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

Sachio Morimoto1, Fumi Yanaga1, Reiko Minakami2, and Iwao Ohtsuki1

1 Department of Clinical Pharmacology, Faculty of Medicine, and 2 School of Health Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812, Japan

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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.

hypertrophy; missense mutation; skinned fibers; calcium sensitivity; heart diseases; ischemia

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

HYPERTROPHIC CARDIOMYOPATHY (HCM) is an autosomal dominant cardiac disease associated with a high incidence of sudden death. Recently it has been shown that HCM is caused by mutations in genes for various cardiac myofibrillar proteins, including beta -myosin heavy chain (beta -MHC) (8), troponin T (TnT) (30), troponin I (TnI) (12), alpha -tropomyosin (30), myosin-binding protein C (2, 29), and myosin light chains (24).

Many studies have been made on the functional changes in MHC caused by the mutations. Mutations in MHC have been shown to decrease the velocity of actin translocation or force output by myosin molecules in vitro (4, 7, 27) and to decrease actin-activated ATPase activity (27) and power output in slow-twitch skeletal muscles expressing the beta -MHC isoform (14) in patients, suggesting that the mutations in beta -MHC impair the contractility of cardiac muscle. Because the impairment of contractility leads to a depression of cardiac performance, the heart is thought to compensate for this by reflexly undergoing hypertrophy.

Several studies have also been made on the functional aspects of mutant cardiac TnT expressed in HCM, and there has been some controversy as to the mechanisms by which TnT mutations cause HCM. A cultured quail skeletal myotube expressing a truncated mutant of human cardiac TnT has been shown to lose its Ca2+-activated maximum force-generating capability to a great extent (31), and Arg92Gln human cardiac TnT has also been reported to impair the contractility of transfected adult feline cardiac myocytes (16), suggesting that these TnT mutations cause compensatory hypertrophy by impairing the contractility of cardiac muscle in the same manner as beta -MHC mutations. In contrast, it has been reported that the Ile91Asn rat embryonic cardiac TnT mutation, which is analogous to the Ile79Asn human cardiac TnT mutation in HCM, does not impair the contractility but increases the velocity of actin translocation in an in vitro motility assay (15), suggesting that the Ile79Asn mutation confers hypercontractility on cardiac muscle.

To explore the definite mechanism(s) underlying the pathogenesis of HCM, we attempted to examine the effects of Ile79Asn and Arg92Gln mutations in human cardiac TnT on the physiological contractile function of cardiac muscle by using the method for exchanging TnT molecules directly into skinned muscle fibers (9, 10). The results indicated that both mutations did not impair the contractility but increased the Ca2+ sensitivity of contraction, suggesting that an increased Ca2+ sensitivity of cardiac muscle contraction is responsible for the pathogenesis of HCM associated with these TnT mutations. Part of this work has been reported previously in abstract form (21, 32).

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cloning and 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 pET3-d, and mutagenesis was carried out by PCR according to the method described by Horton (11); oligonucleotides employed for mutagenesis were 5'-CCT CCC AAG AAC CCC GAT GG-3' for the Ile79Asn mutation and 5'-GAC ATC CAC CAG AAG CGC ATG-3' for the Arg92Gln mutation, and the resultant mutations in the TnT cDNA were confirmed by DNA sequencing.

Expression and purification of recombinant TnTs. Wild-type and mutant TnT cDNAs were constructed in pET3-d and expressed in Escherichia coli BL21(DE3). Cells were grown in LB broth containing 50 µg/ml ampicillin at 37°C. Expression of TnT was induced by adding 100 µg/ml isopropyl beta -D-thiogalactopyranoside at an optical density at 600 nm of 0.6-0.8 and incubating for 14 h. The cells harvested by centrifugation were resuspended in a solution containing 50 mM Tris · HCl (pH 8.0), 1 mM EDTA, 2 mg/ml deoxycholate, and 0.5 mg/ml lysozyme and were incubated for 1 h at 4°C. After repeated sonication on ice, the cell lysate was made to 10 U/ml in DNase I and to 1 mM in MgCl2 and was kept for 10 min at 37°C. The lysate was then made to 5 mM in trans-1,2-cyclohexanediamine-N,N,N',N'-tetraacetic acid (CDTA) and was centrifuged at 15,000 rpm for 25 min at 4°C. Finally, the supernatant was adjusted to 10 mM Tris · HCl (pH 8.0), 6 M urea, 5 mM CDTA, and 15 mM 2-mercaptoethanol and was applied to a RESOURCE Q (6 ml) column (Pharmacia Biotech). TnT was separated with a linear gradient of 0-0.5 M NaCl using a fast-performance liquid chromatography (FPLC) system (Pharmacia Biotech).

Preparation of rabbit cardiac troponin and its components. Troponin and its components were prepared from the left ventricular myocardium of young male albino rabbits (~3 mo old) according to the method of Tsukui and Ebashi (28), using FPLC ion-exchange columns, RESOURCE Q and S (6 ml) and Mono Q and S HR 5/5 (Pharmacia Biotech).

Preparation of skinned trabeculae. Skinned trabeculae were prepared according to a modification of the procedure for disrupting the sarcolemma and sarcoplasmic reticulum of the psoas muscle described by Diffee et al. (5). Small bundles (0.5-1 mm wide and 5-7 mm long) of the left ventricular trabeculae of young male albino rabbits (~3 mo old) were tied to glass capillary tubes and skinned with relaxing solution containing 0.5% (wt/vol) Brij 58 for 30 min at 25°C. The skinned trabeculae were stored at -20°C in relaxing solution containing 50% (vol/vol) glycerol and were used within 3 wk.

Tension measurements. A small fiber (~120 µm in diameter) dissected from a stock skinned trabecula was mounted in a thermostatically controlled chamber with a capacity of 0.2 ml; both ends were attached to stainless steel wire hooks as described by Brandt et al. (3) and further fixed with a fast-setting glue. Fiber length between hooks was ~1 mm. Resting sarcomere length was set to 2.4 µm using laser diffraction. The tension was measured with a strain gauge (UL-2GR; Minebea). The relaxing solution consisted of (in mM) 50 MOPS/KOH (pH 7.0), 100 KCl, 6 MgCl2, 5 Na2ATP, 4 EGTA, 0.5 dithiothreitol (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 (20), to the relaxing solution. All tension measurements were performed at 25°C.

TnT exchange in skinned fibers. TnT exchange was performed by the method of Hatakenaka and Ohtsuki (9, 10), with a slight modification. Fibers mounted to mechanical apparatus were bathed at 25°C, with continuous stirring, in a solution containing (in mM) 50 MES/KOH (pH 6.0), 250 KCl, 4 EDTA, and 0.5 DTT, as well as 0.5 mg/ml purified TnT, for 60 min. The fibers were then reconstituted with purified rabbit cardiac TnI and troponin C (TnC) at 25°C for 40 min in the relaxing solutions containing 0.5 mg/ml of each protein.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Human cardiac TnT cDNA cloned in this study had a nucleotide sequence identical to that reported by Mesnard et al. (17), which lacks an acidic domain of ~10 amino acids that has been identified in the NH2-terminal region of fetal isoforms in various animals. Figure 1 shows SDS-PAGE of the expressed and purified wild-type and mutant human cardiac TnTs. The recombinant human cardiac TnTs showed an electrophoretic mobility similar to that of the major isoform of rabbit adult cardiac TnT. This is consistent with the report showing that the adult major isoforms of human and rabbit cardiac TnTs have almost the same electrophoretic mobility on a similar gel (1).


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Fig. 1.   SDS-PAGE of recombinant wild-type and mutant human cardiac troponin T (TnT). Lane A, tissue-derived rabbit cardiac TnT. Lane B, skinned muscle preparation from adult rabbit hearts. Lane C, wild-type human cardiac TnT. Lane D, Ile79Asn mutant human cardiac TnT. Lane E, Arg92Gln mutant human cardiac TnT. Electrophoresis was performed according to modification by Anderson et al. (1) of the method of Laemmli (13) with 8% separating gel. The gel was stained with Coomassie brilliant blue R-250. MHC, myosin heavy chain; TM, tropomyosin.

It has previously been demonstrated that endogenous TnT · I · C complexes in skinned fibers can be replaced with exogenous TnT when added in an excess amount under acidic and high-ionic-strength conditions (9, 10). With the use of this technique, an attempt was made to exchange the purified recombinant human cardiac TnTs into the skinned rabbit adult cardiac muscle fibers, to investigate their physiological functions. After the skinned fibers were bathed in a solution containing an excess amount (0.5 mg/ml) of human cardiac TnT, a large Ca2+-insensitive tension was developed in the relaxing solution, indicating that substantial amounts of the inhibitory component of the troponin complex, TnI, were lost as a consequence of the replacement of endogenous troponin complexes with the added TnT (Fig. 2A). Two-dimensional isoelectric/SDS-PAGE analyses revealed that ~65% of endogenous rabbit cardiac TnT was displaced by the added human cardiac TnT under these conditions (Fig. 3). One-dimensional SDS-PAGE analysis of the skinned fibers was not able to directly demonstrate the TnT exchange and its extent because of exactly the same electrophoretic mobility of native rabbit and recombinant human TnTs but still allowed us to estimate the extent of TnT exchange from the decrease in the amount of TnI caused by the displacement of endogenous TnT · I · C complexes with recombinant TnT. Thus the estimated amount of TnT exchanged into the fibers was ~65% for all the recombinant TnTs (Fig. 4), in excellent agreement with the extent of exchange that was directly quantified by the two-dimensional gel electrophoresis scans. The levels of Ca2+-insensitive tension caused by removal of TnI after TnT treatment were also 60-65% of total active tension (Fig. 2B), and no statistically significant differences were found between the fibers treated with wild-type and mutant TnTs, providing further evidence that the extents of recombinant TnT exchanged into the skinned fibers were ~65% and were not different between the fiber groups.


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Fig. 2.   Changes in contractile response to Ca2+ of skinned rabbit cardiac muscle fibers after treatment (tr) with recombinant human cardiac TnTs and after subsequent reconstitution (rec) with rabbit cardiac TnI and TnC. A: representative tension recording showing experimental protocol. B: summary of data obtained in 5 different fibers from 4 rabbit hearts. Data are expressed as means ± SE. -Ca, pCa > 9; +Ca, pCa = 5.5; ++Ca, pCa = 4.5. * P < 0.05, ** P < 0.01 vs. wild type (Tukey's multiple-comparison test). NS, not significant.


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Fig. 3.   Two-dimensional isoelectric/SDS-PAGE of fibers before (control) and after treatment with recombinant human cardiac wild-type TnT. Electrophoresis was carried out according to the method of O'Farrell (22) using 6.25% Pharmalyte (pH 4-6.5) on the first dimension as described previously (19), and the gel was stained with a silver staining kit (Pharmacia Biotech). RTnT, rabbit native cardiac TnT; HTnT, human cardiac (wild-type) TnT; Ac, actin; LC1, myosin light chain 1; LC2, myosin light chain 2. Positions of RTnT and HTnT were confirmed by electrophoresis of purified proteins at different ratios as shown (gel was stained with Coomassie brilliant blue R-250). Although TnT/LC1 ratios, estimated by an optical densitometric scan of the gel using Phoretix gel analysis software (Phoretix International), were 0.389 and 0.364 before and after TnT treatment, respectively, the HTnT/(RTnT + HTnT) ratio after TnT treatment was 0.636, indicating that extent of exchange of HTnT into fiber is ~65%. Densitometry tracings for the TnT region are shown on an expanded scale at right. IEF, isoelectric focusing.


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Fig. 4.   SDS-PAGE of skinned rabbit cardiac muscle fibers after treatment with recombinant human cardiac TnTs and after subsequent reconstitution with rabbit cardiac TnI and TnC. Lane A, an untreated control fiber. Lane B, a fiber treated with wild-type human cardiac TnT. Lane C, a fiber treated with Ile79Asn mutant human cardiac TnT. Lane D, a fiber treated with Arg92Gln mutant human cardiac TnT. Lane E, purified rabbit cardiac troponin complex. Lanes F-H, fibers reconstituted with rabbit cardiac TnI and TnC after treatment with human wild-type, Ile79Asn, and Arg92Gln TnT, respectively. Individual skinned fibers were mounted to the mechanical apparatus, and TnT treatment (B-E) or TnT treatment followed by reconstitution with TnI and TnC (F-G) were carried out under same conditions as in tension measurements done in Figs. 2, 5, and 6. Electrophoresis was performed according to method of Laemmli (13) with 12% separating gel. The gel was stained with a silver staining kit (Pharmacia Biotech). Note that amounts of proteins loaded on gel are varied between lanes depending on size of each skinned fiber. TnI/TM ratios estimated by gel scans were 0.472, 0.161, 0.160, 0.169, 0.442, 0.448, and 0.464 in lanes A, B, C, D, F, G, and H, respectively, indicating that extent of TnI extraction and thus extent of TnT exchange is ~65%, and extent of subsequent TnI reconstitution is nearly complete in all cases. TnC is not visible in the fibers because it comigrates with myosin light chain 2 (LC2).

Ca2+-insensitive tension was gradually decreased by bathing the fibers in relaxing solution containing rabbit cardiac TnI and was almost completely inhibited after 40 min of incubation. Subsequent reconstitution with rabbit cardiac TnC restored a Ca2+ regulation of contraction with the level of maximum tension that was an average of 60% of the preextraction value (Fig. 2B and Table 1). SDS-PAGE analysis of the fibers reconstituted with TnI and TnC showed that the reincorporation of TnI into the fibers was nearly stoichiometric (Fig. 4).

                              
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Table 1.   Ca2+ sensitivities, cooperativities, pH sensitivities, and maximum tension in skinned rabbit cardiac muscle fibers after exchange of rabbit native cardiac TnT and recombinant human cardiac TnTs

The tension-pCa relationships of human wild-type TnT-exchanged fibers were then determined at pH 7.0 and compared with those determined in the same preparation before TnT exchange (Fig. 5). The exchange of human wild-type TnT into the rabbit skinned cardiac muscle fibers caused a large decrease in maximal tension by ~40% (Fig. 5A, Table 1) and also caused a slight but significant rightward shift (Delta pCa50 = 0.09, P < 0.05; paired t-test; where pCa50 is pCa required for half-maximum tension development) in the tension-pCa relationship (Fig. 5B), raising the possibility that human cardiac TnT might confer the lower maximal tension and Ca2+ sensitivity when incorporated into the cardiac muscle of a different species, rabbit. However, control experiments performed under the same conditions showed that the exchange of rabbit native cardiac TnT into skinned fibers had exactly the same effects on tension generation (Fig. 5, Table 1), resulting in ~40% reduction of maximum tension with a small but significant rightward shift (Delta pCa50 = 0.09, P < 0.05; paired t-test) in the tension-pCa relationship. These results indicate that the TnT exchange procedure itself has a large nonspecific deteriorative effect on the maximal force-generating capability and a slight nonspecific Ca2+-desensitizing effect on the regulatory system that determines Ca2+ sensitivity and that recombinant human cardiac wild-type TnT is just equivalent to rabbit native cardiac TnT. Treatment with TnT(-) solution had much larger deteriorative effects on maximal tension compared with TnT(+) solution and also caused a considerable increase in resting tension (data not shown), suggesting that the solution has deleterious effects on the fibers and causes more serious damage when endogenous troponin is not replaced by TnT.


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Fig. 5.   Tension-pCa relationships in the same fiber determined at pH 7.0 before (open circle , triangle ) and after (bullet , black-triangle) exchange of recombinant human cardiac wild-type TnT (triangle , black-triangle) or rabbit native cardiac TnT (open circle , bullet ). Data were normalized to maximal tension before TnT exchange (A) and to each maximal tension before and after TnT exchange (B) and are expressed as means ± SE for 5 determinations (5 fibers from 4 different hearts).

Figure 6 compares the tension-pCa relationships of recombinant human cardiac mutant TnT-exchanged fibers with those of recombinant human wild-type TnT-exchanged fibers at pH 7.0, 6.5, and 6.2. Although no differences were observed in the maximal level and cooperativity (nH) of Ca2+-activated tension development between fiber groups (Table 1), tension-pCa relationships in Ile79Asn and Arg92Gln mutant TnT-exchanged fibers were both found to be shifted leftward compared with those in wild-type TnT-exchanged fibers at all pH values (Fig. 6B). Although no significant difference in the free Ca2+ concentrations required for half-maximum tension development (pCa50) was detected between the two mutants, there were statistically significant differences in pCa50 between mutants and wild type (Table 1). Before TnT exchange, there were no significant differences in pCa50 between these fiber groups (Fig. 6A). These results indicate that both mutations have Ca2+-sensitizing effects on cardiac muscle contraction in this pH range.


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Fig. 6.   Tension-pCa relationships in skinned rabbit cardiac muscle fibers determined at pH 7.0, 6.5, and 6.2 after exchange of recombinant wild-type (open circle ), Ile79Asn (bullet ), or Arg92Gln (black-triangle) human cardiac TnT. Note that tension-pCa relationships of individual fiber were first determined at pH 7.0 before TnT exchange (A) and then were sequentially determined at pH 7.0, 6.5, 6.2, and 7.0 after TnT exchange (B). Responses at pH 7.0 after TnT exchange were averaged to compensate for any incidental rundown of the fiber, and tension developed by individual fiber was normalized to maximum tension at pH 7.0. Data are expressed as means ± SE for 5 determinations (5 fibers from 4 different hearts). MOPS was used as a pH buffer at pH 7.0 and 6.5, whereas MES was used at pH 6.2.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

HCM is a genetically heterogeneous disease caused by mutations in various myofibrillar proteins, including MHC and myosin light chains, tropomyosin, myosin-binding protein C, TnI, and TnT. The myosin molecule plays a central role in developing force to contract cardiac myocytes, and this is accomplished by using energy liberated by hydrolysis of ATP on the myosin head that is activated by cyclic interactions of the myosin head with actin molecules. All mutations reported in MHC are localized to the head and the head-rod junction region, so that the mutations can be expected to affect the force-generating interaction between myosin and actin (25). In fact, functional studies using mutant myosin molecules in vitro (4, 7, 27) or using slow-twitch skeletal muscle fibers expressing beta -MHC in patients (14) have demonstrated that the mutations in MHC in general impair the interaction between myosin and actin.

TnT combines with TnI and TnC to form a regulatory complex, troponin, which is localized in the thin filaments. TnI inhibits the force-generating interaction between myosin and actin, whereas TnC removes the inhibitory action of TnI and activates muscle contraction in response to the transient increase in intracellular Ca2+ (23). On the other hand, TnT has no specific regulatory function such as inhibition or activation of contractile interaction between myosin and actin, making it difficult to estimate the functional consequence of TnT mutations in HCM compared with beta -MHC mutations. However, it is known that cardiac hypertrophy produced by TnT mutations is moderate or subtle compared with that produced by beta -MHC mutations, in contrast to the fact that TnT mutations are associated with a higher incidence of death before 30 yr of age and a higher proportion of sudden death compared with beta -MHC mutations (30), suggesting that the mutations in TnT and beta -MHC cause HCM by somewhat different mechanisms.

A study on the Ile79Asn mutation in a fully reconstituted system using an analogous Ile91Asn mutant rat embryonic cardiac TnT has shown that the mutation has no effect on biochemical properties, including affinity of tropomyosin for troponin, the effect of troponin on affinity of tropomyosin for actin, binding of subfragment 1 to the actin-tropomyosin-troponin complex, and Ca2+ regulation or Ca2+ sensitivity of ATPase activity of subfragment 1 regulated by the actin-tropomyosin-troponin complex (15). Only one functional change found in this study was 50% faster than the velocity of actin translocation in an in vitro motility assay, which is similar to the effect observed in the mutation Met149Val in essential myosin light chain (24). On the other hand, a physiological study using a cultured quail skeletal myotube expressing a truncated mutant human cardiac TnT derived from the splice donor mutation in intron 15 has shown that the mutation greatly diminished the Ca2+-activated maximum force of contraction in the myotube (28), which is similar to the effect observed in patients' slow-twitch skeletal muscle fibers expressing beta -MHC mutants Arg403Gln and Gly741Arg (14). Expression of the Arg92Gln mutant human cardiac TnT in a cultured feline cardiac myocyte has also been reported to impair an apparent contractility that is indexed by fractional shortening or peak velocity of shortening of cells that are attached to laminin-coated glass coverslips and stimulated electrically (16). The present study, however, strongly indicates that, compared with wild-type human cardiac TnT, the Ile79Asn and Arg92Gln mutant TnTs, when incorporated into skinned rabbit cardiac muscle fibers, both confer higher Ca2+ sensitivity without impairing maximum force-generating capability. These findings not only provide the first evidence that a heightened Ca2+ sensitivity of cardiac muscle contraction may be involved in the pathogenesis of HCM associated with these TnT mutations but also support the idea that the mutations in TnT and beta -MHC cause HCM by different intracellular mechanisms. Most of the mutations in beta -MHC were shown to impair the contractility of myofilament (4, 7, 14, 27), suggesting that these mutations cause a compensatory cardiac hypertrophy. On the other hand, the TnT mutations examined in this study are expected to enhance the contractility of myofilament by increasing its response to Ca2+, so that it is not easy to anticipate that these TnT mutations cause a compensatory hypertrophic response. In fact, it is known that cardiac hypertrophy produced by TnT mutations is moderate and in some cases appears clinically unimportant, despite the fact that these mutations are associated with a poor prognosis and a high incidence of sudden death (30). Thus the increased Ca2+ sensitivity may be responsible for this distinguishing feature of HCM caused by TnT mutations. The Ile79Asn mutation in TnT has previously been reported to have no effect on Ca2+ sensitivity of actin-activated ATPase activity measured at pH 7.5 (15). On the other hand, a significant Ca2+-sensitizing effect of the Ile79Asn mutation was detected in the pH range of 7.0-6.2, but the effect was relatively small and appeared to be even smaller at higher pH (Fig. 6, Table 1), suggesting that it may be more difficult to detect the effect at alkaline pH.

Figure 2B shows that the Ca2+-insensitive tensions developed by the fibers treated with wild-type and mutant TnTs are completely inhibited after TnI reconstitution and also after subsequent TnC reconstitution and that levels of Ca2+-sensitive maximum tensions developed by the fibers after TnC reconstitution are almost the same. These data indicate that the mutations in TnT do not affect the relaxed and maximally activated tension and also suggest that the extents of TnI and TnC reconstitution are complete and not different between fiber groups. Because the amounts of TnT exchanged into the fibers were also considered not to be varied between fiber groups (Figs. 2B, 3, and 4), the increased Ca2+ sensitivity could not be explained by incomplete reconstitution of troponin components into the thin filaments. However, we cannot completely exclude the possibility that the mutations specifically induce subtle changes in the reconstitution level of thin filaments that affect Ca2+ sensitivity. Although the precise molecular mechanism(s) whereby Ile79Asn and Arg92Gln mutations in TnT increase Ca2+ sensitivity remains to be clarified, one possible mechanism is that these TnT mutations reduce the inhibitory action of TnI by altering the interaction between TnI and actin directly or indirectly to make the contractile response more sensitive to Ca2+. Of interest is that the fibers that are treated with Arg92Gln TnT and reconstituted with TnI develop a significantly larger Ca2+-sensitive tension before TnC reconstitution than the fibers treated with wild-type and Ile79Asn TnT (Fig. 2B). Ca2+-sensitive tension in this state is considered to be developed by Ca2+ binding to the residual rabbit native troponin complexes in skinned fibers. Thus these data suggest that at least Arg92Gln TnT directly weakens the inhibitory action of reconstituted TnI, but the effect is so small as to manifest itself only when the thin filaments are partially activated by residual native troponin complexes in the presence of Ca2+. This is probably consistent with the fact that, after TnC reconstitution, Arg92Gln TnT increases partially activated, submaximal tension at a given Ca2+ concentration, i.e., Ca2+ sensitivity, without affecting maximal or minimal tension.

It is known that acidic pH decreases Ca2+ sensitivity of contraction in cardiac muscle to a greater extent compared with that in fast- and slow-twitch skeletal muscle (6). The greater Ca2+-desensitizing effect of acidic pH on cardiac muscle contraction is considered to play an important cardioprotective role under ischemic conditions in preventing a rapid decay of the ATP level by inhibiting the utilization of ATP by actomyosin ATPase (26). The two mutations in human cardiac TnT examined in this study were both found to increase Ca2+ sensitivity of contraction in cardiac muscle not only at physiological pH 7.0 but also at acidic pH 6.5 and 6.2, mimicking the intracellular pH induced by ischemia (Fig. 6) and suggesting that both mutations offset the cardioprotective effect of acidic pH-induced Ca2+ desensitization. Thus, in patients with these TnT mutations, even a light ischemia may lead to a rapid decrease in intracellular ATP, facilitating the upregulation of expression of immediate early genes (e.g., c-fos, c-jun, Egr-1) during recovery from ischemia (18), products that are known to be involved in determination of cell fate, such as apoptosis, hypertrophy, proliferation, or differentiation. Increased Ca2+ sensitivity of contraction also increases utilization of ATP even under nonischemic conditions and, therefore, increases the possibility of causing an imbalance in energy supply and demand, which might have a fatal effect on the cardiac system in extreme cases. Further studies to clarify these possibilities may shed light on the mechanism for the pathogenesis of HCM.

    ACKNOWLEDGEMENTS

This work was supported by grants-in-aid for Science Research (to S. Morimoto, F. Yanaga, and I. Ohtsuki) from the Ministry of Education, Science, Sports and Culture of Japan and the Uehara Memorial Foundation (to S. Morimoto).

    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: S. Morimoto, Dept. of Clinical Pharmacology, Faculty of Medicine, Kyushu Univ., 3-1-1 Maidashi, Higashi-ku, Fukuoka 812, Japan.

Received 12 January 1998; accepted in final form 2 April 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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