A Mutant Tropomyosin That Causes Hypertrophic Cardiomyopathy Is Expressed In Vivo and Associated With an Increased Calcium Sensitivity

From the Institute of Human Physiology (R.B., M.A.P., C.R.), University of Pavia (Italy); the Institute of Biology and Genetics (D.A.C.), University of Genoa (Italy); the Department of Cardiovascular Medicine (C.S.R., H.W.), University of Oxford (UK); the Minneapolis (Minn) Heart Institute Foundation (B.J.M.); and the Department of Cardiology (P.S.), St Andrea Hospital, La Spezia, Italy.

Abstract—Mutant contractile protein genes that cause familial hypertrophic cardiomyopathy (FHC) are presumed to encode mutant proteins that interfere with contractile function. However, it has generally not been possible to show mutant protein expression and incorporation into the sarcomere in vivo. This study aimed to assess whether a mutant -fast tropomyosin (TM) responsible for FHC is actually expressed and determines abnormal contractile function. Since -fast TM is expressed in heart and skeletal muscle, samples from vastus lateralis muscles were studied from two FHC patients carrying an Asp¹⁷⁵Asn -fast TM mutation and two healthy control subjects. TM isoforms from whole biopsy samples and single fibers were identified by gel electrophoresis and Western blot analysis. An additional faster-migrating TM band was observed in both FHC patients. The aberrant TM was identified as the Asp¹⁷⁵Asn -fast TM by comigration with purified recombinant human Asp¹⁷⁵Asn -fast TM. Densitometric quantification of mutant and wild-type -fast TMs suggested equal expression of the two proteins. Contractile parameters of single skinned muscle fibers from FHC patients and healthy control subjects were compared. Calcium sensitivity was significantly increased in muscle fibers containing Asp¹⁷⁵Asn -fast Tm compared with fibers lacking the mutant TM. No discernible difference was found regarding cooperativity, maximum force, and maximum shortening velocity. This is the first demonstration that the mutant TM that causes FHC is indeed expressed and almost certainly incorporated into muscle in vivo and does result in altered contractile function; this confirms a dominant-negative, rather than null allele, action. Since the mutant TM was associated with increased calcium sensitivity, this mutation might be associated with an enhancement and not a depression of cardiac contractile performance. If so, this contrasts with the hypothesis that FHC mutations induce contractile impairment followed by compensatory hypertrophy.

Key Words: familial hypertrophic cardiomyopathy • tropomyosin • Ca²⁺ sensitivity • skinned muscle fiber

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