Interaction of cardiotonic thiadiazinone derivatives with cardiac troponin C.

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 Ca2+ (100 μM) and little change of the fluorescence in the presence of Mg2+ (2 mM). Kd for the drug-hcTnC interaction in the presence of Ca2+, 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 Ca2+ dependence and stereo-selectivity of EMD 57033 binding were substantiated by a dialysis-based direct binding assay. EMD 57033 was found to interfere with Ca2+-dependent binding of hydrophobic probe 1,1′-bi-(4-anilino)naphthalene-5,5′-disulfonate (bis-ANS) to hcTnC. The relationships between [Ca2+] and Tyr fluorescence of hcTnC and between [Ca2+] and bis-ANS fluorescence in the presence of hcTnC were substantially altered by EMD 57033 in the range of [Ca2+] where Ca2+/Mg2+ sites of hcTnC were titrated by Ca2+. EMD 57033 was found to bind as tightly to 2 Ca2+·hcTnC as to 3 Ca2+·hcTnC. These observations were interpreted as indicating that a EMD 57033-binding site is induced by Ca2+ binding, but not Mg2+ binding, to the Ca2+/Mg2+ sites of hcTnC. The drug-binding site most likely resides in the carboxyl domain of hcTnC.

The molecular mechanism(s) underlying the Ca 2ϩ -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 2ϩ (4,7).
Troponin C (TnC) is the Ca 2ϩ 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 -10) and both belong to the superfamily of helix-loop-helix or EF-hand Ca 2ϩ -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 carboxylterminal domain. While all four are functional Ca 2ϩ -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 2ϩ specifically (K a Ϸ 5 ϫ 10 5 M Ϫ1 ) and are directly involved in Ca 2ϩ -regulation of muscle contraction (15)(16)(17)(18)(19). Sites III and IV bind Ca 2ϩ (K a Ϸ 2 ϫ 10 7 M Ϫ1 ) and Mg 2ϩ (K a Ϸ 5 ϫ 10 3 M Ϫ1 ) competitively, and appear to play primarily a structural role (15,16,20,21). Thus, the NH 2 -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 COOHterminal domain appears to contribute directly to Ca 2ϩ regulation by way of interacting with the inhibitory region of TnI and residues of TnI adjacent to the inhibitory region (see, e.g., Refs. [22][23][24]. Both domains of TnC contains a core of hydrophobic residues, which become more exposed to solvent upon Ca 2ϩ binding to the domains (25,26). The interactions of the hydrophobic patches with TnI are believed to be important for transmission of the Ca 2ϩ 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 2ϩ binding properties (14,(27)(28)(29)(30).

MATERIALS AND METHODS
Expression and Purification of Recombinant hcTnC-Adult human cardiac cDNA (a gift from Dr. M. Jacobson and C. Salvatore) was subjected to polymerase chain reaction using Taq DNA polymerase (Perkin Elmer) and primers TnC5 (5Ј-CCTGGCCATGGATGACATC-TACAAGGC-3Ј) and TnC3 (3Ј-AGTGGGTCTCGACGGATACGGC-CTAGGCC-5Ј). TnC5 contained the first 20 bases (underlined) of the coding sequence of human cTnC cDNA (31) and 7 additional 5Ј nucleotides. The bold bases in TnC5 constitute a recognition site for NcoI. TnC3 contained a 21-base sequence (underlined) complementary to a stretch of the 3Ј-untranslated region of hcTnC cDNA and 8 additional 5Ј nucleotides with a BamHI recognition site (in bold). The polymerase chain reaction products were purified using low melting gel and Gene Clean II (Bio 101), digested with NcoI and BamHI, and ligated into pET3d vector (Novagene) linearized with NcoI and BamHI. The ligation reaction was used to transform competent cells of Escherichia coli strain BL21(DE3) (Novagene). The inserts in plasmids purified from ampicillin-resistant colonies were sequenced by the dideoxy-chain termination method. A plasmid construct containing the correct complete coding sequence for human cTnC was used to direct overexpression of cTnC in E. coli strain BL21(DE3) according to a procedure recommended by Novagene. Recombinant hcTnC was purified using a modified version of the method of Putkey et al. (19). Bacteria from 1 liter of culture were harvested and washed once in distilled water, and then resuspended in 200 ml of 6 M urea, 1 mM EDTA, 50 mM Tris (pH 8.0), 1 mM dithiothreitol (DTT). The suspension was sonicated on ice for 5 min and then centrifuged for 60 min at 40000 rpm in a Beckman ultracentrifuge with a Ti-45 rotor. The supernatant was loaded directly onto an anion exchange column (DE-52, Whatman) pre-equilibrated with the same buffer. The column was eluted with a KCl gradient of 0 -0.5 M made in the above buffer. A crude cTnC peak was eluted at a conductivity of 9 -10 millisiemens/cm. Fractions enriched in cTnC were pooled, dialyzed against 1 M NaCl, 5 mM CaCl 2 , 0.5 mM DTT, 50 mM Tris-HCl (pH 7.5), and then loaded on a phenyl-Sepharose (Pharmacia Biotech Inc.) column equilibrated with 1 M NaCl, 0.1 mM CaCl 2 , 0.5 mM DTT, 50 mM Tris-HCl (pH 7.5). Subsequent to exhaustive wash of the column bed with the above buffer less CaCl 2 , pure cTnC was eluted with a buffer containing 5 mM EDTA, 1 M NaCl, 0.5 mM DTT, and 50 mM Tris-HCl (pH 7.0). About 100 mg of purified hcTnC was obtained from each liter of bacterial culture. The purified recombinant hcTnC was indistinguishable from native bovine cTnC in terms of Ca 2ϩ binding properties and the ability to regulate bovine cardiac actomyosin ATPase.
Fluorescence Measurements-Steady state fluorescence intensities and spectra were acquired using a Fluoromax spectrofluorometer (Spex Industry Inc., Edison, NJ) equipped with a water-jacketed cuvette holder and a magnetic stirrer. Excitation and emission wave lengths were 280 and 305 nm for tyrosine, and 400 and 512 nm for bis-ANS. EMD 57033 and EMD 57439 absorbed light in a wavelength range overlapping parts of the absorption and emission spectra of tyrosine, causing significant inner filter effects when the intrinsic Tyr fluorescence of hcTnC was measured. The inner filter effects were corrected, according to Birdsall et al. (32), by measuring the effect of the compounds on the fluorescence of free tyrosine under the same instrument settings.
Direct Assay of Binding of EMD 57033 or EMD 57439 to cTnC-0.2 ml of 32 M hcTnC was dialyzed for 8 h at 23°C against 15 ml of buffer containing 100 mM KCl, 60 mM MOPS (pH 7.0), 1 mM EGTA, 2 mM MgCl 2 , 2 mM NaN 3 , and drug (EMD 57033 or EMD 57439) with or without 1 mM CaCl 2 . At the end of the dialysis, the drug concentrations in the dialysis buffer and the dialyzed sample were determined from UV absorption as described in the following paragraph. The concentrations of hcTnC in the dialyzed samples were determined by the method of Bradford (33) using hcTnC as standard. The stoichiometry of the binding was calculated as ( Drugs-EMD 57033 and EMD 57439 were provided kindly by E. Merck. Concentrated stock solutions of the compounds were prepared by dissolving the compounds in 90% propylene glycol and 10% dimethyl sulfoxide. The concentration of the drugs was determined by UV ab-sorption using a molar extinction coefficient ⑀ 320 nm 1 cm ϭ 20,000 for both EMD 57033 and EMD 57439.

Binding of EMD 57033 and EMD 57439 to hcTnC Detected from Their Effects on the Intrinsic Fluorescence of hcTnC-
The chemical structures of EMD 57033, the (ϩ)-enantiomer, and EMD 57439, the (Ϫ)-enantiomer, are shown in Fig. 1. When excited at 280 nm, the fluorescence of hcTnC, which does not contain tryptophan, is attributable to tyrosine residues. Fig. 2A shows the effect of EMD 57033 and 57439 on the Tyr fluorescence of hcTnC. In the presence of 2 mM Mg 2ϩ and 100 M free Ca 2ϩ , both compounds caused concentration-dependent quenching of the tyrosine fluorescence. However, at the same concentration, EMD 57033 consistently caused significantly more quenching than did EMD 57439. For example, 50 M EMD 57033 induced approximately 40% quenching of the fluorescence, while 50 M EMD 57439 caused roughly 20% quenching. The double reciprocal plots (1/⌬F versus 1/[EMD 57033] or 1/[EMD 57439]) of the data in the presence of Ca 2ϩ were linear (Fig. 2B), suggesting that the binding of EMD 57033 as well as 57439 to hcTnC was non-cooperative. The K d for the interaction of hcTnC with EMD 57033 and 57439, estimated from the reciprocal plots in Fig. 2B, are approximately 40 and 160 M, respectively. In other words, EMD 57033, the (ϩ)-enantiomer, bound hcTnC 4 times more strongly than the (Ϫ)-enantiomer, EMD 57439. It is also evident from Fig. 2 that the interaction of the drugs with hcTnC was Ca 2ϩsensitive. In the presence of 2 mM Mg 2ϩ and no Ca 2ϩ , EMD 57033 and 57439 had little or no effect on hcTnC fluorescence. Up to 60 M EMD 57439 had no detectable effect on hcTnC fluorescence, while at the highest [EMD 57033] tested (50 M), only a very small effect (a 5% quenching) was observed. Thus, it is clear that EMD 57033 binds to cTnC in a Ca 2ϩ -dependent and stereo-selective manner.
Direct Measurement of the Binding of EMD 57033 and EMD 57439 to hcTnC- Fig. 3 shows the result of an experiment in which the binding of EMD 57033 and EMD 57439 to cTnC was measured directly by equilibrium dialysis. A small number of determinations were made because the drugs were available in limited quantities. In the presence of 100 M free Ca 2ϩ , hcTnC bound considerably more EMD 57033 than EMD 57439, given the same free M EMD 57439. This finding was consistent with the notion that EMD 57033 has higher affinity for hcTnC than does EMD 57439. Based on the dissociation constants of EMD 57033 (40 M) and EMD 57439 (160 M) estimated from fluorescence titrations (Fig. 2), hcTnC would be expected to bind 0.53 mol of EMD 57033/mol of hcTnC at 46 M EMD 57033 and 0.23 mol of EMD 57439/mol of hcTnC at 48 M EMD 57439, assuming that the protein contains a single drug-binding site. Clearly, the equilibrium dialysis data (Fig. 3) are in good agreement with these predications. Furthermore, the data in Fig. 3 confirm that the binding of EMD 57033 and EMD 57439 to hcTnC is Ca 2ϩ -sensitive. In the presence of 39 M EMD 57439, cTnC bound 0.06 mol of EMD 57439/mol of cTnC at pCa 9, compared with 0.23 mol of EMD 57439/mol of cTnC at pCa 4. In the presence of 37 M EMD 57033, the amount of EMD 57033 bound to hcTnC at pCa 9 (0.09 mol of EMD 57033/mol of cTnC) was much less than the level (approximately 0.4 mol of EMD 57033/mol of cTnC) to be expected of Ca 2ϩ -saturated cTnC, based on extrapolation from the data obtained at somewhat higher [EMD 57033] (Fig. 3).
Ca 2ϩ Titration of Tyr Fluorescence of hcTnC in the Presence of EMD 57033-To determine which of the two classes of Ca 2ϩbinding sites of hcTnC was directly linked to the apparent Ca 2ϩ -dependence of the EMD 57033-cTnC interaction, we studied the relationship between [Ca 2ϩ ] and Tyr fluorescence of cTnC in the presence and absence of EMD 57033 (Fig. 4). In the absence of EMD 57033, the Tyr fluorescence intensity increased when [Ca 2ϩ ] was raised gradually from pCa 7.5 to 6, a range where sites III and IV (Ca 2ϩ /Mg 2ϩ sites) were titrated. The fluorescence changed little in the range of pCa 6 to 4, where the Ca 2ϩ -specific site II was titrated. The Ca 2ϩ -induced enhancement of the Tyr fluorescence appeared to result primarily from Ca 2ϩ binding to the Ca 2ϩ /Mg 2ϩ sites of hcTnC. The pCa fluorescence relation in the absence of EMD 57033 was fitted well with a single-term Hill equation, in which pCa 50 ϭ 6.87 and n ϭ 1.8. In the presence of 42 M EMD 57033, the pCa-Tyr fluorescence relation was strikingly different. The intensity of the fluorescence decreased with increasing [Ca 2ϩ ] between pCa 7.5 and 6, and was essentially unchanged between pCa 6 and 4. The relation was fitted well with a Hill equation in which pCa 50 ϭ 6.82 and n ϭ 2.5. The following scenario appears to offer the most straightforward interpretation for the observation. Ca 2ϩ binding to the Ca 2ϩ /Mg 2ϩ sites induced a EMD 57033-binding site in the COOH-terminal domain of hcTnC; the subsequent binding of EMD 57033 to hcTnC induced significant fluorescence quenching, which more than canceled out the fluorescence enhancement caused by Ca 2ϩ binding to the Ca 2ϩ /Mg 2ϩ sites. If instead, Ca 2ϩ -binding to the Ca 2ϩ -specific site were necessary for EMD 57033 binding, the presence of EMD 57033 should not have significantly altered the rising phase (from pCa 7.5 to 6) of the pCa-Tyr fluorescence relation, but should have caused Ca 2ϩ -dependent quenching of the Tyr fluorescence between pCa 6 and 4. Such predications were not borne out in Fig. 4.
Effect of EMD 57033 on the Interaction between hcTnC and Bis-ANS-Bis-ANS, a noncovalent hydrophobic fluorescence probe, has been used widely to study protein conformation. Shown in Fig. 6 are the results of an experiment in which the fluorescence of bis-ANS was titrated by Ca 2ϩ in the presence of hcTnC. The Ca 2ϩ 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 2ϩ binding to the Ca 2ϩ /Mg 2ϩ 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 2 -terminal domain, are induced by Ca 2ϩ binding to the Ca 2ϩ -specific site. The presence of 42 M 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 2ϩ affinities of the Ca 2ϩ /Mg 2ϩ -and Ca 2ϩ -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). Fig. 7 shows 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 2ϩ and absence of Ca 2ϩ , EMD 57033 (up to 50 M) 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 Figs. 2 and 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 2ϩ ⅐TnC since the Ca 2ϩ /Mg 2ϩ sites should be largely saturated by Ca 2ϩ , while the Ca 2ϩ -specific site should be predominantly free of bound Ca 2ϩ . 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 2ϩbinding sites of cTnC were saturated by Ca 2ϩ , 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 app ) equal to the product of K EMD 57033 and a constant, (1 ϩ [bis-ANS]/K bis-ANS ) (where K EMD 57033 and K bis-ANS 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. 7 is due to quenching of the fluorescence of bis-ANS molecules remaining bound to hcTnC, K app would be equal to K EMD 57033 . The values of K app at pCa 6.7 (43 M) and 4.3 (50 M), estimated from the data in Fig. 7, were very similar. Thus, EMD 57033 bound as tightly to 2 Ca 2ϩ ⅐hcTnC as to 3 Ca 2ϩ ⅐hcTnC. The result offered additional support for the notion that the induction of the EMD 57033-binding site requires the Ca 2ϩ /Mg 2ϩ sites to be occupied by Ca 2ϩ . DISCUSSION We have demonstrated for the first time that the Ca 2ϩ 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 (Figs. 2 and 4), noncovalent hydrophobic probe bis-ANS (Figs. 6 and 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 2ϩ -dependent and stereo-selective manner. hcTnC contains a EMD 57033-binding site with a K d of approximately 40 M. The (Ϫ)-enantiomer, EMD 57439, was also found to bind hcTnC, although with much lower affinity (K d Ϸ 160 M). 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 (Figs. 2, 4, 6, and 7) revealed a close link between Ca 2ϩ binding to the Ca 2ϩ /Mg 2ϩ sites of hcTnC and EMD 57033 binding. The data indicate that the EMD 57033-binding site is induced by Ca 2ϩ binding to the Ca 2ϩ /Mg 2ϩ sites and is thus likely to reside in the COOHterminal domain of hcTnC. The drug probably binds directly to the COOH-terminal hydrophobic pocket, which is exposed upon Ca 2ϩ binding to the Ca 2ϩ /Mg 2ϩ 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 2 -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 2ϩbinding loop III and the 10th position of the Ca 2ϩ -binding loop IV. In the crystallographic structure of skeletal TnC whose Ca 2ϩ /Mg 2ϩ sites were occupied by Ca 2ϩ , loops III and IV form a short antiparallel ␤-sheet (12,13). NMR examination of Ca 2ϩ -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 COOHterminal domain of cTnC, it can not be ruled out that EMD 57033, at higher concentrations, also binds to the NH 2 -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 COOHterminal domain of cTnC occurred only when the Ca 2ϩ /Mg 2ϩ sites were occupied by Ca 2ϩ , but not when they were occupied by Mg 2ϩ . The observation is, to our knowledge, the first demonstration that the Ca 2ϩ -saturated and Mg 2ϩ -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 2ϩ or Mg 2ϩ . The finding is in line with previous studies, which revealed conformational differences between the 2 Ca 2ϩ and 2 Mg 2ϩ states of troponin C using diverse techniques including circular dichroism (35), proton magnetic resonance (36 -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 2ϩ and 2 Mg 2ϩ states may have functional significance. Although several TnI inhib- itory peptides bound to the TnC mutant in the presence of either Ca 2ϩ or Mg 2ϩ , it was only in the presence of Ca 2ϩ 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 2ϩ and 2 Mg 2ϩ states of TnC.
The structural differences between the 2 Ca 2ϩ and 2 Mg 2ϩ states of cTnC were also revealed, in the present study, by the characteristics of the interaction between hcTnC and bis-ANS, a hydrophobic probe (Figs. 5 and 6). While Ca 2ϩ binding to the Ca 2ϩ /Mg 2ϩ sites induced bis-ANS-binding site in the COOHterminal domain of cTnC, Mg 2ϩ binding to the Ca 2ϩ /Mg 2ϩ sites failed to do so. The finding implies strongly that when Ca 2ϩ / Mg 2ϩ sites are filled by Mg 2ϩ , the COOH-terminal hydrophobic pocket is in a state substantially different from the state it assumes when the Ca 2ϩ /Mg 2ϩ sites are occupied by Ca 2ϩ .
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 2ϩ occupancy of the Ca 2ϩ /Mg 2ϩ sites of cTnC. Robertson et al. (41) have modeled the time courses of Ca 2ϩ exchange with cardiac troponin under ionic conditions mimicking that in a beating myocyte. They showed that when a quiescent cardiac myocyte, where the [Mg 2ϩ ] i is set at 2.5 mM, is subjected to a train of stimulations (75/min), each inducing a transient rise of [Ca 2ϩ ] i from 10 Ϫ8 M to 2 ϫ 10 Ϫ6 M, the Ca 2ϩ -occupancy of the Ca 2ϩ / Mg 2ϩ sites of cardiac troponin would increase from the resting level (26%) to about 65% during the first Ca 2ϩ transient in the train due to displacement of bound Mg 2ϩ by Ca 2ϩ . Under the assumption that the Ca 2ϩ off-rate of the Ca 2ϩ /Mg 2ϩ sites is 0.33 s Ϫ1 , the analysis predicated that the Ca 2ϩ occupancy of the Ca 2ϩ /Mg 2ϩ sites would decrease only slightly before the onset of the next Ca 2ϩ transient. The Ca 2ϩ occupancy would increase further over several subsequent beats before a steady state is reached. At the steady state, the Ca 2ϩ occupancy of the Ca 2ϩ /Mg 2ϩ sites would oscillate between 85% and 72%. Since the resting [Ca 2ϩ ] i in cardiac myocytes has been shown to be close to 10 Ϫ7 M (42), which is much higher than the value (10 Ϫ8 M) used by Robertson et al. (41) in their calculations, the steady state Ca 2ϩ occupancy of the Ca 2ϩ /Mg 2ϩ sites in a beating heart is certainly to be even higher. There is evidence that Ca 2ϩ off-rate of the Ca 2ϩ /Mg 2ϩ sites of cardiac troponin in an intact myofilament lattice may be many times slower than 0.33 s Ϫ1 . Skinned cardiac or slow twitch muscle fibers and myofibrils contain slowly exchanging Ca 2ϩ -binding sites with an off-rate constant of 2 ϫ 10 Ϫ4 s Ϫ1 (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 2ϩ /Mg 2ϩ sites (44). The extremely slow Ca 2ϩ offrate means that, at a steady state, the Ca 2ϩ occupancy of the Ca 2ϩ /Mg 2ϩ 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 atriaventricular 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 2ϩ 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 2ϩ binding to the Ca 2ϩ -regulatory site of cTnC. This is in agreement with a report (6) that EMD 57033, up to 30 M, did not alter the calcium binding properties of cTnC in skinned fiber preparations. It is conceivable, however, that EMD 57033 may modulate the Ca 2ϩ -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 2ϩ 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 M (4, 6). In isolated atrium and papillary muscle of rat and ferret, EC 50 of EMD 57033 for its positive inotropic effect ranged from 3.2 to 13 M (7, 45). In skinned cardiac muscle fibers, less than 10 M EMD 57033 induced significant increase of the Ca 2ϩ 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 50 values of EMD 57033 and K d (40 M) 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 2 -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 M stimulates deregulated actomyosin ATPase (6), accelerates sliding of unregulated F-actin filaments on a myosin "lawn" (6), and, enhances Ca 2ϩ -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.