Introduction

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Patients with diabetes mellitus exhibit a high incidence of cardiac dysfunction and mortality. Clinical and pathological studies along with the epidemiological data from the Framingham study suggest the existence of a specific diabetic cardiomyopathy, independent of vascular disease (Kannel et al., 1974; Regan et al., 1977). This diabetic cardiomyopathy is associated with impaired cardiac responses to catecholamines. Type I diabetic patients have shown a reduced -adrenoceptor responsiveness of heart beat in isoproterenol infusion study (Berlin et al., 1986). Animal studies have indicated that the diabetic heart is characterized by diminished responsiveness to -adrenoceptor stimulation in association with decreased -adrenoceptor density and alterations in the -adrenoceptor signal transduction pathway (Tomlinson et al., 1992). Furthermore, the diabetic heart responds poorly to PDE inhibitors (Gando et al., 1997). Thus, the efficacy of cyclic AMP-dependent inotropic agents that can be used for the treatment of congestive heart failure is reduced in diabetes. Cardiac glycosides, which have a long history in the treatment of heart failure, show a relatively narrow therapeutic window and significant side effects even in nondiabetic patients, and these drugs might be feared to evoke easily life-threatening arrhythmias in diabetic patients, because diabetic myocardium is more susceptible than normal tissue to develop afterdepolarizations and triggered activity (Nordin et al., 1985). Accordingly, the therapeutic approach to heart failure of diabetic patients using currently available inotropes appears to be still far less than satisfactory.

A new class of inotropes, Ca²⁺ sensitizers, has been developed, which act at the level of the contractile protein to increase the sensitivity of the myofilament to Ca²⁺. These agents have the advantages of avoiding the problems associated with Ca²⁺ loading such as arrhythmias by cardiac glycosides and catecholamines and of enhancing force production without increasing energy utilization. Many of the compounds having a Ca²⁺-sensitizing action have additional cellular effects such as inhibition of PDE III, which may counter some effects of Ca²⁺ sensitizers on cardiac function (Rüegg, 1986; Böhm et al., 1991). However, a number of novel compounds behave as a myofilament Ca²⁺ sensitizer with marginally inhibiting PDE III. These include MCI-154 (Abe et al., 1996), EMD 57033 (Ferroni et al., 1991; Ventura et al., 1992) and EGIS-9377 (Hattori et al., 1999). Although previous works have addressed the question of whether the inotropic effects of Ca²⁺ sensitizers demonstrated in normal cardiac tissues are to be translated into clinically useful effects in diseased hearts (Than et al., 1994; Drake-Holland et al., 1997; Hajjar et al., 1997; Teramura et al., 1997), research concerning the ability of the diabetic heart to respond to Ca²⁺ sensitizers is currently lacking.

We undertook this study to examine the effects of Ca²⁺ sensitizers EMD 57033, MCI-154, and EGIS-9377 on cell length and [Ca²⁺]_i transient in ventricular myocytes isolated from streptozotocin-induced diabetic and age-matched control rat hearts, and to compare these effects with those of pimobendan and isoproterenol. We also studied the effect of EMD 57033 on the Ca²⁺ sensitivity of contractile protein in chemically skinned fibers from diabetic rat hearts. Ca²⁺ sensitizers have the adverse effect of slowing relaxation and elevating diastolic tension in the heart (Hajjar and Gwathmey, 1991). In diabetes, cardiac relaxation is impaired because of abnormal [Ca²⁺]_i handling (Fein et al., 1980; Ren and Davidoff, 1997). We thus determined whether EMD 57033 worsens the relaxation of isometric contraction curve in diabetic rat papillary muscles and LV diastolic function in anesthetized diabetic rats more seriously than in controls of each experimental model.

	Materials and Methods

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Induction of Diabetes. Male Wistar rats, 8 weeks old and 180 to 200 g of body weight, were randomly assigned to two groups. One group of rats (diabetic group) received a single tail-vein injection of streptozotocin (45 mg/kg) under light anesthesia with diethyl ether. Streptozotocin was dissolved in a citrate solution (0.1 M citric acid and 0.2 M sodium phosphate, pH 4.5). Another group (control group) received an equivalent volume of citrate buffer alone. Control and diabetic rats were caged separately but housed under similar conditions. Both groups of animals were fed with the same diet and water ad libitum until they were used 4 to 6 weeks later. This period of diabetes was chosen because previous studies from this laboratory have well characterized cardiac alterations during this period (Gando et al., 1997; Tamada et al., 1998; Hattori et al., 2000). All animals injected with streptozotocin developed severe diabetes, as indicated by increased serum glucose levels. Mean serum glucose levels were 166 ± 6 and 556 ± 17 mg/dl for control rats (n = 32) and diabetic rats (n = 32), respectively. In some experiments, rats were used at 10 and 25 weeks after treatment with streptozotocin. Diabetic rats and age-matched control rats showed serum glucose levels of 500 ± 34 (n = 6) and 163 ± 12 mg/dl (n = 6) at 10 weeks and those of 560 ± 12 (n = 4) and 162 ± 21 mg/dl (n = 4) at 25 weeks, respectively.

Isolation of Ventricular Myocytes. Rats were anesthetized with sodium pentobarbital (150-200 mg/kg i.p.), ventilated with an artificial respirator, and then the heart was removed quickly following opening of the chest. The heart was retrogradely perfused with a modified Tyrode's solution at a temperature of 36°C until its beating rate became stable. The composition of the solution (pH 7.4) was 143 mM NaCl, 5.4 mM KCl, 1.3 mM CaCl₂, 0.5 mM MgCl₂, 0.33 mM NaH₂PO₄, 5.0 mM HEPES, 5.5 mM glucose. The perfusate was changed to a nominally Ca²⁺-free solution for 5 min, resulting in cessation of the heartbeat. The quiescent heart was perfused with a nominally Ca²⁺-free Tyrode's solution containing collagenase (0.03-0.05% w/v; Wako Pure Chemical, Osaka, Japan) for 40 to 60 min. The collagenase solution was washed out with a KB solution that contained 70 mM KOH, 50 mM L-glutamic acid, 40 mM KCl, 20 mM taurine, 20 mM KH₂PO₄, 3.0 mM MgCl₂, 10 mM glucose, 0.5 mM EGTA, 10 mM HEPES (pH 7.4), and 1% bovine serum albumin. The ventricular tissue was cut into small pieces, agitated gently in a small beaker with KB solution, and then filtered through a 100-µm stainless steel mesh.

Simultaneous Measurement of Length and Indo 1 Fluorescence. Single ventricular myocytes bathed in KB solution were loaded with the fluorescent Ca²⁺ probe indo 1 by incubation with 5 µM indo 1-AM (Dojin, Kumamoto, Japan) and 0.02% Pluronic F-127 (Molecular Probes, Eugene, OR) for 10 min at room temperature, followed by washout with KB solution for 60 min. Small aliquots of loaded myocytes were placed in the experimental chamber filled with Tyrode's solution, allowed to settle for 5 min, and superfused with Tyrode's solution for at least 15 min. Myocytes were then field stimulated at a rate of 0.5 Hz by a pair of platinum electrodes connected to an electronic stimulator (SEN-7203; Nihon Kohden, Tokyo, Japan) through an isolation unit (SS-104J; Nihon Kohden).

Experiments on Skinned Cardiac Muscle. Fiber bundles less than 200 µm in diameter were prepared by blunt dissection from ventricular trabecular muscles of control and diabetic rats. These strips were chemically skinned as previously described (Tomita et al., 1997). In brief, the small bundles were treated with the relaxing solution containing 50 µM -escin (Sigma, St. Louis, MO) for 30 min. The relaxing solution contained 87 mM potassium methanesulfonate, 20 mM piperazine-N-N'-bis-(2-ethanesulfonic acid), 5.1 mM Mg(methanesulfonate)₂, 4.2 mM ATP, 10 mM phosphocreatine, 0.5 mg/ml creatine phosphokinase, and 10 mM EGTA (pH 7.0). The skinned fibers were connected to a strain gauge transducer (TB651T; Nihon Kohden) for measurement of isometric tension. Various Ca²⁺ concentrations were prepared by adding the appropriate amount of Ca(methanesulfate)₂ to the relaxing solution. The pH of the solution was adjusted to 7.0 with KOH and the ionic strength was kept constant at 0.2 M by changing the amount of potassium methanesulfonate added. To determine the relation between the Ca²⁺ concentration and force development of the muscle fibers, the bundles were successively immersed in activating solutions containing increasing concentration of Ca²⁺ until the force had reached a stable plateau at each Ca²⁺ concentration. Force was expressed as percentage of the maximal force obtained at 30 µM Ca²⁺ in the same preparation. Experiments were carried out at room temperature (22-25°C).

Organ Bath Experiments. Experiments were performed as described previously (Gando et al., 1997). Briefly, left ventricular papillary muscles were isolated from the hearts of control and diabetic rats. The composition of the bathing solution (pH 7.4) was 119 mM NaCl, 4.8 mM KCl, 1.3 mM CaCl₂, 1.2 mM MgSO₄, 1.2 mM KH₂PO₄, 24.9 mM NaHCO₃, 10.0 mM glucose. The solution in the bath was continuously gassed with 95% O₂ and 5% CO₂, and was kept at a temperature of 35°C. Isometric force of contraction was measured after the muscle was preloaded to 0.5 g. We have confirmed that this resting tension produced >90% maximal force development in papillary muscles from both control and diabetic animals, based on resting tension/developed tension curves (Gando et al., 1997). The muscle was electrically stimulated at 1 Hz with rectangular pulses of 5-ms duration (3F46; Sanei-Sokki, Tokyo, Japan), the voltage being 1.5 times greater than threshold. The preparations were allowed to equilibrate for at least 60 min before any experimental procedure was applied.

Echocardiographic Measurements in Anesthetized Animals. Rats were anesthetized with ketamine (100 mg/kg i.p.). Propranolol (1 mg/kg) and atropine (1 mg/kg) were given intravenously to block sympathetic and parasympathetic effects on the heart. The method for administration of EMD 57033 was essentially the same as that described previously (Haeusler et al., 1997). Thus, EMD 57033 (30 mg/kg) was administered intra-duodenally through a catheter placed in the duodenum via a midline incision of the abdominal wall. The drug was dissolved in a mixture of dimethyl sulfoxide and Tween 80 1:10 and given in a volume of 1 ml/kg. The vehicle alone did not produce any effect on LV functions.

Chemicals. Streptozotocin and l-isoproterenol hydrochloride were purchased from Sigma. EMD 57033 was a gift of Merck KGaA (Darmstadt, Germany), and MCI-154 was from Mitsubishi Chemical Corporation (Yokohama, Japan). EGIS-9377 was synthesized at the Central Pharmaceutical Research Institute, Japan Tabacco Inc. (Takatsuki, Japan). Pimobendan was kindly donated by Dr. K. Thomae (Biberach an der Riss, Germany). Other chemicals used in this study were of the highest purity available from Sigma, Wako Pure Chemical, or Nakalai Tesque (Kyoto, Japan). All drugs except streptozotocin (see above), EMD 57033, and pimobendan were dissolved in distilled water. EMD 57033 was prepared in absolute ethanol, and pimobendan was in dimethyl sulfoxide. Further dilutions to the desired concentrations were made with suitable buffer solution. Ascorbic acid (0.1 mM) was added to the isoproterenol solution to retard the oxidation of the catecholamine.

Statistical Analysis. All values are presented in terms of means ± S.E. Statistical assessment of the data was made by the two-tailed Student's t test. Nonparametric data were analyzed by the Mann-Whitney U test. A P value <0.05 was considered statistically significant.

	Results

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Effects of Ca²⁺ Sensitizers and Other Agents on Myocyte Contractility and [Ca²⁺]_i Transients. As reported in our previous work (Tamada et al., 1998), cell shortening observed in electrically stimulated ventricular myocytes showed no statistically significant difference between the 4- to 6-week diabetic and age-matched control groups. When expressed as a change in length/resting length × 100, cell shortening was 2.9 ± 0.2% (n = 54) for control and 3.2 ± 0.3% (n = 54) for diabetic myocytes. In addition, the two groups of myocytes exhibited qualitatively similar changes in the [Ca²⁺]_i transient. Thus, diastolic [Ca²⁺]_i and peak systolic [Ca²⁺]_i, as monitored by the ratio of the fluorescence at 410 nm to that at 485 nm, did not differ between control (0.289 ± 0.007 and 0.382 ± 0.010, n = 54) and diabetic (0.283 ± 0.008 and 0.357 ± 0.011, n = 54) myocytes.

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Representative traces showing the effect of 10 µM EMD 57033 on cell shortening and [Ca2+]i transients in indo 1-AM-loaded myocytes obtained from control (A) and diabetic (B) rat hearts.

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Concentration-dependent effect of EMD 57033 on cell shortening (A) and [Ca2+]i transients (B) in indo 1-AM-loaded myocytes obtained from control () and diabetic () rat hearts. Points (means ± S.E., n = 4-7) represent net increases in parameters expressed as percentage of basal values recorded before application of EMD 57033.

View larger version (28K): [in this window] [in a new window]   Fig. 3.   Effects of MCI-154, EGIS-9377, pimobendan, and isoproterenol on cell shortening (A) and [Ca2+]i transients (B) in indo 1-AM-loaded myocytes obtained from control () and diabetic () rat hearts. Columns (means ± S.E., n = 5-6) represent net increase in parameters expressed as a percentage of basal values recorded before application of drugs. *P < 0.05, **P < 0.01 versus the corresponding control values.

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Representative traces showing the effect of 100 µM pimobendan on cell shortening and [Ca2+]i transients in indo 1-AM-loaded myocytes obtained from control (A) and diabetic (B) rat hearts.

Effect of EMD 57033 on Resting Tension and Relaxation Time in Papillary Muscles. Figure 5 illustrates the effect of 10 µM EMD 57033 on force of contraction in papillary muscles from control and 4- to 6-week diabetic rats. EMD 57033 increased active force of contraction to a similar extent in control and diabetic myocardium (Table 1). Along with the increase in active force, a rise in resting tension was observed. When the amplitude of the basal force of contraction was assigned a value of 100%, the extent of the rise in resting tension was 12 ± 4% for control and 13 ± 6% for diabetic myocardium. There was no significant difference between these values. Relaxation was also affected by the addition of EMD 57033. Typical examples of the effect of 10 µM EMD 57033 on the duration of isometric contraction curve in control and diabetic papillary muscles are depicted in Fig. 6. The time to 50% relaxation was significantly prolonged by EMD 57033 in both control and diabetic myocardium (Table 1). The time to 50% relaxation of the basal twitch force was significantly greater in diabetic myocardium (Table 1), but EMD 57033 increased the relaxation time to a similar extent in control and diabetic myocardium when the effect was expressed as a percentage of the predrug level (37 ± 5 versus 41 ± 4%).

View larger version (12K): [in this window] [in a new window]   Fig. 5.   Representative traces showing the effect of 10 µM EMD 57033 on force generation in papillary muscles from control (A) and diabetic (B) rat hearts. The muscles were electrically driven at 1 Hz.

View larger version (11K): [in this window] [in a new window]   Fig. 6.   Representative traces showing the effect of 10 µM EMD 57033 on the duration of isometric contraction curve obtained in papillary muscles from control (A) and diabetic (B) rat hearts. The muscles were electrically driven at 1 Hz. Traces of isometric contraction before and 30 min after the addition of EMD 57033 are superimposed.

Effect of EMD 57033 on Myofilament Ca²⁺ Sensitivity in Skinned Cardiac Fibers. The pCa-tension relationships for skinned cardiac preparations from control and 4- to 6-week diabetic rats are shown in Fig. 7. Maximum Ca²⁺-activated tension was not markedly affected by diabetes, with cardiac fibers developing 54 ± 8 (n = 6) and 40 ± 8 mg (n = 6) of tension from control and diabetic rats, respectively. The sensitivity of diabetic preparations to Ca²⁺ tended to be slightly decreased compared with that of control preparations. Thus, the pCa of half-maximum tension generation, i.e., the pCa₅₀, was 5.81 ± 0.05 for control and 5.71 ± 0.08 for diabetic preparations, although the difference between these values was not statistically significant (P > 0.3). The slope of the pCa-tension relation was not significantly different between control (2.12 ± 0.26) and diabetic (2.74 ± 0.55) preparations.

View larger version (15K): [in this window] [in a new window]   Fig. 7.   pCa-tension relationships obtained from -escin-skinned trabecular muscles from control () and diabetic () rat hearts. Relative tension is expressed as percentage of tension achieved at maximum Ca2+ activation (30 µM Ca2+). Points are means ± S.E. of six different preparations. Lines are computer-generated curves fitting the obtained data points.

View larger version (16K): [in this window] [in a new window]   Fig. 8.   pCa-tension relationships in the absence () and presence () of 10 µM EMD 57033 in -escin-skinned trabecular muscles from control (A) and diabetic (B) rat hearts. Points are means ± S.E. of six different experiments. Tension is expressed as percentage of maximal tension obtained at 30 µM Ca2+ without EMD 57033. Lines are computer-generated curves fitting the obtained data points.

Echocardiographic Detection of Effect of EMD 57033 Administration in Anesthetized Rats. When EMD 57033 (30 mg/kg intra-duodenally) was administered to anesthetized rats, its effect appeared evident within 30 min of drug administration. The effect of EMD 57033 on LV systolic function was similar in the two groups. Thus, LV fractional shortening was increased from 60 ± 4 to 68 ± 3% (n = 4) in control and from 63 ± 4 to 71 ± 4% (n = 4) in 4- to 6-week diabetic rats. As shown in Table 2, in diabetic rats, E wave deceleration, an index of LV diastolic filling, was more rapid at baseline (P < 0.01 versus control rats) as assessed by transmitral Doppler recordings, indicating restricted LV diastolic filling. Another index of LV diastolic filling, isovolumic relaxation time, was not significantly affected by diabetes. EMD 57033 administration did not result in any deterioration in these variables of LV diastolic filling in either the control or diabetic group (Table 2).

	Discussion

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In the present study, we showed that EMD 57033, MCI-154, and EGIS-9377 enhanced the twitch amplitude in indo 1-AM-loaded rat ventricular myocytes without a significant effect on the [Ca²⁺]_i transient. These features of the responses to the three agents obtained in single cardiomyocytes are in good agreement with the predictions derived from the experiments on skinned fibers (Kitada et al., 1987; Lues et al., 1993; Hattori et al., 1999). Thus, the positive inotropic effect of these agents is closely associated with an increase in myofilament sensitivity to Ca²⁺. We found that both the potency and the efficacy of EMD 57033 to increase cell shortening were similar in cardiomyocytes from rats with 4- to 6-week streptozotocin-induced diabetes and from age-matched control rats. The positive inotropic effects of MCI-154 and EGIS-9377 did not differ between control and diabetic myocytes. Furthermore, in our skinned fiber preparations, no difference in the effect of EMD 57033 on the myofilament Ca²⁺ sensitivity was found between control and diabetic myocardium. Taken together, these results provide evidence that the Ca²⁺ sensitizers used herein have the potential to produce a positive inotropic effect to the same extent in nondiabetic and diabetic myocardium.

In contrast to the effects of these Ca²⁺ sensitizers, the effect of pimobendan on cell shortening was significantly diminished in myocytes from diabetic rat hearts. Although it has been reported from our and other laboratories that pimobendan enhances the Ca²⁺ sensitivity of tension in skinned cardiac preparations (Rüegg et al., 1984; Tomita et al., 1997; Hattori et al., 1999), the inhibition of PDE III appears to be involved as a major mechanism of pimobendan's positive inotropic effect (Brunkhorst et al., 1989). Thus, the positive inotropic effect of pimobendan is associated with the intracellular concentration of cyclic AMP augmented by preventing its degradation by PDE III. We also observed that the ability of pimobendan to increase the amplitude of the [Ca²⁺]_i transient in cardiomyocytes was less pronounced in diabetes. Additionally, diabetic myocytes showed significant reductions in the cell-shortening and [Ca²⁺]_i responses to isoproterenol. These findings are in accordance with our previous report (Tamada et al., 1998) showing that the cyclic AMP-dependent effect on cell shortening is specifically impaired in cardiomyocytes from diabetic rats in association with the diminished [Ca²⁺]_i responsiveness.

The evaluation of changes in the myofilament Ca²⁺ sensitivity in diabetic myocardium does not reach general agreement. Akella et al. (1995) have demonstrated diminished Ca²⁺ sensitivity of skinned cardiac muscle contractility coincident with troponin T-band shifts in diabetic rats. The diabetes-induced decrease in the Ca²⁺ sensitivity of tension has been also shown in skinned cardiomyocytes (Hofmann et al., 1995). However, Khandoudi et al. (1993) have revealed a significant increase in the Ca²⁺ sensitivity of tension using skinned fibers prepared from diabetic rat papillary muscles. On the other hand, the pCa-tension relations of skinned trabeculae at different sarcomere length have been found to be identical in control and diabetic myocardium (Ishikawa et al., 1999). The reason for the different results is not clear at present, but may be related to the wide variations in the experimental conditions used. In the present study, the Ca²⁺ sensitivity of tension, as reflected by the pCa₅₀, of skinned fibers prepared from diabetic myocardium tended to be slightly decreased compared with that from control myocardium. However, this small decrease in the myofilament Ca²⁺ sensitivity seen in diabetes may have little meaning since it was not a statistically significant change. EMD 57033 caused a great shift in the Ca²⁺ sensitivity of tension of skinned myocardial fibers. No difference in the ability of EMD 57033 to increase the myofilament Ca²⁺ sensitivity was found between control and diabetic myocardium. Therefore, the present experiments on skinned myocardial fibers suggest that the action of EMD 57033 as a myofilament Ca²⁺ sensitizer is qualitatively unaltered in diabetes.

The most prominent functional change in papillary muscles from diabetic rat hearts was a prolonged duration of a single contraction, especially a slower rate of relaxation. The amount of releasable SR Ca²⁺ has been found to be depressed by diabetes (Yu et al., 1994; Tamada et al., 1998). There is evidence that diabetes depresses Ca²⁺ uptake of SR microsomal membranes isolated from heart tissues (Penpargkul et al., 1981) and reduces SR Ca²⁺-ATPase activity (Ganguly et al., 1983). Thus, the slower time course of relaxation in diabetic myocardium could be explained by the dysfunction of the Ca²⁺ uptake into the SR (Ren and Davidoff, 1997). Furthermore, diabetes slows down the cross-bridge cycling rate (Ishikawa et al., 1999) in association with shifts in cardiac myosin heavy chain isoforms from V₁ to V₃ (Dillmann, 1985), which could also contribute to impaired myocardial relaxation. Since the rate of Ca²⁺ release from the myofilaments is delayed in the presence of Ca²⁺ sensitizers (Leijendekker and Herzig, 1992), cross-bridges spend more time in the attached state, resulting in prolongation of relaxation. As observed in our preparations, EMD 57033 had a significant effect of prolonging the time to 50% of relaxation. When the change in the relaxation time was expressed as a percentage of the predrug level, the extent of EMD 57033-induced increase in relaxation was similar in control and diabetic papillary muscles. However, interpretation of the results is complicated by the fact that diabetes essentially exhibits marked prolongation of myocardial relaxation. It has to be considered that the prolonged time course of relaxation in diabetic myocardium might have been further accentuated in the presence of Ca²⁺ sensitizers. Therefore, one may argue that the use of Ca²⁺ sensitizers could worsen the impairment of diastolic filling of the ventricular cavities in diabetes.

An increase in diastolic tonus would be reflective of the prolonged time course of myocardial relaxation. In cardiomyocytes, EMD 57033, MCI-154, and EGIS-9377 caused a slight but significant reduction in diastolic cell length. The reduced diastolic cell length was not accompanied by an elevated level of diastolic [Ca²⁺]_i. This is in agreement with the view that the changes in the [Ca²⁺]_i transient and cell length after application of Ca²⁺ sensitizers are not so obvious straightforwardly because they act primarily on myofilament Ca²⁺ sensitivity (Lee and Allen, 1991). An increase in resting tension in response to EMD 57033 was also observed in papillary muscles. This effect was modest (~10%) even at a high concentration of EMD 57033 that can produce a 2-fold increase in force of contraction. The impairment in myocardial diastolic filling produced by an increase in myofilament Ca²⁺ sensitivity appears evident when the concentrations of Ca²⁺ sensitizers exceed those to be optimal for a positive inotropic action (Lee and Allen, 1991; Ventura et al., 1992). The detrimental effect of EMD 57033 on diastolic tonus was not exaggerated by diabetes. Accordingly, it seems unlikely that the detrimental influences of Ca²⁺ sensitizers on the diastolic properties of diabetic myocardium would be a serious problem in practice. This view could be supported by the whole animal experiments using echocardiography, which indicate that EMD 57033 at appropriate doses may be able to improve systolic function without adverse effects on diastolic function in diabetic rat hearts.

Most of the present experiments were carried out using the animals after 4 to 6 weeks of diabetes because previous studies from this laboratory have well characterized cardiac alterations, including the -adrenoceptor signal transduction pathway, during this period (Gando et al., 1997; Tamada et al., 1998; Hattori et al., 2000; Matsuda et al., 2000). However, some of the cardiac dysfunctions associated with diabetes mellitus may be time-dependent. It may be that the positive inotropic effects of Ca²⁺ sensitizers decrease with the duration of diabetes. Nankervis et al. (1994) have shown that the positive inotropic response to EMD 57033 is significantly diminished in left ventricular papillary muscles, but not in left atria, from rats with 7-week diabetes. In this study, the effect of EMD 57033 was also tested in ventricular myocytes and left ventricular papillary muscles isolated from rats after 10 and 25 weeks of diabetes. No significant difference was found in the contractile response to EMD 57033 between preparations from 10- and 25-week diabetic rats and from age-matched control animals. Furthermore, it should be noted that the changes in the relaxation time in the absence and presence of EMD 57033 were not substantially different between papillary muscles from 4 to 6 week and longer-term diabetic rats.

In conclusion, cardiomyocytes and papillary muscles from streptozotocin-induced diabetic rats exhibited an increase in force of contraction in response to Ca²⁺ sensitizers such as EMD 57033 to the same extent as age-matched controls. This was in contrast with the lesser inotropic responses to -adrenoceptor stimulants and PDE III inhibitors. The present results suggest that Ca²⁺ sensitizers may be helpful for treatment of congestive heart failure in diabetic patients. However, more detailed studies using living animals will be required to accumulate evidence that the benefit of Ca²⁺ sensitizers cannot be disturbed by their possible disadvantages such as a worsening of myocardial diastolic filling.