© 1995 American Heart Association, Inc.
Articles |
Expression of a Mutation Causing Hypertrophic Cardiomyopathy Disrupts Sarcomere Assembly in Adult Feline Cardiac Myocytes
From the Section of Cardiology (A.J.M., Q.-T.Y., D.L.M., R.R.), Baylor College of Medicine, Houston, Tex, and the Departments of Biology and Pathology (F.L.G.), McMaster University, Hamilton, Ontario, Canada.
Correspondence to A.J. Marian, MD, Assistant Professor of Medicine, Baylor College of Medicine, One Baylor Plaza, 543E, Houston, TX 77030.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Abstract Mutations in the ß-myosin heavy chain (ßMyHC) induce hypertrophic cardiomyopathy (HCM), cardiac hypertrophy, and sarcomere disarray, with the latter being the characteristic hallmark. Thus, we sought to determine whether expression of mutant ßMyHC in adult feline cardiac myocytes, a species known to develop HCM with a phenotype identical to that in humans, induces sarcomere disarray. A full-length ßMyHC cDNA was cloned from a human heart cDNA library, and an HCM-causing mutation (Arg403Gln) was induced in the ßMyHC cDNA by site-directed mutagenesis using polymerase chain reaction (PCR). The normal and mutant ßMyHC cDNAs were cloned into p
E1spIB shuttle
vector, downstream from a cytomegalovirus (CMV) promoter.
Replication-deficient recombinant adenoviral constructs
(Ad5/CMV/ßMyHC-N and Ad5/CMV/ßMyHC-403) were generated through
homologous recombination of pE1spIB/CMV/ßMyHC-N or
Ad5/CMV/ßMyHC-403 and pBHG10 after cotransfection in 293 host cells.
Infection of COS-1 cells with the ßMyHC construct resulted in the
expression of a full-length myosin protein. Efficiency of infection of
isolated adult cardiac myocytes was >95%. Expression of the ßMyHC
constructs into mRNA at 48 hours after infection of feline cardiac
myocytes was confirmed by reverse transcription–PCR. The net total
protein and ß-myosin synthesis were determined by using the amount of
incorporation of [3H]phenylalanine into total protein and
ß-myosin, respectively. Although the total amount of protein
synthesis was equal among experimental groups, the net myosin synthesis
at 48 hours was greater in cardiac myocytes infected with normal or
mutant ßMyHC constructs than control myocytes or those infected with
vector alone (P<.05). Electron microscopic examination
showed only minor changes in the structure of sarcomeres in all
experimental groups at 48 hours after infection. However, disruption of
the sarcomeric structures at 120 hours after infection with the mutant
ßMyHC construct was observed in 
50% of the myocytes examined,
whereas the structure of the sarcomeres remained largely intact in
myocytes infected with normal ßMyHC construct, adenoviral vector
alone, or control cardiocytes. Similar results were confirmed by
immunofluorescence using MF-20 antibody to myosin. The results of this
study indicate that disruption of sarcomere assembly and myofibrillar
organization due to mutant ßMyHC protein is the primary defect in
HCM.
Key Words: hypertrophic cardiomyopathy • ß-myosin heavy chain mutation • sarcomere assembly • cardiac myocytes • adenovirus
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Hypertrophic cardiomyopathy (HCM), an inherited autosomal-dominant disease, is genetically heterogeneous and clinically characterized by sudden cardiac death and heart failure.1 The predominant cardiac pathology is myocyte hypertrophy and sarcomere disarray; the former is found in most cardiac diseases, whereas the latter is a hallmark of HCM.2 The ß-myosin heavy chain (ßMyHC) gene, located on chromosome 14, is the most common gene responsible for HCM, in which more than 35 missense mutations have been identified.3 Three different approaches have been taken in the structure-function analysis of the ßMyHC mutations. Cuda et al4 isolated the ßMyHC from the skeletal muscle of patients with HCM and showed, in an in vitro motility assay, that the rate of translocation of actin-coated filaments over mutant myosin was significantly decreased. Sweeney et al5 expressed mutant rat cardiac
-myosin heavy chain
(MyHC) meromyosin constructs containing corresponding ßMyHC
mutations in Sf9 cells and showed actin-activated ATPase activity and
decreased actin-myosin interaction in an in vitro motility assay.
Straceski et al6 expressed normal and mutant MyHC in
COS cells and showed that mutant myosin failed to form filamentous
structures in 30% of the COS cells transfected, whereas only 2% of
cells transfected with normal myosin constructs failed to form such
structures.
In the present study, we specifically examined the effect of
mutations in ßMyHC on cardiac sarcomere assembly in a species more
closely reflective of humans. Normal and mutant human ßMyHC cDNA was
incorporated into recombinant replication-deficient adenoviral
constructs and expressed in adult feline cardiac myocytes.
Replication-deficient adenoviruses provide a highly efficient method of
gene transfer into a variety of cells, including adult cardiac
myocytes.7 8 Adult feline cardiac myocytes offer several
advantages: (1) ßMyHC is the adult cardiac myosin form as in
humans.9 This is in contrast to smaller rodents, such as
mice and rats, in which MyHC is the predominant
myosin.9 (2) They form sarcomeres as their functioning
contractile unit, which remain aligned for a prolonged period of time
in culture (at least 2 weeks).10 (3) HCM is the most
common cardiac disease in cats, with a phenotypic expression identical
to that observed in humans.11
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Cloning of Full-Length ßMyHC cDNA
The full-length human ßMyHC cDNA (6 kb) was cloned as a single fragment for the first time from a normal human cardiac cDNA library. The library was screened with probes constructed to hybridize to the 3' and 5' ends of the ßMyHC cDNA. Probes were radiolabeled to a specific activity of >109 cpm/µg with [32P]dCTP (Amersham) by the random-primer procedure of Feinberg and Vogelstein.12 Two overlapping fragments of the ßMyHC cDNA (one was 5.2 kb and the second was 2 kb) were excised from positive plaques, digested with Nsi I restriction enzyme at position 1019 in an overlapping region, and subsequently ligated to generate the full-length ßMyHC cDNA. The full-length sequence of 6 kb was determined in both directions and found identical to that published previously.13 14
Mutagenesis
A mutant ßMyHC cDNA was constructed to contain the mutation
Arg403Gln, which causes HCM in humans and is associated
with a high incidence of sudden cardiac death.15 16 17 The
Arg403Gln mutation, due to substitution of adenine for
guanine in exon 13 at coding position 1208 of the ßMyHC cDNA, was
introduced in the ßMyHC cDNA by polymerase chain reaction
(PCR)–based oligonucleotide-mediated site-directed
mutagenesis.18 The incorporation of the G
A mutation at
coding position 1208 was confirmed by cycle
sequencing.19
Generation of Recombinant Replication-Deficient Adenoviral
Vectors
To accommodate our ßMyHC cDNA and promoter (7 kb), it was
necessary to construct a new adenoviral vector with greater packaging
capacity that combined extensive deletions in both early region 1 (E1)
and early region 3 (E3). The system used for rescue of the ßMyHC cDNA
into the viral genome is described in detail elsewhere.20
In brief, a chimeric plasmid vector that contains the left end of the
adenoviral genome up to 15.8 map units was used in which polycloning
sites replace the E1 region of the adenoviral genome from map unit 1.0
to 9.8 (pE1spIB). The cloned normal and mutated ßMyHC cDNAs were
excised from pGEM4Z vector and inserted into the HindIII and
Xba I sites at the polycloning region such that the 5' end
of the ßMyHC cDNA was located downstream from a cytomegalovirus
(CMV) promoter. The resulting chimeric constructs
(pE1spIB/CMV/ßMyHC-N and pE1spIB/CMV/ßMyHC-403) were
cotransfected along with plasmid pBHG10 (a construct that carries the
adenoviral genome with E3 deletion) into 293 cells.20
Recombination of the homologous DNA sequences in pBHG10 and
pE1spIB/CMV/ßMyHC-N or pE1spIB/CMV/ßMyHC-403 after
cotransfection of human 293 cells resulted in the production of a
recombinant replication-deficient virus that carries the ßMyHC
expression cassette in place of the original E1 region
(Ad5/CMV/ßMyHC-N and Ad5/CMV/ßMyHC-403) as shown in Fig 1
. The recombinant viruses were propagated, titrated,
and purified in 293 cells according to the protocol published by Graham
and Prevec21 and subsequently used to infect the adult
feline cardiac myocytes.
|
Expression of Recombinant Adenoviral Constructs Into ßMyHC
Protein
COS-1 cells were grown on a 150-mm plate to a confluence of 70%
in the presence of 10% fetal bovine serum (FBS) in DMEM. To
demonstrate expression of recombinant adenoviral constructs into
protein (myosin), COS-1 cells were infected with Ad5E1,
Ad5/CMV/ßMyHC-N, and Ad5/CMV/ßMyHC-403 constructs at a multiplicity
of infection (MOI) of 100:1 for 4 hours, after which viruses were
removed by washing the cells in PBS. The infected COS-1 cells were
cultured for an additional 48 hours. The expressed myosin was extracted
using low-salt and high-salt buffers as described by Bader et
al.22 In brief, cells (20 plates for each construct) as
well as control COS-1 cells were washed twice with PBS and were scraped
for isolation of myosin. COS-1 cells were lysed in 7 mL ice-cold
low-salt buffer containing 150 mmol/L NaCl, 10 mmol/L
NaHPO4, 1% Triton X-100, and 0.1 mmol/L
phenylmethylsulfonyl fluoride (PMSF), pH 7.5 for 5 minutes. Cellular
cytoskeleton was disrupted by using a Dounce homogenizer. The resulting
supernatant was centrifuged at 16 000g for 10 minutes. The
pellet was extracted for 15 minutes in high-salt buffer containing 0.6
mol/L NaCl, 10 mmol/L NaHPO4, 5 mmol/L
MgCl2, 5 mmol/L ATP, and 0.1 mmol/L PMSF, pH 7.2.
Extraction was terminated by centrifugation at 16 000g for
10 minutes. The supernatant was subjected to another cycle of low-salt
and high-salt extraction to further enrich the myosin component. The
concentration of the protein containing myosin was determined by
spectrophotometry using Micro BCA protein assay kit (Pierce).
Western Blotting
Approximately 30 µg of myosin-enriched protein extract was
loaded into each lane on a 7.5% nondenaturing polyacrylamide gel and
was subjected to electrophoresis for 6 hours. The separated proteins
were transferred (in 25 mmol/L Tris, pH 8.3, and 192 mmol/L glycine
with 20% methanol) to a polyvinylidene difluoride membrane (Bio-Rad)
by using a Trans-Blot electrophoretic transfer cell (Bio-Rad). The
membrane was washed twice in PBS for 5 minutes and was incubated in
blocking buffer (0.1% Tween-20 and 1% nonfat dry milk in 1x PBS) at
room temperature for 4 hours. The membrane was incubated with 1:100
dilution (in blocking buffer) of mouse monoclonal IgG2b-
antibody
against adult chicken pectoralis myosin (MF-20) anti-myosin antibody
(Developmental Studies Hybridoma Bank, University of Iowa, Iowa City)
at room temperature for 60 minutes.22 After it was washed,
the membrane was exposed to 1:5000 dilution of goat anti-mouse alkaline
phosphatase conjugate for 60 minutes by using a chemiluminescent
detection system per recommendation of the manufacturer (Western
Exposure Chemiluminescent Detection System, Clontech).
Efficiency of Infection
To determine the efficiency of infection of adult feline cardiac
myocytes with recombinant adenovirus, 104 isolated cardiac
myocytes were cultured on 35-mm plates and infected with recombinant
adenoviruses carrying the Lac-Z reporter gene
(Ad5/CMV/Lac-Z) at MOI values of 1:1, 10:1, 100:1, 500:1,
and 1000:1 for 4 hours. Cardiac myocytes were cultured for an
additional 48 hours and then fixed with 0.5% glutaraldehyde in PBS (pH
7.2) solution for 10 minutes at room temperature. Cardiac myocytes were
rinsed twice with PBS and stained for ß-galactosidase in the buffer
solution of X-gal chromogen containing 5 mmol/L each of
K3Fe(CN)6 and
K4Fe(CN)6, 2 mmol/L
MgCl2, and 1 mg/mL
5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside
overnight at room temperature in the dark.
Infection of Isolated Adult Feline Cardiac Myocytes
Adult feline cardiac myocytes were isolated as we have described
previously.23 24 After 24 hours in culture, the isolated
adult cardiac myocytes were incubated with 106
plaque-forming units (pfu) of control Ad5E1, Ad5/CMV/ßMyHC-N, and
Ad5/CMV/ßMyHC-403 for 4 hours; after which, the cardiac myocytes were
washed to remove unattached viruses and were cultured in medium 199
containing 0.1% human serum albumin in a CO2 incubator at
37°C for an additional 48 or 120 hours. Experiments were performed
with control feline cardiac myocytes (no transduction), feline cardiac
myocytes infected with Ad5/E1 as a control virus, and feline cardiac
myocytes infected with the Ad5/CMV/ßMyHC-N and Ad5/CMV/ßMyHC-403
recombinant viruses.
Reverse Transcription–PCR
To determine whether the ßMyHC cDNA was transcribed into mRNA,
the cultured adult cardiac myocytes were collected 48 hours after
infection, and total RNA was isolated by the guanidinium isothiocyanate
method.25 Reverse transcription–PCR was performed by
using a set of primers to amplify a 320-bp fragment unique to the human
ßMyHC cDNA according to a previously published
protocol.26
Net Protein Synthesis
The amount of newly synthesized total protein 48 hours after
infection with 106 pfu of adenoviruses was
determined from the amount of incorporated radiolabeled
[3H]phenylalanine. A 2-mL suspension of freshly isolated
cardiac myocytes was plated at a final concentration of
1x104 cells per milliliter onto laminin-coated (20 g/mL)
polystyrene Petri dishes. The control or recombinant virus
(106 pfu) was added to each corresponding
experimental group for 4 hours. The unattached virus was then removed
by washing the cultured cardiac myocytes three times with PBS. Cardiac
myocytes were cultured in medium 199 containing 0.1% human serum
albumin. Medium changes were performed on the first and second days of
culture. Beginning on the second day in culture, 0.4 mmol/L of
unlabeled L-phenylalanine was added to the culture medium
to ensure equalized specific activities of the intracellular and
extracellular phenylalanine pools. After allowing the cells to
equilibrate for 1 hour in 0.4 mmol/L L-phenylalanine, the
cells were pulse-labeled for 6 hours with 30 Ci/mL
[3H]phenylalanine. In preliminary control experiments, we
established that incorporation of [3H]phenylalanine was
linear for control as well as experimental groups (r=.99 for
all groups; P<.001), suggesting that protein degradation
was negligible during the 6-hour labeling period. At the conclusion of
the study (48 hours), the incorporation of radiolabeled phenylalanine
was stopped by washing the cultures three times with cold (4°C)
Hanks' balanced salt solution containing 10 mmol/L
L-phenylalanine. Cardiac myocyte proteins were then
solubilized with a buffered SDS sample buffer (4% SDS, 2% glycerol,
and 0.125 mol/L Tris-HCl [pH 6.8]). A portion of the solubilized
sample was taken for analysis of protein content by using a
commercially available assay (BCA, Pierce) with bovine serum albumin
(BSA) used as a standard. The extent of radiolabeling was determined
after acid precipitation for 30 minutes with cold (4°C) 10%
trichloroacetic acid (TCA); the precipitates were then collected on
1.6-mm glass filters, and the filters were washed sequentially with
10% TCA, 5% TCA, and 95% ethanol. The glass filters were air-dried,
and liquid scintillation counting was performed. Protein synthesis was
determined by using the specific activities of medium samples obtained
from direct scintillation counting, as well as the concentration of
phenylalanine (0.4 mmol/L) in the medium, according to the following
formula: phenylalanine incorporation (nmol · g
protein-1 · h-1)=[phenylalanine
incorporated into total cell protein (dpm/g myocyte
protein)/phenylalanine specific activity of medium
(dpm/mmol)]xh-1.
Net Myosin Synthesis
For measurement of specific synthesis of ßMyHC protein,
cardiac myocyte cultures were labeled and prepared exactly as described
above for net protein synthesis. After determining the protein content
for each sample, 2-mercaptoethanol (1%) was added, the samples were
heated for 3 minutes at 95°C, and the samples were layered in equal
protein amounts (20 mg) onto the same gel slab. To facilitate
localization of ßMyHC, purified myosin (5-mg sample) and known
molecular weight standards were prepared as described above and were
electrophoretically separated along with the experimental samples.
Electrophoresis was performed by using 7.5% SDS-PAGE. The resultant
gel slab was copper-stained for 10 minutes (Bio-Rad), the stained bands
of myocyte protein and comigrating standard myosin heavy chain were
aligned, and the ßMyHC band was excised from the gel. Destaining of
the excised gel pieces was performed by using a Tris-glycine buffer
(Bio-Rad), and the ßMyHC protein samples were electroeluted from the
gel in a running buffer consisting of 0.1% SDS, 25 mmol/L Tris, and
190 mmol/L glycine. The eluted protein samples were then precipitated
on ice for 30 minutes by using 1.6 mmol/L sodium deoxycholate and 15%
TCA and then centrifuged at 9000g for 20 minutes. The
resulting pellet was dissolved in a buffer consisting of 0.4% SDS,
12.5 mmol/L Tris-HCl, and 15 mmol/L NaCl, and the sample was split for
protein determination and scintillation counting. Results were
expressed in terms of the net ßMyHC protein synthesized, as described
above.
Indirect Immunofluorescent Staining
Isolated adult cardiac myocytes were cultured on glass
coverslips coated with laminin for 48 or 120 hours after infection with
adenoviruses and were fixed with 100% methanol (chilled at -20°C)
for 5 minutes. Cardiac myocytes were washed with PBS and PBS/1% BSA
twice and left in the blocking buffer (5% BSA, 2% nonfat dry milk, 50
mmol/L Tris, and 0.5 mol/L NaCl) for 30 minutes. After removal of the
blocking buffer, the cardiac myocytes were incubated with the
anti-myosin antibody MF-20 for 30 minutes at room temperature. Samples
were washed in PBS/1% BSA three times for 5 minutes each and then
incubated with the rhodamine-conjugated affinity-purified goat
anti-mouse IgG [F(ab')2 fragment] (Boehringer
Mannheim Co) as the secondary antibody for 30 minutes. A series of
experiments with different dilutions of the primary and secondary
antibodies was performed to determine their optimal concentration.
After antibody treatment, samples were washed in PBS/1% BSA (with a
final wash in water), then dried, and mounted with FluorSave reagent
(Calbiochem).
Electron Microscopy
To study the formation of sarcomeres, adult feline cardiac
myocytes were cultured on glass slide coverslips coated with 1:10
dilution of Matri-gel (Collaborative Research) to increase adhesion.
Cardiac myocytes were infected with 106 pfu of
recombinant and control adenoviral constructs for 4 hours, washed with
PBS three times, and then cultured for an additional 48 or 120 hours.
Electron microscopic examination of the cardiac myocytes was performed
48 and 120 hours after infection according to the method of Brinkley et
al.27
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Rescue of ßMyHC Expression Cassette
To confirm that the CMV/ßMyHC expression cassette was rescued into the replication-deficient adenoviruses, a set of oligonucleotide primers was designed to amplify a 320-bp segment of the human ßMyHC cDNA encompassed between exons 12 and 14 by PCR. The amplified fragment was identified on agarose gel electrophoresis and was sequenced by cycle sequencing.19 The results confirmed the presence of the normal and mutant ßMyHC expression cassette in the recombinant adenoviral constructs (data not shown).
Efficiency of Infection of Adult Feline Cardiac Myocytes With
Recombinant Adenoviral Constructs
Cardiac myocytes were infected with an Ad5/CMV/Lac-Z construct at
MOI values of 1:1, 10:1, 100:1, 500:1, and 1000:1 for 4 hours. The
efficiency of infection was low for an MOI of 1:1 (5%), intermediary
for an MOI of 10:1 (30%), and high (>95%) for an MOI of >100:1 (Fig 2).
|
Western Blotting
Approximately 30 µg of myosin-enriched extract was
electrophoresed on 7.5% polyacrylamide gel for 6 hours, transferred to
a membrane, and probed with anti-myosin antibody MF-20. As shown in Fig 3, myosin-enriched protein preparations from COS-1 cells
infected with Ad5/CMV/ßMyHC-N and Ad5/CMV/ßMyHC-403 showed the
presence of a 220-kD protein migrating at the corresponding myosin
level of the protein size marker, although no myosin band was detected
in lanes representing control COS-1 cells and COS-1 cells
infected with Ad5E1. Thus, these results indicate that the normal
and mutant ßMyHC constructs are expressed into full-length ßMyHC
protein.
|
Expression of ßMyHC mRNA
Agarose gel electrophoresis of the PCR product showed that normal
as well as mutant human ßMyHC was expressed into mRNA 48 hours after
infection (Fig 4). Amplification of the RNA extracted
from control cardiac myocytes or cardiac myocytes infected with control
Ad5E1 failed to show any product indicating that the primers were
specific for human ßMyHC. The results indicate expression of the
mutant and normal human ßMyHC into mRNA.
|
Total Protein Synthesis
There was no significant difference in the total protein synthesis
in cultured control adult feline cardiac myocytes and adult feline
cardiac myocytes infected with Ad5E1 (control virus),
Ad/CMV/ßMyHC-N, or Ad5/CMV/ßMyHC-403 viruses (data not shown).
Net Myosin Synthesis
In keeping with expression of the mutant ßMyHC construct into
the mRNA in feline cardiac myocytes and into protein in COS-1 cells,
the results illustrate that ßMyHC is expressed into protein. The
amount of newly synthesized ßMyHC protein was greater in the cardiac
myocytes infected with Ad5/CMV/ßMyHC-N and Ad5/CMV/ßMyHC-403 than
in control cardiac myocytes or those infected with control Ad5E1.
Net myosin synthesis was 5.2±1.0 nmol phenylalanine per gram protein
in control cardiac myocytes, 5.7±1.9 nmol phenylalanine per gram
protein in cardiac myocytes infected with Ad5E1, 10.9±2.3 nmol
phenylalanine per gram protein in cardiac myocytes infected with
Ad5/CMV/ßMyHC-403, and 13.42±2.8 nmol phenylalanine per gram protein
in cardiac myocytes infected with Ad5/CMV/ßMyHC-N (n=8,
P<.05).
Indirect Immunofluorescent Staining
Final dilutions of 1:500 of MF-20 and 1:1000 of
rhodamine-conjugated goat anti-mouse IgG as the primary and secondary
antibodies, respectively, were used for immunofluorescent staining.
Diffuse staining of the myofibrillar structures was observed in all
cardiac myocytes, indicative of an abundance of myosin protein in adult
feline cardiac myocytes. Only examination of cardiac myocytes under
high magnification (x600) made it feasible to delineate the
myofibrillar structure. Over 100 rod-shaped cardiac myocytes were
examined per each group. There were no significant differences in the
immunofluorescent staining pattern of the myofibrillar structure of
cultured adult cardiac myocytes among experimental groups after 48
hours. Similarly, after 120 hours of culture the myofibrillar structure
appeared to be intact in the control cardiac myocytes and those
infected with Ad5E1 or Ad5/CMV/ßMyHC-N. However, in approximately
half of the cardiac myocytes infected with Ad5/CMV/ßMyHC-403, the
structure of myofibrils showed disarray and lack of appropriate
longitudinal alignment along the cell axis (Fig 5).
|
Sarcomeric Structure
A total of 60 viable adult feline cardiac myocytes were examined
for each experimental group, and the experiments were repeated six
times. Cardiac myocytes were considered viable if the
plasmalemma of the sarcolemma was intact and mitochondria
did not contain amorphous matrix densities. At 48 hours, no significant
differences were observed in the structure of the thin and thick
filaments or the sarcomeres among experimental groups. In the majority
of myocytes examined, the myofibrils were aligned with the long axis of
the cells, and the Z bands were in register throughout the length of
the cardiac myocytes. Disruption of sarcomeric organization was
observed in <10% of the cardiac myocytes in all four experimental
groups. However, 120 hours after infection, in cardiac myocytes
infected with Ad5/CMV/ßMyHC-403, although thick filament formation
appeared to be normal, their assembly into sarcomere was markedly
impaired (Fig 6). In control myocytes as well as in
myocytes infected with Ad5E1 and Ad5/CMV/ßMyHC-N, <20% of the
cells showed evidence of sarcomeric disarray, which was localized
primarily to the ends of the cardiac myocytes and involved <20% of
the total myocyte sarcomeres. In contrast, approximately half of the
cardiac myocytes infected with Ad5/CMV/ßMyHC-403 showed disruption of
the sarcomeric organization, affecting at least 50% of the sarcomeres.
Bundles of thick filaments as well as clusters of Z bands with emerging
rudimentary thick filaments were present in myocytes with
disruption of sarcomeric structures. The percentage of cardiac myocytes
with severe myofibrillar disarray was significantly greater for the
cardiac myocytes infected with the mutant ßMyHC construct than for
cardiac myocytes infected with the normal ßMyHC construct
(P=.03).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This is the first expression study of a ßMyHC cDNA with an HCM mutation in cardiac myocytes. The cardiac myocytes infected with mutant constructs in cell culture exhibited normal filament and sarcomere formation for 48 hours but subsequently exhibited gross impairment of sarcomere assembly despite persistence of normal-appearing filaments. Since the control myocytes and the myocytes infected with the adenoviral vector alone or the normal ßMyHC construct mostly exhibited normal sarcomeres throughout this interval, the Arg403Gln mutation is strongly implicated as the cause of the abnormal sarcomere assembly. The observed normal sarcomere formation for the first 48 hours, even in the mutant infected cells, is also supportive of causality, as evidenced by the time required for turnover of the preformed endogenous normal ßMyHC protein, which has a 5.6-day half-life.28 These findings indicate that the expressed mutant myosin is capable of forming thick myosin filaments but that the filaments do not assemble into sarcomeres and myofibrils. Given the relatively long half-life of ßMyHC (5.6 days),28 the myofibrillar disarray most likely represents ineffective sarcomeric assembly rather than disruption of the formed sarcomeres. Altered stoichiometry of the components of the sarcomeric proteins is also a possible explanation for the observed disarray of the sarcomeric structure. However, this mechanism is unlikely, because the net ßMyHC synthesis was equal in cardiac myocytes infected with normal and mutant ßMyHC constructs, despite a significant disparity in the degree of sarcomeric disruption observed among these two groups of cardiac myocytes. Disruption of sarcomere assembly and selective lysis of the thick filaments are characteristic findings in patients with HCM.2 If one assumes that these findings reflect the primary lesion of HCM, impaired sarcomere assembly would be implicated in the early development of the cardiac pathology.
It is not clear whether the construct was expressed as a normal
full-length protein in adult feline cardiac myocytes. The data showing
increased synthesis of myosin that was of the correct molecular weight
and electrophoretic migration are strongly suggestive. The increased
myosin synthesis occurred in only the feline cells infected with either
normal or mutant ßMyHC constructs but not in those infected with the
vector alone. However, this was confirmed by the protein expressed in
COS cells. These cells do not normally synthesize myosin, but after
infection with the adenoviral construct, they expressed a protein that
was detected by an antibody specific for sarcomeric myosin. This
protein had a molecular weight of 220 kD, identical to that expected
for ßMyHC. We have shown previously,29 from an
analysis of myocardial myosin from a patient that died with the
Arg403Gln mutation, that the quantity of cardiac ßMyHC
myosin was normal (constituting 95% of the myosin) and was normal in
proportion to actin. It is not possible to determine from the
present study whether the mutant ßMyHC molecule is incorporated
and subsequently acts as a poisonous peptide to disrupt the involved
sarcomere as previously suggested30 31 or whether the
sarcomere is not assembled because of some impairment, such as the lack
of proper binding of myosin to other sarcomeric proteins. The inherent
impaired contractility of the mutant myosin suggested by the previous
studies may be secondary to sarcomere assembly. All of the observations
remain compatible with our previous hypothesis26 30 and
that of others31 : the hypertrophy is secondary and
compensatory. It would be speculative to assume that our findings are
indicative of HCM; however, the pathognomonic cardiac lesion of HCM in
humans and felines is sarcomeric and myofibrillar disarray.
Additionally, the results of these studies illustrate the utility of
replication-deficient adenoviruses as expression vectors for the study
of protein function in mammalian cells. The expression cassette cloned
in the vector described in the present study, at 7 kb, is one of
the largest inserts rescued into an Ad5 vector and is close to the
predicted maximum capacity (8 kb) of the system described by Bett et
al.20
In the previous attempt to assess the effect of HCM mutations on
filament formation, the mutation was inserted into MyHC cDNA and
expressed in COS cells. COS cells do not form sarcomeres, but
expression of the normal MyHC did show filamentous structures in
98% of the transfected cells, whereas COS cells transfected with
the mutant MyHC formed nonfilamentous structures in 30% of the
transfected cells. Although the studies are markedly different, the
implication is similar: impaired myofibril or sarcomere formation is
induced by the mutation. We did not perform functional studies, but
decreased myosin-actin interaction would be expected in cells with such
sarcomere disarray and would be compatible with the decreased velocity
of actin-myosin interaction observed by Cuda et al4 and
Sweeney et al5 in an in vitro motility assay.
In the present study, we have shown the following: (1) The
adenovirus vector can be constructed to incorporate the full-length
human ßMyHC cDNA and its promoter. (2) The full-length ß-MyHC
protein is expressed. (3) The ßMyHC cDNA is expressed into mRNA,
resulting in increased myosin protein. (4) Expression of ßMyHC with
Arg403Gln mutation results in myofibrillar disarray and
disruption of sarcomeric structures. This preparation provides an
easily detectable and distinctive morphological phenotype in both the
normal and mutant infected cardiac myocytes, which closely resemble the
pathological hallmark of the disease both in felines and in humans.
These features, in addition to the advantages inherent to feline adult
myocytes previously outlined, make this preparation a much improved and
more appropriate model for future structure-function analysis of
mutations in ßMyHC and other sarcomeric genes responsible for HCM,
such as the recently identified troponin T and
-tropomyosin.31 A limitation of the present study
is the lack of documentation of the incorporation of the mutant myosin
into the filament and, second, the lack of accompanying functional
studies. We specifically did not use an epitope, since the addition of
even a single nucleotide may alter expression or the properties of the
expressed myosin. This is particularly important because more than 36
different missense mutations have been identified in the ßMyHC gene,
each of which impairs cardiac function leading to HCM.3 We
are currently developing combinatorial phage display
antibodies31 with the hope of obtaining a species-specific
antibody against mutant ßMyHC. Studies such as we have previously
performed23 24 are planned to measure indices of
contractility and relaxation in a single-cell preparation to compare
the function of normal and mutant infected cardiac myocytes.
|
Acknowledgments |
|---|
This study was supported in part by grants from the National Heart, Lung, and Blood Institute; Specialized Centers of Research (P50-HL42267-01); the American Heart Association; the Bugher Foundation Center for Molecular Biology (86-2216); the American Heart Association, Texas Affiliate, Inc (93G-1191); the Medical Research Council and the National Cancer Institute of Canada. Dr Graham is a Terry Fox Research Scientist of the National Cancer Institute in Canada. We would like to acknowledge Drs Michael Schneider and Brent French for their critical review of the manuscript and helpful suggestions; Dr Ann Goldstein for review and interpretation of the electron micrographs; J. Rudy, Samir Kapadia, MD, Lorrie Kirshenbaum, PhD, and Dorellyn Lee-Jackson for their excellent technical assistance; and Debora Weaver and Esther Yeager for their secretarial assistance in the preparation of this manuscript and figures. Electron microscopic studies were performed at the EM Core Laboratory of the Department of Cell Biology, Baylor College of Medicine.
Received August 19, 1994; accepted March 17, 1995.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1. Wynne J, Braunwald E. The cardiomyopathies and myocarditides: toxic, chemical, and physical damage to the heart. In: Braunwald E, ed. Heart Disease: A Textbook of Cardiovascular Medicine. 4th ed. Philadelphia, Pa: WB Saunders; 1992:1394-1450.
2. Roberts WC, Ferrans VJ, Kaltenbach M, Epstein SW, eds. Hypertrophic Cardiomyopathy: The Therapeutic Role of Calcium Antagonists. New York, NY: Springer-Verlag; 1982:59.
3. Roberts R, Marian AJ, Bachinski LL. Overview: application of molecular biology to medical genetics. In: Markwald RR, Clark EB, Takao A, eds. Inborn Heart Disease: Developmental Mechanisms. Mount Kisco, NY: Futura Press; 1994:87-111.
4. Cuda G, Fananapazir L, Zhu W, Sellers JR, Epstein ND. Skeletal muscle expression and abnormal function of ß myosin in hypertrophic cardiomyopathy. J Clin Invest. 1993;91:2861-2865.
5.
Sweeney HL, Straceski AJ, Leinwand LA, Tikunov BA,
Faust L. Heterologous expression of a cardiomyopathic myosin
that is defective in its actin interaction. J Biol
Chem. 1994;269:1603-1605.
6.
Straceski AJ, Geisterfer-Lawrance AA, Seidman CE,
Seidman JG, Leinwand LA. Functional analysis of myosin
missense mutations in familial hypertrophic
cardiomyopathy. Proc Natl Acad Sci
U S A. 1994;91:589-593.
7. Kirshenbaum LA, MacLellan WR, French BA, Schneider MD. Highly efficient gene transfer into adult ventricular myocytes by recombinant adenovirus. J Clin Invest. 1993;92:381-387.
8.
French BA, Mazur W, Geske RS, Bolli R. Direct
in vivo gene transfer into porcine myocardium using
replication-deficient adenoviral vectors.
Circulation. 1994;90:2414-2424.
9. Swynghedauw B. Developmental and functional adaptation of contractile proteins in cardiac and skeletal muscles. Physiol Rev. 1986;3:710-760.
10.
Cooper G, Mercer WE, Hoober JK, Gordon PR, Kent RL,
Laurke IK, Marino TA. Load regulation of the properties of adult
feline cardiocytes. Circ Res. 1986;58:692-705.
11. Atkins CE, Gallo AM, Kurzman ID, Cowan P. Risk factors, clinical signs and survival in cats with a clinical diagnosis of idiopathic hypertrophic cardiomyopathy: 74 cases (1985-1989). JAMA. 1992;201:613-618.
12. Feinberg AP, Vogelstein B. A technique for radiolabelling DNA restriction nuclease fragments to high specific activity. Anal Biochem. 1983;132:6-13. [Medline] [Order article via Infotrieve]
13.
Liew CC, Sole MJ, Yamauchi-Takihara K, Kellam B,
Anderson DH, Lin L, Liew JC. Complete sequence and organization
of the human cardiac ß-myosin heavy chain gene. Nucleic
Acids Res. 1990;18:3647-3651.
14. Jaenicke T, Diedrich KW, Haas W, Schleich J, Lichter P, Pfordt M, Bach A, Vosberg HP. The complete sequence of the human heavy chain gene and a comparative analysis of its product. Genomics. 1990;8:194-206. [Medline] [Order article via Infotrieve]
15.
Epstein ND, Cohn GM, Cyran F, Fananapazir L.
Differences in clinical expression of hypertrophic
cardiomyopathy associated with two distinct
mutations in the ß-myosin heavy chain gene: a 908 Leu-Val mutation
and a 403 Arg-Gln mutation. Circulation. 1992;86:345-352.
16. Marian AJ, Yu QT, Mares A Jr, Hill R, Roberts R, Perryman MB. Detection of a new mutation in the ß-myosin heavy chain gene in an individual with hypertrophic cardiomyopathy. J Clin Invest. 1992;90:2156-2165.
17. Watkins H, Rosenzweig A, Hwang D, Levi T, McKenna W, Seidman CE, Seidman JG. Characteristics and prognostic implications of myosin missense mutations in familial hypertrophic cardiomyopathy. N Engl J Med. 1992;326:1108-1114. [Abstract]
18. Nelson RM, Long GL. A general method of site-specific mutagenesis using a modification of the Thermus aquaticus polymerase chain reaction. Anal Biochem. 1989;180:147-151. [Medline] [Order article via Infotrieve]
19. Lee J. Alternative dideoxy sequencing of double stranded DNA by cyclic reaction with Taq DNA polymerase. DNA Cell Biol. 1991;10:67-73. [Medline] [Order article via Infotrieve]
20.
Bett AJ, Haddara W, Prevec L, Graham FL. A
highly efficient and flexible system for construction of human
adenovirus type 5 vectors with insertions or deletions in early region
1 and early region 3. Proc Natl Acad Sci U S A. 1994;91:8802-8808.
21. Graham FL, Prevec L. Manipulation of adenovirus vectors. In: Murray EJ, ed. Methods in Molecular Biology: Gene Transfer and Expression of Protocols. 7th ed. Clifton, NJ: Humana Press; 1991:109-128.
22.
Bader D, Masaki T, Fishman DA. Immunochemical
analysis of myosin heavy chain during avian myogenesis in vivo and
in vitro. J Cell Biol. 1982;95:763-770.
23.
Mann DL, Urabe Y, Kent RL, Vinciguerra S, Cooper G IV.
Cellular versus myocardial basis for the contractile dysfunction
of hypertrophied myocardium. Circ Res. 1991;68:402-415.
24.
Yokoyama T, Vaca L, Rossen RD, Durante W, Hazarika P,
Mann DL. Cellular basis for the negative inotropic effects of
tumor necrosis factor- in the adult mammalian heart. J
Clin Invest. 1993;92:2303-2312.
25. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-choloroform extraction. Anal Biochem. 1987;162:156-169. [Medline] [Order article via Infotrieve]
26.
Yu QT, Ifegwu J, Marian AJ, Mares A Jr, Hill R,
Perryman MB, Bachinski LL, Roberts R. Hypertrophic
cardiomyopathy mutation is expressed in mRNA of
skeletal as well as cardiac muscle.
Circulation. 1993;87:406-412.
27.
Brinkley BR, Murphy P, Richardson LC. Procedure
for embedding in situ selected cells in vitro. J Cell
Biol. 1967;35:279-283.
28.
Zak R, Martin AF, Prior G, Rabinowitz M.
Comparison of turnover of several myofibrillar proteins and
critical evaluation of double isotope method. J Biol
Chem. 1977;252:3430-3435.
29.
Vybiral T, Deitiker PR, Roberts R, Epstein HF.
Accumulation and assembly of myosin in hypertrophic
cardiomyopathy with the 403 Arg to Gln ß-myosin
heavy chain mutation. Circ Res. 1992;71:1404-1409.
30. Perryman MB, Yu QT, Marian AJ, Mares A Jr, Czernuszewicz G, Ifegwu J, Hill R, Roberts R. Expression of a missense mutation in the mRNA for ß-myosin heavy chain in myocardial tissue in hypertrophic cardiomyopathy. J Clin Invest. 1992;90:271-277.
31.
Thierfelder L, Watkins H, MacRae C, Lamas R, McKinna W,
Vosberg HP, Seidman JG, Seidman CE. -Tropomyosin and cardiac
troponin T mutations cause familial hypertrophic
cardiomyopathy: a disease of the sarcomere.
Cell. 1994;77:701-712.[Medline]
[Order article via Infotrieve]
This article has been cited by other articles:
 |
|
 |
|
  J. Davis, M. V. Westfall, D. Townsend, M. Blankinship, T. J. Herron, G. Guerrero-Serna, W. Wang, E. Devaney, and J. M. Metzger Designing Heart Performance by Gene Transfer Physiol Rev, October 1, 2008; 88(4): 1567 - 1651. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. J. Herron, R. Vandenboom, E. Fomicheva, L. Mundada, T. Edwards, and J. M. Metzger Calcium-Independent Negative Inotropy by {beta}-Myosin Heavy Chain Gene Transfer in Cardiac Myocytes Circ. Res., April 27, 2007; 100(8): 1182 - 1190. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Roberts and U. Sigwart Current Concepts of the Pathogenesis and Treatment of Hypertrophic Cardiomyopathy Circulation, July 12, 2005; 112(2): 293 - 296. [Full Text] [PDF]
|
|
|
|
|
|
D. Fatkin and R. M. Graham Molecular Mechanisms of Inherited Cardiomyopathies Physiol Rev, October 1, 2002; 82(4): 945 - 980. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Roberts and U. Sigwart New Concepts in Hypertrophic Cardiomyopathies, Part I Circulation, October 23, 2001; 104(17): 2113 - 2116. [Full Text] [PDF]
|
|
|
|
 |
|
 R. Roberts A perspective: the new millennium dawns on a new paradigm for cardiology--molecular genetics J. Am. Coll. Cardiol., September 1, 2000; 36(3): 661 - 667. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. H. Spencer III and R. Roberts Alcohol Septal Ablation in Hypertrophic Obstructive Cardiomyopathy : The Need for a Registry Circulation, August 8, 2000; 102(6): 600 - 601. [Full Text] [PDF]
|
|
|
|
 |
|
 N. Frey, W. M. Franz, K. Gloeckner, M. Degenhardt, M. Muller, O. Muller, H. Merz, and H. A. Katus Transgenic rat hearts expressing a human cardiac troponin T deletion reveal diastolic dysfunction and ventricular arrhythmias Cardiovasc Res, August 1, 2000; 47(2): 254 - 264. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Sorajja, P.M. Elliott, and W.J. Mckenna The molecular genetics of hypertrophic cardiomyopathy: prognostic implications Europace, January 1, 2000; 2(1): 4 - 14. [PDF]
|
|
|
|
 |
|
 A Muraishi, H Kai, K Adachi, H Nishi, and T Imaizumi Malalignment of the sarcomeric filaments in hypertrophic cardiomyopathy with cardiac myosin heavy chain gene mutation Heart, November 1, 1999; 82(5): 625 - 629. [Abstract] [Full Text]
|
|
|
|
|
|
C. S. Redwood, J. C. Moolman-Smook, and H. Watkins Properties of mutant contractile proteins that cause hypertrophic cardiomyopathy Cardiovasc Res, October 1, 1999; 44(1): 20 - 36. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Bonne, L. Carrier, P. Richard, B. Hainque, and K. Schwartz Familial Hypertrophic Cardiomyopathy : From Mutations to Functional Defects Circ. Res., September 21, 1998; 83(6): 580 - 593. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Kai, A. Muraishi, Y. Sugiu, H. Nishi, Y. Seki, F. Kuwahara, A. Kimura, H. Kato, and T. Imaizumi Expression of Proto-oncogenes and Gene Mutation of Sarcomeric Proteins in Patients With Hypertrophic Cardiomyopathy Circ. Res., September 21, 1998; 83(6): 594 - 601. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. E. Bowles, Q. Wang, and J. A. Towbin Prospects for adenovirus-mediated gene therapy of inherited diseases of the myocardium Cardiovasc Res, September 1, 1997; 35(3): 422 - 430. [Full Text] [PDF]
|
|
|
|
|
|
H. Prentice, N. H Bishopric, M. N Hicks, D. J Discher, X. Wu, A. A Wylie, and K. A Webster Regulated expression of a foreign gene targeted to the ischaemic myocardium Cardiovasc Res, September 1, 1997; 35(3): 567 - 574. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. J. Marian, G. Zhao, Y. Seta, R. Roberts, and Q.-t. Yu Expression of a Mutant (Arg92Gln) Human Cardiac Troponin T, Known to Cause Hypertrophic Cardiomyopathy, Impairs Adult Cardiac Myocyte Contractility Circ. Res., July 19, 1997; 81(1): 76 - 85. [Abstract] [Full Text]
|
|
|
|
 |
|
 M. V. Westfall, E. M. Rust, and J. M. Metzger Slow skeletal troponin I gene transfer, expression, and myofilament incorporation enhances adult cardiac myocyte contractile function PNAS, May 13, 1997; 94(10): 5444 - 5449. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. D. Becker, K. R. Gottshall, R. Hickey, J.-C. Perriard, and K. R. Chien Point Mutations in Human {beta} Cardiac Myosin Heavy Chain Have Differential Effects on Sarcomeric Structure and Assembly: An ATP Binding Site Change Disrupts Both Thick and Thin Filaments, Whereas Hypertrophic Cardiomyopathy Mutations Display Normal Assembly J. Cell Biol., April 7, 1997; 137(1): 131 - 140. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Yokoyama, M. Nakano, J. L. Bednarczyk, B. W. McIntyre, M. Entman, and D. L. Mann Tumor Necrosis Factor-{alpha} Provokes a Hypertrophic Growth Response in Adult Cardiac Myocytes Circulation, March 4, 1997; 95(5): 1247 - 1252. [Abstract] [Full Text]
|
|
|
|
|
|
A.J. Marian and R. Roberts Recent Advances in the Molecular Genetics of Hypertrophic Cardiomyopathy Circulation, September 1, 1995; 92(5): 1336 - 1347. [Full Text]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||