From the Max-Delbrück-Center of Molecular
Medicine, D-13122 Berlin, Germany, the § Institut für
Anästhesiologie und Operative Intensivmedizin,
Universitätsklinikum D-68167 Mannheim, Germany, the ¶ St.
George's Hospital Medical School, British Heart Foundation
Cardiovascular Pathology Unit, London, SW170RE United Kingdom, the
European Molecular Biology Laboratory, D-69012 Heidelberg, and
the ** Institute of Physiology and Pathophysiology, University of
Heidelberg, D-69120 Germany
Received for publication, September 22, 2000, and in revised form, November 1, 2000
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ABSTRACT |
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Myosin-binding protein-C (MyBP-C) is a
component of all striated-muscle sarcomeres, with a well established
structural role and a possible function for force regulation. Multiple
mutations within the gene for cardiac MyBP-C, one of three known
isoforms, have been linked to familial hypertrophic cardiomyopathy.
Here we generated a knock-in mouse model that carries
N-terminal-shortened cardiac MyBP-C. The mutant protein was designed to
have a similar size as the skeletal MyBP-C isoforms, whereas known
myosin and titin binding sites as well as the phosphorylatable MyBP-C
motif were not altered. We have shown that mutant cardiac MyBP-C is readily incorporated into the sarcomeres of both heterozygous and
homozygous animals and can still be phosphorylated by
cAMP-dependent protein kinase. Although histological
characterization of wild-type and mutant hearts did not reveal obvious
differences in phenotype, left ventricular fibers from homozygous
mutant mice exhibited an increased Ca2+ sensitivity
of force development, particularly at lower Ca2+
concentrations, whereas maximal active force levels remained unchanged.
The results allow us to propose a model of how cMyBP-C may
affect myosin-head mobility and to rationalize why N-terminal mutations
of the protein in some cases of familial hypertrophic cardiomyopathy
could lead to a hypercontractile state.
Myosin-binding protein-C
(MyBP-C)1 (1, 2) is a
myofibrillar protein that contributes to the structural integrity of
the sarcomere and possibly is involved in the regulation of contraction (3). Three different isoforms of MyBP-C have been identified: skeletal
(sMyBP-C), fast and slow, and a cardiac-specific variant (cMyBP-C, see
Fig. 1A), each of these being coded for by a distinct gene
(4, 5). All isoforms interact at the C terminus with the rod portion of
myosin (e.g. Ref. 6), as well as with titin (e.g.
Ref. 7), and thus help maintain an ordered thick-filament structure
(reviewed in Ref. 3). MyBP-Cs are modular polypeptides that belong to
the intracellular immunoglobulin (Ig) superfamily. Whereas the skeletal
variants consist of 10 globular domains of the Ig-like or
fibronectin-type-III-like fold (4), cMyBP-C contains an additional
N-terminal Ig module termed C0 (5). Between the Ig domains C1 and C2,
MyBP-Cs also contain a stretch of about 100 residues, the MyBP-C motif
(see Fig. 1A), which in cardiac muscle can be phosphorylated
at three sites by cAMP-dependent protein kinase (8). The
MyBP-C motif was shown to bind to the S-2 segment of myosin, close to
the lever arm domain of the myosin head (9). Interestingly, this
interaction is dynamically regulated by
phosphorylation/dephosphorylation of the MyBP-C motif (10). Moreover,
the controlled interaction with the myosin hinge region appears to
affect the contractile behavior of muscle fibers (11) and thus, could
represent a potential regulatory mechanism of contractility (12). These
insights notwithstanding, direct evidence for a role of cMyBP-C in
force regulation has been difficult to obtain.
Uncovering the functions of cMyBP-C is interesting from a clinical
point of view as the protein is involved in the pathophysiology of
familial hypertrophic cardiomyopathy (FHC, Refs. 13, 14). This
inherited disease occurs in autosomal-dominant fashion and affects
~0.2% of the general population. FHC is known to be a disease of the
sarcomere; mutations in at least eight different sarcomeric protein
genes have been identified as yet (14, 15). Mutations in
cMYBPC account for ~15-20% of genetically defined FHC
cases, but the cMyBP-C-linked types of FHC present as relatively benign
phenotypes with mild hypertrophy at mid-life (16, 17). Most cMyBP-C
lesions show C-terminal truncated polypeptides lacking either the
myosin or myosin and titin binding sites, but some lesions are also
caused by missense mutations occurring in more N-terminal regions of
the protein (16). Genetical engineering approaches have been used to
generate transgenic mice lacking variable numbers of C-terminal domains
of cMyBP-C (18-20). These model systems have demonstrated the
importance of the C terminus of cMyBP-C for a regular sarcomeric
structure and normal contractility of the heart.
In the present study we used knock-out/knock-in technology to generate
mice (hereafter termed knock-in mice) with N-terminal deletion of a
region of cMyBP-C comprising one Ig domain and a linker sequence next
to the MyBP-C motif. The shortened cMyBP-C (see Fig. 1A)
thus has a domain structure similar to that of sMyBP-C. Notably, within
the region affected by the knock-in, a missense mutation has been
described for a family of FHC patients exhibiting a distinct phenotype
(16). We show that the cMyBP-C deletion variant is expressed in both
homozygous and heterozygous mice at the protein level and is readily
incorporated into the sarcomere. Animals carrying the deletion are
viable, show no significant ultrastructural changes of the heart, and
appear to have a normal life span. Mutated cMyBP-C could still be
phosphorylated by cAMP-dependent protein kinase, but
skinned muscle fibers from homozygous mutant hearts revealed a leftward
shift in the force-pCa curve and a decreased slope of that curve. The
increased Ca2+ sensitivity may result from decreased steric
hindrance of myosin-head mobility caused by the expression of the
shorter cMyBP-C. We discuss the possibility that the additional
N-terminal Ig domain present in cardiac versus skeletal
MyBP-C could be included by nature to aid force regulation at the
cross-bridge level in the heart. Our findings also provide a starting
point to explain the development of hypertrophied cardiac tissue in FHC
cases with N-terminal mutations of cMyBP-C.
Gene Targeting--
A P1 clone containing the murine cardiac
MYBP-C sequence was obtained from a mouse 129 P1 genomic library
(Genome Systems, St. Louis, MO). A 9.1-kb EcoRI fragment
from the P1 clone was isolated and subcloned and found to contain the
5'-end of the gene from exon 1-20 (Fig. 1B). A 1.7-kb
StuI/EcoRV fragment (including exon 2) located
upstream of exon 3 and a 5.3-kb NsiI/EcoRI fragment (including exon 7-20) located downstream of exon 6 were used as the 5'
and 3' homology units.
The targeting vector was constructed by standard recombinant
techniques. A genomic fragment of the MYBP-C gene (1.3 kb)
including exons 3-6 was deleted and replaced by a neomycin resistance
gene (Fig. 1B). The vector contained a herpes simplex
thymidine kinase cassette for negative selection of single recombinant
embryonic stem (ES) cell clones. Also, the vector included a unique
ClaI restriction site for linearization of the plasmid.
Homologous recombination between targeting vector and cognate cMyBP-C
locus deleted exons 3-6. Colony selection and target clone
identification were done as described elsewhere (21). Targeting vector
(20 µg) was introduced into 1.2 × 107 ES cells by
electroporation. Genomic DNA was prepared as described (22). Correct
targeting of G418-resistant clones was analyzed by Southern blotting.
Clones were subsequently tested by long PCR assay (Combi Pol/InViTek,
Berlin-Buch). To check for the occurrence of new recognition sites on
the amplificates, Southern blotting was employed. Correctly targeted
clones were microinjected into C57/BL 6 blastocysts, which were
implanted into pseudo-pregnant CB6 mice bred to produce heterozygous or
homozygous mutant animals.
MyBP-C mRNA was assessed by nucleotide sequence analysis of
RT-PCR-amplified DNA fragments according to standard protocols. The
following primer pairs were used: CF 198, GGCTGAGACGGAGCGGTCAGGCG; CR
558, GTCATCAGGGGCTCCCTGATGCTCTGCAGC; CF 198, GGCTGAGACGGAGCGGTCAGGCG; CR1134, CGAAGGTCTGTGACTCCGTGCTGG; CF3923, CAGGATGGCTCCCCAGAGATGGCT; and
CR4195, GCTCCTACACAATGAGCCAGCCAG. Northern blotting of
cardiac/skeletal-muscle RNA was performed as described previously
(23).
Morphology and Microscopy--
Excised hearts were rinsed in 4%
paraformaldehyde and weighed, and cardiac tissue was examined for
pathological alterations (24). Examined parameters included heart
weight, left ventricular wall thickness and cavity size, and myocyte
nuclear size (measured by outlining the nucleus in 150 cardiac cells
cut in their long axis).
Indirect Immunofluorescence Microscopy--
Bundles of
myofibrils prepared from left ventricle essentially as described (25)
were examined under a Zeiss Axiovert 135 microscope. MyBP-C was
visualized by using antibodies against the MyBP-C motif (25).
Cardiac Fiber Mechanics--
Freshly excised mouse hearts were
perfused retrogradely through the aorta with 4 °C rigor buffer (mM:
NaCl, 132; KCl, 5; MgCl2, 1; glucose, 7;
N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid, 10;
pH 7.0; EGTA, 5; leupeptin, 0.1; and 2,3-butanedione monoxime (BDM,
Ref. 20) for 2 min. Papillary muscles or trabeculae from left ventricle
were dissected, tied to thin glass rods, and skinned overnight in
ice-cold relaxing solution (mM: imidazole, 20; pH 6.8; ATP, 7.5;
MgCl2, 10; NaN3, 1; EGTA, 4; leupeptin, 0.1;
BDM, 20; total ionic strength, 130) containing 0.25-0.5% Triton X-100
(26). A relatively low buffer pH used in skinned fiber mechanical
studies was reported to be beneficial for the functional
preservation of the regulatory system (27). After washes with fresh
relaxing buffer, fiber bundles 150-200 µm thin and 3-4 mm long were
mounted isometrically between a position-controlled rigid post and a
force transducer (AME AE 801, Horten Electronics, Norway) with
nitroacetate glue (26). Sarcomere length was adjusted to 2.2 µm by
laser diffractometry. After removal of BDM and addition of an
ATP-regenerating system (creatine phosphate, 10 mM;
creatine kinase, 150 units/ml) to the solution, fibers were activated
by transfer from relaxing to activating buffer, in which EGTA was substituted by Ca2+-EGTA. The desired Ca2+
concentration was calculated as described (26). Experiments were
carried out at room temperature.
The normalized force-pCa relationships, in which force was expressed
relative to the maximum force usually developed at pCa 4.34, were
fitted to the Hill equation,
Gel Electrophoresis and 32P
Autoradiography--
Fiber bundles prepared as described above were
washed with relaxing solution (ATP, 4 mM). Specimens were
incubated with the catalytic subunit of protein kinase A (Sigma, 500 units/ml relaxing buffer) in the presence of [ Generation of Mutant cMyBP-C Mice--
To target the
MYBP-C gene, a 9.1-kb fragment of the murine cardiac MYBP-C
locus encompassing exons 1-20 was isolated and subcloned (Fig.
1B). The targeting construct
was designed to selectively remove exons 3-6 (1.3 kb) thus producing a
deletion of the Ig domain C1 and the linker region between domains C0
and C1 of cMyBP-C (Fig. 1A). Fig. 1B illustrates
the cardiac MYBP-C locus and the gene-targeting construct
containing a neomycin (NEO) resistance gene and a herpes
simplex thymidine kinase cassette (TK) to allow for negative selection.
Fig. 1B (bottom) depicts the cMyBP-C deletion mutant obtained after electroporation of the linearized plasmid into ES
cells and homologous recombination between the targeting vector and the
cognate MYBP-C locus.
Ninety-six G418-resistant ES cell clones were analyzed, and genomic
Southern blotting of DNA from ES cell clones was performed to detect
the targeting event. Correct targeting was found in 6 clones. As shown
in Fig. 2A, a band
corresponding to a 9.1-kb fragment was detected as the wild-type
allele, and a band corresponding to a 2.7-kb fragment as the targeted
allele. Correctly targeted clones were used for blastocyst-mediated
transgenesis and production of chimeric animals. Appropriate breeding
produced mice either homozygous or heterozygous for the cMyBP-C
deletion. These mice were fertile, produced normal litter sizes, and
survived for >1 year. We also tested the correctly targeted clones in
a long PCR assay. Primers were designed such that a 2.2-kb fragment was
produced specific for the wild-type allele and a 1.9-kb fragment
specific for the targeted allele (data not shown). Additionally, a PCR was done with a 4.8-kb product (Fig. 1B, top).
The analysis showed that the restriction enzyme EcoRI cut
the amplificate only of the targeted allele into a 2.8 and a 2.0 kb
fragment (Fig. 2B), indicating the introduction of a new
recognition site.
cMyBP-C Expression in Mutant Mice--
To determine the expression
of cMyBP-C transcripts in mutant mice, we performed RT-PCR analysis
with various primer pairs from different regions of heart cDNA
(Fig. 2C). With a primer pair encompassing domains C0 to C1,
a signal was obtained only for cDNA from wild-type or heterozygous
mice, whereas in homozygous mutant animals, RNA encoding the C1 domain
plus N-terminal adjacent linker was not expressed (Fig. 2C,
panel a). This observation is consistent with the results of
Northern blot analyses (Fig. 2D). No signal was detected
when total RNA, isolated from homozygous hearts, was hybridized with a
C1+linker probe. By contrast, a signal was detectable in heterozygous
hearts. In comparison, when skeletal-muscle RNA was used, no signal was
present (Fig. 2D). Hybridization with a probe of the MyBP-C
motif revealed a normal signal for heart RNA in all types of animals,
but none for skeletal-muscle RNA. Controls with a GAPDH probe showed a
signal in all lanes.
By RT-PCR, using primer pairs encompassing the C0 domain and the MyBP-C
motif, we detected the expected deletion of 474 base pairs (Fig.
2C, panel b). Cloning and sequencing of the
products highlighted by the asterisks (Fig. 2C)
revealed that the upper band corresponds to the wild-type DNA sequence,
whereas the lower band product has the same flanking sequence but
contains the predicted deletion. We note that in competitive PCR, a
shorter product tends to show a larger signal than a longer product, as
seen in Fig. 2C, panel b. This figure, as well as
the RT-PCR at the 3'-untranslated region (Fig. 2C,
panel c), demonstrate that the transgenic RNA is stable and
well expressed; no degradation or lowered expression was
detectable. Thus, regulation at the transcription level seems unlikely.
Both homozygous and heterozygous mice were found to express the
deletion mutant also at the protein level. Western blot analyses with
muscle protein obtained from all types of animals revealed a distinct
band stained by a polyclonal antibody against the MyBP-C motif (Fig.
3A). Moreover, cardiac
myofibrils labeled with fluorophore-marked Histological Characterization--
Hearts from several months (up
to ~1 year) old animals (n = 7, for each animal type)
were examined for histological and morphological abnormalities
(Table I). None of the parameters
investigated differed between animal types in a statistically
significant manner, although one homozygous mutant heart revealed an
abnormal phenotype with strongly increased values for all four
parameters. In general, however, no evidence was found for cardiac
hypertrophy, myocyte loss or inflammation. Thus, the histological
appearance of heterozygous or homozygous mutant hearts appeared to be
normal.
Fibers from Homozygous Mutant Hearts Show Increased
Ca2+ Sensitivity of Force Generation--
Left ventricular
muscle strips obtained from wild-type and mutant, litter-matched,
animals were probed for their contractile properties by measuring the
active force of skinned fiber bundles as a function of the
Ca2+ concentration. Because the Ca2+
sensitivity varies with sarcomere length, laser diffractometry was used
to set this length to 2.2 µm in all experiments. A typical example
demonstrating the force rise with increasing [Ca2+]
(i.e. decreasing pCa) is shown in Fig.
4, inset. Fibers from 5 wild-type, 2 cMyBP-C(+/ The N-terminal Deletion Mutant Is Still Phosphorylated by
cAMP-dependent Protein Kinase--
To find out whether the
altered Ca2+ sensitivity could be related to an altered
response of the mutant cMyBP-C to activation by
cAMP-dependent protein kinase (because the deletion is
close to the MyBP-C motif), we tested the ability of the protein to be
phosphorylated by this kinase (see Refs. 28, 30, 31). As shown in Fig.
5, autoradiography of SDS-polyacrylamide
gels of left ventricular tissue incubated with the catalytic subunit of
cAMK in the presence of [ The structural role of MyBP-C in both skeletal and cardiac
myofibrils is well established (1-7); the C terminus of the protein, providing binding sites for myosin and titin, is essential for the
formation and regular structure of thick filaments. Accordingly, C-terminal truncations of cardiac MyBP-C result in severe changes in
heart ultrastructure and impaired cardiac mechanical performance, both
in transgenic mouse models (18-20) and in FHC-affected humans (14,
16). It is perhaps not surprising that, of the about 30 mutations in
the gene for cMyBP-C (MYBPC3) so far described in families
with FHC, the majority locates to C-terminal domains (14). On the other
hand, some mutations also occur in N-terminal regions of the molecule,
but in these cases the mechanisms leading to FHC are more difficult to
understand. A possibility is that N-terminal domains of cMyBP-C
contribute to the regulation of cardiac-muscle contraction.
The knock-in mouse model presented here was generated in an attempt to
uncover a possible (patho)physiological function of some of the
N-terminal cMyBP-C domains. The knock-in was made bearing in mind that
the cardiac isoform of MyBP-C is distinguished from the skeletal
isoforms by two main features: (i) cMyBP-C contains an additional Ig
domain, the C0 module (4, 5) and (ii) the MyBP-C motif between the Ig
domains C1 and C2 can be phosphorylated by cAMP-dependent
protein kinase and a calmodulin-dependent protein kinase
(8, 28, 30, 31). The (reversible) phosphorylation mediates binding of
the MyBP-C motif to the neck region of myosin (9, 10) and could be
important for the hypothesized regulatory role of cardiac MyBP-C (11).
Whereas the knock-in described here left the MyBP-C motif intact, it
eliminated the N-terminal C1 domain and the linker sequence between C0
and C1 (Fig. 1). Thus, the mutant mice contain a shorter than normal
cMyBP-C molecule whose size and domain architecture resemble those of
the skeletal isoforms.
We bred the mice bearing mutant MyBP-C alleles to homozygosity, because
we expected a relatively mild effect on cardiac structure and/or
function; MyBP-C is not found in the entire A-band but forms 7-9
stripes in the A-band C-zone on either side of the M-line (32, 33).
Indeed, hearts from both heterozygous and homozygous mutant mice showed
no statistically significant changes at the ultrastructural level, and
no differences were found between wild-type and heterozygous animals in
terms of the force response of skinned cardiac fibers to
Ca2+-dependent activation. In contrast, fibers
from homozygous mutant mice showed an increased Ca2+
sensitivity of force production (Fig. 4) whereas maximum force levels
remained unchanged. This finding is consistent with that of an earlier
study reporting that active tension at submaximal Ca2+
concentrations was increased, but maximum tension was not affected, upon partial extraction of MyBP-C from rat skinned cardiomyocytes (34).
We note that extraction of MyBP-C was shown to slightly increase
Ca2+ sensitivity at low to intermediate Ca2+
concentrations also in rabbit psoas muscle fibers, but the effect was
much smaller than in cardiac cells (34). Our results extend the
previous findings, suggesting that at least part of the change in
Ca2+ sensitivity of cardiac sarcomeres may be related to a
functional role of the N-terminal cMyBP-C domains.
One family of FHC patients has been described bearing a missense
mutation (E258K) in the region just N-terminal to the MyBP-C motif
(16). It is not unlikely that an altered function of N-terminal MyBP-C
domains is responsible for the hypertrophy phenotype found in some
members of this family. However, also clinically healthy individuals
can carry the mutant MYBPC3 allele (5, 16). Moreover, mutations in MYBPC3 are frequently characterized by a mild
phenotype particularly in young patients and a delayed age at the onset of symptoms (16, 17). Then, because the physiological background of
human and mouse is very different, it is possible that the life span of
the knock-in mice of this study is not long enough for significant
changes of cardiac ultrastructure (and contractile properties of
heterozygous animals) to occur. On the other hand, we demonstrated that
the mutant protein is expressed and incorporated into the sarcomeres,
which was associated in homozygous mutant animals with enhanced
contractile performance. Taken together, N-terminal cMyBP-C mutations,
if occurring in human heterozygous FHC patients, might in some
instances determine a "hypercontractile" state that could induce
cardiac hypertrophy directly. In this context we point out that a
hypercontractile hypothesis has been put forth for some FHC cases in
which other sarcomeric proteins are mutated, such as What mechanism(s) could be envisioned to explain the observed
functional effect of the N-terminal deletion in cMyBP-C? Although the
exact layout of MyBP-C in the thick filament is still subject to
debate, structural details known to date (3, 10, 37) led us to propose
a plausible model demonstrating the regulatory input by the protein. As
shown in Fig. 6, cMyBP-C binds to the myosin rod (and titin) at the C terminus and also to the myosin neck
region with the MyBP-C motif (in the unphosphorylated state). In the
wild-type protein (Fig. 6A), the C0 domain at the N terminus could well interfere with the myosin head region, either by proximity to the regulatory light chains as proposed (10) or through specific interaction with the head. Indeed, a recent study suggested that the C0
domain of human cMyBP-C contains a novel putative myosin-binding site
(38). Thus, in cardiac sarcomeres, MyBP-C could mechanically constrain
cross-bridge movement in a manner not found in skeletal muscle.
Phosphorylation-induced unbinding of the MyBP-C motif from the myosin
neck region (Fig. 6A, asterisk) would release some constraints from the myosin head, thereby providing cardiac cells
with an additional mechanism to regulate force development. In the case
of N-terminal shortened cMyBP-C (Fig. 6B), the molecule may
not be able to reach the myosin head region, which would change the
flexibility or mobility of the cross-bridge permanently. Even though
the number of myosin heads whose mobility can be affected by cMyBP-C is
limited (because many heads lie outside of the C-zone), mechanical
coupling of cross-bridges within a thick filament (12) would still
produce an effect on contractile properties. A modulation of
cross-bridge cycling rate may then alter the force response to
Ca2+-dependent activation. To summarize, a
permanent increase in cross-bridge mobility caused by a shortened
cMyBP-C would translate into higher force production and explain the
observed mechanical changes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
where HC (the Hill coefficient, a measure of
cooperativity) and Kc are constants.
(Eq. 1)
-32P]ATP
(specific activity, 250 µCi/µM) for 45 min at room
temperature (26, 28). Proteins were then denatured, dissolved, and
electrophoresed on 8% SDS-polyacrylamide gels. Major myofibrillar
proteins were identified by Coomassie staining. 32P
incorporation was visualized by autoradiography, using a 4-12-h exposure time with standard Kodak x-ray film (28, 29).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Structure of cMyBP-C and gene-targeting
strategy. A, domain architecture of normal cMyBP-C and
the N-terminal deletion mutant. The deletion results in loss of the
linker sequence between modules C0 and C1 and of the C1 domain. Shown
are the established binding sites of MyBP-C to the rod (LMM)
portion of myosin, to titin, and to the myosin neck region
(S2), as well as the proposed C0-domain binding site to the
myosin (head). The recognition site of a polyclonal antibody against
the MyBP-C motif (pAB) used in this study is also indicated.
B, schematic of the genomic structure of wild-type MyBP-C
and MyBP-C(Neo) alleles. Exons 1-7 and 19-20 are depicted for each
allele. The mutation removes exons 3-6 (1.3 kb), all other exons are
identical. The bottom part indicates the MyBP-C deletion
mutant obtained after electroporation of the plasmid into ES cells. For
further details on the targeting strategy, see text.
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Fig. 2.
Expression of mutant cMyBP-C.
A, Southern blot analysis of DNA to show correct targeting
in ES cell clones; the 9.1-kb band is specific to the wild-type allele,
the 2.7-kb band to the targeted allele. B, PCR-based
genotypic analysis of wild-type (+/+), heterozygous (+/ ) and
homozygous (
/
) mutant mice. The 4.8-kb amplificate covers the
length indicated in Fig. 1. Only the targeted allele is cut into two
subfragments by the restriction enzyme EcoRI revealing the
introduction of a new recognition site. C, RT-PCR analysis
of cDNA to show expression of cMyBP-C transcripts in wild-type and
mutant mice. Various primer pairs encompassing different regions of
cDNA of cMyBP-C were used, as indicated for each panel;
panel d is a control with tropomyosin. The
asterisks in panel b highlight the products
subsequently cloned and sequenced. D, Northern blot analysis
of RNA from heart and (for comparison) skeletal muscle. The results
confirm the lack of expression of the C0-C1 linker sequence and of the
entire C1 domain of cMyBP-C in homozygous mutant mice. Controls were
done with GAPDH probes.
-MyBP-C antibodies
exhibited the expected staining pattern in the sarcomeric A-band; no
obvious difference in staining intensity or regularity of labeling was
found between wild-type and homozygous mutant animals (Fig.
3B). Thus, mutant mice stably expressed the shortened
cMyBP-C protein.
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Fig. 3.
MyBP-C expression at the protein level.
A, Western blot of wild-type (+/+), heterozygous (+/ ), and
homozygous (
/
) mutant hearts (h), using a polyclonal
antibody against the MyBP-C motif. The antibody cross-reacts with
skeletal-muscle (sm) MyBP-C. B,
immunofluorescence microscopy on cardiac myofibrils, using the
-MyBP-C antibody, which stained the expected two epitopes in the
sarcomeric A-band region (panel a: PC,
phase-contrast image; FL, fluorescence image). The staining
pattern was the same in myofibrils from wild-type (panel b)
and homozygous mutant (panel c) hearts.
Summary of results of morphological measurements
), and homozygous mutant
(
/
) hearts.
), and 6 cMyBP-C(
/
) mice were included in the analysis, and 5-7
fiber bundles per animal were investigated. A summary of results is
presented in Fig. 4. Fibers from wild-type and heterozygous mutant mice
exhibited a similar Ca2+ sensitivity of force development,
with pCa50 values (pCa at half-maximum force) of 5.19 ± 0.01 S.E. and 5.18 ± 0.01 S.E. and Hill coefficients of
3.14 ± 0.06 S.E. and 3.00 ± 0.05 S.E., respectively. In
contrast, homozygous mutant mice showed a statistically significant
increase in Ca2+ sensitivity (pCa50 = 5.26 ± 0.02) and a decreased slope of the force-pCa curve (Hill
coefficient, 2.32 ± 0.20). Thus, the Ca2+ sensitivity
of force generation was particularly increased at low to modest
physiological [Ca2+], the concentrations relevant to
normally working cardiac muscle. No statistically significant
difference was found between animal types with regard to the (absolute)
maximum active force levels (p > 0.05 in unpaired
Student's t test). This indicates that the increase in
relative force observed in the cMyBP-C(
/
) fibers mainly
at lower [Ca2+] is not offset by a change in the maximum
force level.
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Fig. 4.
Cardiac fiber mechanics. Skinned fiber
bundles were activated at a sarcomere length of 2.2 µm at a series of
different Ca2+ concentrations, from pCa 6.0 to pCa 4.34, and forces were recorded (inset). Force was expressed
relative to the maximal force level reached at optimal
[Ca2+]. A summary of results shows that wild-type
(n = 32) and heterozygous mutant fibers
(n = 10) exhibited a similar Ca2+
sensitivity of force development. In contrast, Ca2+
sensitivity of homozygous mutant fibers (n = 38) was
significantly increased at modest to high pCa. The pCa50
value was shifted leftward by 0.07 pCa units; the slope of the curve
was decreased. Statistically significant differences to wild-type
specimens were confirmed by unpaired Student's t test (*,
p < 0.05; **, p < 0.001). Values are
mean ± S.E.
-32P]ATP revealed that
both wild-type and the homozygous mutant cMyBP-C are phosphorylated to
a similar degree (arrowheads in lanes d-g). Thus, the knock-in did not affect the phosphorylation of cMyBP-C, suggesting that the
-adrenergic pathway for this protein based on
phosphorylation/dephosphorylation of the MyBP-C motif may still be
intact.
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Fig. 5.
SDS-polyacrylamide gels (lanes
a-c) and 32P autoradiography (lanes
d-g) of wild-type (+/+) and homozygous mutant fibers
( /
) from the left ventricle. Samples were incubated with the
catalytic subunit of cAMK in the presence of
[
-32P]ATP. st, standard.
Arrowheads point to the position of cMyBP-C. Lanes
d-e correspond to the Coomassie Blue gels shown in lanes
a-b. Lanes f-g are from a different experiment to
demonstrate that major phosphorylation is associated, besides with
cMyBP-C, with troponin I (arrow). Phosphorylation of TnI is
known to cause a distinct decrease in Ca2+ sensitivity of
force.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-tropomyosin
(35, 36). FHC might therefore be a disease induced by mutations causing
either functional cardiac impairment followed by compensatory
hypertrophy (apparently the majority of all cases) or functional
enhancement followed by direct cardiac hypertrophy (14).
View larger version (36K):
[in a new window]
Fig. 6.
Schematic of proposed cMyBP-C influence on
myosin-head mobility. A, the wild-type cMyBP-C
interacts with both the rod portion and the neck region of the myosin
molecule. Phosphorylation of the MyBP-C motif by
cAMP-dependent protein kinase (PKA) releases
cMyBP-C from the myosin neck (asterisk). Interaction of the
cardiac-specific C0 domain of MyBP-C with the myosin head would
constrain cross-bridge movement. B, with the C1 domain plus
adjacent linker missing, the N-terminal deletion mutant of cMyBP-C
lacks the interaction with the myosin head, thereby decreasing steric
hindrance of cross-bridge action permanently. Note that the length of
the mutant cMyBP-C is comparable with that of skeletal MyBP-C isoforms.
MLC, myosin light chain; MHC, myosin heavy chain;
C0-C10, cMyBP-C domain numbers; P,
MyBP-C motif; L, linker region.
An alternative model of cMyBP-C arrangement (not shown here) suggests that three molecules oriented perpendicularly to the fiber axis could overlap and form circular structures that tighten the packing of myosin filaments (3). Phosphorylation of the MyBP-C motif is thought to loosen the packing (39) by decreasing the overlap between the protein's N- and C terminus. The increase in circumference of the ring of cMyBP-C would facilitate regulated changes of actin-myosin interaction. Our results can be readily interpreted based on this "circular model." N-terminal deletion of an Ig domain plus adjacent linker sequence would most likely disrupt the protein ring, thus decreasing the restriction of myosin and improving actomyosin interaction. As in the above model, the actual length of MyBP-C would be important for the protein's regulatory impact. To conclude, molecular-level, mechanical effects of the knock-in generated here are explainable with available models of cMyBP-C arrangement.
In summary, we have produced a mouse mutant of cMyBP-C with N-terminal
deletion whose characterization provided novel insights into the
function of the protein. Knock-in mice stably expressed the deletion
variant in the sarcomere, and cardiac fibers from homozygous mutant
animals exhibited an increased Ca2+ sensitivity of force
production concomitant with no change in maximum active force levels.
The enhanced contractile performance may be caused by decreased steric
hindrance of cross-bridge action by the shortened cMyBP-C. We also
propose that the presence of additional N-terminal domains in the
normal cardiac isoform of MyBP-C, compared with the skeletal isoforms,
may be important to effectively regulate cardiac-muscle contraction at
the cross-bridge level. Extension of these findings to humans suggest a
molecular mechanism by which N-terminal mutations in cMyBP-C could
cause familial hypertrophic cardiomyopathy.
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ACKNOWLEDGEMENTS |
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We would like to thank Dr. Siegfried Labeit for continuous support and Sigrid Milan, Corinna Thiel, Ulla Gaio, and Monika Troschka for expert technical assistance.
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FOOTNOTES |
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* This work was supported by the Max-Delbrück Center for Molecular Medicine (twinning grant), the Deutsche Forschungsgemeinschaft (Li690/5-1, La668/5-1), and the Medical Faculty of the University of Heidelberg (Forschungsförderungsprogramm).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Inst. of Physiology
and Pathophysiology, University of Heidelberg, Im Neuenheimer Feld 326, D-69120, Heidelberg, Germany. Tel.: 49-6221-544130 or 544135; Fax:
49-6221-544049; E-mail: wolfgang.linke@urz.uni-heidelberg.de/
Published, JBC Papers in Press, November 28, 2000, DOI 10.1074/jbc.M008691200
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ABBREVIATIONS |
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The abbreviations used are: MyBP-C, myosin-binding protein-C; PCR, polymerase chain reaction; RT-PCR, reverse transcriptase PCR; TK, thymidine kinase; ES, embryonic stem cell; FHC, familial hypertrophic cardiomyopathy; kb, kilobases.
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