2024 Volume 47 Issue 12 Pages 2138-2142
The effect of a citrus-derived flavonoid, hesperetin, on the automaticity and contraction of isolated guinea pig myocardium was examined. Hesperetin inhibited the rate of ectopic action potential firing of the pulmonary vein myocardium; the slope of the diastolic depolarization was decreased with minimum change in the action potential waveform. The effect was dependent on the concentration; the EC50 value for firing rate was 56.2 ± 14.2 µM and that for the diastolic depolarization slope was 41.6 ± 13.4 µM. In the right atria, hesperetin had no effect on the beating rate at concentrations up to 30 µM, but showed a negative chronotropic effect at 100 µM. In the ventricular papillary muscles, hesperetin had no effect on the contractile force at concentrations up to 30 µM, but showed a slight positive inotropic effect at 100 µM. In isolated pulmonary vein cardiomyocytes, 30 µM hesperetin decreased the firing rate of spontaneous Ca2+ transients. These results showed that hesperetin has a selective inhibitory action on the pulmonary vein automaticity with no inhibitory effect on the ventricular contractile force. Clarification of the mechanism of action of hesperetin as well as further exploration of flavonoids would provide clues for the development of pulmonary vein-selective therapeutic agents.
The contraction of the ventricle, which is responsible for the pumping function of the heart, is triggered by the sinus node, the orthotopic pacemaker located in the right atria. On the other hand, ectopic pacemaker activity arising in various parts of the heart may cause arrhythmia. The pulmonary vein myocardium is one of the sources of such ectopic automatic activity, which is considered to account for most of the causes of atrial fibrillation.1,2) The pulmonary vein cardiomyocytes possess ion channels for depolarization and generate action potentials under the influence of the intracardiac intrinsic neuronal network and various humoral factors.3,4) Atrial fibrillation is initiated when abnormal excitation from the pulmonary vein myocardium triggers re-entry of excitation in the form of multiple wavelets in the atria. Recent studies revealed that both the onset and maintenance of atrial fibrillation are caused by persistent triggers including those in the pulmonary vein.5) At present, the effectiveness of antiarrhythmic drugs used for the treatment of atrial fibrillation, such as class I antiarrhythmic drugs, is not satisfactory.1,6,7) This is probably because these drugs were not developed to inhibit the ectopic automaticity of the pulmonary vein myocardium. Further, these antiarrhythmic drugs often show unwanted cardiosuppressive effects to decrease cardiac output, which limits their use in patients with reduced cardiac function. This is caused by the negative inotropic effects on the ventricular myocardium and chronotropic effects on the sinus node.1,8,9) Thus, drugs that selectively inhibit the automatic firing of the pulmonary vein myocardium would be potential therapeutic agents for the treatment of atrial fibrillation. At present, however, antiarrhythmic drugs with selective inhibitory effects on the pulmonary vein automaticity have not been developed.
Antiarrhythmic drugs such as ajmaline, digoxin, and quinidine have originated from medicinal plants, and preclinical and clinical studies on the antiarrhythmic effects of phytochemicals have been continuously performed.10) Citrus flavonoids like hesperidin, as well as its aglycone hesperetin, are contained in oranges and grapefruits and are well-documented for their beneficial effects against various disorders through their antioxidant effects.11) Epidemiological studies have shown that long-term intake of hesperidin/hesperetin has protective effects against cardiovascular disorders such as coronary heart disease and ischemic stroke. Concerning the acute effects of hesperetin, blockade of ion channels related to myocardial excitation has also been reported.12,13) However, less information is available on the acute effect of hesperetin on the myocardial parameters such as the automatic activity and the contractile force.
In the present study, we examined the acute effects of hesperetin on the automaticity of the pulmonary vein and sinus node, and the contraction of the ventricular myocardium. We found that hesperetin has selective inhibitory effects on the automaticity of the pulmonary vein myocardium.
All experiments were performed by methods basically the same as those in our previous studies.14,15) They were approved by the Toho University Animal Care and User Committee (22-507, March 31). Hearts with lungs were quickly removed from male Hartley guinea pigs weighing 350–450 g (Japan SLC, Inc., Hamamatsu, Japan) under deep isoflurane anesthesia. The four pulmonary veins were detached from the left atria and each was used in the experiments separately. The region of the pulmonary vein close to the orifice was cut open and the preparation was placed in an organ bath luminal side-up. The size of the preparations was approximately 8 × 3 mm. Microelectrodes were inserted into the myocardial layer from the luminal side to measure the firing rate and parameters of spontaneous action potentials. The glass electrodes were pulled from glass capillaries (GD-2; Narishige Co., Tokyo, Japan) and had a resistance of 20 to 30 MΩ when filled with 3 M KCl solution. The output signal of the microelectrode amplifier (MEZ8201; Nihon Kohden Corporation, Tokyo, Japan) was digitized by an A/D converting interface (Power Lab/4SP, AD Instruments, Dunedin, New Zealand), and analyzed with computer software (Chart 7; AD Instruments).
The contractile force of the right atria and right ventricular papillary muscles was recorded isometrically with a force-displacement transducer (TB-611T; Nihon Kohden Corporation) connected to a carrier amplifier (AP-621G; Nihon Kohden Corporation). The sizes of the right atria and papillary muscles were approximately 10 × 8 and 4 × 2 mm, respectively. The analog signal of contractile force was digitized by an A/D converting interface (Power Lab/4SP, AD Instruments), and analyzed with computer software (Chart 7; AD instruments). The spontaneous beating rate of the right atria was calculated from the cycle length of the beatings.
Pulmonary vein cardiomyocytes were obtained by Langendorff perfusion of the hearts with the pulmonary veins attached. After treatment with 0.3 mg/mL collagenase (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) and 0.04 mg/mL protease (type XIV; Sigma-Aldrich, St. Louis, MO, U.S.A.) for about 15 min, the pulmonary vein cardiomyocytes were isolated. The pulmonary vein cardiomyocytes were treated with 5 µM fluo-4/AM (Dojindo, Kumamoto, Japan). A rapid scanning confocal microscope A1R (Nikon, Tokyo, Japan) was used for observation of the cells. The excitation wavelength for fluo-4 was 488 nm and the emission in the wavelength range of 500 to 550 nm was analyzed. Hesperetin was purchased from FUJIFILM Wako Pure Chemical Corporation and dissolved in dimethyl sulfoxide (DMSO). Data were expressed as means ± standard error of the mean (S.E.M.). Statistical significance between means was evaluated by the paired t-test and one-way repeated-measures ANOVA followed by Dunnett’s test. A p-value less than 0.05 was considered significant.
The pulmonary vein myocardium showed spontaneous firing of action potentials; the action potential upstroke was preceded by a diastolic depolarization (Fig. 1A). Hesperetin, 10 to 100 µM, significantly reduced the firing rate through a reduction of the slope of the diastolic depolarization. This effect was dependent on the concentration of hesperetin (Fig. 1D); the EC50 values for reduction of slope and firing rate were 41.6 ± 13.4 and 56.2 ± 14.2 µM, respectively (n = 6). Hesperetin, 30 µM, increased the action potential duration at 90% (APD90), but had no significant effect on other action potential parameters (Table 1).
A: Typical action potential recordings from the pulmonary vein myocardium in the absence (a) and presence of 30 µM hesperetin (b). Partially expanded traces of the diastolic depolarization in the absence (open circle) and presence (closed circle) of 30 µM hesperetin were overlaid (c). B: Typical traces of spontaneous contraction of right atria before (a) and after the application of 30 µM (b) and 100 µM (c) hesperetin. C: Typical recordings of the contraction of ventricular papillary muscles (a). Arrows indicate the application of 10–100 µM hesperetin. Panels b and c show expanded traces obtained before (open circle) and after the application of 30 or 100 µM (closed circle) hesperetin. D: Summarized data for the hesperetin-induced changes in firing rate (a) and the diastolic depolarization slope (b) of the pulmonary vein, the firing rate of the right atria (c), and the contractile force of ventricular papillary muscles (d). Data points and vertical bars indicate the mean ± S.E.M. from 6 experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. before.
| Hesperetin | |||
|---|---|---|---|
| Before | After | p-Value | |
| Maximum diastolic potential (mV) | −70.6 ± 0.7 | −69.7 ± 0.9 | 0.0978 |
| Threshold (mV) | −63.3 ± 0.5 | −62.4 ± 0.5 | 0.1697 |
| Overshoot (mV) | 21.4 ± 2.7 | 20.4 ± 3.5 | 0.4774 |
| APD20 (ms) | 6.4 ± 0.7 | 6.8 ± 0.8 | 0.1905 |
| APD50 (ms) | 22.7 ± 2.7 | 21.7 ± 2.1 | 0.6388 |
| APD90 (ms) | 122.3 ± 5.9 | 143.6 ± 10.6 * | 0.0167 |
| Maximum rate of rise (dV/dt; V/s) | 64.9 ± 10.5 | 55.6 ± 12.6 | 0.1680 |
| Diastolic depolarization slope (mV/s) | 20.6 ± 5.0 | 10.3 ± 2.4 * | 0.0409 |
| Firing rate (Hz) | 1.7 ± 0.2 | 1.1 ± 0.2 ** | 0.0083 |
Parameters were obtained before and after the addition of 30 µM hesperetin. APD20, APD50 and APD90 indicate action potential duration at 20, 50, and 90% repolarization, respectively. Values are the mean ± S.E.M. from 6 experiments. * p < 0.05, ** p < 0.01 vs. before.
The isolated right atria showed spontaneous beating driven by the sinus node with a constant rate of 3.5 ± 0.1 Hz (Fig. 1B). Hesperetin had no effect on the beating rate at concentrations up to 30 µM, but showed an inhibitory effect at 100 µM (Fig. 1D); the beating rate in the presence of 30 and 100 µM hesperetin was 3.4 ± 0.1 and 3.1 ± 0.1 Hz (n = 6), respectively.
In the ventricular myocardium, hesperetin had no effect on the contractile force at concentrations up to 30 µM, but showed a slight positive inotropy at 100 µM (Figs. 1C, D). Hesperetin had no effect on the time course of contraction and relaxation; the time to peak tension and time for 90% relaxation in the absence of hesperetin were 84.5 ± 14.1 and 81.3 ± 6.2 ms, respectively, and those in the presence of 30 µM hesperetin were 85.3 ± 13.2 and 80.0 ± 6.5 ms, respectively (n = 6; The p-value for the time to peak tension was 0.655 and that for the time for 90% relaxation was 0.256).
Isolated pulmonary vein cardiomyocytes loaded with the Ca2+-sensitive fluoroprobe, fluo-4, showed spontaneous Ca2+ transients (Fig. 2); the increase in Ca2+ concentration began at the subsarcolemmal region and spread to the cell center. Hesperetin reduced the firing frequency of the Ca2+ transients; the frequency before and 5 min after the application of 30 µM hesperetin was 0.59 ± 0.05 and 0.24 ± 0.07 Hz, respectively (n = 10; The p-value was 0.002).
A: Typical Ca2+ fluorescence images of spontaneous Ca2+ transients. The images in panel b and c were obtained at 9 ms and 70 ms, respectively, after the image in panel a as indicated in the upper panel of B. B: Typical time course of the fluorescence intensity of the entire cell shown in A before and 5 min after treatment with 30 µM hesperetin.
In the present study, we found that hesperetin inhibits the ectopic firing of action potentials in the pulmonary vein myocardium. The effect was observed not only in pulmonary vein myocardial tissue but also in isolated cardiomyocytes, indicating that the site of action is the cardiomyocyte itself rather than the intracardiac intrinsic neuronal network. Hesperetin suppressed spontaneous activity through the reduction of the diastolic depolarization slope, with minimum change in the waveform of the main body of the action potential (Fig. 1A). The APD90 was increased but this could be attributed to the prolongation of the diastolic depolarization rather than a delay in the repolarization process. Thus, the main action of hesperetin on the pulmonary vein myocardium is the inhibition of some ion channel(s) that contribute to diastolic depolarization.
Among the myocardial ion channel currents reported to be affected by hesperetin,12,13) the persistent component of the voltage-dependent Na+ channel current (late Na+ current) is involved in the diastolic depolarization of the pulmonary vein myocardium.15,16) Hesperetin, 30 µM, blocked the late Na+ current by 17% in the rabbit ventricular cardiomyocytes.13) Further, in the guinea pig pulmonary vein myocardium, GS458967 and NCC-3902, selective inhibitors of the late Na+ current, decreased the slope of the diastolic depolarization similar to the case with hesperetin.15,16) Hesperetin, as well as GS458967 and NCC-3902, had no effect on the spontaneous beating of the right atria and contractile force of ventricular myocardium in the guinea pig (Figs. 1B, C). Thus, hesperetin appears to act through inhibition of the late Na+ current in the pulmonary vein myocardium. However, other ionic currents such as the hyperpolarization-activated current,17) and the Na+-Ca2+ exchanger current14) are also involved in the diastolic depolarization of the pulmonary vein myocardium, but whether hesperetin affects these currents have not been reported. The ionic mechanism of the inhibitory effect of hesperetin remains to be studied by voltage-clamp experiments with pulmonary vein cardiomyocytes.
Hesperetin could be expected to have minimum adverse effects on the pump function of the heart. It showed no negative inotropic effect (Fig. 1C); but rather tended to cause a slight increase in contractile force at 100 µM. This is different from the class I antiarrhythmic drugs whose negative inotropic effect hampers use in patients with comprised cardiac output.1,8) Hesperetin, 30 µM, was reported to affect neither the canine ventricular action potential duration12) nor the RR interval and QTc interval in the rat heart under Langendorff perfusion.13) In the guinea pig right atria, 30 µM hesperetin did not affect the beating rate, but at higher concentrations, it showed a slight decrease in beating rate (Fig. 1B); this is not surprising because hesperetin was reported to block multiple ion currents involved in the pacemaking of the sinus node.12,13)
Concerning the biological availability of hesperetin, it was reported that administration of 100 mg/kg in the rat resulted in blood concentrations of 20–30 µM.18) In humans, a single oral administration of 126 mg hesperetin resulted in a blood concentration of 2.2 µM.19) Further, a hesperidin analogue, glucosyl hesperidin, which has an improved biological availability has been produced.20) Thus, it is not unrealistic that the presently observed effect of hesperetin is observed in humans after hesperidin/hesperetin administration.
The present study showed that hesperetin selectively inhibits the ectopic automaticity of the guinea pig pulmonary vein myocardium; hesperetin appeared to have the ideal characteristics of an effective and safe pharmacological agent for the treatment of atrial fibrillation. Whether hesperetin shows such effects in other animal species and under pathological conditions remains to be investigated. Further, clarification of the mechanism of action of hesperetin as well as exploration of other flavonoids would provide clues for the development of pulmonary vein-selective therapeutic agents.
This study was supported in part by JSPS KAKENHI Grant numbers: JP20K16013, JP20K07299, and JP24K09852. O.R. received Nagai Memorial Research Scholarship from the Pharmaceutical Society of Japan (N-211504).
The authors declare no conflict of interest.
