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In the present study we have analyzed a likely biochemical mechanism underlying the Ca++-sensitizing action of MCI-154 (6-[4-(4'-pyridyl)aminophenyl)-4,5-dihydro-3(2H)-pyridazinone hydrochloride), a novel cardiotonic agent, on the contractile protein system. MCI-154 (10(-7) to 10(-4) M) enhanced the tension development induced by -log molar-free Ca++ concentration (pCa) 5.8 in chemically skinned fiber from the canine right ventricular muscle in a concentration-dependent manner. At pCa 7.0, MCI-154 (10(-7) to 10(-4) M) markedly increased adenosine triphosphatase (ATPase) activities of canine myofibrils and reconstituted actomyosin. In myofibrils and reconstituted actomyosin, MCI-154 (10(-7) to 10(-4) M) caused a parallel shift of the pCa-ATPase activity relation curve to the left without affecting the maximum activity, suggesting an increase in Ca++ sensitivity. MCI-154 (10(-8) to 10(-4) M) had little effect on actin-activated, Mg++, Ca++ and (K+, EDTA)-ATPase activities of myosin. Ca++ binding to cardiac myofibrils or purified cardiac troponin was increased by 10(-4) M MCI-154. These results suggest that MCI-154 enhances Ca++ binding to cardiac troponin C to elevate the Ca++ sensitivity of myofilaments and thus may cause a positive inotropic action in cardiac muscle. MCI-154 may provide a valuable tool for studying the molecular mechanism by which Ca++ regulates the contractile system.

Despite its potential as a key determinant of the functional state of striated muscle, the impact of tropomyosin (Tm) isoform switching on mammalian myofilament activation and regulation in the intact lattice remains unclear. Using a transgenic approach to specifically exchange beta-Tm for the native alpha-Tm in mouse hearts, we have been able to uncover novel functions of Tm isoform switching in the heart. The myofilaments containing beta-Tm demonstrated an increase in the activation of the thin filament by strongly bound cross-bridges, an increase in Ca2+ sensitivity of steady state force, and a decrease in the rightward shift of the Ca2+-force relation induced by cAMP-dependent phosphorylation. Our results are the first to demonstrate the specific effects of Tm isoform switching on mammalian thin filament activation in the intact lattice and suggest an important role for Tm in modulation of myofilament activity by phosphorylation of troponin.

Single fibers of the frog glossopharyngeal nerve respond to MgCl2 at concentrations exceeding 10 mM. NiCl2 at 1 mM enhanced the Mg2+ response. CaCl2 at 0.5-2 mM induced an inhibition of the Ni(2+)-enhanced response to Mg2+ ions. A quantitative explanation for these results is provided by the hypothesis that Ni2+ ions secondarily affect a magnesium receptor (designated X*Mg) that is responsible for the Mg2+ response and that Ca2+ ions inhibit the Ni(2+)-enhanced response to Mg2+ ions by competing with Mg2+ ions for X*Mg. Double-reciprocal plots of the experimental data indicate that Ni2+ ions do not affect the affinities of X*Mg for both Mg2+ ions (agonist) and Ca2+ ions (competitive antagonist) appreciably, and that Ni2+ ions at 1 mM enhanced the maximal response to Mg2+ ions by 270%. It appears that a magnesium receptor interacts with an Ni(2+)-binding element that is affected by Ni2+ ions and, thus, Ni2+ ions can induce an enhancement of the Mg2+ response.

Single water fibers of the frog glossopharyngeal nerve respond to relatively high concentrations of NaCl ( > 80 mM). NiCl2 at 1 mM enhanced the Na+ response and reduced the threshold concentration for NaCl to 20 mM. CaCl2 at 0.5-1 mM induced an inhibition of the Ni2+ -enhanced response to Na+ ions. A quantitative explanations for these results is provided by the hypothesis that Ni2+ ions secondarily affect a sodium receptor or channel (designated XNa*) that is responsible for the Na+ response and that Ca2+ ions inhibit the Ni2+ -enhanced response to Na+ ions by competing with Na+ ions for XNa*. Double-reciprocal plots of the experimental data indicate that the affinity of XNa* for both Na+ ions (agonist) and Ca2+ ions (competitive antagonist) in the presence of 1 mM NiCl2 was five times higher than the previously reported values obtained in the absence of NiCl2 (Kitada, 1991). Ni2+ ions at 1 mM enhanced the maximal response to Na+ ions by 190%. It appears that a sodium receptor (or channel) interacts with a Ni2+ -binding element that is affected by Ni2+ ions and, thus, Ni2+ ions can induce both an increase in the affinity of the sodium receptor for the respective cations and an enhancement of the Na+ response.

The effects of MCI-154, a cardiotonic agent with Ca++ sensitizing actions, on the ischemic contractile failure and myocardial acidosis were studied in the dog heart, in which the left anterior descending coronary artery (LAD) was partially occluded for 90 min, and compared with those of dobutamine, milrinone, pimobendan and isosorbide dinitrate (ISDN). Partial occlusion of LAD decreased segment shortening (measured by sonomicrometry) and myocardial pH (assessed by a micro glass pH electrode) in the ischemic myocardium. MCI-154, when administered i.v. 30 min after ischemia, improved the segment shortening in the ischemic zone, whereas dobutamine, milrinone and pimobendan failed to improve it when the drugs increased peak positive left ventricular dP/dt. Among the cardiotonic agents tested only MCI-154 attenuated myocardial acidosis during ischemia. The degree of the attenuation of acidosis by MCI-154 was equivalent with that by ISDN. However, the improvement of the ischemic zone segment shortening by MCI-154 was more pronounced than that by ISDN. These results suggest that in addition to the attenuation of myocardial acidosis the positive inotropic action of MCI-154, presumably increasing the responses of myofilaments to Ca++, may be possibly responsible for the improvement of regional contractile function in the ischemic myocardium. Thus, MCI-154 may be useful in the management of ischemic heart failure.

In the present study, we have examined the effects of decreasing pH from 7.0 to 6.6 on the tension developed by direct activation of the myofilaments in chemically skinned fibers from guinea pig papillary muscles. We then compared the effects of the novel inotropic agents MCI-154, pimobendan and sulmazole, which have direct action on cardiac myofilaments, on the acidic pH-induced changes in responses of the contractile system to Ca++. The reduction of pH from 7.0 to 6.8 shifted the pCa (-log[Ca++] M)-tension relation curve to the right with no change in maximum tension. However, the reduction of pH from 7.0 to 6.6 shifted the pCa tension relation curve to the right and also depressed maximum force development. These effects were reversible by returning to neutral pH (pH 7.0), but were not overcome by increasing the free [Ca++] (decreasing pCa from 4.4 to 4.0). The amplitude of pMg-ATP (-log[MgATP]M)-tension curve in the absence of free Ca++ (Ca++ less than 1 nM, bell-shaped curve) was shifted downward by reducing pH from 7.0 to 6.6. MCI-154 (1-100 microM) reversed the acidic pH-induced decrease of tension development which was activated by pCa 5.8 in a concentration-dependent manner. Moreover, the acidosis induced reductions of maximum tension (pCa, 4.4) and pMgATP 6.0-activated tension (Ca++ less than 1 nM) were also reversed by MCI-154 (1-100 microM) in a concentration-dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)

MCI-154 is a new positive inotropic agent with vasodilating property. Experiments were carried out in the canine isolated right ventricular muscle in order to elucidate whether or not cyclic AMP is involved in the positive inotropic effect (PIE) of MCI-154. MCI-154 (10(-7) to 10(-4) M) produced a concentration-dependent PIE amounting to 75% of the maximal effect of isoproterenol. MCI-154 did not affect the time to peak tension and had a tendency to shorten the relaxation time and total duration of contraction. Pindolol, reserpine-pretreatment or tetrodotoxin did not modify the PIE of MCI-154. MCI-154 increased the cyclic AMP levels only at 3 X 10(-4) M, whereas CI-914, of which chemical structure is similar to that of MCI-154, elevated definitely the cyclic AMP at the lower concentrations (10(-5) to 10(-4) M). Carbachol at a concentration known to decrease markedly the PIE of amrinone, milrinone and papaverine, did not affect the PIE of MCI-154. MCI-154 inhibited the activity of a crude phosphodiesterase (PDE) from the canine ventricular muscle and it enhanced the PIE of isoproterenol, which implied the involvement of cyclic AMP. However, the maximal inhibition of PDE by MCI-154 remained less than 18%. Amrinone, milrinone and papaverine inhibited more potently the PDE activity than MCI-154. These results suggest that the elevation of cyclic AMP levels is only partially involved in the PIE of MCI-154 in the canine right ventricular muscle, and that MCI-154 may have novel mechanisms of action different from those of amrinone, milrinone and CI-914 that are largely cyclic AMP-dependent.

The effects of MCI-154, a novel cardiotonic agent, on the contractile protein system and the sarcoplasmic reticulum (SR) were investigated by using thin bundles of chemically skinned fibers from the guinea pig papillary muscles. In the skinned muscle fibers treated with 50 micrograms/ml of saponin, MCI-154 shifted the -log[Ca++]M-tension relation curve to the left and upward in the concentration-dependent manner (10(-7) to 10(-4) M). This was confirmed also in the skinned muscle fibers treated with 250 micrograms/ml of saponin which destroyed not only the surface membrane but also the function of SR. Sulmazole (10(-4) M) shifted the -log[Ca++]M-tension relation curve to the left but the effect was about 100 times less potent than that of MCI-154. Unlike MCI-154, sulmazole had little effect on the maximum tension development induced by -log[Ca++]M 4.4. Milrinone did not affect the Ca++-induced tension development in the skinned cardiac fibers. Higher concentration of MCI-154 (10(-4) M) also increased amplitude of -log[Mg-ATP]M-tension-curve in the absence of free Ca++ ion (bell-shaped curve) to the upward. Initial rate and plateau phase of Ca++ uptake by the SR in the skinned fibers treated with 50 micrograms/ml of saponin was increased slightly by MCI-154 at the concentrations of 10(-6) and 10(-4) M. MCI-154 had no effect on the Ca++-induced Ca++ release mechanism in the SR. These results suggest that an increase in Ca++ sensitivity of the contractile protein system is responsible for, at least in part, the mechanism of the positive inotropic action of MCI-154.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of MCI-154 (6-[4-(4'-pyridyl)aminophenyl]-4,5-dihydro-3(2H)- pyridazinone hydrochloride.3H2O), a cardiotonic agent with calcium sensitizing actions, on regional contractile function and myocardial oxygen consumption (MVO2) were studied in the dog hearts with and without partial occlusion of the left anterior descending coronary artery and compared with those of dobutamine. Segment shortening by sonomicrometry, regional myocardial blood flow by microspheres and the oxygen content of coronary venous blood drawn from the ischemic left anterior descending coronary artery area were simultaneously measured. The ischemic zone segment shortening and left ventricular (LV) dP/dtmax were decreased after partial occlusion. The infusion of MCI-154 starting 20 min after ischemia improved the depressed segment shortening and LV dP/dtmax without increasing the ischemic zone MVO2 and regional myocardial blood flow. In the nonischemic hearts, MCI-154 did not increase MVO2 and coronary blood flow despite the augmentation of myocardial contractility. MCI-154 decreased LV end-diastolic pressure and systemic blood pressure. On the other hand, dobutamine failed to increase the ischemic zone segment shortening, but the drug increased MVO2, coronary blood flow and LV dP/dtmax in both ischemic and nonischemic hearts. These results indicate that MCI-154 alleviates the ischemic contractile failure without increasing myocardial oxygen demand. Thus, MCI-154 may be useful in the management of heart failure with reduced coronary reserve.