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

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 alpha-fast tropomyosin (TM) responsible for FHC is actually expressed and determines abnormal contractile function. Since alpha-fast TM is expressed in heart and skeletal muscle, samples from vastus lateralis muscles were studied from two FHC patients carrying an Asp175Asn alpha-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 alpha-fast TM by comigration with purified recombinant human Asp175Asn alpha-fast TM. Densitometric quantification of mutant and wild-type alpha-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 alpha-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.

Rescue of Familial Hypertrophic Cardiomyopathy by Altering Sarcomeric Exposure and Response to Calcium

“…These mice develop a mild hypertrophic phenotype, coupled with systolic and diastolic dysfunction (23). The cardiac myofilaments demonstrate increased calcium sensitivity, which is in agreement with human studies (26). We also found a correlation between an increase in myofilament calcium sensitivity with a decrease in relaxation rate and a blunted response to β-adrenergic stimulation (27).…”

Hypercontractile Properties of Cardiac Muscle Fibers in a Knock-in Mouse Model of Cardiac Myosin-binding Protein-C

“…Taken together, N-terminal cMyBP-C mutations, if occurring in human heterozygous FHC patients, might in some instances determine a "hypercontractile" state that could induce cardiac hypertrophy directly. In this context we point out that a hypercontractile hypothesis has been put forth for some FHC cases in which other sarcomeric proteins are mutated, such as ␣-tropomyosin (35,36). FHC might therefore be a disease induced by mutations causing either functional cardiac impairment followed by compensatory hypertrophy (apparently the majority of all cases) or functional enhancement followed by direct cardiac hypertrophy (14).…”

Molecular Mechanism of the E99K Mutation in Cardiac Actin (ACTC Gene) That Causes Apical Hypertrophy in Man and Mouse

“…It has been widely supposed that mutant protein would be expressed in the heart at up to 50% of total protein and that it exerts its pathological effects by acting as a poison peptide that alters normal function. Direct measurements in human heart tissue have confirmed the poison peptide hypothesis in a few cases (15)(16)(17). The challenge is to explain how over 600 different mutations can cause the same phenotype.…”