© 1997 American Heart Association, Inc.
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Expression of a Mutant (Arg92Gln) Human Cardiac Troponin T, Known to Cause Hypertrophic Cardiomyopathy, Impairs Adult Cardiac Myocyte Contractility
From the Section of Cardiology, Department of Medicine, Baylor College of Medicine, Houston, Tex.
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 Arg92Gln
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 (Ad5
E1) 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.
Key Words: troponin T • hypertrophic cardiomyopathy • sarcomere • contractility • gene transfer
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