inset,
Fig. 5A). At 7 and 14 days
post-injection, DiI was concentrated in cells that were embedded within the
otherwise unlabeled ventricular wall inward from the now faintly labeled
epicardium (Fig. 5A).
Interestingly, labeled cells occasionally appeared to constitute a distinct
layer of 15-20 µm at the junction of compact and trabecular myocardium.
These surprising data indicate that the adult epicardium is a dynamic tissue
that regularly contributes EPDCs to the ventricular wall, presumably through
EMT.
|
).
Interestingly, in uninjured ventricles, tbx18-expressing cells were
also present both in the epicardium and several cell layers deep into the
compact myocardium. Many tbx18-positive cells were located in what
appeared as a distinct layer 15-20 µm subepicardially, a location similar
to the EPDCs revealed by our pulse-chase experiments
(Fig. 5B). For these reasons,
we inferred that tbx18 is a robust marker for emergent EPDCs in the
uninjured ventricular wall. Quantification of these internalized
tbx18-positive EPDCs revealed that the density of EPDCs in RG animals
was almost six times as high as in SG animals (27.4±1.6 vs
4.9±1.0 tbx18-positive cells/mm ventricular wall). Somewhat
unexpectedly, MS animals had more than twice as many tbx18-positive
EPDCs as SG animals (11.9±1.8; Fig.
5C). This may be a consequence of age differences between these
groups, and/or the possibility that tbx18 is a cumulative marker of
EPDCs that have emerged in the ventricular wall over weeks to months. The
latter idea is supported by the observation that regenerated muscle possesses
cells robustly expressing tbx18 for 30 days or more after injury
(Lepilina et al., 2006). Given
the role of epicardial tissue during embryonic heart growth and the appearance
of tbx18-positive cells coincident with new vascular tissue during
regeneration, we postulate that tbx18-positive EPDCs contribute to
neovascularization of emergent myocardial tissue and/or vascular maintenance
in uninjured animals. Indeed, we could observe in some cases cells positive
for tbx18 that had the appearance of vascular tissue
(Fig. 5B, red arrowhead and
inset), similar to DiI-labeled cells in pulse-chase experiments. Thus, the
adult epicardium supports cardiac homeostasis by regular contribution of new
EPDCs to the ventricular wall.
|
;
Morabito et al., 2001). During
zebrafish heart regeneration, the injured zebrafish myocardium increases
expression of fgf17b, while the receptors fgfr2 and
fgfr4 are expressed in EPDCs. Furthermore, abrogation of Fgf
signaling disrupts EPDC supplementation of the wound and new muscle during
regeneration, inhibiting neovascularization and myocardial renewal
(Lepilina et al., 2006). We
found that fgf17b, fgfr2 and fgfr4 were all present in SG
and RG ventricles, although no differences in expression between the two
groups were detectable by in situ hybridization (data not shown). Based on
these findings, we suspected that Fgf signaling may also be important for EPDC
supplementation of the ventricle during cardiac homeostasis.
To test this idea, we placed zebrafish transgenic for a heat-inducible
dominant-negative Fgf receptor (hsp70:dn-fgfr1) and their wild-type
clutchmates in RG conditions for 14 days, and applied a daily heat-shock
(Lee et al., 2005). Transgenic
animals appeared healthy during the experiment, although they grew less than
wild-type animals under RG conditions (see Fig. S3 in the supplementary
material). Although tbx18 expression was comparable between
transgenics and wild types in the epicardium itself, the density of
tbx18-positive EPDCs within the ventricular walls of
hsp70:dn-fgfr1 animals was less than half that in wild-type animals
(Fig. 6A,B; 24.6±2.1
EPDCs/mm in wild type, versus 11.5±2.1, in hsp70:dn-fgfr1).
Fgfr inhibition also resulted in a slight reduction in ventricular growth
under RG conditions, with transgenic animals exhibiting 31% less of a
growth increase than wild-type clutchmates
(Fig. 6C). Thus, normal
homeostatic supplementation of the ventricular wall by EPDCs requires Fgf
signaling. We suspect that the partial phenotype is due to the continued
presence of other factors that act on EPDCs, incomplete inhibition of Fgf
signaling, and/or the possibility mentioned above that tbx18
expression reflects cumulative events of EPDC contribution.
As pulse-chase and tbx18 expression experiments indicated ongoing
EPDC contribution even in the absence of significant cardiac growth, we tested
the requirements for Fgf signaling in mature adult zebrafish. We delivered a
long-term block of Fgf signaling by daily heat-shocks to
hsp70:dn-fgfr1 transgenics for 60-70 days, with identical heat-shocks
given to wild-type clutchmates. We found that 15% of
hsp70:dn-fgfr1 fish developed extensive collagen deposition within
the ventricular wall after this period, indicating the presence of scar tissue
(Fig. 6D; n=39 fish).
No wild-type animals displayed scarring (n=44 fish; Fisher-Irwin
test, P<0.01). We suspect that these fibrotic events resulted from
reduced efficiency of incremental EPDC supplementation over the 2-month
period, although marker examination revealed no obvious defects (data not
shown). These results demonstrate that Fgf signaling is essential for normal
homeostatic growth and maintenance of cardiac tissue in the adult
zebrafish.
|
DISCUSSION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
;
Dor et al., 2004;
Hsieh et al., 2007;
Meilhac et al., 2003).
Moreover, it will be critical to define homeostatic signals that trigger
cardiomyogenesis and how these signals are distributed and calibrated in the
adult animal.
As a thin epithelium covering the entire surface of the heart, the
epicardium has an ideal architecture for relaying such homeostatic signals.
Numerous studies support this idea, because: (1) the epicardium serves as a
source of mitogens in the embryonic heart
(Chen et al., 2002;
Merki et al., 2005;
Reese et al., 2002), (2)
organ-wide developmental gene expression is activated within the adult
zebrafish epicardium within hours of cardiac injury
(Lepilina et al., 2006), and
(3) during regenerative cardiogenesis as well as homeostatic cardiac growth,
developmentally active epicardium is present at sites of cardiogenesis
(Lepilina et al., 2006).
Indeed, consistent with this idea are the findings here that manipulations of
the pericardial environment that mimic growth-induced changes in pericardial
space cause epicardial responses similar to those elicited by homeostatic
growth. Because epicardial retinoic acid (RA) synthesis is regulated in each
of these scenarios and is known to influence CM proliferation in embryos
(Chen et al., 2002;
Stuckmann et al., 2003), this
molecule is a strong candidate for homeostatic regulation of cardiogenesis. We
have found that the endocardium also increases RA synthesis during adult
growth and regeneration. It is possible that endocardial cell activity
regulates growth and/or remodeling of the inner trabecular muscle, which would
appear to have reduced access to epicardial signaling molecules.
In addition to serving as a source of RA, we found that the epicardium also regularly contributes cells to the compact myocardial wall of the ventricle. Thus, the epicardium is by no means a static tissue in adult animals, but rather one that actively sustains the ventricle. This conclusion is bolstered by our finding that sustained inhibition of Fgf signaling, which disrupts EPDC supplementation in rapidly growing fish, led to spontaneous scar formation in animals maintaining their animal and organ size. Further exploration of the fate and function of these adult EPDCs, using tissue-specific fate-mapping and ectopic expression tools, will illuminate the roles of the epicardium in cardiac homeostasis. From what is known of embryonic heart development, it is likely that the adult zebrafish myocardial wall requires new EPDCs to replenish vasculature, or to neovascularize newly created muscle, or both. It will also be fascinating to learn to what extent the epicardium participates in homeostasis of the adult mammalian ventricular wall.
Cardiac growth, maintenance and regeneration
Our study exposes important similarities between cardiac homeostasis and
cardiac regeneration. In each case, new myocardial and epicardial-derived
tissues are generated in amounts appropriate to animal size, involving
expression of a suite of cardiogenic developmental markers. Whereas
regeneration is characterized by an intense focus of cardiogenesis at the
injury site, homeostasis involves organ-wide cell addition in doses dependent
on the vigor of animal growth. Thus, the adult zebrafish not only has the
ability to generate new cardiac tissues, but can also control the intensity
and the spatial distribution of these cardiogenic events to select replacement
of a portion of tissue, rapid chamber-wide growth, or occasional exchange of a
few cells (Fig. 7).
|
; Hofmann et al.,
1999). Thus, one reason why cardiac regeneration persists in
zebrafish, and probably other teleosts, may be the accessibility of injured
tissue to cellular and molecular machinery that is normally employed for rapid
indeterminate growth. This notion makes sense in light of extremely weak
hyperplasia in the injured or uninjured hearts of adult mammals, species with
determinate growth. Continued study of cardiac regeneration and homeostasis in
zebrafish, including comparisons with mammalian cardiac dynamics, will expose
critical mechanisms by which cardiac tissues are rejuvenated.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/1/183/DC1
|
ACKNOWLEDGMENTS |
|---|
|
REFERENCES |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Beltrami, A. P., Barlucchi, L., Torella, D., Baker, M., Limana, F., Chimenti, S., Kasahara, H., Rota, M., Musso, E., Urbanek, K. et al. (2003). Adult cardiac stem cells are multipotent and support myocardial regeneration. Cell 114,763 -776.[CrossRef][Medline]
Borowsky, R. L. (1973). Social control of adult size in males of Xiphophorus variatus. Nature 245,332 -335.[CrossRef]
Cai, C. L., Liang, X., Shi, Y., Chu, P. H., Pfaff, S. L., Chen, J. and Evans, S. (2003). Isl1 identifies a cardiac progenitor population that proliferates prior to differentiation and contributes a majority of cells to the heart. Dev. Cell 5, 877-889.[CrossRef][Medline]
Chen, T. H., Chang, T. C., Kang, J. O., Choudhary, B., Makita, T., Tran, C. M., Burch, J. B., Eid, H. and Sucov, H. M. (2002). Epicardial induction of fetal cardiomyocyte proliferation via a retinoic acid-inducible trophic factor. Dev. Biol. 250,198 -207.[CrossRef][Medline]
Conlon, I. and Raff, M. (1999). Size control in animal development. Cell 96,235 -244.[CrossRef][Medline]
Dor, Y., Brown, J., Martinez, O. I. and Melton, D. A. (2004). Adult pancreatic beta-cells are formed by self-duplication rather than stem-cell differentiation. Nature 429,41 -46.[CrossRef][Medline]
Farrell, A. P., Johanson, J. A. and Graham, M. S. (1988). The role of the pericardium in cardiac performance of the trout (Salmo gairdneri). Physiol. Zool. 61,213 -221.
Flink, I. L. (2002). Cell cycle reentry of ventricular and atrial cardiomyocytes and cells within the epicardium following amputation of the ventricular apex in the axolotl, Amblystoma mexicanum: confocal microscopic immunofluorescent image analysis of bromodeoxyuridine-labeled nuclei. Anat. Embryol. 205,235 -244.[CrossRef][Medline]
Hofmann, H. A., Benson, M. E. and Fernald, R. D.
(1999). Social status regulates growth rate: consequences for
life-history strategies. Proc. Natl. Acad. Sci. USA
96,14171
-14176.
Hsieh, P. C., Segers, V. F., Davis, M. E., Macgillivray, C., Gannon, J., Molkentin, J. D., Robbins, J. and Lee, R. T. (2007). Evidence from a genetic fate-mapping study that stem cells refresh adult mammalian cardiomyocytes after injury. Nat. Med. 13,970 -974.[CrossRef][Medline]
Jordan, D. S. (1905). A Guide to the Study of Fishes. New York: Henry Holt.
Laugwitz, K. L., Moretti, A., Lam, J., Gruber, P., Chen, Y., Woodard, S., Lin, L. Z., Cai, C. L., Lu, M. M., Reth, M. et al. (2005). Postnatal isl1+ cardioblasts enter fully differentiated cardiomyocyte lineages. Nature 433,647 -653.[CrossRef][Medline]
Lavine, K. J., White, A. C., Park, C., Smith, C. S., Choi, K.,
Long, F., Hui, C. C. and Ornitz, D. M. (2006). Fibroblast
growth factor signals regulate a wave of Hedgehog activation that is essential
for coronary vascular development. Genes Dev.
20,1651
-1666.
Lee, Y., Grill, S., Sanchez, A., Murphy-Ryan, M. and Poss, K.
D. (2005). Fgf signaling instructs position-dependent growth
rate during zebrafish fin regeneration. Development
132,5173
-5183.
Lepilina, A., Coon, A. N., Kikuchi, K., Holdway, J. E., Roberts, R. W., Burns, C. G. and Poss, K. D. (2006). A dynamic epicardial injury response supports progenitor cell activity during zebrafish heart regeneration. Cell 127,607 -619.[CrossRef][Medline]
Li, F., Wang, X., Capasso, J. M. and Gerdes, A. M. (1996). Rapid transition of cardiac myocytes from hyperplasia to hypertrophy during postnatal development. J. Mol. Cell. Cardiol. 28,1737 -1746.[CrossRef][Medline]
Mably, J. D., Mohideen, M. A., Burns, C. G., Chen, J. N. and Fishman, M. C. (2003). heart of glass regulates the concentric growth of the heart in zebrafish. Curr. Biol. 13,2138 -2147.[CrossRef][Medline]
Martin, C. M., Meeson, A. P., Robertson, S. M., Hawke, T. J., Richardson, J. A., Bates, S., Goetsch, S. C., Gallardo, T. D. and Garry, D. J. (2004). Persistent expression of the ATP-binding cassette transporter, Abcg2, identifies cardiac SP cells in the developing and adult heart. Dev. Biol. 265,262 -275.[CrossRef][Medline]
Meilhac, S. M., Kelly, R. G., Rocancourt, D., Eloy-Trinquet, S.,
Nicolas, J. F. and Buckingham, M. E. (2003). A retrospective
clonal analysis of the myocardium reveals two phases of clonal growth in the
developing mouse heart. Development
130,3877
-3889.
Merki, E., Zamora, M., Raya, A., Kawakami, Y., Wang, J., Zhang,
X., Burch, J., Kubalak, S. W., Kaliman, P., Belmonte, J. C. et al.
(2005). Epicardial retinoid X receptor alpha is required for
myocardial growth and coronary artery formation. Proc. Natl. Acad.
Sci. USA 102,18455
-18460.
Molkentin, J. D. and Markham, B. E. (1993).
Myocyte-specific enhancer-binding factor (MEF-2) regulates alpha-cardiac
myosin heavy chain gene expression in vitro and in vivo. J. Biol.
Chem. 268,19512
-19520.
Morabito, C. J., Dettman, R. W., Kattan, J., Collier, J. M. and Bristow, J. (2001). Positive and negative regulation of epicardial-mesenchymal transformation during avian heart development. Dev. Biol. 234,204 -215.[CrossRef][Medline]
Oberpriller, J. O. and Oberpriller, J. C. (1974). Response of the adult newt ventricle to injury. J. Exp. Zool. 187,249 -253.[CrossRef][Medline]
Oh, H., Bradfute, S. B., Gallardo, T. D., Nakamura, T., Gaussin,
V., Mishina, Y., Pocius, J., Michael, L. H., Behringer, R. R., Garry, D. J. et
al. (2003). Cardiac progenitor cells from adult myocardium:
homing, differentiation, and fusion after infarction. Proc. Natl.
Acad. Sci. USA 100,12313
-12318.
Olivey, H. E., Compton, L. A. and Barnett, J. V. (2004). Coronary vessel development: the epicardium delivers. Trends Cardiovasc. Med. 14,247 -251.[Medline]
Poss, K. D., Wilson, L. G. and Keating, M. T.
(2002). Heart regeneration in zebrafish.
Science 298,2188
-2190.
Potter, C. J. and Xu, T. (2001). Mechanisms of size control. Curr. Opin. Genet. Dev. 11,279 -286.[CrossRef][Medline]
Reese, D. E., Mikawa, T. and Bader, D. M.
(2002). Development of the coronary vessel system.
Circ. Res. 91,761
-768.
Shabetai, R., Abel, D. C., Graham, J. B., Bhargava, V., Keyes, R. S. and Witztum, K. (1985). Function of the pericardium and pericardioperitoneal canal in elasmobranch fishes. Am. J. Physiol. 248,H198 -H207.[Medline]
Smart, N., Risebro, C. A., Melville, A. A., Moses, K., Schwartz, R. J., Chien, K. R. and Riley, P. R. (2007). Thymosin beta4 induces adult epicardial progenitor mobilization and neovascularization. Nature 445,177 -182.[CrossRef][Medline]
Soonpaa, M. H. and Field, L. J. (1997). Assessment of cardiomyocyte DNA synthesis in normal and injured adult mouse hearts. Am. J. Physiol. 272,H220 -H226.[Medline]
Soonpaa, M. H., Kim, K. K., Pajak, L., Franklin, M. and Field, L. J. (1996). Cardiomyocyte DNA synthesis and binucleation during murine development. Am. J. Physiol. 271,H2183 -H2189.[Medline]
Spodick, D. H. (1997). The Pericardium: A Comprehensive Textbook. New York: Marcel Dekker.
Stuckmann, I., Evans, S. and Lassar, A. B. (2003). Erythropoietin and retinoic acid, secreted from the epicardium, are required for cardiac myocyte proliferation. Dev. Biol. 255,334 -349.[CrossRef][Medline]
Taub, R. (2004). Liver regeneration: from myth to mechanism. Nat. Rev. Mol. Cell Biol. 5, 836-847.[CrossRef][Medline]
Tsai, S. B., Tucci, V., Uchiyama, J., Fabian, N. J., Lin, M. C., Bayliss, P. E., Neuberg, D. S., Zhdanova, I. V. and Kishi, S. (2007). Differential effects of genotoxic stress on both concurrent body growth and gradual senescence in the adult zebrafish. Aging Cell 6,209 -224.[CrossRef][Medline]
van Tuyn, J., Atsma, D. E., Winter, E. M., van der Velde-van
Dijke, I., Pijnappels, D. A., Bax, N. A., Knaan-Shanzer, S., Gittenberger-de
Groot, A. C., Poelmann, R. E., van der Laarse, A. et al.
(2007). Epicardial cells of human adults can undergo an
epithelial-to-mesenchymal transition and obtain characteristics of smooth
muscle cells in vitro. Stem Cells
25,271
-278.
Warren, K. S., Baker, K. and Fishman, M. C.
(2001). The slow mo mutation reduces pacemaker current and heart
rate in adult zebrafish. Am. J. Physiol. Heart Circ.
Physiol. 281,H1711
-H1719.
Yelon, D., Ticho, B., Halpern, M. E., Ruvinsky, I., Ho, R. K., Silver, L. M. and Stainier, D. Y. (2000). The bHLH transcription factor hand2 plays parallel roles in zebrafish heart and pectoral fin development. Development 127,2573 -2582.[Abstract]
This article has been cited by other articles:
|
|
 |
|
  A. A. Wills, A. R. Kidd III, A. Lepilina, and K. D. Poss Fgfs control homeostatic regeneration in adult zebrafish fins Development, September 15, 2008; 135(18): 3063 - 3070. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Smart and P. R. Riley The Stem Cell Movement Circ. Res., May 23, 2008; 102(10): 1155 - 1168. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Chen, S. A. Carney, R. E. Peterson, and W. Heideman Comparative genomics identifies genes mediating cardiotoxicity in the embryonic zebrafish heart Physiol Genomics, April 21, 2008; 33(2): 148 - 158. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. M. Tanaka and G. Weidinger Micromanaging regeneration Genes & Dev., March 15, 2008; 22(6): 700 - 705. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||
