Introduction

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Most inotropic agents that are used for the treatment of congestive heart failure act via cAMP-dependent or cAMP-independent mechanisms. The efficacy of cAMP-dependent inotropic effects is reduced in human failing myocardium because of a down-regulation of beta adrenoceptors (Bristow et al., 1982), an increase of Gi (Feldman et al., 1988; Böhm et al., 1990) and reduced cAMP concentrations (Feldman et al., 1987; Danielsen et al., 1989). Furthermore, PDE inhibitors which constitute the main group of cAMP-dependent inotropic agents have deleterious effects on survival in patients with end-stage heart failure (Packer et al., 1991).

A new class of inotropic agents, Ca⁺⁺ sensitizers, has been developed, which act at the level of the contractile proteins to increase the sensitivity of the myofilaments to Ca⁺⁺ (Fujino et al., 1988; Hajjar et al., 1988; v.Meel et al., 1988; Honerjäger et al., 1989; Lee and Allen, 1991; Ferroni et al.; 1991, Ventura et al., 1992; Kawabata and Endoh, 1993; Lues et al., 1993; Gambassi et al., 1993; Solaro et al., 1993; Hgashiyama et al., 1995; Neumann et al., 1995). These agents have the advantage of enhancing force production without increasing energy utilization (Grandis et al., 1995). However, they have the adverse effect of slowing relaxation and elevating diastolic tension (Hajjar and Gwathmey, 1991). In heart failure, relaxation is impaired because of abnormal [Ca⁺⁺]_i handling (Gwathmey et al., 1987, 1988, 1990; Gwathmey and Hajjar, 1990; Beuckelmann et al., 1992); therefore, Ca⁺⁺ sensitizers may potentially increase diastolic force further in these failing hearts. However, most of the calcium-sensitizing agents have additional cellular effects such as inhibition of PDE III which may counter the effects of Ca⁺⁺ sensitizers on relaxation.

We undertook this study to examine the effects of Ca⁺⁺ sensitization in the presence and absence of PDE inhibition on force generation and intracellular Ca⁺⁺ handling in ventricular preparations from nonfailing and failing human hearts with use of two newly developed inotropic agents EMD 57033 and ORG 30029. EMD 57033, a cardiotonic thiadiazone derivative, has been reported to have potent myofilament Ca⁺⁺-sensitizing effects with negligible PDE inhibitory effects at low concentrations (<1 µmol/l) and weak PDE inhibitory effects at higher concentrations (Ferroni et al., 1991; Lues et al., 1993; Solaro et al., 1993). ORG 30029 is a cardiotonic agent which increases the sensitivity of the myofilaments to Ca⁺⁺ and selectively inhibits PDE III (Kawabata and Endoh, 1993).

	Materials and Methods

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Human hearts. Experiments were performed on isolated, electrically stimulated trabeculae carneae from human left ventricular myocardium. Tissue was obtained from human hearts during cardiac transplantation (n = 7, 1 female, 6 males; age range, 20-61 years; 3 with dilated cardiomyopathy and 4 with ischemic cardiomyopathy). All the patients suffered from heart failure clinically classified as NYHA IV with ejection fractions <25%. All patients were receiving diuretics, nitrates, angiotensin converting enzyme inhibitors and cardiac glycosides at the time of transplantation. Nonfailing human myocardium was obtained from 7 brain-dead donors (3 car accidents and 4 hemorrhagic strokes; 4 males, 3 females; age range, 19-54 years). There was no clinical and echocardiographic evidence of left ventricular dysfunction. These hearts could not be transplanted for either technical reasons or because of age.

Isolated muscle preparations. Muscle fibers running approximately parallel with the length of the preparations (diameter: 0.6-1.0 mm) of uniform size were dissected in an oxygenated bathing solution at room temperature (see below) under microscopic control with sharp scissors. The preparations were electrically stimulated by a bipolar platinum electrode located at the base. The bathing solution used was a modified Krebs-Henseleit solution containing (in mmol/l): NaCl, 120; KCl, 5.9; CaCl₂, 2.5; MgCl₂, 1.2; NaH₂PO₄, 1.2; NaHCO₃, 25; dextrose, 11.5; with 95% O₂ and 5% CO₂ and maintained at 37°C, pH 7.4. Isometric force of contraction was measured with an inductive force transducer attached to a Gould recorder. The preparations were electrically paced at 1 Hz with square wave pulses of 5-msec duration (Grass stimulator SD 9), at threshold voltage. Muscles were stretched incrementally until there was no further increase in peak twitch force. Force measurements were normalized to the cross-sectional area of the muscles (g/mm²), and t_80% was measured from peak twitch force. All preparations were allowed to equilibrate at least 90 min in the bathing solution until complete mechanical stabilization.

Intracellular Ca⁺⁺ measurements. The muscles were loaded with the bioluminescent Ca⁺⁺ indicator aequorin with a modified chemical-loading procedure described previously (Pesaturo and Gwathmey, 1990). Aequorin was introduced into the muscle via macroinjection through a glass micropipette. The aequorin solution contained (mmol/l): EGTA, 0.1; Na₂ATP, 5; KCl, 120; MgCl₂, 2; TES, 20; 0.5 mg/ml aequorin. The muscle was then placed in Ca⁺⁺-free standard Krebs' solution (as described above) at 20°C, and Ca⁺⁺ was re-added to this solution at 20-min intervals in increasing concentrations: 0.025, 0.25, 1.25 and 2.5 mmol/l. The muscle was then gradually rewarmed to 37°C. Calcium and force responses from aequorin-loaded muscles were recorded simultaneously by a force transducer and a specially designed light-collecting apparatus (Blinks, 1984). Force and [Ca⁺⁺]_i responses were digitally acquired. For each response studied 10 to 50 signals were averaged depending on the brightness of an individual muscle preparation to improve the signal-to-noise ratio. Light signals were converted to intracellular [Ca⁺⁺] with an in vitro calibration curve modeled by the following equation: where L is the luminescence signal, L_max is the maximum light emitted after the lysis of the muscle preparation exposed to saturating [Ca⁺⁺] with 2% Triton X-100. K_R and K_TR are constants. L_80% is the time from peak to 80% relaxation of the [Ca⁺⁺]_i signal.

Skinned fibers preparations. Thin muscle fibers (diameter <400 µm) were dissected and handled as described above. The electrical stimulation was turned off and the bath temperature was then lowered to 22°C. The fibers were then exposed to a skinning solution composed of (mmol/l): Na₂ATP, 5; MgCl₂, 7; EGTA, 60; KCl, 50; MOPS, 12; phosphocreatine, 12; creatine phosphokinase, 15 U/ml; saponin, 250 µg/ml. The muscles were exposed to this solution for 30 min. After skinning, the muscles were placed in a relaxing solution (pCa > 8.0) and then activated over a full range of pCa values (8.0-4.0). The relaxation and activating solutions were calculated with the program of Fabiato (1988). The activating and relaxing solutions were prepared at 22°C and contained (mmol/l): MOPS, 50; EGTA, 10; TES, 30; adjusted for a pMg of 2.5, pMgATP of 2.5, an ionic strength of 160 mmol/l, and pH of 7.1. EMD 57033 and ORG 30029 were added at all pCa values throughout the activation cycle. Each Ca⁺⁺-activation experiment (with or without drugs) yielded a [Ca⁺⁺]-force relationship which was then fitted individually to the Hill equation: where F is force developed, [Ca⁺⁺] is the Ca⁺⁺ concentration in the solution, n is the Hill parameter, [Ca⁺⁺]_50% is the [Ca⁺⁺] required for 50% activation and F_max is the maximal Ca⁺⁺-activated force. The Hill parameters (F_max, n and [Ca⁺⁺]_50%) are then calculated by nonlinear regression analysis for that one individual experiment. The Hill parameters are then averaged for the different groups of experiments (i.e., no drug, +EMD 57033, +ORG 30029), yielding a mean ± S.E.M. for the Hill parameters of each group (Moiescu and Thieleczek, 1978; Harrison and Bers, 1989). We define differences in myofilament calcium sensitivity as a change in [Ca⁺⁺]_50% as opposed to differences in myofilament calcium responsiveness which may involve differences in F_max and the Hill coefficient with and without changes in [Ca⁺⁺]_50%.

Materials. EMD 57033 was kindly provided by E. Merck, Darmstadt, Germany and ORG 30029 was kindly provided by Dr. M. Endoh (Yamagata University School of Medicine, Yamagata, Japan). All other chemicals were obtained from Sigma Chemical Co. (St Louis, MO).

Statistics. The data shown are represented as mean ± S.E.M. Student t test was performed to test for statistical differences with P < .05 considered significant. [Ca⁺⁺]-force relationships were fitted individually to the Hill equation and Hill parameters were then averaged and expressed as mean ± S.E.M. Analysis of variance was then performed to test for statistical differences between pre- and postdrug effect on Hill parameters from the experimental groups.

	Results

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Effect of EMD 57033 on active force, diastolic force of contraction and t_80% in nonfailing and failing human myocardium. The peak twitch force generated by the muscles at 1 Hz were 2.17 ± 0.22 g/mm² (nonfailing, n = 6) and 2.32 ± 0.29g/mm² (failing, n = 6). Figure 1 shows the effect of 50 µmol/l EMD 57033 on the force of contraction in failing human myocardium. An increase in active force is observed along with a significant increase in diastolic force. As shown in figure 2A, increasing the concentration of EMD 57033 increases the active force of contraction in human ventricular trabeculae to a similar extent in both nonfailing and failing myocardium (280 ± 27% vs. 305 ± 30%, n = 6, P > .2). However, as shown in figure 2B, EMD 57033 increased the diastolic force (% control active force) in failing myocardium to a greater extent than in nonfailing myocardium (65 ± 12% vs. 28 ± 8%, n = 6, P < .05) in a concentration-dependent manner. Relaxation was also affected by the addition of EMD. The t_80% was increased in the presence of EMD in a concentration-dependent manner as shown in figure 2C. EMD 57033 increased t_80% to a greater extent in failing myocardium than in nonfailing myocardium (378 ± 56% vs. 278 ± 45%, n = 6, P < .05).

View larger version (11K): [in this window] [in a new window]   Fig. 1.   The effect of adding 50 µmol/l EMD 57033 on force generation in an electrically driven muscle from a failing heart stimulated at 1 Hz with a square pulse of 5 msec at 37°C in Krebs' buffer.

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Effect of increasing concentrations of EMD 57033 on active force (A), diastolic force of contraction (B) and t80% (C) in failing and nonfailing human myocardium. The muscle preparations were stimulated at 1 Hz with a square pulse of 5 msec at 37°C in Krebs' buffer. * P < .05 compared with nonfailing myocardium.

Effect of ORG 30029 on active force, diastolic force of contraction and t_80% in nonfailing and failing human myocardium. Figure 3 shows the effect of 5 mmol/l ORG 30029 on the force of contraction in failing human myocardium. As was observed with EMD, there is an increase in active force along with an increase in diastolic force and prolongation of the twitch time course. However, ORG 30029 had a smaller effect on all of these parameters when compared with the effects of EMD. As shown in figure 4A, increasing the concentration of ORG 30029 increased active force of contraction to a similar extent in both nonfailing and failing myocardium (94 ± 13% vs. 88 ± 2%, n = 6, P > .1). However, as shown in figure 4B, ORG 30029 increased the diastolic force (% of control active force) in failing myocardium to a greater extent than in nonfailing myocardium (24 ± 5% vs. 12 ± 3%, n = 6, P < .05). Relaxation was also affected by the addition of ORG. The t_80% was increased in the presence of ORG 30029 in a concentration-dependent fashion as shown in figure 4C. ORG 30029 increased t_80% to a greater degree in failing myocardium than in nonfailing myocardium (230 ± 26% vs. 155 ± 21%, n = 6, P < .05).

View larger version (9K): [in this window] [in a new window]   Fig. 3.   The effect of adding 5 mmol/l ORG 30029 on force generation in an electrically driven muscle from a failing heart stimulated at 1 Hz with a square pulse of 5 msec at 37°C in Krebs' buffer.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Effect of increasing concentrations of ORG 30029 on active force (A), diastolic force of contraction (B) and t80% (C) in failing and nonfailing human myocardium. The muscle preparations were stimulated at 1 Hz with a square pulse of 5 msec at 37°C in Krebs' buffer. * P < .05 compared with nonfailing myocardium.

Effect of EMD 57033 and ORG 30029 on intracellular Ca⁺⁺ transients. Intracellular Ca⁺⁺ was measured in fibers from nonfailing and failing human hearts. As shown in table 1, at a stimulation of 1 Hz, there were no significant differences in peak intracellular [Ca⁺⁺]_i between nonfailing and failing fibers. However, both the diastolic [Ca⁺⁺]_i was elevated and the time course of the calcium transients was prolonged in failing myocardium as compared to nonfailing myocardium. Figure 5, A and B, demonstrates the effects of 50 µmol/l EMD 57033 and 5 mmol/l ORG 30029 on intracellular calcium transients as detected by aequorin. EMD 57033 (50 µmol/l) did not significantly change the peak of the [Ca⁺⁺]_i transient; however, it prolonged the time course of the [Ca⁺⁺]_i transient and increased diastolic [Ca⁺⁺]_i in both nonfailing and failing myocardium as shown in table 1. ORG 30029 (5 mmol/l) increased the peak of the Ca⁺⁺ transient without affecting diastolic [Ca⁺⁺]_i in both nonfailing and failing human myocardium (fig. 5B) and prolonged the time course of the [Ca⁺⁺]_i transient in failing myocardium as shown in table 1.

View larger version (16K): [in this window] [in a new window]   Fig. 5.   Effects of 5 and 50 µmol/l EMD 57033 on the light transients in a muscle from a failing heart loaded with aequorin (A). Effect of 5 mmol/l ORG 30029 on the light transient in a muscle from a failing heart loaded with aequorin (B). Both preparations were in Krebs' buffer stimulated at 1 Hz with a square pulse of 5 msec at 37°C.

Effect of EMD 57033 and ORG 30029 on force of contraction and diastolic force in the presence of isoproterenol. In isolated fibers from human myocardium, isoproterenol has been reported to increase the force of contraction and to shorten the time to relaxation by increasing intracellular cAMP and thereby increasing the rate of Ca⁺⁺ uptake by the SR. To test whether 1 µmol/l isoproterenol would reverse the detrimental effects on relaxation by both compounds, we added isoproterenol to preparations pretreated with either EMD 57033 or ORG 30029. As seen in figure 6A, the active force of contraction remained unchanged after the addition of isoproterenol in the presence of 50 µmol/l EMD 57033; however, diastolic force decreased to pre-EMD levels. In contrast, isoproterenol further increased active force of contraction in the presence of 5 mmol/l ORG 30029 while decreasing diastolic force to pre-ORG levels (fig. 6B).

View larger version (22K): [in this window] [in a new window]   Fig. 6.   Effect of EMD 57033 (A) and ORG 30029 (B) on active force and diastolic force in the presence of isoproterenol in intact muscle preparations of failing human myocardium (n = 4). The muscle preparations were stimulated at 1 Hz with a square pulse of 5 msec at 37°C in Krebs' buffer. *P < .05 compared with basal levels; P < .05 compared with ORG 30029 (5 mmol/l).

Effect of EMD 57033 and ORG 30029 on myofilament Ca⁺⁺ responsiveness in skinned fibers preparations from human myocardium. To test whether EMD and ORG increase the sensitivity of the myofilaments to Ca⁺⁺, we examined the effects of these two agents in skinned fiber preparations from nonfailing and failing human myocardium. EMD 57033 and ORG 30029 both shifted the force-pCa relationship to the left (EMD 57033 pCa, 0.14; ORG 30029 pCa, 0.06) in nonfailing human preparations (fig. 7A and table 2). Similar effects were observed in failing human myocardium (EMD 57033 pCa, 0.16; ORG 30029 pCa, 0.09) (fig. 7B and table 2). As shown in table 2, ORG 30029 had no significant effect on the maximal Ca⁺⁺-activated force, whereas EMD 57033 increased the maximal Ca⁺⁺-activated force by 15 ± 10% (n = 4) and 21 ± 12% (n = 6) in nonfailing and failing myocardium, respectively.

View larger version (28K): [in this window] [in a new window]   Fig. 7.   Effect of ORG 30029 and EMD 57033 on the[Ca++]-force relationship in skinned fibers preparations of human nonfailing (A) and failing (B) myocardium. Each Ca++-activation experiment (with or without drugs) was fitted individually to the Hill equation: F/Fmax = [Ca++]n/[Ca++]n + [Ca++]50%n; the Hill parameters Fmax, n and [Ca++]50% were then obtained by nonlinear regression analysis for that one individual experiment. The Hill parameters were then averaged for the different groups of experiments (i.e., no drug, +EMD 57033, +ORG 30029), yielding a mean ± S.E.M. for the Hill parameters of each group. The curves depicted in this figure were derived by inputting the mean of the Hill parameters from each group into the Hill equation. The data points represent the mean force generated at that pCa in each individual group.

Correlation between Ca⁺⁺ sensitivity and increase in t_80% and diastolic force. To test whether Ca⁺⁺-sensitizing effects of EMD and ORG contribute to the increase in relaxation time and diastolic force we related the increase in t_80% and diastolic force to the Ca⁺⁺-sensitizing effects of EMD and ORG. For EMD 57033 there was a strong correlation between increase in the [Ca⁺⁺]_50% and increase in t_80% (R² = .98). ORG 30029 also demonstrated a strong correlation between the increase in the [Ca⁺⁺]_50% and the increase in t_80% (R² = .69). For EMD 57033 there was also a strong correlation between increase in the [Ca⁺⁺]_50% and diastolic force (R² = .98). ORG 30029 also demonstrated a strong correlation between the increase in the [Ca⁺⁺]_50% and diastolic force (R² =.85).

	Discussion

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Effects of EMD 57033 and ORG 30029 on force and relaxation. The potential benefit of calcium sensitizers in the treatment of heart failure is that their actions would bypass defects at the level of receptor-coupled membranes in failing myocytes and target the myofilaments directly to enhance the force of contraction while having little effect on energy utilization. However, several studies have shown that agents targeted at the myofilaments may have differential effects in nonfailing versus failing myocardium (Hajjar et al., 1988; Baudet and Ventura-Clapier, 1990; Gwathmey and Hajjar, 1992). These included DPI 201-106, which demonstrated a greater calcium sensitization in failing human myocardium (Hajjar et al., 1988), and caffeine, which also demonstrated a greater calcium sensitization in pressure-overloaded hypertrophied ferret hearts (Baudet and Ventura-Clapier, 1990) and myopathic turkey hearts (Gwathmey and Hajjar, 1992).

Mechanisms of action of ORG 30029 and EMD 57033. Ca⁺⁺ sensitizers act directly on the myofilaments to exert their inotropic actions. There is little known about the mechanism of action of ORG 30029 on the myofilaments; however, the thiadiazones have been studied extensively (Solaro et al., 1993; Grandis et al., 1995; Pan and Johnson, 1996). It has been suggested by Solaro et al. (1993) that EMD 57033's action may be directed at a site on the actin-myosin interface which would promote the interaction of myosin with the thin filaments increasing myofibrillar ATPase rate. Furthermore, Solaro et al. (1993) found that actin sliding velocity was increased in the presence of EMD 57033 with a motility assay. Increasing cross-bridge rate would, however, decrease the apparent Ca⁺⁺ sensitivity of the myofilaments (Brenner, 1988) and would also enhance relaxation, effects that are opposite to the ones observed with EMD 57033. It is unclear whether results from the motility assay, which is not regulated by troponin-tropomyosin, can be related to regulated isometrically contracting muscles (Solaro et al., 1993). More recently, with recombinant human TnC, Pan and Johnson (1996) found that EMD 57033 binds to the Ca⁺⁺/Mg⁺⁺ sites of TnC in a Ca⁺⁺-dependent and stereo-selective manner. Furthermore, Leijendekker and Herzig (1992) found that a compound related to EMD 57033 (EMD 53998) actually decreases the rate of actin-cross-bridge reaction in isolated fibers, and Strauss et al. (1992) found that the same compound (EMD 53998) antagonizes phosphate action on cross-bridges. From our present studies, it is not possible to deduce whether EMD 57033 changes cross-bridge kinetics. However, because failing hearts have decreased energy reserve, Ca⁺⁺-sensitizing agents that increase cross-bridge kinetics would increase energy consumption and would therefore further impair the energy balance in these hearts (Ingwall et al., 1993; Gwathmey and Ingwall, 1995).

Limitations of the study. In our studies we used aequorin to measure intracellular [Ca⁺⁺]_i. As a calcium indicator, aequorin is characterized by a high sensitivity, a high signal-to-noise ratio, a fast response time and no Ca⁺⁺-buffering action (Blinks, 1982). However, aequorin has a nonlinear [Ca⁺⁺] versus light relationship with the curve flattening at low [Ca⁺⁺] in the range of 100 to 150 nmol/l. In our preparations, diastolic [Ca⁺⁺]_i was measured to be 100 to 300 nmol/l, which is well within the linear range of the aequorin calibration curve and agrees well with reports using microelectrodes and other calcium indicators (Blinks et al., 1982). Furthermore this range of diastolic Ca⁺⁺ (200-300 nmol/l) is in good agreement with measurements of diastolic Ca⁺⁺ in human myocardium with the fluorescent indicator, Fura-2 (Beuckelmann et al., 1992).

Conclusion. Taken together, the data presented herein provide evidence that Ca⁺⁺ sensitizers have the potential to increase the force of contraction to the same extent in nonfailing and failing myocardium. However, the benefit in treatment of heart failure may be limited by a worsening of diastolic relaxation and increased diastolic force in the presence of such agents. A combination of agents that increase cAMP and sensitize the myofilaments to Ca⁺⁺ may be helpful in the therapy of heart failure because of their ability to increase the force of contraction and to prevent an increase in diastolic [Ca⁺⁺]_i and force.