Abstract
The mechanism(s) by which mutations in sarcomeric proteins cause hypertrophic cardiomyopathy (HCM) remains unknown. A leading hypothesis proposes that mutant sarcomeric proteins impair cardiac myocyte contractility, providing an impetus for compensatory hypertrophy. To test this hypothesis, we determined the impact of expression of a mutant (Arg92Gln) human cardiac troponin T (cTnT), known to cause HCM in humans, on adult cardiac myocyte contractility. A full-length human cTnT cDNA was cloned, and the Arg92-Gln mutation was induced. Recombinant adenoviruses Ad5/CMV/cTnT-N and Ad5/CMV/cTnT-Arg92Gln were generated through homologous recombination. Adult feline cardiac myocytes were infected with recombinant adenoviruses or a control viral vector (Ad5ΔE1) at a multiplicity of infection of 100. Expression levels of the full-length normal and mutant cTnT proteins were equal on Western blots. Expression of the exogenous cTnT proteins in cardiac myocytes was also shown by immunocytochemistry and immunofluorescence, and their incorporation into myofibrils was confirmed by Western blotting on myofibrillar extracts. Electron microscopy showed intact sarcomere structure in rod-shaped cardiac myocytes in all groups. Cell fractional shortening 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 shortening were significantly reduced (24% [P<.001] and 26% [P<.001], respectively) in cardiac myocytes in the Ad5/CMV/cTnT-Arg92Gln compared with the Ad5/CMV/cTnT-N groups. The magnitude of the reductions was greater at 72 hours after transduction (45% and 39%, respectively; P<.001). Our results indicated that expression of the mutant (Arg92Gln) cTnT, known to cause HCM in humans, impaired intact adult cardiac myocyte 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.