6‐Gingerol, an active pungent component of ginger, inhibits L‐type Ca2+ current, contractility, and Ca2+ transients in isolated rat ventricular myocytes

Abstract Ginger has been widely used as a flavor, food, and traditional medicine for centuries. 6‐Gingerol (6‐Gin) is the active components of ginger and offers some beneficial effects on cardiovascular diseases. Here, the effects of 6‐Gin 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. The 6‐Gin decreased the ICa‐L of normal and ischemic ventricular myocytes by 58.17 ± 1.05% and 55.22 ± 1.34%, respectively. 6‐Gin decreased ICa‐L in a concentration‐dependent manner with a half‐maximal inhibitory concentration (IC50) of 31.25 μmol/L. At 300 μmol/L, 6‐Gin reduced the cell shortening by 48.87 ± 5.44% and the transients by 42.5 ± 9.79%. The results indicate that the molecular mechanisms underlying the cardio‐protective effects of 6‐Gin may because of a decreasing of intracellular Ca2+ via the inhibition of ICa‐L and contractility in rat cardiomyocytes.

External Ca 2+ enters the cell by passing through calcium channels. Ca 2+ can also be released from internal Ca 2+ stores including the endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) (Berridge, 1993;Clapham, 1995). This area houses protein synthesis and transport to membranous networks. The Ca 2+ mainly enters through L-type Ca 2+ channels (LTCCs), which are essential to cardiac excitability and excitation-contraction coupling (Ferrier & Howlett, 2001).
L-type Ca 2+ channels are related to Ca 2+ influx (Liu et al., 2016). Therefore, LTCCs blockers generally protect against myocardial ischemic injury via the inhibition of calcium channels. Previous studies have emphasized the inhibitory effect of verapamil (VER) on myocardial contraction and the protective effect on excess calcium overload (Song et al., 2017). Hence, drugs that can weaken I Ca-L are promising for myocardial protection (Song et al., 2016).
Recent reports have detailed the cardio-protective effect of 6-Gin against ischemia-reperfusion injury in rats (Lv et al., 2018); however, the precise mechanism underlying the cellular Ca 2+ homeostasis remains poorly understood. The pathogenesis of ischemic disease is related to Ca 2+ signaling and cardiac function; thus, it is important to explain the direct action of 6-Gin on Ca 2+ homeostasis and contractility in cardiomyocytes as well as the potential character of 6-Gin on treatment of Ca 2+ -related cardiac disease. This work systematically characterized the regulatory effects of 6-Gin on Ltype Ca 2+ current (I Ca-L ), contractility, and Ca 2+ transients in isolated rat ventricular myocytes via the patch-clamp technique and the Ion Optix system. It further explored the possible cellular mechanism of 6-Gin for the management of ischemic cardiac diseases.
Type Ⅱcollagenase was bought from Worthington Biochemical Corporation (USA). VER was from Hefeng Pharmaceutical Co., Ltd.
(China). Other chemicals and reagents were acquired from Sigma (USA) and were of analytical grade.

| Animals
Male Sprague-Dawley rats (180-220 g) were from the National Experimental Animal Center of Hebei, National Science Council.
They were housed in cages at a constant temperature of 25 ± 1°C and supplied with food and water (approval number: 1803064; approval date: March 7, 2018).
The hearts were then quickly excised and perfused at 6 ml/min with Ca 2+ -free Tyrode solution for 4 min and Ca 2+ -free Tyrode's solution containing CaCl 2 (34 μmol/L) and collagenase (500 mg/L) for 15-20 min via Langendorff equipment. The hearts were then washed with Tyrode's solution after the digestion. The freshly dissociated cells were stored in Kreb's buffer solution.
Rats were injected with vasopressin via tail vein (1.5 IU/kg, i.v.) to induce cardiac ischemia (Li et al., 2014). After 10 min of ischemia, the heart was removed as above to isolate normal rat ventricular myocytes.

| Measurement of I Ca-L
The Ca 2+ -current was recorded via the whole cell patch-clamp10.0 software using an Axon patch 200B amplifier (Axon Instrument, F I G U R E 1 Chemical structure of 6-Gin F I G U R E 2 Confirmation of I Ca-L in cardiomyocytes. (a) Exemplary traces and (b) pooled data showed the representative I Ca-L recordings with application of VER (10 μmol/L). Data are expressed as mean ± SEM (n = 5 cells). **p < 0.01 versus control USA). The patch electrodes were pulled with a pipette puller (Sutter Instruments, USA). By recording the I Ca-L , the external solution contained (in mmol/L) TEACl 140, MgCl 2 2, CaCl 2 1.8, glucose 10, and HEPES 10, and the pH was adjusted to 7.4 with CsOH. The intracellular pipette solution contained (in mmol/L) CsCl 120, tetraethylammonium chloride (TEACL) 20, HEPES 10, Mg-ATP 5, and EGTA 10, and the pH was adjusted to 7.2 with CsOH. Drugs were dissolved in Tyrode's solution.
F I G U R E 3 Reversible effects of 6-Gin on I Ca-L in normal ventricular myocytes and ischemic ventricular myocytes. Exemplary traces (a, d), pooled data (b, e), and time course (c, f) of I Ca-L were measured under the treatment of 6-Gin (300 μmol/L) and during washout. (g) Exemplary traces and (h) time course of I Ca-L in exposure to 3, 10, 30, 100, 300 μmol/L 6-Gin or 10 μmol/L VER. (i) Concentration-response curves of 6-Gin. Data are expressed as mean ± SEM (n = 6-8 cells). **p < 0.01, versus control

| Measurement of contractility
The contractions of ventricular myocytes were recorded with a videobased edge-detection system (Ion Optix, USA). Cells were placed on the stage of inverted microscope, and contractility was induced at a frequency of 0.5 Hz. Clear myocytes were selected to measure contractions.

| Measurement of Ca 2+ transients
Fura-2/AM (1 mmol/L) was fitted with a 340 or 380 nm optical filter and used to study ventricular myocyte [Ca 2+ ] i dynamics and associated myocyte contractile function. Ventricular myocyte was loaded with the fluorescent dye in the dark and measured with a fluorescence system (Ion Optix). The contractility of the myocytes was stimulated with a 0.5 Hz field.

| Data analysis
The results were presented as mean ± SEM. Comparisons were analyzed via one-way analysis of variance (ANOVA) followed by the Student's t test using Origin Pro version 9.1 software. p < 0.05 was considered to be statistically significant.

| Effects of 6-Gin on transients
The changes of the 6-Gin on Ca 2+ transients are shown in Figure 7.

| Effects of 6-Gin on contractile and relaxation function
The time to 50% of the peak (Tp) describes the speed of myocyte shortening or Ca 2+ elevation, the time to 50% of the baseline (Tr) is a parameter of cellular relaxation or Ca 2+ reuptake. 6-Gin at 300 μmol/L decreased the Tp and Tr (p < 0.05) (Figure 8). Also, 6-Gin at 300 μmol/L decreased the maximum velocity of contraction-relaxation (±dL/dt) (p < 0.05 or p < 0.01) (Figure 8).

| D ISCUSS I ON
Ginger is a food and traditional medicine used for centuries. 6-Gin is a major active ingredient in ginger and possesses a variety of interesting pharmacological effects. However, the molecular mechanisms of 6-Gin on cardio-protection have yet been reported to the best of our knowledge. This work reports intracellular I Ca-L , contractility, and Ca 2+ transients in isolated rat ventricular myocytes to detail the molecular mechanisms of 6-Gin underlying its cardio-protective effects. The isolated myocyte model provides a specific opportunity to observe physiological adaptations of cardiac function. Calcium is a ubiquitous signal that is responsible for a broad range of cell activities (Berridge, Bootman, & Roderick, 2003;Clapham, 2007). Ca 2+ is rapidly removed from the cytoplasm via pumps (Pozzan, Rizzuto, Volpe, & Meldolesi, 1994) and exchangers (Blaustein & Lederer, 1999), for example, the Ca 2+ -ATPase pumps and Na + /Ca 2+ exchangers. This is then reported via signals. Internal calcium stores are held in the ER or SR membrane systems of muscle cells (Berridge, Lipp, & Bootman, 2000). Calcium ion release is then controlled by various channels including the inositol-1, 4, 5-trisphosphate receptor (InsP3R) and ryanodine receptor (RYR) families (Berridge, 1993;Clapham, 1995). Ca 2+ passing through the calcium channel is important for cardiac electrical activity and the excitation-contraction coupling of cardiac muscle. The principal activator of these channels is Ca 2+ itself.
There is a depolarizing current when calcium ions flow into cells and calcium current flow after the calcium channels open. Other mechanisms for influx of Ca 2+ , for example, Na + /Ca 2+ exchange, can also lead to depolarization and increase cytosolic calcium. A trace of calcium entry from the calcium channel causes more release of Ca 2+ from the SR, that is, Ca 2+ -induced Ca 2+ release (CICR). The CIRC hypothesis (Fabiato, 1983) states that the release of calcium from the SR is not only promoted by a rapid elevation of the Ca 2+ activity (d[Ca 2+ ]j/dt) but also inactivated by a moderate or prolonged elevation of [Ca 2+ ] i . Myocardial contractility was trigged mainly by cytosolic calcium ions entry through calcium channels (Blaustein & Lederer, 1999), which can mediate excitation-contraction coupling.
Cardiac muscle is activated by the depolarization-dependent Ca 2+ current and the release of calcium from SR that elevates myoplasmic calcium and allows the myofilaments to contract (Atwater, Rojas, & Vergara, 1974 Ca 2+ current in cardiac cells does not act primarily as a direct activator of the contractile filaments but that it acts indirectly by releasing Ca 2+ from the SR. A wave of depolarization opens the T-type channels first followed by LTCCs. Calcium antagonists (CCAs) act by changing the mode of channel opening from long-duration to shorts.
Thus, CCAs lower the rate at which Ca 2+ enters via the LTCCs. VER is a CCA and interfered with the calcium-dependent processes.
Our data suggest that 6-Gin reduces the I Ca-L (Figure 3) in a concentration-dependent manner with an IC 50 of 31.25 μmol/L in cardiomyocytes. Figure 4 shows that the I-V relationship or the reversal potential of I Ca-L did not change. Furthermore, the contractility and Ca 2+ transients were inhibited by 6-Gin (Figures 6 and 7).
Also, 6-Gin at 300 μmol/L reduced the I Ca-L in ischemic ventricular myocytes (Figure 3d-f). Ischemia causes membrane depolarization, calcium influx in ischemic cells is increased. Elevated intracellular calcium accelerates the activity of several ATP-consuming enzymes, which further depletes already marginal cellular energy stores, making the heart even more susceptible to ischemic damage (Undrovinas & Maltsev, 1998). Our data suggest that 6-Gin could inhibit the increase in [Ca 2+ ] i via decreasing the extracellular Ca 2+ influx. Excitation-contraction coupling in all cardiac cells requires Ca 2+ influx, therefore the inhibitory effects of 6-Gin on contractility may through the reduction on Ca 2+ influx. Collectively, these results detail the cardio-protective effects of 6-Gin on rat ventricular myocytes as well as and its cellular mechanism.

| CON CLUS IONS
These results clearly indicate that 6-Gin inhibits the Ca 2+ transients and contractility of cardiomyocytes. This is mainly via inhibition of the L-type Ca 2+ . This restricts Ca 2+ flow into the ventricle myocytes and decreases [Ca 2+ ] i . The findings of the present study provide new perspectives for further research on pharmacology of 6-Gin as a possible candidate for the treatment of cardiovascular diseases.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

E TH I C A L S TATEM ENT
All animal care and experimental protocols were ethically reviewed and approved by the Ethics Committee of Hebei University of Chinese Medicine.