Different Effect of the Ca Sensitizers EMD 57033 and CGP 48506 on Cross-Bridge Cycling in Human Myocardium

Ca sensitizers may be advantageous for treatment in human heart failure by increasing cardiac force without increasing the Ca transient or energy consumption. To study the mode of action of the Ca sensitizers EMD 57033 (EMD) and CGP 48506 (CGP), their influence on butanedione monoxime (BDM)mediated depression of cross-bridge cycling was analyzed in human myocardium (explanted hearts, dilated cardiomyopathy, n 5 19). In Triton X (1%)-skinned fiber preparations of left ventricular myocardium from patients suffering from dilated cardiomyopathy, troponin I was extracted by vanadate (10 mM) treatment, resulting in a Ca-independent contraction. In troponin I-depleted fibers BDM (5–50 mM) was applied in the absence and presence of EMD (10 mM) or CGP (10 mM). To analyze the influence on cross-bridge kinetics, tension cost (ratio of ATPase activity and tension development) was studied. BDM exerted a dose-dependent force inhibition in troponin I-depleted fibers (IC50 5 7.22 mM), which was antagonized by EMD (IC50 of BDM 1 EMD 5 19.97 mM) and CGP (IC50 of BDM 1 CGP 5 15.30 mM). EMD increased Ca sensitivity of force and maximal force in Triton X-skinned fibers. The Ca-sensitizing effect of CGP was accompanied by an increased Ca sensitivity of myosin-ATPase activity, an increased slope of the Ca force and Ca ATPase curve, as well as a reduced maximal myosin ATPase activity. CGP and EMD reduced tension cost. In conclusion, EMD and CGP antagonize the BDMmediated relaxation in troponin I-depleted cardiac muscle fibers. The Ca-sensitizing effect of CGP seems to be dependent on an improvement of the myofilament cooperativity, whereas EMD seems to operate by increasing the force per cross-bridge. 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 Ca-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 (Pi) release step and stabilizing the actomyosin-ADP-Pi intermediate cross-bridge state (actomyosin-ADP-Pi) (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-(3,4dimethoxybenzoyl)-1,2,3,4-tetrahydro-6-quinolyl]-6-methyl-3,6dihydro-2H-1,3,4-thiadiazin-2-one) and its (1)-enantiomer EMD 57033 are classified as Ca 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 crossbridge attachment in the strongly bound force-generating state (Simnett et al., 1993a; Arner et al., 1995), whereas the crossbridge detachment rate (gapp), 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 Pi 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 Pi release step, perhaps by lowering the Pi Received for publication March 8, 2000. 1 This work was supported by Deutsche Forschungsgemeinschaft (R.H.G.S.) and Köln Fortune (K.B.). ABBREVIATIONS: BDM, 2,3-butanedione monoxime; Pi, inorganic phosphate; EMD, EMD 57033; CGP, CGP 48506; gapp, cross-bridge attachment rate; Triton X, Triton X-100. 0022-3565/00/2953-1284$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 295, No. 3 Copyright © 2000 by The American Society for Pharmacology and Experimental Therapeutics 2680/866634 JPET 295:1284–1290, 2000 Printed in U.S.A. 1284 at A PE T Jornals on A ril 9, 2017 jpet.asjournals.org D ow nladed from affinity for myosin (Strauss et al., 1994). Therefore, its action should be antagonistic to that of BDM. CGP 48506 (GCP), the (1)-entantiomer of the racemic mixture 1,5-benzodiazocine derivative 5-methyl-6-phenyl-1,3,5,6tetrahydro-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). Ca sensitizers may be advantageous for the treatment in human heart failure because they increase force of contraction without increasing the intracellular Ca 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 Myocardial Tissue. Experiments were performed on human left ventricular myocardium. Tissue was obtained during cardiac transplantation (n 5 19, 8 female, 11 male; age 44 6 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, b-adrenoceptor or Ca 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 220°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 (2log[Ca]) 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 Ca dependence of force in each preparation. After this procedure, fibers were again maximally contracted (pCa 5 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 Ca regulated, i.e., they contracted maximally even at 10 nM free Ca. 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 mM), respectively, CGP 48506 (10 mM). 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 a-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 a-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 220°C until the sarcomeric length measurement. The measurement of sarcomeric length was performed using a Zeiss Axiovert 135 fluorescence microscope (filterset 15 Zeiss; excitation BP 546/16, emission LP 590), a Sony three chip camera, and computer-assisted imaging software (Optimas 6.01). For investigation of the sarcomeric length, the skinned fibers were fixed at slack position in relaxation solution. The distance of 10 to 15 actinin/Cy3labeled Z-lines was measured at 10 different areas of each skinned fiber using a 403 Neofluar objective (Zeiss, Oberkochen, Germany). The sarcomeric length was calculated by dividing the measured distance by the number of spaces between labeled Z-lines. The mean of sarcomeric length for each skinned fiber was calculated from all investigated areas. Average sarcomere length was 2.01 6 0.08 mm. 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 59) 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 59) 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 Ca was successively increased every 15 s. Free Ca 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 ATP2000 EMD 57033 and CGP 48506 in Human Myocardium 1285 at A PE T Jornals on A ril 9, 2017 jpet.asjournals.org D ow nladed from 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 a-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 6 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 5 [Ca]/([pCa50] H 1 [Ca]), where Y is the fractional force, or myosin-ATPase activity, pCa is the Ca concentration giving half-maximal activation (inhibition), and H is an index of cooperativity (Hill coefficient). The concentration needed for half-maximal Ca activation of tension development or myosin ATPase activity (EC50 for Ca ), 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).