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 (Arg(403)Gln) 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 [H-3]phenylalanine into total protein and beta-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 beta MyHC 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 beta MyHC construct was observed in approximate to 50% of the myocytes examined, whereas the structure of the sarcomeres remained largely intact in myocytes infected with normal beta MyHC 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 beta MyHC protein is the primary defect in HCM.