Download Species-specific differences of myosin content in the developing

THE ANATOMICAL RECORD 268:27–37 (2002)
Species-Specific Differences of
Myosin Content in the Developing
Cardiac Chambers of Fish, Birds,
and Mammals
DIEGO FRANCO,1* ALEJANDRO GALLEGO,2 PETRA E.M.H. HABETS,1
V. SANS-COMA,2 AND ANTOON F.M. MOORMAN1
1
Experimental and Molecular Cardiology Group, Cardiovascular Research Institute
Amsterdam, Academic Medical Centre, University of Amsterdam,
Amsterdam, The Netherlands
2
Department of Animal Biology, Faculty of Science, University of Malaga,
Malaga, Spain
ABSTRACT
Key morphogenetic events during heart ontogenesis are similar in different vertebrate species. We report that in
primitive vertebrates, i.e., cartilaginous fishes, both the embryonic and the adult heart show a segmental subdivision
similar to that of the embryonic mammalian heart. Early morphogenetic events during cardiac development in the
dogfish are long-lasting, providing a suitable model to study changes in pattern of gene expression during these stages.
We performed a comparative study among dogfish, chicken, rat, and mouse to assess whether species-specific qualitative and/or quantitative differences in myosin heavy chain (MyHC) distribution arise during development, indicative of
functional differences between species. MyHC RNA content was investigated by means of in situ hybridisation using an
MyHC probe specific for a highly conserved domain, and MyHC protein content was assessed by immunohistochemistry. MyHC transcripts were found to be homogeneously distributed in the myocardium of the tubular and embryonic
heart of dogfish and rodents. A difference between atrial and ventricular MyHC content (mRNA and protein) was
observed in the adult stage. Interestingly, differences in the MyHC content were observed at the tubular heart stage
in chicken. These differences in MyHC content illustrate the distinct developmental profiles of avian and mammalian
species, which might be ascribed to distinct functional requirements of the myocardial segments during ontogenesis.
The atrial myocardium showed the highest MyHC content in the adult heart of all species analysed (dogfish (S.
canicula), mouse (M. musculus), rat (R. norvegicus), and chicken (G. gallus)). These observations indicate that in the
adult heart of vertebrates the atrial myocardium contains more myosin than the ventricular myocardium. Anat Rec
268:27–37, 2002. © 2002 Wiley-Liss, Inc.
Key words: myosin heavy chain; heart morphogenesis; cardiac performance
The early stages of heart development are essentially
similar between different vertebrate species (Icardo, 1996;
Fishman and Chien, 1997). The cardiac crescent fuses in
the midline of the antero-posterior embryonic axis to give
rise to a straight cardiac tube. At this stage, myosin isoforms are first expressed and the cardiac patterns of expression are different—at least in avian and rodent species (for review, see Franco et al., 1998). In mice, rats, and
humans, two myosin heavy chains (MyHCs) are expressed
along the tubular heart: ␣MyHC shows an postero-anterior gradient, whereas ␤MyHC displays an antero-posterior gradient (De Groot et al., 1989; Moorman and Lamers,
1994). In chicken, a single ␤MyHC-like (VMHC1) gene has
been reported with essentially the same developmental
pattern as its rodent homologue (Bishaba and Bader,
1991). In contrast, two ␣MyHC-like genes have been reported: CC2SV mRNA, which shows a similar pattern to
©
2002 WILEY-LISS, INC.
the mouse ␣MyHC mRNA (Oana et al., 1995), and
AMHC1 mRNA, which is restricted to the future atrial
Grant sponsor: DGES, Ministerio de Educación y Cultura;
Grant number: PB 98-1418-C02-01; Grant sponsor: NWO; Grant
number 902-16-219; Grant sponsor: Dutch Heart Foundation;
Grant number: 97206.
Diego Franco and Alejandro Gallego contributed equally to this
work.
*Correspondence to: Diego Franco, Department of Experimental Biology, University of Jaén, Paraje Las Lagunillas s/n, 23071
Jaen, Spain. Fax: ⫹34-953-012141. E-mail: [email protected]
Received 26 February 2002; Accepted 25 April 2002
DOI 10.1002/ar.10126
Published online 00 July 2002 in Wiley InterScience
(www.interscience.wiley.com).
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FRANCO ET AL.
myocardial cells at the tubular heart stage (Yutzley et al.,
1994). Recently, a new MyHC isoform (CMHC1) was
cloned, which is expressed in both the atrial and ventricular myocardium (Croissant et al., 2000). Moreover, the
neonatal skeletal MyHC is transiently expressed in the
embryonic chicken heart, predominantly in the primary
myocardium and ventricular conduction system (Machida
et al., 2000). At the protein level, it has been suggested
that up to five different MyHC isoforms are expressed in
the chicken heart, each one having a specific expression
profile within the atrial and ventricular myocardial components (Evans et al., 1988; De Jong et al., 1988). At
present, no information is available concerning myosin
composition and distribution in the dogfish heart.
With further development, five different morphological
and functional areas are present in the embryonic heart of
birds and mammals: the inflow tract, atria, atrioventricular canal, ventricle, and outflow tract (Moorman and
Lamers, 1994). Each of these regions represents a distinct
transcriptional domain (Franco et al., 1997; Kelly et al.,
1999). Concomitant with the formation of a regionalised
embryonic heart, MyHC isoforms become confined to distinct compartments (Lyons, 1994; Franco et al., 1998).
␣MyHC becomes restricted to the atrial/inflow tract myocardium, whereas ␤MyHC becomes restricted to the ventricular/outflow tract myocardium. Coexpression of both
isoforms is observed in the “primary myocardium,” i.e., the
inflow tract, atrioventricular canal, and outflow tract (for
reviews, see Moorman and Lamers, 1994; Franco et al.,
1998).
The hearts of birds and mammals acquire separate left
and right atrial/ventricular chambers during development. The timing of ventricular chamber subdivision differs substantially between avian and mammalian species.
In the chick, the left and right ventricular primordia become recognisable after cardiac looping. In contrast, the
formation of left and right ventricles, including the ventricular septum, occurs concomitant with cardiac looping
in mice and rats. The acquisition of left and right ventricular segments is concomitant with transient differences in
left and right sarcomeric gene expression (Kelly et al.,
1999, Zammit et al., 2000) and transcriptional potential
(Kelly et al., 1995, 1999; Ross et al., 1996; Franco et al.,
1997).
Cardiac performance is directly related to conductive
and contractile characteristics of cardiomyocytes. In mammals, impulse conduction is mainly achieved via gap junctional communication. Little is known about gap junction
protein distribution in avian and fish hearts (Beyer, 1990;
Satchell, 1991; Minkoff et al., 1993; Gourdie et al., 1993).
The main determinants of contractile performance in striated muscle are differences in MyHC composition. One
can envisage that cardiac performance is related to total
MyHC content, and that its regional heterogeneity contributes to differential chamber-specific cardiac performance during ontogenesis.
Currently, there is no information concerning myosin
composition and distribution in the heart of primitive
vertebrates, such as elasmobranchs, that maintain single
atrial and ventricular chambers in the adult stage. Therefore, we selected the dogfish (Scyliorhinus canicula) as an
animal model in which to examine changes in total MyHC
content during the transition from a tubular heart into a
segmented heart. Comparison with chicken, rat, and
mouse embryonic stages enabled us to study MyHC con-
tent during the change from a single circulatory cardiac
system into a double-pumping heart.
In the present study, we analysed the expression pattern profile of MyHC content in three stages: 1) an early
looping stage; 2) an embryonic stage in which five morphological segments can be distinguished (inflow tract,
atrium, atrioventricular canal, ventricle, and outflow
tract); and 3) the adult stage.
MATERIALS AND METHODS
Embryos
Fertilised eggs and adult specimens of the spotted dogfish (Scyliorhinus canicula) were maintained in captivity
under standard conditions as previously described (Gallego et al., 1997). In our experience, total length (TL)
represents the most suitable parameter for describing the
developmental stage of these embryos. Embryos of 20 and
40 mm TL, and adult heart samples were examined. Samples employed for in situ hybridisation were dissected in
0.1 M phosphate buffer (pH 7.3) and fixed in 4% freshly
prepared formaldehyde in 0.1 M phosphate buffer (pH
7.3). After they were washed in PBS, the samples were
dehydrated in increasing concentrations of ethanol and
embedded in paraplast. Serial sections were cut at 7 ␮m,
mounted onto RNAse-free aminopropyltriethosixylanecoated glasses (for in situ hybridisation) or onto polylysine-coated glasses (for immunohistochemistry) and
stored at room temperature.
Fertilised chicken eggs were obtained from a local
hatchery (Drost BV, Nieuw Loosdrecht, The Netherlands),
incubated at 37°C in a moist atmosphere, and automatically rotated every hour. After appropriate incubation
times, embryos of stages 14, 20, 24, and 30 (Hamburger
and Hamilton, 1951) were isolated and processed for in
situ hybridisation or protein immunohistochemistry as
previously described. Adult chicken hearts were also obtained from a local hatchery and quickly processed for in
situ hybridisation or protein immunohistochemistry.
Wistar rat embryos of embryonic day (E) 12.5, E14.5,
E16.5, and E18.5; mouse C57BL6/J embryos of E10.5,
E12.5, E14.5, and E16.5; and rat and mouse adult hearts
were examined. The day of vaginal plug was taken as
E0.5. Embryos were excised from the uterus and fixed
either in 4% freshly-prepared formaldehyde in phosphatebuffered saline (PBS) overnight at 4°C for in situ hybridisation or in methanol : acetone : water (2:2:1) at 4°C for
immunohistochemistry. Samples used for protein immunohistochemistry were handled as previously described
for other species.
In Situ Hybridisation
We used a cDNA probe coding for the highly conserved
ATP binding site of the human ␤MyHC gene (nucleotides
460 – 643 (Jaenicke et al., 1990)) as a general marker for
MyHC content. The length of the probe is 184 nucleotides,
and it shows a high homology to the same region of the
MyHC genes in rat (90%), mouse (90%), chicken (88%),
and carp (84%; see Fig. 1). This highly conserved homology
is also shared with skeletal muscle-specific isoforms at
both amino acid and nucleotide sequences (Habets et al.,
1999).
The human ␤MyHC probe was linearised with BamHI
and transcribed with T7 RNA polymerase. Complementary RNA probe was made with 35S-CTP (single-labelled)
MYOSIN CONTENT IN DEVELOPING VERTEBRATE HEART
29
Fig. 1. Nucleotide comparison of the ATP binding cassette of the
human ␤MyHC gene to the corresponding region of different MyHC
isoforms in rat (␣MyHC and ␤MyHC; accession numbers X15938 and
X15939, respectively) chicken (neonatal, embryonic, skeletal, and fast
white; AB021180, U87231, J02714, and M13516, respectively) and carp
(accession number D89992). The identity comparison for rat ␣MyHC
(161/181) is 89%; for rat ␤MyHC (160/181) it is 88%. The identity
comparison is 88% for chicken neonatal MyHC (157/179), 88% for
chicken embryonic MyHC (157/179), 89% for chicken skeletal MyHC
(159/179), and 86% for chicken fast white MyHC (113/131). The identity
comparison for carp MyHC (79/94) is 84%. Comparisons with AMHC1,
VMHCH1, CMHC1, and CCSV2 could not be included because the
nucleotide sequences of the homologous region of these genes are not
available.
or with 35S-UTP and 35S-CTP (double-labelled) by in vitro
transcription according to standard protocols (Melton et
al., 1984). Complementary RNA probes against chicken
AMHC1 (Yutzley et al., 1994), chicken VMHC1 (Bishaba
and Bader, 1991), rat ␣MyHC (Schiaffino et al., 1989;
Boheler et al., 1992) and rat ␤MyHC (Boheler et al., 1992)
mRNAs were used as positive controls on the hybridisation assays in chicken and mouse embryos. Hybridisation
conditions were as described elsewhere (Moorman et al.,
1995, 2000; Franco et al., 2001). Briefly, the sections were
deparaffinated, rinsed in absolute ethanol, and dried in an
air stream. Pretreatment of the sections was as follows: 20
min 0.2 N HCl, 5 min bidistilled water, 20 min in 2⫻ SSC
(70°C), 5 min bidistilled water, 2–20 min digestion in 0.1%
pepsin dissolved in 0.01 N HCl (37°C), 30 sec in 0.2%
glycine/PBS, two 30-sec rinses in PBS, 20 min postfixation
in 4% freshly-prepared formaldehyde, 5 min in bidistilled
water, 5 min in 10 mM DTT, and finally drying in an air
stream. The prehybridisation mixture contained 50% formamide, 10% dextran-sulphate, 2⫻ SSC, 2⫻ Denhardt’s
solution, 0.1% Triton X-100, 10 mM DTT, and 200 ng/␮l
heat-denatured herring sperm DNA. The sections were
hybridised overnight at 52°C and washed as follows: a
rinse in 1⫻ SSC, 30 min in RNAse A (10 ␮g/ml), 10 min 1⫻
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FRANCO ET AL.
SSC, 10 min 0.1⫻ SSC, and dehydration in 50%, 70%, and
90% ethanol containing 0.3 M ammonium acetate. The
sections were then dried and immersed in nuclear autoradiographic emulsion G5 (Ilford, UK). The exposure time
ranged from 7 to 14 days, and the development times from
4 to 8 min. Images were obtained using a Photometrics
camera attached to a Zeiss Axiophot microscope. Digital
images were composed using Adobe PhotoShop 5.0 and
Microsoft Power Point 7.0 software packages.
Whole-Mount In Situ Hybridisation
Complementary RNA probe against the human ␤MyHC
was labelled with digoxigenin-UTP by in vitro transcription according to standard protocols (Hogan et al., 1994;
Henrique et al., 1997). Hybridisation conditions were as
described by Henrique et al. (1997) with slight modifications (Christoffels et al., 2000).
Protein Immunohistochemistry
Monoclonal primary antibodies against human ␣MyHC
(Wessels et al., 1991), human ␤MyHC (Wessels et al.,
1991), chicken ␣MyHC (De Groot et al., 1987), and chicken
␤MyHC (De Groot et al., 1987), and polyclonal primary
antibody against all MyHC isoforms (L53) were used. The
L53 polyclonal antibody was generated as described by
Sanders et al. (1984) and its specificity was characterised
by Western blot analysis. Sections were deparaffinated,
hydrated in decreasing concentrations of ethanol, and
rinsed in PBS. Subsequently, sections were treated for 30
min with 3% hydrogen peroxide in PBS to reduce endogenous peroxidase activity, followed by incubation in
TENG-T (10 mM Tris, 5 mM EDTA, 150 mM NaCl, 0.25%
gelatine, 0.05% Tween-20, pH 8.0) for 30 min, and finally
incubated overnight in primary antibody. Binding of the
primary antibody was detected using peroxidase-avidin/
biotin-complex- or alkaline phosphatase (AP)-coupled secondary antibodies.
After application of the secondary biotinylated antibody
for 2 hr, the signal was visualised by incubation with a 0.5
mg/ml diaminobenzidine (DAB) solution (sp2001; Vector
Laboratories, Burlingame, CA) for 2–10 min, following the
manufacturer’s protocol. Incubation with secondary AP-
Fig. 2. In situ hybridisation using a probe against the ATP binding
domain of the human ␤MyHC gene in the developing dogfish heart at
(A) 20 mm TL and (B and C) 40 mm TL. Panel B represents a colour
scale conversion. A colour scale bar is provided. Yellow indicates low
expression and blue indicates high expression. A: MyHC transcripts
are evenly expressed along the antero-posterior axis of the looped
cardiac tube in the 20 mm TL dogfish heart. B: Expression of MyHC
transcripts at the 40 mm TL stage is almost absent in the sinus
venosus (sv), but is evenly expressed in the atrial myocardium (a),
including the atrial side of the sinoatrial valve (arrowhead), as well as
in the atrioventricular (avc), ventricular (v), and conal (co) myocardia.
B and C: Within the ventricular myocardium, no differences are observed between the innermost and outermost layers. B: Expression of
MyHC transcripts is also observed in skeletal muscle (arrows). C:
Note that the hybridisation signal is absent in the endocardial cushions (arrowhead) and the aorta (ao), but is present in the myocardial
sleeve of the AVC. D and E: Protein immunohistochemistry using
polyclonal L53 in the developing dogfish heart. Expression of MyHC
protein at the 40 mm TL stage is similar to that observed at the mRNA
level (see for comparison panels B and C). Bar: (A) 120 ␮m, (B–E) 240
␮m.
coupled antibody was performed for 2 hr. Endogenous AP
activity was inhibited by adding 5 mM levamisole in all
incubation and washing solutions. After primary and secondary antibody incubations, the sections were heated for
20 min at 55°C for further blocking of the remaining
endogenous AP activity. Visualisation of the AP activity
was performed by incubation in NBT/BCIP (#1681451;
Roche, Basel, Switzerland) solution for approximately 20
min. The specificity of the antigen-antibody reaction in
different species was supported by the fact that incubations without primary antibody led to no detectable signal
in all cases.
Image Analysis
Gray-scale radioactive in situ hybridisation signals
were converted into colour-scaled images to easily identify
quantitative differences in gene expression within different myocardial compartments, using the methodology described by Moorman et al. (2000).
Cardiac Nomenclature
In birds and mammals, the embryonic heart consists of
five segments: the inflow tract, atrium, atrioventricular
canal, ventricle, and outflow tract. The inflow tract is the
myocardial region upstream of the atrium (Franco et al.,
1998); it becomes incorporated into the atrium and eventually develops into different myocardial structures, including the caval veins, the coronary sinus, the crista
terminalis, and the Thebesian valve (Rogers, 1986; Dor
and Corone, 1991; Moorman and Lamers, 1994; De Ruiter
et al., 1995; Tasaka et al., 1996; Webb et al., 1998). The
outflow tract is the myocardial region at the arterial pole
of the heart lined by endocardial cushions (Franco et al.,
1999); it becomes divided into pulmonary and aortic outlets by the formation of the outlet septum, and contributes
to the formation of the left and right ventricular infundibuli (De la Cruz et al., 1989; Franco et al., 1997).
In the dogfish, the embryonic heart shows the five segments mentioned above (Gallego et al., 1998). The inflow
tract differentiates early into a well defined chamber, the
sinus venosus, which remains as a single distinct entity in
the adult heart (Santer, 1985; Satchell, 1991; Muñoz-Chá-
Fig. 3. A: Schematic representation of the adult dogfish heart. B–E: In
situ hybridisation using a probe against the ATP binding domain of the
human ␤MyHC gene in the adult dogfish heart. Panels D and G represent
colour scale conversions. Colour scale bars are provided: (D) yellow indicates low expression and blue indicates high expression; (G) blue indicates
low expression and yellow indicates high expression. B and C: Expression
of the MyHC transcripts is higher in the atrial (a) than in the ventricular (v)
and conal (co) (arrowheads) myocardia. E: Expression of MyHC transcripts
is confined to the muscular layer of the sinus venosus (sv) wall (arrows). D:
Expression in the atrioventricular canal myocardium (arrowhead) is lower
than in the flanking atrial and ventricular myocardium. C: The conal myocardium expression (arrowhead) shows just slightly lower levels than the
ventricular myocardium. F–H: MyHC protein expression in the adult dogfish
heart. F: The MyHC protein expression in the conal myocardium is similar to
that observed in the ventricular myocardium. G: Note that expression of
MyHC protein is slightly higher in the atrial than in the ventricular myocardium, whereas expression in the atrioventricular myocardium (avc) is just
slightly weaker. H: The expression of MyHC protein in the sinus venous is
confined to the myocardial layer (arrows). Bar: (B) 400 ␮m, (C–E) 240 ␮m, (F
and G) 240 ␮m, (H) 120 ␮m.
MYOSIN CONTENT IN DEVELOPING VERTEBRATE HEART
C
O
L
O
R
Figure 2.
C
O
L
O
R
Figure 3.
31
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FRANCO ET AL.
puli et al., 1994; Gallego et al., 1997). Both the atrium and
ventricle remain as single myocardial chambers. Until
now, the existence of a distinct myocardial AVC region has
not been fully acknowledged in the embryonic dogfish
heart (see Results), although previous descriptive works
have emphasised the presence of a discrete zone interposed between the atria and the ventricles, which is characterised by anchoring endocardial cushions (Muñoz-Chápuli et al., 1994). The outflow tract appears early in
development, having an outer myocardial wall; it is involved in the formation of the conal endocardial cushions,
and gives rise to the adult conus arteriosus or bulbus
cordis (Gegenbaur, 1866), without any change of its original position or substantial remodelling (Muñoz-Chápuli
et al., 1994; Sans-Coma et al., 1995). Thus, the heart of the
adult dogfish is composed of five myocardial compartments: the sinus venosus, atrium, atrioventicular canal,
ventricle, and conus arteriosus (bulbus cordis).
RESULTS
Dogfish Cardiac Development
The developmental stages of the dogfish heart are similar to the early stages observed during mouse embryogenesis (Muñoz-Chápuli et al., 1994). Early in development,
the heart tube loops rightward and acquires a left-dorsal
inflow region and a right-ventral outflow region (Gallego
et al., 1998). At around the 20 mm TL stage, the heart is
almost fully looped and the different cardiac regions begin
to be distinguished from each other, with the atrial and
ventricular chambers being the most prominent. Total
MyHC transcripts are evenly expressed along the myocardium at this stage (Fig. 2A). A similar pattern is observed
at the protein level using the polyclonal antibody against
all MyHC isoforms (data not shown).
At the 40 mm TL stage the interregional differences
have been established and the dogfish heart has acquired
five fully distinct morphological and molecular myocardiac
compartments: the sinus venosus, atrium, atrioventricular canal, ventricle, and conus arteriosus. The ventricle
exhibits two layers: a thin outer compact myocardial layer
and a more developed inner trabeculated layer. The conus
arteriosus and atrioventricular canal are lined by endocardial cushions (Muñoz-Chápuli et al., 1994). Total
MyHC transcript expression is weak in the sinus venosus,
and evenly strong in the atrial face of the sinoatrial
valves, the atrial myocardium, the atrioventricular canal,
the ventricular myocardium (which is compact and trabeculated), and the conal myocardium (Fig. 2B and C). Hybridisation to the highly conserved ATP-binding domain
cassette of the MyHC gene is also observed in all skeletal
muscle cells (Fig. 2B). Immunolocalisation of MyHC protein using the polyclonal antibody L53 displays a similar
pattern to that observed at the mRNA level, including the
skeletal muscle-specific expression (Fig. 2D and E).
The anatomical configuration of the adult dogfish heart
is essentially similar to that observed in the foetal dogfish
heart. Five different myocardial segments can still be
traced morphologically: the sinus venosus, atrium, atrioventricular canal, ventricle, and conus arteriosus. At this
stage, the conal and atrioventricular endocardial cushions
have been remodelled into two transversal rows of conal
valves and four atrioventricular valve leaflets (SansComa et al., 1995) (A. Gallego and B. Buch, unpublished
data). Total MyHC expression is dramatically changed at
this stage as compared to the embryonic stages. The sinus
venosus shows a clear hybridisation signal confined to the
thin myocardial middle layer of the sinus wall (Fig. 3E
and H). This observation is in line with previous reports
that described a single muscular layer in the sinus venosus surrounded by connective and neural components
(Saetersdal et al., 1975; Ramos et al., 1996; Gallego et al.,
1997). Expression in the atrial myocardium is higher than
in the ventricular/conal myocardium (Fig. 3B–D). The
atrioventricular myocardium displays a weaker hybridisation signal than the atrial and the ventricular myocardium (Fig. 3D). The expression of total MyHC transcripts
in the conal myocardium is just slightly weaker than in
the ventricular myocardium (Fig. 3C). No differences in
MyHC expression were observed between the trabeculated and compact myocardial layers of the ventricles.
Similar results were obtained at the protein level (Fig.
3F–H). The atrioventricular canal displays a slightly
weaker MyHC protein level than the atrial and ventricular myocardia, whereas the MyHC protein level of the
conus arteriosus is similar to that of the ventricular myocardium (Fig. 3F and G). Thus, according to our observations, the atrial and ventricular myocardia contain more
MyHC transcripts and are flanked by myocardial segments that contain less MyHC transcripts, namely the
atrioventricular canal and the conus arteriosus. At the
protein level, the differences are less sharp than those
observed at the mRNA level.
Chicken Heart Development
The early stages of cardiac organogenesis in chicken are
essentially similar to those observed in the dogfish heart.
At H/H14, the heart can still be considered as a linear
tube, with the myocardium separated from the endocardium by an acellular cardiac jelly. At this stage, thickening of the cardiac jelly can already be observed in the
prospective outflow tract (conal) region and the atrioventricular canal region. In contrast to the dogfish (20 mm
TL), expression of total MyHC transcripts is higher in the
venous pole than in the arterial pole of the heart in
chicken at similar stages (H/H14) (Fig. 4C).
With further development (H/H24), the heart acquires
five morphological and functional segments: the inflow
tract, atrium, atrioventricular canal, ventricle, and outflow tract (Argüello et al., 1986; De Jong et al., 1992;
Moorman and Lamers, 1994). Expression of MyHC transcripts is higher in the inflow tract/atrium region than in
the ventricles/outflow tract region (Fig. 4B and D), while
no differences exist between the inflow tract and the atrial
myocardium. Expression in the outflow tract is similar to
that observed in the ventricular myocardium. No differences are observed in the compact and trabeculated components of the ventricular myocardium (Fig. 4D). At the
protein level, the MyHC pattern resembles that obtained
at the mRNA level (Fig. 4E). Control markers such as
AMHC1 and VMHC1 (at both the mRNA and protein
level) display an expression pattern confined to the atrial
and ventricular myocardia, respectively, as previously described (De Jong et al., 1992; Yutzley and Bader, 1995). In
the adult chicken heart, expression of MyHC mRNA and
protein is similar to that observed in the embryonic stage,
with higher expression being observed in the atrial myocardium as compared to the ventricular myocardium (Fig.
5A and B).
MYOSIN CONTENT IN DEVELOPING VERTEBRATE HEART
Fig. 4. A: Schematic representation of the foetal chicken heart at stage
HH30. In situ hybridisation using a probe against the ATP binding
domain of the human ␤MyHC gene in the developing chicken heart at (B)
H/H22 in whole-mount, and at (C) H/H14 and (D) H/H24 in tissue sections. Panel B is a left lateral view of an HH22 embryonic chicken heart.
Panel D represents a colour scale conversion. A colour scale bar is
provided. Yellow indicates low expression, and blue indicates high expression. C: Expression of the MyHC transcripts is higher in the venous
pole as compared to the arterial and prospective ventricular region at
33
H/H14. B and D: At the embryonic stage (H/H22–24), the expression of
MyHC transcripts remains higher in the atrial myocardium as compared
to the ventricular myocardium. E: Protein immunohistochemistry using
L53 polyclonal antibody in the developing chicken heart. MyHC protein
distribution is similar to that observed at the mRNA level at H/H24. a,
atrium; avc, atrioventricular canal; ift, inflow tract; la, left atrium; lv, left
ventricle; o, outflow region; oft; outflow tract; ra, right atrium, rv, right
ventricle; v, ventricle. Bar: (B) 200 ␮m, (C) 200 ␮m (D) 240 ␮m, (E) 450 ␮m.
Rodent Heart Development
Fig. 5. A: In situ hybridisation using a probe against the ATP binding
domain of the human ␤MyHC gene in the adult chicken heart. Expression of the MyHC transcript is higher in the atrial than in the ventricular
myocardium. B: Protein immunohistochemistry using a polyclonal antibody against all MyHC isozymes. Observe that expression of total MyHC
protein is higher in the atrial than in the ventricular myocardium. la, left
atrium; lv, left ventricle; mv, mitral valve. Bar: (A and B) 50 ␮m.
Expression of total MyHC content at both the mRNA
and protein level was analysed in rat and mouse embryos
at heart development stages similar to those considered in
the dogfish and chicken. No differences were detected
between rats and mice. Similar to the situation in the
dogfish heart, the expression of MyHC transcripts is homogeneous through the myocardium at both the tubular
heart stage (mouse E8.5; rat E10.5) and the embryonic
heart stage (mouse E10.5; rat E12.5) (Fig. 6B–D). Similar
results were obtained at the protein level, using the polyclonal L53 antibody (data not shown). Control experiments designed for the localisation of ␣MyHC and ␤MyHC
(mRNA and protein) displayed a regionalised expression
pattern as previously described (for review, see Franco et
al., 1998).
In the adult rodent heart, as in the dogfish and chicken
adult hearts, expression of total MyHC content was higher
in the atrial than in the ventricular myocardium (Fig. 6E).
A similar pattern was observed at the protein level (data
not shown).
DISCUSSION
A comparison of the amino acid (data not shown) and
nucleotide sequences (Fig. 1) of different mammalian (human, mouse, rat, pig, and hamster), avian (chicken and
34
FRANCO ET AL.
Fig. 6. A: Schematic representation of an embryonic (E12.5) mouse
heart. B: Whole-mount in situ hybridisation using a probe against the
ATP binding domain of the human ␤MyHC gene corresponding to a
E12.5 mouse heart. In situ hybridisation using a probe against the ATP
binding domain of the human ␤MyHC gene in the (B–D) developing and
(E) adult mouse heart in tissue sections. Panel D represents a colour
scale conversion. A colour scale bar is provided. Yellow indicates low
expression and blue indicates high expression. C: Expression of the
MyHC transcripts in the embryonic heart (E9.0) is homogeneous along
the inflow tract, and the atrial and ventricular myocardia. D: At E12.5,
expression of MyHC transcripts remains similar in the atrial and ventricular myocardia as well as in the inflow tract derivatives, pulmonary veins
(pv), and caval veins (lscv). E: In the adult mouse heart, expression of
MyHC transcripts is higher in the atrial myocardium than in the ventricular myocardium. ao, aortic valve; avc, atrioventricular canal; cv, caval
veins; ift, inflow tract; la, left atrium; lv, left ventricle; ra, right atrium; rv,
right ventricle; rscv, right superior caval vein. Bar: (B) 300 ␮m, (C) 100
␮m, (D) 240 ␮m, (E) 500 ␮m.
quail), and fish (carp) isozymes corresponding to the ATP
binding site of the MyHC gene used to localise cardiac
MyHC transcripts shows that this region of the MyHC
gene is highly conserved among species. Such a high homology guarantees that the hybridisation signal obtained
according to our standard hybridisation conditions (Moorman et al., 1995, 2000; Franco et al., 2001) would react
with all MyHC transcripts, and that it can therefore be
used as a parameter to estimate total MyHC content. The
pattern of expression of total MyHC protein, as revealed
by using the L53 polyclonal antibody, is consistent with
the data obtained at the transcriptional level.
Although distinct MyHC isoforms are expressed in each
cardiac compartment of the chicken and mammalian
hearts (e.g., ␣MyHC and ␤MyHC in the atria and ventricles, respectively), the L53 antibody reacts with both the
atrial and ventricular myocardium in dogfish, chicken,
rat, and mouse, and can thus be considered as a panMyHC antibody. Furthermore, L53 antibody reacts similarly in cardiac and skeletal muscle in dogfish, chicken,
rat, and mouse tissues. Although we did not perform
epitope mapping of the binding affinity of this antibody,
the fact that the L53 antibody reacts in all species and in
all striated muscles suggests that it recognises a highly
conserved epitope, which is shared by MyHC isoforms
among different vertebrate species.
We have demonstrated herein the existence of regional
differences in total MyHC content in different vertebrate
species during ontogenesis. MyHC content is homogeneous along the myocardium in the dogfish and rodent
tubular and embryonic heart stages, whereas differences
in MyHC content occur at the tubular heart stage in
chicken, being higher in the venous pole than in the arterial pole. However, in the adult heart, the atrial myocardium contains more MyHC than the ventricular myocardium in all species studied. This suggests that although
there are differences in the MyHC content profile during
ontogenesis of each species, the adult MyHC distribution
pattern persists throughout the phylogenetic tree of all
vertebrates. Thus, the vertebrate cardiac design implies
an atrial myocardium with higher MyHC content than the
ventricular myocardium in the adult heart.
MYOSIN CONTENT IN DEVELOPING VERTEBRATE HEART
The species-specific differences observed during ontogenesis may be related to differential patterns of expression of the distinct MyHC isoforms (␣MyHC and ␤MyHC)
existing in avian and mammalian hearts. In rodents, opposite gradients of expression for ␣MyHC and ␤MyHC
have been described as early as at the tubular heart stage
(De Groot et al., 1989). In contrast, expression of AMHC1
in the chicken heart is confined to the venous pole of the
heart at the tubular stage, and precedes in time the ubiquitous expression of VMHC1 in the myocardium (Yutzley
et al., 1994; Yutzley and Bader, 1995). CCSV2 is more
broadly expressed than AMHC1, but it is mainly confined
to the atrial myocardium (Oana et al., 1995). Recently, a
new MyHC isoform (CMHC1) was cloned, which is expressed similarly in both the atrial and ventricular myocardia (Croissant et al., 2000). Interestingly, neonatal
skeletal MyHC is also transiently expressed in the embryonic heart (Machida et al., 2000). The analysis of MyHC
protein distribution suggests the presence of as many as
five protein isozymes in the developing avian heart (De
Jong et al., 1987, 1988, 1990; Evans et al., 1988), in contrast to two isozymes expressed in the mammalian heart
(De Groot et al., 1989; Wessels et al., 1991; Lyons, 1994;
Franco et al., 1998). Several skeletal muscle-specific
MyHC isoforms have been reported in bony fishes (Rowlerson et al., 1985; Gerlach et al., 1989; Chanoine et al.,
1990). However, few data are available concerning the
cardiac MyHC composition in teleosts and elasmobranch
species (Vornanen, 1994; Yelon et al., 1999), which makes
correlation with the present findings difficult. The differential MyHC content observed between atrial and ventricular myocardia during all stages of chicken cardiogenesis
suggests that the distinct atrial and ventricular functional
requirements may be mediated by the total MyHC content, although formally we can not exclude an isoformspecific contribution to the chamber-specific functional
modulation.
The adult dogfish heart presents five different morphological segments (Gallego et al., 1998), resembling those of
chicken and rodent embryos (Moorman and Lamers, 1994;
Franco et al., 1998). In the latter, the atria and ventricles
are fast-conducting segments (high abundance of gap
junctional proteins, mainly connexin 43) which are
flanked by slow-conducting segments (absence of connexin
43), namely the inflow tract, atrium, atrioventricular canal, ventricle, and outflow tract (Van Kempen et al., 1991,
1996).
In the adult dogfish heart, a distinct atrioventricular
segment is interposed between the atrium and the ventricle. The low MyHC content in the myocardium of this
segment suggests that this myocardium may play a role in
the coordination of the cardiac cycle. Moreover, the AVC
myocardium may provide a delay in the propagation of the
cardiac impulse throughout the heart, thereby allowing
the integrated performance of the atrial and ventricular
myocardia. However, further investigations into the distribution of gap junctional communication genes are required to verify this hypothesis.
Interestingly, a weaker expression of MyHC is observed
in the sinus venosus vs. the atrium of the embryonic
dogfish heart. In contrast, the expression in the inflow
tract is similar to that observed in the atrium of embryonic
avian and mammalian hearts. Such differences may be
related to the fact that in the dogfish, the sinus venosus
acts as a passive drainage pool of blood early in develop-
35
ment and retains this function in adult life (Satchell,
1970, 1991). In contrast, avian and mammalian inflow
tracts are transient structures that mainly become incorporated into the right atrial chamber, and eventually also
contribute to the remodelling of the right and left inlets to
the atrial chambers in the adult heart (De Ruiter et al.,
1995; Tasaka et al., 1996; Webb et al., 1998; Franco et al.,
2000). The weak MyHC expression in the adult sinus
venosus wall of the dogfish heart is attributed to the fact
that neural and connective tissues are intermingled with
a thin, poorly contractile myocardial layer (Santer, 1985;
Ramos et al., 1996; Gallego et al., 1997) which acts as the
nodal tissue (these animals lack a morphologically distinguishable conduction system).
The observation that the conal myocardium has just a
slightly weaker total MyHC content as compared to the
ventricular myocardium suggests that it plays an active
role in contraction during the cardiac cycle. In fact, the
adult conus arteriosus of elasmobranchs is known to display a peristaltic pattern of contraction that plays a critical role in contributing an “extra systole” (Johansen,
1965) in Squalus suckleyi, and in allowing the conal valvular apparatus to function correctly (Satchell and Jones,
1967) in Heterodontus portusjacksoni.
In summary, the different levels of total MyHC distribution in distant vertebrate species support the notion
that the functional requirements of the avian heart diverge from those of dogfish and rodents as early as at the
linear cardiac tube stage, although, curiously, there is a
similar pattern in adulthood. The homogeneous expression along the embryonic slow- and fast-conducting segments argues in favour of the notion that differences in
contractile properties in the distinct cardiac segments are
dictated more by the MyHC isoform composition than by
the total MyHC content, whereas in the adult heart, the
atrium has a higher MyHC content than the ventricles,
despite having a single or double circuitry. Moreover, the
adult vertebrate cardiac design appears to require a
higher MyHC content in the atrial than in the ventricular
myocardium.
ACKNOWLEDGMENTS
We thank Robert Kelly (Pasteur Institute, Paris) for his
critical reading of the manuscript, Dr. Marina Campione
for sharing unpublished data, Belén Buch for the design of
the schematic representations, and Corrie de Gier-de
Vries for excellent technical support.
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