Contraction of cardiac muscle is regulated by a change in intracellular Ca2+ concentration ([Ca2+]i), and Ca2+ binding to the regulatory site of troponin-C is an important step for tension development (1). After Ca2+ binds to troponin-C, the cross-bridges attach to thin filaments and contraction is initiated. The various types of inotropic drugs available can be classified as follows: (a) drugs that increase Ca2+ transients [e.g., digitalis, β-adrenoceptor stimulation (2)] or prolong the time course of Ca2+ transients; (b) drugs that increase the affinity of troponin-C for Ca2+ [e.g., caffeine (3), α-adrenoceptor stimulation (4)]; and (c) drugs that alter cross-bridge kinetics [e.g., EMD 53998 (5)]; furthermore (d) some drugs are expected to increase maximal tension.
Some of the phosphodiesterase type III inhibitors increase the Ca2+ sensitivity of the contractile elements accompanying the increase in Ca2+ transients (5-7), although others do not increase the Ca2+ sensitivity (8). Pimobendan (UD-CG 115 BS; 4,5-dihydro-6-[2-(pethoxyphenyl)-5-benzimidazolyl]-5-methyl-3(2H)-pyridazinone), a phosphodiesterase inhibitor, is already used clinically and has been reported to improve the hemodynamic state remarkably without serious side effects in patients with heart failure (9). The cardiotonic effect of pimobendan is considered to be due to both an increase in the Ca2+ sensitivity (6,10,11) and in the intracellular cyclic AMP concentration (6,12). However, orally administered pimobendan is converted to its active form UD-CG 212 Cl (Fig. 1) by hepatic demethylation (13). In contrast to pimobendan, the mechanism of the effects of UD-CG 212 Cl on mammalian cardiac muscles has not been fully clarified. Therefore, we investigated the effects of UD-CG 212 Cl on intracellular Ca2+ transients and contraction in ferret cardiac muscles using the aequorin method. Preliminary results were reported previously in abstract form (14,15).
The study conformed to the Guide for the Care and Use of Laboratory Animals (US NIH publication no. 85-23, revised 1985).
Ferrets (body weight 800-1,200 g) were anesthetized with pentobarbital [intraperitoneal (i.p.) injection, 100 mg/kg], and the heart was quickly removed. After the blood was washed retrogradely, thin papillary muscles (diameter 0.4-1.0 mm) were dissected from the right ventricle in a bath perfused with normal Tyrode's solution at 30 °C. Both ends of the preparation were tied with silk threads, and the preparation was mounted horizontally in an experimental chamber. One end of the preparation was connected to a fixed hook; the other end was connected to the arm of a tension transducer (BG-10, Kulite, NJ, U.S.A.). A pair of platinum black electrodes placed in parallel with the preparation in the experimental chamber was used for electrical stimulation. The experimental chamber was continuously perfused with Tyrode's solution, and the temperature was maintained at 30 ° ± 0.5 °C. The preparation was stimulated with a 5-ms rectangular pulse of 1.2-fold threshold and slowly stretched to Lmax, the length at which developed tension became maximal. The stimulation frequency was 0.2 Hz. To produce tetanic contraction, the preparation was treated with ryanodine (5 μM) and repetitively stimulated at 10 Hz for >6 s (pulse duration 40 ms, strength three-fold threshold) (2,16,17). Length and diameter of the preparation were 3.9 ± 0.2 mm and 0.72 ± 0.04 mm (mean ± SEM, n = 23), respectively.
Measurement of intracellular Ca2+transients
The calcium-sensitive photoprotein aequorin, which was dissolved in 150 mM HEPES solution with a final aequorin concentration of 50-100 μM, was microinjected into 100-200 superficial cells of the preparation. Aequorin light signal was detected with a photomultiplier (EMI 9789A, Ruislip, U.K.) in a small housing, according to the method of Allen and Kurihara (18). All data were stored in a computer (PC-9801, NEC, Tokyo, Japan) for later analysis. To improve the signal/noise ratio, 64-128 signals were averaged in twitch and 2-4 signals were averaged in tetanic contraction.
The light signal of aequorin was converted into intracellular Ca2+ concentration ([Ca2+]i) by in vitro calibration (19). The constants used in this experiment were as follows: n, 3.14; KR, 4.025 · KTR, 114.6 (2).
Normal Tyrode's solution, used for dissecting the preparations and for superfusing the muscle preparations during the injection of aequorin, was composed of the following (in mM): Na+ 135, K+ 5, Ca2+ 2, Mg2+ 1, Cl- 102, HCO3- 20, HPO42- 1, SO42- 1, acetate 20, glucose 10, and insulin 5 U/L, pH, 7.35 at 30 °C when equilibrated with 5% CO2/95% O2. In most experiments, Tyrode's solution was buffered with HEPES (HEPES-Tyrode's solution) (in mM): Na+ 128, K+ 5, Ca2+ 2, Mg2+ 1, Cl- 117, SO42- 1, acetate 20, HEPES 5, glucose 10, and insulin 5 U/L; pH was adjusted to 7.40 with NaOH at 30 °C. When extracellular Ca2+ concentration ([Ca2+]o) was decreased, the concentration of CaCl2 was decreased without compensating osmotic pressure (2). The solution was equilibrated with 100% O2. The temperature of the solution was maintained at 30 ° ± 0.5 °C.
To evaluate the effects of UD-CG 212 Cl, the following parameters were measured: (a) peaks of Ca2+ transients and tension; (b) the time to peak light (TPL) and time to peak tension (TPT), which are the times between the onset of stimulus and the peak of light or tension; (c) the decay time of Ca2+ transients, the time for the aequorin light to decay from the peak to 50% (TL50), and the time for the aequorin light to decay from 75 to 25% of the peak (TL75-25); (d) the relaxation time, the time for the tension to decrease from the peak to 50% (TR50) and the time for the tension to decrease from 75 to 25% of peak (TR75-25). Because these parameters were also measured in previous reports, the effects of UD-CG 212 Cl in the present study could be compared with those of isoproterenol, epinephrine, EMD 53998, 3-isobutyl-1-methylxanthine (IBMX), and DB cyclic AMP (2,5,7,17,20,21).
Aequorin was purchased from Dr. J. R. Blinks (Friday Harbor, WA, U.S.A.). UD-CG 212 Cl was a gift from Nippon Boehringer Ingelheim (Kawanishi, Hyogo, Japan), and the stock solution was made by dissolving UD-CG 212 Cl in dimethyl sulfoxide (DMSO) (Wako Pure Chemical Industries, Osaka, Japan), which was stored at 4 °C. The maximum concentration of DMSO was 0.05%; we previously confirmed that DMSO at a concentration <0.1% does not significantly affect aequorin light and tension at steady state (22). Ryanodine was purchased from Agri System (Wind Gap, PA, U.S.A.). Stock solution of ryanodine was made by dissolving ryanodine in distilled water, which was stored at 0 °C.
Student's t test (for paired samples) was performed, and the statistically significant change was verified at p < 0.05 (two-tailed).
Effects of UD-CG 212 Cl at 2 mM[Ca2+]o
UD-CG 212 Cl was added to the solution after stabilization of the preparation in the HEPES-Tyrode's solution was confirmed. Aequorin light signals reached the steady state within 10-20 min and tension within 20-30 min after the application of UD-CG 212 Cl. The time lag between the change in the light signal and tension was the result of the slow diffusion of UD-CG 212 Cl into the core of the preparation, as the aequorin light signal was recorded from the superficial cells and tension was recorded from the entire preparation. Therefore, we recorded the aequorin light signal and tension after tension reached the steady state. Because the effects of UD-CG 212 Cl on the aequorin light signal and tension did not recover soon after the removal of the drug, UD-CG 212 Cl was added to the solution in a cumulative manner.
The increase in tension induced by UD-CG 212 Cl applied to the solution with 2 mM [Ca2+]o (extracellular Ca2+ concentration) was small, and the change was not statistically significant except for the concentration of 10-5M(Fig. 2B). With [Ca2+]o of 2 mM, the control values of the peaks of Ca2+ transients and tension were 1.36 ± 0.14 μM (n = 6) and 44.4 ± 4.5 mN/mm2 (n = 7), respectively. Because the maximal tension in tetanic contraction was ≈50-60 mN/mm2 (described in the Discussion section), tension before the application of UD-CG 212 Cl was already near the maximal level (Fig. 2).
We considered that the potentiating effect of inotropic agents was barely observable because the tension of the control was near the maximal level. Therefore, in the subsequent experiments, [Ca2+]o was decreased to 0.5 mM to decrease the peaks of Ca2+ transients and tension in control.
Effects of UD-CG 212 Cl at 0.5 mM [Ca2+]o
When [Ca2+]o was decreased to 0.5 mM, the peaks of Ca2+ transients and tension were significantly decreased. The peaks of Ca2+ transients and tension at 0.5 mM [Ca2+]o were 1.20 ± 0.10 μM (n = 7) and 24.5 ± 4.6 mN/mm2 (n = 7), respectively. UD-CG 212 Cl potentiated contraction, with an increase in the peak of Ca2+ transients (Figs. 3 and 4). Although the peak of Ca2+ transients increased slightly at low concentrations of UD-CG 212 Cl, the change was not statistically significant. However, at the higher concentration of UD-CG 212 Cl (10-5-10-4M), the peak of Ca2+ transients was increased from 1.20 ± 0.10 μM (n = 7) (control) to 1.60 ± 0.09 μM (n = 7, p < 0.05) (10-5M) and to 2.03 ± 0.10 μM (n = 7, p < 0.01) (10-4M). The peak tension was increased from 24.5 ± 4.6 mN/mm2 (n = 7) (control) to 27.8 ± 4.9 mN/mm2 (n = 6, p < 0.01) (10-7M), 33.0 ± 5.2 mN/mm2 (n = 6, p < 0.01) (10-6M), 37.8 ± 5.2 mN/mm2 (n = 7, p < 0.01) (10-5M), and 45.1 ± 6.6 mN/mm2 (n = 7, p < 0.01) (10-4M).
We also evaluated the change in the time course of Ca2+ transients and tension altered by UD-CG 212 Cl (Figs. 5 and 6). UD-CG 212 Cl shortened TPL from 52.7 ± 2.5 ms (n = 7) (control) to 49.2 ± 3.0 ms (n = 6, p < 0.05) (10-6M) and to 45.3 ± 1.4 ms (n = 7, p < 0.01) (10-5M). UD-CG 212 Cl shortened the decay time of Ca2+ transients (TL50, TL75-25) at 10-6-10-4M. TL50 was shortened from 46.4 ± 2.1 ms (n = 7) (control) to 39.0 ± 1.9 ms (n = 6, p < 0.01) (10-6M), 39.6 ± 2.2 ms (n = 7, p < 0.01) (10-5M), and 36.7 ± 2.6 ms (n = 7, p < 0.01) (10-4M). TL75-25 was shortened from 48.9 ± 2.3 ms (n = 7) (control) to 42.7 ± 2.6 ms (n = 6, p < 0.05) (10-6M), 40.6 ± 2.5 ms (n = 7, p < 0.01) (10-5M), and 39.7 ± 2.8 ms (n = 7, p < 0.01) (10-4M). UD-CG 212 Cl also shortened TPT from 195 ± 7 ms (n = 7) (control) to 187 ± 6 ms (n = 6, p < 0.05) (10-8M), 180 ± 7 ms (n = 6, p < 0.05) (10-6M), 178 ± 7 ms (n = 7, p < 0.05) (10-5M), and 170 ± 6 ms (n = 7, p < 0.01) (10-4M). However, the relaxation time (TR50, TR75-25) varied among preparations, and no significant change was observed.
As shown in Fig. 3F, a hump was observed in the Ca2+ transients at the highest concentration (10-4M) of UD-CG 212 Cl. A similar hump has been reported with a higher concentration of epinephrine or isoproterenol (2,17,18).
Relation between [Ca2+]iand tension in tetanic contraction
When UD-CG 212 Cl concentration was increased from 10-7 to 10-6M, the increase in tension was greater than that in the peak of Ca2+ transients, which is shown when the peak of Ca2+ transients was simply plotted against the peak tension (Fig. 7). We considered this to be due to an increase in the Ca2+ sensitivity of the contractile elements. Therefore, we investigated the effects of 10-6M UD-CG 212 Cl on the relation between [Ca2+]i and tension at steady state in tetanic contraction.
When the preparation was repetitively stimulated after the treatment with 5 μM ryanodine, sustained tetanic contraction was produced (2,16,17). [Ca2+]i and tension were altered by changing [Ca2+]o. The relation between [Ca2+]i and tension was measured at 6 s after the onset of stimulation and fitted with Hill equation: T = Tmax·[Ca2+]iH/([Ca2+]iH + K½H), where T is the measured tension, Tmax is maximal tension, K½ is the [Ca2+]i which causes 50% of Tmax and H is the Hill coefficient. UD-CG 212 Cl (10-6M) did not alter the [Ca2+]i-tension relation at steady state (Fig. 8C). None of the parameters differed in the absence (Tmax, 57.8 ± 10.1 mN/mm2; K½, 1.14 ± 0.16 μM;H, 4.7 ± 0.4) or presence of 10-6M UD-CG 212 Cl (Tmax, 56.7 ± 9.5 mN/mm2; K½, 1.12 ± 0.12 μM;H, 4.7 ± 0.4) (n = 4).
We demonstrated that a metabolite of pimobendan, UD-CG 212 Cl, potentiated twitch tension at lower [Ca2+]o in ferret papillary muscles. The peak of twitch tension induced by 0.2-Hz stimulation in the solution containing 2 mM [Ca2+]o was almost the maximum level in ferret ventricular muscle, as indicated by comparison of the peak twitch tension (44.4 mN/mm2, n = 7) and the tetanic tension at 20 mM [Ca2+]o (≈50-60 mN/mm2). Therefore, the twitch-potentiating effect of UD-CG 212 Cl was not clearly observed at 2 mM [Ca2+]o, because tension was almost saturated. Similarly, the control tension level was previously reported to be critical in inducing the twitch-potentiating effect of isoproterenol in rat ventricular muscles (23). The twitch-potentiating effects at the higher UD-CG 212 Cl (10-5-10-4M) in the HEPES-Tyrode's solution containing 0.5 mM [Ca2+]o accompanied a significant increase in the peak of Ca2+ transients. In addition to these changes (the peaks of Ca2+ transients and tension), UD-CG 212 Cl altered the time courses of the Ca2+ transients and tension; in particular, significant changes were observed in the TPT and the decay time of Ca2+ transients. These changes were qualitatively similar to those that occur when β-adrenoceptor is stimulated by isoproterenol or epinephrine (2,17,18,23).
Because UD-CG 212 Cl selectively inhibits phosphodiesterase type III (11,24,25), the increase in cyclic AMP concentration (12) and the activation of protein kinase A are the most likely mechanisms to explain these changes induced by UD-CG 212 Cl, which is similar to the case of β-adrenoceptor stimulation. When the cyclic AMP concentration is increased by UD-CG 212 Cl, protein kinase A is activated by cyclic AMP, and L-type Ca2+ channel protein, phospholamban, and troponin-I are phosphorylated (26,27).
Phosphorylation of L-type Ca2+ channel protein increases Ca2+ influx (26), which enhances the release of Ca2+ from sarcoplasmic reticulum (SR). The increased Ca2+ influx through the Ca2+ channels is removed by SR at a faster rate, which secondarily increases the Ca2+ content of the SR. These factors are considered to increase the peak of Ca2+ transients. A significant increase in the peak of the Ca2+ transients was observed even at similar low UD-CG 212 Cl concentrations when [Ca2+]o was 2 mM, but the increase was not observed at lower UD-CG 212 Cl concentrations when [Ca2+]o, was 0.5 mM. At higher [Ca2+]o, the gradient of Ca2+ concentration between inside and outside the surface membrane is larger than that at 0.5 mM [Ca2+]o, and more Ca2+ influx through the Ca2+ channel is expected at higher [Ca2+]o. Therefore, Ca2+ influx through the Ca2+ channel is one of the factors responsible for the increase in the Ca2+ transients. In addition, the increase in the Ca2+ content of the SR, which is subsequently induced by the increased Ca2+ influx, generates a greater Ca2+ gradient through the SR membrane. This larger gradient of the Ca2+ concentration is probably related to the faster TPL (2). The faster TPT (Fig. 5B) can be explained by an increase in cyclic AMP that increases the cross-bridge cycling rate as well as by the faster TPL, as in the case of β-adrenoceptor stimulation (20,21,28,29).
Phosphorylation of phospholamban enhances the Ca2+ uptake by the SR (27), which shortens the decay time of Ca2+ transients (Fig. 6A)(2,17,21,23). The enhanced Ca2+ uptake by the SR also contributes to the increase in the peak of Ca2+ transients, as already described. The increase in the Ca2+ transients induced by UD-CG 212 Cl is much smaller than the increase induced by isoproterenol. Although isoproterenol and UD-CG 212 Cl increase the cyclic AMP concentration, the difference in the effects of isoproterenol and UD-CG 212 Cl is probably due to the quantitative difference in the increase in cyclic AMP concentration.
The twitch-potentiating effect of UD-CG 212 Cl was observed at 10-7M when [Ca2+]o was 0.5 mM. However, the peak of Ca2+ transients was not significantly increased at the same concentration. An apparent dissociation of the increase in tension and the peak of Ca2+ transients was observed at 10-7-10-6M. Therefore, UD-CG 212 Cl apparently increases the Ca2+ sensitivity of the contractile elements. A similar apparent dissociation between the change in Ca2+ transients and cell shortening is reported in isolated myocytes of guinea pigs (30). However, in addition to the changes in the peaks of Ca2+ transients and contraction, the time courses of contraction and Ca2+ transients were altered by UD-CG 212 Cl [tension in the present study and cell shortening in the study of Van Meel and colleagues (30)]. Therefore, direct comparison of the relation between the peaks of [Ca2+]i and tension is difficult in the assessment of the change in the Ca2+ sensitivity. Therefore, we wished to determine whether the relation between [Ca2+]i and tension in tetanic contraction is altered at 10-6M UD-CG 212 Cl; the relation was not influenced. If the cyclic AMP concentration is increased by UD-CG 212 Cl and troponin-I is phosphorylated, a decrease in the Ca2+ sensitivity of the contractile elements is expected (2). Therefore, the relation between [Ca2+]i and tension in tetanic contraction should be shifted to the right as in the case of β-adrenoceptor stimulation induced by a high concentration of isoproterenol (2). However, no significant change in this relation was observed at 10-6M UD-CG 212 Cl. Similarly, when a low concentration of isoproterenol was used, the peaks of Ca2+ transients and tension were increased but the Ca2+ sensitivity was not altered (2). Therefore, the increase in the cyclic AMP concentration induced by 10-6M UD-CG 212 Cl is probably not sufficient to decrease the Ca2+ sensitivity but is sufficient to increase the Ca2+ transients and tension. Another possible explanation for the unchanged relation between [Ca2+]i and tension at 10-6M UD-CG 212 Cl is that the decrease in the Ca2+ sensitivity induced by cyclic AMP (phosphorylation of troponin-I; described herein) is antagonized by the direct Ca2+-sensitizing effect of UD-CG 212 Cl. However, the changes in the time courses of Ca2+ transients and tension are slight, even as compared with those induced by low concentrations of isoproterenol (2). The slight increase in cyclic AMP could also explain the fact that relaxation time was not consistently altered by UD-CG 212 Cl (Fig. 6B), although many factors are related to the relaxation time (2). Therefore, numerous possible factors must be considered in the explanation of the twitch-potentiating effect of UD-CG 212 Cl.
Our conclusion is supported by the observation that UD-CG 212 Cl does not increase the Ca2+ sensitivity of the skinned preparation of human failing hearts (11). If UD-CG 212 Cl does not increase the Ca sensitivity, the most plausible explanation for the positive inotropic effect in intact preparation is attributable to the slight increase in the cyclic AMP concentration, as in the case of OPC compound (8). Recently, very low concentrations of UD-CG 212 Cl were reported to sensitize the myofilaments for Ca2+, whereas higher concentrations desensitize the myofilaments, as proved in skinned preparations of dogs (31). However, in the present study, in intact ferret papillary muscles, UD-CG 212 Cl increased tension in a concentration-dependent manner (Fig. 4A) and we noted no significant inotropic effect at very low concentrations of UD-CG 212 Cl. The different results might be due to the different preparations used (intact vs. skinned, ferret vs. dog). In addition, inorganic phosphate, which inhibits tension, is proposed to be another factor related to the inotropic effect of UD-CG 212 Cl. However, we noted no inotropic effect even with a higher phosphate concentration (5 mM) in the Tyrode's solution containing 2 mM [Ca2+]o (K. Komukai and S. Kurihara, unpublished observations). That the more marked inotropic effect was observed at higher concentrations of UD-CG 212 Cl, in contrast to the direct effect of the drug on the skinned preparation (31), further supports the view that the positive inotropic effect of UD-CG 212 Cl in intact preparations is caused by an increase in the Ca2+ transients. Therefore, the positive inotropic effect of UD-CG 212 Cl is not due to the direct effect on the myofilaments but is primarily attributable to the change in Ca2+ transients, which is induced by the slight increase in the cyclic AMP concentration.
Acknowledgment: We thank Nippon Boehringer Ingelheim for providing UD-CG 212 Cl, Mary Beth Sibuya for reading the manuscript, and Naoko Tomizawa for skilled technical assistance. K.K. thanks Professors Tetsuo Okamura and Seibu Mochizuki for their encouragement.
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