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
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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.
calcium sensitivity; cooperativity; truncated mutant; skinned fiber
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INTRODUCTION |
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
-myosin heavy chain (
-MHC) (8), troponin T (TnT) (33), troponin I (TnI) (11),
-tropomyosin (
-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
-MHC
mutations with a comparably poor prognosis (1, 6, 24, 34). Mutations in
the
-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
(TnT
14), and the other mutant
lacks 21 amino acids because of the replacement of the normal 28 terminal amino acids with 7 novel residues
(TnT
28(+7)). It has been
shown that TnT
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
-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 TnT
28(+7) slightly
decreased the maximum
Ca2+-activated force,
TnT
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.
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MATERIALS AND METHODS |
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 TnT
28(+7)
cDNA, PCR was first carried out to create
Spl I and
EcoR I sites by using oligonucleotides 5'-GGT GGT GGA AG
A AGA GG-3'
(18F; Spl I site
underlined) and 5'-CGG
AGA ACA TT
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
TnT
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
A AGA GG-3' (18F;
Spl I site underlined) and
5'-CCC GCG G
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 2.
Effect of TnI phosphorylation on Ca2+ sensitivity,
cooperativity, and maximum force in recombinant human cardiac
TnT-exchanged fibers
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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.
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RESULTS |
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
TnT 28(+7); lane 4, human cardiac truncated
mutant TnT 14; lane 5, human cardiac wild-type
TnT plus truncated mutant TnT 28(+7); lane 6,
human cardiac wild-type TnT plus truncated mutant TnT 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, TnT 28(+7), and
TnT 14 were loaded in lanes 2-4,
respectively.
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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
TnT
28(+7) and
TnT
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, TnT
28(+7), and
TnT
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.
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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
TnT
14-exchanged fibers was not
statistically significantly different from that generated by the
wild-type TnT-exchanged fibers, the
TnT
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 ( ),
TnT 28(+7) ( ), and
TnT 14 ( ) 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.
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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
-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, TnT
28(+7) conferred a
significantly lower maximum force than wild-type TnT and
TnT
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,
TnT 28(+7)-exchanged fiber
reconstituted with phosphorylated TnI; lane
6,
TnT 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- ( , ),
TnT 28(+7)- ( , ), and
TnT 14 ( , )-treated fibers
were reconstituted with TnI that had been dephosphorylated by
phosphatase ( , , ) or phosphorylated by PKA ( , , )
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.
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DISCUSSION |
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,
TnT
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
-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 TnT
28(+7) (28). The
-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
-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 TnT
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
TnT
14 exchanged in the absence
of Ca2+ even before the
reconstitution with TnI and TnC (Fig.
2A). Because the extent of
TnT
28(+7) exchanged was no
different from the extents of wild-type TnT and
TnT
14 exchanged, this may indicate that TnT
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
TnT
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
-adrenergic stimulation.
However, the inhibitory effect of
TnT
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
TnT
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
-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 TnT2
);
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, TnT2
,
which lacks the COOH-terminal 17 residues (26, 27). It has been shown that the regulatory function retained in
TnT2 (or
TnT2
) is lost in
TnT2
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, TnT
14 and TnT
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
-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
-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.
 |
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