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

Hiroyuki Nakaura1,2, Sachio Morimoto1, Fumi Yanaga1, Masashi Nakata3, Hirofumi Nishi2, Tsutomu Imaizumi2, and Iwao Ohtsuki1

1 Department of Clinical Pharmacology, Faculty of Medicine, Kyushu University, Higashi-ku, Fukuoka 812-8582; and 2 Third Department of Medicine and 3 Cardiovascular Research Institute, Kurume University School of Medicine, Kurume, Fukuoka 830-0011, Japan


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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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.

calcium sensitivity; cooperativity; truncated mutant; skinned fiber


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

FAMILIAL HYPERTROPHIC CARDIOMYOPATHY (HCM) is an autosomal dominant cardiac disease associated with a high incidence of sudden death. HCM is a genetically heterogeneous disease that has been shown to be caused by mutations in the genes for a variety of cardiac myofibrillar proteins, including beta -myosin heavy chain (beta -MHC) (8), troponin T (TnT) (33), troponin I (TnI) (11), alpha -tropomyosin (alpha -TM) (33), myosin-binding protein C (3, 32), and myosin light chains (22).

HCM is also clinically variable, and the mutations in cardiac TnT are known to be associated with a high risk of sudden death despite an incomplete disease penetrance with moderate hypertrophy (33), in contrast to the high penetrance with more severe hypertrophy of beta -MHC mutations with a comparably poor prognosis (1, 6, 24, 34). Mutations in the beta -MHC gene in general impair the interaction between myosin and actin so that they depress cardiac performance and lead to a compensatory cardiac hypertrophy (5, 7, 13, 25). 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 (21). We have previously shown that two missense mutations, the substitution of Asn for Ile at position 79 (Ile79Asn) and Arg92Gln, have a Ca2+-sensitizing effect on the contraction of cardiac muscle without affecting the maximum Ca2+-activated force and the cooperativity (15), suggesting that the mutations in TnT are not a stimulus for compensatory cardiac hypertrophy and that Ca2+ sensitization may be responsible for the distinguishing feature of HCM caused by TnT mutations, i.e., incomplete disease penetrance and poor prognosis.

The clinical phenotype of the splice donor site mutation in intron 15 of the cardiac TnT gene has been shown to be similar to those of the missense mutations Ile79Asn and Arg92Gln (33). This mutation results in aberrant splice products encoding two truncated TnT molecules lacking COOH termini (29). One truncated mutant TnT simply lacks the COOH-terminal 14 amino acids (TnTDelta 14), and the other mutant lacks 21 amino acids because of the replacement of the normal 28 terminal amino acids with 7 novel residues (TnTDelta 28(+7)). It has been shown that TnTDelta 28(+7) can be incorporated into troponin complexes within a sarcomere when expressed in a cultured, transfected quail skeletal myotube and that it greatly diminishes the maximum Ca2+-activated force (35), suggesting that the splice donor site mutation in the TnT gene acts as a dominant-negative allele that impairs cardiac contractile performance and leads to a compensatory hypertrophy as in the case of the beta -MHC mutations. However, in this quail skeletal myotube expression system, it was reported to be technically impossible to control the extent of mutant TnT incorporation into the sarcomeres, and the contractile response to the submaximal Ca2+ concentrations (e.g., Ca2+ sensitivity and cooperativity) remains to be determined because of a dramatic depression of maximum force. In the present study, we attempted to determine the functional effects of this splice donor site mutation on the cardiac muscle contraction by exchanging the two truncated human cardiac TnT molecules directly into rabbit skinned cardiac muscle fibers. The extent of mutant TnTs exchanged into the skinned fibers was 50-55%, which was close to the heterozygous state expected in most patients. Both mutants increased the Ca2+ sensitivity, as in the case of the missense mutations Ile79Asn and Arg92Gln, and also decreased the cooperativity of the contraction of skinned cardiac muscle fibers. Whereas TnTDelta 28(+7) slightly decreased the maximum Ca2+-activated force, TnTDelta 14 had no significant effect on the maximum force under these conditions. The results suggest that these functional changes in cardiac muscle, an increase in Ca2+ sensitivity and decreases in cooperativity and maximum force, might be involved in the pathogenesis of HCM associated with the cardiac TnT splice donor site mutation.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Mutagenesis of human cardiac TnT cDNA. Human cardiac TnT cDNA was amplified by RT-PCR of human heart mRNA purchased from Clontech (Palo Alto, CA). The PCR products were subcloned into the pUC119 vector for screening by restriction analysis and DNA sequencing. The obtained wild-type TnT cDNA was then constructed in the pET3-d vector, and mutagenesis was carried out by PCR. To generate TnTDelta 28(+7) cDNA, PCR was first carried out to create Spl I and EcoR I sites by using oligonucleotides 5'-GGT GGT GGA AG<OVL>C</OVL> <OVL>GTA:CG</OVL>A AGA GG-3' (18F; Spl I site underlined) and 5'-CGG AGA ACA TT<OVL>G AAT TC</OVL>A TAT TTC-3' (794R; EcoR I site underlined). The obtained PCR product was then digested by Spl I and EcoR I and connected to oligonucleotides 5'-CAT GTC TGA CAT CGA AGA AGT GGT GGA AGA-3', to be ligated to the Nco I site of the pET-3d vector, and 5'-AAT TGC AAG ACC CGC GGG AAG GCT AG-3', to replace the COOH-terminal 28 amino acids with 7 novel amino acids. To generate TnTDelta 14 cDNA, PCR was carried out to introduce a stop codon and a BamH I site at the position of Ser-275 by using oligonucleotides 5'-GGT GGT GGA AG<OVL>C:GTA:CG</OVL>A AGA GG-3' (18F; Spl I site underlined) and 5'-CCC GCG G<OVL>GG:ATC</OVL> <OVL>C</OVL>TA GAC TTT CTG G-3' (837R; BamH I site underlined). The obtained PCR product, digested by Spl I and BamH I, was connected to oligonucleotide 5'-CAT GTC TGA CAT CGA AGA AGT GGT GGA AGA-3' to be ligated to the Nco I site of the pET-3d vector and thereby was joined with the vector. The complete nucleotide sequences of the mutant TnT cDNAs were confirmed by DNA sequencing.

Purification of proteins. Expression and purification of the recombinant human cardiac TnTs were performed as described previously (15). Recombinant TnT was purified with a fast-performance liquid chromatography (FPLC) ion-exchange column, RESOURCE Q (6 ml; Pharmacia Biotech), with a linear gradient of 0-0.5 M NaCl in the presence of 20 mM Tris · HCl (pH 8.0), 6 M urea, 5 mM trans-1,2-cyclohexanediamine-N, N,N',N'-tetraacetic acid (CDTA), and 15 mM 2-mercaptoethanol; recombinant TnT was eluted at 0.3-0.4 M NaCl. Native troponin and its subunits, TnT, TnI, and troponin C (TnC), were prepared from the left ventricular myocardiums of young male albino rabbits (~3 mo old) according to the method of Tsukui and Ebashi (30), by using FPLC ion-exchange columns RESOURCE Q and RESOURCE S (6 ml) and Mono Q and Mono S HR 5/5 (Pharmacia Biotech).

Dephosphorylation and phosphorylation of TnI. Purified native cardiac TnI (0.5 mg/ml) was dephosphorylated by incubation with 50 U/ml alkaline phosphatase, type VII NL (Sigma Chemical), for 10 min at 25°C in 5 ml of a solution containing 20 mM Tris · HCl (pH 9.8), 0.25 M KCl, 1 µg/ml pepstatin A, and 0.2 mM phenylmethylsulfonyl fluoride (PMSF). The reaction was stopped by the addition of 6 M urea, and dephosphorylated TnI was purified by FPLC cation-exchange column POROS HS/M (PerSeptive Biosystems), with a linear gradient of 0-0.5 M NaCl in the presence of 20 mM Tris · HCl (pH 8.0), 6 M urea, 5 mM CDTA, and 15 mM 2-mercaptoethanol; dephosphorylated TnI was eluted at 0.3-0.4 M NaCl.

The phosphorylation of TnI was performed by incubating the dephosphorylated TnI (0.25 mg/ml) with 200 U/ml catalytic subunit of cAMP-dependent protein kinase (PKA; Sigma Chemical) for 20 min at 25°C in 5 ml of a solution containing (in mM) 50 KH2PO4-KOH (pH 7.0), 250 KCl, 10 ATP, 10 MgCl2, 30 NaF, and 40 dithiothreitol (DTT), as well as 1 µg/ml pepstatin A and 0.2 mM PMSF. The reaction was stopped by the addition of 6 M urea, and the phosphorylated TnI was purified by a POROS HS/M column with a linear gradient of 0-0.5 M NaCl in the presence of 20 mM Tris · HCl (pH 8.0), 6 M urea, 5 mM CDTA, and 15 mM 2-mercaptoethanol; phosphorylated TnI was eluted at 0.3-0.4 M NaCl.

Preparation of skinned fibers and force measurements. Skinned fibers were prepared from the left ventricular trabeculae of young male albino rabbits (~3 mo old), and force measurements were performed as described previously (15). Briefly, 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 -20°C in relaxing solution containing 50% glycerol. A small fiber (~120 µm in diameter) dissected from the stock skinned trabecula was mounted in a thermostatically controlled chamber with a capacity of 0.2 ml. The fiber length between hooks was ~1 mm, and the resting sarcomere length was set to 2.3 µm by laser diffraction. The force was measured with strain gauge UL-2GR (Minebea). The relaxing solution consisted of (in mM) 50 MOPS-KOH (pH 7.0), 100 KCl, 6 MgCl2, 5 ATP, 4 EGTA, 0.5 DTT, and 10 creatine phosphate, as well as 35 U/ml creatine kinase. Activating solutions with desired free Ca2+ concentrations were prepared by adding appropriate amounts of CaCl2, calculated as described previously (14), to the relaxing solution. All force measurements were done at 25°C.

To determine the pCa value at half-maximal force generation (pCa50) and the Hill coefficient (nH), the force was normalized to the maximum force generated in the same fiber and the relationship between relative force and pCa in each fiber was fitted to the following form of the Hill equation by the Marquardt nonlinear least-squares method (Prism 2.01; GraphPad Software): relative force (%) = 100/[1 + 10(pCa - pCa50)nH]. Average values of pCa50 and nH were then calculated and are summarized in Tables 1 and 2. The value R2 (square of the multiple-correlation coefficient), a measure of goodness of fit, was in the range of 0.98-1.0 in all determinations.

                              
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Table 1.   Ca2+ sensitivity, cooperativity, and maximum force in recombinant human cardiac TnT-exchanged fibers


                              
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Table 2.   Effect of TnI phosphorylation on Ca2+ sensitivity, cooperativity, and maximum force in recombinant human cardiac TnT-exchanged fibers

TnT exchange in skinned fibers. TnT exchange in the skinned fibers was performed by the method described previously (15).

SDS-PAGE and immunoblot analyses. SDS-PAGE was carried out at 8 or 12% acrylamide concentration according to the method of Laemmli (12). The fiber samples were lysed in Laemmli's sample buffer by heating for 4 min at 95°C after having been frozen (-80°C) and thawed several times. The gel was stained with silver with a staining kit (Pharmacia Biotech) or with Coomassie brilliant blue R-250. Immunoblot analysis was performed after electrophoretically transferring proteins from unstained SDS-12% polyacrylamide gels to polyvinylidene difluoride membranes (0.2 µm; Bio-Rad). After fixation with 0.2% glutaraldehyde (31), the blots were blocked with 3% nonfat dry milk and reacted with primary antibody; primary antibodies used in the present study were monoclonal anti-cardiac TnT antibody 1F2 (HyTest), monoclonal anti-cardiac TnI antibody 8E10 (HyTest), monoclonal anti-tropomyosin (sarcomeric) antibody CH1 (Sigma Chemical), and a rabbit polyclonal anti-phosphoserine antibody that should not differentiate between mono- and diphosphorylated forms of TnI (Zymed Laboratories). After being washed, the membranes were reacted with an alkaline phosphatase-conjugated second antibody, and the antigens of primary antibodies were visualized by a chemiluminescent detection reagent (CDP-Star; NEN Life Science Products). An optical densitometric scan was performed with Phoretix International gel analysis software calibrated by a photographic step tablet (21 steps; density range 0.05-3.03; Eastman Kodak). The linearity of the densitometric readings from immunoblots was confirmed by changing the amount of sample applied on the gel in steps.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Figure 1A shows the results of SDS-PAGE of the bacterially expressed wild-type and HCM-causing truncated mutants of human adult cardiac TnT. The recombinant human wild-type cardiac TnT showed an electrophoretic mobility similar to that of the native cardiac TnT purified from the hearts of adult rabbits, consistent with the study reporting that the major isoforms of adult human and rabbit cardiac TnTs show almost the same electrophoretic mobilities on a similar gel (2). Both the two truncated mutant cardiac TnTs showed slightly higher electrophoretic mobility than wild-type TnT, as expected.


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Fig. 1.   A: SDS-PAGE of purified recombinant wild-type, and truncated mutants of, human cardiac troponin T (TnT). Lane 1, rabbit cardiac tissue-derived (native) TnT; lane 2, human cardiac wild-type TnT; lane 3, human cardiac truncated mutant TnTDelta 28(+7); lane 4, human cardiac truncated mutant TnTDelta 14; lane 5, human cardiac wild-type TnT plus truncated mutant TnTDelta 28(+7); lane 6, human cardiac wild-type TnT plus truncated mutant TnTDelta 14. Acrylamide concentration of separating gel was 8%, and gel was stained with Coomassie brilliant blue R-250. B: SDS-PAGE of rabbit skinned cardiac muscle fibers treated with human recombinant cardiac TnTs and reconstituted with rabbit native cardiac troponin I (TnI) and troponin C (TnC). Acrylamide concentration of separating gel was 12%, and gel was stained with a silver staining kit (Pharmacia Biotech). LC2, myosin light chain 2; TM, tropomyosin. C: immunoblot analysis of TnT-treated fibers before reconstitution with rabbit native cardiac TnI and TnC probed with primary antibodies against TnT, TM, and TnI. D: immunoblot analysis of TnT-treated fibers after reconstitution with rabbit native cardiac TnI and TnC probed with primary antibodies against TnT, TM, and TnI. In B-D, untreated (control) rabbit cardiac skinned fibers were loaded in lane 1 and fibers treated with human cardiac wild-type TnT, TnTDelta 28(+7), and TnTDelta 14 were loaded in lanes 2-4, respectively.

It has previously been demonstrated that endogenous TnT-TnI-TnC complexes in skinned muscle fibers are selectively replaced with exogenous TnT when the fibers are treated with an excess amount of TnT under acidic and high-ionic-strength conditions (9, 10, 15). To investigate the functional consequence of the splice donor site mutation in the TnT gene, an attempt was made in the present study to directly exchange the two human recombinant truncated TnTs, as well as the recombinant wild-type TnT, into adult rabbit cardiac skinned muscle fibers by this technique. SDS-PAGE analysis of the TnT-treated fibers after reconstitution with rabbit native cardiac TnI and TnC indicated that the two truncated TnTs were incorporated into the fibers without changing the stoichiometry of other myofibrillar constituents (Fig. 1B). Immunoblot analysis of the TnT-treated fibers using monoclonal antibodies against TnT, TM, and TnI directly demonstrated that the two truncated TnTs were exchanged into the skinned fibers (Fig. 1C); ratios of truncated TnT to total TnT in lanes 3 and 4 estimated by optical densitometric scans were 0.50 and 0.54, respectively. Quantification of the relative amounts of native and truncated TnT in a subgroup of fibers indicated that the extents of TnTDelta 28(+7) and TnTDelta 14 exchanged were 54.5 ± 2.3 and 56.8 ± 2.8% (means ± SE of measurements on 3 fibers). The immunoblot analysis of the TnT-treated fibers also showed that the TnT treatment caused a decrease in the amount of native TnI, an inhibitory subunit of the troponin complex, in all fibers, indicating that endogenous TnT-TnI-TnC complexes were just displaced by exogenously added TnT (Fig. 1C); TnI-to-TM ratios in lanes 1, 2, 3, and 4 were 1.41, 0.65, 0.70, and 0.63, respectively. Quantification of the decreased amount of TnI in a subgroup of fibers by densitometric scans indicated that the extents of the endogenous troponin complex displaced by TnT and thus the extents of TnT exchanged into the fibers were 54.9 ± 2.6, 51.4 ± 2.1, and 55.8 ± 1.2% (means ± SE of measurements on 3 fibers) for wild-type TnT, TnTDelta 28(+7), and TnTDelta 14, respectively. Consistent with this, the skinned fibers treated with these three recombinant TnTs developed a large force even in the absence of Ca2+ (Fig. 2A). Reconstitution of the TnT-treated skinned fibers with rabbit native cardiac TnI inhibited the Ca2+-insensitive force generation completely (Fig. 2B). Immunoblot analysis indicated that the reincorporation of TnI was nearly stoichiometric in all the skinned fibers treated with the three recombinant TnTs (Fig. 1D); TnI-to-TM ratios in lanes 1, 2, 3, and 4 were 1.43, 1.39, 1.47, and 1.31, respectively. Subsequent reconstitution of the skinned fibers with rabbit native cardiac TnC restored Ca2+-dependent force generation (Fig. 2C). The TnC-to-myosin light chain 2 ratios estimated by optical densitometric scans of the SDS-PAGE gel in B were 0.25, 0.23, 0.23, and 0.24 in lanes 1, 2, 3, and 4, respectively, indicating that the reincorporation of TnC is also stoichiometric in all cases.


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Fig. 2.   Changes in force response to Ca2+ by TnT exchange into rabbit skinned cardiac muscle fibers. A: changes after treatment with recombinant human cardiac TnTs. B: changes after reconstitution with rabbit cardiac TnI. C: changes after reconstitution with rabbit cardiac TnC. Forces measured in absence of Ca2+ (pCa > 9; open bars) and in presence of a saturating amount of Ca2+ (pCa < 5.5; solid bars) were normalized to maximum force developed by same fiber before TnT treatment and are expressed as means ± SE of measurements on 8-10 fibers.

A previous study showed that the exchange of the human wild-type cardiac TnT into rabbit cardiac skinned fibers caused a decrease in the maximum force and also caused a slight rightward shift in, and a decrease in the steepness of, the normalized force-pCa relationship (15). However, the previous study also demonstrated that the exchange of tissue-derived rabbit native cardiac TnT into rabbit skinned cardiac muscle fibers had exactly the same effects on these parameters. This indicates that although the TnT exchange procedure itself has a nonspecific deteriorative effect on both contractile and regulatory systems to some extent, the displacement of the endogenous troponin complex in rabbit skinned cardiac muscle fibers with recombinant human cardiac TnT and the subsequent reconstitution with rabbit cardiac TnI and TnC constitute an effective approach for investigating the functional effects of the HCM-causing mutations in human cardiac TnT on cardiac muscle contraction. In the following experiments, the wild-type human cardiac TnT-exchanged fiber was taken as a control to detect a specific functional effect exerted on the fibers by mutant human cardiac TnT, as in the previous study (15).

Figure 3 compares the force-pCa relationships of the fibers into which the two truncated mutants of human cardiac TnT were exchanged with the force-pCa relationship of the fibers into which the human wild-type cardiac TnT was exchanged. Table 1 summarizes the Ca2+ sensitivity (pCa50) and cooperativity (nH) in the force-pCa relationships, as well as the normalized maximum force. Although the maximum force generated by the TnTDelta 14-exchanged fibers was not statistically significantly different from that generated by the wild-type TnT-exchanged fibers, the TnTDelta 28(+7)-exchanged fibers developed a slightly but statistically significantly lower level of maximum force than the wild-type TnT-exchanged fibers (Fig. 3 and Table 1). Both these two truncated TnTs conferred a higher Ca2+ sensitivity and a lower cooperativity than did wild-type TnT, as demonstrated by a significant increase in pCa50 and a significant decrease in nH (Table 1). Before TnT exchange, there were no statistically significant differences in Ca2+ sensitivity and cooperativity between these three fiber groups (Fig. 3, inset).


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Fig. 3.   Effect of exchanging truncated mutant TnT into skinned cardiac muscle fibers on force-pCa relationships. Force-pCa relationships determined after exchange of recombinant human cardiac wild-type TnT (open circle ), TnTDelta 28(+7) (black-triangle), and TnTDelta 14 (black-down-triangle ) were compared; forces were normalized to averaged maximum force in fibers with wild-type TnT exchanged and are means ± SE of measurements on 8-10 fibers. Inset, force-pCa relationships determined in respective fibers before TnT exchange; forces were normalized to maximum force developed by each fiber.

It is known that phosphorylation of TnI by PKA results in a decrease in the Ca2+ sensitivity of cardiac muscle contraction, which may play an important role in the positive inotropic effect of beta -adrenergic stimulation. To see whether the truncated mutant TnT affects this Ca2+-desensitizing effect of TnI phosphorylation by PKA, the wild-type TnT- and the two truncated TnT-treated fibers were reconstituted with dephosphorylated or phosphorylated TnI and TnC. Figure 4A shows an immunoblot analysis of the dephosphorylated or phosphorylated TnI with antibodies against TnI and phosphoserine, which confirms the phosphorylation and dephosphorylation of TnI used for reconstitution. Immunoblot analyses of the fibers reconstituted with dephosphorylated or phosphorylated TnI with antibodies against TnI and TM also confirmed that both the phosphorylated and dephosphorylated TnIs were incorporated stoichiometrically into the fibers (Fig. 4B). In both the wild-type and truncated TnT-exchanged fibers, force-pCa relationships conferred by the reconstitution with phosphorylated TnI were significantly shifted rightward compared with those conferred by the reconstitution with dephosphorylated TnI (Fig. 4B), indicating that the Ca2+ sensitivity is decreased by TnI phosphorylation in the presence of truncated TnT as well as in the presence of wild-type TnT; it should be noted that the pCa50 values for the fibers reconstituted with untreated TnI in Fig. 3 (Table 1) are intermediate between the values for the fibers reconstituted with phosphorylated and dephosphorylated TnI (Table 2), in agreement with the fact that the untreated TnI has already been phosphorylated considerably in vivo (Fig. 4A). The truncated TnT-exchanged fibers always showed significantly higher Ca2+ sensitivity and lower cooperativity than did the wild-type TnT-exchanged fibers irrespective of whether they were reconstituted with phosphorylated or dephosphorylated TnI (Fig. 4B and Table 2). Before TnT exchange, there were no statistically significant differences in Ca2+ sensitivity and cooperativity between these four fiber groups (data not shown). These results indicate that the two truncated mutant TnTs increase the Ca2+ sensitivity and decrease the cooperativity independently of the phosphorylation of TnI by PKA. Although the two truncated mutant TnTs had no significantly different effect on these two parameters, TnTDelta 28(+7) conferred a significantly lower maximum force than wild-type TnT and TnTDelta 14 when reconstituted with phosphorylated TnI.


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Fig. 4.   A: immunoblot analysis of dephosphorylated and phosphorylated TnI. Purified TnI was dephosphorylated by treatment with alkaline phosphatase or phosphorylated by cAMP-dependent protein kinase (PKA) after dephosphorylation as described in MATERIALS AND METHODS, and subjected to immunoblotting with anti-TnI antibody and anti-phosphoserine antibody. Lane 1, untreated TnI; lane 2, phosphorylated TnI after dephosphorylation; lane 3, dephosphorylated TnI. B, top: immunoblot analysis of TnT-treated fibers after reconstitution with dephosphorylated or phosphorylated TnI and TnC probed with primary antibodies against TM and TnI. Lanes 1 and 4, untreated (control) rabbit cardiac skinned fibers; lane 2, wild-type TnT-exchanged fiber reconstituted with phosphorylated TnI; lane 3, wild-type TnT-exchanged fiber reconstituted with dephosphorylated TnI; lane 5, TnTDelta 28(+7)-exchanged fiber reconstituted with phosphorylated TnI; lane 6, TnTDelta 28(+7)-exchanged fiber reconstituted with dephosphorylated TnI. TnI-to-TM ratios in lanes 1-6, estimated by optical densitometric scans, were 0.90, 0.88, 0.98, 0.95, 0.87, and 0.88, respectively. Data are representative of those from 3 other experiments. Bottom: effect of TnI phosphorylation on force-pCa relationships in wild-type and truncated mutant TnT-exchanged skinned fibers. Wild-type TnT- (open circle , ), TnTDelta 28(+7)- (triangle , black-triangle), and TnTDelta 14 (down-triangle, black-down-triangle )-treated fibers were reconstituted with TnI that had been dephosphorylated by phosphatase (open circle , triangle , down-triangle) or phosphorylated by PKA (, black-triangle, black-down-triangle ) and were further reconstituted with TnC. Forces were normalized to maximum force developed by each fiber and are means ± SE of measurements on 3-5 fibers.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present study demonstrates that both the two truncated cardiac TnTs produced by the HCM-causing splice donor site mutation increase the Ca2+ sensitivity and decrease the cooperativity of the Ca2+-activated force generation in cardiac muscle and that the short truncated mutant, TnTDelta 28(+7), furthermore decreases the maximum force. By the same method used in the present study, we have previously shown that the HCM-causing missense mutations Ile79Asn and Arg92Gln in the TnT gene also increase the Ca2+ sensitivity but do not alter the cooperativity and maximum force (15). Because the increase in Ca2+ sensitivity is expected to increase the force generated at submaximal intracellular Ca2+ concentrations, these HCM-causing TnT mutations may have a common functional effect of directly enhancing the cardiac performance, in contrast to the missense mutations in the beta -MHC gene, which are considered to cause a depression of cardiac performance that leads to a compensatory hypertrophy.

Because the increased Ca2+ sensitivity would also involve an increase in the myofibrillar ATPase activity (i.e., an increase in the utilization of ATP by actomyosin ATPase) at submaximal intracellular Ca2+ concentrations, these mutations in TnT may facilitate the rapid exhaustion of the intracellular ATP under severe stress, leading to an imbalance in energy supply and demand in the heart that might be responsible for sudden death (23). The increased Ca2+ sensitivity may also cause a diastolic dysfunction by impairing relaxation, as has recently been shown in hearts from transgenic mice expressing the human cardiac truncated mutant TnTDelta 28(+7) (28). The beta -adrenergic stimulation activates PKA and causes a decrease in Ca2+ sensitivity through the phosphorylation of TnI, which plays a cardioprotective role in preventing the overstimulation of myofilaments by a dramatic increase in the intracellular Ca2+ concentration (23). The present study shows that the truncated TnT has a Ca2+-sensitizing effect independent of the phosphorylation of TnI by PKA, suggesting that these unfavorable effects of the increased Ca2+ sensitivity may be augmented by the positive inotropic effect of beta -adrenergic stimulation. On the other hand, the decrease in cooperativity caused by the truncated TnT is expected to make the force response of cardiac muscle to changes in intracellular Ca2+ concentration less steep, but it is difficult to predict its impact on cardiac performance.

Analysis of a quail skeletal myotube expression system has shown that the truncated mutant TnTDelta 28(+7) is incorporated into the sarcomeres of myotubes in vivo and greatly inhibits (by ~80%) the maximum force generation (35). This indicates that the splice donor site mutation in the TnT gene acts as a dominant-negative allele but not as a null allele. The present study indicates that this truncated TnT can also be incorporated into the sarcomeres of cardiac muscle in vitro as effectively as wild-type TnT and inhibits the maximum force generation. Of interest is the fact that the fibers with this mutant TnT exchanged produce significantly less force than the fibers with wild-type TnT or TnTDelta 14 exchanged in the absence of Ca2+ even before the reconstitution with TnI and TnC (Fig. 2A). Because the extent of TnTDelta 28(+7) exchanged was no different from the extents of wild-type TnT and TnTDelta 14 exchanged, this may indicate that TnTDelta 28(+7) itself has a direct inhibitory effect on the force-generating interaction of the thin filament with myosin cross bridges. Another interesting finding is that the inhibition of the maximum force by TnTDelta 28(+7) has only been observed in the fibers reconstituted with phosphorylated TnI (Table 2). This suggests that the maximum force-generating capability of cardiac muscle in patients might be impaired by beta -adrenergic stimulation. However, the inhibitory effect of TnTDelta 28(+7) on the force generation in skinned rabbit cardiac muscle observed in the present study was quite small (~20%) compared with that observed in the quail skeletal myotube expression system. This discrepancy is probably due to a difference in the amount of the truncated TnT incorporated into the sarcomere; replacement of endogenous TnT with truncated TnT was nearly complete in the quail skeletal myotube expression system, whereas it was 50-55% in the skinned cardiac muscle system in the present study. Because HCM is an autosomal dominant disorder, most patients with the splice donor site mutation in the TnT gene are expected to be heterozygous, with the normal and the two truncated mutant proteins being expressed and probably incorporated into the sarcomere in the ratio of 2:1:1 (29). The present study showed that TnTDelta 14 has no effect on maximum force-generating capability so that the splice donor site mutation in the TnT gene could not have a large effect on the maximum force-generating capability of cardiac muscle in patients. Because intact cardiac muscle is generally thought to be never activated beyond about half-maximal level (23) and because both truncated mutant TnTs are expected to just increase the force at the submaximal Ca2+ concentration (Fig. 3), the splice donor site mutation in the TnT gene would potentially have an enhancing effect on the force generation in the living heart.

The contraction of vertebrate striated muscle is regulated by a specific regulatory protein complex, troponin, located at regular intervals in the thin filament. TnT has an important structural role in anchoring the other constituents of the troponin complex, TnI and TnC, to TM (17). Previous studies of the rabbit fast-twitch skeletal TnT molecule have revealed that a highly alpha -helical region of 81 amino acid residues at the NH2-terminal side of the molecule constitutes a primary, strong TM-binding site and that a COOH-terminal region of the molecule constitutes a second, weak TM-binding site (20, 27). Chymotryptic digestion is known to split the fast-twitch skeletal TnT into two subfragments designated TnT1 and TnT2 (or TnT2alpha ); TnT1 is the NH2-terminal three-fifths of TnT containing the primary TM-binding region, and TnT2 is the remaining COOH-terminal two-fifths of TnT constituting the second TM-binding region and retaining a partial regulatory function (16, 26). TnT2 is further digested into a smaller subfragment, TnT2beta , which lacks the COOH-terminal 17 residues (26, 27). It has been shown that the regulatory function retained in TnT2 (or TnT2alpha ) is lost in TnT2beta such that the troponin complex cannot inhibit the contractile response in the absence of Ca2+ (18, 19), indicating that the COOH-terminal 17 residues of rabbit fast-twitch skeletal TnT play an important role in allowing the troponin complex to inhibit muscle contraction in the absence of Ca2+. It should be noted that these COOH-terminal 17 amino acid residues of rabbit fast-twitch skeletal TnT are just homologous to the COOH-terminal 14 amino acid residues of human cardiac TnT, which are lost in the HCM-causing truncated mutants, TnTDelta 14 and TnTDelta 28(+7). Thus the splice donor site mutation in cardiac TnT associated with HCM may impair the inhibitory action of the troponin complex, leading to an increase in the Ca2+ sensitivity. The present study also showed that both the truncated mutant TnTs in HCM conferred a lower cooperativity than wild-type TnT, providing the first evidence that this COOH-terminal region of cardiac TnT is involved in the cooperative Ca2+ regulation of contraction.

Recently, missense mutation Asp175Asn in the alpha -TM gene has been shown to increase the Ca2+ sensitivity without changing the cooperativity and maximum force-generating capability in skeletal muscle fibers of HCM patients (4), suggesting that the increased Ca2+ sensitivity may be a common functional effect of the HCM-causing mutations in the two distinct thin-filament regulatory proteins, cardiac TnT and alpha -TM. Further studies on the function of other mutations in the TnT gene would clarify this possibility.


    ACKNOWLEDGEMENTS

This study was supported in part by grants-in-aid (07458222, 0868089, 08770071, 109670771 and 10557073) from the Ministry of Education, Science, Sports and Culture of Japan; the Ministry of Health and Welfare of Japan; and the Uehara Memorial Foundation.


    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: S. Morimoto, Dept. of Clinical Pharmacology, Faculty of Medicine, Kyushu Univ., 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan (E-mail: morimoto{at}clipharm.med.kyushu-u.ac.jp).

Received 21 December 1998; accepted in final form 29 April 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Anan, R., G. Greve, L. Thierfelder, H. Watkins, W. J. McKenna, S. Solomon, C. Vecchio, H. Shono, S. Nakao, and H. Tanaka. Prognostic implications of novel beta cardiac myosin heavy chain gene mutations that cause familial hypertrophic cardiomyopathy. J. Clin. Invest. 93: 280-285, 1994[Medline].

2.   Anderson, P. A., N. N. Malouf, A. E. Oakeley, E. D. Pagani, and P. D. Allen. Troponin T isoform expression in humans. A comparison among normal and failing adult heart, fetal heart, and adult and fetal skeletal muscle. Circ. Res. 69: 1226-1233, 1991[Abstract].

3.   Bonne, G., L. Carrier, J. Bercovici, C. Cruaud, P. Richard, B. Hainque, M. Gautel, S. Labeit, M. James, and J. Beckmann. Cardiac myosin binding protein-C gene splice acceptor site mutation is associated with familial hypertrophic cardiomyopathy. Nat. Genet. 11: 438-440, 1995[Medline].

4.   Bottinelli, R., D. A. Coviello, C. S. Redwood, M. A. Pellegrino, B. J. Maron, P. Spirito, H. Watkins, and C. Reggiani. A mutant tropomyosin that causes hypertrophic cardiomyopathy is expressed in vivo and associated with an increased calcium sensitivity. Circ. Res. 82: 106-115, 1998[Abstract/Free Full Text].

5.   Cuda, G., L. Fananapazir, W. S. Zhu, J. R. Sellers, and N. D. Epstein. Skeletal muscle expression and abnormal function of beta -myosin in hypertrophic cardiomyopathy. J. Clin. Invest. 91: 2861-2865, 1993[Medline].

6.   Epstein, N. D., G. M. Cohn, F. Cyran, and L. Fananapazir. Differences in clinical expression of hypertrophic cardiomyopathy associated with two distinct mutations in the beta-myosin heavy chain gene. A 908Leuright-arrowVal mutation and a 403Argright-arrowGln mutation. Circulation 86: 345-352, 1992[Abstract].

7.   Fujita, H., S. Sugiura, S. Momomura, M. Omata, H. Sugi, and K. Sutoh. Characterization of mutant myosins of Dictyostelium discoideum equivalent to human familial hypertrophic cardiomyopathy mutants. Molecular force level of mutant myosins may have a prognostic implication. J. Clin. Invest. 99: 1010-1015, 1997[Abstract/Free Full Text].

8.   Geisterfer-Lowrance, A. A., S. Kass, G. Tanigawa, H. P. Vosberg, W. McKenna, C. E. Seidman, and J. G. Seidman. A molecular basis for familial hypertrophic cardiomyopathy: a beta  cardiac myosin heavy chain gene missense mutation. Cell 62: 999-1006, 1990[Medline].

9.   Hatakenaka, M., and I. Ohtsuki. Replacement of three troponin components with cardiac troponin components within single glycerinated skeletal muscle fibers. Biochem. Biophys. Res. Commun. 181: 1022-1027, 1991[Medline].

10.   Hatakenaka, M., and I. Ohtsuki. Effect of removal and reconstitution of troponins C and I on the Ca2+-activated tension development of single glycerinated rabbit skeletal muscle fibers. Eur. J. Biochem. 205: 985-993, 1992[Abstract].

11.   Kimura, A., H. Harada, J. E. Park, H. Nishi, M. Satoh, M. Takahashi, S. Hiroi, T. Sasaoka, N. Ohbuchi, T. Nakamura, T. Koyanagi, T. H. Hwang, J. A. Choo, K. S. Chung, A. Hasegawa, R. Nagai, O. Okazaki, H. Nakamura, M. Matsuzaki, T. Sakamoto, H. Toshima, Y. Koga, T. Imaizumi, and T. Sasazuki. Mutations in the cardiac troponin I gene associated with hypertrophic cardiomyopathy. Nat. Genet. 16: 379-382, 1997[Medline].

12.   Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685, 1970[Medline].

13.   Lankford, E. B., N. D. Epstein, L. Fananapazir, and H. L. Sweeney. Abnormal contractile properties of muscle fibers expressing beta -myosin heavy chain gene mutations in patients with hypertrophic cardiomyopathy. J. Clin. Invest. 95: 1409-1414, 1995[Medline].

14.   Morimoto, S., and I. Ohtsuki. Ca2+- and Sr2+-sensitivity of the ATPase activity of rabbit skeletal myofibrils: effect of the complete substitution of troponin C with cardiac troponin C, calmodulin, and parvalbumins. J. Biochem. (Tokyo) 101: 291-301, 1987[Abstract].

15.   Morimoto, S., F. Yanaga, R. Minakami, and I. Ohtsuki. Ca2+-sensitizing effects of the mutations at Ile-79 and Arg-92 of troponin T in hypertrophic cardiomyopathy. Am. J. Physiol. 275 (Cell Physiol. 44): C200-C207, 1998[Abstract/Free Full Text].

16.   Nakamura, S., K. Yamamoto, K. Hashimoto, and I. Ohtsuki. Effect of chymotryptic troponin T subfragments on the Ca2+ sensitivity of superprecipitation. J. Biochem. (Tokyo) 89: 1639-1641, 1981[Medline].

17.   Ohtsuki, I., K. Maruyama, and S. Ebashi. Regulatory and cytoskeletal proteins of vertebrate skeletal muscle. Adv. Protein Chem. 38: 1-67, 1986[Medline].

18.   Ohtsuki, I., K. Yamamoto, and K. Hashimoto. Effect of two C-terminal side chymotryptic troponin T subfragments on the Ca2+ sensitivity of superprecipitation and ATPase activities of actomyosin. J. Biochem. (Tokyo) 90: 259-261, 1981[Abstract].

19.   Onoyama, Y., and I. Ohtsuki. Effect of chymotryptic troponin T subfragments on the calcium ion-sensitivity of ATPase and superprecipitation of actomyosin. J. Biochem. (Tokyo) 100: 517-519, 1986[Abstract].

20.   Pearlstone, J. R., and L. B. Smillie. Binding of troponin-T fragments to several types of tropomyosin. Sensitivity to Ca2+ in the presence of troponin-C. J. Biol. Chem. 257: 10587-10592, 1982[Abstract].

21.   Perry, S. V. Troponin T: genetics, properties and function. J. Muscle Res. Cell Motil. 19: 575-602, 1998[Medline].

22.   Poetter, K., H. Jiang, S. Hassanzadeh, S. R. Master, A. Chang, M. C. Dalakas, I. Rayment, J. R. Sellers, L. Fananapazir, and N. D. Epstein. Mutations in either the essential or regulatory light chains of myosin are associated with a rare myopathy in human heart and skeletal muscle. Nat. Genet. 13: 63-69, 1996[Medline].

23.   Rüegg, J. C. Calcium in Muscle Activation. Berlin: Springer-Verlag, 1986.

24.   Solomon, S. D., S. Wolff, H. Watkins, P. M. Ridker, P. Come, W. J. McKenna, C. E. Seidman, and R. T. Lee. Left ventricular hypertrophy and morphology in familial hypertrophic cardiomyopathy associated with mutations of the beta-myosin heavy chain gene. J. Am. Coll. Cardiol. 22: 498-505, 1993[Medline].

25.   Sweeney, H. L., A. J. Straceski, L. A. Leinwand, B. A. Tikunov, and L. Faust. Heterologous expression of a cardiomyopathic myosin that is defective in its actin interaction. J. Biol. Chem. 269: 1603-1605, 1994[Abstract/Free Full Text].

26.   Tanokura, M., Y. Tawada, and I. Ohtsuki. Chymotryptic subfragments of troponin T from rabbit skeletal muscle. I. Determination of the primary structure. J. Biochem. (Tokyo) 91: 1257-1265, 1982[Abstract].

27.   Tanokura, M., Y. Tawada, A. Ono, and I. Ohtsuki. Chymotryptic subfragments of troponin T from rabbit skeletal muscle. Interaction with tropomyosin, troponin I and troponin C. J. Biochem. (Tokyo) 93: 331-337, 1983[Abstract].

28.   Tardiff, J. C., S. M. Factor, B. D. Tompkins, T. E. Hewett, B. M. Palmer, R. L. Moore, S. Schwartz, J. Robbins, and L. A. Leinwand. A truncated cardiac troponin T molecule in transgenic mice suggests multiple cellular mechanisms for familial hypertrophic cardiomyopathy. J. Clin. Invest. 101: 2800-2811, 1998[Abstract/Free Full Text].

29.   Thierfelder, L., H. Watkins, C. MacRae, R. Lamas, W. McKenna, H. P. Vosberg, J. G. Seidman, and C. E. Seidman. alpha -Tropomyosin and cardiac troponin T mutations cause familial hypertrophic cardiomyopathy: a disease of the sarcomere. Cell 77: 701-712, 1994[Medline].

30.   Tsukui, R., and S. Ebashi. Cardiac troponin. J. Biochem. (Tokyo) 73: 1119-1121, 1973[Medline].

31.   Van Eldik, L. J., and S. R. Wolchok. Conditions for reproducible detection of calmodulin and S100 beta in immunoblots. Biochem. Biophys. Res. Commun. 124: 752-759, 1984[Medline].

32.   Watkins, H., D. Conner, L. Thierfelder, J. A. Jarcho, C. MacRae, W. J. McKenna, B. J. Maron, J. G. Seidman, and C. E. Seidman. Mutations in the cardiac myosin binding protein-C gene on chromosome 11 cause familial hypertrophic cardiomyopathy. Nat. Genet. 11: 434-437, 1995[Medline].

33.   Watkins, H., W. J. McKenna, L. Thierfelder, H. J. Suk, R. Anan, A. O'Donoghue, P. Spirito, A. Matsumori, C. S. Moravec, and J. G. Seidman. Mutations in the genes for cardiac troponin T and alpha -tropomyosin in hypertrophic cardiomyopathy. N. Engl. J. Med. 332: 1058-1064, 1995[Abstract/Free Full Text].

34.   Watkins, H., A. Rosenzweig, D. S. Hwang, T. Levi, W. McKenna, C. E. Seidman, and J. G. Seidman. Characteristics and prognostic implications of myosin missense mutations in familial hypertrophic cardiomyopathy. N. Engl. J. Med. 326: 1108-1114, 1992[Abstract].

35.   Watkins, H., C. E. Seidman, J. G. Seidman, H. S. Feng, and H. L. Sweeney. Expression and functional assessment of a truncated cardiac troponin T that causes hypertrophic cardiomyopathy. Evidence for a dominant negative action. J. Clin. Invest. 98: 2456-2461, 1997[Abstract/Free Full Text].


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