(Received for publication, September 15, 1995)
From the
The cardiotonic effects of thiadiazinone derivative EMD 57033
are mediated by direct actions on myofilaments (Lues, I., Beier, N.,
Jonas, R., Klockow, M., and Haeusler, G. J. (1993) Cardiovasc.
Pharmacol. 21, 883-892). Cardiac troponin C has been
postulated to be a potential target of the drug (White, J., Lee, J. A.,
Shah, N., and Orchard, C. H.(1993) Circ. Res. 73,
61-70). This study tested whether EMD 57033 interacts directly
with recombinant human cardiac TnC (hcTnC). EMD 57033 caused
concentration-dependent quenching of tyrosine (Tyr) fluorescence of
hcTnC in the presence of Ca (100 µM) and
little change of the fluorescence in the presence of Mg
(2 mM). K
for the
drug-hcTnC interaction in the presence of Ca
,
determined by Tyr fluorescence titrations, was approximately 40
µM. The binding of EMD 57033 was stereo-selective: the
optical isomer of EMD 57033 bound hcTnC much more weakly. The
Ca
dependence and stereo-selectivity of EMD 57033
binding were substantiated by a dialysis-based direct binding assay.
EMD 57033 was found to interfere with Ca
-dependent
binding of hydrophobic probe
1,1`-bi-(4-anilino)naphthalene-5,5`-disulfonate (bis-ANS) to hcTnC. The
relationships between [Ca
] and Tyr
fluorescence of hcTnC and between [Ca
] and
bis-ANS fluorescence in the presence of hcTnC were substantially
altered by EMD 57033 in the range of [Ca
]
where Ca
/Mg
sites of hcTnC were
titrated by Ca
. EMD 57033 was found to bind as
tightly to 2 Ca
hcTnC as to 3
Ca
hcTnC. These observations were interpreted as
indicating that a EMD 57033-binding site is induced by Ca
binding, but not Mg
binding, to the
Ca
/Mg
sites of hcTnC. The
drug-binding site most likely resides in the carboxyl domain of hcTnC.
Thiadiazinone derivative EMD
57033()(1, 2) , developed by Pharmaceutical
Research, E. Merck (Darmstadt, Germany), is a novel cardiotonic agent
that induces positive inotropy in intact cardiac myocytes without a
significant effect on [Ca
]
transients(1, 2, 3, 4, 5, 6, 7) .
The pharmacological effects of the compound are thought to be mediated
by enhanced responsiveness of the contractile filaments to
Ca
(2, 3, 4, 5, 6, 7) .
EMD 57033 has been found to increase Ca
sensitivity
of both myofibrillar ATPase (2, 6) and force
development by skinned muscle fibers(2) . The myofibrillar
Ca
-sensitizing effects of EMD 57033 are remarkably
stereo-specific. The(-)-enantiomer, EMD 57439, exhibits no such
activity(2, 6) .
The molecular mechanism(s)
underlying the Ca-sensitizing effects of EMD 57033 is
not well understood. The compound appears to have a direct effect on
actin/myosin interactions(6) . It has also been hypothesized
that EMD 57033 may bind cardiac troponin C and increase its affinity
for Ca
(4, 7) .
Troponin C (TnC)
is the Ca binding subunit of troponin, a
hetero-trimeric protein containing, in addition, inhibitory subunit
(TnI) and tropomyosin binding subunit. Troponin, together with
tropomyosin, constitutes a thin filament-based regulatory protein
complex. The two known isoforms of TnC, i.e. TnC found in
cardiac and slow twitch skeletal muscles (cTnC) and that found in fast
skeletal muscles (sTnC) share extensive sequence homology (8, 9, 10) and both belong to the superfamily
of helix-loop-helix or EF-hand Ca
-binding
proteins(11) . Crystallographic structure of avian sTnC
revealed a dumb bell-shaped molecule consisting of two globular domains
connected by a long central helix(12, 13) . cTnC is
thought to have a similar three dimensional structure(14) .
Both sTnC and cTnC contain four helix-loop-helix motifs (I-IV),
two (I/II) in the amino-terminal domain, and two (III/IV) in the
carboxyl-terminal domain. While all four are functional
Ca
-binding sites in sTnC, only three (II, III, and
IV) are functional in cTnC. Site I in sTnC and II in both sTnC and cTnC
bind Ca
specifically (K
5
10
M
)
and are directly involved in Ca
-regulation of muscle
contraction (15, 16, 17, 18, 19) .
Sites III and IV bind Ca
(K
2
10
M
)
and Mg
(K
5
10
M
) competitively,
and appear to play primarily a structural
role(15, 16, 20, 21) . Thus, the
NH
- and COOH-terminal domains of TnC are often described as
the regulatory and structural domains, respectively. In addition to its
well documented structural role in anchoring TnC to the thin filament,
the COOH-terminal domain appears to contribute directly to
Ca
regulation by way of interacting with the
inhibitory region of TnI and residues of TnI adjacent to the inhibitory
region (see, e.g., (22, 23, 24) ).
Both domains of TnC contains a core of hydrophobic residues, which
become more exposed to solvent upon Ca binding to the
domains(25, 26) . The interactions of the hydrophobic
patches with TnI are believed to be important for transmission of the
Ca
signal and the stability of troponin complex.
These hydrophobic residues appear to also participate in interaction of
TnC with small organic ligands, such as trifluoperazine and bepridil,
which bind TnC and modulate its Ca
binding
properties(14, 27, 28, 29, 30) .
In the present report, we demonstrate that EMD 57033 interacts
directly with cTnC in a Ca-dependent and
stereo-selective manner. We show that cTnC contains a EMD 57033-binding
site, which is induced by Ca
binding, but not by
Mg
binding, to the
Ca
/Mg
sites of cTnC. The
drug-binding site most likely resides in the COOH-terminal domain of
cTnC. In addition, the report offers new evidence that the
COOH-terminal domain of cTnC, when saturated by Mg
,
is in a conformational state substantially different from the state it
assumes when occupied by Ca
.
Figure 1: The chemical structures of EMD 57033 and EMD 57439.
Figure 2:
Titration of tyrosine fluorescence of cTnC
with EMD 57033 and 57439. A, Tyr fluorescence intensity of
hcTnC as a function of [EMD 57033] (squares) and
[EMD 57439] (circles). The conditions were 2.9
µM hcTnC, 100 mM KCl, 2 mM Mg, 60 mM MOPS (pH 7.0), 1 mM
EGTA, pCa 9 (open symbols) or 4 (closed
symbols). Excitation wavelength, 280 nm; emission wavelength, 305
nm. The data are expressed as fractions of the fluorescence intensity
in the absence of the drugs. B, the data at pCa 4
replotted as double-reciprocal plots. The symbols are the same as in A. Also shown are the linear regression lines of the
data.
Figure 3:
Direct
measurement of binding of EMD 57033 and EMD 57439 to hcTnC by
equilibrium dialysis. The conditions were 100 mM KCl, 60
mM MOPS (pH 7.0), 2 mM Mg, 2 mM NaN
, pCa 9 or 4. Each bar represents
average of two or three measurements.
Figure 4:
Effect of EMD 57033 on the relationship
between [Ca] and Tyr fluorescence of hcTnC.
Tyr fluorescence of hcTnC (5.25 µM) was titrated with
Ca
in the presence of 100 mM KCl, 2 mM EGTA, 100 mM MOPS, 0.5 mM NaN
with (circles) or without (triangles) 42 µM EMD 57033. F
is defined as the
fluorescence (F) at pCa 9.0. The lines represent the
least square fits of the data with the following equation: F/F
= 1 +
F
[Ca
]
/(K
+ [Ca
]
) where n and K are
Hill coefficient and [Ca
] at
0.5
F
. The
[Ca
]-fluorescence relation in the absence
of EMD 57033 was fitted well with a Hill equation with K = 0.13
µM and n = 1.8. In the presence of 42
µM EMD 57033, the relation was fitted well with a Hill
equation with K = 0.15 µM and n =
2.5.
Figure 5:
Fluorescence spectra of bis-ANS. The
spectra a, b, c and d were obtained sequentially. a: bis-ANS (5
µM) in 100 mM KCl, 100 mM MOPS (pH 7.0),
2 mM EGTA, 0.5 mM azide; b: after 5 µM apo-hcTnC was added; c: after addition of 2 mM MgCl; d: after addition of CaCl
to pCa 4.3.
Shown in Fig. 6are the results of an experiment in
which the fluorescence of bis-ANS was titrated by Ca in the presence of hcTnC. The Ca
titration
curve was biphasic, consisting of a steep rising phase between pCa 7.5 and 6.5, and a less steep rising phase between pCa 6.5 and 4.3. The data strongly suggest that Ca
binding to the Ca
/Mg
sites
induced bis-ANS-binding site(s), presumably in the COOH-terminal domain
of hcTnC, and that additional bis-ANS-binding site(s), presumably in
the NH
-terminal domain, are induced by Ca
binding to the Ca
-specific site. The presence
of 42 µM EMD 57033 in the titration (Fig. 6) caused
a significant decrease of the magnitude of the steep rising phase of
the pCa-bis-ANS fluorescence relation, with little effect on
the second phase. An obvious interpretation of the observation is that
EMD 57033 inhibited the binding of bis-ANS to the COOH-terminal domain
of hcTnC. In the presence of EMD 57033, the Ca
affinities of the Ca
/Mg
- and
Ca
-specific sites obtained from fitting the data with
a two-term Hill equation were not significantly different from those in
the absence of the drug (see legend for Fig. 6).
Figure 6:
Effect of EMD 57033 on the relationship
between [Ca] and bis-ANS fluorescence in
the presence of hcTnC. The fluorescence titrations were conducted in
the presence of 5 µM bis-ANS, 5 µM hcTnC, 100
mM KCl, 100 mM MOPS (pH 7.0), 2 mM EGTA and
0.5 mM azide with (triangles) and without (circles) 42
µM EMD 57033. The lines are the best fit of the data with
the following equation, which describes two classes of binding sites: F/F
= 1 + A
[Ca
]
Fig. 7shows the results of experiments in which the
fluorescence of bis-ANS was titrated with EMD 57033 in the presence of
hcTnC. In the presence of 2 mM Mg and
absence of Ca
, EMD 57033 (up to 50 µM)
had little or no effect on the fluorescence. This was expected since
there was little binding of EMD 57033 to cTnC under such conditions
(see Fig. 2and Fig. 3). At pCa 6.7, EMD 57033
caused a concentration-dependent decrease of bis-ANS fluorescence. At pCa 6.7, a majority of cTnC molecules should be in the form of
2 Ca
TnC since the
Ca
/Mg
sites should be largely
saturated by Ca
, while the
Ca
-specific site should be predominantly free of
bound Ca
. The effect of EMD 57033 at pCa 6.7
probably resulted from interference by EMD 57033 of bis-ANS binding to
the COOH-terminal hydrophobic patch of hcTnC. At pCa 4.3,
where all the three Ca
-binding sites of cTnC were
saturated by Ca
, the relationship between [EMD
57033] and the fluorescence change was very similar to that
observed at pCa 6.7. The double-reciprocal plots of the
titration data (1/
F versus 1/[EMD 57033]) at pCa 6.7 and 4.3 were linear. In principle, the EMD
57033-induced bis-ANS fluorescence changes at pCa 6.7 or 4.3 (Fig. 7, A and B) could result either from 1)
displacement of bis-ANS from hcTnC, or 2) a quenching of the
fluorescence of bis-ANS bound to hcTnC. The available data do not allow
us to distinguish between the two possibilities. If EMD 57033
competitively inhibits binding of bis-ANS to cTnC, the ratio of the
slope and the intercept on 1/
F axis for each of the plots
in Fig. 7B would be an apparent constant (K
) equal to the product of K
and a constant, (1 +
[bis-ANS]/K
) (where K
and K
stand
for the dissociation constants of hcTnC-EMD 57033 complex and
hcTnC-bis-ANS complex). On the other hand, if the effect of EMD 57033
shown in Fig. 7is due to quenching of the fluorescence of
bis-ANS molecules remaining bound to hcTnC, K
would be equal to K
. The values of K
at pCa 6.7 (43 µM) and
4.3 (50 µM), estimated from the data in Fig. 7,
were very similar. Thus, EMD 57033 bound as tightly to 2
Ca
hcTnC as to 3
Ca
hcTnC. The result offered additional support
for the notion that the induction of the EMD 57033-binding site
requires the Ca
/Mg
sites to be
occupied by Ca
.
Figure 7:
Titration of bis-ANS fluorescence by EMD
57033 in the presence of hcTnC. A, bis-ANS fluorescence as a
function of [EMD 57033]. The common conditions for the three
titrations were 5 µM bis-ANS, 5 µM hcTnC, 100
mM KCl, 100 mM MOPS (pH 7.0), 2 mM EGTA, 0.5
mM azide. The unique conditions for the titrations were,
respectively, 2 mM Mg (circles), pCa 6.7 (triangles), and pCa 4.3 (squares). B, the data at pCa 4.3 and 6.7
(from Fig. 6A) replotted as double-reciprocal plots.
The symbols are the same as in A. The lines represent
the least square fits.
We have demonstrated for the first time that the
Ca sensitizer EMD 57033 interacts directly with
isolated cardiac troponin C. The binding of EMD 57033 to recombinant
human cardiac TnC was determined using techniques which employed
intrinsic Tyr fluorescence of hcTnC ( Fig. 2and Fig. 4),
noncovalent hydrophobic probe bis-ANS ( Fig. 6and Fig. 7), and direct binding assay (Fig. 3). The data
obtained with the three different techniques were in good agreement,
showing that EMD 57033 binds hcTnC in a Ca
-dependent
and stereo-selective manner. hcTnC contains a EMD 57033-binding site
with a K
of approximately 40 µM.
The(-)-enantiomer, EMD 57439, was also found to bind hcTnC,
although with much lower affinity (K
160
µM). The chirality of the thiadiazinone derivatives
appears to be an important determinant of their affinity for hcTnC. The
thiadiazinone group, which contains the chiral center of EMD 57033 (Fig. 1), is likely involved in bonding with hcTnC.
The
effects of EMD 57033 on Tyr fluorescence of hcTnC and on the
interaction of bis-ANS with cTnC (Fig. 2, Fig. 4, Fig. 6, and Fig. 7) revealed a close link between
Ca binding to the Ca
/Mg
sites of hcTnC and EMD 57033 binding. The data indicate that the
EMD 57033-binding site is induced by Ca
binding to
the Ca
/Mg
sites and is thus likely
to reside in the COOH-terminal domain of hcTnC. The drug probably binds
directly to the COOH-terminal hydrophobic pocket, which is exposed upon
Ca
binding to the Ca
/Mg
sites. In view of the substantial quenching of Tyr fluorescence
of hcTnC induced by EMD 57033, one may further speculate that the
drug-binding site is in the vicinity of Tyr-111 and Tyr-150. hcTnC
contains 3 Tyr residues: 1 (Tyr-5) in the NH
-terminal
domain and 2 (Tyr-111 and Tyr-150) in the COOH-terminal
domain(31) . Tyr-111 and Tyr-150 are, respectively, in the 7th
position of the Ca
-binding loop III and the 10th
position of the Ca
-binding loop IV. In the
crystallographic structure of skeletal TnC whose
Ca
/Mg
sites were occupied by
Ca
, loops III and IV form a short antiparallel
-sheet(12, 13) . NMR examination of
Ca
-saturated cTnC in solution showed that the
-sheet is formed by Tyr-111-Ile-112-Asp-113 and
Arg-147-Ile-148-Asp-149(34) . Thus Tyr-111 and
Tyr-150 are closely positioned in space, and both may be in proximity
to the bound EMD 57033.
Although our data strongly suggest that EMD
57033, at the concentrations studied here, primarily binds to the
COOH-terminal domain of cTnC, it can not be ruled out that EMD 57033,
at higher concentrations, also binds to the NH-terminal
domain of cTnC. To resolve the issue, it would be necessary to
determine accurately the stoichiometry of EMD 57033 binding. In
principle, this may be achieved by conducting the dialysis-based direct
binding assay as described above (Fig. 3) over a much wider
range of drug concentrations.
We found that the binding of EMD 57033
to the COOH-terminal domain of cTnC occurred only when the
Ca/Mg
sites were occupied by
Ca
, but not when they were occupied by
Mg
. The observation is, to our knowledge, the first
demonstration that the Ca
-saturated and
Mg
-saturated COOH-terminal domain of cTnC exhibit
substantially different affinities for a non-peptide organic ligand.
The implication of the finding is that the region of the COOH-terminal
domain involved in anchoring EMD 57033 assumes different structures
depending on whether sites III and IV are occupied by Ca
or Mg
. The finding is in line with previous
studies, which revealed conformational differences between the 2
Ca
and 2 Mg
states of troponin C
using diverse techniques including circular dichroism(35) ,
proton magnetic resonance (36, 37, 38) ,
Fourier transform infrared spectroscopy(39) , and x-ray
solution scattering(40) . Using a sTnC mutant containing a
tryptophan in position 154, Chandra et al.(24) showed
that the structural difference between the 2 Ca
and 2
Mg
states may have functional significance. Although
several TnI inhibitory peptides bound to the TnC mutant in the presence
of either Ca
or Mg
, it was only in
the presence of Ca
that the binding of the peptides
induced significant changes of the Trp fluorescence of the TnC
mutant(24) . The findings suggest that the region of
COOH-terminal domain of TnC involved in interaction with the inhibitory
region of TnI is not the same structurally for the 2 Ca
and 2 Mg
states of TnC.
The structural
differences between the 2 Ca and 2 Mg
states of cTnC were also revealed, in the present study, by the
characteristics of the interaction between hcTnC and bis-ANS, a
hydrophobic probe ( Fig. 5and Fig. 6). While
Ca
binding to the Ca
/Mg
sites induced bis-ANS-binding site in the COOH-terminal domain of
cTnC, Mg
binding to the
Ca
/Mg
sites failed to do so. The
finding implies strongly that when Ca
/Mg
sites are filled by Mg
, the COOH-terminal
hydrophobic pocket is in a state substantially different from the state
it assumes when the Ca
/Mg
sites are
occupied by Ca
.
Assuming that the COOH-terminal
EMD 57033-binding site of cTnC remains accessible when cTnC is an
integrative part of the myofibrils in a myocyte, the occupancy of the
drug-binding site by EMD 57033 would be directly proportional to the
Ca occupancy of the
Ca
/Mg
sites of cTnC. Robertson et al. (41) have modeled the time courses of
Ca
exchange with cardiac troponin under ionic
conditions mimicking that in a beating myocyte. They showed that when a
quiescent cardiac myocyte, where the
[Mg
]
is set at 2.5 mM,
is subjected to a train of stimulations (75/min), each inducing a
transient rise of [Ca
]
from
10
M to 2
10
M, the Ca
-occupancy of the
Ca
/Mg
sites of cardiac troponin
would increase from the resting level (26%) to about 65% during the
first Ca
transient in the train due to displacement
of bound Mg
by Ca
. Under the
assumption that the Ca
off-rate of the
Ca
/Mg
sites is 0.33
s
, the analysis predicated that the Ca
occupancy of the Ca
/Mg
sites
would decrease only slightly before the onset of the next
Ca
transient. The Ca
occupancy
would increase further over several subsequent beats before a steady
state is reached. At the steady state, the Ca
occupancy of the Ca
/Mg
sites
would oscillate between 85% and 72%. Since the resting
[Ca
]
in cardiac myocytes has
been shown to be close to 10
M(42) , which is much higher than the value
(10
M) used by Robertson et al. (41) in their calculations, the steady state
Ca
occupancy of the
Ca
/Mg
sites in a beating heart is
certainly to be even higher. There is evidence that Ca
off-rate of the Ca
/Mg
sites
of cardiac troponin in an intact myofilament lattice may be many times
slower than 0.33 s
. Skinned cardiac or slow twitch
muscle fibers and myofibrils contain slowly exchanging
Ca
-binding sites with an off-rate constant of 2
10
s
(43) . These
sites were not found in skinned fast twitch skeletal muscle fibers.
However, replacement of the native sTnC with exogenous cTnC bestowed
the slowly exchanging sites on the skinned fast twitch fibers, implying
that these sites are most likely to reside in cTnC(44) . There
is good reason to believe that these sites are identical with the
Ca
/Mg
sites(44) . The
extremely slow Ca
off-rate means that, at a steady
state, the Ca
occupancy of the
Ca
/Mg
sites will be near 100% and
unchanged over the entire cardiac cycle, even when the heart rate is
abnormally slow, such as occurs during bradycardia resulting from
atria-ventricular block. Thus it seems safe to assume that the
COOH-terminal domain of most, if not all, of cTnC molecules in a
beating heart is always occupied by Ca
and, most
probably, capable of binding EMD 57033.
Our data (Fig. 6)
suggest that the binding of EMD 57033 is not accompanied by enhanced
Ca binding to the Ca
-regulatory
site of cTnC. This is in agreement with a report (6) that EMD
57033, up to 30 µM, did not alter the calcium binding
properties of cTnC in skinned fiber preparations. It is conceivable,
however, that EMD 57033 may modulate the Ca
-dependent
interaction of cTnC with the inhibitory region of cTnI. By doing so,
the drug may alter the cooperative properties of the thin filament and,
as result, increase the apparent Ca
sensitivity of
the contractile system.
EMD 57033 is known to exert detectable
positive inotropic effects on isolated cardiac myocytes at
concentrations as low as 1 µM(4, 6) . In
isolated atrium and papillary muscle of rat and ferret, EC of EMD 57033 for its positive inotropic effect ranged from 3.2 to
13 µM(7, 45) . In skinned cardiac muscle
fibers, less than 10 µM EMD 57033 induced significant
increase of the Ca
sensitivity of force
development(2) . At these low drug concentrations, only a very
small fraction of cTnC would be occupied by EMD 57033 if its affinity
for cTnC in intact myofilaments were the same as for pure cTnC in
solution. The rather large difference between the EC
values of EMD 57033 and K
(40 µM) of
cTnC-EMD 57033 complex makes it difficult to assign cTnC-EMD 57033
binding a principal role in mediating the biological effects of EMD
57033. One needs to consider the possibility that EMD 57033 exerts its
effects through binding to other sites in the contractile apparatus.
For instance, the NH
-terminal domain of the thin
filament-bound cTnC may bind EMD 57033. There is evidence that the
positive inotropic effects of EMD 57033 are, at least, in part mediated
by its direct actions on myosin cross-bridge/actin interactions. The
concept is supported by reports that EMD 57033 at concentrations below
10 µM stimulates deregulated actomyosin ATPase (6) , accelerates sliding of unregulated F-actin filaments on a
myosin ``lawn''(6) , and, enhances
Ca
-independent force production by TnI-depleted
skinned cardiac muscle fibers(46) . It is not yet known whether
EMD 57033 binds myosin, actin, or both.
At this juncture, it would be premature to regard cTnC-EMD 57033 interaction as merely a test tube phenomenon, since it is possible that cTnC in myofilaments may have a higher affinity for EMD 57033 than does cTnC in solutions. Further experiments are necessary to characterize the binding of EMD 57033 to cTnC complexed with other thin filament proteins and to cTnC in intact myofilaments.