Introduction

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2,3-Butanedione monoxime (BDM) has been characterized as a nucleophilic oxime with a phosphatase-like activity (Coulombe et al., 1990). On the level of the contractile apparatus, BDM exerts a Ca²⁺-desensitizing effect on cardiac as well as on skeletal skinned muscle fibers (Fryer et al., 1988; Gwathmey et al., 1991). Accordingly, BDM has been shown to inhibit the actomyosin ATPase (Higuchi and Takemori, 1989; McKillop et al., 1994). From experiments demonstrating that BDM decreases contractile force of skinned fibers at maximal calcium activation, but less than immediate stiffness, it was concluded that BDM increases the population of strongly attached "preforce-generating" cross-bridges, possibly by slowing and inhibiting the inorganic phosphate (P_i) release step and stabilizing the actomyosin-ADP-P_i intermediate cross-bridge state (actomyosin-ADP-P_i) (Zhao and Kawai, 1994). Experiments on frog muscle have shown that BDM decreases the rate of cross-bridge attachment and the force per cross-bridge (Bagni et al., 1992). From simultaneous measurements of force and myosin ATPase activity in skinned cardiac rat trabeculae, it was concluded that BDM not only affects cross-bridge formation but also causes an increase in the apparent rate of cross-bridge detachment (Ebus and Stienen, 1996).

The thiadiazinone derivative EMD 53998 (EMD, 5-[1-(3,4-dimethoxybenzoyl)-1,2,3,4-tetrahydro-6-quinolyl]-6-methyl-3,6-dihydro-2H-1,3,4-thiadiazin-2-one) and its (+)-enantiomer EMD 57033 are classified as Ca²⁺ sensitizers (Beier et al., 1991; Solaro et al., 1993). Both compounds seem to directly interact with the actomyosin contractile system (Strauss et al., 1992a; Solaro et al., 1993). EMD 53998 increases the rate of cross-bridge attachment in the strongly bound force-generating state (Simnett et al., 1993a; Arner et al., 1995), whereas the cross-bridge detachment rate (g_app), is not affected (Simnet et al., 1993b; Strauss et al., 1994). The transition into the force-generating state that is accelerated by EMD 53998 is associated with the release of P_i from the actomyosin-ADP-Pi ternary complex (Barth et al., 1995). Because EMD 53998 also antagonizes the inhibitory effect of inorganic phosphate in skinned fibers (Strauss et al., 1992a), it seems likely that EMD 53998 accelerates the P_i release step, perhaps by lowering the P_i affinity for myosin (Strauss et al., 1994). Therefore, its action should be antagonistic to that of BDM.

CGP 48506 (GCP), the (+)-entantiomer of the racemic mixture 1,5-benzodiazocine derivative 5-methyl-6-phenyl-1,3,5,6-tetrahydro-3,6-methano-1,5-benzodiazocine-2,4-dione, has been shown to shift the force-pCa curve to the left in porcine skinned right ventricular muscle fibers (Palmer et al., 1996). This action was paralleled by an increase in the ATPase activity. From these experiments it was concluded that CGP 48506 does not alter the apparent detachment rate of the cross-bridges (Palmer et al., 1996).

Ca²⁺ sensitizers may be advantageous for the treatment in human heart failure because they increase force of contraction without increasing the intracellular Ca²⁺ transient or energy expenditure. The kind of action by which EMD 57033 or CGP 48506 influence cross-bridge interaction in human failing myocardium is not fully understood. Thus, by studying the interference of EMD 57033 and CGP 48506 on the known inhibitory action of BDM on force generation in troponin I-depleted fibers from left ventricular human myocardium, the molecular mechanisms of EMD 57033 or CGP 48506 should be elucidated. To analyze the influence on cross-bridge kinetics, tension cost, i.e., the ratio of ATPase activity and tension development, was studied after application of EMD 57033, CGP 48506, and BDM.

	Experimental Procedures

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Myocardial Tissue. Experiments were performed on human left ventricular myocardium. Tissue was obtained during cardiac transplantation (n = 19, 8 female, 11 male; age 44 ± 4 years). Patients suffered from heart failure clinically classified as New York Heart Association class IV on the basis of clinical symptoms and signs as judged by the attending cardiologist shortly before operation. All patients gave written informed consent before surgery. Medical therapy consisted of diuretics, nitrates, angiotensin-converting enzyme inhibitors, and cardiac glycosides. Patients receiving catecholamines, -adrenoceptor or Ca²⁺ antagonists were withdrawn from the study. Drugs used for general anesthesia were flunitrazepam and pancuroniumbromide with isoflurane. Cardiac surgery was performed on cardiopulmonary bypass patients with cardioplegic arrest during hypothermia. The cardioplegic solution (a modified Bretschneider solution) contained 15 mM NaCl, 9 mM KCI, 4 mM MgCl₂, 180 mM histidine, 2 mM tryptophan, 30 mM mannitol, and 1 mM potassium dihydrogen oxoglutarate. The study was approved by the local ethics committee.

Skinned Fibers. Left ventricular muscle fibers were prepared according to previously published procedures (Schwinger et al., 1994). Briefly, the fiber bundles (diameter <0.2 mm) were dissected and permeabilized at 4°C for 20 h in a solution containing 50% (v/v) glycerol, 1% Triton X, and in 10 mM NaN₃, 5 mM ATP, 5 mM MgCl₂, 4 mM EGTA, 2 mM 1,4-dithioerythritol, and 20 mM imidazole (pH 7.0). Afterward the fibers were stored in a similar solution but without Triton X at 20°C.

Troponin I Washout Experiments. The relaxation solution contained 20 mM imidazole, 10 mM ATP, 12.5 mM MgCl₂, 10 mM creatine phosphate, 5 mM NaN₃, 5 mM EGTA, 1 mM 1,4-dithioerythritol, and 350 U/ml creatine kinase (pH 7.0). The ionic strength was adjusted to 130 mM by adding KCl. In the contraction solution EGTA was replaced by 5 mM calcium EGTA. The pCa (log[Ca²⁺]) was varied by mixing contraction and relaxation solution as appropriate and pCa values were calculated according to Fabiato and Fabiato (1979), using the stability constants given by Fabiato (1981). For force measurements, fibers were mounted isometrically between a force transducer and a rigid post attached to a micrometer for length adjustment (Scientific Instruments, Heidelberg, Germany). In relaxation solution, fiber length was adjusted to an extent where resting tension was just threshold (slack position). An initial test contraction-relaxation cycle was performed on all fibers to ensure Ca²⁺ dependence of force in each preparation. After this procedure, fibers were again maximally contracted (pCa = 4.5) to establish a control level of maximum isometric tension. After a tension plateau had been reached, fibers were incubated for 10 min in relaxing solution containing 10 mM sodium vanadate. After incubation with vanadate, fibers were transferred to fresh relaxing solution to remove the vanadate. After washing out the vanadate, fibers were no longer Ca²⁺ regulated, i.e., they contracted maximally even at 10 nM free Ca²⁺. Experiments were performed according to Strauss et al. (1992a). Contracted fibers were then exposed to increasing concentrations of BDM (5-50 mM), and solutions containing BDM (5-50 mM) with and without the addition of EMD 57033 (10 µM), respectively, CGP 48506 (10 µM).

Immunocytochemistry in Skinned Fibers of Human Cardiac Muscle and Measurement of Sarcomere Length. Skinned fibers of failing hearts, prepared as described above, were used for immunocytochemical labeling of Z-lines by -actinin staining. After three washes in 0.1 M PBS buffer, the skinned fiber preparations were incubated in a 1:800 dilution of mouse anti-rat -actinin antibody for 1 h at room temperature, followed by treatment with a secondary biotinylated goat anti-mouse antibody (1:400) for 1 h and subsequent Cy3-labeled extravidin (1:600) for 1 h (Ji et al., 1999). Then the skinned fibers were washed with 0.1 M Tris-buffered saline and stored at 20°C until the sarcomeric length measurement.

Force and ATPase Activity Measurements. In a second type of experiment, force and ATPase activity were simultaneously measured (Güth and Wojciechowski, 1986; experimental setup, Scientific Instruments). Relaxation solution contained 20 mM imidazole, 10 mM ATP, 5 mM NaN₃, 5 mM EGTA, 12.5 mM MgCl₂, and 0.2 mM P₁,P₅-di(adenosine 5') pentaphosphate. The contraction solution contained calcium EGTA (5 mM) instead of EGTA. The ATP concentration was stabilized with an ATP-regenerating system, phosphoenolpyruvate (12.5 mM), and pyruvate kinase (100 U/ml). ATPase activity and force were simultaneously measured using a linked NADH fluorescence assay (0.6 mM NADH, 140 U lactate dehydrogenase). The relaxation solution contained 20 mM imidazole, 10 mM Na₂ATP, 5 mM NaN₃, 5 mM EGTA, 12.5 mM MgCl₂, 5 mM phospho(enol)-pyruvate, 0.6 mM NADH, and 0.2 mM P₁,P₅-di(adenosine 5') pentaphosphate (myokinase inhibitor), 25 mM cyclopiazonic acid, together with 100 units/ml pyruvate kinase and 125 units/ml lactate dehydrogenase. The contraction solution contained calcium EGTA (5 mM) instead of EGTA. Both solutions were mixed by a gradient mixer so that Ca²⁺ was successively increased every 15 s. Free Ca²⁺ concentration was determined by calculator programs designed for experiments in skinned muscle cells by Fabiato and Fabiato (1979). Measurement of developed tension and myosin ATPase activity started 3 s after the solution was exchanged. Developed tension and myosin ATPase activity had reached a stable plateau at that time. Experiments were performed as described previously (Brixius and Schwinger, 2000). All experiments were performed in slack position. By subtracting the basal ATPase activity obtained in the relaxation solution from the measured ATPase activity, the suprabasal ATP-splitting rate was obtained. The ratio of suprabasal ATPase activity and force was assumed as a measure for the "tension cost".

Materials. EMD 57033 was generously provided by Merck (Darmstadt, Germany). CGP 48506 was a gift from Ciba-Geigy (Wehr, Germany). All other chemicals were of analytical grade or the best grade commercially available. Mouse anti-rat -actinin antibody and Cy3-labeled extravidin were obtained from Sigma (Deisenhofen, Germany), and biotinylated goat anti-mouse antibody was from Dako Corp. (Carpinkia, Canada).

Statistics. All values are means ± S.E.M. unless otherwise noted. Student's t test or paired t test were used to test significance. P values of <.05 were accepted as significant. pCa-force as well as pCa-myosin ATPase activity relationships were fitted by a modified Hill equation (Hill, 1910) as follows: Y = [Ca²⁺]^H/([pCa₅₀]^H + [Ca²⁺]^H), where Y is the fractional force, or myosin-ATPase activity, pCa is the Ca²⁺ concentration giving half-maximal activation (inhibition), and H is an index of cooperativity (Hill coefficient). The concentration needed for half-maximal Ca²⁺ activation of tension development or myosin ATPase activity (EC₅₀ for Ca²⁺), the concentration of BDM needed to achieve a half-maximal decline of tension development (IC₅₀ for BDM), all Hill-coefficients, and the tension cost (ratio of ATPase activity and tension development) were analyzed by GraphPad Prism (GraphPad, San Diego, CA).

	Results

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BDM, EMD 57033, CGP 48506, and Tension Cost in Human Myocardium. To investigate whether EMD or CGP may interact with the BDM-mediated influence on the cross-bridge detachment rate in human myocardium, Ca²⁺-activated tension development and myosin-ATPase activity were simultaneously studied in the presence of BDM (10 mM), EMD (10 µM), and CGP (10 µM). All fibers were set in "slack position", i.e., fiber length was adjusted to an extent where resting tension was just threshold. In this situation a basal ATPase activity was measured of 78.7 ± 12.2 µmol ADP/s. Application of BDM reduced basal ATPase activity (39.3 ± 4.7 µmol ADP/s). EMD (82.2 ± 5.6 µM ADP/s) or CGP (64.5 ± 6.8 µM ADP/s) did not influence basal myosin ATPase activity. The results obtained for the maximal Ca²⁺-activated force (F_max) are summarized in Fig. 1A. Under basal conditions (without drug application), F_max was 25.0 ± 1.0 mN/mm² in Triton X-skinned fiber preparations of left ventricular human failing myocardium. BDM (10 mM) depressed F_max (8.0 ± 1.2 mN/mm²), and the Ca²⁺ sensitizer EMD increased F_max (31.4 ± 2.9 mN/mm²). Figure 1B shows the Ca²⁺-force curves under basal conditions as well as after application of BDM (10 mM), EMD (10 µM), and CGP (10 µM). Under basal conditions, the concentration of Ca²⁺ needed to achieve half-maximal force (EC₅₀ Ca²⁺) was 1.74 µM (95% CI = 1.10-2.38 µM). The negative inotropic effect of BDM was accompanied by a Ca²⁺ desensitization of the myofilaments as indicated by an increased Ca²⁺ concentration needed to achieve half-maximal force [EC₅₀ Ca²⁺ + BDM (10 mM) = 5.24 µM, CI = 4.22-6.25 µM, P < .05], i.e., after application of BDM more Ca²⁺ was necessary for the same relative tension development (Fig. 1B). The Hill coefficient of the force-Ca²⁺ curve (H_F) was increased after the application of BDM [control: 1.8 ± 0.1 + BDM (10 mM) = 2.4 ± 0.1, P < .05]. The two Ca²⁺ sensitizers reduced the Ca²⁺ concentration needed for half-maximal force [EC₅₀ Ca²⁺ + EMD (10 µM) = 0.98 µM, CI = 0.76-1.67 µM, P < .05; EC₅₀ Ca²⁺ + CGP 48506 (10 µM) = 0.70 µM, CI = 0.60-0.79 µM, P < .05], so that, in comparison to the control, less Ca²⁺ was necessary to increase tension development in the presence of EMD or CGP (Fig. 1B). CGP significantly increased H_F (2.8 ± 0.2).

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Influence of BDM (10 mM, n = 6), EMD 57033 (10 µM, n = 6), and CGP 48506 (10 µM, n = 11) on maximum Ca2+-dependent force (A) and Ca2+ sensitivity of force (B) of skinned fiber preparations from human failing myocardium (control, n = 7).

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Influence of BDM (10 mM, n = 6), EMD 57033 (10 µM, n = 6), and CGP 48506 (10 µM, n = 11) on maximum Ca2+-activated myosin ATPase activity (A) and Ca2+ sensitivity of myosin ATPase activity (B) of skinned fiber preparations of human failing myocardium (control, n = 7).

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Influence of BDM (10 mM, n = 6), EMD 57033 (10 µM, n = 6), and CGP 48506 (10 µM, n = 11) on tension cost of skinned fiber preparations of human failing myocardium (control, n = 7) at increasing Ca2+ concentrations (0.01-32 µM free Ca2+). A, typical recordings from single experiments. B, summary of the results. The lines were obtained by a linear regression to all individual data points of each experimental group.

Force Development of Troponin I-Depleted Fibers. Figure 4 presents an original tracing of the force development before and after troponin I depletion by vanadate extraction in skinned fiber preparations of human myocardium. Initially, all fibers demonstrated a Ca²⁺-dependent contraction. After incubation in a solution of 10 mM vanadate and transfer to normal relaxation solution without vanadate, a force development was observed, which was 93% of the F_max measured before the vanadate treatment. Under the same conditions, myosin ATPase activity was 93% of the ATP_max measured before vanadate treatment. No additional Ca²⁺-dependent force or myosin ATPase activity was obtained when the free Ca²⁺ concentration was increased in the solution. Thus, Ca²⁺-dependent cross-bridge-interaction was no longer present in these fibers.

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Original tracing of the isometric force development during the troponin I washout experiments and after application of BDM in skinned fiber preparations of human failing myocardium. After testing the Ca2+ dependence with an initial maximal contraction (free Ca2+ 11.5 µM), followed by a subsequent relaxation (free Ca2+ 10 nM), the skinned fibers were incubated in vanadate (10 mM) to extract troponin I. After vanadate (10 mM) treatment, Ca2+-dependent contraction was lost indicated by a maximal contraction of the fiber even at very low Ca2+ concentrations (10 nM). Contracted fibers were then immersed into solutions with increasing concentrations of BDM (5-50 mM), resulting in a decrease of the developed tension.

Effect of BDM, EMD 57033, and CGP 48506 on Ca²⁺-Independent Tension in Troponin I-Depleted Fibers. To investigate whether the negative inotropic effect of BDM can be directly attributed to an interaction of BDM with the cross-bridge cycle in human myocardium, the concentration-dependent effect of BDM (5-50 mM) was studied in troponin I-depleted, Ca²⁺-unregulated skinned fibers. The results are summarized in Fig. 5. Contractile force of the vanadate-treated, troponin I-depleted cardiac fibers was concentration dependently decreased by BDM. The concentration of BDM needed to achieve a 50% decrease in basal force (IC₅₀) was 7.22 mM (CI = 4.41-10.0 mM). The negative inotropic action of BDM was partly antagonized by the application of EMD 57033 (10 µM); EMD 57033 produced a significant rightward shift of the BDM concentration-response curve [IC₅₀ of BDM in the presence of EMD 57033 (10 µM) = 19.97 mM, CI = 17.59-22.36 mM, P < .05]. EMD did not change the slope of the force-BDM curve. In the absence of BDM, EMD 57033 increased maximal developed tension in troponin I-depleted fibers by +22.8 ± 4.0% (P < .05). In the presence of CGP 48506 (10 µM) a significant rightward shift of the BDM concentration-response curve was observed [IC₅₀ of BDM in the presence of CGP 48506 (10 µM) = 15.30 mM, CI = 12.83-17.77 mM, Fig. 5]. CGP 48506 did not influence the slope of the BDM-force curve. CGP decreased F_max in troponin I-depleted fibers by 11.1 ± 1.8%, in the absence of BDM.

View larger version (18K): [in this window] [in a new window]   Fig. 5.   The inhibitory effect of BDM () on troponin I-depleted skinned cardiac fibers of human failing myocardium in the presence of EMD 57033 (10 µM, , n = 9) or CGP 48506 (10 µM, , n = 9).

	Discussion

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Ca²⁺ sensitizers may be advantageous for the treatment in human heart failure because they increase force of contraction without increasing the intracellular Ca²⁺ transient or energy expenditure. The mode of action of the novel developed Ca²⁺ sensitizers EMD and CGP in human failing myocardium is yet not fully understood. In contrast to these Ca²⁺ sensitizers, the nucleophilic oxime BDM has been shown to depress contractile force independently of changes in intracellular calcium (Backx et al., 1994). From simultaneous measurements of force and myosin ATPase activity in skinned cardiac rat trabeculae, it has been concluded that BDM causes a marked increase in the apparent rate of cross-bridge detachment (Ebus and Stienen, 1996). Therefore, to further understand the mechanism of action of Ca²⁺ sensitizers, the present study investigated whether the inhibitory action of BDM could be antagonized by the addition of the Ca²⁺ sensitizers EMD or CGP to human myocardium. For the direct study of cross-bridge-interaction troponin I-depleted skinned fiber preparations were used, in which a Ca²⁺-unregulated tension development is taking place (Strauss et al., 1992b). To analyze the influence of EMD, CGP, and BDM on cross-bridge kinetics, tension cost (ratio of myosin ATPase activity and tension development) was studied as well. The tension cost is supposed to be proportional to the cross-bridge detachment rate (g_app; Brenner, 1988, 1990).

Effect of EMD 57033 on Cross-Bridge Cycling. EMD decreases tension cost in human failing myocardium and additionally increases maximum tension development (Table 1). Additionally, as shown in the present study, the increase of maximum tension development by EMD is paralleled by an unchanged maximum Ca²⁺-activated myosin ATPase activity compared with control conditions (in the absence of EMD, Table 1). These results may indicate that EMD increases the probability of the cross-bridges to be in the force-generating state, thereby increasing the force per cross-bridge. Accordingly, there are no observable changes in cross-bridge kinetics or fiber stiffness of rabbit fast skeletal myofilaments (Kraft et al., 1994). In chemically skinned cells from rat ventricle EMD induced marked increases in the rate of tension redevelopment (k_tr) after brief slack release/restretch (Vannier et al., 1997). This indicates the existence of an increased number of attached cross-bridges during one cycle. An effect of EMD on the distribution of force-producing, preforce-, and nonforce-producing cross-bridges has been also proposed (Strauss et al., 1994).

Effect of CGP 48506 on Cross-Bridge Cycling. In the present study obtained in left ventricular Triton X-skinned fiber preparations of human failing myocardium, CGP significantly decreased maximal Ca²⁺-activated myosin ATPase activity without depressing maximal Ca²⁺-activated force (Table 1), which may indicate that the Ca²⁺-sensitizing effect of CGP is partly due to an increase of the force development per cross-bridge. In contrast to EMD, the Ca²⁺-sensitizing effect of CGP is accompanied by an increased Ca²⁺ sensitivity of the myosin ATPase. There are two hypotheses for the Ca²⁺-sensitizing mechanism of CGP proposed (Palmer et al., 1996). First, the Ca²⁺-sensitizing mechanism of CGP may promote of the change from the detached to the weakly attached cross-bridge state, thereby shifting a greater proportion of the cross-bridges ready to shift to the force-producing state when actomyosin interaction is promoted through Ca²⁺ binding to troponin C. This hypothesis may supported by the finding of the present study that CGP 48506 changes the Ca²⁺ sensitivity of both force and myosin ATPase activity. Consistently, we found an increased Hill coefficient for both the Ca²⁺ force and the ATPase-Ca²⁺ relationship, indicating that CGP increases the cooperativity of the myofilaments and thereby improves the Ca²⁺ affinity of troponin C. However, as shown by the present study, the Ca²⁺-sensitizing effect of CGP was also present in Ca²⁺ (and thus troponin C)-unregulated cardiac muscle fibers. Therefore, additional mechanisms seem to contribute to the Ca²⁺-sensitizing effect of CGP. CGP significantly reduced tension cost in human failing myocardium. Because alterations of tension cost reflect changes of the cross-bridge detachment rate, the present study supports the hypothesis of Herold et al. (1995) that CGP 48506 increases Ca²⁺ sensitivity by a reduction of the dissociation constant g_app.

Antagonistic Effects of EMD 57033 and CGP 48506 on BDM-Mediated Depression of Cross-Bridge Cycling. The inhibitory effect of BDM in troponin I-depleted skinned fiber preparations from human myocardium was antagonized by the Ca²⁺ sensitizers EMD and CGP. These results suggest that the action of EMD and CGP, like that of BDM, occur at the level of the actin cross-bridge reaction (Wolska et al., 1996). Consistently, it has been shown that EMD 53998 accelerates the transition into the force-generating state by increasing the release of P_i from the actomyosin-ADP-P_i ternary complex. This mechanism may also hold true for EMD 57033. EMD 57033 may enhance the stability of the attached cross-bridge state, thereby slowing the rate of detachment and increasing the rate of attachment. These actions may reverse the suppressive actions of BDM on the cross-bridge cycle, which have been attributed to a depression in the number of force-generating cross-bridges as well as to a facilitation of P_i release in the conversion of the weakly attached state into the force-generating state (Gwathmey et al., 1991).

Limitations of the Present Study. It cannot be excluded that diseased human myocardium taken from explanted hearts may show intramyocardial damage. However, special care was taken to check the used tissue by electron microscopic techniques. Figure 6 shows an electronic microscopic picture taken from a Triton X-skinned fiber preparation of the presented experimental studies. This picture shows distinct sarcomeric structures, so that detrimental experimental damage to the preparation can be excluded. In addition, all studies of this publication are done in failing human myocardium. This article does not intend to work out differences in the action of EMD 57033 and CGP 48506 between normal and diseased states because pharmacological treatment is focused on failing hearts.

View larger version (154K): [in this window] [in a new window]   Fig. 6.   Electromicroscopic longitudinal picture taken from a Triton X-skinned fiber preparation from human left ventricular myocardium.