Functional changes in troponin T by a splice donor site mutation that causes hypertrophic cardiomyopathy

Abstract

A splice donor site mutation in intron 15 of the cardiac troponin T (TnT) gene has been shown to cause familial hypertrophic cardiomyopathy (HCM). In this study, two truncated human cardiac TnTs expected to be produced by this mutation were expressed in Escherichia coli and partially (50–55%) exchanged into rabbit permeabilized cardiac muscle fibers. The fibers into which a short truncated TnT, which lacked the COOH-terminal 21 amino acids because of the replacement of 28 amino acids with 7 novel residues, had been exchanged generated a Ca2+-activated maximum force that was slightly, but statistically significantly, lower than that generated by fibers into which wild-type TnT had been exchanged when troponin I (TnI) was phosphorylated by cAMP-dependent protein kinase. A long truncated TnT simply lacking the COOH-terminal 14 amino acids had no significant effect on the maximum force-generating capability in the fibers with either phosphorylated or dephosphorylated TnI. Both these two truncated TnTs conferred a lower cooperativity and a higher Ca2+ sensitivity on the Ca2+-activated force generation than did wild-type TnT, independent of the phosphorylation of TnI by cAMP-dependent protein kinase. The results demonstrate that the splice donor site mutation in the cardiac TnT gene impairs the regulatory function of the TnT molecule, leading to an increase in the Ca2+ sensitivity, and a decrease in the cooperativity, of cardiac muscle contraction, which might be involved in the pathogenesis of HCM.

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.…”

“…The cloning and mutagenesis of human cTnT cDNA were carried out as described previously (14). To generate the ⌬K210 and ⌬E160 cDNAs, mutageneses were first carried out by PCR according to the GeneSOEing method described by Horton (27) by using the following oligonucleotide primers: 5Ј-GGT GGT GGA AGC GTA CGA AGA GG-3Ј (18F, SplI site is underlined), 5Ј-CCT CTC AGC CAG AAT CTT CTT CTT TTC CCG (645R), 5Ј-AAG AAG ATT CTG GCT GAG AGG AGG AAG GTG (622F), and 5Ј-GCT GCA GGA TCC TAT TTC CAG CGC CCG G (878 R, BamHI site is underlined) for ⌬K210; 5Ј-GGT GGT GGA AGC GTA CGA AGA GG-3Ј (18F, SplI site is underlined), 5Ј-CCT GTT CTC CTC CTC TCG TCG AGC CCT CTC (495R), 5Ј-CGA CGA GAG GAG GAG AAC AGG AGG AAG GCT (472F), and 5Ј-GCT GCA GGA TCC TAT TTC CAG CGC CCG G (878 R, BamHI site is underlined) for ⌬E160.…”

Inherited Cardiomyopathies as a Troponin Disease

“…It has been shown that the mutations in cTnI associated with HCM modify the effect of cTnI phosphorylation by PKA. Deng et al have shown that R145Q mutation increases the Ca 2+ sensitivity of myosin subfragment-1 ATPase activity and the in vitro filament motility independently of the phosphorylation state of cTnI [79], suggesting that this mutation impairs the mechanism involving cardioprotection through the phosphorylation of cTnI, as with a cTnT mutation [80]. The same group has also reported that the C-terminal mutation K206Q abolishes the sensitivity of myofilaments to the phosphorylation of cTnI by PKA [81].…”