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Vol. 14, Issue 8, 3180-3191, August 2003
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* Department of Biology, Faculty of Science, Chiba University, Yayoi-cho,
Inage-ku, Chiba 263-8522, Japan;
Graduate School of Science and Technology, Chiba University, Yayoi-cho,
Inage-ku, Chiba 263-8522, Japan; and
Department of Physiology and Functional Genomics, College of Medicine,
University of Florida, Gainesville, Florida 32610-0274
Submitted October 25, 2002;
Revised March 3, 2003;
Accepted April 9, 2003
Monitoring Editor: Mary Beckerle
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Cardiac myosin-binding protein-C (MyBP-C), also known as C-protein, is one
of the major myosin-binding proteins in vertebrate striated muscles with an
approximate molecular mass of 140 kDa
(Offer et al., 1973
;
Pepe and Drucker, 1975).
MyBP-C is localized to the central cross-bridge zone in each half of the
A-band region of myofibrils (Pepe and
Drucker, 1975; Craig and Offer,
1976). MyBP-C modulates myosin assembly
(Offer et al., 1973),
actin–myosin interaction in sarcomeres
(Moos et al., 1978;
Hartzell, 1985), and
stabilizes thick filaments (Moos et
al., 1978). In addition, MyBP-C interacts with
connectin/titin, which is important for the precise arrangement of
actin-myosin filaments in sarcomeres
(Maruyama, 1986;
Fürst et al.,
1992; Koretz et al.,
1993).
MyBP-C, a member of the intracellular immunoglobulin (Ig) superfamily, has
three isoforms, two skeletal (fast- and slow-) and one cardiac type encoded by
three distinct genes. The two skeletal types of MyBP-C are composed of a total
of 10 domains: seven IgI domains and three fibronectin (FN) type III repeats
(Einheber and Fischman, 1990;
Fürst et al.,
1992; Okagaki et al.,
1993; Weber et al.,
1993; Harpaz and Chothia,
1994; Kurasawa et
al., 1999), whereas, cardiac MyBP-C has 11 domains
(C0–C10): eight IgI domains (instead of seven in the skeletal types) and
three FN type III repeats (Figure
1A). In addition, cardiac MyBP-C has a unique phosphorylation
domain between the C1 and C2 domain
(Kasahara et al.,
1994; Gautel et al.,
1995; Yasuda et al.,
1995; Yang et al.,
1998). Through these domains, MyBP-C interacts with other
sarcomeric proteins; C1–C2 domains interact with the S2 segment of
myosin, C10 domain with light meromyosin (LMM), and C8–C10 with
connectin/titin.
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All isoforms of MyBP-C are specific for different muscle fiber types and
different developmental stages (Obinata
et al., 1984;
Obinata, 1985;
Bähler et al.,
1985; Kawashima et
al., 1986; Fougerousse
et al., 1998; Gautel
et al., 1998;
Kurasawa et al.,
1999). In humans and mice, expression of the cardiac-type isoform
is restricted to the heart through development, and no splicing variants of
the cardiac isoform have been reported
(Fougerousse et al.,
1998; Gautel et al.,
1998; Kurasawa et
al., 1999). On the other hand, the chicken cardiac isoform
containing the specific phosphorylation domain is abundantly expressed in
cardiac muscle through development; however, an alternative spliced variant
lacking the phosphorylation domain is expressed dominantly but transiently in
embryonic skeletal muscle (Bähler
et al., 1985; Yasuda
et al., 1995; Mohamed
et al., 1998).
Mutations in the gene MYBPC3, encoding human cardiac-type MyBP-C,
have been identified to cause modest and late-onset familial hypertrophic
cardiomyopathy (Bonne et al.,
1995; Watkins et al.,
1995; Carrier et al.,
1997; Rottbauer et
al., 1997; Yu et
al., 1998). Familial hypertrophic cardiomyopathy is an
autosomal dominant disease with typical morphological changes, including
cardiomyocyte hypertrophy and/or myofibrillar disarray with fibrosis (Maron
et al.,
1987a,b;
Seidman and Seidman, 2001).
Most of the mutations found in the MYBPC3 are predicted to lead to
the altered mRNA sequence and to produce the carboxyl-terminal truncation of
the cardiac MyBP-C polypeptides lacking the C10 myosin binding site, or in
some cases, lacking the C8–C10 connectin/titin binding site
(Bonne et al., 1995;
Watkins et al., 1995;
Carrier et al., 1997;
Kimura et al., 1997;
Rottbauer et al.,
1997; Yu et al.,
1998). These findings demonstrate that the expression of mutant
sarcomeric proteins, such as the truncated cardiac MyBP-C, induce cardiac
dysfunction and alteration of myofibrils in cardiomyocytes. However, the
precise mechanism for this negative effect has not yet been determined.
Herein, we identified a novel isoform of mouse cardiac MyBP-C mRNA, namely, MyBP-C(+), that has an additional 30 nucleotides by alternative splicing and encodes exactly the same amino acid sequence as previously reported, but contains an additional 10 amino acids in the connectin/titin binding region. MyBP-C(+) decreased binding to myosin filaments and connectin/titin in vitro. Green fluorescent protein (GFP)-tagged MyBP-C(+) did not correctly localize to sarcomeres, and it disrupted the A-band formation in chicken cardiomyocytes. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that mRNA encoding MyBP-C(+) was dominantly expressed in the aged mouse atrium.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). The newly isolated clone and previously isolated clone were
sequenced with a SequiTherm EXCEL2 Long-Read DNA sequencing kit-LC (Epicentr,
Madison, WI) or a Thermo Sequenase Cy5.5 dye terminator cycle sequencing kit
(Amersham Biosciences UK, Little Chalfont, Buckinghamshire, United Kingdom).
Analysis of DNA sequence data was carried out with the GENETYX program (SDC
Software, Tokyo, Japan). Consequently, we found some errors on the nucleotide
and amino acid sequences encoding MyBP-C(–) reported previously and then
corrected them here; T441 was revised to C (no change in the
encoded amino acid sequence), a G was inserted at 2638, and a C was deleted at
2700; therefore, the amino acid sequence QSMPWECPGPALPLSPSCLL (844–863)
was replaced with AVNAVGMSRPSPASQPFMPI.
PCR and RT-PCR Samples and Primers
In this study, we used two cDNA libraries: one was prepared from total RNA
isolated from 8-wk-old ICR mouse hearts by using reverse-transcription with
oligo-dT primers (Chomczynski and Sacchi,
1987) and the other was prepared from 8-wk-old C57BL/6J
(SuperScript mouse heart cDNA library; Invitrogen, Carlsbad, CA).
Messenger RNAs were extracted using a QuickPrep MicromRNA purification kit (Amersham Biosciences UK) from ventricles and atria of newborn, 4-wk-, 8-wk-, and >6-mo-old ICR mice. These mRNAs were reverse-transcripted with oligo-dT primers. The following primers were used for PCR reactions: primer F, 5'-TCAATGTGGCTCTGGAGTGG-3' (3335–3354); primer R, 5'-GAAGACCCGGAAGTAGTAGC-3' (3527–3546); and primer I, 5'-AGGATGGTGAAGGAAGGGAG-3' (3433–3452).
Genomic tail DNA was prepared from ICR mice according to the method
described by Blin and Stafford
(1976). By a PCR reaction with
a primer F + R set, the genomic DNA fragment was isolated, sequenced, and a
comparison with human cardiac MyBP-C genomic DNA sequence (accession no.
U91629
[GenBank]
, Entrez) was made to define the exon-intron structure.
Generation of Recombinant MyBP-Cs by Using a Baculovirus Expression
System and the Binding Assay
Both of the XhoI-EcoRI fragments encoding full-length
MyBP-C(–) and MyBP-C(+) were subcloned into pBlueBacHis2A (Invitrogen).
Preparation of recombinant viral DNA was performed using a Bac-N-Blue
transfection kit (Invitrogen) according to the technical bulletin. Consequent
recombinant MyBP-Cs with histidine tags were purified from lysates of sf9
cells by using a HiTrap affinity column (Amersham Biosciences UK), which was
saturated with nickel ions, and then dialyzed against binding buffer (0.1 M
KCl, 20 mM imidazole-HCl, pH 7.0). The supernatant separated by centrifugation
at 50,000 rpm for 30 min (TLA-100; Beckman Coulter, Fullerton, CA) was used
for the binding reactions.
Crude myosin was prepared from rabbit ventricles as described previously
(Perry, 1955), and the myosin
was purified using DEAE-Sephadex A-50
(Richards et al.,
1967). Reconstitution of myosin filaments was performed during a
dialysis against the binding buffer. 
-Connectin prepared from chicken
pectoralis muscle (Kimura and Maruyama,
1983), which was kindly provided by Dr. S. Kimura (Chiba
University, Chiba, Japan), was dialyzed against the binding buffer.
Quantitations of the MyBP-Cs, reconstituted myosin filaments, and
-connectin were performed by a bicinchoninic acid protein assay reagent
kit (Pierce Chemical, Rockfold, IL).
For binding assays, recombinant MyBP-C(–) or MyBP-C(+) at varying
concentrations was incubated with reconstituted myosin filaments (0.5 µM)
or -connectin (0.04 µM) for 30 min at 4°C, and then centrifuged
at 50,000 rpm for 30 min. Supernatants and pellets were subjected to SDS-PAGE,
stained with Coomassie Blue, scanned with a densitograph (Atto, Tokyo, Japan),
and analyzed using NIH Image.
Vector Constructs
Using a Transformer site-directed mutagenesis kit (BD Biosciences
Clontech), each A106 in the cDNA encoding mouse cardiac
MyBP-C(–) and MyBP-C(+) was substituted to G. The
XhoI-EcoRI fragment encoding the full length of
MyBP-C(–) open reading frame and that of MyBP-C(+) open reading frame
were subcloned into pEGFP-C3 (BD Biosciences Clontech), and the vector was
named pGMC(–) or pGMC(+), respectively. To construct pGMC
7-10,
the pGMC(–) was partially digested with ApaI and then ligated.
As for the pGMC9-10, the pGMC(–) was digested at the
EcoRV site and the BamHI site and then blunted and ligated
by a Blunting Kit (Takara Bio., Kyoto, Japan). To construct
pGMC10(–) and pGMC10(+), the pGMC(–), and the
pGMC(+) were digested, respectively, with HindIII and BamHI,
and then blunted and ligated by the blunting kit.
Cell Culture and Transfection
Cardiac myocytes were prepared from chicken embryos at embryonic day
9–10, according to Lin et al.
(1989). Chick ventricles were
incubated in 4 ml of trypsin solution (0.125% trypsin in phosphate-buffered
saline (PBS) containing 1 mM EGTA) for 5 min at room temperature. The
supernatant containing dispersed cells was placed into 25 ml of growth medium
(10% fetal calf serum, 10 U/ml penicillin, 10 µg/ml streptomycin in minimal
essential medium with L-glutamine and Earle's salts; Invitrogen).
The remaining tissue fragments were incubated in 4 ml of the fresh trypsin
solution as described above, and the dispersed cells were harvested for an
additional two times. The cell suspension was filtered to remove large clumps
of cells and then centrifuged for 5 min. Cells were plated for 1 h at 37°C
in a humidified 5% CO2 incubator
(Hyde et al., 1969).
The supernatant containing floating myocytes was collected and centrifuged for
5 min. The cells were resuspended in growth medium, grown on glass coverslips
at an initial density of 1–2 x 105 cells/35-mm dish.
The following day, cells were transfected with the expression vector using
Effectene transfection reagent (QIAGEN, Tokyo, Japan).
Indirect Immunofluorescence Microscopy and Immunoblotting
Three days after transfection, cells were rinsed with PBS and fixed with
10% formalin in PBS for 15 min. The cells were then permeabilized with 0.2%
Triton X-100 in PBS for 10 min. Atrial and ventricular tissues isolated from
ICR mice >20 mo of age were prefixed with 4% paraformaldehyde in PBS for 30
min, followed by immersion in liquid nitrogen-cooled isopentane. The
longitudinal frozen sections (3 µm) were fixed with 4% paraformaldehyde in
PBS for 10 min. After rinsing repeatedly with PBS, the cells and the sections
were blocked with 1% bovine serum albumin in PBS and incubated with primary
antibodies for 1 h. After repeated washing with 1% bovine serum albumin in
PBS, they were incubated with rhodamine-conjugated goat anti-rabbit IgG or
anti-mouse IgG as secondary antibodies for 1 h. After a final washing with
PBS, the specimens were mounted in 50% glycerol in PBS. All steps were carried
out at room temperature. Samples were examined with an Axioskop (Carl Zeiss,
Jena, Germany) with a cooled charge-coupled device camera, CoolSNAP (Nippon
ROPER, Chiba, Japan,) or with an Axiovert 135 TV (Carl Zeiss) with a cooled
charge-coupled device camera, PXL37 (Photometrics, Tucson, AZ).
For immunoblotting, proteins were transferred to nitrocellulose membranes
(Towbin et al.,
1979). Membranes were treated with 5% skimmed milk in
tris-buffered saline for 1 h and then incubated with primary antibody followed
by treatment with alkaline phosphatase-conjugated secondary antibody. The
membrane was washed with Tris-buffered saline and stained with nitroblue
tetrazolium chloride and 5-bromo-4-chloro-3-indolylphosphate
p-toluidine salt in + phosphatase buffer (0.1 M NaCl, 2 mM
MgCl2, 0.1 M Tris-HCl, pH 9.5).
Primary Antibodies
The following primary antibodies were used: C315, a monoclonal antibody
specific for chicken cardiac MyBP-C
(Kawashima et al.,
1986); 
ccp, a polyclonal antibody that recognizes both
chicken and mouse cardiac MyBP-C and reacts with both MyBP-C(–) and
MyBP-C(+) (Yasuda et al.,
1995); A4.1045, a monoclonal antibody against the sarcomeric
myosin heavy chain developed by Helen M. Blau (obtained from the Developmental
Studies Hybridoma Bank developed under the auspices of the National Institute
of Child Health and Human Development and maintained by The University of
Iowa, Department of Biological Sciences, Iowa City, IA); and Pc72C, a
polyclonal antibody against the carboxyl-terminal (M-line) region of
connectin/titin was kindly provided from Dr. S. Kimura (Chiba University)
(Soeno et al.,
1999).
Statistical Analysis
To study the influence of the expression of MyBP-C variants on the
endogenous cross-striated sarcomeres in cardiomyocytes, cells were transfected
with plasmids and 3 d later, processed for immunostaining as described above.
The percentage of cells expressing the MyBP-Cs and exhibiting alteration of
sarcomeres as observed after staining with A4.1045 was determined for each
cover glass. The data obtained from three independent transfections were
analyzed. For each construct, three coverglasses were examined and between 240
and 394 transfected cells were scored. The data were analyzed by one-way
analysis of variance followed by the multiple comparison analysis with the
test of Ryan (Ryan, 1959,
1960).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). The novel cDNA of 
4.2 kb, encodes
identical eight IgI and three FN type III repeats, except for an additional 30
bases insertion located at the 5' end of the exon 31, which was
previously reported as an intron sequence between exon 30 and exon 31
(Figure 1B, exon 31A, sequence
in the box). The insertion does not result in a frame-shift mutation, nor does
it contain a termination codon. Therefore, this cDNA encodes the entire amino
acids of the mouse cardiac-type MyBP-C with an extra10 amino acids in the C9
domain (Figure 1A, insertion),
which is important for connectin/titin binding
(Freiburg and Gautel, 1996)
and incorporation into sarcomeres (Gilbert
et al., 1996). We term the previously reported cardiac
MyBP-C as insert-minus or MyBP-C(–) and the novel variant MyBP-C
containing the extra amino acids as insert-plus or MyBP-C(+). To confirm that the MyBP-C(+) cDNA was not an artifact of the cDNA library used, two additional cDNA libraries prepared from whole hearts of 8-wk-old C57BL/6J mice or ICR mice were screened by PCR. As shown in Figure 1B, a combination of primer F and R was designed to recognize both the insert-plus (212-base pair products) and the insert–minus cDNA (182-base pair products) and a combination of primer F and I was designed only for amplifying the insert-plus cDNA (118 base pairs). As shown in Figure 1C (lane 1), the PCR reaction with an F + R primer set amplified a 182-base pair PCR product representing MyBP-C(–) with an additional few bands with higher molecular weight, including a 212-base pair product in the C57BL/6J cDNA library. The nucleotide sequence confirmed that the 212-base pair PCR product contains exon 31A. When we used an F + I primer set, a 118-base pair PCR product from MyBP-C(+) was clearly amplified in the same cDNA library (Figure 1C, lane 2). Although the 212-base pair MyBP(+) products were hardly detected in the ICR cDNA library (Figure 1C, lane 3), the 118-base pair products were clearly detected (Figure 1C, lane 4). These results demonstrate the expression of cardiac-type MyBP-C(+) in all the cDNA libraries examined and its expression is significantly lower than that of MyBP-C(–).
Genomic Southern blotting demonstrated that a single gene encodes mouse
cardiac MyBP-C (Harris et al.,
2002). Therefore, MyBP-C(+) and MyBP-C(–) are expected be
generated from a single gene, with an identical sequence except for the
30-base pair nucleotides insertion in MyBP-C(+), which is coded in the genomic
region previously reported as an intron. In addition, two consensus splice
acceptor sequences CAG (Figure
1B, *) were located next to the newly identified exon 31A and the
known exon 31B with a consensus 5' splice donor sequence gt
(Figure 1B, *) located at the
5' end of intron next to the coding sequence (exon 30), which is
identical to the sequence of MyBP-C(–) as we described previously
(Kasahara et al.,
1994). Therefore, we concluded that both MyBP-C(+) and
MyBP-C(–) mRNAs were encoded by a single gene and generated by an
alternative splicing (Figure
1D).
MyBP-C(+) Reduced the Binding Affinity to Myosin Filaments and
Connectin/Titin
The 10 amino acid insert encoded by exon 31A is located at the C9 domain, a
domain that interacts with connectin/titin
(Figure 1A). Therefore, we
examined whether this insert affects its binding to connectin/titin.
Full-length MyBP-C(–) or MyBP-C(+) protein were produced in sf9 cells by
using a baculovirus expression system and were used for a cosedimentation
assay with reconstituted myosin filaments as well as -connectin.
MyBP-C(–) or MyBP-C(+) was mixed with reconstituted myosin filaments prepared from rabbit cardiac muscles for 30 min at 4°C. Representative results obtained using 0.5 µM cardiac myosin and 0.15 µM recombinant MyBP-C(–) or 0.17 µM MyBP-C(+) are shown in Figure 2A. Before mixing, the majority of myosin fractionated to pellets (P) (lane 2), whereas MyBP-C fractionated to supernatants (S) (lanes 3 and 7). When MyBP-C was mixed with reconstituted myosin, MyBP-C bound to myosin fractionated in pellets (lanes 6 and 10) and unbound MyBP-C stayed in supernatants (lanes 5 and 9). Densitometric analysis showed that 40% of MyBP-C(–) or 20% of MyBP-C(+) was associated with the myosin filaments under this experimental condition.
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To analyze the binding of MyBP-C proteins more precisely, various
concentrations of MyBP-C proteins were mixed with a constant concentration of
myosin (0.5 µM). Baculovirus derived MyBP-C(–) was found to bind to
myosin with a molar ratio of 0.5:1 at the saturated concentration which
is similar to that of native MyBP-C (0.6:1) as reported previously
(Alyonycheva et al.,
1997) (Figure 2B,
left). At a concentration between 0 and 0.4 µM, which is below the
saturating concentration, MyBP-C(–) binds myosin with higher affinity
than that of MyBP-C(+) (Figure
2B, right).
Similar results were obtained using -connectin purified from chicken
pectoralis muscle. Using 0.04 µM -connectin mixed with 0.1 µM
MyBP-C(–) or 0.13 µM MyBP-C(+) demonstrated that MyBP-C(+)
(Figure 2C, lane 10) had a
weaker interaction with -connectin than that of MyBP-C(–)
(Figure 2C, lane 6). These
results demonstrate that an insertion of 10 amino acids into the
connectin/titin binding domain of MyBP-C resulted in decreased affinity to
connectin/titin as well as myosin.
MyBP-C(+) Does Not Effectively Assemble into Sarcomeres
Multiple reports described that carboxyl-terminus deletion mutants of
cardiac as well as skeletal MyBP-C, either deleting the myosin binding domain
(C10) or the connectin/titin binding domain (C8–C10), resulted in
diffusely localized MyBP-C protein impairing the endogenous sarcomeric
structure in transfected cells (Gilbert
et al., 1996; McConnell et al.,
1999,
2001; Yang et al.,
1998,
1999;
Flavigny et al.,
1999). Therefore, we asked whether this 10 amino acid insertion in
C9 domain modifies its assembly into sarcomeres. GFP-tagged expression vectors
encoding MyBP-C(+) or MyBP-C(–), as well as a series of
carboxyl-terminus deletion mutants were generated
(Figure 3A): GMC(+) represents
GFP-tagged full-length MyBP-C(+), GMC(–), GFP-tagged full-length
MyBP-C(–), GMC7–10, GMC(–) with deletion of C7 to
C10, GMC9-10, a deletion mutant without C9 and C10, GMC10(+),
GMC(+) with a deletion of C10 and GMC10(–), GMC(–) with a
deletion of C10.
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These vectors were transfected into chicken cardiomyocytes and protein
expression was confirmed by Western blotting by using anti-MyBP-C antibody
(ccp). GFP-tagged full-length MyBP-C(–) [GMC(–)] or
MyBP-C(+) [GMC(+)] proteins migrated at a slightly higher molecular weight
(arrowheads) than that of endogenous chicken cardiac MyBP-C (arrow). A series
of carboxyl-terminus deletion mutants were also detected with anti-MyBP-C
antibody (ccp) (Figure
3B, arrowheads) and anti-GFP antibody (our unpublished data).
After confirming the molecular size of the GFP-fused various MyBP-C protein constructs, we examined the intracellular localization of these proteins. First, we examined the localization of nonfused GFP proteins. The majority of GFP proteins were diffusely localized in the cytoplasm (Figure 4A). Some nonfused GFP proteins were broadly incorporated into the sarcomere structure and showed overlapping staining with myosin (Figure 4B, red) that was observed in 51% of the total of 240 transfected cells (Figure 4, A and B and Table 1). However, GFP signals were not completely colocalized with myosin (red color), and the strong green GFP signals were easily detected at the gap between the A-bands (M lines) (enlarged image in Figure 4B). When GFP proteins were detected in myofibrils, 93% of cells showed organized sarcomere structure (Figure 4, A and B, and Table 1). This indicates that GFP proteins nonspecifically associate with myofibrils, but these proteins do not have deleterious effects on sarcomere organization.
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In contrast, in 73% of the cells expressing GFP-MyBP-C(–) protein, GFP signals were colocalized with sarcomeric myosin at A-bands; therefore, the gap between the A-bands (M-lines) were clearly observed as fluorescent negative areas (Figure 4, C and D versus A and B). Coimmunostaining with anti-connectin/titin antibody (red), a marker for the M-line, showed a narrow red line (M-line), indicating MyBP-C(–) is localized exclusive from the M-line. Of note, fluorescent signals from GFP and connectin/titin overlapped besides the M-line (yellow). However, we assume this is an artifact from the strong fluorescent signals. Indeed, anti-MyBP-C antibody recognized endogenous MyBP-C proteins exclusive from the M-line, colocalizing with GMC(–) proteins (Figure 4, G and H).
Interestingly, in the 73% of cells expressing GFP-MyBP-C(+) proteins, they diffusely localized in the cytoplasm without forming striated sarcomere structures (Figure 5, A and C, and Table 1). However, GFP-MyBP-C(+) proteins were detected as filamentous structure and were not concentrated at the M-line (Figure 4, A and B versus Figure 5, E and J). In rest of the cells (27%), GFP-MyBP-C(+) proteins colocalized with myofibrils (Figure 5, E and H).
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When GFP-MyBP-C(+) proteins were diffusely localized, 57% of the cells showed well-organized sarcomeric myosin staining (Figure 5B) and 43% of cells did not (Figure 5D). When GFP-MyBP-C(+) were detected in the myofibrils, only 21% of cells showed organized sarcomere structure and 79% of these cells displayed somewhat disorganized sarcomere structure detected with anti-myosin antibody (Figure 5, E–G) and anti-connectin/titin antibody (Figure 5, H–J). In addition, we sometimes detected condensed GFP signals in the cytoplasm (our unpublished data). These data are summarized in Table 1.
These results indicate that GFP-MyBP-C(–) was effectively incorporated into sarcomere structures and colocalized with the endogenous MyBP-C protein; however, GFP-MyBP-C(+) was not effectively targeted to A-bands. When GFP-MyBP-C(+) proteins were incorporated myofibrils along with myosin filaments or connectin/titin (Figure 5, E–J, arrow), sarcomere structures were significantly disturbed (p = 0.0004, multiple analyses using the test of Ryan; Table 1). This suggests that MyBP-C(+) has dominant negative effects on sarcomere organization.
Alteration of Sarcomere Structure by MyBP-C(+) or Carboxyl-Terminus
Deletion Mutants
To further examine the effects of MyBP-C(+) on sarcomere structure, we
compared it to several carboxyl-terminus deletion mutants that have been shown
to disorganize sarcomere structure (Figure
3A) (Gilbert et al.,
1996; Flavigny et
al., 1999). Consistent with previous reports, 60–74% of
transfected cells showed diffusely localized carboxyl-terminus deletion
mutants (Table 1). Altered
sarcomere structure was also detected in 53% of the cells expressing
GMC7-10 mutants, 57% of GMC9-10, 65% of GMC10(+), and 77%
of GMC10(–) (Figure
6, *p < 0.001). Accordingly GMC10(–) showed the
most severe effects on sarcomere disorganization, which was significantly
higher than GMC9–10, GMC7–10, and GFP-MyBP-C(+)
(Figure 6, 
p <
0.01).
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MyBP-C(+) mRNA Is Dominantly Expressed in the Atria of Aged Mice
According to PCR amplification from an 8-wk-old whole heart cDNA library,
expression levels of mRNA of MyBP-C(+) is lower compared with MyBP-C(–)
(Figure 1C). To further examine
MyBP-C(+) expression levels during different stages of heart development, we
performed RT-PCR with RNA isolated from ICR mouse atria or ventricles at the
newborn stage, 4 wk, 8 wk, and >6 mo of age
(Figure 7A). In atria, the
level of the expression of MyBP-C(–) mRNA detected as a 182-base pair
product was dominant at the newborn stage and 4 wk of age, and then it
gradually decreased at 8 wk and >6 mo of age. In contrast, the expression
of the 212-base pair products representing MyBP-C(+) gradually increased and
was dominant >6 mo of age. In ventricles, 182-base pair product of
MyBP-C(–) mRNA was dominantly expressed at all stages examined and
212-base pair product of MyBP-C(+) was detected only at 8 wk of age
(Figure 7A, top). This was
confirmed by another primer set amplifying MyBP-C(+) cDNA as a 118-base pair
product (Figure 7A, bottom).
Although, the proportion of the expression of MyBP-C(–) versus MyBP-C(+)
is different among individual animals examined, MyBP-C(–) is dominantly
expressed in ventricles in all postnatal developmental stages, whereas
MyBP-C(+) is dominantly expressed in the atria of older mice.
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Expression of Cardiac MyBP-C Protein in the Aged Mouse Heart
To further examine the expression of MyBP-C(+) protein in the aged mouse
atria, we tried to raise a specific antibody for MyBP-C(+) recognizing the
unique 10 amino acid insertion but we were unsuccessful after several
immunizations. Western blotting using an anti-MyBP-C antibody (ccp),
which recognizes both of MyBP-C(–) and MyBP-C(+) shows that the total
amount of ventricular MyBP-C expression is increased at 12 wk of age and then
decreased at >20 mo of age. In contrast, the expression of the total amount
of atrial MyBP-C, MyBP-C(–) plus MyBP-C(+), was constant at all three
stages examined (Figure
7B).
In aged mouse hearts (> 20 mo of age), ventricular MyBP-C exhibited striated sarcomeric pattern colocalizing with myosin (Figure 8, A and B); however, atrial MyBP-C was diffusely localized in the cytoplasm despite the striated sarcomere formation stained with anti-myosin antibody (Figure 8, C and D). These results indicate that the localization of MyBP-C is specifically stage and tissue (aria versus ventricles) regulated. According to the dominant expression of MyBP-C(+) in aged mouse demonstrated by RT-PCR, diffusely localized MyBP-C proteins in aged atria are likely the MyBP-C(+) form.
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|
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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;
reviewed in Breitbart et al.,
1987; Magnuson et
al., 1991), including human and mouse fibronectin
(Tamkun et al., 1984;
Kornblihtt et al.,
1985; reviewed in
Schwarzbauer, 1991).
Biochemical analyses demonstrated several unique characteristics of
MyBP-C(+) compared with MyBP-C(–). In a cosedimentation assay,
baculovirus-derived MyBP-C(+) decreased binding affinity to connectin/titin
and myosin filaments, whereas MyBP-C(–) showed a very similar binding
affinity as native MyBP-C(–) described previously
(Alyonycheva et al.,
1997). These results indicate the baculovirus derived
MyBP-C(–) and the native MyBP-C(–) have very similar protein
structures, but the 10 amino acid insertion at C9 domain changes its
structure.
Six of 10 amino acid residues are hydrophobic, and these hydrophobic
residues likely disrupt the -helical stretch of MyBP-C(–) and
replace it with beta-sheets at the C9 domain according to the Chou-Fasman
protein secondary analyses of a GENETYX program. Because C8, C9, and C10
domains of MyBP-C are known to directly bind to a subset of Ig domains of
connectin/titin (Freiburg and Gautel,
1996), conformational changes due to the 10 amino acid insertion
will decrease the association between MyBP-C and connectin/titin. In addition,
our experiments showed that the insertion into the C9 domain significantly
decreased the binding to myosin filaments. The major myosin-binding domain of
cardiac and skeletal MyBP-C was mapped at the carboxyl-terminal C10 domain
(Okagaki et al.,
1993; Alyonycheva et
al., 1997), but a recent report from Welikson and Fischman
demonstrated that C7–C9 domains of the chicken skeletal MyBP-C also have
an ability to bind with myosin (Welikson
and Fischman, 2002). Therefore, we assume that the inserted 10
amino acids in the C9 domain modify the structure of MyBP-C, resulting in
alteration of conformation or direction of the C10 domain, which is important
for binding with myosin, or the inserted 10 amino acids directly reduced
binding to myosin filaments.
Supporting these in vitro binding assays, transfection experiments showed
that GFP-MyBP-C(+) was not effectively incorporated into sarcomere structures
compared with GFP-MyBP-C(–). Because MyBP-C(+) could still bind to
myosin filaments and connectin/titin with lower affinity, it sometimes
associated with myofibrils, but it could not fully compete with and replace
the endogenous chick MyBP-C protein, which does not include the 10 amino acid
insertion (Yasuda et al.,
1995). We could not fully explain why MyBP-C(+) is sometimes
incorporated into myofibrils and sometimes diffusely distributed in the
cytoplasm. It may be due to the different accumulation level of the MyBP-C(+)
in different cells or the timing of association of MyBP-C(+) with sarcomeric
components during sarcomere assembly.
The carboxyl-terminus end of MyBP-C is known to be important for sarcomere
assembly, and expression of the carboxyl-terminus deletion mutants impaired
the endogenous sarcomeric structure in the transfected myocytes
(Gilbert et al.,
1996; McConnell et al.,
1999,
2001;
Flavigny et al.,
1999; Yang et al.,
1998,
1999). We confirmed these
results in >50% of the cells transfected with the several carboxyl-terminus
deletion mutants. In our hands, GMC10(–) showed the strongest
effect on sarcomere disorganization in chicken cardiomyocytes followed by
GMC10(+), GMC9-10, and then GMC7-10, indicating that the
C10 deletion mutants have the strongest effects and that further truncation
decreases the effects. Because MyBP-C binds to connectin/titin through
C8–C10 domains, the C10 deletion mutant is expected to bind
connectin/titin with higher affinity than other deletion mutants.
We hypothesized that the lack of myosin binding but the preserved
connectin/titin binding may cause the severe effects on sarcomere
disorganization. These mutants could be replaced with the endogenous MyBP-C
more effectively because of partially reserved binding to connectin/titin.
Accordingly, GMC7-10, which lacks both connectin/titin and myosin
binding, showed the mildest effects on sarcomere disorganization, but still
>50% of the transfected cells with GMC7-10 showed disorganized
sarcomere structure. We assume that interaction with other sarcomeric proteins
through domain C0–C6 of GMC7-10 is responsible for the effects,
including the N-terminal myosin S2 binding site
(Gruen and Gautel, 1999).
Surprisingly, MyBP-C(+) disorganizes sarcomere structure as effectively as
carboxyl-terminus deletion mutant GMC7-10, showing a similar
deleterious effect on sarcomeres. As assessed by mRNA levels at least,
MyBP-C(+) is dominantly expressed in aged mouse atria; therefore, it may act
as a disruptive polypeptide via a dominant-negative effect on remaining
MyBP-C(–) or it may partially compensate for the decreased expression of
MyBP-C(–) in aged atria. It is generally accepted that the aged heart is
associated with a number of characteristic morphological, histological,
biochemical, and functional changes, including a reduction in the number of
myocytes, the development of cardiac fibrosis, diastolic dysfunction, and a
decrease in the intracellular response to -adrenergic stimulation
(reviewed in Roffe, 1998).
These changes are associated with hypertrophy as a result of a net decrease in
the number of cardiomyocytes (Fleg,
1986) and isoform transition of myosin heavy chain (MHC) from
-MHC to -MHC in rat ventricles
(Lompre et al., 1984;
Schuyler and Yarbrough, 1990;
Wahr et al., 2000).
We have not examined the molecular mechanisms explaining the switch from
MyBP-C(–) to MyBP-C(+) and its effects on aged atrium nor have we
investigated whether any novel cardiac MyBP-C isoform is expressed in other
animals besides the mouse. But interestingly, it has been demonstrated that
senescent rat atrial myocytes displayed abnormal myofilament arrays
(Feldman and Navaratnam,
1981).
In summary, we found a novel alternatively spliced form of cardiac MyBP-C that is abundantly expressed in aged mouse atria. This isoform, MyBP-C(+), shows a decreased binding to myosin filaments and connectin/titin and a decreased incorporation into sarcomere structure, suggesting it is involved in the morphological and functional changes of cardiac muscle during aging.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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-connectin and Pc72C. This research was supported by research grants
from the Ministry of Education, Culture, Sports, Science and Technology of
Japan and the National Center of Neurology and Psychiatry of the Ministry of
Health, Labour and Welfare of Japan. |
Footnotes |
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Corresponding author. E-mail address:
sato{at}bio.s.chiba-u.ac.jp
or
tobinata{at}bio.s.chiba-u.ac.jp.
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