Ca²⁺-sensitizing effects of the mutations at Ile-79 and Arg-92 of troponin T in hypertrophic cardiomyopathy

Abstract

Several mutations in human cardiac troponin T (TnT) gene have been reported to cause hypertrophic cardiomyopathy (HCM). To explore the effects of the mutations on cardiac muscle contractile function under physiological conditions, human cardiac TnT mutants, Ile79Asn and Arg92Gln, as well as wild type, were expressed in Escherichia coli and exchanged into permeabilized rabbit cardiac muscle fibers, and Ca2+-activated force was determined. The free Ca2+ concentrations required for tension generation were found to be significantly lower in the mutant TnT-exchanged fibers than in the wild-type TnT-exchanged fibers, whereas no significant differences were found in tension-generating capability under maximal activating conditions and in cooperativity. These results suggest that a heightened Ca2+ sensitivity of cardiac muscle contraction is one of the factors to cause HCM associated with these TnT mutations.

Ca 2+ -desensitizing effect of a deletion mutation ΔK210 in cardiac troponin T that causes familial dilated cardiomyopathy

“…skinned cardiac muscle fibers before and after exchanging native cTnT with recombinant human wild-type or ⌬K210 cTnT. Treatment of skinned fibers with an excess amount of recombinant human cTnT resulted in a decrease in the amount of endogenous cTnI and cTnC (lanes 3 and 4) because of displacement of the Tn complex with recombinant human cTnT, as demonstrated in our previous studies (10,14,(30)(31)(32). Subsequent incubation with rabbit cTnI and cTnC accomplished the exchange of recombinant human cTnT into rabbit cardiac muscle fibers (lanes 5 and 6).…”

“…The decrease in Ca 2ϩ sensitivity of force generation also is expected to facilitate the relaxation of the cardiac muscle at diastole and might directly explain the cardiac dilation itself to some extent. In contrast, we have previously shown that the mutations in cTnT associated with HCM have Ca 2ϩ -sensitizing effects on force generation in cardiac muscle (10)(11)(12)(13)(14)(15). The increase in Ca 2ϩ sensitivity of force generation is expected to impair the relaxation of cardiac muscle and thus cause a diastolic dysfunction, which might be directly involved in the high incidence of sudden death despite mild or no hypertrophy in HCM patients associated with cTnT mutations.…”

“…We have already examined the effects of eight HCM-linked cTnT mutations (I79N, R92Q, ⌬E160, E244D, R278C, and two truncated mutants produced by a splice donor site mutation Int15G 1 3A) and six HCM-linked cTnI mutations (R145G, R145Q, R162W, ⌬K183, G203S, and K206Q) on the contractile functions of cardiac muscle by using a technique for exchanging the exogenous Tn complex into skinned muscle fibers and isolated myofibrils. We found that Ca 2ϩ sensitization in cardiac muscle contractility is a common effect caused by HCM-linked mutations in cTnT and cTnI (10)(11)(12)(13)(14)(15)(16). Several groups also have reported Ca 2ϩ -sensitizing effects of the HCM-linked mutations in cTnT and cTnI.…”

“…Rabbit cardiac skinned muscle fibers were prepared from the left ventricular trabeculae of young male albino rabbits (Ϸ3 mo old) as described (10). In brief, small bundles (0.5-1 mm wide and 5-7 mm long) of trabeculae tied to glass capillary tubes were skinned with relaxing solution containing 0.5% Brij-58 for 30 min at 25°C and were stored up to 3 wk at Ϫ20°C in relaxing solution containing 50% glycerol.…”

“…Expression and purification of the recombinant human cTnTs were performed as described previously (10). The wild-type and mutant cTnTs were eluted at 0.3-0.4 M NaCl on a fast-performance liquid chromatography (FPLC) ion-exchange column, RESOURCE Q (6 ml) (Amersham Pharmacia Biotech), with a linear gradient of 0-0.5 M NaCl in the presence of 20 mM Tris⅐HCl (pH 8.0)͞6 M urea͞5 mM trans-1, 2-cyclohexanediamine-N,N,NЈ,NЈ-tetraacetic acid͞and 15 mM 2-mercaptoethanol.…”