EXPRESSION OF A MUTANT (ARG(92)GLN) HUMAN CARDIAC TROPONIN-T, KNOWN TO CAUSE HYPERTROPHIC CARDIOMYOPATHY, IMPAIRS ADULT CARDIAC MYOCYTE CONTRACTILITY

The mechanism(s) by which mutations in sarcomeric proteins cause hyper trophic cardiomyopathy (HCM) remains unknown. A leading hypothesis pro poses that mutant sarcomeric proteins impair cardiac myocyte contracti lity, providing an impetus for compensatory hypertrophy. To test this hypothesis, we determined the impact of expression of a mutant (Arg(92 )Gln) human cardiac troponin T (cTnT), known to cause HCM in humans, o n adult cardiac myocyte contractility. A full-length human cTnT cDNA w as cloned, and the Arg(92)Gln mutation was induced. Recombinant adenov iruses Ad5/CMV/cTnT-N and Ad5/CMV/cTnT-Arg(92)Gln were generated throu gh homologous recombination. Adult feline cardiac myocytes were infect ed with recombinant adenoviruses or a control viral vector (Ad5 Delta E1) at a multiplicity of infection of 100. Expression levels of the fu ll-length normal and mutant cTnT proteins were equal on Western blots. Expression of the exogenous cTnT proteins in cardiac myocytes was als o shown by immunocytochemistry and immunofluorescence, and their incor poration into myofibrils was confirmed by Western blotting on myofibri llar extracts. Electron microscopy showed intact sarcomere structure i n rod-shaped cardiac myocytes in all groups. Cell fractional shortenin g and the peak velocity of shortening were not significantly different among the groups 24 hours after transduction. However, 48 hours after transduction, both fractional shortening and the peak velocity of sho rtening were significantly reduced (24% [P<.001] and 26% [P<.001], res pectively) in cardiac myocytes in the Ad5/CMV/cTnT-Arg(92)Gln compared with the Ad5/CMV/cTnT-N groups. The magnitude of the reductions was g reater at 72 hours after transduction (45% and 39%, respectively; P<.0 01). Our results indicated that expression of the mutant (Arg(92)Gln) cTnT, known to cause HCM in humans, impaired intact adult cardiac myoc yte contractility. Our data also show that both normal and mutant cTnT were incorporated into myofibrils. These results provide a potential mechanism by which mutations in sarcomeric proteins cause HCM.