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 |
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 |
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
-myosin heavy chain (
-MHC) (8), troponin T (TnT) (30),
troponin I (TnI) (12),
-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
-MHC isoform (14) in patients,
suggesting that the mutations in
-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
-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 |
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
-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 |
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).

View larger version (51K):
[in this window]
[in a new window]
|
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.

View larger version (13K):
[in this window]
[in a new window]

View larger version (26K):
[in this window]
[in a new window]
|
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.
|
|

View larger version (29K):
[in this window]
[in a new window]
|
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.
|
|

View larger version (74K):
[in this window]
[in a new window]
|
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).
View this table:
[in this window]
[in a new window]
|
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
(
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 (
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.

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 5.
Tension-pCa relationships in the same fiber determined at pH 7.0 before
( , ) and after ( , ) exchange of recombinant human cardiac
wild-type TnT ( , ) or rabbit native cardiac TnT ( , ). 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.

View larger version (20K):
[in this window]
[in a new window]
|
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 ( ), Ile79Asn ( ), or Arg92Gln ( ) 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 |
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
-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
-MHC mutations. However, it is known that cardiac hypertrophy
produced by TnT mutations is moderate or subtle compared with that
produced by
-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
-MHC
mutations (30), suggesting that the mutations in TnT and
-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
-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
-MHC cause HCM by different intracellular mechanisms. Most of
the mutations in
-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 |
1.
Anderson, P. A. W.,
N. N. Malouf,
A. E. Oakeley,
E. D. Pagani,
and
P. D. Allen.
Troponin T isoform expression in humans.
Circ. Res.
69:
1226-1233,
1991[Abstract].
2.
Bonne, G.,
L. Carrie,
J. Bercovici,
C. Cruaud,
P. Richard,
B. Hainque,
M. Gautel,
S. Labeit,
M. James,
J. Beckmann,
J. Weissenbach,
H. P. Vosberg,
M. Fiszman,
M. Komajda,
and
K. Schwarz.
Cardiac myosin binding protein C gene splice acceptor site mutation is associated with familial hypertrophic cardiomyopathy.
Nat. Genet.
11:
438-440,
1995[Medline].
3.
Brandt, P. W.,
R. N. Cox,
and
M. Kawai.
Can the binding of Ca2+ to two regulatory sites on troponin C determine the steep pCa/tension relationship of skeletal muscle?
Proc. Natl. Acad. Sci. USA
77:
4717-4720,
1980[Abstract].
4.
Cuda, G.,
L. Fananapazir,
W.-S. Zhu,
J. R. Sellers,
and
N. D. Epstein.
Skeletal muscle expression and abnormal function of
-myosin in hypertrophic cardiomyopathy.
J. Clin. Invest.
91:
2861-2865,
1993[Medline].
5.
Diffee, G. M.,
M. L. Greaser,
F. C. Reinach,
and
R. L. Moss.
Effects of a non-divalent cation binding mutant of myosin regulatory light chain on tension generation in skinned skeletal muscle fibers.
Biophys. J.
68:
1443-1452,
1995[Abstract].
6.
Donaldson, S. K. B.,
and
L. Harmansen.
Differential, direct effects of H+ on Ca2+-activated force of skinned fibers from the soleus, cardiac and adductor magnus muscles of rabbits.
Pflügers Arch.
376:
55-65,
1978[Medline].
7.
Fugita, H.,
S. Sugiura,
S. Momomura,
M. Omata,
H. Omata,
H. Sugi,
and
K. Suto.
Characterization of mutant myosins of dictyostelium discoideum equivalent to human familial hypertrophic cardiomyopathy mutants
molecular force level of mutant myosins may have a prognostic implication.
J. Clin. Invest.
99:
1010-1015,
1997[Abstract/Free Full Text].
8.
Geisterfer-Lowrance, A. A. T.,
S. Kass,
G. Tanigawa,
H. P. Vosberg,
W. McKenna,
C. E. Seidman,
and
J. G. Seidman.
A molecular basis for familial hypertrophic cardiomyopathy: a
cardiac myosin heavy chain gene missense mutation.
Cell
62:
999-1006,
1990[Medline].
9.
Hatakenaka, M.,
and
I. Ohtsuki.
Replacement of three troponin components with cardiac troponin components within single glycerinated skeletal muscle fibers.
Biochem. Biophys. Res. Commun.
181:
1022-1027,
1991[Medline].
10.
Hatakenaka, M.,
and
I. Ohtsuki.
Effect of removal and reconstitution of troponins C and I on the Ca2+-activated tension development of single glycerinated rabbit skeletal muscle fibers.
Eur. J. Biochem.
205:
985-993,
1992[Abstract].
11.
Horton, R. M.
In vitro recombination and mutagenesis of DNA.
Methods Mol. Biol.
15:
251-261,
1993.
12.
Kimura, A.,
H. Harada,
J. E. Park,
H. Nishi,
M. Satoh,
M. Takahashi,
S. Hiroi,
T. Sasaoka,
N. Ohbuchi,
T. Nakamura,
T. Koyanagi,
T. H. Hwang,
J. A. Choo,
K. S. Chung,
A. Hasegawa,
R. Nagai,
O. Okazaki,
H. Nakamura,
M. Matsuzaki,
T. Sakamoto,
H. Toshima,
Y. Koga,
T. Imaizumi,
and
T. Sasazuki.
Mutations in the cardiac troponin I gene associated with hypertrophic cardiomyopathy.
Nat. Genet.
16:
379-382,
1997[Medline].
13.
Laemmli, U. K.
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature
227:
680-685,
1970[Medline].
14.
Lankford, E. B.,
N. D. Epstein,
L. Fananapazir,
and
H. L. Sweeney.
Abnormal contractile properties of muscle fibers expressing b-myosin heavy chain gene mutations in patients with hypertrophic cardiomyopathy.
J. Clin. Invest.
95:
1409-1414,
1995[Medline].
15.
Lin, D.,
A. Bobkova,
E. Homsher,
and
L. S. Tobacman.
Altered cardiac troponin T in vitro function in the presence of a mutation implicated in familial hypertrophic cardiomyopathy.
J. Clin. Invest.
97:
2842-2848,
1996[Abstract/Free Full Text].
16.
Marian, A. J.,
G. Zhao,
Y. Seta,
R. Roberts,
and
Q. Yu.
Expression of a mutant (Arg92Gln) human cardiac troponin T, known to cause hypertrophic cardiomyopathy, impairs adult cardiac myocyte contractility.
Circ. Res.
81:
76-85,
1997[Abstract/Free Full Text].
17.
Mesnard, L.,
D. Logeart,
S. Taviaux,
S. Diriong,
J.-J. Mercadier,
and
F. Samson.
Human cardiac troponin T: cloning and expression of new isoforms in the neonatal and failing heart.
Circ. Res.
76:
687-692,
1995[Abstract/Free Full Text].
18.
Mizukami, Y.,
K. Yoshioka,
S. Morimoto,
and
K. Yoshida.
A novel mechanism of JNK1 activation
nuclear translocation and activation of JNK1 during ischemia and reperfusion.
J. Biol. Chem.
272:
16657-16662,
1997[Abstract/Free Full Text].
19.
Morimoto, S.,
and
Y. Ogawa.
Ca2+-insensitive sustained contraction of skinned smooth muscle after acidic ADP treatment.
Am. J. Physiol.
268 (Cell Physiol. 37):
C21-C29,
1995[Abstract/Free Full Text].
20.
Morimoto, S.,
and
I. Ohtsuki.
Ca2+ and Sr2+ sensitivity of the ATPase activity of rabbit skeletal myofibrils: effect of the complete substitution of troponin C with cardiac troponin C, calmodulin, and parvalbumins.
J. Biochem.
101:
291-301,
1987[Abstract].
21.
Morimoto, S., F. Yanaga, R. Minakami, and I. Ohtsuki.
Physiological function of human cardiac troponin T mutants that
cause familial hypertrophic cardiomyopathy (Abstract).
Jpn. J. Physiol.
47, Suppl. 2: S42,
1997.
22.
O'Farrell, P. H.
High resolution two-dimensional electrophoresis of proteins.
J. Biol. Chem.
250:
4007-4021,
1975[Abstract].
23.
Ohtsuki, I.,
K. Maruyama,
and
S. Ebashi.
Regulatory and cytoskeletal proteins of vertebrate skeletal muscle.
Adv. Protein Chem.
38:
1-67,
1986[Medline].
24.
Poetter, K.,
H. Jiang,
S. Hassanzadeh,
S. R. Master,
A. Chang,
M. C. Dalakas,
I. Rayment,
J. R. Sellers,
L. Fananapazir,
and
N. D. Epstein.
Mutations in either the essential or regulatory light chains of myosin are associated with a rare myopathy in human heart and skeletal muscle.
Nat. Genet.
13:
63-69,
1996[Medline].
25.
Rayment, I.,
H. M. Holden,
J. R. Sellers,
L. Fananapazir,
and
N. D. Epstein.
Structural interpretation of the mutations in the
-cardiac myosin that have been implicated in familial hypertrophic cardiomyopathy.
Proc. Natl. Acad. Sci. USA
92:
3864-3868,
1995[Abstract/Free Full Text].
26.
Rüegg, J. C.
Calcium in Muscle Activation. Berlin: Springer-Verlag, 1986.
27.
Sweeney, H. L.,
A. J. Straceski,
L. A. Leinwand,
B. A. Tikunov,
and
L. Faust.
Heterologous expression of a cardiomyopathic myosin that is defective in its actin interaction.
J. Biol. Chem.
269:
1603-1605,
1994[Abstract/Free Full Text].
28.
Tsukui, R.,
and
S. Ebashi.
Cardiac troponin.
J. Biochem.
73:
1119-1121,
1973[Medline].
29.
Watkins, H.,
D. Conner,
L. Thierfelder,
J. A. Jarcho,
C. MacRae,
W. J. McKenna,
B. J. Maron,
J. G. Seidman,
and
C. E. Seidman.
Mutations in the cardiac myosin binding protein C gene on chromosome 11 cause familial hypertrophic cardiomyopathy.
Nat. Genet.
11:
434-437,
1995[Medline].
30.
Watkins, H.,
W. J. McKenna,
L. Thierfelder,
H. J. Suk,
R. Anan,
A. O'Donoghue,
P. Spirito,
A. Matsumori,
C. S. Moravec,
J. G. Seidman,
and
C. E. Seidman.
Mutations in the genes for cardiac troponin T and
-tropomyosin in hypertrophic cardiomyopathy.
N. Engl. J. Med.
332:
1058-1064,
1995[Abstract/Free Full Text].
31.
Watkins, H.,
C. E. Seidman,
J. G. Seidman,
H. S. Feng,
and
H. L. Sweeney.
Expression and functional assessment of a truncated cardiac troponin T that causes hypertrophic cardiomyopathy.
J. Clin. Invest.
98:
2456-2461,
1996[Abstract/Free Full Text].
32.
Yanaga, F., S. Morimoto, R. Minakami, F. Shiraishi, T. Goto, and
I. Ohtsuki. The expression and characterization of human cardiac
troponin T mutants in familial hypertrophic cardiomyopathy (Abstract).
Jpn. J. Pharmacol. 73, Suppl. I: 47, 1997.
Am J Physiol Cell Physiol 275(1):C200-C207
0002-9513/98 $5.00
Copyright © 1998 the American Physiological Society