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Influence of Crocetin, a Natural Carotenoid Dicarboxylic Acid in Saffron, on L-Type Ca2+ Current, Intracellular Ca2+ Handling and Contraction of Isolated Rat Cardiomyocytes
Zhifeng ZhaoBin ZhengJinghan LiZiheng WeiSijie ChuXue HanLi Chu Hongfang WangXi Chu
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2020 Volume 43 Issue 9 Pages 1367-1374

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Abstract

Crocetin is a major bioactive ingredient in saffron (Crocus sativus L.) and has favorable cardiovascular effects. Here, the effects of crocetin on L-type Ca2+ current (ICa-L), contractility, and the Ca2+ transients of rat cardiomyocytes, were investigated via patch-clamp technique and the Ion Optix system. A 600 µg/mL dose of crocetin decreased ICa-L 31.50 ± 2.53% in normal myocytes and 35.56 ± 2.42% in ischemic myocytes, respectively. The current voltage nexus of the calcium current, the reversal of the calcium current, and the activation/deactivation of the calcium current was not changed. At 600 µg/mL, crocetin abated cell shortening by 28.6 ± 2.31%, with a decrease in the time to 50% of the peak and a decrease in the time to 50% of the baseline. At 600 µg/mL, crocetin abated the crest value of the ephemeral Ca2+ by 31.87 ± 2.57%. The time to half maximal of Ca2+ peak and the time constant of decay of Ca2+ transient were both reduced. Our results suggest that crocetin inhibits L-type Ca2+ channels, causing decreased intracellular Ca2+ concentration and contractility in adult rat ventricular myocytes. These findings reveal crocetin's potential use as a calcium channel antagonist for the treatment of cardiovascular disease.

INTRODUCTION

Saffron (Crocus sativus L.) is a herb that originates in Iran and approximately 85% of the world saffron now is cultivated in Iran.1) Saffron as a medicinal plant has many therapeutic effects. The content of this herb includes proteins (12%), sugars (63%), crude fiber (5%), fat (5% (w/w)), minerals (5%) and moisture (10%). The major bioactive components in saffron include crocetin, crocin, picrocrocin, and safranal.2) Crocetin is one of the most important components in saffron. It has a polyunsaturated conjugated enoic acid structure and is a type of carotenoid. Crocetin has advantageous cardiovascular effects, such as reducing oxidative stress3) and inhibiting the development of insulin resistance,4,5) atherosclerosis,6) hypertension,7,8) cardiac hypertrophy,9) and related diseases. Several studies have revealed that crocetin can effectively protect against cardiac damage that is caused by myocardial infarction through several mechanisms. These include reducing oxidative stress and inflammatory cytokines, reducing lipid peroxidation and Ca2+ levels,10) increasing superoxide dismutase (SOD) activity and glutathione (GSH) content,11) and thus reducing the occurrence of myocardial infarction and maintaining cardiac function.12,13)

From earlier in vitro studies, negative inotropic and bradycardic effects of crocetin have been reported. There have been few studies focused on the role of crocetin in the regulation of contractility and Ca2+ signaling in cardiac muscle. Ca2+ plays an important role in cardiac electrophysiology. When cardiac myocytes are excited, changes in cell membrane potential activate (and open) L-type calcium channels (LTCCs), allowing influx of extracellular Ca2+ into the cytoplasm. This further activates the sarcoplasmic reticulum to release Ca2+ into the cytoplasm, and cytoplasmic concentration rise rapidly. Calcium, combined with troponin, induce muscle wire to central, sarcomere myocardial contraction, complete an excitation-contraction coupling.14) Experiments (in vivo and in vitro) reveal that substances that inhibit calcium channels ameliorate intracellular Ca2+ homeostasis through the mitigation of Ca2+ overload.15,16) This mechanism underlies the cardioprotective effect and reduces hypoxic cardiomyocyte damage.17)

In many physiological and pathological conditions, for instance, glycolysis, oxidant stress, and myocardial ischemia leads to calcium influx, triggering an increase in the release of solute calcium ions from the sarcoplasmic reticulum. This leads to an immediate increase in diastolic calcium concentration and calcium overload, as well as arrhythmia and cellular injury. Therefore, decreasing the diastolic calcium concentration is of great importance for improving the normal physiological function of cardiac myocytes.

Our previous results showed that crocin, another component of saffron, inhibits calcium currents and reduces the intracellular calcium concentration.18) In recent years, many studies have investigated the anti-myocardial injury of crocetin,19) and its mechanism of action have been conducted.11) However, the influence of crocetin and its role in Ca2+ homeostasis and contraction remain unexplored. The purpose of this investigation is to determine whether crocetin is capable of inhibiting the calcium current, cytosolic Ca2+ concentration, and myocyte contractility in rat cardiac myocytes. Whole cell patch clamp, dual excitation, ion fluorescence photomultiplier system and edge detection (based on video) techniques were used to conduct this investigation.

MATERIALS AND METHODS

Materials

Crocetin was purchased from Yuanye Bio-Technology Co., Ltd. (Songjiang, Shanghai, China), and selection of collagenase type II (Lakewood, NJ, U.S.A.). Nicardipin (Nic), Verapamil (Ver) were purchased from Hefeng Pharmaceutical Co., Ltd. (Pudong, Shanghai, China). Unless otherwise noted, additional chemicals were obtained from Sigma-Aldrich (St. Louis, MO, U.S.A.). Solutions used in these experiments were of analytical purity.

Solutions

Calcium-free Tyrode’s aqueous solution, Kreb’s buffer aqueous solution, and regular Tyrode’s aqueous solution were prepared as reported previously.20,21) The enzymatic hydrolysate resembled calcium-free Tyrode’s aqueous solution, it included 0.5 mg/mL of bovine serum albumin, 0.6 mg/mL of collagenase type II, and 30 µmol/L of CaCl2. The intracellular pipette solution contained 5 mmol/L of Mg-ATP, 10 mmol/L of N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES), 10 mmol/L of ethylene glycol bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA), 120 mmol/L of CsCl and 20 mmol/L of tetraethylammonium chloride, pH was brought to 7.2–7.3 using CSOH. The external solution for whole-cell patch-clamp equipment included 2 mmol/L of MgCl2, 140 mmol/L of tetraethylammonium chloride, 10 mmol/L of glucose, 10 mmol/L of HEPES, and 1.8 mmol/L of CaCl2, and pH was brought to 7.3–7.4 using CsOH. Crocetin in its free-acid form is insoluble in water and most organic solvents, except for dimethyl sulfoxide (DMSO) and pyridine.22) Crocetin was dissolved in DMSO and diluted in the external solution to achieve concentrations of 100, 200, 300, 400, 500, 600 µg/mL, prepared daily and protected from light. The final concentration of DMSO was 0.02%. This amount of DMSO was also added to the normal external solution as a control and had no effects on calcium current, calcium transient and myocardial contraction.

Experimental Animals

Adult Sprague-Dawley rats (6–8 weeks old, 180–220 g) were obtained from the Laboratory Animal Center of Hebei Medical University. The rat were housed in rust-free cages at 22–25°C and 45–60% relative humidity on a 12 h light–dark cycle, and spontaneous intake of normal granular diet and tap water. In addition, veterinarians and researchers were have monitored animal health and behavior twice a day at 8:00 and 17:00. All experiments conformed to the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH Publication, 8 th Edition, 2011). This study was carried out following the recommendations of the Declaration of Helsinki. The protocol was approved by the Hebei University of Chinese Medicine Committee on Animal Care. All experiments were approved by the Ethics Committee of Hebei University of Chinese Medicine.

Acute Isolation of Animal Cardiomyocytes by Enzymatic Disaggregation

Normal cardiomyocytes—The isolation of cardiac muscle cells from rats was completed using enzymatic disaggregation.23) Rats were anesthetized with an intraperitoneal injection of 1.0 g/kg ethyl carbamate. Heparin (500 IU/kg) was injected intraperitoneally to prevent blood coagulation. Rapid cardiac resection was carried out and the rats were intubated and perfused with a langendorff device. The blood was removed from the main artery at a speed of 4 mL/min using calcium-free tyrode aqueous solution. The blood was removed for 3 min until spontaneous contractions stopped. Perfusion of the heart was then performed with the enzymatic hydrolysate for about 20 min until the heart relaxed.

In the last step, the calcium-free tyrode aqueous solution was used to elute the enzymatic hydrolysate. After completion of the perfusion, the ventriculus sinister was cut into small pieces and immediately placed in petri dishes. Cardiomyocytes were kept in Kreb’s solution at 25°C for at more than one hour before they were tested. All solutions used in the separation process were bubbled using 100% oxygen for more than 30 min and the whole operation was carried out after ten hours of being separated.

Ischemic cardiomyocytes—In short, ISO (90 mg/kg) was subcutaneously injected into rats to induce myocardial ischemia.24) After induction of myocardial ischemia for two consecutive days, the heart was removed and used for experiments, similar to normal rat ventricular myocytes above.

Whole Cell Patch Clamp Method

The calcium current was measured in cardiac muscle cells isolated from rats at 23–25°C. The myocardial cells were placed in an observation chamber and observed under an inverted microscope. Cells were kept in an extracellular fluid at a flow rate of 2–3 mL/min for ten minutes. When filled with the intracellular pipette solution, patch electrodes had a resistance ranging from 3–5 MΩ and were made from borosilicate glass using a Micropipette Puller Model 97 (Sutter Instrument, Novato, CA, U.S.A.). Only rod-type shaped cells with a clear margin and striation were used in these experiments. Junction potentials of the electrode were adjusted to the proper value for a formation of a gigaseal. A suction pulse was used to rupture the membrane to establish whole-cell voltage-clamp recordings after obtaining a gigaseal. Membrane capacitance and series resistance were compensated after membrane rupture to minimize the duration of the capacitive current. Series resistance was compensated to the maximum extent (about 50–70%). The capacitive transient was caused by a 0 mv depolarizing step 10 ms duration from a holding potential (HP) of −80 mv. It can estimate membrane capacitance (Cm) from the capacitive transient and calculate according to Cm = τm×IoVm(1−Iss/Io), τm is the membrane time constant, Io is the maximal amplitude of the capacitive current spike, Iss is the current at the end of the 10 ms pulse (steady state) and Vm is the amplitude of the voltage step (Cm = 156.3 ± 22.5 pF, n = 60).25)

The whole cell current was stimulated for 200 µs depolarizing pulses at a frequency of 0.5 Hz, and the holding potential was kept at −80 mV and depolarized to 0 mV. According to the steady-state activation protocol used, the current evoked by this depolarizing pulse (for 200 µs).25,26) The steady-state inactivation curves were obtained by applying a one-second prepulse to voltages between −60–60 mV, at 10 mV intervals, before the −80–0 mV 200 µs test pulse. A patch clamp amplifier (Axopatch 200B, Axon Instruments, Union City, CA, U.S.A.) was used to record the transmembrane current. The sampling and data analyses were done using pCLAMP 10.2 software (Axon Instruments).

Monitoring of Ventricular Myocyte Contraction

The mechanical properties of the cardiac myocytes were evaluated using the video-based edge-detection system (IonOptix) (Milton, MA, U.S.A.). The cells were placed in a recording chamber on an inverted microscope table, and normal tyrode solution with 1.8 mmol/L of CaCl2 was perfused at a rate of 1 mL/min. Cell contraction was induced via stimulation with an electric field at a frequency of 0.5 Hz at a strength of two times the Shrinkage threshold.

Measurement of Cytosolic Calcium Ion Concentration

Myocytes were loaded with the fluorescent indicator fura-2 AM (100 nM to 5 mM) at 23–25°C in the dark, and the fluorescence was measured using a dual-excitation fluorescence photomultiplier system (IonOptix; Milton, MA, U.S.A.). Myocardial cells were imaged by fluorescence using a 4 × oil objective lens, and irradiated alternately with 340 or 380 nm filters (bandwidth + 15 nm) and stimulated in situ at 0.5 Hz, contracting once every two milliseconds. The fluorescence produced was emitted at 510 nm and the fluorescence ratio of the two wavelengths (340/380) was calculated to obtain the index of cytoplasmic calcium ion concentration.27)

Data Analysis

Results are presented in the form of mean (± standard error). Multiple comparisons were analyzed using a one-way ANOVA, followed by a least significance difference test performed using the social science statistics package 19.0 software. A p-value <0.05 threshold was used to determine statistical significance.

RESULTS

Identification of Calcium Current

Calcium current in rat cardiomyocytes was used to measure the voltage-dependence of steady-state activation. The current can be almost eliminated by using 0.01 mmol/L Nic,28) which revealed that these electrical currents were calcium ion electrical currents (Fig. 1A) (p < 0.01). Ver, with a concentration of 0.01 mmol/L is a specific LTCCs blocker, which can block nearly all calcium currents, revealing that these currents are calcium mediated (Fig. 1B) (p < 0.01).29)

Fig. 1. Confirmation of Calcium Current

The typical calcium current before and after the steady-state activation protocol was found to be similar under Nic (0.01 mmol/L) (A) and Ver (0.01 mmol/L) (B). The calcium current in rat cardiomyocytes was almost entirely interdicted by Ver and Nic. The results are expressed as mean (± standard error; n = 12 cells ** p < 0.01 vs. control).

Effects of Crocetin on the Calcium Current in Ventricular Myocytes

Figure 2 shows the structure of crocetin. Crocetin (C20H24O4; molecular weight 328). The maximum calcium current was notably depressed after exposure to 600 µg/mL of crocetin (p < 0.01). After elution of the crocetin solution, the calcium current was recovered (Figs. 3A, B), indicating that the effect of crocetin on the calcium current is reversible. The rates of inhibition of by crocetin at 100, 200, 300, 400, 500, and 600 µg/mL were 2.32 ± 1.84, 10.42 ± 1.77, 12.57 ± 1.11, 18.09 ± 1.82, 24.0 ± 2.41, and 31.5 ± 2.53%, respectively (Fig. 3C).

Fig. 2. The Structural Formula of Crocetin
Fig. 3. The Effects of Crocetin on Calcium Current of Cardiomyocytes (A, B) Sample Records, Aggregated Information and Time Course of Calcium Currents Were Recorded under Controlled Conditions Using Crocetin (600 µg/mL) and during Washout

The results are expressed as mean (±standard error; n = 12 cells; ** p < 0.01, vs. control). (C) The concentration response curve indicates the rate of inhibition caused by crocetin. The results are expressed as mean (± standard error; n = 10–12 cells).

Effects of Crocetin on the Current–Voltage Relationship of Calcium Current

Figure 4A shows the current–voltage relationship with and without crocetin exposure (400 and 600 µg/mL) and 0.01 mmol/L of Ver. Figure 4B illustrates the current produced at different test voltages from −60 to +60 mV. The amplitude of calcium current increases at 221220 mV and reaches its maximum between 0 and 10 mV. Nevertheless, the current–voltage relationship and reversal potential of the calcium current did not change significantly.

Fig. 4. The Effects of Crocetin on Voltage-Dependence of Activation of the Ca2+ Current

(A) Representative traces of steady-state activation with crocetin (400 and 600 µg/mL) or 0.01 mmol/L Ver. (B) The averaged I–V relationships of the calcium current in cardiac muscle cell in the deficiency (□) or the existence of 400 µg/mL of crocetin (○), 600 µg/mL of crocetin (△), or 0.01 mmol/L of Ver (▽). The results are expressed as mean (±standard error; n = 10–12 cells).

Effects of Crocetin on L-Type Ca2+ Current (ICa-L) in Ventricular Myocytes of Normal Rats and Myocardial Ischemic Rats

Representative current recordings with the activation protocol after the sequential handles of 400 µg/mL and 600 µg/mL crocetin are shown in Fig. 5. Figures 5A and C are exemplary traces of ICa-L in ventricular myocytes of normal rats and myocardial ischemic rats, respectively. Figure 5B shows that the peak amplitude of ICa-L in normal rats was decreased by 17.52 ± 2.87 and 30.52 ± 2.56% by crocetin at 400 and 600 µg/mL, respectively (p < 0.01). Figure 5D shows that the peak amplitude of ICa-L in myocardial ischemic rats was decreased by 21.37 ± 0.92 and 35.56 ± 2.42% by crocetin derivatives at 400 and 600 µg/mL, respectively (p < 0.01).

Fig. 5. The Effects of Crocetin on ICa-L in Ventricular Myocytes of Normal Rats and Myocardial Ischemic Rats

Exemplary traces (A, C) and pooled data (B, D) of ICa-L were recorded under control conditions and during exposure to 400 µg/mL, 600 µg/mL crocetin. (A, B) Effects of crocetin on ICa-L in normal rat ventricular myocytes (±standard error; n = 12 cells, ** p < 0.01, vs. control). (C, D) Effects of crocetin on ICa-L in myocardial ischemic rat ventricular myocytes (±standard error; n = 12 cells, ** p < 0.01, vs. control).

Effects of Crocetin on Calcium Current Steady Activation and Inactivation

Figure 6 exhibits the voltage dependence of steady-state activation and inactivation of calcium current in the presence and absence of crocetin (400 µg/mL and 600 µg/mL). The current density–voltage (I–V) curve was obtained from the steady-state activation curve and fitted using a Boltzmann function with the formula G/Gmax = 1/{1 + exp(V1/2 − V)/k}.

Fig. 6. The Effects of Crocetin on the Stable Activation and Inactivation of Calcium Current

(A) The steady-state activation curves based on standardized conductance of the I–V curves in the deficiency (□) or the existence of 400 µg/mL of crocetin (O) or 600 µg/mL of crocetin (△). (B) Normalized steady-state inactivation of calcium current in the deficiency (O) or after 400 µg/mL of crocetin (□) or 600 µg/mL of crocetin (△). The results are expressed as mean (±standard error) (n = 8–10 cells).

The V1/2 value of the standardized activation conductivity curve was found to be −10.98 ± 0.76 mV, the slope (k) was 7.09 ± 0.69 mV, the control group was −8.87 ± 0.54 mV, the k value was 7.11 ± 0.48, the concentration of crocetin was 400 µg/mL, the k value was −7.46 ± 0.35 mV, 600 µg/mL was 7.39 ± 0.31 mV, in fits of the steady-state inactivation curve. I/Imax = 1/{1 + exp(V – V1/2)/k} is the formula of Boltzmann function, whereas k represents the slope and V1/2 represents half-deactivation voltage. Steady-state inactivation (V1/2) value was −32.45 ± 0.16 mV, k value was 5.16 ± 0.14 mV, control group was −33.21 ± 0.07 mV, k value was 4.78 ± 0.06, 400 µg/mL of crocetin was −34.09 ± 0.30 mV, 600 µg/mL of crocetin was 5.32 ± 0.26 mV.

Effects of Crocetin on Cell Shortening and the Index of Cell Contraction Time

Figures 7A and B show that the typical cell shortening before and after 600 µg/mL crocetin. Figure 7C indicates that crocetin notably inhibits cell shortening by 28.6 ± 2.31%, at a concentration of 600 µg/mL (p < 0.01). The time to 50% of the peak (Tp) is an important index of cell contraction velocity. Furthermore, the time to 50% of the baseline (Tr) is an important index of cell laxity. Figures 7D and E show that crocetin decreased Tp (p < 0.01) and Tr for myocyte shortening (p < 0.05), respectively.

Fig. 7. The Effects of Crocetin on Cell Contraction and the Index of Cell Contraction Time

(A) Effects of 600 µg/mL crocetin on cell contraction in cardiac myocytes. (B) Single typical trace recorded under control, crocetin (600 µg/mL) and wash out. (C) Summary of contraction data of cardiomyocytes under control, crocetin (600 µg/mL) and wash out. (D) The time to 50% of the peak. (E) The time to 50% of the baseline. The results are expressed as mean (±standard error; * p < 0.05, ** p < 0.01, vs. control, n = 10 cells).

Effects of Crocetin on Calcium Ion Transients and the Index of Calcium Transient Time

Figure 8A shows the change in the Ca2+ transient in the presence of crocetin (600 µg/mL). Figure 8B shows the typical Ca2+ transients in the absence and presence of crocetin (600 µg/mL). As shown in Fig. 8C, the results indicate that the transient amplitude of Ca2+ is decreased by 31.87 ± 2.57% (p < 0.01). Figures 8D and E show that crocetin decreased the time to half maximal of Ca2+ peak (p < 0.05) and the time constant of decay of Ca2+ transient, respectively. Crocetin decreased the time constant from 611 ± 23 to 487 ± 19 ms (p < 0.01).

Fig. 8. The Effects of Crocetin on Calcium Ion Transients and the Index of Calcium Transient Time

(A) Effects of 600 µg/mL crocetin on calcium ion transients in cardiac myocytes. (B) Single typical trace recorded under control, crocetin (600 µg/mL) and wash out. (C) Summary data of Ca2+ transients in cardiomyocytes recorded under control, crocetin (600 µg/mL) and wash out. (D) The time to half maximal of Ca2+ peak. (E) The time constant of decay of Ca2+ transient. The results are expressed as mean (± standard error; * p < 0.05, ** p < 0.01, vs. control, n = 10 cells).

DISCUSSION

In recent years, the increased demand for herbal medicines as natural, effective and safe medicines has led to the discovery of many natural products that have great therapeutic value in preventing or treating various diseases. Crocetin can effectively control myocardial injury and maintain the function of the heart.13,24) Although it has been reported that crocetin shows comparatively beneficial effects on cardiovascular injury, to the best of our knowledge, there has been no systematic study on its effect on LTCC, Ca2+ influx and myocontraction. In the present investigation, the encouraging findings indicated that crocetin possesses the basic feature of calcium channel antagonists (CCAs), it describes crocetin as a candidate for medicinal applications. It is generally recognized that CCAs are widely used for the treatment of cardiovascular diseases such as cardiac ischemia, hypertension, arrhythmias, and atherosclerosis.30) Many studies have shown that LTCCs play an important role in the Ca2+ influx of cardiomyocytes, and it has become a hotspot to regulate LTCCs activity.31,32) In this study, we describe the effects of crocetin on ventricular myocytes and studied how crocetin affects ion channels. We investigated the effects of crocetin on the L-type Ca2+ current, Ca2+ transients and the contractility of rat cardiomyocytes. The results of these experiments reveal that crocetin can inhibit myocardial contractility and affects calcium ion inflow through calcium channels, which are similar pharmacological effects caused by calcium antagonists.

Crocetin and its digentiobiosyl ester, crocin are the two important active constituents in saffron, of which components, characteristics, and activities have been investigated by many researchers.33) The present results confirm that crocetin possesses the basic features of CCAs. Interestingly, our previous research has shown that crocin, which consist of a group of crocetin, also has an inhibitory effect on LTCC. However, the maximum inhibition rate of L-type calcium current by crocin is 72.195 ± 1.54%, which is much higher than that of crocetin.18) This gap may be due to the different physical properties of crocetin and crocin. Contrary to crocetin, crocins are water-soluble carotenoids.34) Crocetin contains a carboxyl group at each end of the polyene chain, in its free-acid form, crocetin is insoluble in water and most organic solvents, except for pyridine and dimethylsulfoxide,33) which perhaps limits its pharmacological activities in vitro experiments. In addition, previous experiments have indicated that crocetin has myocardial protective effects, while our experiments provide a theoretical basis for confirming that crocetin has a calcium antagonistic effect and anti-myocardial ischemia effect.

These results reveal that crocetin inhibits LTCCs in rat ventricular myocytes, and this inhibition is reversible (Fig. 3). In these experiments, ICa-L continues to decrease during long-lasting recordings under whole-cell patch-clamp. This phenomenon is called “run-down,” it is a common problem in recording calcium ion channel currents. “Run-down” of ICa-L can partially be explained by a rise in intracellular Ca2+ concentration and a loss of high energy compounds. To avert the interference of “run-down” of calcium currents, we added EGTA (10 mmol/L) and Mg-ATP (5 mmol/L) to the intracellular pipette solution, similarly to Belles et al.35) The beneficial effects of ATP include an increased capacity of the cells to either extrude or sequester intracellular Ca2+, and is protective against enzymatic proteolysis. EGTA (in the intracellular pipette solution) prolongs the Ca2+ current survival. The survival of ICa-L was significantly prolonged when EGTA and ATP were added to the intracellular pipette solution. Currents were recorded five to twenty-five minutes after membrane rupture.36) The partial recovery (after scouring) ensured that the effect of crocetin on calcium current is not the result of “run-down.” Our results indicate that it is effective to record ICa-L. After washout, the currents partially recovered, indicating that the effect of crocetin on ICa-L was not a consequence of ICa-L rundown.

LTCCs can typically be recorded in secretory cells, along with muscle cells. LTCCs cause the contraction of cardiac muscle cells and are indispensable in the process of shrunk coupling. LTCCs are characterized by slow inactivation and have slow kinetics for opening, which is the primary pathway of intracellular calcium ion influx during excitation. Blocking this channel can lead to negative myodynamic effects on cardiac myocytes.37) The opening of voltage-gated LTCCs, induced by the excitation of cardiomyocytes, facilitates Ca2+ flow into the cells, and subsequently leads to an increase in intracellular Ca2+. Experiments here reveal that crocetin can reduce the calcium current in a concentration-dependent manner. But it did not affect the voltage-dependence of activation or the reversal potential of calcium (Fig. 4). Crocetin was found to inhibit the maximum value calcium current without changing the steady state activation and inactivation of the calcium current (Fig. 6). These results reveal that crocetin inhibits the calcium current mainly by reducing the amplitude of the calcium current.

Ca2+ enters the cell via LTCCs, which stimulates the sarcoplasmic reticulum to release Ca2+ and bind to troponin. After that, myosin binds to troponin, which is an important step for myocardial contraction. In the process of contraction, extracellular calcium influx and intracellular calcium ion concentrations are increased, thereby activating the calcium pump in the sarcoplasmic reticulum, and reducing the Ca2+ concentration after regulation, and this process is ephemeral Ca2+.14,38) In these experiments, the prohibitive effects of crocetin on LTCCs caused a decrease in heart ionotropic, while the calcium ion instantaneous condition and contractibility drop in the midst of crocetin (Figs. 7, 8). Myocardial contraction is related to the concentration of solute Ca2+ and intracellular proteins, and also to contraction (myosin and actin) and regulatory mechanisms (troponin, proglobulin and protomyosin). The effect of crocetin on cell shortening seems to be more transient than that of calcium.39) The mechanism of action of crocetin on myocardial contraction needs further study.

In the experiments presented here, crocetin reduced Tp and Tr, in which Tp and Tr represent the determinant of cell contraction and relaxation speed (Fig. 7). Contrary to crocetin, our previous research results indicated that crocin increased Tp. This gap may also be due to the differences of chemical and physical properties between crocetin and crocin, the comparative evaluation of inhibitory activities on Ca2+ influx is required in future. Crocetin may also induce the delivery of calcium ions in the sarcoplasmic reticulum by inhibiting the activity of LTCCs. These results in the reduction of available cytoplasmic calcium ions, which are shown by the transient decrease of calcium ions in the sarcoplasmic reticulum.40)

In our study, we first detected the suppressive effect of crocetin on isolated adult-rat healthy and ischemic cardiomyocytes. This reveals that crocetin restrained cardiac calcium ion content to prevent calcium ion influx. Intracellular calcium ion concentration can further aggravate myocardial tissue injury. Blocking calcium ion channels in ischemic cardiomyocytes rapidly depressed myocardial electrical motion and contractile motion, which preserves energy for later repair and plays a protective role in the myocardium.41) Accordingly, our research indicates that crocetin may play an active role as a LTCCs blocker for the protection of the musculus cardiacus and is preventative for ischemic injury. Nevertheless, the exact mechanism of inhibiting calcium current by crocetin is unexplored.

CONCLUSION

Taken together, this study clearly shows that crocetin can suppress Ca2+ transients and contractility in cardiac myocytes. This occurs mainly by inhibiting LTCCs, which inhibits the influx of calcium ions into cardiac myocytes and reduces the concentration of calcium ions in cell solutes. These findings provide new perspectives for further research on the pharmacology of crocetin as a LTCCs inhibitor for the treatment of cardiovascular diseases.

Acknowledgment

This work was supported by the Research Fund of Administration of Traditional Chinese Medicine of Hebei Province, China (No. 2015030).

Conflict of Interest

The authors declare no conflict of interest.

REFERENCES
 
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