Expression of a mutation causing hypertrophic cardiomyopathy disrupts sarcomere assembly in adult feline cardiac myocytes.
Mutations in the beta-myosin heavy chain (beta MyHC) 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 beta MyHC 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 beta MyHC cDNA was cloned from a human heart cDNA library, and an HCM-causing mutation (Arg403Gln) was induced in the beta MyHC cDNA by site-directed mutagenesis using polymerase chain reaction (PCR). The normal and mutant beta MyHC cDNAs were cloned into p delta E1spIB shuttle vector, downstream from a cytomegalovirus (CMV) promoter. Replication-deficient recombinant adenoviral constructs (Ad5/CMV/beta MyHC-N and Ad5/CMV/beta MyHC-403) were generated through homologous recombination of p delta E1spIB/CMV/beta MyHC-N or Ad5/CMV/beta MyHC-403 and pBHG10 after cotransfection in 293 host cells. Infection of COS-1 cells with the beta MyHC construct resulted in the expression of a full-length myosin protein. Efficiency of infection of isolated adult cardiac myocytes was > 95%. Expression of the beta MyHC constructs into mRNA at 48 hours after infection of feline cardiac myocytes was confirmed by reverse transcription-PCR. The net total protein and beta-myosin synthesis were determined by using the amount of incorporation of [3H]phenylalanine into total protein and beta-myosin, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Marian
A J
AJ
Section of Cardiology, Baylor College of Medicine, Houston, Tex. 77030, USA.
Yu
Q T
QT
Mann
D L
DL
Graham
F L
FL
Roberts
R
R
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D015153:Blotting, Western; D002312:Cardiomyopathy, Hypertrophic / Q000235:genetics*; D002415:Cats; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000235:genetics; D004587:Electrophoresis, Agar Gel; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D008854:Microscopy, Electron; D009154:Mutation; D009206:Myocardium / Q000166:cytology* / Q000378:metabolism; D009218:Myosins / Q000032:analysis / Q000235:genetics*; D010641:Phenotype; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics; D012518:Sarcomeres / Q000235:genetics* / Q000648:ultrastructure
D018076:DNA, Complementary; D012333:RNA, Messenger; D009218:Myosins