Institute of Molecular Medicine and Department of Medicine, School of Medicine, University of California-San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0641, USA
* Author for correspondence (e-mail: juchen{at}ucsd.edu)
Accepted 27 August 2003
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SUMMARY |
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Key words: Myosin light chain 2a, MLC2a, Atria, Cardiac morphogenesis, Angiogenesis, Embryogenesis
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Introduction |
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Cardiac muscle expresses two major isoforms of myosin light chain 2, MLC2v
(MYL2 Mouse Genome Informatics) and MLC2a (MYLC2A Mouse Genome
Informatics). During cardiogenesis, MLC2v is expressed exclusively in
ventricular and atrio-ventricular junction myocardium. Ablation of MLC2v
results in disruption of ventricular function at embryonic day (ED) 11.5 and
embryonic lethality at ED12.5 (Chen et al.,
1998). MLC2a is initially expressed throughout the heart at ED7.5
and becomes restricted to the atria after ED12.5
(Kubalak et al., 1994
).
Despite its early expression throughout the heart until ED12.5, MLC2a protein
is only incorporated into myofibrillar structures in atria, not in ventricle
(Chen et al., 1998
). As MLC2a
is the only isoform expressed in atria, we reasoned that disruption of MLC2a
would interrupt embryonic atrial function, and would therefore allow us to
investigate the role of atrial function in embryogenesis. Our results have
demonstrated that ablation of MLC2a disrupts the earliest functioning of the
heart, demonstrating an early requirement for atrial function. Additionally,
these results have allowed us to address longstanding questions in development
concerning the role of embryonic heart function in cardiac morphogenesis and
angiogenesis.
Recently, it has been shown that the embryonic heart in early chick embryos
begins to beat substantially prior to a requirement for convective bulk
transport to deliver oxygen and nutrients for growth
(Burggren et al., 2000).
Similar observations have been made for zebrafish, clawed frog and salamander
embryos (Mellish et al., 1994
;
Pelster and Burggren, 1996
;
Territo and Burggren, 1998
).
These findings are consistent with the hypothesis that early blood flow may
play a role not only as a transportation fluid, but also as a physical factor
in development, perhaps influencing normal morphogenesis of the heart and
angiogenesis.
Hemodynamic parameters are known to be a determinant of myocardial growth,
structure and function in the adult heart, and may be important in the
etiology of heart failure (Hutchins et
al., 1978; Rockman et al.,
1994
; Zak, 1974
).
It has been postulated that normal flow and hemodynamics are also crucial
determinants of normal cardiac morphogenesis, and may play a causative role in
congenital heart disease (Sedmera et al.,
1998
). Previously, this issue has been addressed by ligation
experiments to perturb blood flow in chick embryos
(Hogers et al., 1997
;
Icardo, 1989
;
Sedmera et al., 1999
). By
technical necessity, these experiments perturbed blood flow in relatively late
stage embryos, between HH stages 21-24, a time when considerable cardiac
morphogenesis has already taken place. Because of the technically demanding
nature of these experiments, small numbers of experimental embryos can be
obtained, and from these variable phenotypes are observed
(Broekhuizen et al., 1999
).
Additionally, mechanical intervention to perturb blood flow may also restrict
cell migration, or may inflict non-specific tissue damage, complicating the
interpretation of any effects on growth or morphogenesis. Genetic ablation of
MLC2a has allowed us to selectively inactivate atrial function, altering
hemodynamics from the earliest stages of heart development. Prior to any
growth retardation, MLC2a mutants exhibit consistent phenotypic abnormalities
in embryonic cardiac morphology. Our results unequivocally demonstrate that
normal growth and morphogenesis of the heart are dependent on normal heart
function from the earliest stages, and strongly suggest that altered
hemodynamics during embryonic development can have significant morphologic
consequences for heart development. Embryonic atrial insufficiency in MLC2a
mutants has severe consequences for multiple aspects of chamber and looping
morphogenesis, suggesting that a less severe perturbation could similarly
affect these morphogenetic processes and give rise to congenital anomalies of
the heart in humans.
Many mouse mutants have been described that simultaneously affect early
heart function and angiogenesis (Bi et al.,
1999; Gerety and Anderson,
2002
; Li et al.,
1999
; Lin et al.,
1998
; Lin et al.,
1997
; Lyons et al.,
1995
; Regan et al.,
2002
; Stanley et al.,
2002
; Tanaka et al.,
1999
; Yamagishi et al.,
2000
). However, each of these genes is expressed in both
developing vasculature and heart, making it difficult to assess the
independent effects of embryonic heart malfunction on the developing
vasculature. The expression of MLC2a exclusively in developing myocardium
allowed us to examine this question in our MLC2a knockouts, and has clearly
demonstrated a requirement for cardiac function in both extra- and
intraembryonic angiogenesis.
Together, our data imply that alterations in blood flow during early development, as a result of either genetic or environmental influences, may have an etiological role in congenital cardiovascular disease.
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Materials and methods |
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DNA analysis
DNA was extracted from G418-resistant ES cell clones, yolk sac and mouse
tails, as previously described (Moens et
al., 1993). ES cell DNA was digested with SstI,
electrophoresed on a 0.8% (w/v) agarose gel, and subsequently blotted onto
nitrocellulose. A 400 bp fragment, corresponding to the 5' untranslated
region of the MLC2a gene, was generated by polymerase chain reaction (PCR)
using mouse genomic DNA and specific MLC2a primers (forward primer,
5'-GTGAGCCACTGAGAATGGTTGT-3'; reverse primer,
5'-CTTAGAACCCCACTTCATCCCT-3'), and subsequently radiolabeled using
[32P]dATP by random priming (Invitrogen, San Diego, CA). DNA blots
were hybridized to the radiolabeled probe at 68°C, washed twice for 15
minutes each time at 60°C in 0.1xSSC with 0.1% sodium dodecyl
sulphate (SDS), and visualized by autoradiography. DNA from yolk sac and mouse
tails was also subjected to PCR, using both Cre (forward primer,
5'-GTTCGCAAGAACCTGATGGACA-3'; reverse primer,
5'-CTAGAGCCTGTTTTGCACGTTC-3') and MLC2a (forward primer,
5'-GGAACAGAGACCAGCCACA-3'; reverse primer,
5'-GGTCTGATTTGCAGATGATC-3') specific primers, and products were
visualized by ethidium bromide staining.
Whole-mount in situ hybridization analysis
In situ hybridization was performed on MLC2a-null and somite-matched
embryos for Mlc2a, Mlc2v, Nkx2.5, dHand, eHand and Tbx5,
using digoxigenin-labeled antisense riboprobes, essentially as previously
described (Wilkinson, 1992)
with modifications. Briefly, plasmids containing Mlc2v, Nkx2.5 and
Tbx5 cDNAs were linearized with NotI, HindIII and
SpeI, respectively. T7 RNA polymerase (Invitrogen) was used to
synthesize RNA ribprobes by in vitro transcription in the presence of
digoxigenin-11-dUTP (Roche). Fixed embryos were subsequently pre-hybridized in
50% formamide, 5xSSC (pH 5), 50 ug/ml yeast RNA, 1% SDS and 50 µg/ml
heparin at 68°C overnight, and subsequently hybridized with riboprobes at
68°C for 18 hours. After vigorous washing, embryos were incubated with
alkaline-phosphatase-conjugated anti-digoxigenin antibodies at 4°C
overnight (Roche), vigorously washed again, and incubated in a NTMT buffer
(Promega), including freshly added nitroblue tetrazolium (NBT; 4.5 µl) and
5-bromo-4-chloro-3-indolyl phosphate (BCIP; 3.5 µl) per ml NTMT. Embryos
were subsequently fixed in 4% paraformaldehyde for 1-2 hours, until color
development had occurred to the desired extent, and stored at 4°C in
phosphate buffered saline (PBS) to terminate the color reaction. Whole embryos
were analyzed and photographed on a ZEISS SV-5 dissecting microscope with a
Nikon C-mount 35 mm camera.
Protein blot analysis
Total protein extracts were prepared from hearts of MLC2a-null, as well as
wild-type, embryos, and protein analysis was performed as previously described
(Chen et al., 1998).
Immunodetection of MLC2a (28.2 kDa) and MLC2v (34.7 kDa) was performed in
cardiac atria and ventricular samples by using a rabbit polyclonal antibody to
MLC2a (1:500), as previously described
(Chen et al., 1998
). The blot
was subsequently incubated with horseradish peroxidase-conjugated anti-rabbit
Ig (1:1000; Sigma). Results were visualized by using enhanced
chemiluminescence (Amersham).
Histology
Embryos and yolk sacs from MLC2a-null and wild-type, somite-matched embryos
were fixed, dehydrated and embedded in paraffin wax. Serial transverse
sections were obtained at 10 µm intervals. Sections were stained with
Hematoxylin and Eosin, and photographed as previously described
(Zhou et al., 2001).
Immunohistochemistry
Whole embryos and yolk sacs were fixed in 4% paraformaldehyde overnight at
4°C, and then washed in a series of methanol washes and bleached in 5%
H2O2/100% methanol for 4 hours at room temperature (RT).
Subsequently, embryos were rinsed in 100% methanol, rehydrated and incubated
in PBSMT (3% milk and 0.1% Triton X-100 in PBS) for 2 hours at RT. Embryos
were then incubated with (1) platelet endothelial cell adhesion molecule
(PECAM; 1:75; Sigma) or (2) fetal liver kinase 1 (FLK1; 1:400; Sigma)
overnight at 4°C, washed in PBSMT and incubated with anti-mouse
horseradish peroxidase (1:1000) overnight at 4°C. Subsequently, embryos
were washed in PBSMT and briefly rinsed in PBT (0.2% BSA and 0.1% Triton X-100
in PBS). For color development, embryos were incubated in 0.2 mg/ml DAB
(Sigma) and 0.35% NiCl2 in PBT at RT, until the desired intensity
was achieved. The reaction was stopped with the addition of 0.03%
H2O2. The embryos were rinsed several times in PBT and
PBS, and fixed in a 2% paraformaldehyde/0.1% gluteraldehyde solution overnight
at 4°C. The next day, embryos were rinsed several times in PBS, and
equilibrated at RT in 50% glycerol and then 70% glycerol for one hour each.
Samples were analyzed and photographed on a ZEISS SV-5 dissecting microscope
with a Nikon C-mount 35 mm camera.
Transmission electron microscopy
Embryos were fixed in 0.1 M cacodylate, containing 2% paraformaldehyde and
2% gluteradehyde, overnight at RT, and subsequently processed for transmission
electron microscopy as previously described
(Chen et al., 1998).
Assessment of heart rate
ED9.5 embryos (with deciduas and fetal blood vessels left attached) were
dissected from anesthetized mothers (n=3) in 37°C DMEM containing
5% fetal bovine serum (Invitrogen), and placed within a closed 35 mm dish on
the stage of a dissecting microscope connected to an image processor. Light
was transmitted through a region of the atrioventricular junction and was
detected by the image processor. Using the computer monitor, both atrial and
ventricular beating rates were determined three times, simultaneously by two
people within a three minute period.
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Results |
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We also performed whole-mount in situ hybridization analysis of MLC2a-null embryos and control littermates, using digoxigenin-labeled antisense riboprobes for a number of myocardial markers, including TBX5 (left ventricle and atria), MLC2v (ventricle) and NKX2.5 (entire myocardium). No differences were observed between MLC2a mutants and their control littermates (data not shown).
MLC2a-null embryos display lack of atrial myofibrillar organization
and function
Ultrastructural analysis revealed that MLC2a-null atrial cardiomyocytes
have a complete absence of myofibrillar organization, a lack of normal
parallel alignment of thick and thin filaments when compared with
somite-matched wild-type controls (Fig.
4A,C). By contrast, cardiomyocytes from the left ventricle of
MLC2a-null embryos exhibited normal parallel alignment of thick and thin
filaments, and Z-line formation, similar to somite-matched wild-type controls
(Fig. 4B,D). In terms of
function, severely diminished atrial beating was observed in MLC2a-null
embryos (Fig. 5). By contrast,
no significant difference in the rate of ventricular beating was observed
between MLC2a-null and somite-matched wild-type controls
(Fig. 5).
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Discussion |
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MLC2a-null mice died at approximately ED10.5-11.5 of cardiovascular
insufficiency, as indicated by pericardial edema. The proximal cause of death
was atrial malfunction. Consistent with the ablation of the atrial specific
isoform of myosin light chain 2, the atrial chamber of null embryos contracted
infrequently and sporadically, and displayed a lack of myofibrillar assembly.
By contrast, the left ventricle contracted normally, and displayed normal
myofilament assembly. These results are consistent with previous studies from
our laboratory, which demonstrated that although MLC2a is initially expressed
throughout the linear heart tube before becoming restricted to the atria at
ED12.5 (Kubalak et al., 1994),
MLC2a protein is not incorporated into the sarcomeric structure of the
ventricles (Chen et al.,
1998
).
We have utilized the fact that atrial function is disrupted from the beginning of cardiogenesis to investigate longstanding questions as to the role of early heart function in cardiac morphogenesis, and in both extra- and intraembryonic vasculogenesis. Our studies clearly demonstrate that disruption of embryonic heart function has severe consequences on crucial aspects of morphological development of the heart, and on angiogenesis. These results have important implications for the etiology of congenital heart disease.
Despite our demonstration that myofibrillogenesis in extra-atrial
myocardium was intact in MLC2a-null mutants, defects in chamber morphogenesis
were observed throughout the heart, beginning at the early linear heart tube
stage. Just prior to this stage, cardiac contraction has initiated, and
embryonic and extraembryonic circulations have amalgamated
(Kaufman, 1998). Linear heart
tubes of MLC2a mutants appeared relatively large and amorphous relative to
control littermates. At subsequent stages of heart development, looping
morphogenesis occurred in mutant hearts, but was aberrant in a number of
respects, including the length, shape and size of each cardiac segment, and
their geometrical relationship to each other. Histological analysis revealed
multiple abnormalities within myocardial and endocardial lineages of MLC2a
homozygous-knockout embryos. The ventricular myocardium of MLC2a mutants was
relatively thin, with fewer trabeculae, and left ventricular (LV) dilation may
have occurred secondary to diminished cardiac function.
Similar secondary effects on ventricular morphogenesis consequent to atrial
dysfunction have been made in a zebrafish line carrying a mutation in an
atrial myosin heavy chain, weak atrium (wea) (see
Berdougo et al., 2003). As with
the left ventricle in MLC2a mouse mutants, ventricles in wea mutants exhibit a
decreased ventricular lumen.
A crucial aspect of cardiac morphogenesis is the formation of septa, which
divide the mature heart into four chambers. As an initial stage in the
division of the atrial and ventricular chambers, endothelial cells in the
region of the atrioventricular canal become activated to invade the cardiac
jelly (Markwald et al., 1999).
The seeding of cardiac jelly by endocardial cells occurs at approximately ED10
in the mouse. In MLC2a homozygous-knockout embryos, this seeding was not
observed. This observation suggests that septation and valve formation, among
the most common manifestations of congenital heart disease
(Epstein and Buck, 2000
), can
be critically affected by aberrations of early myocardial function.
Our observations have demonstrated that absence of atrial contraction can
impact multiple aspects of cardiac morphogenesis, and can affect both
myocardial and endocardial lineages. These effects may be secondary to
alterations in hemodyamic fluid forces, or secondary to oxygen or nutrient
delivery. We favor the explanation that the first alterations in cardiac
morphology observed in MLC2a mutants can be attributed to changes in
hemodynamic forces for the following reasons. Initial aberrations in mutant
heart morphologies are observed shortly after embryonic and extraembryonic
circulations have merged. Oxygen-carrying red blood cells are not present
within the bloodstream of the mouse embryo until the 20 to 25 somite stage
(Palis et al., 1999),
rendering it unlikely that insufficient circulation of blood affects tissue
oxygenation prior to this stage. Growth retardation in MLC2a embryos is not
apparent until approximately 16 somites, making it unlikely that nutrients are
limiting at early heart tube stages. Consistent with these observations,
experiments in a number of vertebrate species have demonstrated that heart
beat and circulation considerably precede the demand for oxygen or nutrient
delivery (Burggren et al.,
2000
). Alterations in fluid forces, distinct from effects on
oxygen or nutrient delivery, have previously been shown to be crucial for
kidney morphogenesis (Serluca et al.,
2002
). Our observations suggest that fluid forces also play a
crucial role at the earliest stages of cardiac morphogenesis.
A number of previous studies have demonstrated that alterations in fluid
forces experienced by endothelial cells, including those of the endocardium,
can result in changes in their alignment and changes in gene expression
(Icardo, 1989;
Resnick et al., 2000
;
Shyy and Chien, 2002
;
Topper and Gimbrone, 1999
).
Genes that are regulated within endothelium in response to hemodynamic sheer
stress include those involved in extracellular matrix remodeling and growth
factor pathways. In this light, it is interesting to note the changes observed
in both cardiac jelly and extra-atrial myocardium in the MLC2a mutant, where
alterations in hemodyamic sheer stress experienced by the endocardium would be
expected to occur owing to the lack of atrial function.
In addition to defects in cardiac architecture, MLC2a-null mice also
displayed defects in both yolk sac and intraembryonic angiogenesis. In MLC2a
mutants, the initial vascular plexus of the yolk sac forms, but does not
remodel into a vascular network of larger vessels. Similar secondary effects
of cardiac malfunction on yolk sac angiogenesis have been observed in a mouse
knockout of the sodium calcium exchanger, and in a cardiac specific rescue of
an N-cadherin-null mouse, where a previous defect in yolk sac angiogenesis was
rescued by expression of either N- or E-cadherin in heart
(Koushik et al., 2001;
Luo et al., 2001
;
Wakimoto et al., 2000
).
However, in addition to observed effects on yolk sac angiogenesis, MLC2a
mutants also show defects in intraembryonic vasculogenesis. Initial stages of
angiogenesis appear normal, but subsequent remodeling of vessels does not
occur normally in the MLC2a homozygous-null embryos. These results demonstrate
aberrations in intraembryonic angiogenesis in MLC2a mutants, which do not
reflect an intrinsic defect in endothelial or smooth muscle cells, but rather
are secondary to cardiac malfunction. Similar defects in vasculogenesis have
been reported in a number of knockout mice that are mutant for genes that are
expressed both in myocardium and in vascular cells, or in vascular endothelium
and endocardium (Puri et al.,
1999). Our results uniquely demonstrate that cardiac function is
required for normal maturation of intraembryonic vessels. Our results also
caution that an intrinsic requirement for gene function in vascular cell types
cannot be inferred in cases where defects in vascular maturation are
accompanied by defects in myocardial function. The latter could arise owing to
defects within myocardium itself, as seen here with the MLC2a knockout, or
within cell lineages that affect the myocardium, including the endocardium
(Puri et al., 1999
).
In summary, our analysis of the MLC2a mutant has demonstrated that ablation of atrial function can have drastic secondary consequences on cardiac morphology and vascular development. They also suggest that more minor perturbations of atrial or cardiac contractile function, including perturbations of flow during development, could have less drastic but serious consequences for normal cardiovascular development in the human fetus. In particular, our findings suggest that normal alignment of cardiac chambers and cushion formation, crucial aspects in septation of the normal heart that are perturbed in the majority of cases of congenital cardiac disease, can be perturbed consequent to aberrant cardiac function.
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ACKNOWLEDGMENTS |
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