Positive Lusitropic Effect of Quercetin on Isolated Ventricular Myocardia from Normal and Streptozotocin-Induced Diabetic Mice

Abstract

The lusitropic effect of quercetin was examined on isolated ventricular myocardial tissue preparations from normal and streptozotocin-induced diabetic mice. The time required for 90% relaxation of the myocardium, which was prolonged in the diabetic mice, was shortened by quercetin in both normal and diabetic myocardia. This effect of quercetin was completely inhibited by cyclopiazonic acid but not by SEA0400. These results indicated that quercetin accelerates myocardial relaxation through activation of the sarco–endoplasmic reticulum Ca²⁺-ATPase.

INTRODUCTION

Diastolic dysfunction is a major cardiac deficit underlying heart failure accompanying hypertension, coronary artery disease, and diabetes mellitus.¹⁾ More than half of heart failure patients have normal or preserved left ventricular ejection fraction but increased left ventricular end-diastolic pressure, which indicates an impaired diastolic function of the myocardium. Diastolic dysfunction has also been reported in various animal models of heart failure.^2,3) The myocardium of streptozotocin-induced diabetic animals displays evident diastolic dysfunction with or without systolic dysfunction. Diastolic dysfunction is accompanied by abnormalities in cellular Ca²⁺ handling caused by a reduction in the expression of the Na⁺–Ca²⁺ exchanger (NCX) and the sarco–endoplasmic reticulum Ca²⁺ pump (SERCA).^4,5) Although the information on the mechanisms for diastolic dysfunction is accumulating, therapeutic agents targeting diastolic dysfunction are not yet clinically available. Thus, novel agents, which improve myocardial relaxation, are much anticipated, and searches among synthetic compounds and natural products are in progress.^6,7)

Quercetin, 2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-chromen-4-one, is one of the major flavonoid compounds contained in fruits and vegetables and is considered to have antioxidant, antiviral, and antitumor activity.^8–10) In the cardiovascular system, the long-term application of quercetin was reported to have beneficial effects such as blood pressure lowering and cardioprotection. Concerning the heart failure induced by diabetes mellitus, quercetin was reported to prevent cholesterol accumulation and alleviate oxidative stress resulting in the attenuation of diastolic dysfunction in diabetic rats.¹¹⁾ While these beneficial effects of quercetin have been mostly attributed to its antioxidant effects, quercetin was also reported to affect functional proteins related to intracellular signal transduction and Ca²⁺ handling.^12–15) Thus, quercetin possibly has unexplored therapeutic potential for various cardiovascular diseases.

In the present study, we examined whether quercetin accelerates myocardial relaxation in isolated ventricular tissue preparations from normal and streptozotocin-induced diabetic mice. As we found that quercetin indeed accelerates myocardial relaxation, we further determined the transporter involved using inhibitors of the NCX and SERCA.

MATERIALS AND METHODS

The present study was conducted in accordance with the Guiding “Principles for the Care and Use of Laboratory Animals Approved by The Japanese Pharmacological Society” and the Guide for the Care and Use of Laboratory Animals at Faculty of Pharmaceutical Sciences, Toho University. Experiments were performed as described in our previous reports.^5,16)

Male ddY mice (4 weeks old) were made diabetic by a single injection of streptozotocin (200 mg/kg) into the peritoneal cavity. Age-matched control mice (normal mice) received an equivalent volume of citrate buffer (0.1 M, pH 4.5). Streptozotocin-induced diabetic mice (diabetic mice) and normal mice were maintained on the same diet until they were used 4–6 weeks later. Blood samples were drawn from the tail vein, and blood glucose levels were determined by the glucose pilot system (NGP-01B; Iwai Chemicals, Tokyo, Japan). The blood glucose levels in normal and diabetic mice were 154.4 ± 5.3 mg/dL (n = 18) and 559.2 ± 6.2 mg/dL (n = 18), respectively.

Hearts were removed from mice under deep isoflurane anesthesia. The right ventricular free wall preparations were placed in an organ bath filled with a physiological salt solution of the following composition: 118.4 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl₂, 1.2 mM MgSO₄, 1.2 mM KH₂PO₄, 24.9 mM NaHCO₃, and 11.1 mM glucose (pH = 7.4, 37 °C). For the measurement of contractile force, the preparations were attached to a force-displacement transducer (TB-611T, Nihon Kohden, Tokyo, Japan) connected to a carrier amplifier (AP-621G, Nihon Kohden). The myocardium was stimulated at 1 Hz by 3 ms square pulses of 1.5× threshold voltage. The output of the amplifiers was digitized and analyzed (Power Lab System, AD Instruments, Dunedin, New Zealand). Contractile parameters, including the contractile force, the time to peak contraction (contraction time), and the time required for 90% relaxation (relaxation time), were measured before and at 30 min after treatment with pharmacological agents.

All experimental data were expressed as the mean ± standard error of the mean (S.E.M.). The statistical significance of differences between means was evaluated by student’s t-test for basal values (in the text) and one-way repeated-measures ANOVA followed by Dunnett’s test for multiple comparisons for the effects of quercetin (in Fig. 1 and Table 1). A p-value less than 0.05 was considered significant. Citric acid, trisodium citrate, cyclopiazonic acid (CPA), and streptozotocin were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). Quercetin was purchased from Tokyo Chemical Industry (Tokyo, Japan). SEA0400 was synthesized in our faculty.

Fig. 1. Effect of Quercetin on Myocardial Relaxation

A: Typical traces for the contraction and relaxation of ventricular myocardium from normal (a, c, e) and diabetic mice (b, d, f) in the presence of 10 µM SEA0400 (c, d) or 3 µM cyclopiazonic acid (e, f), and their absence (a, b). Open and closed circles indicate the absence and presence of 30 µM quercetin, respectively. The force was normalized to the peak value in each trace. B: Summarized results showing the effects of 10 and 30 µM quercetin alone (a) or in the presence of 10 µM SEA0400 (b) or 3 µM cyclopiazonic acid (c). Open and closed squares indicate data from normal and diabetic mice, respectively. Symbols and vertical bars indicate the mean ± standard error of the mean (S.E.M.) from 6 experiments. Asterisks indicate significant differences from the value obtained before the addition of quercetin. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Table 1. Changes in the Contractile Parameters Induced by Quercetin

		Contractile force (mg/mm2)		Contraction time (ms)		Relaxation time (ms)
		Normal	Diabetic	Normal	Diabetic	Normal	Diabetic
Control	Before	130.0 ± 15.6	69.4 ± 12.1	49.3 ± 1.1	52.3 ± 1.7	68.8 ± 1.5	78.1 ± 0.5
	Quercetin 10 µM	118.5 ± 13.7**	65.6 ± 11.3	47.9 ± 1.2	49.9 ± 1.0	65.4 ± 1.0*	73.4 ± 0.8*
	Quercetin 30 µM	99.3 ± 12.1***	63.9 ± 11.0	45.7 ± 1.2***	48.1 ± 0.7*	63.7 ± 1.7*	69.5 ± 0.5***
	ΔChanges at 30 µM	−30.7 ± 3.6	−5.5 ± 3.7	−3.6 ± 0.4	−4.2 ± 1.1	−5.1 ± 1.2	−8.6 ± 0.8
SEA0400	Before	110.9 ± 9.2	74.7 ± 12.5	49.3 ± 1.3	47.9 ± 0.9	68.8 ± 2.1	76.6 ± 2.2
	Quercetin 10 µM	96.9 ± 9.2*	67.6 ± 12.1*	47.3 ± 1.2*	47.6 ± 0.8	64.3 ± 1.7*	70.5 ± 2.4**
	Quercetin 30 µM	83.5 ± 7.3***	66.0 ± 11.3*	45.5 ± 1.0***	46.7 ± 0.7	62.2 ± 1.6*	66.1 ± 2.9***
	ΔChanges at 30 µM	−27.4 ± 3.4	−8.7 ± 2.7	−3.7 ± 0.4	−1.2 ± 0.7	−6.6 ± 1.8	−10.5 ± 1.0
CPA	Before	87.5 ± 16.2	67.3 ± 6.7	54.1 ± 1.3	56.4 ± 2.1	87.0 ± 3.0	96.7 ± 2.5
	Quercetin 10 µM	80.3 ± 16.0**	62.8 ± 6.2	52.7 ± 1.1	53.8 ± 2.1	86.7 ± 3.2	96.7 ± 2.6
	Quercetin 30 µM	74.0 ± 15.5***	59.3 ± 5.8	52.6 ± 1.5	52.9 ± 2.0***	86.9 ± 3.2	96.6 ± 2.6
	ΔChanges at 30 µM	−13.5 ± 1.3	−8.0 ± 2.7	−1.5 ± 0.6	−3.5 ± 0.4	−0.1 ± 0.2	−0.1 ± 0.2

Values are the mean ± S.E.M. from 6 ventricular preparations. Asterisks indicate significant differences from corresponding values before the application of quercetin. * p < 0.05, ** p < 0.01, and *** p < 0.001.

RESULTS

The contractile force of isolated ventricular free wall preparations from diabetic mice was smaller than that of normal mice; the contractile force in normal and diabetic mice was 114.9 ± 8.8 mg/mm² (n = 18) and 85.2 ± 7.4 mg/mm² (n = 18), respectively (p < 0.05). The rate of contraction was slower in diabetic mice than in normal mice; the contraction time in normal and diabetic mice was 47.9 ± 0.7 ms (n = 18) and 52.2 ± 0.7 ms (n = 18), respectively (p < 0.001). The rate of relaxation was slower in diabetic mice than in normal mice (Fig. 1); the relaxation time in normal and diabetic mice was 67.5 ± 0.8 ms (n = 18) and 78.0 ± 0.8 ms (n = 18), respectively (p < 0.001).

Quercetin (10 and 30 µM) caused a slight decrease in the contractile force and the contraction time in both normal and diabetic mice (Table 1). Quercetin accelerated the relaxation in both normal and diabetic mice; the effect was dependent on the concentration (Fig. 1, Table 1).

SEA0400, an inhibitor of the sarcolemmal NCX,¹⁷⁾ scarcely affected relaxation; the relaxation time in normal and diabetic mice was increased by 2.8 ± 1.0 ms (n = 6) and 3.7 ± 1.6 ms (n = 6), respectively, after the application of 10 µM SEA0400. The effects of quercetin on the contractile parameters were not affected by SEA0400 (Fig. 1, Table 1).

CPA, an inhibitor of the SERCA,¹⁸⁾ significantly delayed relaxation. The relaxation time in normal and diabetic mice was prolonged by 20.3 ± 3.2 ms (n = 6) and 17.4 ± 2.1 ms (n = 6), respectively, after the application of 3 µM CPA. The decreases in the contractile force and contraction time induced by quercetin were not affected by CPA (Table 1). The quercetin-induced acceleration of relaxation was completely inhibited by CPA (Fig. 1, Table 1).

DISCUSSION

In the present study, we demonstrated that quercetin accelerates myocardial relaxation in isolated ventricular tissue preparations from normal and diabetic mice, and determined the transporter involved using selective inhibitors of the NCX and SERCA.

In cardiomyocytes, the NCX is expressed on the sarcolemma, including the T-tubules, and is considered to be the main pathway for transsarcolemmal Ca²⁺ extrusion from the cytoplasm. The expression level of the NCX is reduced in the diabetic myocardium.^3,4) Concerning myocardial relaxation, the present observation that SEA0400 had no effect on the relaxation time indicated that transsarcolemmal Ca²⁺ extrusion by the NCX does not affect myocardial relaxation rate.

The SERCA is considered to be the major transporter for the uptake of cytoplasmic Ca²⁺ into the sarcoplasmic reticulum; its activity was reported to be reduced in the diabetic myocardium.^3,4) In our present and previous⁵⁾ studies, CPA markedly prolonged myocardial relaxation in both normal and diabetic mice, providing functional evidence that the SERCA activity is indeed a major determinant of myocardial relaxation under both normal and diabetic conditions.

Quercetin accelerated relaxation in both normal and diabetic myocardium. Concerning the acute myocardial effect of quercetin, conflicting results have been reported.^12–15) Positive and negative effects on the Ca²⁺ current amplitude and maximal cellular shortening were observed. Concerning lusitropic effects, both an acceleration of the Ca²⁺ transient decay and a lack of effects on the maximum relaxing velocity have been reported in isolated mouse ventricular cardiomyocytes. These discrepancies may be attributed to factors such as variations among cardiomyocytes and disturbance of Ca²⁺ measurements by the fluorescence of quercetin. The present results, free from such factors, showed that quercetin exerts positive lusitropic effects on the myocardium. The effect was completely inhibited by CPA, indicating that it is mediated by activation of the SERCA.

The precise mechanism of action of quercetin remains to be clarified. Quercetin reduced the contractile force and tended to accelerate the rate of contraction in both normal and diabetic mice, and these effects were not affected by SEA0400 and CPA. Quercetin was reported to inhibit the L-type Ca²⁺ channel^11,19) and the ryanodine receptor,²⁰⁾ which play important roles in the action potential-triggered increase in the cytoplasmic Ca²⁺ concentration, the so-called Ca²⁺-induced Ca²⁺ release. Its inhibition by quercetin may decrease the maximum Ca²⁺ concentration and the time to reach it. The overall effect of quercetin on the myocardium is different from β-adrenoceptor agonists, which show both positive inotropic and lusitropic effects.¹⁶⁾ This implies that quercetin is less likely to increase myocardial oxygen demand, which may be an advantage in the treatment of failing myocardium. Although pharmacological activation of the SERCA is anticipated to be effective for the treatment of various myocardial disorders accompanied by impaired myocardial relaxation, a specific activator of the SERCA is not clinically available at present. Naturally occurring compounds that accelerate myocardial relaxation through activation of the SERCA include ellagic acid and gingerol,⁵⁾ which were effective in both normal and diabetic myocardia. Further investigation on the pharmacological property and clinical potential of quercetin and related compounds may lead to the development of novel therapeutic agents.

Acknowledgments

This work was supported by JSPS KAKENHI Grant Nos. JP20K16013, JP20K07299, and JP20K07091.

Conflict of Interest

The authors declare no conflict of interest.

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