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The thiadiazinone derivatives EMD 60417, EMD 66430, and EMD 66398 were developed as class III antiarrhythmic agents. Their chemical structure is closely related to that of their calcium-sensitizing congener |+|-EMD 60263, and EMD 66398 possesses the methylsulfonylaminobenzoyl moiety present in the prototypical I Kr blocker E-4031, We compared the electrophysiologic effects of these compounds with standard drugs (almokalant, E-4031, quinidine) in cardiac myocytes from guinea-pig ventricle and human atrium by whole-cell patch-clamp technique. The test compounds' class III action, which is related to impairment of K + channel function, was confirmed by action potential measurements. EMD 60417, EMD 66430, EMD 66398, and almokalant (1 μM each) reversibly prolonged the action potential duration in guinea-pig myocytes. In the same cells, the rapidly activating component I Kr of the delayed rectifier K + current, which has been defined by its sensitivity to E-4031, was reduced by EMD 60417, EMD 66430, EMD 66398, and almokalant. Inhibition of I Kr was concentration-dependent as determined by attenuation of tail currents. The slowly activating component I Ks of the delayed rectifier K + current was not affected. The inward rectifier K + current I K1 was not influenced at potentials close to the reversal potential. Transient and sustained outward K + currents (I to , I so ) measured in human atrial myocytes were not altered by any EMD compound. L-type Ca 2+ current was hardly affected at concentrations of 1-10 μM, but sodium current was decreased. Action potential prolongation by EMD 60417, EMD 66430, and EMD 66398 is due to block of I Kr . I Na is inhibited at higher concentrations by EMD 66430 and EMD 60417. EMD 66398 is more potent and selective for I Kr than EMD 60417 and EMD 66430, and thus resembles E-4031 in structure and function.

Calcium sensitizers increase myocardial contractile function without affecting Ca2+ homeostasis, which might be beneficial in the treatment of patients with heart failure. However, it remains uncertain whether Ca sensitizers induce quantitatively similar inotropic responses in control and failing hearts. To compare their effects in normal versus failing hearts at the cellular level, shortening mechanics and intracellular calcium ([Ca2+]i) transient were simultaneously measured in the left ventricular myocytes isolated from normal dogs (n = 8) and dogs with rapid pacing-induced heart failure (n = 7). CGP 48506 and EMD 57033 exerted a positive inotropic effect in a dose (0.1-3 microM)-dependent manner in both normal and heart failure myocytes. The percent increase of cell shortening magnitude was comparable between the two groups. CGP 48506 and EMD 57033 did not affect the diastolic cell length and resting [Ca2+]i level. They did not affect the duration of [Ca2+]i transient dynamics. Thus Ca2+ sensitizers exerted comparable positive inotropic effects without affecting the rest cell length and rest [Ca2+]i in normal and heart failure myocytes.

Levosimendan is a novel positive inotropic drug targeted to increase contraction force of the heart through its calcium-dependent binding to troponin C (cTnC). We investigated the calcium-sensitizing effect of levosimendan on contractile proteins as well as its positive inotropic and lusitropic effects in paced guinea pig papillary muscle. We also studied the effect on energy consumption of myosin-actin crossbridges in a myosin ATPase assay. The calcium sensitization induced by levosimendan in fibers skinned with saponin was dependent on the perforation velocity of cell membranes. Levosimendan was almost ineffective in slowly perforated fibers, but was the most potent calcium sensitizer in fibers with rapidly perforated cells. The perforation-dependent calcium sensitization was probably due to changes in phosphorylation state of contractile proteins during the slow dissection of fibers. It is noteworthy that the calcium-sensitizing effect of levosimendan was not affected by acidic pH. Levosimendan at therapeutically relevant (0.3-10 microM) concentrations markedly increased calcium sensitivity both at pH 6.7 and 7.0, being more potent than EMD 53998, pimobendan, and MCI-154. The lack of effect of levosimendan on maximum tension supports the hypothesis that levosimendan increases calcium sensitivity through its action on cTnC. Unlike EMD 53998, levosimendan did not increase myosin ATPase activity, indicating that it did not increase the cycling rate of myosinactin crossbridges. In paced papillary muscles, levosimendan induced positive inotropic effect without changing relaxation time. Thus, levosimendan was devoid of the main negative factors described for calcium sensitizers.

In papillary muscles from reserpinized guinea pigs, EMD 57033, the most potent Ca 2+ -sensitizer known, effectively increased force of contraction (FOC) and concomitantly increased duration of contraction. In isolated 32 P-labeled guinea pig ventricular cardiomyocytes, EMD 57033 increased the phosphorylation state of phospholamban as well as the inhibitory subunit of troponin, which are usually linked to reductions in contraction time. Therefore, EMD 57033 dissociates phospholamban-phosphorylation and effects on contractile parameters, probably owing to its strong Ca 2+ -sensitizing properties

Calcium sensitizers may influence myocardial energetics by their action on calcium turnover and on crossbridge behavior. Using a myothermal method, the effects of the Ca2+ sensitizer EMD-53998 on calcium cycling, crossbridge behavior, and myocardial energy turnover were compared with the effects of an increase in extracellular calcium from 1.25 to 7.5 mM and with the effects of the catecholamine isoproterenol. All three inotropic interventions increased isometric force development in right ventricular rabbit papillary muscles. Relaxation time was decreased with isoproterenol, unchanged with high calcium, and increased with EMD 53998. Calcium cycling-related energy consumption, as measured by tension-independent heat, increased by 234% with high calcium, by 439% with isoproterenol, and by 77% with EMD 53998. In contrast to high calcium and isoproterenol, EMD 53998 increased economy of crossbridge cycling by increasing the force-time integral of the individual crossbridge cycle. The data indicate that EMD 53998 acts by phosphodiesterase inhibition and myofilament calcium sensitization. The latter effect is in part mediated by alteration of crossbridge behavior. Because of its effects on calcium cycling and crossbridge function myocardial energy turnover was reduced significantly with EMD 53998, whereas energy turnover was unchanged with high calcium and was increased with isoproterenol. The new calcium sensitizer levosimendan was investigated in isolated failing human myocardium. Levosimendan dose-dependently increased isometric tension. The inotropic effect was associated with increased rate of relaxation and reduced relaxation time. Measurements of intracellular calcium using the photoprotein aequorin suggest that levosimendan acts by increasing myofilament calcium sensitivity and by increasing cAMP due to phosphodiesterase inhibition. However, the contribution of the cAMP system to the action of levosimendan appears to be rather small. Therefore, the finding of a positive lusitropic effect of levosimendan may be consistent with the notion that levosimendan binds to troponin-C and increases calcium sensitivity only at high (systolic) intracellular calcium concentrations.

The two isomers of the positive inotropic compound EMD 53998, (+)EMD 57033 and (-)EMD 57439, possess selective calcium sensitizing and phosphodiesterase (PDE) inhibitory properties, respectively. We measured the pharmacological responses to both enantiomers in isolated rat cardiac and vascular tissues and in muscles from severely failing human hearts. We also measured positive inotropic and chronotropic responses to EMD 57033 in cardiac tissues from rats with thyroid dysfunction, diabetes, or hypertension. Both compounds increased force of contraction in isolated rat cardiac tissues, although the ventricular response to EMD 57439 was only approximately 10% that of calcium chloride. Forskolin pretreatment potentiated responses to both compounds in atria but only to EMD 57439 in ventricles. Hyperthyroidism increased ventricular responses to EMD 57033 relative to calcium chloride; hypothyroidism and diabetes decreased these responses. Ventricular responses were unchanged in hypertensive rats. Both enantiomers produced positive inotropy in human isolated right atrial trabeculae, although the maximal increases were only 14% (EMD 57033) and 26% (EMD 57439) that of calcium chloride. In rat thoracic aortic rings, both enantiomers produced relaxation; the responses due to EMD 57033 were endothelium dependent. Thus, calcium sensitization produces positive inotropy and vascular relaxation in rats. Positive chronotropic responses to EMD 57033 are most likely due to PDE inhibition. The limited inotropic response in severely failing human myocardium, together with possible vasorelaxation, may provide cardiac support in heart failure without an excessive increase in cardiac O2 demand.

The effect of a new cardiotonic agent Y-20487 [6-(3,6-dihydro-2-oxo-2H-1,3,4-thiadiazin-5-yl)-3,4-dihydro-2(1H)- quinolinone] on cyclic AMP levels of rat ventricular cardiomyocytes and the contractile force of papillary muscles was investigated in comparison with selective cyclic AMP phosphodiesterase (PDE) inhibitors, milrinone (PDE-III selective), rolipram and Ro 20-1724 (PDE-IV selective), and a nonselective inhibitor 3-isobutyl-1-methylxanthine (IBMX). Rolipram and Ro 20-1724 did not elicit cyclic AMP accumulation and positive inotropy, but they potentiated the isoproterenol (ISO)-induced cyclic AMP accumulation more effectively than IBMX. Rolipram was more effective than Ro 20-1724 in enhancing ISO-induced cyclic AMP accumulation but was less effective in enhancing the ISO-induced positive inotropic effect, indicating that these agents produce a differential action on cyclic AMP metabolism and inotropy. Milrinone and Y-20487 elicited cyclic AMP accumulation and positive inotropy by themselves. Whereas milrinone scarcely affected the ISO-induced effects, Y-20487 shifted the concentration-response curve for the positive inotropic effect of ISO to the left to the same extent that IBMX did. Y-20487, however, was much less effective than IBMX in enhancing the ISO-induced cyclic AMP accumulation. The present results indicate that in rat ventricular myocardium, PDE-IV may play a crucial role in breakdown of cyclic AMP generated by beta-adrenoceptor stimulation, whereas other types of PDE isoenzymes, including PDE-III, may be responsible for the cyclic AMP accumulation and direct positive inotropic effect induced by PDE inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)

EMD 53 998, a novel thiadiazinone derivative, increases the contractile force of cardiac tissue in vitro through both an inhibition of phosphodiesterase III (PDE III) and a sensitization of cardiac contractile proteins to Ca2+. Guinea pig ventricular PDE III is selectively inhibited by EMD 53 998 (IC50 = 60 nM) without major effects on other PDE isoenzymes. Consonant with this is an increase in cAMP content of rat ventricular cells and a potentiation by EMD 53 998 of the cAMP-elevating action of isoprenaline (increase by 50% at 1.3 microM). Sensitization to Ca2+ by EMD 53 998 (3-30 microM) finds its expression in a leftward shift of the Ca2+ response curve for force generation in skinned fibers from porcine ventricular muscle and failing human heart as well as for activation of bovine cardiac myofibrillar actomyosin ATPase. Interestingly, EMD 53 998 elevates the maximum of the Ca(2+)-response curve for both parameters. Pimobendan studied under identical conditions was 100 times less potent than EMD 53 998. EMD 53 998 increases force development of guinea pig papillary muscle in a concentration-dependent manner with an EC50 of 3.6 microM, thus being 10 times more potent than pimobendan. In contrast to pimobendan, the positive inotropic effect of EMD 53 998 is barely affected by carbachol. Further evidence for a Ca(2+)-sensitizing effect of EMD 53 998 is provided by an additional increase in force generation in the presence of supramaximal isoprenaline concentrations. It is concluded that the positive inotropic action of EMD 53 998 is mediated through both cAMP-independent and cAMP-dependent mechanisms, with the former probably prevailing. We are not aware of other compounds with a similarly high Ca(2+)-sensitizing potency. On these grounds. EMD 53 998 appears to be a promising inotropic agent.

Although one of the salient abnormalities in signal transduction of failing myocardium is downregulation of the beta-adrenergic receptor, the extent of presentation of downstream pathways distal to beta-receptors is misunderstood. We addressed this question in tachypacing-induced canine failing heart by assessing changes in myofibrillar Ca2+ sensitivity and troponin I phosphorylation. At a basal state, no significant difference in myofibrillar Ca2+ sensitivity was found between normal and failing hearts. Isoproterenol 8-bromo-cylic adenosine monophosphate (cAMP), and 8-bromo-cAMP isobutylmethylxantine all significantly decreased the Ca2+ sensitivity in the normal, but not in the failing, heart. EMD57033 (10 microM ), a myofibrillar Ca2+ sensitizer increased the Ca2+ sensitivity to a similar extent in both groups. The troponin I phosphorylation levels were significantly decreased in the failing heart. These results suggest that abnormalities of the beta-adrenergic signaling system exist not only at the receptor level but also at downstream steps after cAMP production.

The thiadiazinone enantiomers [+]-EMD 60263 and [-]-EMD 60264 ((+)-5-(1-(α-ethylimino-3,4-dimethoxybenzyl)-1,2,3,4-tetrahydroquinoline-6-yl)-6-methyl-3,6-dihydro-2H-1,3,4-thiadiazine-2-on) exhibit distinct stereoselectivity for Ca 2+ -sensitizing action ([+]-enantiomer) and phosphodiesterase inhibition ([-]-enantiomer). However, in isolated guinea pig papillary muscle, both compounds cause an action-potential prolongation that has been related to a nonselective depression of the delayed rectifier potassium current. Because [-]-EMD 60264 did not increase force of contraction despite phosphodiesterase inhibition, we postulated that one or several additional actions may oppose the anticipated positive inotropic effect. Therefore we investigated whether other membrane currents were also affected in voltage-clamped ventricular cardiomyocytes. Both [+]-EMD 60263 and [-]-EMD 60264 reduced sodium current as well as L-type calcium current in guinea pig ventricular myocytes, but steady-state inactivation or conductance curves of I Na and I ca were not shifted along the voltage axis. Inward rectifier and transient outward current were studied in rat myocytes. but neither current was affected. We conclude that the positive inotropic action of [+]-EMD 60263 can be explained by prevalence of the Ca 2+ -sensitizing effect over its inhibitory actions on Na + and Ca 2+ current, whereas the negative inotropic effect of [-]-EMD 60264 may be caused by inhibition of I Ca predominating over PDE inhibition.

The previous separation of the racemic cardiotonic thiadiazinone derivative EMD 53 998 yielded two enantiomers with different pharmacologic properties: EMD 57 033, a potent Ca 2+ sensitizer with some residual phosphodiesterase III (PDE III) inhibition, and EMD 57 439, a pure PDE III inhibitor. Although numerous in vitro studies demonstrated the ability of EMD 57 033 to increase the responsiveness of cardiac contractile proteins to Ca 2+ , in vivo evidence for such an action is lacking. Because there is no possibility of directly proving Ca 2+ sensitization in vivo, we attempted to exclude PDE III inhibition as a major contributing component of the positive inotropic action of EMD 57 033. In anesthetized rats, EMD 57 033 increased left ventricular (LV) first derivative of change in systolic pressure over time (dP/dt max) without affecting blood pressure. In contrast, the PDE III-inhibitory enantiomer EMD 57 439 decreased blood pressure. The pattern of hemodynamic effects in anesthetized dogs revealed similar differences between EMD 57 033 and PDE inhibitors. Thus the increase in LV dP/dt max in response to EMD 57 033 was not accompanied by changes of heart rate and blood pressure. As expected for PDE inhibitors, pimobendan and milrinone increased cardiac contractile force in dogs, concomitant, however, with tachycardia, hypotension, and a decrease in total peripheral resistance. When regional contractility was measured separately in two different areas of the dog myocardium, the positive inotropic action of the PDE inhibitors pimobendan and milrinone was antagonized by local coronary infusion of acetylcholine. The cardiotonic effect of the Ca 2+ sensitizer EMD 57 033 was entirely resistant to inhibition by acetylcholine. In conscious dogs, β-blockade markedly attenuated the increase in LV dP/dt max produced by two different doses of the PDE III inhibitor EMD 57 439. In contrast, a dose of EMD 57 033 equieffective in positive inotropic action with the lower dose of EMD 57 439 remained unaffected by

This review compares the mechanisms of action of the calcium-sensitizing agents levosimendan, pimobendan, MCI-154, and EMD 53998. By using purified human recombinant troponin-C (cTnC), the role of cTnC as a target protein for these compounds was investigated. Accordingly, the calcium-dependent binding to cTnC in a cTnC-high-performance liquid affinity chromatography (HPLAC) column and the stabilizing effects of the compounds on the calcium-induced conformation of dansylated cTnC were studied. Only levosimendan showed calcium-dependent action on cTnC. Of the studied compounds, levosimendan was the most potent calcium sensitizer in skinned fiber experiments. Furthermore, EMD 53998 and MCI-154, but not levosimendan and pimobendan, increased myosin ATPase activity, indicating that they may enhance the cycling rate of myosin-actin crossbridges. By analyzing the velocity (dT/dt) of isometric tension development in paced papillary muscles, it was shown that levosimendan probably enhances the association rate but decreases the dissociation rate of myosin-actin crossbridges. These effects occurred before the peak twitch tension was achieved. Therefore, levosimendan does not seem to affect the actual relaxation phase. The other calcium sensitizers, however, appear to act mainly by decreasing the dissociation rate of crossbridges. The weak calcium-sensitizing effect of pimobendan may be based on indirectly mediated increase in affinity of cTnC for calcium. MCI-154 might act in a similar way but, like EMD 53998, MCI-154 also acts on myosin-actin crossbridges. We suggest that levosimendan binds in a calcium-dependent manner to the N-terminal domain of cTnC, which magnifies the extent of the contraction produced by cTnC when it is calcium-activated.

The novel cardiotonic EMD 53,998 increases contractile force in vitro through both inhibition of phosphodiesterase III (PDE III) activity and increase in the responsiveness of the contractile proteins to calcium ("calcium sensitization"). Because EMD 53,998 is a racemate, the possibility arose that the two modes of action do not reside equally in the enantiomers. Therefore, the effects of the racemate and its two enantiomers [(+)EMD 57,033 and (-)EMD 57,439] were analyzed in guinea pig and rat cardiac tissue with respect to Ca2+ sensitization (Ca(2+)-induced force development in skinned cardiac myofibers and myofibrillar ATPase activity) and PDE III inhibition (isolated PDE isoenzymes and cyclic AMP level in isolated cardiac myocytes). In addition, the positive inotropic effects were compared in isometrically contracting papillary muscles. Enhancement of force of contraction (Fc) in submaximally activated skinned fibers showed a selectivity for the (+)enantiomer with EC50 = 1.7, 4.8, and > 100 microM for EMD 57,033, EMD 53,998, and EMD 57,439, respectively. Ca2+ concentration for half-maximal activation was decreased by 0.5 log units, and Cmax was increased by 15% at 10 microM EMD 57,033. Similarly, myofibrillar ATPase activity was most potently enhanced by the (+)enantiomer, with EC50 values of 1.8, 2.5, and > 30 microM for EMD 57,033, EMD 53,998, and EMD 57,439, respectively. PDE III activity was inhibited with greater potency by the (-)enantiomer, with IC50 values of 0.05, 0.06, and 1.94 microM for EMD 57,439, EMD 53,998, and EMD 57,033, respectively. The cyclic AMP content of isoprenaline-stimulated rat cardiac myocytes was increased by 50% at 13.6 and 0.71 microM for EMD 57,033 and EMD 57,439, respectively. In intact guinea pig papillary muscle, the positive inotropic effect of the (+)enantiomer was insensitive to isoprenaline pretreatment; in contrast, the (-)enantiomer showed only a weak positive inotropic action which was strongly enhanced in the presence of isoprenaline. We conclude that one of the two different mechanisms underlying the overall positive inotropic activity of EMD 53,998 can be assigned, almost exclusively, to one of the two enantiomers. Thus, the (-)enantiomer EMD 57,439 is a "pure" PDE III inhibitor with almost no Ca2+ sensitizing activity; the (+)enantiomer EMD 57,033 is a potent Ca2+ sensitizer with only a weak PDE III inhibitory activity as compared with the racemate. In contrast to other compounds with mixed activity, EMD 57,033 is unique in possessing both a high absolute potency at the level of the contractile elements and a favorable relation of Ca2+ sensitization to PDE inhibition.