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The Novel Insulinotropic Mechanism of Pimobendan: Direct Enhancement of the Exocytotic Process of Insulin Secretory Granules by Increased Ca²⁺ Sensitivity in ß-Cells¹

Department of Metabolism and Clinical Nutrition, Kyoto University Faculty of Medicine, Kyoto 606–01; and the Department of Internal Medicine, Kitano Hospital (K.T.), Osaka 530, Japan

Address all correspondence and requests for reprints to: Shimpei Fujimoto, M.D., Department of Metabolism and Clinical Nutrition, Kyoto University Faculty of Medicine, 54 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606, Japan. E-mail: fujimoto{at}metab.kuhp.kyotou.ac.jp

	Abstract

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Pimobendan is a new class of inotropic drug that augments Ca²⁺ sensitivity and inhibits phosphodiesterase (PDE) activity in cardiomyocytes. To examine the insulinotropic effect of pimobendan in pancreatic ß-cells, which have an intracellular signaling mechanism similar to that of cardiomyocytes, we measured insulin release from rat isolated islets of Langerhans. Pimobendan augmented glucose-induced insulin release in a dose-dependent manner, but did not increase cAMP content in pancreatic islets, indicating that the PDE inhibitory effects may not be important in ß-cells. This agent increased the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in the presence of 30 mM K⁺, 16.7 mM glucose, and 200 µM diazoxide, but failed to enhance the 30 mM K⁺-evoked [Ca²⁺]_i rise in the presence of 3.3 mM glucose. Insulin release evoked by 30 mM K⁺ in 3.3 mM glucose was augmented. Then, the direct effects of pimobendan on the Ca²⁺-sensitive exocytotic apparatus were examined using electrically permeabilized islets in which [Ca²⁺]_i can be manipulated. Pimobendan (50 µM) significantly augmented insulin release at 0.32 µM Ca²⁺, and a lower threshold for Ca²⁺-induced insulin release was apparent in pimobendan-treated islets. Moreover, 1 µM KN93 (Ca²⁺/calmodulin-dependent protein kinase II inhibitor) significantly suppressed this augmentation. Pimobendan, therefore, enhances insulin release by directly sensitizing the intracellular Ca²⁺-sensitive exocytotic mechanism distal to the [Ca²⁺]_i rise. In addition, Ca²⁺/calmodulin-dependent protein kinase II activation may at least in part be involved in this Ca²⁺ sensitization for exocytosis of insulin secretory granules.

	Introduction

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PIMOBENDAN [4,5-dihydro-6–2-(4-methoxyphenyl)-1H-benzimidazole-5-yl-5-methyl-3(2H)-pyridazinone] is a new class of nondigitalis inotropic drug and is used clinically in the treatment of heart failure. The agent is reported to act by two different mechanisms. First, it inhibits mainly phosphodiesterase III (PDE III) activity and elevates the intracellular cAMP concentration. The resultant activation of cAMP-dependent protein kinase phosphorylates many target proteins, including Ca²⁺ channels in the plasma membrane, thereby leading to more enhanced Ca²⁺ influx, a greater accumulation of intracellular Ca²⁺ ([Ca²⁺]_i), and, hence, more tension in the cardiac muscle (1). Secondly, it has a Ca²⁺-sensitizing effect. That is, it increases myocardial contractility, in that more force per given amount of cytoplasmic Ca²⁺ was generated (2). This Ca²⁺-sensitizing effect is not necessarily related to the PDE III inhibitory effect, because milrinone, a known, more selective PDE III inhibitor than pimobendan (1), was found to exhibit no Ca²⁺-sensitizing effect (2). It has been established that this sensitizing effect is derived from pimobendan’s direct effect to increase in the Ca²⁺ affinity of troponin C, the calcium-binding protein that plays a regulatory role in cardiac muscle contraction (3, 4).

In pancreatic ß-cells, the uptake and metabolism of glucose and other nutrients lead to the increase in the ATP concentration or ATP/ADP ratio that results in closure of the ATP-sensitive K⁺ channels (K_ATP channels) (5, 6). The decreased membrane permeability for K⁺ caused by K_ATP channel closure induces depolarization of the plasma membrane, thus activating the voltage-dependent Ca²⁺ channels (VDCC), leading to Ca²⁺ entry and the rise in [Ca²⁺]_i that triggers insulin secretion (7, 8). Although how the [Ca²⁺]_i rise regulates the complex series of steps that results in insulin release remains largely unknown, it is now established that Ca²⁺-binding proteins, including protein kinase C (PKC) (9), calmodulin (10, 11), and calcyclin (12), are important mediators of the late step of stimulation-secretion coupling after the rise in [Ca²⁺]_i.

The stimulation-contraction coupling of cardiomyocytes and the stimulation-secretion coupling of ß-cells share some common features. It is well known that [Ca²⁺]_i plays a very important role in both couplings. The elevation of [Ca²⁺]_i triggers a physiological effect, contraction in the case of cardiomyocytes and insulin secretion in the case of pancreatic ß-cells, in which Ca²⁺-binding proteins are thought to be involved in both contractile and exocytotic events distal to the rise in [Ca²⁺]_i. Moreover, the elevation of [Ca²⁺]_i produces phosphorylation of proteins that are involved in their effects via intracellular protein kinase activation (2, 13, 14).

In this study, we found that pimobendan, an agent characterized by both a PDE inhibitory effect and a calcium-sensitizing effect in cardiomyocytes, has an insulinotropic effect in pancreatic islets. The mechanism by which the calcium-sensitizing effect of pimobendan operates was also investigated.

	Materials and Methods

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Animals
Male Wistar rats were obtained from Shimizu Co. (Kyoto, Japan). The animals were fed standard lab chow ad libitum and allowed free access to water in an air-conditioned room with a 12-h light, 12-h dark cycle until used in the experiments. All experiments were carried out with rats aged 8–12 weeks.

Measurement of insulin release from intact islets
Islets of Langerhans were isolated from Wistar rats by collagenase digestion as described previously (15). Insulin release from intact islets was monitored using either static incubation or perifusion conditions. For static incubation experiments, the islets were preincubated at 37 C for 30 min in Krebs-Ringer bicarbonate buffer (KRBB) supplemented with 3.3 mM glucose and 0.2% BSA. Groups of islets were then batch incubated for 30 min in 0.7 ml KRBB with test materials. At the end of the incubation period, islets were pelleted by centrifugation (10,000 x g, 180 sec), and aliquots of the buffer were sampled. For perifusion experiments, groups of islets were placed in each of the parallel chambers (400 µl each) of a perifusion apparatus and perifused with the KRBB supplemented with 3.3 mM glucose, 0.2% BSA, and 10 mM HEPES adjusted to pH 7.4 at a rate of 0.7 ml/min at 37 C. The medium was continuously gassed with 95% O₂ and 5% CO₂. Islets usually were perifused for 30 min to establish a stable insulin secretory rate at the basal level of glucose and then were exposed to test substances. Perifusate samples were collected at the times indicated in the figures. The amount of immunoreactive insulin was determined by RIA, using rat insulin as standard (16). Experiments using the same protocol were repeated at least three times to ascertain reproducibility.

Measurement of cAMP contents
Groups of islets were batch incubated for 30 min in 0.4 ml KRBB with test materials. At the end of the incubation period, they were placed on ice, and each of the batch-incubated islet groups was mixed with 0.1 ml 30% trichloroacetic acid. After removing the trichloroacetic acid by water-saturated diethyl ether, cAMP contents were determined by RIA (Yamasa Shoyu Co., Chiba, Japan) after the succinylation step.

Measurement of intracellular Ca²⁺
Isolated islets were washed in PBS and incubated with 0.25% trypsin and 1 mM EDTA solution (Life Technologies, Grand Island, NY) for 3 min at 37 C. Digestion was terminated by rinsing the cells in cold PBS. They were washed in PBS again, placed on small glass coverslips (15 x 4 mm), coated with Cell-Tak (Collaborative Biomedical Products, Bedford, MA) to accelerate cell adhesion, and incubated in KRBB supplemented with 3.3 mM glucose and 0.2% BSA in humidified air containing 5% CO₂ at 37 C until used for experiments. Fura-2/acetoxymethyl ester (fura-2/AM; 1 µM) was loaded in freshly dispersed islet cells for 30 min at 37 C. A heat-controlled chamber on a stage of an inverted microscope kept at 36 ± 1 C was superfused with KRBB supplemented with 3.3 mM glucose and 10 mM HEPES adjusted to pH 7.4. Ratiometry of the emission light (510 nm) elicited by dual wave excitation light (340 and 380 nm) was performed on an ARGUS-50 image analyzing system (Hamamatsu Photonics, Hamamatsu, Japan). The 340 nm (F₃₄₀) and 380 nm (F₃₈₀) fluorescence signals were detected every 10 sec, and the ratios (F₃₄₀/F₃₈₀) were calculated. In vivo calibration was performed according to the equation: [Ca²⁺]_i = K_dß(R - R_min)/(R_max - R), where R_max is the ratio (F₃₄₀/F₃₈₀) after treating dispersed cells with 20 µM ionomycin in the presence of 10 mM calcium, R_min is the ratio in the presence of 20 µM ionomycin and 10 mM EGTA, R is the measured F₃₄₀/F₃₈₀, ß is the ratio of F_{380 min} and F_{380 max}, and K_d is the dissociation constant of fura-2 and Ca²⁺. The calibration values in this study were 3.49, 0.45, 2.20, and 224 nM for R_max, R_min, ß, and K_d respectively.

Measurement of insulin release from permeabilized islets (17)
After preincubation as described above, the islets were washed twice in cold potassium aspartate buffer (KA buffer) containing 140 mM potassium aspartate, 7 mM MgSO₄, 5 mM ATP, 3.3 mM glucose, 2.5 mM EGTA, 30 mM HEPES, and 0.5% BSA (pH 7.0), with CaCl₂ added to give a Ca²⁺ concentration of 30 nM. The islets were then permeabilized by high voltage discharge (four exposures each of 450-µsec duration to an electrical field of 4.0 kV/cm) in KA buffer and washed once with the same buffer. Groups of electrically permeabilized islets were then batch incubated for 30 min at 37 C in 0.4 ml KA buffer with various concentrations of Ca²⁺ and test materials. At the end of the incubation period, permeabilized islets were pelleted by centrifugation (15,000 x g, 180 sec), and aliquots of the buffer were sampled for immunoreactive insulin determination, as described above. The Ca²⁺ concentrations in the KA buffer were calculated using the dissociation constants of Martell and Smith (18). Experiments using the same protocol were repeated three times to ascertain reproducibility.

Materials
Pimobendan was donated by Nippon Boehringer Ingelheim (Hyogo, Japan), and wortmannin was provided by Kyowa Hakko (Tokyo, Japan). Nitrendipine, diazoxide, potassium aspartate, and 12-O-tetradecanoylphorbol-13-acetate (TPA) were obtained from Sigma Chemical Co. (St. Louis, MO). 2-[N-(2-hydroxyethyl)-N-(4-methoxybenzenesulfonyl)]-aminoN-(4-chlorocinnamyl)-N-methylbenzylamine (KN93) was obtained from Seikagaku Kogyo (Tokyo, Japan), bisindolylmaleimide was purchased from Calbiochem (La Jolla, CA), fura-2/AM was obtained from Molecular Probes (Eugene, OR), ATP was purchased from Kohjin (Tokyo, Japan), and rat insulin was obtained from Novo Nordisk (Bagsvaerd, Denmark). All other reagents were of analytical grade and were obtained from Nacalai Tesque (Kyoto, Japan). Pimobendan, 3-isobutyl-1-methylxanthine (IBMX), diazoxide, nitrendipine, TPA, bisindolylmaleimide, and wortmannin were first prepared in dimethylsulfoxide (DMSO) and then diluted to 1:1000 with buffer. The final concentration of DMSO did not exceed 0.2%, and the same concentration of DMSO was added to the control.

Statistical analysis
Results were expressed as the mean ± SE. Statistical significance was evaluated by unpaired Student’s t test. P < 0.05 was considered significant.

	Results

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Enhancement of glucose-stimulated insulin release by pimobendan in pancreatic islets
Pimobendan augmented 8.3 mM glucose-stimulated insulin release from rat islets in a dose-dependent manner (Fig. 1). The EC₅₀ for the potentiating activity was approximately 25 µM. Maximum effects were seen at concentrations of about 100 µM, and this concentration increased insulin release about 2.5-fold from 0.88 ± 0.10 to 2.38 ± 0.25 ng/islet·30 min (n = 5; P < 0.01 vs. control). Pimobendan (25 µM) did not increase insulin release with the basal (3.3 mM) concentration of glucose. Its effect was apparent only at glucose concentrations above 5.5 mM. High (16.7 mM) glucose-stimulated insulin release was still significantly augmented by pimobendan [4.57 ± 0.76 ng/islet·30 min (n = 5; control) vs. 6.72 ± 0.23 ng/islet·30 min (n = 5; pimobendan), P < 0.05; Fig. 2]. In perifusion experiments, 25 µM pimobendan did not affect insulin release in the presence of 3.3 mM glucose, but augmented both first phase (0–6 min) and second phase (after 7 min) insulin release induced by 16.7 mM glucose (Fig. 3B). This enhancement was still significant at 3 min [at 3 min, 1.01 ± 0.11 ng/10 islets·min (n = 6, pimobendan) vs. 0.18 ± 0.07 ng/10 islets·min (n = 6; control), P < 0.01; Fig. 3A]. The pimobendan effect was reversible for 15 min after cessation of pimobendan stimulation (Fig. 3B).

View larger version (42K): [in this window] [in a new window]   Figure 1. Concentration-dependent effects of pimobendan on 8.3 mM glucose-stimulated insulin release. Values represent the mean ± SE of five determinations in the same experiment. *, P < 0.01, vs. control (no pimobendan).

View larger version (36K): [in this window] [in a new window]   Figure 2. Effects of 25 µM pimobendan on insulin release in the presence of various concentration of glucose. Values represent the mean ± SE of five determinations in the same experiment. *, P < 0.01; **, P < 0.05 (vs. respective controls).

View larger version (20K): [in this window] [in a new window]   Figure 3. Effects of 25 µM pimobendan on biphasic 16.7 mM glucose-induced insulin release. Two groups of islets were perifused for 30 min with 3.3 mM glucose (G3.3) and for an additional 30 min with 16.7 mM glucose (G16.7) with or without 25 µM pimobendan. Pimobendan was introduced 10 min before the period of 16.7 mM glucose stimulation. A, Time course of insulin release during the first 9 min after exposure to 16.7 mM glucose. B, Time course of insulin release during 60 min after administration of 16.7 mM glucose, which was performed in the same experiment as that shown in A. It also shows the withdrawal effect of pimobendan for 30 min. Values represent the mean ± SE of six determinations in the same experiment. *, P < 0.01 vs. respective controls.

View larger version (35K): [in this window] [in a new window]   Figure 4. Effects of 25 µM pimobendan on 8.3 mM glucose-stimulated insulin release without diazoxide (a), with diazoxide (b), with diazoxide and a depolarizing concentration (30 mM) of K+ (c), and with diazoxide, a depolarizing concentration of K+, and nitrendipine (d). Values represent the mean ± SE of five determinations in the same experiment. *, P < 0.01 vs. respective controls. , P < 0.01 vs. control in a. ¶, P < 0.01 vs. control in b. §, P < 0.01 vs. control in c. NTD, Nitrendipine.

View larger version (23K): [in this window] [in a new window]   Figure 5. Effects of 25 µM pimobendan on the time course of a depolarizing concentration (30 mM) of K+-stimulated insulin release in the presence of 3.3 mM glucose for 30 min. K+ (30 mM) and pimobendan (25 µM) were introduced simultaneously. Values represent the mean ± SE of six determinations in the same experiment. *, P < 0.01 vs. respective controls. G, Glucose.

Effects of pimobendan on glucose stimulated-insulin release in the presence of TPA
Pimobendan (25 µM) still augmented the 8.3 mM glucose-stimulated insulin release that was enhanced by 10 nM or 50 nM TPA, a known PKC activator. The degrees of these enhancements were both about 2.2-fold. TPA (5 nM), which could not enhance the 8.3 mM glucose-stimulated insulin release, did augment that enhanced by 25 µM pimobendan (Table 2).

View larger version (23K): [in this window] [in a new window]   Figure 6. Fluorescence measurement of [Ca2+]i and responses to 25 µM pimobendan compared with controls under two different conditions in dispersed islet cells. A, A further increase in [Ca2+]i induced by pimobendan in the presence of 30 mM K+, 16.7 mM glucose, and 200 µM diazoxide. The glucose concentration was raised from 3.3 to 16.7 mM from 2 min. Cells that responded to 16.7 mM glucose were regarded as ß-cells. Then, diazoxide and a depolarizing concentration of K+ were introduced from 10 min. This trace during the first 15 min indicates the average [Ca2+]i of 15 such cells. Traces from 15–30 min indicate the average [Ca2+]i of 8 cells to which pimobendan was administrated and 7 control cells of these 15 cells. Vertical bars represent ±SE of 8 or 7 determinations at 17, 20, 25, and 30 min from four experiments, respectively. *, P < 0.01 vs. corresponding control. , P < 0.05 vs. corresponding control. G, Glucose. B, No effect of pimobendan on 30 mM K+-evoked [Ca2+]i rise. In the presence of 30 mM K+, 3.3 mM glucose and pimobendan were introduced from 0 min. Traces indicates the average [Ca2+]i of 16 cells to which pimobendan was administrated and 8 control cells. Vertical bars represent ±SE of 16 or 8 determinations from four experiments at 1, 2, 5, 10, and 15 min. No significant changes were observed compared with controls. G, Glucose.

View larger version (17K): [in this window] [in a new window]   Figure 7. Effects of 50 µM pimobendan on the Ca2+ dose response of insulin release in electrically permeabilized islets. Values represent the mean ± SE of five determinations in the same experiment. *, P < 0.05 vs. corresponding control. , P < 0.01 vs. corresponding control. ¶, P < 0.05 vs. 0.03 µM, pimobendan. §, P < 0.05 vs. 0.03 µM, control.

View larger version (13K): [in this window] [in a new window]   Figure 8. Curve of the dose-dependent effects of pimobendan on 0.32 µM Ca2+-induced insulin release from electrically permeabilized islets. Values represent the mean ± SE of five determinations in the same experiments. *, P < 0.01 vs. control.

Wortmannin (3 µM), a known myosin light chain kinase (MLCK) inhibitor (20), did not alter the control level of insulin release and did not attenuate that which was augmented by 50 µM pimobendan (Table 3). Wortmannin (10 µM) decreased the basal level of insulin release significantly (P < 0.01), increased its degree of enhancement to about 150%, and did not decrease the augmentation of pimobendan.

View larger version (42K): [in this window] [in a new window]   Figure 9. Inhibitory effects of KN93 on insulin release enhanced by 50 µM pimobendan at 0.32 µM Ca2+ in electrically permeabilized islets. *, P < 0.05; **, P < 0.01 (vs. pimobendan without KN93). Values represent the mean ± SE of five determinations in the same experiment. , P < 0.01 vs. respective control.

	Discussion

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Pimobendan was found to significantly enhance insulin release at certain concentrations of Ca²⁺ in electrically permeabilized islets, and it has been shown for the first time that pimobendan has calcium-sensitizing effects in pancreatic ß-cells. This calcium-sensitizing effect is unique; it is effective only at a level of Ca²⁺ concentration (0.18 and 0.32 µM) near the threshold for initiation of insulin release, and no significant augmentation was detected above this concentration. Moreover, pimobendan lowered the threshold level of Ca²⁺-induced insulin secretion. The similar sensitization near the threshold Ca²⁺ level and the lowered threshold have been also observed by pimobendan in reconstituted cardiac thin filament (4). This feature is quite different from the calcium-sensitizing effect of TPA or folskolin previously reported, in which Ca²⁺ sensitization is observed in a broad range (from 0.01–10 µM in TPA; from 1–10 µM in folskolin) (9, 24). Thus, it is possible that components other than the activation of protein kinase A or C are involved in the augmentation of Ca²⁺-induced insulin release by pimobendan.

Pimobendan has been reported to inhibit PDE III activity and to elevate intracellular cAMP in cardiomyocytes (1). In pancreatic ß-cells, cAMP augments insulin release by increasing the sensitivity to Ca²⁺ in a late step in exocytotic systems (25, 26). These observations are compatible with the possibility that pimobendan acts as a PDE inhibitor with increasing intracellular levels of cAMP, thereby enhancing Ca²⁺ sensitivity in pancreatic islets. However, as pimobendan did not increase the cAMP contents in our experiments, we conclude that the PDE inhibitory effects do not play a crucial part in its Ca²⁺-sensitizing effects. However, other PDE III-selective inhibitors have been reported to increase insulin release from pancreatic islets without any detectable elevation of cAMP content (27), although their Ca²⁺-sensitizing effects have not yet been studied. In addition, we observed preliminarily that UD-CG 212 Cl, a metabolite of pimobendan that was reported to have a more potent PDE III inhibitory effect than pimobendan in cardiomyocytes (1), has less insulinotropic effect than pimobendan. Further investigation into the details of the relationship between the Ca²⁺-sensitizing effect and PDE III inhibition in ß-cells is needed.

The glucose-induced second phase of insulin secretion dose not depend simply on [Ca²⁺]_i, but also involves an increase in the efficacy of the Ca²⁺ signal, which may be brought about by protein kinase stimulation (19, 28). Because pimobendan augments the second phase of secretion and increases the efficacy of Ca²⁺ in permeabilized islets, it might stimulate some kind of protein kinase. To determine whether a protein kinase is involved in the increased efficacy of the Ca²⁺ signal caused by pimobendan, we examined the effects of various kinds of protein kinase inhibitors on the suppression of its effect in electrically permeabilized islets.

PKC activator is another known Ca²⁺ sensitizer in pancreatic ß-cells (26, 29), and PKC is known to be one of the Ca²⁺-binding proteins (30). Bisindolylmaleimide, a known PKC-selective inhibitor, suppressed the Ca²⁺-sensitizing effect of TPA, but not that of pimobendan. Moreover, pimobendan increased TPA-enhanced insulin release from intact islets. This suggests that PKC activation is not involved in the insulinotropic mechanism of pimobendan and that it acts on a target different from that of pimobendan.

Calmodulin is a Ca²⁺-binding protein closely related to troponin C, and the Ca²⁺-calmodulin complex activates protein kinases, including MLCK and CaM kinase II (28). MLCK has been reported in pancreatic ß-cells (31) and has been suggested to be involved in the insulin exocytotic process (32). CaM kinase II also has been identified in ß-cells (13) and regulates exocytosis distal to the elevation of [Ca²⁺]_i (33, 34). Accordingly, we also investigated the involvement of MLCK and CaM kinase II. Wortmannin, a known MLCK inhibitor, did not attenuate the enhancement by pimobendan, whereas KN93, a CaM kinase II inhibitor, did suppress it. Although the CaM kinase II inhibitor has been reported to influence glucokinase (34, 35) and Ca²⁺ channel activity (36), these effects are not thought to affect insulin release at the basal concentration of glucose observed in electrically permeabilized islets. In addition, it is unlikely that this inhibitory effect is nonspecific, because KN93 did not suppress insulin release from control islets. Therefore, CaM kinase II activation would seem to be involved in the pimobendan-sensitive pathway. As its suppression was found to be incomplete, however, other protein kinases or calcium-binding proteins also may play a part in its effect, and identification of its target protein still remains.

Pimobendan evokes a [Ca²⁺]_i elevation in the presence of a stimulatory concentration of glucose, probably due to augmented Ca²⁺ influx, but not in the presence of basal glucose. The reason is not clear, but one possible explanation is that pimobendan also links the phenomenon of VDCC augmentation observed in ß-cells through intracellular glucose metabolism (37).

We have shown that pimobendan enhances insulin release by acting directly on the exocytotic mechanism following the elevation of [Ca²⁺]_i and that it increases Ca²⁺ sensitivity. In addition, CaM kinase II activation is at least in part involved in this enhancement of Ca²⁺ sensitization for exocytosis of insulin secretory granules from pancreatic ß-cells. In pancreatic ß-cells, a PDE inhibitory effect, even if occurs, play a less important role than in cardiomyocytes.

	Footnotes

 
¹ This work was supported by Grants-in Aid for Scientific Research from the Ministry of Education, Science and Culture; the Committee of Experimental Models of Intractable Diseases of the Ministry of Health and Welfare of Japan; a grant from the Japan Diabetes Foundation; and a grant from the Research for the Future Program of the Japan Society for the Promotion of Science (JSPS-RFTF97100201).

Received June 6, 1997.

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