* Department of Medicine, Center for Molecular Genetics, and American Heart Association Bugher Foundation Center for
Molecular Biology, University of California, San Diego, La Jolla, California 92093; and Institute for Cell Biology, Swiss Federal
Institute of Technology, 8093 Zurich, Switzerland
Hypertrophic cardiomyopathy is a human
heart disease characterized by increased ventricular
mass, focal areas of fibrosis, myocyte, and myofibrillar
disorganization. This genetically dominant disease can
be caused by mutations in any one of several contractile proteins, including cardiac myosin heavy chain
(
MHC). To determine whether point mutations in human
MHC have direct effects on interfering with filament assembly and sarcomeric structure, full-length
wild-type and mutant human
MHC cDNAs were
cloned and expressed in primary cultures of neonatal
rat ventricular cardiomyocytes (NRC) under conditions
that promote myofibrillogenesis. A lysine to arginine change at amino acid 184 in the consensus ATP binding
sequence of human
MHC resulted in abnormal subcellular localization and disrupted both thick and thin
filament structure in transfected NRC. Diffuse
MHC
K184R protein appeared to colocalize with actin throughout the myocyte, suggesting a tight interaction
of these two proteins. Human
MHC with S472V mutation assembled normally into thick filaments and did
not affect sarcomeric structure. Two mutant myosins
previously described as causing human hypertrophic cardiomyopathy, R249Q and R403Q, were competent
to assemble into thick filaments producing myofibrils
with well defined I bands, A bands, and H zones. Coexpression and detection of wild-type
MHC and either
R249Q or R403Q proteins in the same myocyte showed these proteins are equally able to assemble into the sarcomere and provided no discernible differences in subcellular localization. Thus, human
MHC R249Q and
R403Q mutant proteins were readily incorporated into
NRC sarcomeres and did not disrupt myofilament formation. This study indicates that the phenotype of myofibrillar disarray seen in HCM patients which harbor either of these two mutations may not be directly due to
the failure of the mutant myosin heavy chain protein to
assemble and form normal sarcomeres, but may rather
be a secondary effect possibly resulting from the chronic stress of decreased
MHC function.
A sarcomere, the functional unit of muscle, is composed of a precise arrangement of at least 20 known proteins ordered in nearly crystalline fashion. Complex interactions of a few of these proteins,
mainly actin and myosin, produce the force necessary for
muscular contraction (for review see Squire, 1986 The discovery that mutations in sarcomeric proteins cause
an inherited form of human heart disease has brought significant attention towards understanding muscle protein
function. Familial hypertrophic cardiomyopathy (HCM)1
is an autosomal dominant, genetically heterogeneous heart
disease with variable penetrance and an assortment of
clinical phenotypes ranging from mild syncope to sudden
death. The hallmark of this disease is unexplained left ventricular hypertrophy in which dilation of the ventricular
chamber is absent (Maron, 1988 Mutations in the To address this question, we have developed an in vivo
competition assay that will allow the direct evaluation of the
ability of an epitope tagged mutant human cardiac Isolation of Human A human heart (ventricular) specific cDNA library (Stratagene, La Jolla,
CA) was screened with end labeled oligonucleotide probes derived from
the Tissue Culture and Transfections
Neonatal rat cardiac cells were isolated from hearts of 1-2-d-old neonatal
rats, purified on a discontinuous percoll gradient (Zhu et al., 1991 Nonmuscle cells (CHO) were grown in two-well chamber slides and
were transfected as described for the neonatal rat ventricular myocytes. 48 h
after transfection the cells were fixed in 3% paraformaldehyde and were
immunostained.
Antibodies, Immunostaining, and Microscopy
For indirect immunofluorescence staining, the cells were washed briefly in
relaxation buffer A (0.1 M KCl, 5 mM EDTA, 1 mM EGTA), fixed in
fresh 3% paraformaldehyde (made in buffer A), neutralized with 50 mM
NH4Cl, and immunostained with the appropriate antibodies. Sequential
staining using two mouse monoclonals as primary antibodies was performed as follows: after incubation with the first primary antibody and
subsequent washing with buffer A, the immunostained cells were incubated with an FITC conjugated goat anti-mouse affinity purified F(ab)
fragment diluted 1:50 for 40 min. Washing in buffer A was followed by the
second primary antibody, applied for 1 h. The second secondary was lissamine rhodamine (LRSC) AffiniPure donkey anti-mouse diluted 1:100.
The anti-rat myosin light chain 2v antibody was produced in our lab by
Dr. S. Kubalak, as previously described (Kubalak et al., 1996 Cloning and Characterization of Wild-type and Mutant
Myosin cDNAs
A full-length human
An epitope tag was inserted in the NH2-terminal region
of human
Four missense mutations were generated in the It is possible that insertion of an epitope tag or mutations at specific amino acids may render the Epitope Tagged Wild-type Human Transfected NRC display two distinct classes of phenotype
relevant to the organization of the myofibrils in immunostained cells. One third of the cells display highly organized myofibrils having well defined sarcomeres identifiable
by clear H zones, A bands, and I bands. The remaining
transfected cells were not well developed or were too faint
to be analyzed. Both classes of cellular phenotype were
observed in nontransfected as well as in transiently transfected NRC fixed and stained for either endogenous or exogenous myofibrillar proteins. NRC cultures exposed to
calcium phosphate transfection with the backbone vector
pCB6 produce ~40% of the cells filled with highly organized myofibrils. Cells were only scored if some level of
sarcomeric organization was present. This eliminated contaminating fibroblasts from the data set.
Human Table I.
Morphology of Transfected NRC
). The
association of actin and myosin is tightly regulated by the
troponin complex, tropomyosin, and flux of Ca2+ ions. Productive muscular contraction is dependent upon proper spatial relationships of muscle structural proteins within
the sarcomere. Genetic analysis in Drosophila and Caenorhabditis has shown that mutations in several of the sarcomeric proteins disrupt myofibrillar organization and affect muscle function (for review see Epstein and Bernstein,
1992
). However, for most of these mutations the underlying biochemical/functional defect has not been elucidated. Thick and thin filaments can form independently of one
another, yet apparently equal stoichiometric quantities of
actin and myosin are required for proper sarcomeric order
(Beall et al., 1989
).
) and an increase in myocyte disarray associated with abnormal myofilament structure in hearts with HCM is apparent. Genetic mapping has
identified a minimum of six loci which cosegregate with
the disease, any of which can be responsible for the dominant phenotype. The genes identified as responsible for
HCM all encode different structural components of the sarcomere,
myosin heavy chain (
MHC; Geisterfer-Lowrance et al., 1990
), cardiac troponin T,
tropomyosin
(Thierfelder et al., 1994
), C protein (Bonne et al., 1995
;
Watkins et al., 1995a
) as well as the cardiac essential and
regulatory myosin light chains (Poetter et al., 1996
). Interestingly, mutations in the myosin light chains produce
skeletal muscle defects and are associated with a rare form
of HCM which displays midventricular hypertrophy. However, the connection between mutations in sarcomeric proteins and the pathogenesis of HCM is not understood.
MHC gene are present in ~30% of
the HCM families currently characterized (Watkins et al.,
1995b
). Nearly all of the
MHC mutations reported are in
the S1 head domain and occur at amino acids that are
highly conserved throughout MHC phylogeny (Warrick
and Spudich, 1987
; Mornet et al., 1989
). The crystal structure of S1 shows that many of the HCM mutations contribute to functional or structural domains within the globular
myosin head (Rayment et al., 1995
). Reduced function has
been demonstrated for a few of these mutations (Cuda et
al., 1993
; Sweeney et al., 1994
; Lankford et al., 1995
), yet it
remains to be elucidated how decreased myosin function
may lead to the morphological disarray observed in HCM
hearts. The possibility exists that the mutations in
MHC
are directly responsible for the myofibrillar disarray causing defective assembly of the mutant protein and/or interference with the assembly of the wild-type proteins by a
dominant negative function. In this regard, a class of missense mutations in the S1 portion of Caenorhabditis muscle MHC displays dominant phenotypes that dramatically
disrupt sarcomere organization (Bejsovec and Anderson,
1988
, 1990
). Although none of the C. elegans mutations correspond to those described in HCM, it is possible that
the human
MHC mutant proteins have a similar deleterious affect on myofibril assembly and/or structure.
MHC
protein to effectively compete with a wild-type
MHC protein for assembly into endogenous sarcomeric units in living myocardial cells. In this paper we report the cloning
and construction of full length human
MHC cDNAs encoding wild-type and mutant proteins. Expression of these clones either individually or in combination within transiently transfected primary cultures of neonatal rat ventricular cardiomyocytes (NRC) was used to analyze the effect of
MHC mutations on subcellular localization and
myofibril assembly. A missense mutation in the consensus
ATP binding sequence dramatically affected sarcomere structure, disrupting myofibril assembly at an early stage.
However,
MHC with either of two HCM mutations, R249Q
or R403Q, assemble normally. This indicates the myofibrillar disarray observed in HCM patients may not be the
direct result of mutant contractile proteins altering myofilament assembly. Furthermore, the data presented here
support previous information suggesting that biochemical defects in the human
MHC proteins produce physiological dysfunction and pathological alterations in the heart.
Materials and Methods
MHC cDNA Clones
MHC published sequence (Jaenicke et al., 1990
; Liew et al., 1990
).
End labeling and library screening were performed as per standard protocols (Sambrook et al., 1989
). Selected cDNAs were sequenced using cDNAsequence-specific primers (synthesized on a Pharmacia LKB Gene Assembler Plus; Pharmacia Biotech, Piscataway, NJ) in conjunction with the
Sequenase kit (United States Biochemicals, Cleveland, OH). Restriction
enzymes used for mapping and subcloning were obtained from GIBCO
BRL (Gaithersburg, MD). Site-directed mutagenesis was performed with
the Sculptor kit (Amersham Corp., Arlington Heights, IL) or by PCR as
described by Higuchi et al. (1988)
. A small portion of the final PCR product was excised from the full length product by treatment with appropriate
restriction enzymes and subsequent gel purification (GeneClean; Bio 101, La Jolla, CA). The small fragment was sequenced in its entirety to confirm
that the only change(s) present were the desired sequence insertion or
mutation. Correct subclones were then used to replace the wild-type sequence in the full length
MHC cDNA. To eliminate the possibility of a
second mutation affecting the K184R phenotype, this construct was made
independently a second time and subsequently tested. There was no difference in the phenotype observed for both clones with this mutation.
), and
were plated on chamber slides (NUNC, Naperville, IL) coated with laminin (Sigma Chemical Co., St. Louis, MO). After ~24 h the cells were transiently transfected using a modified calcium phosphate method (Chen and
Okayama, 1988
) in DME plus 15% serum (10% horse serum, 5% fetal calf
serum). Approximately 3 µg of DNA was used per well for a two-well
chamber slide. The precipitate was allowed to be in contact with the cells
for 12-20 h. After transfection the cells were washed with PBS, changed to DME with 1% horse serum, 10
4 M phenylephrine, and were subsequently cultured for 2-5 d. Alternatively, the washed cells were cultured
in 15% serum (10% horse serum, 5% fetal calf serum) for 2-5 d. No difference was observed between these two culture conditions.
). The epitope specific glu-glu (EE) antibody, derived from polyoma virus medium
T antigen, was a generous gift of Dr. G. Walter (University of California,
San Diego, CA) and was used at a 1:50 dilution of hybridoma supernatant.
MHC, 4H3, monoclonal antibody was a generous gift of Dr. J. Leger (Institut National de la Santé et de la Recherche Médicale, Montpellier,
France) and was diluted 1:20. Anti-HA antibody was purchased from
Boehringer Mannheim (Indianapolis, IN) and was diluted 1:10. Antibody
to actin (A20) was a generous gift of Dr. J. Lin (University of Iowa, Iowa
City, IA) and was diluted 1:20. Phalloidin-rhodamine was purchased from Molecular Probes Inc. (Eugene, OR) and was diluted 1:1,000. Secondary antibodies conjugated to FITC or LRSC (1:100 to 1:200 dilution) were
purchased from Jackson Laboratories (Bar Harbor, ME). Stained cells
were mounted using gelvitol with 2.5% DABCO (1,4-diazabicyclo-[2.2.2] octane). Laser scanning confocal microscopy was performed using the
Bio-Rad MRC1024 equipped with LaserSharp software. All confocal pictures were derived from a z-series which has been projected to produce
the final image shown. Composite images were given pseudocolor and
merged in Adobe Photoshop.
Results
MHC cDNA was constructed from
the overlapping clones using restriction enzyme sites endogenous to the
MHC sequence (Fig. 1). The reconstructed
full-length cDNA was completely sequenced on both strands
to ensure that no mutations occurred in the encoded wildtype protein. Small portions of the wild-type
MHC cDNA
were used as templates to generate nucleotide changes encoding desired missense mutations as well as the insertion of epitope tags. These fragments were sequenced to ensure the only change present was the preferred mutation
and were subsequently exchanged into the full length
cDNA. The resulting clones were sequenced across any restriction sites used during construction to ensure the desired mutation was the only change present. The open
reading frame of all the
MHC clones was confirmed via
in vitro transcription and translation (data not shown).
Fig. 1.
Cloning and construction of human MHC cDNA expression plasmids. (A) The three overlapping cDNA clones
(
cDNA4, 13, and 10) used for construction of the 6-kbp, fulllength human
MHC cDNA are shown below the representation
of the full-length cDNA (
MHC cDNA). The restriction sites
SmaI (S), BamHI (B), and AatII (A) were used to assemble the
full-length clone. Subclones KS5
, c13-5
, and c13-M were used to
insert the epitope tag sequences or for generation of the mutant
codons. Also listed are the full-length, epitope tagged clones used.
(B) Single letter amino acid sequence of wild-type myosin from
several species are compared. The numbering is based upon the
published human
MHC sequence (Liew et al., 1990
). The underlined area is involved in ATP binding. 1, human
MHC; 2,
chick sarcomeric MHC (Molina et al., 1987); 3, rat embryonal sarcomeric MHC (Strehler et al., 1986
); 4, Ceanorhabditis elegans MHC
A (Karn et al., 1983
); 5, chick smooth muscle MHC (Yanagisawa et al., 1987
); 6, Dictyostelium discoideum (Warrick et al., 1986
); 7,
Acathamoeba castellanii myosin II (Hammer et al., 1987
).
[View Larger Version of this Image (38K GIF file)]
MHC to facilitate detection of exogenous protein in transfected NRC. Exogenous
MHC proteins containing either haemaglutinin (HA-1 = YPYDVPDYA;
Wilson et al., 1984
) or glu-glu (EE = EEYMPME; Grussenmeyer et al., 1985
) epitopes were specifically recognized
by their respective monoclonal antibodies in transiently
transfected NRC (Fig. 2). Each epitope was positioned between amino acids 3 and 4 of the
MHC molecule, a domain with no ascribed function. As noted in previous studies, the crystal structure of the myosin S1 head reveals that
the NH2 terminus is free in solution, existing as a random
coil on the surface molecule (Rayment et al., 1993a
,b), making this region a good target for the insertion of short
sequences. The exogenous
MHC protein was incorporated into myofibrils in a pattern indistinguishable from
the endogenous MLC2v (data not shown), a molecule
known to bind MHC.
Fig. 2.
Epitope tagged wild-type human MHC protein assembles into normal myofibrils in transiently transfected NRC. Cells were
transfected with plasmids containing the wild-type human
MHC cDNA tagged with either the EE (A-C) or HA (D-F). The cells were
stained for the presence of actin filaments using rhodamine-phalloidin (A and C) and were costained with the anti-EE (B) or anti-HA (E) specific antibodies. Sarcomeres containing human
MHC have well defined A bands (a), I bands (i), and M lines (m). The composite images (C and F) show that the exogenous
MHC (green) fills the A band, except the H zone as expected, in a pattern that is complementary and partially overlapping with F-actin (red, C and F). Bar, 20 µm (inset enlarged 3×).
[View Larger Version of this Image (50K GIF file)]
MHC
coding sequence via PCR (see Materials and Methods).
Two of the
MHC mutations generated were based upon
corresponding mutants that remove functions of the Dictyostelium MHC II. The lysine at amino acid 185 in Dictyostelium MHC is in the ATP binding pocket (Fisher et al.,
1995
), and mutation of this amino acid to arginine blocks
ATP exchange of Dictyostelium MHC resulting in a "rigorlike" molecule (Spudich, J., personal communication). The
second mutation is a serine to valine change at amino acid
472 in human
MHC. Dictyostelium myosin with this mutation has slightly higher than normal actin activated ATPase yet in an in vitro motility assay this myosin can move
F-actin at only 10% of the rate for wild-type MHC (Ruppel and Spudich, 1996
). If actin motility is a requirement
for sarcomeric assembly, then
MHC protein with this
mutation may be expected to disrupt myofilament structure. The other two changes, R249Q and R403Q, are
representative of the class of
MHC mutations found associated with human hypertrophic cardiomyopathy (Geisterfer-Lowrance et al., 1990
; Rosenzweig et al., 1991
). In
families with these two mutations the disease has high
penetrance and a high incidence of sudden death (Epstein
et al., 1992
; Watkins et al., 1992
). Furthermore, the R403Q
mutant protein has been shown to display defective contractile function in a variety of in vitro assays (Cuda et al.,
1993
; Sweeney et al., 1994
; Lankford et al., 1995
).
MHC molecule unstable once expressed in cultured cells. However,
transiently transfected cultures of COS cells immunostained with either 4H3 (anti-
MHC) or the epitope specific antibodies show detectable levels of
MHC protein in
~25% of the cells for all constructs (data not shown). Furthermore, the frequency of transfection in NRC is the
same for all of the
MHC expression plasmids (
1% of
NRC was transfected). Taken together, these studies indicate that the epitope tagged wild-type and mutant proteins
were synthesized at comparable levels.
MHC Assembles in
a Normal Sarcomeric Pattern in Transfected Neonatal
Rat Cardiomyocytes
MHC protein accumulates and was detectable
in NRC transiently transfected with the expression constructs containing the
MHC epitope tagged cDNA. In
transfected NRC, the human wild-type
MHC protein
tagged with either EE or HA epitopes assembled into highly ordered sarcomeres in cells with organized myofilaments (Fig. 2). Ordered myofibrils containing exogenous
human
MHC have defined H zones in addition to A
band periodicity complementary to thin filament staining
within the same cell. Clearly defined H zones visible in
cells immunostained for human
MHC showed that the
globular head of the tagged myosin was excluded from the
H zone which indicated that these thick filaments have
normal structure. This result was similar to the previously
described distribution of epitope tagged myosin light chain
(Soldati and Perriard, 1991
), which decorated the A band
and was excluded from the H zone, a region devoid of myosin heads. The thick filament marker myomesin was found
at the M line in mature myofibrils containing wild-type human
MHC (data not shown). Furthermore, the staining
pattern of human
MHC was coincident with endogenous
MHC as well as exogenous epitope tagged myosin light chain
(data not shown). Similar to the control cultures, endogenous thin and thick filament markers detected in transfected cells displayed the same degree of organization observed for the exogenous wild-type protein (Table I). These data indicate that the exogenous human
MHC protein
assembled normally into sarcomeres in transfected NRC,
and the presence of the glu-glu or HA epitope tag between amino acids 3 and 4 did not inhibit this process nor
disrupt sarcomeric organization.
The K184R Mutation Disrupts Sarcomeric Structure in Transfected NRC
The K184R mutation in MHC resulted in a severe alteration in the subcellular localization of this mutant protein
in transfected cardiomyocytes (Fig. 3). The distribution of
K184R protein was visualized as diffuse and somewhat
uniform throughout the cell or occurred as cable-like structures. Alternatively, K184R accumulated as brightly staining globules of various size. NRC with large amounts of
K184R protein (i.e., stain very brightly for this molecule) were small and irregularly shaped with many pointed
lamellipodia (data not shown). As seen in Fig. 3 B, K184R
mutant protein formed cable-like structures similar to those
described as the premyofibril (Rhee et al., 1994
). In this
same cell, endogenous MHC (Fig. 3 A) displayed nearly
complete loss of sarcomeric organization. The presence of
K184R
MHC in cardiomyocytes caused disorganization of endogenous myomesin (Fig. 3, C and D), indicating that
thick filament structure is disturbed. Thin filament order
was also disrupted in cells expressing K184R (Fig. 3, E
and F). In a series of experiments, >2,000 individual cells
successfully transfected with K184R were analyzed by immunostaining. In more than half of these cells sarcomeric
organization of thick or thin filament markers was completely absent. The remaining K184R positive cells contained few sarcomeres, except for rare cells in which the
entire cell volume was filled with highly organized myofibrils (Table I). In NRC with a few recognizable myofilaments, K184R
MHC sometimes appeared to decorate
thin filaments or occurred in A band regions, albeit in a
sporadic pattern that never filled the entire sarcomere. Even though organized phalloidin staining was lost in
many cardiomyocytes with K184R, G actin was readily detectable (Fig. 4). The scattered distribution of K184R protein within the cell appears to colocalize with G actin, indicating that these molecules may be in close association
with one another.
Mutations at Amino Acids 249, 403, and 472 Do Not Alter MHC Assembly
There was no detectable difference in the distribution of
phenotypes comparing expression of wild-type MHC with
the three
MHC mutations R249Q, R403Q, or S472V in
NRC. All three of these mutant proteins assembled into
extremely organized myofibrils (Fig. 5) at the same frequency as observed for the wild-type
MHC protein (Table I). R249Q, R403Q, and S472V mutant proteins assembled into A band regions as defined via costaining for
endogenous myofilament proteins (Figs. 5 and 6). This result indicated that the three mutants are readily incorporated into thick filaments, and such inclusion did not produce an observable defect in sarcomere organization. Unlike
the K184R mutant there was little excess accumulation or abnormal subcellular localization of the R249Q, R403Q,
or S472V molecules even in cells with little sarcomeric
structure. These three mutant proteins always colocalized
with the endogenous MHC in transfected cells.
Coexpression of Mutant and Wild-type MHC in the
Same Cell
Expressing both mutant and wild-type MHC within the
same transfected cell provided a sensitive measure of detecting minor differences in subcellular localization of the
two proteins. Utilization of two different epitopes inserted
at the same location within the
MHC sequence facilitated
immunostaining of both mutant and wild-type
MHC proteins within the same cell. However, the two epitopes that
were useful in the
MHC context were both recognized by
mouse monoclonal antibodies. Therefore a sequential staining procedure was used to detect signal specific to each
primary antibody (see Materials and Methods). Fig. 7, A
and B shows that little or no bleed through signal was detected in NRC expressing only one wild-type
MHC construct that was stained with both epitope specific antibodies. In cotransfected cells, both wild-type constructs were
observed, and as would be expected, the subcellular localization was identical for the two wild-type proteins present
within the same cell (Fig. 7, C and D). Both proteins were
coincorporated into A bands in a pattern that was complementary to thin filament staining.
Coexpression of wild-type MHC with either R249Q,
R403Q, or S472V showed that the assembly characteristics
of the mutant proteins are equivalent to the wild-type molecule. All three mutant proteins coassembled into thick filaments with the same localization as the wild-type
MHC
within the same cell (Fig. 7, E and F). There was no abnormal distribution or accumulation of mutant in relation to
wild-type
MHC in NRC expressing these human proteins. In contrast, coexpression of K184R disrupts the sarcomeric organization of wild-type human
MHC within
the same cell (data not shown).
In this paper we describe an in vivo system that should
prove useful in the molecular dissection of structure-function relationships between specific residues in contractile
proteins and their role in myofibrillar assembly in cardiac
muscle cells. The in vivo competition assay can determine
the sorting characteristics of individual contractile protein
isoforms, elucidating subtle differences in subcellular localization within the cardiomyocyte (Komiyama et al., 1996).
As an initial model system we have focused on human cardiac
MHC proteins since point mutations in this protein
are implicated as causative in hypertrophic cardiomyopathy. Furthermore, since MHC are highly conserved, insights from studies of invertebrate and nonmuscle myosins
can be analyzed for effects in mammalian cells.
Full length cDNAs expressed MHC protein when transiently transfected into primary cultures of neonatal rat
ventricular cardiomyocytes. The distribution of the wildtype human protein is not the result of sticking to myofilaments, based upon the observed highly ordered nature of
the immuno-stained sarcomeres containing human
MHC. Thick filaments containing human
MHC are normal in
structure based upon the A band localization and the distribution of myomesin staining. Furthermore, the lack of
any fluorescent signal found within the H zone strongly
suggests the exact incorporation of human
MHC into
thick filaments. Previous studies have shown that a truncated form of MHC containing the S1 and S2 domains does not incorporate into sarcomeres in a defined pattern (Johnson et al., 1988
), making it unlikely that a premature stop
codon is present in the
MHC cDNAs expressed in the
current studies.
A critical aspect of this in vivo, dual epitope competition
assay is that the presence of the EE or HA epitopes does
not affect the assembly of the human MHC molecule
into normal sarcomeres of neonatal rat cardiomyocytes.
Slightly more than one-third of the NRC transfected with
either wild-type epitope tagged construct is filled with
highly ordered myofibrils which have incorporated the exogenous protein throughout their length. This is nearly the
same number of highly organized cells observed in NRC
cultures transfected with the backbone vector. NRC transfected with the epitope tagged myosin light chain construct,
MLC3f-vsv, also show a similar pattern in which approximately one third of the transfected cells have perfectly ordered myofibrils throughout the cell (data not shown). It
should be noted that previous studies have demonstrated that the epitope tag on MLC3f-vsv does not affect subcellular localization of this molecule in cultures of micro-injected
adult rat (Soldati and Perriard, 1991
) or in transfected NRC
(Komiyama et al., 1996
).
A point mutation in the ATP binding domain of human
cardiac MHC acts as a dominant negative on myofilament structure. The subcellular localization of human
MHC protein in transfected NRC is dramatically affected by the K184R mutation. The corresponding mutant
in Dictyostelium MHC (K185R) binds actin "normally"
but will not release in the presence of ATP (Spudich, J.,
personal communication). In vivo the Dictyostelium mutant can form thick filaments, however the mutant-containing cells have the same phenotype as cells in which the
myosin gene is deleted. The K184R mutation is located in
the consensus nucleotide binding sequence (GlyXXXXGlyLys; Walker et al., 1982
). The lysine in this sequence contributes to nucleotide binding via neutralization of one of
the phosphate groups as proposed from the crystal structure of two GTP binding proteins, EF-Tu and Ha-ras (Jurnak, 1985
; Pai et al., 1989
). The inability to hydrolyze or
bind nucleotide has been described for mutations in other
amino acids within the ATP binding consensus in several
proteins such as ras (Clanton et al., 1987
), herpes virus thymidine kinase (Liu and Summers, 1988
), and Escherichia coli adenylate kinase (Reinstein et al., 1988
). Since the nucleotide binding sequence is so highly conserved it would
be predicted that the K184R mutation in the human
MHC protein is a "rigor-like" molecule which binds
strongly to actin, resulting in the inability to complete the
energy transduction cycle. This is consistent with the observed mutant myosin distribution as well as the colocalization of human K184R and endogenous actin. In vitro
analysis should elucidate the biochemical characteristics of
this mutant myosin.
Human K184R MHC may be incorporated into thick
filaments in transfected NRC however sarcomeric organization is disrupted in these cells. A similar effect occurs
when cytoplasmic actins are over expressed in cardiomyocytes leading to the decay of thin filaments (von Arx et al.,
1995). In contrast to the K184R mutant, cardiac cells containing cytoplasmic actin maintain the framework of the
myofibril including Z lines, A bands, and M lines. Some sarcomeric assemblages can be found in K184R expressing
NRC, however the predominant phenotype for K184Rpositive cardiomyocytes is the lack of thick and thin filament
organization (Figs. 3 and 4). This suggests a dominant negative action for the mutant K184R myosin. Mutations in
the glycine residues of the consensus ATP binding sequence
in C. elegans MHC B are dominant, disrupting thick filament assembly and muscle function (Bejsovec and Anderson, 1988
, 1990
). Thin filament number and organization
are normal in these animals. Only small amounts of the
mutant MHC B protein accumulates in the body wall muscles, which may account for the presence of thin filaments.
To our knowledge this is the first report of a myosin mutation, K184R, that destroys the structure of both thick and
thin myofilaments.
Sarcomeric disorder in NRC expressing K184R may be
the result of blocking nascent myofilament assembly. K184R
can form short strings of cable-like structures (Fig. 3) similar to the phenotype of loosely aligned MHC filaments
found in developing sarcomeres (Lu et al., 1992). Myomesin, which is found only in mature myofibrils (Lin et al.,
1994
), is not associated with K184R
MHC protein outside of organized sarcomeres. In cells costained for epitope tagged
MHC and actin, the K184R mutant colocalizes with actin throughout the cell (Fig. 4). This suggests
that premyofibril thin filaments (Rhee et al., 1994
), or I-Z-I
structures (Schultheiss et al., 1990
), are present in association with the mutant myosin that may be irreversibly
bound to actin, and this aggregation blocks the maturation
of the myofibril. Alternatively, K184R may destroy the cytoarchitecture after incorporation into sarcomeric thick filaments. It is possible that both nascent assembly is blocked
and the destruction of myofilaments is occurring in the presence of K184R. The abnormal distribution of K184R
in transfected NRC validates the use of this system to explore sequence-specific requirements on myosin assembly
through single amino acid changes created in vitro.
The S472V change does not disrupt assembly of human
MHC, documenting the specificity of the effect of the
K184R mutation on sarcomeric assembly. The corresponding Dictyostelium mutant myosin, S465V, has normal actin
binding and actin activated ATPase activities yet moves
actin filaments at only 10% of the rate of wild-type MHC
(Ruppel and Spudich, 1996
). This amino acid is part of a
random coil that is highly conserved in all myosins and
which connects the upper and lower domains of the 50-kD
fragment in the myosin head (Rayment et al., 1993a
). It is
thought that movement of these two domains occurs during the transition from weak to strong actin binding, which
may decrease affinity for the products of ATP hydrolysis
thus initiating the power stroke (Rayment et al., 1993b
).
Dictyostelium myosin with this mutation partially rescues
the myosin null phenotype and has increased basal ATPase activity yet has severely impaired ability to mobilize
actin filaments in vitro (Ruppel and Spudich, 1996
). It is
plausible that a similar effect on
MHC function is produced by the S472V human
MHC mutation, which would
suggest that productive acto-myosin interaction is not required for sarcomere assembly. Alternatively, mutation of this highly conserved amino acid does not alter function in
the human
MHC protein.
In contrast to the K184R ATP binding site mutation, hypertrophic cardiomyopathy mutations, R249Q and R403Q,
in human MHC display normal assembly and have no
dominant negative effects on sarcomeric structure. The
subcellular localization of these two proteins is coincident
with endogenous thick filament markers myomesin, MHC
and myosin light chain. In the cotransfection assay HCM
mutant MHCs colocalized with wild-type human
MHC,
and both proteins assembled into structurally normal myofilaments. This is consistent with the observation for
MHC
R403Q/+ mice in which mutant
MHC accumulates normally and cardiac dysfunction precedes histopathologic
changes (Geisterfer-Lowrance et al., 1996
). It is unclear,
however, why Marian et al. (1995)
have observed sarcomere disarray when expressing human
MHC R403Q in
adult feline cardiomyocytes. These authors used an adenoviral vector to infect cultured adult cardiomyocytes and
described sarcomere disorganization in cells 5 d after being exposed to high MOI of virus containing
MHC cDNA under the control of the CMV promoter. Marian et al.
(1995)
are unable to show that
MHC protein is incorporated into sarcomeres of infected cells. Furthermore, adult
rat cardiomyocytes dedifferentiate with prolonged exposure to culture conditions (Messerli et al., 1993
), and this
may also be the case for the feline system. Prolonged culture of NRC transfected with our human
MHC constructs does not change the observations presented and,
expression of our R403Q construct in regenerating adult
rat cardiomyocytes does not interfere with normal myofilament structure (data not shown). However, it is possible
that myofibrils in adult feline myocytes are more sensitive
to disruption by the R403Q mutant protein. In addition, the NRC system may not accurately mimic the mechanical
load found in vivo which may be necessary to produce disarray.
The data presented in this paper support the interpretation that a decrease in MHC function is the primary defect in the etiology of hypertrophic cardiomyopathy. These
results are consistent with the idea that MHC functional
insufficiency is the primary stimulus for the hypertrophic
cardiomyopathy phenotype. Therefore, compensatory hypertrophy and sudden death may be secondary responses to chronic decreased contractile function. R403Q
MHC
protein purified from HCM patients has impaired ability
to mobilize actin filaments in vitro (Cuda et al., 1993
), and
skeletal muscle fibers from such patients have reduced
power output as well as decreased unloaded maximal velocity of shortening (Lankford et al., 1995
). Decreased
functional capabilities have also been observed for other
HCM
MHC mutations. Future studies employing analysis of single cell function in live transfected NRC should
address the relationship between the biochemical defect in
human
MHC and its effect on contractility.
It is not necessary to propose a poison effect of the
MHC molecule on the thick filament, as a functional insufficiency would not interfere with the wild-type
MHC
present in HCM patients, yet could still present the stimulus for hypertrophy, which is the hallmark of this disease.
Transgenic mice overexpressing oncogenic ras in the heart
have ventricular hypertrophy, selective diastolic dysfunction (Hunter et al., 1995
), increased systolic function, myocyte disarray, fibrosis, and increased juvenile mortality
(Gottshall et al., 1997
). This suggests that chronic stimulation of a second messenger system in the heart produces
all the overt characteristics of hypertrophic cardiomyopathy in humans caused by mutations in sarcomeric proteins.
The data presented in this paper suggest that cellular dysmorphology is also secondary to biochemical dysfunction of the sarcomere producing decreased heart function.
Received for publication 14 October 1996 and in revised form 14 January 1997.
The authors are very much indebted to Mahmoud Itani for excellent technical assistance in preparation of neonatal rat cardiomyocytes. The authors thank the Institute for Biomedical Engineering Confocal and Imaging Core for the use of the confocal microscope. We also thank Drs. Sanford Bernstein, Pieter Doevendans, Peter Gruber, Kirk Knowlton, and Howard Rockman for discussions and critical reading of the manuscript.HCM, hypertrophic cardiomyopathy; MHC, myosin heavy chain; NRC, neonatal rat cardiomyocytes.