(Circulation Research. 1998;83:580-593.)
© 1998 American Heart Association, Inc.
Familial Hypertrophic Cardiomyopathy
From Mutations to Functional Defects
Gisèle Bonne,
Lucie Carrier,
Pascale Richard,
Bernard Hainque,
, Ketty Schwartz
From the INSERM Unit 153 (G.B., L.C., K.S.), the Service de Biochimie B
(P.R., B.H.), and the IFR de Physiologie et Génétique
Cardiovasculaire (G.B., L.C., P.R., B.H., K.S.), Groupe Hospitalier
Pitié-Salpêtrière, Paris, France.
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Abstract
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AbstractHypertrophic
cardiomyopathy is characterized by left and/or
right ventricular hypertrophy, which is usually
asymmetric and involves the interventricular septum.
Typical morphological changes include myocyte hypertrophy
and disarray surrounding the areas of increased loose connective
tissue. Arrhythmias and premature sudden deaths are common.
Hypertrophic cardiomyopathy is familial in the
majority of cases and is transmitted as an autosomal-dominant trait.
The results of molecular genetics studies have shown that familial
hypertrophic cardiomyopathy is a disease of the
sarcomere involving mutations in 7 different genes encoding proteins of
the myofibrillar apparatus: ß-myosin heavy chain,
ventricular myosin essential light chain,
ventricular myosin regulatory light chain, cardiac troponin
T, cardiac troponin I,
-tropomyosin, and cardiac myosin binding
protein C. In addition to this locus heterogeneity,
there is a wide allelic heterogeneity, since numerous
mutations have been found in all these genes. The recent development of
animal models and of in vitro analyses have allowed a better
understanding of the pathophysiological mechanisms
associated with familial hypertrophic
cardiomyopathy. One can thus tentatively draw the
following cascade of events: The mutation leads to a poison polypeptide
that would be incorporated into the sarcomere. This would alter the
sarcomeric function that would result (1) in an altered cardiac
function and then (2) in the alteration of the sarcomeric and myocyte
structure. Some mutations induce functional impairment and support the
pathogenesis hypothesis of a "hypocontractile" state followed by
compensatory hypertrophy. Other mutations induce cardiac
hyperfunction and determine a "hypercontractile" state that would
directly induce cardiac hypertrophy. The development of
other animal models and of other mechanistic studies linking the
genetic mutation to functional defects are now key issues in
understanding how alterations in the basic contractile unit of the
cardiomyocyte alter the phenotype and the function
of the heart.
Key Words: familial hypertrophic cardiomyopathy genetic mutation mouse model sarcomeric protein alteration in cardiac contractility
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Introduction
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Observations of myocardial diseases that can reasonably
be interpreted as hypertrophic cardiomyopathy were
made in the middle of the last century at the Hôpital La
Salpêtrière in Paris by A. Vulpian, who called what he saw
at the macroscopic level a "rétrécissement de l'orifice
ventriculo-aortique" or "sub-aortic
stricture."1 However, it was only in the late
1950s that the unique clinical features of hypertrophic
cardiomyopathy were systematically described. It is
characterized by left and/or right ventricular
hypertrophy, which is usually asymmetric and which can
affect different regions of the ventricle. The
interventricular septum is most commonly affected, with or
without involvement of either the anterior wall or the posterior wall
in continuity. A particular form of regional involvement affects the
apex but spares the upper portion of the septum (apical
hypertrophy).2 Typically, the left
ventricular volume is normal or reduced. Systolic
gradients are common. Typical morphological changes include myocyte
hypertrophy and disarray surrounding the areas of increased
loose connective tissue. Arrhythmias and premature sudden
deaths are common.3 4 Although hypertrophic
cardiomyopathy has been regarded largely as a
relatively uncommon cardiac disease, the prevalence of
echocardiographically defined hypertrophic
cardiomyopathy in a large cohort of apparently
healthy young adults selected from a community-based general population
was reported 3 years ago to be as high as 0.2%.5
Familial disease with autosomal-dominant inheritance predominates, and
the first large pedigree of familial hypertrophic
cardiomyopathy (FHC) was reported in
1960.6 None of the previous hypotheses of the
pathophysiological mechanisms would have predicted
that defects in sarcomeric genes could be a possible molecular basis
for the disease. The results of molecular genetic studies have
nevertheless shown that all mutations found so far concern sarcomeric
proteins: 3 myofilament proteins, the ß-myosin heavy chain
(ß-MyHC), the ventricular myosin essential light chain 1
(MLC-1s/v), and the ventricular myosin regulatory light
chain 2 (MLC-2s/v); 3 thin-filament proteins, cardiac troponin T
(cTnT), cardiac troponin I (cTnI), and
-tropomyosin (
-TM); and,
finally, 1 myosin binding protein, the cardiac myosin binding protein C
(cMyBP-C) (Table
). These genes certainly do not
represent the whole spectrum of FHC disease genes since an
additional locus was reported,7 and one might
reasonably hypothesize that disease genes yet to be identified include
additional components of the sarcomere. One of the next challenges is
to decipher the mechanisms through which the disease results from
sarcomeric gene defects. The focus of this article is to review the
current state of knowledge involving (1) the organization and mutations
of FHC disease genes and proteins and (2) the in vitro and in vivo
functional consequences of these mutations.
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Organization and Mutations of FHC Disease Genes
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All the disease genes code proteins that are part of the
sarcomere, which is a complex structure with an exact stoichiometry and
multiple sites of protein-protein interactions. Myosin is the molecular
motor that transduces energy from the hydrolysis of ATP into directed
movement and that, by doing so, drives sarcomere shortening and muscle
contraction. Cardiac myosin is a conventional class II myosin that
consists of 2 heavy chains (MyHCs) and 2 pairs of light chains (MLCs),
referred to as essential (or alkali) light chains (MLC-1) and
regulatory (or phosphorylatable) light chains (MLC-2), respectively
(reviews in References 8 and 98 9 ). The myosin molecule is highly
asymmetric, consisting of 2 globular heads joined to a long rodlike
tail. The light chains are arranged in tandem in the head-tail
junction. Their function is not fully understood. Neither MLC type is
required for the ATPase activity of the myosin head, but they probably
modulate it in presence of actin and contribute to the rigidity of the
neck, which is hypothesized to function as a lever arm for generating
an effective power stroke. Mutations were found in the heavy chain and
in the 2 types of ventricular light chains. The troponin
complex and tropomyosin constitute the calcium-sensitive switch
that regulates the contraction of cardiac muscle fibers. Mutations were
found in the
-TM and in 2 of the subunits of the troponin complex:
cTnI, the inhibitory subunit, and cTnT, the tropomyosin
binding subunit. As for cMyBP-C, its function is uncertain, but for a
decade, evidence has existed to indicate both structural and regulatory
roles. Partial extraction of cMyBP-C from rat skinned cardiac myocytes
and rabbit skeletal muscle fibers alters calcium-sensitive
tension,10 and it was shown that
phosphorylation of cMyBP-C alters myosin crossbridges
in native thick filaments, suggesting that cMyBP-C can modify force
production in activated cardiac
muscles.11 Myosin and MyBP-C are part of the
thick filaments of the sarcomere, with MyBP-C being located at the
level of the transverse stripes, 43 nm apart, seen by electron
microscopy in the sarcomere A-band (Figure 1
).12 Troponins and
tropomyosin are located in the thin filaments. Each of these proteins
is encoded by multigene families, which exhibit tissue-specific,
developmental, and physiologically regulated
patterns of expression.

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Figure 1. Schematic organization of the sarcomeric proteins
associated with FHC. FHC disease genes encode contractile proteins of
the thick filament (in green), associated proteins of the thin filament
(in blue), and a myosin binding protein (in red). The Z lines and the
titin filament are indicated in gray and black, respectively. C and M
correspond to the C zone and M zone of band A of the sarcomere,
respectively.
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The organization of the human FHC disease genes and the sequences of at
least all exon-intron boundaries are known for MYH7,
MYL3, MYL2, TNNT2, TNNI3,
and MYBPC3, which encode ß-MyHC, MLC-1s/v, MLC-2s/v, cTnT,
cTnI, and cMyBP-C, respectively. For all these genes, mutational
analysis in FHC can therefore be performed on genomic DNA. As
for TPM1, which encodes
-TM, the genomic organization is
known only in the rat. The genomic and protein organizations
presented below were computed from the GENBANK, GDB, EMBL, PIR,
and SWISSPROT databanks. The nomenclature of the genes and proteins
varies from one databank to the other. For the sake of simplicity and
clarity, we have used the nomenclature of GDB for the genes and the
nomenclature of Schiaffino and Reggiani9 and of
Gulick et al13 for the proteins. The amino acid
numbering includes the NH2-terminal methionine
without taking into account possible posttranslational
modifications.
Human MYH7
ß-MyHC is the major isoform of the human
ventricle14 15 and of slow-twitch skeletal fibers
(review in Reference 99 ). It is also expressed in the human atria. The
other cardiac isoform is
-MyHC, which is predominantly expressed in
the human atria and which, in mouse and rat hearts, predominates in
both atria and ventricles. The
-MyHC isoform is encoded by the
MYH6 gene. The MYH6 and MYH7 genes are
organized in tandem in a cluster on chromosome 14q11.2-q13, with
MYH7 being located 4 kb upstream from
MYH6.16 17 As shown on Figure 2
, MYH7 is composed of 40
exons, 38 of which are coding, and encompasses
23 kb of
DNA.18 19 It encodes a protein of 1935 amino
acids. The globular amino-terminal part (subfragment 1 [S1])
corresponds to the motor domain that contains the ATP binding site and
the actin binding site. Recent studies conducted on a skeletal/smooth
chimeric myosin showed that the ATPase cycle rate (thus, the ATP
hydrolysis rate or phosphate release rate) is solely determined by the
globular head domain.20 The neck region of MyHC,
which consists of a long 8.5-nm
-helix, is associated with the light
chains (review in Reference 88 ). The COOH-terminal rod, which has a
characteristic
-helical coiled-coil structure with 7-residue and
28-residue repeats, is responsible for the assembly of myosin into
thick filaments. MYH7 contains 2 polymorphic
dinucleotide repeats, one in the promoter region and one in
the 24th intron, that we have named MYOI and MYOII, respectively, and
that allow easy linkage analysis in FHC
families.21 22

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Figure 2. Schematic representation of
MYH7 and ß-MyHC. Top, Genomic structure with location
of the mutations. The translation start codon (ATG) is indicated, and
the exons containing mutations are numbered and indicated by gray
boxes. Bottom, Protein structure. The major proteolytic fragments S1,
S2, and light meromyosin (LMM) are indicated. The functional regions
involved in ATP and actin binding, the "active thiols" (SH1 and
SH2), and the S2 hinge [H(S2)] are indicated.
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At least 50 mutations were found in unrelated families with
FHC23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 (Figure 2
), and 3 hot spots for
mutations were identified, codons 403,23 32
719,37 38 and 741.33 44 All
but 3 of these mutations are missense mutations located either in the
head or in the head-rod junction of the molecule. The 3 exceptions are
two 3-bp deletions that do not disrupt the reading frame, one of codon
1046 and the other of codon
930,52 and a 2.4-kb deletion in the 3'
region.28 In the kindred with the latter
mutation, only the proband had developed clinically diagnosed
hypertrophic cardiomyopathy at a very late onset
(age, 59 years). Finally, a termination codon at position 54 was found
by chance in the mother (38 years old) and the grandmother (70 years
old) of an affected child (16 years old) who had also inherited from
his affected father a missense mutation in exon
22.47 The allele corresponding to the
nonsense mutation should encode a short variant of the ß-MyHC
comprising only the first 53 residues of the molecule, yet it does not
present the characteristics of a disease-causing mutation, since
the 2 women were clinically unaffected.
To examine the structural consequences of the mutations in the
MYH7 gene, 29 mutations have been precisely positioned on
the 3-dimensional structure of chicken skeletal myosin S1, which is
expected to be very similar to that of the human ß-MyHC
S1.48 Twenty-four mutations do not appear to
occur randomly in the structure but, rather, to cluster to 4 discrete
localizations: the actin binding interface, around the
nucleotide binding site, adjacent to the region that
connects 2 reactive cysteine residues, and, finally, in proximity to
the interface of the heavy chain with the essential light chain. The
remaining 5 are in the myosin rod.
Human MYL3
The ventricular myosin essential (or alkali) light
chain, MLC-1s/v, is expressed both in the ventricular
myocardium and in the slow-twitch
muscles.54 It belongs to the superfamily of
EF-hand proteins, which includes calmodulin and troponin C
(review in Reference 5555 ). Thus, it folds into a dumbbell-like structure
shape, with 2 helix-loop-helix structural motifs in each half of the
molecule, which indicate the existence of 4 putative calcium binding
sites (review in Reference 5656 ). Indeed, in scallop myosin (but not in
human ventricular myosin), calcium binding by the myosin
essential light chain switches the motor on.57 In
chicken skeletal muscle, removal of the myosin essential light chain
slows the velocity at which skeletal muscle myosin moves actin in a
motility assay58 and reduces isometric force by
>50%.59 Synthetic peptides corresponding to the
NH2 terminus of human MLC-1s/v were recently
shown to increase contractility of intact and
chemically skinned human heart fibers60 and to
induce supramaximal stimulation of rat myofibrillar ATPase activity
under specific stoichiometric conditions.61 These
data strongly suggest that MLC-1s/v potently produces an inotropic
effect through a cooperative mechanism that may involve activation of
the entire thin filament.
MYL3 is located on chromosome 3p21.2-p21.3. It is composed
of 7 exons, 6 of which encode a polypeptide of 195 amino
acids62 (Figure 3
).
Six functional domains were putatively characterized, an actin binding
site, a proline-rich region, and 4 helix-loop-helix regions. Because
the 3-dimensional structure of chicken S1 demonstrated that this MyHC
region bearing a cluster of mutations represents an interface
with MLC-1s/v,48 Poetter et
al63 hypothesized that mutations in this MLC
might result in similar abnormalities in a subset of families with FHC.
By screening DNA from representatives of 383 unrelated
families with FHC, these authors found 2 missense mutations in exon 4,
one in all affected members of a family and the other in an unrelated
individual (Figure 3
). Half of these patients (7 of 14) exhibited a
rare phenotype involving mid left ventricular
chamber thickening.

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Figure 3. Schematic representation of genomic and
protein structures of MLCs. For each protein, the genomic structure
with intron-exon boundaries is shown at the top of each panel.
Noncoding regions are indicated in dark gray; location of mutations, by
stars. The exons containing mutations are numbered. The protein
structure with mutations is shown at the bottom of each panel. Arrows
indicate the different structural domains, and the amino-acid numbers
correspond to the limits of the structural domains. EF indicates the
EF-hand domain; S15, phosphorylation site.
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Human MYL2
The ventricular myosin regulatory light chain,
MLC-2s/v, is expressed both in the ventricular
myocardium and in the slow-twitch
muscles.64 Like the essential MLC, it belongs to
the superfamily of EF-hand proteins, and its removal significantly
decreases the velocity of actin movement on skeletal
myosin.58 However, at variance with MLC-1,
removal of skeletal MLC-2 has little effect on isometric
force.59 Phosphorylation of MLC-2
by MLC kinase is essential for actin-myosin interaction in smooth
muscles, but it has only a modulator role in striated
muscles.65 Recent studies conducted in the mouse
highlighted the role of MLC-2s/v.13 66 The
partial transgene-driven replacement of MLC-2s/v with the skeletal
isoform reduces both left ventricular
contractility and relaxation, although the unloaded
shortening velocity of isolated ventricular
cardiomyocytes is not significantly
different.13 The disruption of the MLC-2s/v gene
results in sarcomeric disassembly and in an embryonic form of dilated
cardiomyopathy, indicating that there is a
selective requirement for MLC-2s/v in the normal development of
ventricular cardiac myocyte structure and
function.66
MYL2 is located on chromosome
12q23-q24.3,67 and we have recently refined its
localization on the genetic map, in an interval of 6 centimorgans
containing 6 informative microsatellites.68 MYL2 encompasses
12 kb of genomic DNA; it is composed of 7
exons, all of which are coding ones (Figure 3
). The encoded polypeptide
is composed of 166 amino acids. Five active sites have been
characterized, a phosphorylation site on serine 15 in
exon 2 and 4 EF-hand domains, one of which has retained the ability to
bind a metal ion and which is most likely occupied by
Mg2+ in vivo.69 Five
mutations were reported (Figure 3
)63 68 : 2 of
them (E22K and A13T) are associated with the same rare
phenotype seen with mutations in MYL3 (see above)
involving mid left ventricular chamber
thickening,63 whereas mutations F18L and R58Q are
associated with familial and classical forms of hypertrophic
cardiomyopathy.68
Human TNNT2
In human cardiac muscle, multiple isoforms of cTnT have been
described that are expressed in the fetal, adult, and diseased heart
and that result from alternative splicing of the single gene
TNNT2.70 71 72 The precise
physiological relevance of these isoforms is
currently poorly understood. TNNT2 was mapped by somatic
cell hybrid analysis and by fluorescent in situ
hybridization to chromosome 1q32.71 73 The
structural organization and the complete nucleotide
sequence have been determined in the rat by Jin et
al,74 and the first mutations reported in FHC
were numbered according to this rat structure.75
We have partially established the organization of the human gene, and
this allows us now to precisely identify the position of the mutations
within exons, including those alternatively spliced during development,
and also to use an amino acid numbering that reflects the full coding
potential of human TNNT2.76
TNNT2 is composed of 17 exons spread over 17 kb and
gives rise to multiple isoforms by the use of both alternative exons
and alternative acceptor sites (Figure 4
). The cardiac isoform shares the
overall structure and function of other troponin isoforms. It is an
asymmetric molecule of
37 kDa whose elongated
NH2-terminal part extends for a considerable
length along tropomyosin and spans the head-to-tail overlap of
tropomyosin (review in Reference 7777 ). It contains several putative
functional domains: a phosphorylation site in the
NH2-terminal region, an
-tropomyosin binding
region between exons 9 and 12, and in the COOH-terminal part a region
that binds in a calcium-dependent manner
-tropomyosin, troponin C,
and troponin I and that contains 3 phosphorylation
sites.77 The phosphorylation of
cTnT results in an inhibition of the maximum ATPase rate of
reconstituted thin-filament preparations showing that cTnT plays a role
in the regulation of crossbridge kinetics (review in Reference
7777 ). Eleven mutations were found in unrelated FHC
families,75 76 78 79 80 3 of which are located in a
hot spot (codon 102) (Figure 4
).75 76 79 Ten
mutations are missense ones located between exons 9 and
17,75 76 78 79 80 one mutation is a 3-bp deletion
located in exon 12 that does not disrupt the coding
frame,78 and the last is located in the intron 16
splice donor site and is predicted to produce a truncated protein in
which the C-terminal binding sites are
disrupted.75

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Figure 4. Schematic representation of genomic and
protein structures of cTnT, cTnI, and -TM. For each
protein, the genomic structure with intron-exon boundaries is shown at
the top of each panel. Noncoding regions are indicated in black;
alternative spliced exons, in gray; and location of mutations, by
stars. The exons containing mutations are numbered. The protein
structure with mutations is shown at the bottom of each panel. Arrows
indicate the different binding domains, and the amino acid numbers
correspond to the limits of the structural domains. The cTnT amino acid
numbering corresponds to that of Forissier et al76 and
takes into account the full coding potential of the gene. The EMBL
accession numbers of the human genomic sequence of TNNT2
are X98477, X98478, X98479, X98480, X98481, X98482, X98483, Y09626,
Y09627, and Y0628. S2, T204, T213, and T294 are
phosphorylation sites of cTnT, and S23, S24, S42, and
S44 are phosphorylation sites of cTnI.
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Human TNNI3
The cTnI isoform is expressed only in cardiac
muscles.81 TNNI3, located on
chromosome 19p13.2-q13.2,82 comprises 8 exons
contained within 6.2 kb of genomic DNA (Figure 4
). It encodes a
polypeptide of 210 amino acids that shows a high degree of homology
with the fast-skeletal and slow-skeletal isoforms in the COOH-terminal
region, whereas the NH2-terminal region is more
divergent.83 84 Cooperative binding of cTnI to
actin-tropomyosin is a unique property of the cardiac variant (review
in Reference 8585 ). cTnI contains several functional domains: (1) the
NH2-terminal extension that contains 2 sites at
serine residues 23 and 24, the phosphorylation of which
alters calcium sensitivity and eliminates cooperative binding to actin,
(2) the near NH2-terminal domain that binds to
the COOH terminus of cardiac troponin C and that contains 2 sites at
serine residues 42 and 44, the phosphorylation of which
reduces the maximum ATPase rate, (3) the inhibitory region
that binds to actin and to cardiac troponin C and that causes
relaxation through inhibition of the actomyosin interaction, and (4)
the COOH-terminal domain, which is essential for the calcium
sensitivity of the myofilaments.85 86 Six
mutations were recently identified (Figure 4
).84
Five are missense mutations located in exons 7 and 8, and one is a
K183
mutation that does not disrupt the coding frame.
Human TPM1
TPM1 encodes several isoforms generated by alternative
splicing (review in Reference 8787 ). The cardiac isoform is expressed
both in the ventricular myocardium and in
fast-twitch skeletal muscles. It shares the overall structure of other
tropomyosins that are rodlike proteins that possess a simple dimeric
-coiled-coil structure in parallel orientation along their entire
length. The coiled-coil structure is based on a repeated pattern of 7
amino acids, with hydrophobic residues at the first and fourth
positions. These dimers are arranged in a head-to-tail fashion, lie in
the major groove of actin filaments, and span 7 actin monomers.
The chromosomal localization of TPM1 is known in humans
(15q22) and not in rats,75 88 but its complete
organization has been determined in rats and not in humans (review in
Reference 8787 ). TPM1 is composed of 14 exons, with exons 1a,
2b, 3, 4, 5, 6b, 7, 8, 9a, and 9b being expressed in the cardiac tissue
(Figure 4
). The encoded polypeptide is composed of 284 amino acids and
contains 2 putative troponin T binding domains that attach
-TM to
the troponin complex, one near C190, where troponin C and troponin I
occur, and one along the COOH-terminal stretch of the molecule (Figure 4
).77 Four missense mutations were found in
unrelated FHC families.75 89 90 91 Two of them,
A63V and K70T, are located in exon 2b within the consensus pattern of
sequence repeats of
-TM and could alter tropomyosin binding to
actin.89 90 Mutations D175N and E180G are both
located within constitutive exon 5, in a region near the C190 and near
the calcium-dependent troponin T binding
domain.75 89 91
Human MYBPC3
Human MYBPC3 was localized by fluorescent
in situ hybridization on chromosome 11p11.2, and its precise position
was determined by radiation hybrid mapping between loci
D11S4133 and D11S1326.92 93
We have recently determined its organization and sequence (Figure 5
).94 It comprises
>21 kb and contains 35 exons, out of which 34 are coding. Two exons
are unusually small in size, 3 bp each. The full-length 4.5-kb cDNA
encodes a 1173-residue polypeptide that shares the overall modular
architecture of the intracellular immunoglobulin superfamily and
contains 8 IgI modules and 3 fibronectin-3 domains. Three distinct
regions are specific to the cardiac isoform: the
NH2-terminal domain C0 IgI containing 101
residues, the MyBP-C motif (a 105-residue stretch linking the C1 and C2
IgI domains), and a 28-residue loop inserted in the C5 IgI
domain.93 The MyBP-C motif is not specific to the
cardiac isoform, but the alignment of skeletal and cardiac sequences
revealed the addition of a 9-residue loop in the cardiac variant, which
is the key substrate site for phosphorylation by both
protein kinase A and a calmodulin-dependent protein kinase
associated with the native protein.93
We95 and others96 recently
showed that cMyBP-C is specifically expressed in the heart during human
and murine development. This result is at variance with what has been
described in the chicken, where cMyBP-C is expressed in both cardiac
and skeletal muscle during embryonic
development.97 This probably reflects a
difference in the organization of the promoter region of the avian and
the mammalian genes. The major myosin binding site of MyBP-C resides in
the COOH-terminal C10 IgI domain and is mainly restricted to the last
102 amino acids. The titin binding site is also located in the
COOH-terminal region, spanning the C8 to C10 IgI domains of the
molecule.98 99

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Figure 5. Schematic representation of
MYBPC3 and cMyBP-C and consequences of the mutations at
the protein level. Top, Genomic and normal protein structures with
locations of mutations. The translation start codon (ATG) and the
phosphorylation, titin, and myosin binding sites are
indicated. The exons or introns containing mutations are numbered, and
the aberrantly spliced exons are in black. Mutations producing either a
mutated protein or a truncated one are indicated in bold. Bottom,
Predicted protein structures resulting from the different mutations.
Seventeen mutations result in truncated proteins (intron 7 D1g-a,
intron 7 D5g-a, intron 11 A-2a-g, exon 18 1-593, intron 20 A-2a-g,
exon 22 1-698, intron 23 D1g-a, intron 23 D1g-t, exon 24 I1-791,
exon 25 5-845, exon 27 2-955, exon 27 Q969+, exon 29 I2-1042,
intron 30 D5g-c, intron 31 D1g-a, intron 32 D1g-a, and exon 33
X12/ 4-1220); 7 result in mutated or deleted proteins (exon 6 E258K,
exon 17 R495Q, exon 17 R502Q, exon 21 R654H, exon 23 N755K, intron 27
D1g-a, and exon 33 X18-1254); and 3 result either in truncated protein
or mutated one (exon 15 E451Q, exon 17 E542Q, and intron 23 BPa-g). D1
and D5 indicate donor splice site mutations at positions 1 and 5,
respectively; A-2, acceptor splice site mutation at position minus 2;
x, deletions of x bp; Ix, insertion of x bp; BP, branch-point
mutation; X, duplication; +, stop codon; g, guanine; a, adenine; and c,
cytosine. The numbers correspond to the amino acid
number.
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Twenty-seven MYBPC3 mutations were found in unrelated
families with FHC84 92 94 100 101 102 103 104 (Figure 5
).
Seventeen of them result in aberrant transcripts that are predicted to
encode COOH-terminal truncated cardiac MyBP-C polypeptides lacking at
least the myosin binding
domain.84 92 94 100 101 104 Seven others result
in mutated or deleted proteins without disruption of the reading frame:
5 are missense mutations in exons 6, 17, 21, and
23,102 103 104 one is a splice donor site mutation
in intron 27,84 and one is an 18-residue
duplication in exon 33.100 Finally, 3 mutations
are predicted to produce either a mutated protein or a truncated one: 2
are missense mutations in exons 15 and 17,94 104
and one is a branch-point mutation in intron
23.94
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Putative Molecular Mechanisms by Which Mutations Cause FHC
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Any consideration of the molecular mechanisms by which mutations
cause FHC must be consistent with the dominant mode of disease
inheritance. Most mutations found in the MYH7,
MYL3, MYL2, TPM1, TNNT2,
and TNNI3 genes are missense ones or small deletions without
a frameshift that are predicted to lead to mutant proteins, whereas
most of those found in the MYBPC3 gene are splicing
consensus sites, deletions, insertions, or nonsense mutations that are
predicted to lead to truncated proteins. Mutant proteins are presumably
present in the tissue and act in a dominant fashion, probably as
poison polypeptides; ie, they are incorporated in the sarcomere and
change the function of the wild-type protein and/or the assembly of the
sarcomeric filaments.105 Resolution of mutant
from wild-type protein has typically not been possible because of the
subtle nature of most mutations and the limited access to myocardial
specimens. The incorporation of mutant protein in vivo has been
nevertheless demonstrated for 2 mutations, the R403Q MYH7
mutation that could be distinguished in extracts from skeletal muscle
because it induces the loss of an arginine specific endoproteinase
digest site31 and the D175N TPM1
mutation because it presents a specific electrophoretic band in
skeletal muscle from affected patients.106 This
poison polypeptide hypothesis is also supported by a variety of results
obtained in vitro (see below) and by findings in nematodes in which
missense mutations produce stable polypeptides that are incorporated
into myofibrils and disrupt the sarcomere
assembly.107 108
The situation is more complex when mutations leading to truncated
proteins are involved. The mutations could induce "null
alleles" potentially leading to haploinsufficiency: the
production of insufficient quantities of a normal sarcomeric
protein would produce an imbalance in the stoichiometry of the thick-
or the thin-filament components that would be sufficient to alter the
sarcomeric structure and function. In this case, the null alleles
would exhibit a dominant phenotype. This is what occurs in
Drosophila, where heterozygotes for actin or myosin null
alleles have complex myofibrillar defects, whereas double
heterozygotes for both actin and myosin null alleles where
stoichiometry is maintained have nearly normal
myofibrils.109 This is also what occurs in
heterozygous mice for
-MyHC null alleles that have severe
impairment of both contractility and
relaxation.110 However, none of the available
data in FHC are consistent with a mechanism of
haploinsufficiency. A nonsense mutation has been found in the
MYH7 gene that is predicted to encode a short variant of
ß-MyHC protein (53 residues) in 2 healthy individuals (38 and 70
years old).47 This indicates that the single
normal human cardiac MYH7 allele is sufficient to
compensate for the heterozygous defect of the null allele. Two
other recent studies addressed the null allele hypothesis, one by
expressing truncated human cTnT in quail
myotubes111 and the other by characterizing the
transcripts and proteins present in an
endomyocardial biopsy of a patient with a cMyBP-C
splice donor site mutation.101 None of the
results were consistent with a mechanism of haploinsufficiency.
The cTnT mutation appeared to function as a dominant-negative
allele, maybe because the truncated cTnT still contained one
tropomyosin binding site. Whether the truncated cTnT protein is
expressed and stable in myocardial tissue from affected patients with
FHC remains to be determined. As for the cMyBP-C, the authors suggest
that the mutation could produce a misfolded RNA template that may
interfere with the formation of ordered sarcomeres already on the mRNA
level.101 More studies are necessary to
understand the molecular mechanisms by which mutations predicted to
lead to truncated proteins cause FHC.
 |
How Mutations Alter Sarcomere Function and Lead to
Hypertrophic Cardiomyopathy
|
|---|
The fact that mutations in different components of the sarcomere
appear to produce the same phenotype suggests that the
mutations share functional consequences that lead to a common mode of
pathogenesis of FHC. One of the major problems in our understanding of
FHC has been the difficulty in obtaining cardiac specimens from
affected patients. This has generated the development of animal models
and of in vitro analyses to study the pathophysiology of
mutations.
Mouse Models of FHC
Two mouse models of FHC have been published as full studies. They
were produced by the introduction of the R403Q MYH7 mutation
into the mouse MYH6 gene (the adult mouse ventricle contains
only
-MyHC and no ß-MyHC).112 113 In one
model, the R403Q mutation was introduced by homologous recombination;
thus, heterozygous mice carry one normal gene and one mutated
gene.112 The other model was generated by the
transgenic expression of a rat
-MyHC carrying the R403Q mutation
associated with a deletion in the actin binding site and thus giving
the transgenic mice 2 normal genes plus a mutated
one.113 Both models represent
heterologous systems, because the human ß-MyHC gene mutation was
introduced into the mouse
-MyHC.
-MyHC (high ATPase activity) is
adapted to the mouse heart, and ß-MyHC (low ATPase activity) is
adapted to the human heart. The functional consequences of the
mutations observed in the mouse myocytes may not thus represent
exactly what occurs in human myocytes. In spite of this,
analysis of these genetically different animal models allows us
to make hypotheses regarding the pathophysiological
mechanisms of the mutated
-MyHC molecules and to extrapolate them to
the ß-MyHC molecules.
In
-MyHC403/+ mice, the R403Q MYH7
gene mutation was introduced in the mouse MYH6 gene by a
"hit-and-run" homologous recombination
technique.112 Homozygous mutant mice died 7 days
after birth. Heterozygous mice survived and presented cardiac
dysfunction at 5 weeks of age, characterized by a modification of the
left ventricular pressure and a prolonged relaxation time.
Histological alterations similar to those seen in FHC
patients appeared at 15 weeks of age, including myocyte disarray,
myocyte hypertrophy, and fibrosis, suggesting that the
altered mechanical properties may lead to the
histological changes. At 15 weeks of age, no
ventricular hypertrophy was observed, whereas
atrial enlargement was present, which is at variance with what
occurs in FHC patients. Further investigations of electrocardiological
and electrophysiological
parameters demonstrated a modified QRS axis,
heterogeneous ventricular conduction, and
arrhythmia.114 All these modifications
evolved with time to a more pronounced phenotype in males than
in females, but without ventricular
hypertrophy. Since humans with FHC may experience
life-threatening events with exercise, Geisterfer-Lowrance et
al112 evaluated the impact of physical activity
on the phenotype by a swimming test in a subset of five
-MyHC403/+ mice. One mouse died suddenly while
swimming; its heart was grossly enlarged with a thrombus in the dilated
atrium, mild right ventricular hypertrophy, and
marked asymmetric left ventricular
hypertrophy.
In the transgenic mice, the transgene carrying both the R403Q
MYH7 mutation and a deletion in the actin binding site of
-MyHC was transcribed at 26% to 50% of the endogenous
-MyHC mRNA level.113 Although the mutant
protein represented only 1% to 12% of the heart's
myosin, the mice exhibited the cardiac histopathology seen in FHC
patients, namely, myocyte disarray and hypertrophy,
sarcomeric disorganization, and, finally, left and right
ventricular hypertrophy. In addition, 2
molecular markers of compensatory hypertrophy, ANF and
-skeletal actin genes, were upregulated. Evolution of the
phenotype with age showed differences between males and
females, with an evolution toward a severe left ventricular
hypertrophy for the females, whereas males developed
ventricular dilation. In contrast to the
-MyHC403/+ model, no atrial enlargement was
found.
These 2 mouse models give us several clues. First, they confirm the
hypothesis of the dominant-negative effect of the R403Q MYH7
gene mutation. Indeed, for the 2 models, the mutant protein is
synthesized, stable, and present, although in variable
amounts.112 113 The second message is that
modifications of MYH6 expression result in alterations of
the normal cardiac function and structure. This is in agreement with
what was found in the in vitro analyses of the R403Q
MYH7 mutation, which showed an impairment of the function
(see below).31 105 111 115 116 117 144 One can thus
tentatively draw the cascade of events that would lead from the R403Q
myosin mutation to the phenotype. Incorporation of a poison
protein would alter sarcomeric function, which would result first in an
altered cardiac function and then in the alteration of the sarcomeric
and myocyte structures. Obviously, the alteration of the sarcomeric
structure would contribute to the emphasis of the cardiac dysfunction,
since sarcomeres presented both abnormal structure and
function. Finally, both abnormal structure and cardiac dysfunction lead
to the compensatory response of the heart that develops
hypertrophy, the last manifestation observed in the 2
models. The third message is the incomplete penetrance of the
phenotypes as well as the sex-related evolution of the disease.
This suggests the implication of other "actors," such as modifier
genes and/or environmental factors, that could modulate the phenotypic
expression of the mutated genes.
In Vitro Analyses of the Consequences of FHC Mutations
Structural Aspects
The expression of ß-MyHC and cTNT mutant proteins in primary
cultures of ventricular cardiomyocytes or quail
myotubes does not appear to have major structural
consequences.111 117 118 119 Indeed the R403Q
and the R249Q MYH7 mutations were cloned and expressed in
primary cultures of neonatal rat
cardiomyocytes.119 Mutants were
readily incorporated into cardiac cells and did not disrupt
myofilaments even after prolonged exposure. In another study, however,
the R403Q MYH7 mutation was expressed in adult feline
cardiac myocytes and resulted in a disruption of the sarcomere
structure in 50% of the myocytes 5 days after being exposed to a high
dose of adenoviral vector; whether the ß-MyHC mutant protein is
incorporated into sarcomeres had not been
determined.118 It is unclear why the same mutant
ß-MyHC, R403Q, produced different in vitro structural consequences.
Becker et al119 suggest that myofibrils in adult
feline myocytes may be more sensitive to disruption by the R403Q mutant
ß-MyHC or that the neonatal rat cardiomyocytes system may
not accurately mimic the mechanical load found in vivo, which may be
necessary to produce disarray. As for cTnT, the expression in adult
feline cardiomyocytes of the R102Q TNNT2
mutation located in one of the
-TM binding domains showed an
integration of the cTnT mutant in the sarcomere without significant
disruption of its structure 48 hours after transfection with adenoviral
vector.117 Almost similar conclusions were drawn
by Watkins et al,111 who showed an integration of
the COOH-terminal truncated cTnT mutant in the primary culture of quail
myotubes and only focal disruption of sarcomeric organization in 14%
to 21% of the cells. That study111 is at
variance with the study involving the analogous Drosophila
mutation, which induced a rapid degradation of flight muscle troponin T
leading to myofibrillar misassembly.120
Functional Aspects
In contrast to structural analyses, several in vitro
functional studies have shown that FHC mutants alter sarcomere
function, either by decreasing the translocating filament activity
and/or force leading to a reduction of power production by
cardiac cells31 105 111 115 116 117 144 or, on the
contrary, by increasing in vitro motility rates of filament sliding
and/or force.63 106 121 122 123
Nine MYH7 mutations, T124I, Y162C, R249Q, G256E, R403Q,
R453C, V606 M, R870H, and L908V, resulted in a decrease of the sliding
of the actin filament in the in vitro motility
assay.116 144 Cuda et al116
suggest that several mechanisms could explain this slower motility: (1)
the mutant might affect the crossbridge cycling rate by interfering
with the kinetic rate constant that limits sliding velocity; (2) the
crossbridge kinetics might be unaltered, but the distance in which the
actin filament is moved per crossbridge stroke might be shortened; and
(3) the mutated head of myosin might be noncycling yet still able to
bind to actin and act as an internal load on the movement of the normal
crossbridges present in the assay. Other studies performed on
skinned slow skeletal muscle fibers from affected patients with FHC
have shown that fibers containing the G741R or the R403Q
MYH7 mutations exhibit decreased maximum shortening of
velocity and decreased isometric force
generation.115 The calcium-activated
force of contraction was also found depressed in quail myotubes
expressing a C-terminal truncated cTnT mutant
protein.111 This latter study demonstrates that
in contrast to what was found for the analogous Drosophila
mutation,120 the truncated cTnT that causes FHC
does not act as a null allele but probably acts as a
dominant-negative allele, blocking full calcium activation of the
thin filament. Another expressed TNNT2 mutation, R102Q,
located in the
-TM binding domain, has been shown to result in an
impairment of the contractile performance of adult feline
cardiomyocytes (reduced fractional shortening and peak
velocity of shortening).117 Finally, mutants of
Dictostelium discoideum myosin II equivalent to 6 human FHC
mutations, R403Q, F513C, G584R, G716R, R719Q, and R719W, were generated
by site-directed mutagenesis.124 The mutant
Dictostelium discoideum myosins showed impaired function
determined by reduction of force, affinity to actin, and ATPase
activity, and these data could be correlated to the prognosis of
individuals affected by the corresponding FHC mutation. The authors
have thus proposed that the force level of the mutant myosin molecule
may be one of the key factors for pathogenesis responsible for the
prognosis of human FHC. All these data suggest that some FHC mutations
induce functional impairment rather than disruption of the sarcomeric
structure and support the FHC pathogenesis hypothesis of a
"hypocontractile" state, in which mutations induce functional
cardiac impairment followed by compensatory
hypertrophy.
Not all FHC mutations result in decreased in vitro motility filament
translocation. Ventricular myosin from patients with either
the R719Q MYH7 or the M149V MYL3 mutations
displayed faster than normal in vitro actin sliding
rates.63 The R719Q MYH7 mutation lies
near the interface of the ß-MyHC with the MLC-1s/v, suggesting that
the topography of the mutation may be an important factor contributing
to the variability of function alteration. For MYL2 gene
mutations, preliminary data showed that ventricular myosin
from patients with the E22K mutation gives actin translocation
velocities that are indistinguishable from those of control
myosin.63 This mutation is close to the MLC-2s/v
phosphorylation site, and this result is at variance
with what was found in Drosophila melanogaster, in which
mutations of the phosphorylation sites of the skeletal
muscle MLC-2s/v are associated with a large reduction of both the power
output and force in isolated skinned fibers.125
The authors suggest that MLC-2s/v phosphorylation may
be required for forming the myosin-actin interaction or that it may
also affect the mobility or position of the myosin head to promote
actomyosin interaction. A recombinant rat cTnT mutant, corresponding to
the I89N TNNT2 mutation in humans, which is located in the
5' end of the gene outside of the
-TM binding region, also resulted
in a 50% faster thin-filament movement over a surface coated with
heavy meromyosin in in vitro motility assays121 ;
however, neither the binding of the troponin complex with the other
thin-filament components nor the interactions between thick and thin
filaments were altered.121 As for
-TM, its
D175N mutant (affected near the calcium-dependent cTnT binding domain)
increased the velocity of actin translocation in an in vitro motility
assay.122 Other data obtained on skinned fibers
or with recombinant mutant proteins did not show any impairment of the
sarcomeric function. This is the case for the G256E MYH7
mutation, which had contractile properties that were indistinguishable
from normal,115 as well as for the skinned
skeletal fibers of 2 patients containing the D175N TPM1
mutation, which showed an increase of the calcium sensitivity of the
skeletal muscle fibers,106 and for the
recombinant D175N mutant protein, which showed a reduction of the actin
affinity with altered conformation when bound to actin in the
S1-induced on-state of the thin filament.123 So
the mutant protein is expressed and almost certainly incorporated into
muscle in vivo and does result in altered contractile function; this
confirms a dominant-negative rather than a null allele action.
Because the mutant
-TM was associated with an increased calcium
sensitivity, this mutation might be associated more with an enhancement
than a depression of cardiac contractile performance. Taken
altogether, these data suggest that some mutations might determine a
"hypercontractile" state that would directly induce cardiac
hypertrophy.
It should be pointed out that not only the hypercontractile hypothesis
but also the more common hypocontractile hypothesis are still somewhat
tentative and incomplete. Indeed, in most studies and for practical
reasons, the in vitro motility assay has been used to determine the
function of mutant proteins. Because velocity is determined in the
absence of load, it is not clear what a decrease or increase in
velocity in motility assays would necessarily mean in terms of force
generation or power output under load, since the heart always works
under load in vivo. Only 3 studies have reported force measurements,
and even though these results involved human skinned muscle fibers,
which to date correspond to the "most
physiological" conditions, they should be
considered with caution since the experiments were carried out at low
temperature.106 111 115 Finally, these data
further emphasize the need to analyze the
contractility for other FHC mutants and the development
of other animal models to better understand the pathogenesis of
FHC.
 |
Does the Genotypic Heterogeneity of FHC Account for
its Phenotypic Heterogeneity?
|
|---|
Before genetic studies, it was well known that the pattern and
extent of left ventricular hypertrophy vary
greatly even in first-degree relatives and that a high incidence of
sudden deaths occurs in selected families.126
Genetic studies have provided insights into the
heterogeneity of FHC clinical features. However, the
results must be seen as preliminary, because the available data relate
to only a few hundred individuals, and it is obvious that although a
given phenotype may be apparent in a small family, examining
large or multiple families with the same mutation is required before
drawing unambiguous conclusions. Nevertheless, several concepts begin
to emerge, at least for mutations in the MYH7,
TNNT2, and MYBPC3 genes. For MYH7, it
is clear that prognosis for patients with different mutations varies
considerably30 127 (for review see Reference
128128 ). For example, the R403Q mutation appears to be associated with
markedly reduced survival,30 whereas some other
mutations, such as V606M, appear more benign.127
The disease caused by TNNT2 mutations is usually associated
with a 20% incidence of nonpenetrance, a relatively mild and sometimes
subclinical hypertrophy, but a high incidence of sudden
death, which can occur even in the absence of significant clinical left
ventricular
hypertrophy.78 79 80 In one family with
a TNNT2 mutation, however, penetrance is complete,
echocardiographic data show a wide range of
hypertrophy, and there has been no sudden cardiac
death.76 Mutations in MYBPC3 seem to
be characterized by specific clinical features with a mild
phenotype in young subjects, a delayed age at the onset of
symptoms, and a favorable prognosis before the age of
40.104 129 Genetic studies have also revealed the
presence of clinically healthy individuals carrying the mutant
allele, which is associated in first-degree relatives with a
typical phenotype of the disease.32 94 In
some cases, as many as a quarter of genetically affected individuals do
not express the disease. Several mechanisms could account for the large
variability of the phenotypic expression of the mutations: the degree
of functional impairment caused in the sarcomere by the mutation (that
may vary markedly with the position of the mutation in the molecule and
the type of protein involved), the role of environmental differences
and acquired traits (eg, differences in lifestyle, risk factors, and
exercise), and the existence of modifier genes and/or polymorphisms
that could modulate the phenotypic expression of the disease. The only
significant results obtained so far concern the influence of the
angiotensin Iconverting enzyme insertion/deletion
polymorphism. Association studies showed that the D allele is
more common in patients with hypertrophic
cardiomyopathy and in patients with a high
incidence of sudden cardiac death than in a control
population.130 131 We recently showed that the
association between the D allele and hypertrophy is
observed in the case of MYH7 R403 codon mutations but not
with MYBPC3 mutation carriers,132
raising the concept of multiple genetic modifiers in FHC.
The genetic status begins to be used as the criterion of reference to
reassess diagnostic criteria and penetrance. The diagnosis
of FHC is usually based on ECG and
echocardiography, and it is generally considered
that echocardiography is a more accurate technique
than ECG for the diagnosis in adults.3
Analysis of a large genotyped population recently
showed that, in fact, ECG and echocardiography have
similar diagnostic values for FHC in adults, with an
excellent specificity and a lower sensitivity.133
As for penetrance, it was a much-debated issue. Before molecular
genetic analyses, several studies have indicated either a full
penetrance134 or an incomplete
one.135 136 We have recently reassessed the
penetrance of FHC in a large genotyped population and found
that it is incomplete, age-related, and greater in males than in
females.52 These latter data have very important
implications for genetic counseling, especially for women under the age
of 50. The transgenic mouse model of FHC also shows a gender
difference,113 and it now provides a good genetic
model for determining the direct role of sexual hormones on
myocardium and for studying the role of putative modifier
genes on sexual chromosomes.
 |
Conclusions and Future Directions
|
|---|
Although we have only begun to dissect the molecular mechanisms
leading to FHC, this review illustrates that mutations in sarcomeric
genes are now recognized as a principal cause of this disease. The
present focus on sarcomeric proteins should not, however, preclude
the search for other genetic origins: the finding that both
hypertrophic and dilated cardiomyopathies in
hamsters are caused by mutations in the
-sarcoglycan gene encoding a
protein of the dystrophin-associated glycoprotein
complex137 could provide significant new insights
into the pathogenesis of hypertrophic
cardiomyopathy in humans. Conversely, it was
recently reported that mutations in another sarcomeric gene, cardiac
actin, cause dilated cardiomyopathy in
humans.138 These 2 sets of data raise the
important issue of whether hypertrophic and dilated
cardiomyopathies are inherently independent
diseases or whether dilation is part of the FHC spectrum. The demand
for molecular genetic testing will certainly increase as physicians and
patients become better educated as to how these data can influence
patient care and family-planning decisions. Given the large genetic
heterogeneity of the disease, new strategies of genetic
testing need to be developed. Since the disease genes that cause FHC
have been identified only recently, the continued characterization of
patient mutations and their phenotypes is necessary to
establish clinically meaningful correlations. Finally, the development
of other animal models and of other mechanistic studies linking the
genetic mutation to functional defects are now key issues in
understanding how alterations in the basic contractile unit of the
cardiomyocyte alter the phenotype and the function
of the heart.
 |
Note Added in Proof
|
|---|
Tardiff et al have created transgenic mice expressing a cTnT
allele with a carboxyl-terminal truncation (Tardiff JC, Factor SM,
Tompkins TE, Hewett TE, Palmer BM, Moore RL, Schwartz S, Robbins J,
Leinwand L. A truncated cardiac troponin T molecule in transgenic mice
suggests multiple cellular mechanisms for familial hypertrophic
cardiomyopathy. J Clin Invest. 1998;101:28002811). The
hearts of these animals are smaller than wild types because of a
primary loss of myocytes and a decrease in myocyte cell size. In
addition, these animals exhibit significant diastolic dysfunction. This
novel mouse model of FHC provides a model system to investigate the
relationship between geometric changes in the heart and the severity of
the phenotype.
 |
Acknowledgments
|
|---|
We are grateful to Dr Marc Fiszman for a careful reading of
the manuscript.
 |
Footnotes
|
|---|
This manuscript was sent to Laurence H. Kedes, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
Correspondence to Dr Ketty Schwartz, INSERM UR 153, Institut de Myologie, Batiment Babinski, 47 boulevard de l'Hôpital, 75651 Paris Cedex 13, France.
Received February 11, 1998;
accepted June 24, 1998.
 |
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