© 1999 American Heart Association, Inc.
UltraRapid Communications |
A Subpopulation of Cardiomyocytes Expressing 
-Skeletal Actin Is Identified by a Specific Polyclonal Antibody
From the Department of Pathology, University of Geneva-CMU, Geneva, Switzerland.
Correspondence to Prof Giulio Gabbiani, University of Geneva-CMU, Department of Pathology, 1 rue Michel-Servet, 1211 Geneva 4, Switzerland. E-mail giulio.gabbiani{at}medecine.unige.ch
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Abstract |
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Abstract—The NH2-terminal decapeptide of
-skeletal actin that
contains a primary sequence specific for this isoform was used to raise
a polyclonal antibody in rabbits. Using sequential affinity
chromatography, we recovered from serum antibodies
reacting exclusively with -skeletal actin when tested by
immunoblotting and
immunofluorescence. Epitope mapping by means of
competition assays with synthetic peptides indicated that the acetyl
group and the first 9 amino acids are essential for specificity. The
monospecific antibody was then used to investigate the distribution of
-skeletal actin in the myocardium of newborn and normal
or hypertensive (with or without fibrotic areas) adult rats.
Immunostaining of normal heart revealed that
-skeletal actin is diffusely distributed within practically all
myocardial fibers of the newborn rat, whereas it is restricted to a
small proportion of adult rat cardiomyocytes, which appear
intensely stained. A correlation, albeit not complete, was found
between the distribution of -skeletal actin and ß-myosin heavy
chain. During cardiac hypertrophy induced by aortic
ligature between the renal arteries, the expressions of -skeletal
actin mRNA and protein were increased. The distribution of
immunostaining had a focal pattern similar to that of
normal adult rats, reactive fibers being more numerous and more
intensely stained compared with normal myocardium. Positive
fibers were particularly abundant at the periphery of fibrotic areas.
Using this antibody, we have demonstrated for the first time the
differential distribution of -skeletal actin in heart tissues.
Changes in the distribution of this isoform in hypertrophic heart
provide new insight into the mechanisms by which the heart adapts to
work overload. This antibody will prove useful in exploring the
mechanisms of expression of -skeletal actin and in defining its role
in physiological and pathological situations.
The full text of this article is available at
http://www.circresaha.org.
Key Words: actin isoform • cardiac hypertrophy • fibrosis • myocardium • hypertension
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The 6 actin isoforms found in mammalians constitute a family of closely related proteins expressed in a tissue-specific way.
-Skeletal and -cardiac actins are the preponderant actin isoforms
detected in striated muscles.1 Their sequences are almost
identical, differing by only 4 out of 375 amino acids.2
Two of these differences consist of an inversion of glutamic and
aspartic acid at residues 2 and 3 in the
NH2-terminus of the protein. The nucleic acid
sequences of mRNA coding regions are similarly well conserved; however,
these 2 isoactin mRNAs markedly differ in their 5' and 3' untranslated
regions, which can be used to unambiguously distinguish between them.
Numerous studies have taken advantage of these differences to show that
the expression of each mRNA varies quantitatively with species, muscle
type, development, or pathological situations.3 4 5 6 It has
been shown that the 2 sarcomeric mRNAs are coexpressed in skeletal and
cardiac muscle: -cardiac actin mRNA has been found in developing
mouse,7 rat,8 and chick9
skeletal muscles. Conversely, the myocardium of various
species contains the mRNA encoding skeletal actin, in proportions
varying during development and/or pathological situations. In the rat
heart, both isogenes are expressed in utero; the proportion of total
actin mRNA due to -skeletal actin mRNA is 28% at 17 to 19
days of fetal development, 40% one week after birth and 5% in the
adult.5 An increase in skeletal actin mRNA expression also
has been demonstrated in a variety of experimental models of
hypertension with consequent myocardial
hypertrophy.3 10 11 12 At the protein level,
studies on actin isoform distribution and quantification have been
carried out using electrophoresis and by analysis of the
partially hydrolyzed NH2-terminal
peptides.1 13 Using these methods, it was shown that both
-skeletal and -cardiac actins are simultaneously
expressed in various types of striated muscle and that a good
correlation between the relative protein and mRNA expressions generally
is observed. However, no description of the distribution of these
isoforms at the cellular level heretofore has been presented
because of the lack of an antibody recognizing unambiguously one of
them, despite the existence of antibodies specific for other actin
isoforms.14 15 16 This has left open questions regarding the
distribution of -skeletal actin protein expression in muscle
tissues, as well as the degree to which the 2 -striated muscle
actins colocalize in the same cell and within the sarcomere.
In the present work, we report the production,
immunopurification, and characterization of an antibody raised in the
rabbit against a synthetic decapeptide corresponding to the
NH2-terminal sequence of -skeletal actin.
Using this antibody, we have established the localization of
-skeletal actin in the normal rat myocardium during
postnatal development and provided new insight regarding its
distribution during the evolution of an experimental model of
hypertension.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Peptide Synthesis
Decapeptides with the NH2-terminal sequences of the 6 mammalian actin isoforms were synthesized as previously described.17 Shorter
-skeletal actin
peptides with 5, 7, 8, and 9 amino acids at the
NH2-terminal extremity were synthesized by
Research Genetics.
Preparation and Purification of the Anti–-Skeletal Actin
Antibody (Anti–-SKA1)
The NH2-terminal decapeptide of
-skeletal actin (Ac-DEDETTALVC-COOH) was coupled to
maleimide-activated keyhole limpet hemocyanin (KLH, Pierce)
through its cysteine residue according to the instructions of the
manufacturer. The coupled peptide was used for antiserum
production in a rabbit (performed by Biodesign
International).
The antiserum was first affinity purified using Sulfolink beads
(Pierce) coupled with the -skeletal decapeptide. Antibodies bound to
the column were eluted with 0.2 mol/L glycine-HCl pH 2.8 and 0.5 mol/L
NaCl, dialyzed overnight against phosphate-buffered saline solution
(PBS), and then loaded on a Sulfolink column coupled with the
NH2-terminal decapeptide of ß-cytoplasmic actin
followed by a Sulfolink column coupled with the
NH2-terminal decapeptide of 
-smooth muscle
actin to remove the population of antibodies cross-reacting with these
2 isoforms. Specificity of the fractions was tested by Western
blotting.
Electrophoretic and Immunoblot Analysis
Anti–-SKA1 specificity was determined by
immunoblotting of the following whole tissue
homogenates: rat striated muscle, rat
myocardium, human blood platelets, rat aorta, and
chicken gizzard (40 µg/lane were loaded for Coomassie blue staining
and 2 µg/lane for immunoblotting). Sodium
dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) was carried out in 5% to 20%
gradient polyacrylamide gels,18 and the proteins
were electroblotted to nitrocellulose according to Towbin et
al.19 Nitrocellulose membranes were incubated with
anti–-SKA1 diluted in Tris-buffered saline solution (TBS)
containing 3% BSA and 0.1% Triton X-100 for 2 hours at room
temperature. After 3 washes with TBS, a second incubation was performed
with peroxidase-conjugated affinity purified goat anti-rabbit IgG
(Jackson Immunoresearch Laboratories) at a dilution of 1:10 000 in TBS
containing 0.1% BSA and 0.1% Triton X-100. Peroxidase activity was
developed using the enhanced chemiluminescence Western blotting
system (Amersham) according to the instructions of the manufacturer.
Blots were scanned (Arcus II; Agfa), and the intensity of the bands was
quantified by means of the ImageQuant Program (Image Quant
Analysis, Molecular Dynamics).
Sedimentation Assays and Fab Fragment Preparation
Purification of actin from rabbit skeletal muscle acetone powder
was carried out according to Spudich and Watt.20 The
effect of anti–-SKA1 on -skeletal actin polymerization was
examined as described by Chaponnier et al.17
Fab fragments were prepared by a slight modification of the method of
Coulter and Harris.21 Whole immunoglobulins were separated
from other serum proteins on a Protein A-Sepharose 4B column
(Pharmacia). After dialysis against 20 mmol/L phosphate and
10 mmol/L EDTA (pH 7.0), cysteine-HCl was added at a final
concentration of 10 mmol/L, and whole immunoglobulins were
processed with immobilized papain (Pierce, 0.25 mL gel/mg
immunoglobulin) overnight at 37°C. Papain beads were then separated
from the digestion mixture and the undigested immunoglobulins were
removed by chromatography on a Protein A column leaving
in the flow through fraction a mixture of Fab and Fc fragments. The Fab
fraction was affinity purified as described above for
anti–-SKA1.
Experimental Models of Hypertension
A ligature was placed between renal arteries below the superior
mesenteric artery of 300 g male Wistar rats.22
This procedure results in a rapid development of a renin-dependent
hypertension.23 Animals were anesthetized with
CO2 and killed by cervical dislocation 3 or 7 days after
ligature. Hearts were collected and used for RNA and protein
extraction and immunofluorescence staining (see
below). The experiments were approved by the Ethical Committee of
Geneva Medical faculty.
RNA Extraction and Reverse Transcription-Polymerase Chain
Reaction (RT-PCR)
Total RNAs were isolated from muscle tissues by Tri-Reagent
(Molecular Research Center), according to the manufacturer’s
instruction. Each RNA sample was quantified spectrophotometrically at
260 nm before RT-PCR. The following primers were selected at the
extremities of the 3' untranslated region of -skeletal actin because
this region is isotype specific24 : the sense primer was
based on the complete gene sequence number 2985 to 3008
5'-CTCTCTCTCCTCAGGACGACAATC-3' and the antisense number 3168 to 3191
5'-CAGAATGGCTGGCTTTAATGCTTC-3'. Two micrograms of total RNA were first
reverse-transcribed in the presence of 5 mmol/L of random primers
p(dN)6 (Boehringer-Mannheim AG) using the conditions previously
described by Andreutti et al.25 PCR in the linear
range of amplification was performed to allow comparative
analysis of numerous cDNA samples. The optimal reaction
conditions chosen for the -skeletal actin–specific primers were the
following: 2 µL of the cDNA mixture were added to a master mix
containing 1.25 mmol/L MgCl2; 50 mmol/L
KCl; 10 mmol/L Tris-HCl, pH 9.0; 0.1% Triton X-100; 0.02
mmol/L of each dNTP; 1.25 nmol/L of both sense and reverse primers; and
1U of Taq-DNA Polymerase (Promega). The PCR cycling
parameters consisted of 20 seconds denaturation at 95°C,
30 seconds annealing at 60°C, and 30 seconds extension at 72°C for
25 cycles. Parallel ß2-microglobulin amplification was performed
(annealing temperature 58°C, 25 cycles) with the following
primers: 5'-ATCTTTCTGGTGCTTGTCTC-3' (sense) and
5'-AGTGTGAGCCAGGATGTAGT-3' (antisense) (amplified fragment of 243-bp
length). In each RT run, negative controls included the use of
sterile water in place of the RNA to control the purity of the reagents
and the absence of genomic DNA contamination (no amplified products
were detected in this procedure, thus confirming the purity of RNA
preparation). PCR products were then analyzed by
electrophoresis on 2.5% agarose gel; 1 µg of pGEM DNA (Promega) was
used as marker. The amount of amplified products was quantified for
each sample from scanned Polaroid negatives using ImageQuant software.
Each value was determined as the mean of 3 densitometric readings. The
final amount of PCR was expressed as the ratio of -skeletal actin
gene amplified to that of ß2-microglobulin to account for any
differences in starting amounts of RNA.
Indirect Immunofluorescence, Confocal Laser
Scanning Microscopy, and Light Microscopy
Tissue samples were embedded in OCT 4583 (Miles Scientific) and
frozen in precooled liquid isopentane. Three-µm cryostat sections
were fixed in acetone at -20°C for 5 minutes and air-dried for 2
hours at room temperature. Sections were stained with purified
anti–-SKA1 (1:5 dilution in PBS) alone or combined with monoclonal
antibodies against either human desmin (Clone 33, Dako), connexin 43
(clone CX1B1, Zymed Laboratories), ß-myosin heavy chain (clone
169-II-A2, kindly provided by Dr A Moorman, University of Amsterdam,
The Netherlands,26 27 ) or -smooth muscle
actin.14 Subsequently, tissues were incubated with
tetrarhodamine isothiocyanate–conjugated anti-rabbit and
fluorescein-labeled anti-mouse antibodies (both from
Jackson ImmunoResearch Laboratories).
Tissue sections were mounted in buffered polyvinyl alcohol28 and observed with a confocal laser scan fluorescence inverted microscope (model LSM 410, Carl Zeiss). Images were stored on optical disks (Sony Corp) and printed with a digital Fujifilm Pictography 4000 printer (Fujifilm).
For light microscopy, tissue samples were fixed in 10%
neutral-buffered formol and embedded in paraffin. Four-µm sections
were stained with hematoxylin-eosin and Masson’s trichrome or
processed for immunochemistry using anti–-SKA1 (1:10 dilution in
TBS) or anti–-smooth muscle actin (1:50 dilution in TBS).
Immunoperoxidase staining was performed as previously
described.29 Sections were observed using a Zeiss Axiophot
photomicroscope. Images were acquired with a high sensibility Photonic
Coolview color camera (Zeiss), stored, and printed as described above.
Images were subsequently analyzed using the software KS400
(Kontron System, Zeiss Vision). Using this software, any structure
could be selected on the basis of the pixel intensity values in each
color channel. To measure the intensity of
immunostaining, the image was transformed in gray
luminance values ranging from 0 (corresponding to black) to 255
(corresponding to white). Results were given as the percentage of pixel
number corresponding to a value below 200 (values above 200 were
considered as background; Bochaton-Piallat, personal communication,
1998).
Statistical Analysis
Results are shown as mean±SEM. For statistical comparison, the
results were analyzed by the Students’s t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Anti–
-SKA1 Purification and CharacterizationImmunization with hemocyanin-coupled
-skeletal actin
NH2-terminal decapeptide resulted in high titer
antibodies showing an important cross-reactivity with all actin
isoforms (data not shown). The first step of purification consisted in
loading the immune serum on Sulfolink beads coupled with the
decapeptide used for immunization. To test the cross-reactivity of the
resulting antibodies with actin isoforms, we used extracts of rat
striated muscle for -skeletal actin; rat myocardium for
-cardiac actin; human blood platelets for ß and
cytoplasmic actins, known to be present at the ratio of 5 to
130 ; rat aorta for -smooth muscle actin,
representing 70% of total actin; and chicken gizzard for
-smooth muscle actin.31 Immunoblotting
analysis (Figure 1b
) revealed
that the population of antibodies eluted from the beads coupled with
the -skeletal decapeptide reacted with -skeletal actin but also
with the other isoforms.
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To remove antibody populations recognizing epitopes common to the 6
isoforms, we chose to first eliminate those reacting with the
NH2-terminal decapeptide of ß-cytoplasmic actin
that shares the amino acid sequence ALV with the
NH2-terminal decapeptide of -skeletal actin
(see Figure 2B) and then to remove
antibodies reacting with the NH2-terminal
decapeptide of -smooth muscle actin. The resulting purified antibody
population recognized the actin isoform present in the striated
muscle extract but not actin from platelets, aorta, and gizzard
(Figure 1c
). The antibody also reacted with the cardiac muscle
homogenate, albeit to a clearly lesser extent (Figure 1c, lane 2). -Skeletal actin has been shown to be coexpressed
with -cardiac actin and to represent 5% of the sarcomeric
actin mRNA in the normal adult rat cardiac tissue.5 Thus,
because all extracts were blotted after loading with similar amounts of
actin (Figure 1a), it appeared likely that the reactivity of
purified anti–-SKA1 with the cardiac muscle extract is due to the
fraction of myocardial -skeletal actin.
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To further establish the specificity of purified anti– -SKA1,
we then investigated the amino acid residues involved in the
interaction with the antibody by incubating anti–-SKA1 with
synthetic peptides corresponding to the
NH2-terminal sequences of the 6 isoforms before
immunoblotting analysis (Figure 2). The
decapeptide of -skeletal actin used originally to raise the
anti–-SKA1 antiserum completely blocked the reactivity of purified
anti–-SKA1; however, peptides corresponding to the
NH2-terminal sequence of the other vertebrate
actins, including -cardiac actin, did not (Figure 2).
Moreover, incubation of anti–-SKA1 with the decapeptide of
-skeletal actin inhibited the reactivity with myocardial extract
(data not shown).
To identify the antibody epitope, a series of short peptides
corresponding to the NH2-terminal sequence of
-skeletal actin were synthesized (Figures 2A and 2B).
Only the acetylated nonapeptide was able to fully compete with
the antibody for actin. Thus the epitope recognized by the
antibody includes the acetyl group and the first 9 amino acids of the
-skeletal actin sequence.
Because an antibody against -smooth muscle actin recognizing the
epitope Ac-EEED has been shown to specifically interfere with
-smooth muscle actin in vitro polymerization,17 the
effect of anti–-SKA1 on -skeletal actin polymerization was
investigated. The protocol followed for this purpose has been
previously described.17 Briefly, we compared the amount of
actin present in the pellets and supernatants after sedimentation
assays performed either in physiological salt
conditions (100 mmol/L KCl, 2 mmol/L
MgCl2) or in the presence of anti–-SKA1
(1 µmol/L final concentration) in the same salt conditions. In
salt conditions, 25% of -skeletal actin partitioned in the
insoluble fraction. -Skeletal actin polymerization was significantly
enhanced when anti–-SKA1 was included in the incubation mixture
(with more than 70% of the protein recovered in the pellet, data not
shown). However, the anti–-SKA1 Fab, with only 1 actin-binding
site, had no significant effect on actin polymerization.
The specificity of purified anti–-SKA1 was then tested by means of
immunofluorescence on adult rat
stretched32 skeletal muscle. As shown in Figure 3, the only positive staining was located
in muscle fibers with the classical banding pattern. Confocal
microscopy demonstrated that, as expected, desmin and anti–-SKA1
stainings are located in the Z- and I-bands of myocytes, respectively
(Figure 3b).
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Immunolocalization of -Skeletal Actin in Myocardium
Anti–-SKA1 immunostaining was again localized
in myocardial fibers with a banding pattern. Unexpectedly, however,
-skeletal actin was restricted to a subpopulation of
ventricular cardiomyocytes randomly distributed
throughout the sections (Figure 4a).
Double staining with anti–-SKA1 and anti–connexin 43, the
principal gap junction protein located in intercalated disks further
confirmed the specific localization of -skeletal actin in distinct
cardiomyocytes (Figure 4c). Staining intensity was
variable in different cells, suggesting a spectrum of -skeletal
actin expression. Positively-stained cells were consistently
more abundant in the left compared with the right ventricle and
staining was not detectable in the atria (data not shown).
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A similar pattern of focal staining has been described for cardiac
myosin isoforms by immunochemical studies as well.33 34 35
To investigate a possible colocalization of -skeletal actin and
ß-myosin heavy chain, we performed double staining with
anti–-SKA1 and an antibody specific for ß-myosin heavy
chain.26 27 An important degree of coexpression was
observed (56.0±4.1% of ß-myosin heavy chain positive cells
expressed also -skeletal actin); however, cardiomyocytes
expressing only 1 of the 2 proteins were also present (Figures 4a and 4b).
Evaluation of -Skeletal Actin mRNA and Protein Content and
Distribution in Normal Newborn and Adult, and in Hypertensive Rat
Myocardium
-Skeletal actin mRNA is known to represent up to 40%
of the total sarcomeric actin mRNA in newborn rats and then to decrease
to <5% at 2 months of age.5 The availability of
anti–-SKA1 allowed us to investigate the modulation of -skeletal
actin during postnatal development at the protein level by means of
immunoperoxidase staining and Western blot analysis (Figure 5). Unlike what is observed in normal
adult cardiac muscle (Figure 5A, panel b), the
myocardium of 3-day-old rats displayed a uniform pattern of
reactivity (Figure 5A, panel a). Thus focal distribution of
-skeletal actin in myocardial fibers takes place during development.
Western blot analysis confirmed that -skeletal actin content
is 4-fold higher in newborn compared with adult heart (Figure 5B). Analysis of RNA expression by RT-PCR corroborated
the results obtained for the protein in that the ratio of PCR
product of -skeletal actin mRNA/ß2-microglobulin mRNA obtained
for newborn sample also was approximately 4-fold greater than that of
normal adult (Figure 5C).
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Acute hypertension induced by aortic ligature between renal arteries
resulted in cardiac hypertrophy (ventricular
wet weight/body weight 4.48±0.61 mg/g compared with 2.47±0.09 in
control rats P<0.001 and see also Reference 3434 ).
Immunohistochemical staining with anti–-SKA1 revealed that the
number of positive myocardial fibers was significantly increased 3 and
7 days after aorta ligature (Figure 5A, panel c) compared with
the number of positive fibers present in normal heart. The area of
positive staining increased from 4.2±0.5% in the normal heart to
31.5±2.4% in the hypertensive heart 7 days after aortic ligature
(P<0.001). Reactivity to anti–-SKA1 also occurred in
left atrium, where it was undetectable in the normal situation (data
not shown). At the same time, reactivity remained negative in the right
atrium of hypertensive animals (data not shown). The increase of
protein expression was also confirmed by means of
immunoblotting (Figure 5B, 4-fold higher
compared with normal adult) and at the mRNA level (Figure 5C, 2-fold increase of the signal). The expression of ß-myosin heavy
chain was increased during hypertension; however, in contrast to what
was observed for anti–-SKA1, the staining pattern for ß-myosin
became diffuse throughout the ventricular
myocardium confirming the findings of Gorza et
al34 (data not shown).
Spots of fibrosis were visible 7 days after the beginning of the
experiment on examination of Masson’s trichrome–stained sections
(Figure 6a). These fibrotic areas were
characterized by the expression of -smooth muscle actin, the actin
isoform typical of myofibroblastic cells (Figure 6b), which are
known to be abundant and persistent in granulation tissue developing
after cardiac infarction.36 37 Interestingly, fibers
surrounding the fibrotic areas in the hypertensive animals
consistently demonstrated an important staining for
-skeletal actin (Figure 6c).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous attempts to develop antibodies specific for
-skeletal
or -cardiac actin isoforms have not so far allowed to discriminate
between these isoforms,38 39 40 41 with the exception of the
anti–-cardiac actin antibody raised by Franke et al,16
the specificity of which is dependent of the experimental conditions.
This is not unexpected because the divergences among the 2 sequences
are minimal. The experimental strategy we used here shows that it is
possible to produce in the rabbit and immunopurify an antibody that
reacts specifically with -skeletal actin. This suggests the
possibility of obtaining monospecific antibodies for other actin
isoforms insofar as they are not presently available because the
sequences of -cardiac and -skeletal actins are the most difficult
to discriminate. The fractions obtained after the first step of
immunopurification (on beads coupled with the -skeletal actin
decapeptide used for immunization) resulted in several antibody
populations, indicating that the decapeptide contains several epitopes.
The further and successful purification we used was based on the
removal of antibody populations recognizing the sequence ETTALVC
(positions 4 to 10) common to -skeletal, -cardiac, and -smooth
muscle actins, and the resulting antibody showed a high specificity for
-skeletal actin. These results are compatible with the assumption
that the Ac-DED sequence represents a good candidate for the
-skeletal actin–specific epitopic activity, as it corresponds to
the only difference between the NH2-terminal end
of -skeletal actin and that of -smooth muscle actin (Ac-EEE) (see
Figure 2B). However, inhibition experiments with synthetic
peptides show that the first 9 amino acids are essential for antibody
recognition. Possibly, a particular 3D conformation is involved in this
process. The blocking acetyl group appears to be an integral part of
the epitope because the nonacetylated decapeptide does not
block anti–-SKA1 reactivity. Incubation of the Fab fragment of
anti–-SKA1 does not influence -skeletal actin polymerization,
differently to what we have previously reported for the corresponding
fragment of -smooth muscle actin antibody,17 indicating
that for polymerization, the epitopic region of this antibody is not as
important as that of -smooth muscle actin reacting with the specific
antibody.
To date, it is not clear whether the 2 striated muscle actins have
different physiological properties. Studies with
probes for mRNAs, the only tools available so far to distinguish
between -skeletal and -cardiac actin isoforms, have shown that
both sarcomeric actin transcripts are expressed in skeletal and cardiac
muscles and that their expression is tightly regulated during
development.5 These results provide a strong argument in
favor of functional differences between these isoforms. Further
investigations on their cellular distribution should lead to a greater
insight into their respective biological functions during muscle
development or pathology. As a first approach to such a study, in situ
hybridization procedures performed by Schiaffino et al11
did not detect -skeletal actin mRNA labeling in normal adult rat
heart; this labeling, however, was important and was uniformly
distributed throughout the ventricle soon after aortic ligature. Our
investigations at the protein level confirm the increased expression of
this isoform during cardiac hypertrophy. However, we show
that -skeletal actin protein is focally present in normal adult
myocardial fibers. The reasons for this apparent discrepancy between in
situ hybridization and immunolabeling are presently not clear.
Our results indicate that there may be a relationship between the
expression of -skeletal actin and ß-myosin. Thus a high percentage
of myocytes expresses both proteins, although this is not uniformly the
case. Furthermore, during the development of hypertrophy,
the distribution of -skeletal actin remains focal, whereas it is
known that the distribution of ß-myosin is more
diffuse.34 Further work along this line may help in
understanding the physiological role of both
proteins.
Several studies have demonstrated that there is increased expression of
-skeletal actin during the evolution of cardiac
hypertrophy.3 4 42 One of the explanations
that has been offered is that elements of the genetic programming
necessary during development may be reactivated, which could
result in the re-expression of fetal genes. Our investigations of
postnatal development show that the distribution of -skeletal actin
is uniform within the myocardium of newborn rats and
becomes focal as development proceeds. During hypertension-induced
hypertrophy, however, the pattern of -skeletal actin
reactivity does not become diffuse but rather remains focal. This
suggests that the mechanisms of -skeletal actin gene regulation
during development are different from those occurring during the
evolution of hypertrophy. It has also been proposed that
the activation of the -skeletal actin gene during the development of
hypertrophy reflects a temporary requirement for large
quantities of striated muscle actin when muscle volume is increasing
rapidly.1 The particular pattern of -skeletal actin
distribution suggests that the expression of this actin isoform is
associated with specialized functions of certain
cardiomyocytes. Taken together with our observation that
-skeletal actin–positive cells are more abundant in the left
compared with the right ventricle and that their number increases
during hypertrophy, our results are in accordance with the
hypothesis that this actin isoform may be required to achieve a higher
degree of myocardial contractility as indicated by the
findings of Hewett et al,43 showing that increased levels
of -skeletal actin mRNA are significantly correlated with increased
heart contractility in BALB/c mice. The observation
that fibers close to fibrotic areas express high amounts of
-skeletal actin during hypertrophy are compatible with
the possibility that these particular fibers are submitted to stronger
local physical forces and/or to particular cytokines or growth
factors liberated in the fibrotic areas. In this respect it is
noteworthy that locally produced transforming growth factor ß1
(TGF-ß1) plays an important role in the establishment of fibrotic
changes (for review see Reference 4444 ); TGF-ß1 has also been shown to
induce a selective upregulation of the -skeletal actin
promoter.45 46 47
In conclusion, our success in producing a specific -skeletal actin
antibody suggests that the strategy used in the present work may be
attempted for other actin isoforms. Our results indicate that during
development, there is a modulation of -skeletal actin expression
such that uniform distribution in the newborn gives way to a focal
pattern in the adult. The pattern of expression during development of
hypertrophy also is focal; moreover, the reactivity to
anti– -SKA1 is preferentially associated with regions of
fibrosis. Further studies with this antibody will be useful in the
understanding of the relationship between -skeletal actin expression
and striated muscle function.
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Acknowledgments |
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This work was supported by the Swiss National Science Foundation, grants No. 31-50568.97 and 31-54048.98. We wish to thank Dr A.F. Moorman and Dr P. Meda for providing the anti–ß-myosin heavy chain antibody and the anti–connexin 43 antibody, respectively. We are grateful to Dr R.B. Low for his valuable advice throughout the project and for critical reading of the manuscript. We gratefully acknowledge Dr G. Benzonana for preparation of anti–
-SKA1 Fab
fragment; F. Gabbiani for performing aortic ligatures; P. Henchoz, A.
Hiltbrunner-Maurer, M. Redard, and P. Ropraz for technical help; and
J.-C. Rumbeli for photographic work. Received September 23, 1999; accepted September 29, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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