Functional analyset of mutations in human cardiac troponin T that cause familial hypertrophic cardiomyopathy

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Familial hypertrophic cardiomyopathy (HCM) is an autosomal dominant cardiac disease associated with a high incidence of sudden death. HCM is a genetically heterogeneous disease which has been shown to be caused by mutations in the genes for a variety of cardiac myofibrillar proteins, including β-myosin heavy chain (β-MHC), subunits of Ca^<2+>- receptor troponin (troponin T and troponin I), α-tropomyosin, myosin-binding protein C, and myosin light chains. HCM is also clinically variable and the mutations in cardiac troponin T (TnT) are known to be associated with a high risk of sudden death despite an incomplete disease penetrance with moderate hypertrophy, in contrast to the high penetrance with more severe hypertrophy of β-MHC mutations with a comparable poor prognosis.At least 13 different mutations in the TnT gene have been identified ; 11 missense mutations, a mutation involving a deleted codon and a splice donor site mutation. To explore mechanism (s) underlying the pathogenesis o … More f HCM, several mutants of human cardiac TnT associated with HCM [five missense mutants (Ile79Asn, Arg92Gln, Phe110Ile, Glu244Asp, Arg278Cys), one deletion mutant (ΔGlu160), and two truncated mutants produced by a splice donor site mutation] were expressed in E.Coli, exchanged into permeabilized rabbit cardiac muscle fibers, and Ca^<2+>-activated force was determined.The missense mutations Ile79Asn and Arg92Gln and the mutation involving a deleted codon ΔGlu160 had a Ca^<2+>- sensitizing effect on the contraction of the skinned cardiac muscle fibers without affecting the maximum force and the cooperativity. The missense mutations Glu244Asp and Arg278Cys and the splice donor site mutation also had a Ca^<2+>-sensitizing effect, but the Glu244Asp mutation was also found to increase the maximum force remarkably and the missense mutation Arg278Cys and the splice donor site mutation decreased the cooperativity significantly. The missense mutation Phe110Ile, the only TnT mutation which has ever been shown to be associated with a favorable prognosis, had no Ca^<2+>-sensitizing effect and did not change the cooperativity, but was found to increase the maximum force remarkably. The results indicate that these HCM-linked mutations in TnT can be classified into at least four groups according to their functional effects on the Ca^<2+>-activated contraction, which are Ca^<2+>-sensitization (Ile79Asn, Arg92Gln, ΔGlu160), potentiation of the maximum force (Phe110Ile), a mixture of Ca^<2+>-sensitization and potentiation of the maximum force (Glu244Asp), and a mixture of Ca^<2+>-sensitization and reduction in the cooperativity (Arg278Cys and splice donor site mutation).These results indicate that TnT plays a critical role in the thin filament regulatory mechanism involving the Ca^<2+> sensitivity, cooperativity and maximum force-generating capability of cardiac muscle contraction and that an increased Ca^<2+>-sensitivity might be responsible for the distinguishing feature of HCM caused by TnT mutations, i.e. incomplete disease penetrance and poor prognosis. Less