Ca2+ Sensitization and Potentiation of the Maximum
Level of Myofibrillar ATPase Activity Caused by Mutations of Troponin T
Found in Familial Hypertrophic Cardiomyopathy*
Fumi
Yanaga
,
Sachio
Morimoto, and
Iwao
Ohtsuki
From the Department of Clinical Pharmacology, Faculty of Medicine,
Kyushu University, 3-1-1 Maidashi, Higashi-ku,
Fukuoka 812-8582, Japan
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ABSTRACT |
Human wild-type cardiac troponin T, I, C and five
troponin T mutants (I79N, R92Q, F110I, E244D, and R278C) causing
familial hypertrophic cardiomyopathy were expressed in
Escherichia coli, and then were purified and incorporated
into rabbit cardiac myofibrils using a troponin exchange technique. The
Ca2+-sensitive ATPase activity of these myofibrillar
preparations was measured in order to examine the functional
consequences of these troponin mutations. An I79N troponin T mutation
was found to cause a definite increase in Ca2+ sensitivity
of the myofibrillar ATPase activity without inducing any significant
change in the maximum level of ATPase activity. A detailed analysis
indicated the inhibitory action of troponin I to be impaired by the
I79N troponin T mutation. Two more troponin T mutations (R92Q and
R278C) were also found to have a Ca2+-sensitizing effect
without inducing any change in maximum ATPase activity. Two other
troponin T mutations (F110I and E244D) had no
Ca2+-sensitizing effects on the ATPase activity, but
remarkably potentiated the maximum level of ATPase activity. These
findings indicate that hypertrophic cardiomyopathy-linked
troponin T mutations have at least two different effects on the
Ca2+-sensitive ATPase activity,
Ca2+-sensitization and potentiation of the maximum level
of the ATPase activity.
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INTRODUCTION |
The contraction of striated muscle is regulated by
Ca2+ through two specific regulatory proteins, troponin
(Tn)1 and tropomyosin, which
are located on the thin filament. Tn is a complex of three subunits,
troponin C (TnC), troponin I (TnI), and troponin T (TnT). TnI inhibits
myosin-actin interaction and TnC suppresses the inhibitory effect of
TnI by binding of Ca2+. TnT binds to tropomyosin and
integrates the whole Tn complex into the thin filament. All three Tn
subunits are required for the contraction regulated by Ca2+
(1).
Familial hypertrophic cardiomyopathy (HCM) is an autosomal dominant
cardiac disease associated with a high incidence of sudden death
(2-4). This disease has been reported to be caused by mutations in
cardiac sarcomeric proteins including TnT (5-7), TnI (8),
-myosin
heavy chain (9, 10),
-tropomyosin (5, 11), myosin-binding protein C
(12, 13), and myosin light chains (14). Previous genetic analyses have
shown that eight different point mutations in TnT to be associated with
HCM. However, it is still not clear as to how these single point
mutations are functionally related to and cause this disease.
This study was carried out to investigate the functions of five
HCM-linked TnT mutants in cardiac myofibrils under physiological conditions. We prepared recombinant human cardiac Tn subunits (wild-type TnC, TnI, and TnT) and five HCM-linked TnT mutants, and
these recombinant proteins were then incorporated into rabbit cardiac
myofibrils using our previously reported TnT treatment procedure
(15-17). The Ca2+-sensitive ATPase activity of these
reconstituted myofibrils was examined. We thus found the HCM-linked TnT
mutants to have at least two different effects on ATPase activity,
which are Ca2+-sensitization (I79N, R92Q, and R278C) and
the potentiation of the maximum level (F110I and E244D). The
Ca2+-sensitizing effect of I79N and R92Q TnT mutants is
consistent with our previous study using skinned fibers (18). These
findings suggest that an enhanced contractility of the cardiac muscle, which occurs as a result of mutations in both groups, may be a common
mechanism for the pathogenesis of HCM associated with these TnT mutations.
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EXPERIMENTAL PROCEDURES |
Cloning and Mutagenesis of Human Cardiac Tn cDNAs--
Human
cardiac Tn (TnC, TnI, and TnT) cDNAs were amplified by reverse
transcriptase-polymerase chain reaction of human heart mRNA
purchased from CLONTECH (Palo Alto, CA). The
polymerase chain reaction products were subcloned into the pUC 119 vector for screening by a restriction analysis and DNA sequencing. The
obtained wild-type Tn cDNAs were then constructed into a pET-3d
vector for expression. To obtain mutant TnTs, mutagenesis was carried
out by polymerase chain reaction according to the method described by
Horton (19). Oligonucleotides employed for mutagenesis were:
5'-CCTCCCAAGAACCCCGATGG-3' for the I79N mutation,
5'-GACATCCACCAGAAGCGCATG-3' for the R92Q mutation,
5'-GAGGCTCACATTGAGAACAGG-3' for the F110I mutation, 5'-TATAACTTGGATGCAGAGAAG-3' for the E244D mutation, and
5'-CTCCAAGACCTGCGGGAAGG-3' for the R278C mutation.
The changed bases were underlined. The results of the mutations in TnT
cDNA were confirmed by DNA sequencing.
Expression and Purification of Recombinant Tns--
The
wild-type Tn and mutant TnTs were expressed in Escherichia
coli BL21(DE3) and purified as described previously (18) with
slight modifications. The induction of protein expression by
isopropyl-
-D-thiogalactopyranoside was continued for
5.0 h, and then the cells were harvested and lysed. The lysate was
then centrifuged at 15,000 rpm for 25 min at 4 °C. For the
purification of TnT and TnC, 10 mM Tris/HCl (pH 8.0), 6 M urea, 5 mM CDTA, and 15 mM 2-ME
were added to the supernatant and the samples were applied to RESORCE Q
column (Pharmacia Biotech Inc.) using a fast protein liquid
chromatography system. For the purification of TnI, the pellet was
resuspended with the same solution described above and the sample was
applied to a Poros HS/M column (PerSeptive Biosystem).
Preparation of Myofibrils--
To prepare myofibrils, fresh
rabbit cardiac left ventricular muscle was cut in 5-mm slice and soaked
in a solution containing 1% Triton X-100, 100 mM KCl, 20 mM MOPS (pH 7.0), and 0.5% 2-ME for 6 h at 4 °C.
The solution was then changed to another solution containing 50%
glycerol, 100 mM KCl, 20 mM MOPS (pH 7.0), and 0.5% 2-ME and the slices were kept in it overnight at 4 °C. The following morning, these slices were transferred to the new solution and stored at
20 °C. Myofibrils were prepared from the kept
samples according to the method of Solaro et al. (20).
Tn Exchange in Myofibrils--
Tn exchange was carried out
according to our previously reported method (15-17) with slight
modifications. Myofibrils (300 µg/ml) were incubated in a solution
containing 20 mM MOPS/KOH (pH 6.2), 265 mM KCl,
2 mM EDTA, 2 mM MgCl2, 0.5 mM 2-ME and 37.5 µg/ml recombinant human cardiac TnT at
25 °C for 1 h while shaking. These myofibrils were washed with
a solution containing 0.5% Briji-58, 20 mM MOPS (pH 7.0),
265 mM KCl, 2 mM MgCl2, 0.5 mM 2-ME, and the myofibrils were resuspended in a solution
(300 µg/ml) containing 60 mM KCl and 1 mM
sodium bicarbonate after washing with the same solution. The myofibrils
were then reconstituted with recombinant TnI (15.2 µg/ml) and TnC
(11.6 µg/ml) on ice for 1 h.
ATPase Activity Measurement--
The reaction mixture (200 µl)
for the ATPase assay consisted of 90 mM KCl, 5 mM MgCl2, 20 mM MOPS (pH 7.0), 1 mM Ca2+-EGTA, 4 mM ATP, 40 µg of
myofibrils. The reaction was started by adding ATP at 25 °C and was
terminated by adding 1.6 ml of a mixture of 50% acetone, 2.5 mM
(NH4)6Mo7O24·4H2O
and 1.25 N H2SO4. The contents were
mixed carefully and 160 µl of 1 M citric acid were added
to tubes and the yellow color was measured at OD355 (21).
Ca2+ concentrations in the reaction mixtures were
calculated as described previously (22).
Electrophoresis--
SDS-PAGE was performed according to the
procedure of Laemmli (23), with an acrylamide concentration of 12%.
Alkaline gel electrophoresis was carried out according to the procedure
described previously (24). Alkaline urea gel electrophoresis was done by the procedure of Pan and Solaro (25), with an acrylamide concentration of 13.5% at pH 8.5. The gel was stained with Coomassie Brilliant Blue R-250 and an optical densitometric scan was performed using Phoretix gel analysis software (Phoretix International Ltd.).
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RESULTS |
Comparison of Recombinant Human Cardiac Tn Subuint and Native
Rabbit Cardiac Tn Subuint--
Three subunits of human cardiac Tn
(TnT, TnI, and TnC) were cloned by the reverse transcriptase-polymerase
chain reaction technique and expressed using a pET-3d vector. As shown
in Fig. 1A, recombinant human
cardiac TnT and TnC had exactly the same electrophoretic mobility as
rabbit cardiac TnT and TnC on 12% SDS-PAGE gel. Recombinant human
cardiac TnI, however, had a little faster mobility than rabbit cardiac
TnI. An alkaline gel electrophoresis analysis showed the recombinant
human cardiac Tn subunit combined with the native rabbit cardiac Tn
subunit to form binary or ternary complexes under nondenaturing
conditions (Fig. 1B). As a result, the recombinant human
cardiac Tn subunits had the ability to form a Tn complex in the same
way as the native rabbit cardiac Tn subunits.

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Fig. 1.
Comparison of the gel electrophoresis
patterns of the recombinant human cardiac Tn subunit and the native
rabbit cardiac Tn subunit. A, SDS-PAGE pattern. All samples
were separated on 12% SDS-PAGE gel. Lane 1, rabbit cardiac
TnC; lane 2, recombinant human cardiac TnC; lane
3, rabbit cardiac TnI; lane 4, recombinant human
cardiac TnI; lane 5, rabbit cardiac TnT; lane 6, recombinant human cardiac TnT. B, alkaline gel
electrophoresis pattern. TnT, TnI, and TnC were mixed at a molar ratio
of 1:1:2 and the mixture was kept at 4 °C for 30 min with 2 mM CaCl2 to form a binary or ternary complex.
All samples were applied on alkaline gel (pH 8.5) in a nondenaturing
condition. Lane 1, rabbit cardiac TnC; lane 2, recombinant human cardiac TnC; lane 3, rabbit cardiac TnC + recombinant human cardiac TnI; lane 4, recombinant human
cardiac TnC + rabbit cardiac TnI; lane 5, rabbit cardiac TnC + TnI + TnT; lane 6, recombinant human cardiac TnC + rabbit
cardiac TnI, TnT; lane 7, recombinant human cardiac TnC + TnI + rabbit cardiac TnT; lane 8, recombinant human cardiac
TnC + TnI + TnT. The data are representative of four other
experiments.
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Effect of Wild-type TnT and I79N TnT Treatment on the ATPase
Activity of Rabbit Cardiac Myofibrils--
We previously reported the
endogenous Tn complexes in myofibrils were replaced with exogenous TnT
after treatment with an excessive amount of purified TnT in acidic
solution and the ATPase activity of the treated myofibrils becomes
insensitive to Ca2+ (15-17). Fig.
2 shows the ATPase activity of rabbit
cardiac myofibrils treated with different amounts of recombinant TnT in
the presence (pCa 5.18) and absence (pCa 7.05) of
Ca2+. The ATPase activity in the presence of
Ca2+ did not change after the treatment with TnT at any
concentrations, whereas the ATPase activity in the absence of
Ca2+ increased in a concentration-dependent manner
and reached almost a maximum level after treatment with 20 µg/ml TnT
(Fig. 2). The maximum ATPase activities of wild-type TnT-treated
myofibrils were 22 ± 1.6 nmol·Pi/mg/min in the
absence of Ca2+ and 32 ± 0.5 nmol·Pi/mg/min in the presence of Ca2+
(mean ± S.E., n = 4). The maximum ATPase
activities of I79N TnT-treated myofibrils were 22 ± 1.5 nmol·Pi/mg/min in the absence of Ca2+ and
33 ± 3.0 nmol·Pi/mg/min in the presence of
Ca2+ (mean ± S.E., n = 4). As a
result, the maximum ATPase activity in the absence of Ca2+
increased up to approximately 65% of the maximum activity in the
presence of Ca2+ by the treatments with either wild-type or
I79N TnT at 37.5 µg/ml. In the following experiments, myofibrils were
treated with this concentration of TnT.

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Fig. 2.
The ATPase activity of rabbit cardiac
myofibrils treated with recombinant TnT. Myofibrils were treated
with different amounts of wild-type ( , ) or I79N mutant ( ,
) TnT and the ATPase activity of these myofibrils were measured in
the presence (pCa 5.18, open symbols) or absence
of Ca2+ (pCa 7.05, closed symbols).
The results are the mean ± S.E. of four experiments performed in
triplicate.
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Determination of the Exchange Rate of Tn Complex in Myofibrils
Treated with Wild-type TnT and I79N TnT--
To determine the extent
of the exchange of Tn complex in the myofibrils, both SDS-PAGE and
alkaline urea gel electrophoresis were carried out. The densitometry of
TnI bands in the SDS-PAGE pattern of the myofibrils showed that
65.3 ± 3.2% (mean ± S.E., n = 5) and
63.8 ± 4.0% (mean ± S.E., n = 5) of the
native TnI was removed by wild-type TnT treatment and I79N TnT
treatment, respectively (Fig.
3A). Although native rabbit
cardiac TnT and recombinant human cardiac TnT had exactly the same
electrophoretic mobility on 12% SDS-PAGE gel (Fig. 3A, lanes
1 and 2), the recombinant human cardiac TnT had a
slightly faster mobility than rabbit cardiac TnT on alkaline urea gel
(Fig. 3B, lanes 1 and 2). A densitometry scan on
alkaline urea gel was performed to estimate the amount of TnT exchanged
into myofibrils. On the typical scanning patterns of TnT-treated and
untreated myofibrils (Fig. 3C), the density of both native
rabbit TnT and TnC bands decreased by treatment with wild-type TnT or
I79N TnT of which mobility were the same as actin. Although a
quantitative analysis of TnTs could not be carried out because of the
high background density, the increase in the density of the actin peak
after TnT treatment indicated the incorporation of human recombinant
TnTs into the myofibrils. The densitometry of the TnC bands showed the
percentage of native rabbit TnC removed from myofibrils to be 65.0 ± 4.0% (mean ± S.E., n = 5) by wild-type TnT
treatment and 63.9 ± 2.1% (mean ± S.E., n = 5) by I79N TnT treatment. These gel scan analyses, taken together with the ATPase assays, provided strong evidence that the extent of the
recombinant human cardiac TnT incorporated into the myofibrils were
approximately 65% and that the wild-type and I79N mutant TnTs were
also equally effective in replacing the endogenous Tn complex in
myofibrils.

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Fig. 3.
The gel electrophoresis pattern of
TnT-treated and TnI·C-reconstituted myofibrils. A,
SDS-PAGE pattern. The myofibrils were treated with 37.5 µg/ml
wild-type or I79N mutant TnT and reconstituted with recombinant human
TnI (15.2 µg/ml) and TnC (11.6 µg/ml). The samples were lysed in
Laemmli's sample buffer and separated on 12% SDS-PAGE. Lane
1, recombinant human cardiac TnT, I, C-mixture; lane 2, purified rabbit cardiac TnT, I, C-mixture; lane 3, untreated
myofibrils; lane 4, wild-type TnT-treated myofibrils;
lane 5, reconstituted myofibrils after treatment with
wild-type TnT; lane 6, I79N TnT-treated myofibrils;
lane 7, reconstituted myofibrils after treatment with I79N
TnT. The data are representative of four other experiments.
B, the alkaline urea gel electrophoresis pattern of
TnT-treated myofibrils. The recombinant TnT-treated myofibrils were
separated on alkaline gel (pH 8.5) containing 6 M urea and
13.5% acrylamide. Lane 1, recombinant human wild-type TnT;
lane 2, native rabbit TnT; lane 3, untreated
myofibrils; lane 4, wild-type TnT-treated myofibrils;
lane 5, I79N TnT-treated myofibrils. All data are
representative of four other experiments. C, the
densitometry tracings for the alkaline urea gel electrophoresis pattern
of the myofibrils. The gels stained with Coomassie Brilliant Blue R-250
were analyzed by an optical densitometric scan using Phoretix gel
analysis software. (i) untreated myofibrils; (ii)
wild-type TnT-treated myofibrils, (iii) I79N TnT-treated
myofibrils. The tracing is typical of four other experiments.
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Effect of Recombinant Human Cardiac TnI and TnC on the Myofibrils
Treated with Wild-type and I79N TnTs--
As shown in Fig.
4, the addition of the recombinant human
cardiac TnI resulted in a decrease in the Ca2+-insensitive
ATPase activity of the myofibrils treated with wild-type or I79N TnT in
a dose-dependent manner and were completely inhibited at
15.2 µg/ml (Fig. 4A). The amounts of TnI required for a
50% inhibition (IC50) were 0.80 ± 0.05 µg/ml and
1.74 ± 0.21 µg/ml (mean ± S.E., n = 3)
for wild-type TnT-treated and I79N TnT-treated myofibrils,
respectively. This indicated that the inhibitory effect of TnI on
actin-tropomyosin was significantly smaller in the I79N mutant
TnT-treated myofibrils than the wild-type TnT-treated myofibrils. In
the presence of Ca2+, recombinant human cardiac TnC
activated the suppressed ATPase activity of the TnI-reconstituted
myofibrils in a dose-dependent manner and fully neutralized
the ATPase activity at the concentration of 11.6 µg/ml (Fig.
4B). No significant difference in the neutralizing effect of
TnC was detected between the myofibrils containing wild-type TnT and
I79N TnT.

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Fig. 4.
The effects of recombinant human cardiac TnI
and TnC on the myofibrils treated with wild-type or I79N TnT.
A, inhibitory effect of TnI on ATPase activity of
TnT-treated myofibrils. The myofibrils (300 µg/ml) were treated with
37.5 µg/ml wild-type ( ) or I79N mutant ( ) TnT for 1 h at
25 °C with a shake and then were reconstituted with various amounts
of TnI at 4 °C for 1 h. The ATPase activity of the treated
myofibrils was measured in the absence of Ca2+
(pCa 7.05). The results are expressed as a percentage of the
ATPase activity of TnT-treated myofibrils without adding TnI and are
the mean ± S.E. of three experiments performed in triplicate.
B, the effect of TnC on the restoration of the
Ca2+ sensitivity of TnT-treated and TnI-reconstituted
myofibrils. The myofibrils treated with wild-type or I79N TnT were
reconstituted with 15.2 µg/ml TnI and various amounts of TnC, and
then ATPase activity of the reconstituted myofibrils were measured in
the presence of Ca2+ (pCa 5.18). The results are
expressed as a percentage of the maximum ATPase activity of the
reconstituted myofibrils and are the mean ± S.E. of three
experiments performed in triplicate.
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pCa-ATPase Activity Relationships of the Myofibrils Treated with
Wild-type TnT and I79N TnT, and Reconstituted with Recombinant Human
Cardiac TnI and TnC at Different pH Values (pH 6.5, 7.0, and
7.5)--
Although no significant difference was seen in the
pCa-ATPase activity relationships at pH 7.5, the
Ca2+ sensitivity (pCa50,
pCa at half-maximum activation) of the myofibrils reconstituted with I79N mutant TnT was significantly higher than the
Ca2+ sensitivity of myofibrils reconstituted with wild-type
TnT at pH 7.0 and 6.5 (Fig. 5). The
difference in the Ca2+ sensitivity of the reconstituted
myofibrils was thus larger at pH 6.5 than at pH 7.0 (Table
I). However, no significant difference was observed in the maximum ATPase activity and the Hill coefficient (nH, an index of cooperativity) between the
myofibrils reconstituted with wild-type TnT and reconstituted with I79N
mutant TnT at any pH.

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Fig. 5.
The Ca2+-activated ATPase
activity of myofibrils reconstituted with human cardiac TnI and TnC
after treatment of recombinant TnT. Myofibrils were treated with
wild-type ( ) or I79N mutant ( ) TnT and then reconstituted with
TnI and TnC. ATPase activity of the reconstituted myofibrils were
measured at pH 7.5( ), pH 7.0 (···), and pH 6.5 (- - -). The
maximum ATPase activities of wild-type TnT-treated myofibrils were
15.8 ± 1.2, 32.3 ± 3.6, and 37.7 ± 0.67 (nanomole of
Pi/mg/min, mean ± S.E., n = 3) for pH
6.5, 7.0, and 7.5, respectively. The maximum ATPase activities of I79N
TnT-treated myofibrils were 16.2 ± 0.4, 31.0 ± 0.8, and
36.3 ± 1.5 (nanomole of Pi/mg/min, mean ± S.E.,
n = 3) for pH 6.5, 7.0, and 7.5, respectively. The
results are expressed as a percentage of the maximum ATPase activity
stimulated by Ca2+ and are the mean ± S.E. of three
experiments performed in triplicate.
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Table I
The Ca2+ sensitivity of ATPase activity of myofibrils after
being reconstituted with recombinant human cardiac Tns at different
pH levels
pCa values at half-maximum ATPase activity
(pCa50) at each pH were calculated by fitting the
data shown in Fig. 4 to the Hill equation. The data are the mean ± S.E. of three or four independent experiments done in triplicate.
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An SDS-PAGE Analysis of TnT Mutants, R92Q, F110I, E244D, and
R278C--
Four other missense mutations, R92Q, F110I, E244D, and
R278C mutant TnTs, were generated by the overlap extension methods. An
SDS-PAGE analysis showed all of these mutants to have the same electrophoretic mobility on 12% SDS-PAGE gel (Fig.
6A). The densitometric scan of
the SDS-PAGE gel was performed to determine the amount of native TnI
removed from the myofibrils after treatment with mutant TnTs (Fig.
6B, Table II). No significant
difference was found in the amounts of native TnI removed from
myofibrils by treatment with mutant TnTs. This result indicated that
the extent of the removal of native TnT and hence the incorporation of
these mutant TnTs into the myofibrils were almost the same as the
wild-type and I79N TnTs.

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Fig. 6.
SDS-PAGE pattern of mutant TnTs.
A, purified recombinant TnT mutants. Samples were separated
on 12% SDS-PAGE gel. Lane 1, wild-type TnT; lane
2, I79N TnT; lane 3, R92Q TnT; lane 4, F110I
TnT; lane 5, E244D TnT; lane 6, R278C TnT.
B, mutant TnTs treated and reconstituted myofibrils. The
myofibrils were treated with 37.5 µg/ml mutant TnTs and reconstituted
with recombinant human TnI (15.2 µg/ml) and TnC (11.6 µg/ml). The
samples were lysed in Laemmli's sample buffer and separated on 12%
SDS-PAGE. Lane 1, untreated myofibrils; lanes 2 and 3, R92Q TnT-treated myofibrils; lanes 4 and
5, F110I TnT-treated myofibrils; lanes 6 and
7, E244D TnT-treated myofibrils; lanes 8 and
9, R278C TnT-treated myofibrils. Lanes 2, 4, and
6, before reconstitution; lanes 3, 5, and
7, after reconstitution with TnI and TnC. All data are
representative of three other experiments.
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Table II
Comparison of the effects of mutant TnTs on myofibrils
Myofibrils were treated with wild-type or mutant TnT, and an SDS-PAGE
analysis or ATPase activity measurement in the presence of Ca2+
(pCa5.18) was carried out before and after reconstitution.
The gels stained with Coomassie Brilliant Blue R-250 were analyzed by
an optical densitometric scan using Phoretix gel analysis software to
determine the amount of TnI removed from myofibrils by the treatment
with TnTs. Data are the mean ± S.E. of five or four independent
experiments. pCa values at half-maximum ATPase activity
(pCa50) and Hill coefficient (nH)
were calculated from the data shown in Fig 7. The data are the
mean ± S.E. of three or four experiments done in triplicate.
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The Effect of TnT Mutants on the Ca2+-sensitive ATPase
Activity--
The pCa-ATPase activity relationships were
examined at pH 7.0 using myofibrils which were treated with these TnT
mutants and then reconstituted with recombinant human cardiac TnI and
TnC. As shown in Fig. 7, an increase in
the Ca2+ sensitivity of the ATPase activity was observed
with R92Q and R278C TnT mutants, with no significant effect on the
maximum ATPase activity. On the other hand, an increase in the maximum
ATPase activity was observed with F110I and E244D TnT mutants, without any change in the Ca2+ sensitivity of the ATPase activity
(Fig. 8). No significant change in the
cooperativity was observed between the wild-type TnT-treated myofibrils
and mutant TnT-treated myofibrils (Table II). The summary in Table II
clearly indicates these HCM-related TnT mutants to have the same
ability as wild-type TnT to exchange native TnT in the myofibrils and
are thus classified into at least two groups according to their effects
on the Ca2+-sensitive ATPase activity.

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Fig. 7.
Ca2+-sensitizing effect on the
ATPase activity caused by R92Q and R278C TnT mutants. The
myofibrils were treated with wild-type ( ), R92Q ( ), or R278C
( ) TnT (37.5 µg/ml) for 1 h at 25 °C and reconstituted
with recombinant human cardiac TnI (15.2 µg/ml) and TnC (11.2 µg/ml) for 1 h at 4 °C. Ca2+-sensitive ATPase
activity of the reconstituted myofibrils was measured at pH 7.0. The
results are the mean ± S.E. of three experiments performed in
triplicate.
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Fig. 8.
The potentiation of the maximum ATPase
activity caused by F110I and E244D TnT mutants. Myofibrils were
treated with wild-type ( ), F110I ( ), or E244D ( ) TnT (37.5 µg/ml) for 1 h at 25 °C and reconstituted with recombinant
human cardiac TnI (15.2 µg/ml) and TnC (11.2 µg/ml) for 1 h at
4 °C. The Ca2+-activated ATPase activity of the
reconstituted myofibrils was measured at pH of 7.0. The results are the
mean ± S.E. of three experiments performed in triplicate.
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DISCUSSION |
This is the first report to evaluate the properties of recombinant
human cardiac TnT·I·C complex in myofibrils under physiological conditions. The results of the present study indicated the recombinant human cardiac Tn subunits and native rabbit cardiac Tn subunits to be
almost identical. These findings led us to analyze the effects of
HCM-linked TnT mutants on the Ca2+-sensitive ATPase
activity of rabbit cardiac myofibrils. We already reported that I79N
and R92Q TnT mutants in HCM have a Ca2+ sensitizing effect
on the tension development of skinned fibers prepared from the rabbit
ventricular muscle (18). In the present study, the Ca2+
sensitizing effects of I79N and R92Q mutant TnTs on the myofibrillar ATPase activity were thus confirmed.
Regarding the molecular mechanism of action of these TnT mutants, the
inhibitory effect of TnI on the Ca2+-insensitive ATPase
activity of myofibrils with I79N mutant TnT was found to be smaller
than that with wild-type TnT, while the neutralizing effect of
wild-type TnC was not affected by this mutation (Fig. 3, A
and B). These findings suggest that the I79N mutant TnT
reduced the affinity of TnI to actin-tropomyosin. The same mechanisms
on the action of R92Q TnT mutant was also suggested in our previous
study using skinned fibers (18). In this report, we showed that a
mutation at Arg278 in TnT, in addition to the mutations at
Ile79 and Arg92 in the TnT mentioned above,
thus caused an increased Ca2+ sensitivity of the ATPase
activity without any change in the maximum level. It was also recently
reported that the HCM-linked
-tropomyosin mutant, D175N, is
associated with an increase in the Ca2+ sensitivity (26).
It is therefore highly conceivable that an increase in the
Ca2+ sensitivity is one of the characteristic features of
the HCM caused by the regulatory proteins associated with the thin filament.
The results of both the present and previous studies showing the
Ca2+ sensitizing effects of R92Q mutant on the ATPase
activity of myofibrils and the tension development of skinned fibers
apparently conflict with the results of a recent study by Marian
et al. (27). They reported that the expression of R92Q
mutant human cardiac TnT in cultured adult feline cardiac myocytes
impaired the contractile performance as indicated by both fractional
cell shortening and the peak velocity of shortening of the myocytes
(27). Although the reason for this discrepancy in the results is not
known, it might be due to the difference of the design of the
experiments. No significant difference has been reported between the
effects of I91N mutation in rat embryonic cardiac TnT, thus
corresponding to I79N mutation in human, and wild-type TnT on
Ca2+ sensitivity of the thin filaments examined by S-1
Mg-ATPase activity at pH 7.5 (28). While we found this mutant to behave
in a manner similar to a wild-type at pH 7.5, the mutant had a
Ca2+ sensitizing effect on the actomyosin ATPase activity
at pH 7.0 and 6.5, which was more significant at an acidic pH 6.5, which is also consistent with the previous observations on skinned
fibers (18). This finding suggests that the I79N mutant may have a strong effect on the contractile performance during myocardial ischemia, although the effects of acidic pH on other TnT mutants have
not yet been examined.
The most significant finding of the present study was that the maximum
ATPase activity of the myofibrils was potentiated by the mutations at
Phe110 and Glu244 in TnT (Fig. 8 and Table II).
The potentiation effects of F110I and E244D TnT mutants were not
observed after the TnT treatment but were observed only after
subsequent reconstitution with TnC and TnI (Table II). This strongly
suggests that the potentiation of the maximum ATPase activity is caused
through a change in the interaction of TnT and TnC·I.
In the present study, we found that the effects of HCM-linked TnT
mutations can be classified into at least two different types. The
I79N, R92Q, and R278C TnT mutants enhanced Ca2+ sensitivity
of myofibrillar ATPase activity without affecting the maximum level of
ATPase activity, whereas the F110I and E244D TnT mutants enhanced a
maximum level of ATPase activity without affecting the Ca2+
sensitivity. These two groups of missense mutations in TnT might cause
HCM in different manners. However, it is noteworthy that the mutations
which cause an increase in the maximum ATPase activity alone also have
an apparent Ca2+ sensitizing effect at a submaximal
concentration of Ca2+ due to a scale-up of the overall
ATPase activity. Since the intact cardiac muscle has been reported to
never be activated beyond the half-maximal level (29), an enhanced
myofilament response to the submaximal Ca2+ may be a common
phenomenon for the pathogenesis of HCM associated with these missense
mutations in TnT. In several reports HCM-linked
-myosin heavy chain
mutations and TnT mutations have been shown to impair cardiac
performance and cause compensatory cardiac hypertrophy (30-32).
However, our findings suggest that certain mutations might determine a
hypercontractile state and thus appear to induce hypertrophy directly,
instead of causing it indirectly via the mechanism of compensatory hypertrophy.
 |
ACKNOWLEDGEMENTS |
We thank Dr. K. Maeda and Dr. R. Minakami for
technical advice.
 |
FOOTNOTES |
*
This work was supported by a research grant from the
Ministry of Education, Culture, Sports and Science of Japan.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. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Clinical
Pharmacology, Faculty of Medicine, Kyushu University, 3-1-1 Maidashi,
Higashi-ku, Fukuoka 812-8582, Japan. Fax: 81-92-642-6084; E-mail:
yanaga{at}clipharm.med.kyushu-u.ac.jp.
 |
ABBREVIATIONS |
The abbreviations used are:
Tn, troponin;
HCM, familial hypertrophic cardiomyopathy;
CDTA, trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic
acid;
PAGE, polyacrylamide gel electrophoresis;
pCa, -log
[Ca2+].
 |
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