Expression of Mutation Causing Hypertrophic Cardiomyopathy Disrupts Sarcomere Assembly in Adult Feline Cardiac Myocytes

Mutations in the β myosin heavy chain (βMHC) gene cause hypertrophic cardiomyopathy (HCM), disease characterized by sudden death, cardiac hypertrophy and sarcomere disarray. To determine the primary defect, we sought to express mutant βMHC gene in adult feline cardiac myocytes. The feline model was selected since HCM is common disease in cats with phenotype identical to that of man and the cardiac myosin is βMHC as in man in contrast to αMHC in the mouse. full length human βMHC cDN was cloned for the first time and an HCM causing mutation (Arg403Gln) was induced by site-directed mutagenesis. The adenoviral vector was selected for its high efficiency of gene transfer and reconstructed to accommodate the 7 kb βMHC cDN and promoter. The normal and mutant βMHC cDNAs were cloned into shuttle vector (ΔE1sp1B) downstream to cytomegalovirus promoter and the 7 kb fragment was incorporated through homologous recombination during co-transfection of pΔE1sp1B/CMV/βMHC and pBHG10 in 293 host cells. Adult feline cardiac myocytes were transfected with the recombinant viruses and examined at 48 and 120 hours after transfection. Expression of the βMHC into mRN was confirmed by RT-PCR. Net myosin synthesis, determined by 3H phenylalanine incorporation (48 hr), was greater in myocytes with normal or mutant βMHC constructs than control myocytes or those infected with vector alone (p < 0.05). Electron microscopy showed normal sarcomere assembly at 48 hours in >90% of myocytes, however, at 120 hours sarcomere disruption was evident in >50% of myocytes infected with mutant βMHC but remained intact in control myocytes and those infected with normal βMHC or vector alone (p = 0.03). In this study, we successfully incorporated 7 kb βMHC/promoter construct into an adenoviral vector and showed its expression in adult feline cardiac myocytes. The mutant βMHC induced sarcomere and filament disarray similar to the lesion seen in man. This would indicate sarcomere disarray is the primary defect in HCM and the hypertrophy is compensatory.