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© 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 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
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