Abstract
Mutations in the β-myosin heavy chain (βMyHC) 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 βMyHC 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 βMyHC cDNA was cloned flora a human heart cDNA library, and an HCM-causing mutation (Arg403Gln) was induced in the βMyHC cDNA by site-directed mutagenesis using polymerase chain reaction (PCR). The normal and mutant βMyHC cDNAs were cloned into pΔE1sp1B shuttle vector, downstream from a cytomegalovirus (CMV) promoter. Replication-deficient recombinant adenoviral constructs (Ad5/CMV/βMyHC-N and Ad5/CMV/βMyHC-403) were generated through homologous recombination of pΔE1sp1B/CMV/βMyHC-N or Ad5/CMV/βMyHC-403 and pBHG10 after cotransfection in 293 host cells. Infection of COS-I cells with the βMyHC construct resulted in the expression of a full-length myosin protein. Efficiency of infection of isolated adult cardiac myocytes was >95%. Expression of the βMyHC constructs into mRNA at 48 hours after infection of feline cardiac myocytes was confirmed by reverse transcription-PCR. The net total protein and β-myosin synthesis were determined by using the amount of incorporation of [3M]phenylalanine into total protein and β-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 βMyHC 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 βMyHC construct was observed in ≃50% of the myocytes examined, whereas the structure of the sarcomeres remained largely intact in myocytes infected with normal βMyHC 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 βMyHC protein is the primary defect in HCM.