Introduction

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The sarcoplasmic reticulum (SR) plays a central role in excitation-contraction coupling and relaxation in the cardiac muscle (Arai et al., 1994; Bers, 2000). While the release of calcium (Ca²⁺) from the SR increases intracellular Ca²⁺ concentration ([Ca²⁺]_i) to induce contraction, the uptake of Ca²⁺ into the SR decreases [Ca²⁺]_i to induce relaxation. The uptake of Ca²⁺ into the SR is mediated by a Ca²⁺ pump, SR Ca²⁺-ATPase. This process requires the hydrolysis of one molecule of ATP for two calcium ions that are pumped against a large concentration gradient into the lumen of the SR (de Tombe, 1998). The SR Ca²⁺ uptake activity is reduced in the failing myocardium in humans (Flesch et al., 1996) and animals (Gupta et al., 1997). The reduction in the Ca²⁺ uptake activity may be due to decreased protein level of SR Ca²⁺-ATPase (Hasenfuss et al., 1997), although this finding appears to be inconsistent. In fact, reduction in SR Ca²⁺ uptake activity is observed in the human failing myocardium with unchanged protein level of SR Ca²⁺-ATPase (Flesch et al., 1996). Decrease in SR Ca²⁺ uptake activity is associated with prolonged relaxation of the cardiac muscle (Sen et al., 2000). This is supported by prolonged relaxation time in the cardiac muscle treated with pharmacological SR Ca²⁺-ATPase inhibitors (Takahashi et al., 1995) and cardiac myocytes with overexpressed phospholamban, an endogenous inhibitor of SR Ca²⁺-ATPase (Davia et al., 1999).

Enhancement of SR Ca²⁺ uptake could be attained by cAMP-dependent phosphorylation of phospholamban by cAMP-increasing drugs such as -adrenergic agonists and phosphodiesterase inhibitors (Johnson, 1998). However, sustained increase in [Ca²⁺]_i as a result of increased influx of Ca²⁺ through L-type Ca²⁺ channel by cAMP-increasing drugs could be seriously toxic to cardiac myocytes (Mann et al., 1992). Furthermore, chronic stimulation of -adrenergic receptors could result in down-regulation and desensitization of the receptors themselves (Zhao et al., 1996). The SR Ca²⁺ uptake could also be enhanced by overexpression of cardiac SR Ca²⁺-ATPase (del et al., 1999) and vector-mediated expression of phospholamban antisense RNA (Eizema et al., 2000), although further basic studies are required before these gene-based approaches come into clinical practice. Thus, drugs that enhance SR Ca²⁺ uptake in a cAMP-independent manner could be ideal for the treatment of heart failure with lusitropic abnormality, including diastolic heart failure.

MCC-135, 5-methyl-2-(1-piperazinyl) benzenesulfonic acid monohydrate (Fig. 1), is a new potent compound with beneficial effects in heart failure. Chronic treatment with MCC-135 improved cardiac function (Puley et al., 1998; Satoh et al., 1999) and reduced mortality (Satoh et al., 1999) in animal models of heart failure. Although MCC-135 was demonstrated to restore Ca²⁺-ATPase activity of the SR isolated from the myocardium acutely exposed to ischemia and reperfusion in vitro (Kawasumi et al., 1999), the effect of MCC-135 on the SR function of the cardiac muscle with chronic heart failure remains to be studied. In this study, we investigated the effects of MCC-135 on 1) contraction and relaxation, and 2) SR function, in the left ventricular muscles isolated from rats with chronic heart failure due to diabetic cardiomyopathy. The animals of this model have manifest diastolic dysfunction accompanied by prolonged relaxation (Litwin et al., 1990; Hoit et al., 1999), which is associated with SR dysfunction (Lagadic-Gossmann et al., 1996).

View larger version (8K): [in this window] [in a new window]   Fig. 1.   Chemical structure of MCC-135.

	Materials and Methods

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Diabetic Rats. Male Wistar rats (7 weeks old, 160-200 g) were made diabetic by a single injection of streptozotocin (40 mg/kg i.v.) into the tail vein. This dose of streptozotocin is reported to result in reduced cardiac performance (Yamamoto and Nakai, 1988). Streptozotocin was dissolved in a citrate buffer (0.05 M citric acid and 0.05 M sodium phosphate, pH 4.5). Age-matched normal control rats received an equivalent volume of the citrate buffer alone. Diabetic and normal animals were maintained on the same diet until they were used 7 months later. This period of diabetes was chosen because our preliminary study has characterized alterations in mechanical function of the ventricular muscle during this period (data not shown).

Tension Measurement. The left ventricular papillary muscle was obtained from diabetic and normal rats anesthetized with pentobarbital sodium (50 mg/kg i.p.). The base of the papillary muscle was fixed to a muscle holder and the tendinous end was connected to a strain-gauge tension transducer (Minebea UL-2GR, Tokyo, Japan) for the measurement of changes in isometric tension. The papillary muscle was kept in Krebs' buffer (115 mM NaCl, 4.8 mM KCl, 1.0 mM MgSO₄ · 7H₂O, 2.0 mM CaCl₂ · 2H₂O, 25 mM NaHCO₃, 1.2 mM KH₂PO₄, 10 mM glucose 10; pH 7.4, 30°C) aerated with 95% O₂ + 5% CO₂. The papillary muscle was stimulated via platinum electrode at a voltage 10% above threshold (2 Hz, 3-ms duration) with a stimulator (NEC Medical Systems 2907, Tokyo, Japan). The papillary muscle was equilibrated for a period of 1 h, during which the muscle was gradually lengthened to attain maximal developed tension. Developed tension (DT), time from the onset of twitch to peak tension (TTP), and time from peak tension to 80% relaxation (TR80) were measured. Developed tension is presented as the value normalized by the cross-sectional area of the fiber.

SR Ca²⁺ Uptake, Release, and Leakage. Preparation of skinned fibers of the left ventricular papillary muscle and protocols used were similar to those in previous reports (Kitada et al., 1987). Small bundles (diameter, 0.25 mm; length, 2.75-3.75 mm) of the left ventricular papillary muscle fibers were dissected free from the left ventricle. The preparations were mounted between two hooks in a small chamber. One hook was fixed to a fiber holder and the other to a strain-gauge tension transducer (Acers AE801, Horten, Norway) for the measurement of changes in isometric tension. The chamber was usually filled with a relaxing buffer [128 mM K-methanesulfonate, 5.1 mM Mg-methanesulfonate, 4.2 mM ATP-Na₂, 2 mM EGTA, 20 mM piperazine-N,N'-bis(2-ethanesulfonic acid); pH 7.0]. The preparations were treated with saponin (50 µg/ml) for 20 min in the relaxing buffer. This treatment produces skinned fibers that preserve the ability of the SR to accumulate and release Ca²⁺ (Endo and Iino, 1980). All the experiments were performed at 20°C. 1) Ca²⁺ uptake: the SR was loaded with Ca²⁺ in a buffer of pCa 6.7 for various lengths of loading period in the absence or presence of MCC-135. MCC-135 was present only during this loading period. The amount of Ca²⁺ loaded in the SR was estimated by the transient contraction induced by caffeine (50 mM). Caffeine at this concentration releases Ca²⁺ loaded in the SR almost completely. In some experiments, protein kinase A and cAMP were applied instead of MCC-135 for comparison. 2) Ca²⁺ release: the SR was loaded with fixed amount of Ca²⁺ in a buffer of pCa 6.7 for 2 min. The Ca²⁺ was then released from the SR by applying various levels of Ca²⁺ for 30 s in the absence or presence of MCC-135 by Ca²⁺-induced Ca²⁺ release mechanism. The amount of Ca²⁺ remaining in the SR was estimated by the transient contraction induced by caffeine (50 mM). The difference in the amount of Ca²⁺ remaining in the SR between runs with and without giving the Ca²⁺ stimulus was taken as SR Ca²⁺ release. 3) Ca²⁺ leakage: the SR was loaded with fixed amount of Ca²⁺ in a buffer of pCa 6.7 for 2 min. The fiber was then immersed in relaxing buffer with and without MCC-135 for 2 min. The amount of Ca²⁺ remaining in the SR was estimated by the transient contraction induced by caffeine (50 mM). If Ca²⁺ leak is inhibited during exposure to MCC-135, the amount of Ca²⁺ remaining in the SR should increase and the magnitude of caffeine-induced contraction with MCC-135 treatment should be greater than that without MCC-135 treatment. The caffeine-induced contraction in the Ca²⁺ uptake, release, and leakage studies was measured as the area under the caffeine-induced contraction by a planimeter with image analysis (NIH Image). The area measured is presented as the value normalized by the cross-sectional area of the fiber.

Protein Levels of SR Ca²⁺-ATPase and Phospholamban. Preparation of protein samples and Western blot analysis were performed as described previously (Satoh et al., 2000) with modifications. The left ventricular papillary muscle was homogenized with a glass homogenizer in lysis buffer (20 mM Tris-HCl, 1 mM EDTA, 1 mM dithiothreitol, 0.001 mM leupeptin, 0.1 mM phenylmethylsulfonyl fluoride, pH 7.4) on ice. For the determination of SR Ca²⁺-ATPase protein level, protein samples were denatured by heating to 100°C in sample buffer (62.5 mM Tris-HCl, 2% SDS, 25% glycerol, 0.01% bromophenol blue) with mercaptoethanol for 5 min and subjected to SDS-polyacrylamide gel electrophoresis (12.5% running gel) electrophoresis. Samples for phospholamban were left over at room temperature in sample buffer without mercaptoethanol for 30 min before SDS-polyacrylamide gel electrophoresis (12.5% running gel). Proteins were transferred to nitrocellulose membranes (Hybond-ECL, 0.45 µm; Amersham, Buckinghamshire, UK) by semidry electrophoretic blotting with transfer buffer [25 mM Tris-HCl, pH 8.3, 192 mM glycine, 20% (v/v) methanol]. The membrane was stained with Ponceau red to confirm equal loading of the samples. The membrane was incubated for 1 h in phosphate buffer solution buffer (137 mM NaCl, 2.7 mM KCl, 1.5 mM KH₂PO₄, 8.1 mM Na₂HPO₄, pH 7.4) with 5% nonfat milk for SR Ca²⁺-ATPase, in Tris buffer solution buffer (200 mM Tris-HCl, 150 mM NaCl, pH 7.3) with 5% nonfat milk for phospholamban. The membrane was then incubated overnight with mouse monoclonal antibody specific to SR Ca²⁺-ATPase (Affinity Bioreagents, Golden, CO) diluted (1:1,000) in phosphate buffer solution buffer or with mouse monoclonal antibody specific to phospholamban (Affinity Bioreagents) diluted (1:2000) in Tris buffer solution buffer containing 5% nonfat milk and 0.05% Tween 20 at 4°C. The membrane was then incubated for 1 h with a 1:1000 dilution of peroxidase-conjugated goat antibody raised against mouse IgG (Amersham) at room temperature for immunodetection. After repeated washes, detection was performed with the enhanced chemiluminescence kit (Western blot detection reagent; Amersham). The membrane was then exposed to X-ray film. The signals were quantified by densitometric analysis (ATTO AE-6920 M, Tokyo, Japan).

Chemicals. MCC-135, 5-methyl-2-(1-piperazinyl) benzenesulfonic acid monohydrate, was synthesized in Mitsubishi-Tokyo Pharmaceuticals, Inc. (Tokyo, Japan). Streptozotocin, isoproterenol HCl, protein kinase A, cAMP, and Ponceau red were purchased from Sigma (St. Louis, MO). Caffeine was purchased from Wako Pure Chemical Industries (Osaka, Japan). Saponin was purchased from NBC (Cleveland, OH). Mouse monoclonal antibodies against SR Ca²⁺-ATPase and phospholamban were purchased from Affinity Bioreagents. Goat anti-mouse IgG was purchased from Amersham.

Statistical Analysis. The results are presented as mean ± S.E.M. When two groups were compared, Student's t test was used. When more than two groups were compared, one-way analysis of variance was used, and multiple comparisons were performed by Dunnett's test. When groups with two or more factors were compared, two-way analysis of variance was used followed by multiple comparisons by Dunnett's test. Differences were considered significant at a value of p < 0.05.

	Results

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Characteristics of Experimental Animals. Diabetes was confirmed on the basis of plasma glucose measurement on the day before sacrifice. The plasma glucose levels were markedly elevated in the rats treated with streptozotocin compared with the rats treated with the vehicle (636 ± 32 versus 164 ± 10 mg/dl, p < 0.001). This change verified that diabetes was induced by the treatment.

Effects of MCC-135 on Twitch of Left Ventricular Papillary Muscles. Basal DT (19.5 ± 1.5 versus 31.2 ± 1.9 mN/mm²; n = 14; p < 0.001) was significantly smaller in diabetic rats compared with normal rats. TTP (120 ± 2 versus 95 ± 2 ms; n = 14; p < 0.001) and TR80 (134 ± 4 versus 105 ± 2 ms; n = 14; p < 0.001) were significantly greater in diabetic rats compared with normal rats.

View larger version (27K): [in this window] [in a new window]   Fig. 2.   Effects of MCC-135 and isoproterenol on twitch parameters of isolated left ventricular papillary muscles of normal and diabetic rats. Isometric tension of the muscle was measured under electrical stimulation (2 Hz, 3-ms duration, 30°C). Values are mean ± S.E.M. n = 7 in each group. *p < 0.05, **p < 0.01, ***p < 0.001 versus respective control (C).

Effects of MCC-135 on SR Ca²⁺ Uptake, Release, and Leakage. The SR Ca²⁺ uptake is plotted against various lengths of uptake time (Fig. 3A). The SR Ca²⁺ uptake was significantly smaller at any given uptake time in diabetic rats compared with normal rats. MCC-135 had minimal effects on SR Ca²⁺ uptake in normal rats, that was observed as increased SR Ca²⁺ uptake at uptake time of 20 and 30 s at the highest concentration of 10⁵ M (Fig. 3B). In diabetic rats, MCC-135 increased SR Ca²⁺ uptake all over the range of uptake time. Both initial rate of SR Ca²⁺ uptake and the amount of Ca²⁺ accumulated in the SR with longer uptake time were increased by MCC-135 (Fig. 3B). The combination of protein kinase A (50 µg/ml) and cAMP (10⁴ M) increased SR Ca²⁺ uptake significantly at uptake time of 180 s in normal rats (Fig. 4). However, protein kinase A and cAMP was devoid of any effect on SR Ca²⁺ uptake in diabetic rats (Fig. 4).

View larger version (18K): [in this window] [in a new window]   Fig. 3.   A, Ca2+ uptake by the SR of skinned fibers of the left ventricular papillary muscles from normal and diabetic rats. The SR of skinned fibers treated with saponin (50 µg/ml) was loaded with Ca2+ at pCa 6.7 for various lengths of uptake time. The SR Ca2+ uptake was estimated by the transient contraction (the area under the contraction curve) induced by caffeine (50 mM). Values are mean ± S.E.M. n = 7 in each group. **p < 0.01, ***p < 0.001 versus normal. B, effect of MCC-135 on Ca2+ uptake by the SR of skinned fibers of the left ventricular papillary muscles from normal (left) and diabetic (right) rats. MCC-135 was present only during the uptake period. Values are mean ± S.E.M. n = 7 in each group. *p < 0.05, **p < 0.01, ***p < 0.001 versus control.

View larger version (14K): [in this window] [in a new window]   Fig. 4.   Effect of combination of protein kinase A and cAMP on Ca2+ uptake by the SR, at uptake time of 180 s, of skinned fibers of the left ventricular papillary muscles from normal (left) and diabetic (right) rats. Protein kinase A and cAMP were present only during the uptake time of 180 s. Values are mean ± S.E.M. n = 7 in each group. **p < 0.01 versus control.

View larger version (13K): [in this window] [in a new window]   Fig. 5.   A, Ca2+ release from the SR of skinned left ventricular papillary muscles of normal and diabetic rats. The SR of the skinned fiber treated with saponin (50 µg/ml) was loaded with fixed amount of Ca2+ at pCa 6.7 for 2 min followed by exposure to various levels of Ca2+ for 30 s. The SR Ca2+ release was estimated by the difference in transient contraction induced by caffeine (50 mM), an index of the amount of Ca2+ remaining in the SR, between runs with and without giving the Ca2+ stimulus. Values are mean ± S.E.M. n = 7 in each group. *p < 0.05, **p < 0.01 versus normal. B, effect of MCC-135 on Ca2+ release from the SR of skinned left ventricular papillary muscles of normal (left) and diabetic (right) rats. MCC-135 was present only during exposure to various levels of Ca2+. Values are mean ± S.E.M. n = 7 in each group.

View larger version (12K): [in this window] [in a new window]   Fig. 6.   A, Ca2+ leakage from the SR of skinned left ventricular papillary muscles of normal and diabetic rats. The SR of skinned fibers treated with saponin (50 µg/ml) was loaded with fixed amount of Ca2+ at pCa 6.7 for 2 min followed by immersion in relaxing buffer with and without MCC-135 for 2 min. The SR Ca2+ leakage was estimated by the transient contraction induced by caffeine (50 mM), an index of the amount of Ca2+ remaining in the SR. Values are mean ± S.E.M. n = 7 in each group. **p < 0.01 versus normal. B, effect of MCC-135 on Ca2+ leakage from the SR of skinned left ventricular papillary muscles of normal (left) and diabetic (right) rats. MCC-135 was present only during immersion in relaxing buffer for 2 min after Ca2+ loading. Values are mean ± S.E.M. n = 7 in each group. **p < 0.01, ***p < 0.001 versus control (C).

Protein Levels of SR Ca²⁺-ATPase and Phospholamban. The protein levels of SR Ca²⁺-ATPase and phospholamban in the left ventricular papillary muscles of normal and diabetic rats were analyzed by Western blot (Fig. 7) and quantified (Table 2). The protein level of SR Ca²⁺-ATPase was significantly lower in diabetic rats compared with normal rats, while there was no significant difference in the protein level of phospholamban between normal and diabetic rats. Due to the reduction in SR Ca²⁺-ATPase level, the ratio of SR Ca²⁺-ATPase to phospholamban was significantly lower in diabetic rats than normal rats.

View larger version (14K): [in this window] [in a new window]   Fig. 7.   Representative Western blot analysis of SR Ca2+-ATPase and phospholamban in the left ventricular papillary muscles of normal and diabetic rats. Immunochemical detection revealed protein bands at 110 kDa for SR Ca2+-ATPase and at 25 kDa for phospholamban.

	Discussion

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This study demonstrates that MCC-135 accelerated relaxation without significant effect on contractility in the left ventricular muscles of rats with diabetic cardiomyopathy. This effect of MCC-135 on relaxation was associated with improved SR function, including enhanced SR Ca²⁺ uptake and reduced Ca²⁺ leakage from the SR.

Characteristics of Experimental Animals. Higher plasma glucose level and higher ratios of the left ventricular weight to body weight, the right ventricular weight to body weight and the lung weight to body weight in streptozotocin-treated rats suggest that these rats have typical congestive heart failure due to diabetes, although in vivo cardiac function was not assessed in this study. Although the differences between normal and diabetic ventricular muscles observed in the current study should essentially result from pathophysiological alterations due to diabetic cardiomyopathy, we cannot exclude the possibility that some failure of maturation could contribute to the differences, considering much smaller body weight and the ventricular weights in diabetic rats.

Effects of MCC-135 on Twitch of Left Ventricular Muscles. MCC-135 exerted positive lusitropic effect, as evidenced by accelerated relaxation, in the left ventricular muscles of diabetic rats without significant effect on contractility. Of particular interest, MCC-135 had no significant effects either on relaxation or contractility of the left ventricular muscles of normal rats. This profile of MCC-135 suggests that MCC-135 is entirely different from inotropic drugs with known mechanisms of actions such as cardiotonic agents or cardiodepressants. MCC-135 increased SR Ca²⁺ uptake in the left ventricular muscles of diabetic rats, but not in normal rats. The lack of significant effect on the SR Ca²⁺ uptake in normal rats is consistent with the lack of significant effect on relaxation in normal rats. Thus, the acceleration of relaxation by MCC-135 could be associated with enhanced SR Ca²⁺ uptake in the left ventricular muscle of diabetic rats. The lack of effect of MCC-135 on SR Ca²⁺ uptake in normal rats, although its reason is not clear, would suggest that MCC-135 may affect regulation of SR Ca²⁺ uptake that is altered in diabetic rats compared with normal rats, speculatively including reduced phosphorylation of phospholamban (Schwinger et al., 1999) and augmented inhibition of SR Ca²⁺-ATPase activity by phospholamban due to decreased ratio of SR Ca²⁺-ATPase-to-phospholamban proteins (Table 2). Since the increase in SR Ca²⁺ uptake by MCC-135 was observed only a few minutes after the drug application, this action is more likely to be based upon enhancement of activity of the existing SR Ca²⁺-ATPase or increase in affinity of SR Ca²⁺-ATPase to Ca²⁺ rather than increase in protein level of SR Ca²⁺-ATPase. Since enhancement of SR Ca²⁺ uptake could refill more Ca²⁺ in the SR, more Ca²⁺ might be released upon the next stimulation to induce stronger contraction (Luo et al., 1994). However, that was not the case for MCC-135. The ventricular muscle in the twitch study was electrically stimulated at 2 Hz, which allows as brief as 0.5 s for SR Ca²⁺ uptake. Although the increased rate of SR Ca²⁺ uptake by MCC-135 at the brief uptake time of 0.5 s of shortened relaxation, the increased amount of Ca²⁺ accumulated in the SR might not be enough to be reflected as increased contractility on the next beat. Alternatively, a decrease in tension induced by MCC-135 at pCa between 7.0 and 5.5 in the skinned ventricular muscle preparation without functional SR (unpublished observation) could offset the possible potentiation of contraction due to enhanced SR Ca²⁺ uptake and thereby provide an explanation for the lack of potentiation of contraction by MCC-135 in the diabetic heart. MCC-135 had another effect of inhibiting Ca²⁺ leakage from the SR. The Ca²⁺ leakage from the SR is suggested to decrease SR Ca²⁺ loading and elevate basal [Ca²⁺]_i during diastole, leading to contractile and relaxation dysfunction (Yano et al., 2000). Thus, prevention of SR Ca²⁺ leakage could contribute to enhancement of SR Ca²⁺ uptake by keeping Ca²⁺ in the SR effectively until the next stimulation.