draft - university of toronto t-space · draft crocin, a carotenoide component of crocus sativus l,...

Draft

Crocin, A Carotenoide Component of Crocus Sativus L,

Exerts Inhibitory Effects on L-type Ca2+ Current, Ca2+

Transient and Contractility in Rat Ventricular Myocytes

Journal: Canadian Journal of Physiology and Pharmacology

Manuscript ID cjpp-2015-0214.R1

Manuscript Type: Article

Date Submitted by the Author: 02-Aug-2015

Complete List of Authors: Liu, Tao; Hebei Medical University,

Chu, Xi; The Fourth Hospital of Hebei Medical University, Wang, Hua; Hebei Medical University, Zhang, Xuan; Hebei University of Chinese Medicine, Zhang, Yuanyuan; Hebei University of Chinese Medicine, Guo, Hui; Hebei University of Chinese Medicine, Liu, Zhenyi; Hebei University of Chinese Medicine, Department of Pharmacology Dong, Yongsheng; Hebei University of Chinese Medicine, Department of Pharmacology Liu, Hongying; Hebei General Hospital, Liu, Yang; Hebei Medical University Chu, Li; Hebei University of Chinese Medicine,

Zhang, Jianping; Hebei University of Chinese Medicine,

Keyword: crocin, patch clamp, L-type Ca2+ current, myocyte shortening, Ca2+ transient

https://mc06.manuscriptcentral.com/cjpp-pubs

Canadian Journal of Physiology and Pharmacology

Draft

1

Crocin, A Carotenoide Component of Crocus Sativus L, Exerts Inhibitory Effects on L-type

Ca2+

Current, Ca2+

Transient and Contractility in Rat Ventricular Myocytes

Tao Liu1,#

, Xi Chu2,#

, Hua Wang1, Xuan Zhang

3, Yuanyuan Zhang

3, Hui Guo

3, Zhenyi Liu

3,

Yongsheng Dong4 , Hongying Liu5, Yang Liu1, Li Chu1,3,*a, Jianping Zhang1,3,*b

1 Hebei Medical University, No.361, East Zhongshan Road, Shijiazhuang 050017, Hebei, China.

2 The Fourth Hospital of Hebei Medical University, No.12, Jiankang Road, Shijiazhuang 050011,

Hebei, China.

3 Hebei University of Chinese Medicine, No.3, Xingyuan Road, Shijiazhuang 050200, Hebei, China.

4 Intensive Care Unit, Air Force General Hospital, No.30, Fucheng Road, Haidian, 100142, Beijing,

China.

5 Department of Infectious Diseases, Hebei General Hospital, Shijiazhuang, Hebei 050051,

Shijiazhuang, China

# These authors contributed equally to this work.

Corresponding author: *a Li Chu, Tel.: +86 311 86265130; fax: +86 311 86265174. E-mail address:

[email protected] ; *b Jianping Zhang, Tel.: +86 311 86265319; fax: +86 311 86265174. E-mail

address: [email protected]

Page 1 of 25



Draft

2

Abstract. Crocin, a carotenoide component of Crocus sativus L. belonging to the Iridaceae family,

has demonstrated cardiovascular protective effects. In order to investigate the cellular mechanisms of

these cardioprotective effects, here we studied the influence of crocin on L-type Ca2+ current (ICa-L),

intracellular Ca2+ ([Ca2+]i) and contraction of isolated rat cardiomyocytes by using the whole cell patch

clamp technique and video-based edge detection and dual excitation fluorescence photomultiplier

systems. Crocin inhibited ICa-L in a concentration-dependent manner with the half-maximal inhibitory

concentration (IC50) of 45 µM and the maximal inhibitory effect of 72.195 ± 1.54%. Neither

current-voltage relationship, reversal potential of ICa-L nor the activation/inactivation of ICa-L was

significantly changed. Crocin at 1 µM reduced cell shortening by 44.64 ± 2.12% and the peak value of

the Ca2+

transient by 23.66 ± 4.52%. Crocin significantly reduced amplitudes of myocyte shortening

and [Ca2+]i with an increase in the time to reach 10% of the peak (Tp) and a decrease in the time to

10% of the baseline (Tr). Thus, the cardioprotective effects of crocin may be attributed to the

attenuation of [Ca2+]i through the inhibition of ICa-L in rat cardiomyocytes and negative inotropic

effects on myocardial contractility.

Keywords: crocin, patch clamp, L-type Ca2+ current, myocyte shortening, Ca2+ transient

Page 2 of 25



Draft

3

Introduction

Saffron, the dried stigma of the plant Crocus sativus L., belongs to the Iridaceae family and the

Liliaceae line, which is cultivated in Azerbaijan, France, Greece, India, Italy, Spain, China, Israel,

Morocco, Turkey, Egypt and Mexico(Alavizadeh et al. 2014). The use of saffron dates back almost

3000 years on many continents, civilizations and cultures (Melnyk et al. 2010). Saffron is a spice that

adds a faint, delicate aroma with a pleasing flavor and brilliant yellow color to enhance palatability.

Saffron is not only popularly known as the “Golden Condiment”, but it also has been successfully

used in traditional Chinese medicine as an antispasmodic, anticatarrhal, nerve sedative, stimulant,

etc.(Zheng et al. 2007)

The saffron extract contains more than 150 components as determined by chemical analysis

(Bathaie et al. 2010), the major characteristic components of which are crocin, picrocrocin and

safranal (Pfander et al. 1982). As one of the major bioactive constituents of saffron (Alavizadeh et al.

2014), crocin forms deep red crystals with a melting point 186 °C (Bolhassani et al. 2014), and its

structural formula has been determined (Fig. 1) (Akhtari et al. 2013). In recent studies, crocin has

demonstrated various medicinal activities, such as antioxidant (Ochiai et al. 2004), antitumorigenic

(Abdullaev 2002; Escribano et al. 1996; Mousavi et al. 2011), antidepressant/anxiolytics (Wang et al.

2010), genoprotective (Hosseinzadeh et al. 2007), antitussive (Hosseinzadeh et al. 2006),

neuroprotective (Essa et al. 2012) and cardioprotective (Goyal et al. 2010) effects. In some studies,

crocin has demonstrated potent inhibitory effects on the contractility and rate of guinea pig hearts

(Boskabady et al. 2008). However, studies that have systematically demonstrated cellular mechanisms

of cardioprotective effects of crocin are limited.

Page 3 of 25



Draft

4

Enhanced or overload of intracellular Ca2+

([Ca2+

]i) is known to both cause increased

contractility and contribute to the associated pathological changes such as hypertrophy (Frey et al.

2003) and apoptosis (Chen et al. 2005). Increased cardiomyocyte contractility is the central feature of

the cardiac response to ischemic myocardial diseases (Gao et al. 2014) . In addition, the opening of

L-type calcium channels (LTCCs) are associated with Ca2+ influx (Piper et al. 1998), and LTCC

blockers generally have been proven to have cardioprotective effect during the ischemic period

(Cohen et al. 1987). In this study, we examined the influences of crocin on L-type Ca2+ current (ICa-L),

Ca2+

transient and contractility in rat ventricular myocytes under physiological conditions to expound

the cellular mechanisms of its cadioprotective effects by using the whole-cell patch-clamp technique

and video-based edge detection and dual excitation fluorescence photomultiplier systems.

Materials and methods

Materials

Collagenase type II was purchased from Worthington (Lakewood, NJ, USA). Verapamil (Ver) was

purchased from Hefeng Pharmaceutical Co., Ltd. (Shanghai, China). Unless otherwise stated, other

chemical reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA). All solvents used were

of analytical purity.

Solutions

The Ca2+

-free Tyrode’s solution, the normal Tyrode’s solution and the Kreb’s buffer (Hockberger et

al.) solution were prepared as described previously (Gao et al. 2014). The enzyme solution was similar

to the Ca2+-free Tyrode’s solution except that it contained 0.6 mg/mL collagenase type II, 0.5 mg/mL

bovine serum albumin and 30 µmol/L CaCl2. The intracellular pipette solution contained (in mmol/L)

CsCl 120, tetraethylammonium chloride (TEACL) 20, HEPES 10, Mg-ATP 5 and EGTA 10, and the

Page 4 of 25



Draft

5

pH was adjusted to 7.2 with CsOH. The external solution for the patch-clamp experiments contained

(in mmol/L) TEACl 140, MgCl2 2, CaCl2 1.8, glucose 10 and HEPES 10, and the pH was adjusted to

7.4 with CsOH. Crocin was dissolved in the external solution to obtain the appropriate concentrations.

Animals

Adult Sprague-Dawley rats, weighing 200–240 g, were provided by the Experimental Animal

Center at Hebei Medical University. All animal experiments were conducted in compliance with the

Guidelines of Animal Experiments from the Committee of Medical Ethics National Health

Department of China.

Isolation of adult rat ventricular myocytes

Single rat ventricular myocytes were isolated from the hearts by enzymatic dissociation as

described preciously (Gao et al. 2014). The rats were injected intraperitoneally (i.p.) with heparin (500

IU/kg) and anesthetized with sodium pentobarbital (40 mg/kg). The heart was quickly removed,

cannulated and perfused on a Langendorff apparatus with Ca2+

-free Tyrode’s solution via the aorta at a

rate of 4 mL/min for 5 min to remove the blood until spontaneous contractions ceased and the efflux

was clear. Subsequently, the heart was perfused with the enzyme solution for 20–25 min until the heart

was flaccid. Finally, Ca2+

-free Tyrode’s solution was perfused through the heart to wash out the

enzyme solution. After perfusion, the left ventricles were dissected into smaller pieces and placed in a

beaker gently. The cells were stored in KB solution at room temperature for at least 1 h before

measurements. All solutions used during the isolation were bubbled with 100% O2, and all

experiments were implemented within 10 h after isolation.

Whole-cell patch clamp recordings

Recording of ICa-L in isolated ventricular myocytes was performed at room temperature (22–24 °C).

Page 5 of 25



Draft

6

The isolated ventricular myocytes were placed into the recording chamber, which was mounted on the

stage of an inverted microscope (Olympys IX71, Japan), and bathed with an external solution at a flow

rate of 2–3 mL/min for 10 min. Patch electrodes, with a resistance ranging from 3–5 MΩ when filled

with the pipette solution, were made of borosilicate glass with a Micropipette Puller Model 97 (Sutter

Instrument, Novato, CA, USA). Only rod-type shaped cells with a clear margin and striation were

used in the experiments. Junction potentials of the electrode were adjusted to zero prior to formation

of the gigaseal. A suction pulse was used to rupture the membrane to establish the whole-cell

voltage-clamp configuration after the gigaseal. Membrane capacitance and series resistance were

compensated after membrane rupture to minimize the duration of the capacitive current.

Whole-cell currents were elicited by a 200 ms depolarizing pulse from a holding potential of -80 to

0 mV at a frequency of 0.5 Hz. For the steady-state activation protocol, currents were elicited by a 200

ms depolarizing pulse applied at 0.5 Hz from a holding potential of -80 mV(Carre et al. 2014; Shi et al.

2010), in 10 mV increments between -60 and +60 mV. Steady-state inactivation curves were obtained

by applying 1 s prepulses within the range of –60 to 60 mV in 10 mV increments before a test pulse

for 200 ms from a holding potential of -80 to 0 mV. Transmembrane currents were recorded with a

patch-clamp amplifier (Axopatch 200B, Axon Instruments, Union city, CA, USA). Sampling and data

analyses were performed using pClamp 10.2 software (Axon Instruments).

Measurements of myocyte contractions

Mechanical properties of ventricular myocytes were assessed with the video-based edge-detection

system (IonOptix, Milton, MA, USA). In brief, the cells were settled on a recording chamber mounted

on the stage of an inverted microscope and perfused with normal Tyrode’s solution with 1.8 mM

CaCl2 at a rate of 1 mL/min. Cell contractions were elicited by field stimulation at a frequency of 0.5

Page 6 of 25



Draft

7

Hz (2-msec duration) and intensity twice that of the contraction threshold.

Measurement of [Ca2+

]i

Myocytes were loaded with the fluorescent indicator fura-2 AM (1 mM) at room temperature in

the dark as described previously (Gao et al. 2014) , and fluorescence measurements were recorded

with a dual-excitation fluorescence photomultiplier system (IonOptix). The myocytes were imaged

through a Fluor 40× oil objective and alternately illuminated with a 340 nm or 380 nm filter

(bandwidth ± 15 nm) while being field stimulated at 0.5 Hz to contract every 2 ms. The resulting

fluorescence was emitted at 510 nm, and the ratio of the emitted fluorescence at the two wavelengths

(340/380) was calculated to provide an index of [Ca2+]i (Salem et al. 2010).

Data analysis

Results were expressed as the mean ± SEM. Statistical comparisons were performed using either

the independent sample t test or one-way ANOVA followed by Bonferroni corrected t tests for

multiple comparisons, as appropriate. P < 0.05 was considered to indicate a significant difference.

Results

Confirmation of ICa-L

The steady-state activation protocol was confirmed to elicit ICa-L in rat ventricular myocytes.

Nicardipin (0.01mM), could almost abolished currents (Ren et al. 2012), which indicated that these

currents were Ca2+

currents (Fig. 2A) (P < 0.01). NiCl2 (0.1 mM), a specific T-type calcium channel

blocker, did not affect the currents, which indicated that these currents were not T- type Ca2+ currents

(Fig. 2B). Ver (10 µM), a specific LTCC blocker, could nearly completely block the ICa-L, which

indicated that these currents were L-type Ca2+

currents (Tao et al. 2004) (Figure 2C) (P<0.01). Peak

calcium current measured at 0 mV in the presence of drugs was normalized to the peak current

Page 7 of 25



Draft

8

measured in the absence of drugs.

Effects of crocin on ICa-L of ventricular myocytes

The peak of ICa-L was significantly reduced after exposure to 300 µM (P < 0.01). After washing out

crocin with the external solution, the ICa-L partially recovered (Fig. 3A), indicating that the effect of

crocin on ICa-L was reversible. Peak calcium current measured at 0 mV in the presence of verapamil

was normalized to the peak current measured in the absence of verapamil. Fig. 3C is a time course at

300 µM and washout. Representative current recordings according to the activation protocol at

different concentrations of crocin (1, 3, 10, 30, 100, 300 µM) are shown in Fig. 3D and E. Crocin

obviously decreased the current density of ICa-L in a concentration-dependent manner. As reflected by a

logistical equation, the half-maximal inhibitory concentration (IC50) of crocin was 43 µM. The rates of

inhibition rates by crocin at 1, 3, 10, 30, 100 and 300 µM were 9.53 ± 0.87%, 13.04 ± 1.03%, 29.66 ±

1.5%, 39.11 ± 1.85%, 60.23 ± 1.90%, and 72.20 ± 1.54%, respectively (Fig. 3F).

Effects of crocin on current-voltage relationship of ICa-L

Taking the test voltage as the X axis and current density (pA/pF) as the Y axis, current-voltage

relationship curves in the absence and presence of crocin (3, 30 and 300 µM) and 0.01 mM Ver are

shown in Figure 4A. Figure 4B shows the current generated at different test voltages in the range -60

to +60 mV. The amplitude of ICa-L began to increase at -20 mV and reached a maximum between 0 and

10 mV. However, the current-voltage relationship and reversal potential of ICa-L were not significantly

changed. Peak calcium current measured at 0 mV in the presence of verapamil was normalized to the

peak current measured in the absence of verapamil.

Effects of crocin on steady-state activation and inactivation of ICa-L

Fig. 5 shows the voltage dependence of steady-state activation and inactivation of ICa-L in the

Page 8 of 25



Draft

9

absence and presence of crocin (3 µM and 30 µM). Steady-state activation curves were obtained from

the I-V curves and were fitted with the Boltzmann function: G/Gmax = 1/1+exp[(V1/2-V)/k], where G

is conductance (from the function G = I/(V-Vrev), I and V are current and holding potential, Vrev is the

reverse potential that is found using extrapolation from the linear part of the I-V relation, V1/2 is

half-active voltage, and k is the slope. Values at V1/2 for the normalized activation conductance curves

were -10.93 ± 0.66 mV with a slope factor (k) of 7.04 ± 0.590 mV for control, -8.75 ± 0.59 mV with a

k value of 7.23 ± 0.53 mV for 3 µM and -6.88 ± 0.49 mV with a k value of 7.53 ± 0.44 mV for 30 µM.

Steady-state inactivation curves were fitted with the Boltzmann function: I/Imax = 1/ 1+exp

[(V-V1/2)/k], where V1/2 is the half-inactive voltage, and k is the slope. Values of V1/2 for the

steady-state inactivation was -34.22 ± 0.09 mV with a k value of 4.88 ± 0.08 mV for control, -33.27 ±

0.17 mV with a k value of 5.05 ± 0.10 mV for 3 µM crocin, and -34.05 ± 0.14 mV with a k value of

5.33 ± 0.12 mV for 30 µM crocin. These data show that crocin did not alter the activation and

inactivation of gating properties of the cardiac Ca2+

channel (P > 0.05). There is no significant

differences between values of V1/2 in the absence and presence of crocin for the normalized activation

and inactivation (P > 0.05).

Effects of crocin on cell shortening and Ca2+

transient

The representative cell shortening and Ca2+ transient recording before and after administration of

crocin (1 µM) are shown in Fig. 6. The results indicated that crocin significantly inhibited cell

shortening by 44.64 ± 2.12% at the concentration of 1 µM (P < 0.05). Meanwhile, amplitudes of the

Ca2+ transient decreased by 23.66 ± 4.52% (P < 0.05).

Effects of crocin on time parameters of cell shortening

The time to 10% of the peak (Tp) is an important parameter for the speed of cell contraction. In

Page 9 of 25



Draft

10

addition, the time to 10% of the base line (Tr) is an important parameter of cellular relaxation.

Compared with the control group, crocin markedly increased the Tp by 40.92 ± 15.83% (P < 0.01),

and at the same time significantly decreased the Tr by 7.47 ± 1.79% (P < 0.05) (Figure 7).

Discussion

Some studies have shown that crocin has protective effects against cardiovascular conditions, such

as atherosclerosis (He et al. 2005). Few studies on the mechanism of crocin at the cellular level or ion

channel have been reported thus far. In our study, we used the whole-cell patch-clamp technique and

video-based edge detection and dual excitation fluorescence photomultiplier systems to demonstrate

that crocin suppresses ICa-L of rat myocytes in a concentration-dependent manner. In order to avoid

interference from the run-down phenomenon of ICa-L, which has been reported to be a common

problem when recording the Ca2+

channel current (Zhen et al. 2006), we added Mg-ATP (5 mmol/L)

and EGTA (10 mmol/L) in the pipette solution as described in the study by Belles et. al (Belles et al.

1988). The current was recorded 5–25 min after rupture of the membrane (Tao et al. 2005), and it

partially recovered after washout, ensuring that the effect of crocin on ICa-L was not the consequence of

the run-down phenomenon.

Ca2+

, an important intracellular messenger, regulates many physiological activities, such as heart

pace-making, excitation-contraction coupling, releasing of neurotransmitters. Many

pathophysiological conditions, such as hypertension, glycuresis, ischemic reperfusion injury and

oxidant stress, can increase the calcium influx, and triggers endo-calcium releasing of sarcoplasmic

reticulum, which leads to the increasing of diastolic calcium concentration and alternately to cellular

calcium overload. As a result, subsequent arrhythmia (Zhou et al. 2011) and cellular damage (Soler et

al. 2011) occur. Therefore, it is significant to reduce the rise of diastolic calcium concentration to

Page 10 of 25



Draft

11

improve normal physiological functions of cardiomyocytes. LTCCs, mainly present in muscle cells

and secretory cells, can cause contraction of myocardial cells because they are integral in the

processing of excitement-contraction coupling. LTCCs, with properties such as slow inactivation and

long opening duration, provide the main avenue for intracellular Ca2+ influx during excitation, and

blocking this channel can cause a negative inotropic effect in cardiomyocytes (Guan Bingcai 2013). In

our study, we found that crocin could significantly decrease the magnitude of the ICa-L in a

concentration-dependent manner at concentrations of 1, 3, 10, 30, 100 and 300 µM, and the inhibition

was partially reversible. However, it did not affect the current-voltage relationship or the reversal

potential of ICa-L (Fig. 4). Crocin suppressed the peak value of ICa-L without altering the steady-state

activation and inactivation of ICa-L (Fig. 6). These results indicate that crocin inhibited the ICa-L

primarily by reducing the Ca2+

current amplitude.

Excitatory-contraction coupling mediated by Ca2+ is the critical beginning link of cardiocyte

contractility. And the opening of voltage dependent LTCC was induced by excitation of cardiocyte

contractility (Shutt et al. 2006). In this study, the inhibitory effects of crocin on LTCCs resulted in

reduced cardiac contractility, while the Ca2+ transient and contractility significantly decreased in the

presence of crocin (Fig. 6) We observed that crocin seems to have a stronger effect on cell shortening

than on calcium transient, that is because myocardial contraction is a complex process which is not

only related to intracellular Ca2+

concentration, but also intracellular proteins, and which involved in

contraction (actin and myosin) , or regulation (troponin, tropomyosin, and tropomodulin) (Adamcova

et al. 2006). The more detailed mechanisms of effects of crocin on myocardial contraction need to be

explored in future studies. Tp and Tr are the parameters for the speed of cell contraction and cell

relaxation. In this study crocin also markedly increased the TP but decreased the Tr (Fig. 7). Crocin

Page 11 of 25



Draft

12

may indirectly suppress Ca2+

release from the SR possibly via inhibiting the activity of the LTCC,

thereby leading to a decrease in intracellular free Ca2+ as represented by a reduction of the Ca2+

transient. There are three factors that affect the myocardial oxygen consumption (MVO2), i.e.,

myocardial contractile state (myocardial contractility and the speed of contractility), cardiac muscle

tension and heart rate (Sonnenblick et al. 1971). Both developed tension and contractile state are

significant factors in the regulation of MVO2 (Graham et al. 1968), and the decrease of myocardial

oxygen consumption after the reduction of contractility appear to be the most important cellular

mechanism for clinical treatment of myocardial ischemia (Crystal et al. 2013). In this study, crocin

could significantly reduce cardiac contractility, which can lead to decrease of MVO2.

This report is the first to electrophysiologically detail the inhibitory effects of crocin in adult rat

cardiomyocytes. The data show that crocin could inhibit cardiac Ca2+

content in order to prevent

cardiac Ca2+ influx and inhibit myocardial contraction. The ultimate effect is the reduction of

myocardial oxygen consumption which protects the cardiomyocytes, since mitochondrial Ca2+

overload occurs after persistent increases in cytosolic Ca2+ concentration that leads to myocardial

injury under pathological conditions such as ischemia–reperfusion (Shintani-Ishida et al. 2012).

Furthermore, the increased [Ca2+

]i accelerated several ATP-consuming activities and further depleted

the cellular energy reserves, which could facilitate the heart damage caused by ischemia (Balaban

2002). Free radical damage and [Ca2+

]i influx caused by myocardial ischemia can further aggravate

cardiac tissue damage. Blockage of the Ca2+ channel in ischemic myocardial cells can quickly reduce

the cardiac electrical activity and contractile activity, which saves energy for subsequent repair and

thus play a role in myocardial protection (Saida et al. 1994). Furthermore, a sustained increase in Ca2+

influx via LTCCs can lead to apoptosis through the mitochondrial death pathway (Chen et al. 2005).

Page 12 of 25



Draft

13

Therefore, our study demonstrated that crocin as a LTCC blocker may play an active role in protecting

the myocardium against ischemic damage and even apoptosis. However, the precise mechanism of the

inhibitory effects of crocin on ICa-L will need to be confirmed.

In conclusion, the present study clearly demonstrated the significant inhibitory effects of crocin as

a Ca2+ antagonist on ICa-L, [Ca2+]i and contraction of adult rat cardiomyocytes. By demonstrating that

crocin could decrease the ICa-L and reduce the Ca2+

flow into cardiomyocytes, this study provides new

ionic evidence of a possible link between cardiac effects of crocin and LTCCs and may help to expand

clinical treatments for cardiovascular disease.

Acknowledgements

This work was supported by the Research Foundation of Administration of Traditional Chinese

Medicine of Hebei Province, China (No. 2015030), Nature Fund of Hebei Province (No.

C2011206025) and Research Foundation of Education Bureau of Heibei Province (QN20131046 to

XZ).

References

Abdullaev, F. I. 2002. Cancer chemopreventive and tumoricidal properties of saffron (Crocus sativus L.). Exp

Biol Med (Maywood). 227(1): 20-25. doi. PMID: 11788779.

Adamcova, M., Sterba, M., Simunek, T., Potacova, A., Popelova, O.and Gersl, V. 2006. Myocardial regulatory

proteins and heart failure. Eur J Heart Fail. 8(4): 333-342. doi: 10.1016/j.ejheart.2005.09.007. PMID:

16309957.

Akhtari, K., Hassanzadeh, K., Fakhraei, B., Fakhraei, N., Hassanzadeh, H.and Zarei, S. A. 2013. A density

functional theory study of the reactivity descriptors and antioxidant behavior of Crocin. Computational

and Theoretical Chemistry. 1013: 123-129. doi: http://dx.doi.org/10.1016/j.comptc.2013.03.015.

Alavizadeh, S. H.and Hosseinzadeh, H. 2014. Bioactivity assessment and toxicity of crocin: a comprehensive

review. Food Chem Toxicol. 64: 65-80. doi: 10.1016/j.fct.2013.11.016. PMID: 24275090.

Balaban, R. S. 2002. Cardiac energy metabolism homeostasis: role of cytosolic calcium. J Mol Cell Cardiol.

34(10): 1259-1271. doi. PMID: 12392982.

Bathaie, S. Z.and Mousavi, S. Z. 2010. New applications and mechanisms of action of saffron and its important

ingredients. Crit Rev Food Sci Nutr. 50(8): 761-786. doi: 10.1080/10408390902773003. PMID:

20830635.

Page 13 of 25



Draft

14

Belles, B., Malecot, C. O., Hescheler, J.and Trautwein, W. 1988. "Run-down" of the Ca current during long

whole-cell recordings in guinea pig heart cells: role of phosphorylation and intracellular calcium.

Pflugers Arch. 411(4): 353-360. doi. PMID: 2456513.

Bolhassani, A., Khavari, A.and Bathaie, S. Z. 2014. Saffron and natural carotenoids: Biochemical activities and

anti-tumor effects. Biochim Biophys Acta. 1845(1): 20-30. doi: 10.1016/j.bbcan.2013.11.001. PMID:

24269582.

Boskabady, M. H., Shafei, M. N., Shakiba, A.and Sefidi, H. S. 2008. Effect of aqueous-ethanol extract from

Crocus sativus (saffron) on guinea-pig isolated heart. Phytother Res. 22(3): 330-334. doi:

10.1002/ptr.2317. PMID: 18058985.

Carre, G., Carreyre, H., Ouedraogo, M., Becq, F., Bois, P., Thibaudeau, S., et al. 2014. The hypotensive agent

dodoneine inhibits L-type Ca2+ current with negative inotropic effect on rat heart. Eur J Pharmacol.

728: 119-127. doi: 10.1016/j.ejphar.2014.01.059. PMID: 24508520.

Chen, X., Zhang, X., Kubo, H., Harris, D. M., Mills, G. D., Moyer, J., et al. 2005. Ca2+ influx-induced

sarcoplasmic reticulum Ca2+ overload causes mitochondrial-dependent apoptosis in ventricular

myocytes. Circ Res. 97(10): 1009-1017. doi: 10.1161/01.res.0000189270.72915.d1. PMID: 16210547.

Cohen, N. M.and Lederer, W. J. 1987. Calcium current in isolated neonatal rat ventricular myocytes. J Physiol.

391: 169-191. doi. PMID: 2451004.

Crystal, G. J., Silver, J. M.and Salem, M. R. 2013. Mechanisms of increased right and left ventricular oxygen

uptake during inotropic stimulation. Life Sci. 93(2-3): 59-63. doi: 10.1016/j.lfs.2013.05.011. PMID:

23747965.

Escribano, J., Alonso, G. L., Coca-Prados, M.and Fernandez, J. A. 1996. Crocin, safranal and picrocrocin from

saffron (Crocus sativus L.) inhibit the growth of human cancer cells in vitro. Cancer Lett. 100(1-2):

23-30. doi. PMID: 8620447.

Essa, M. M., Vijayan, R. K., Castellano-Gonzalez, G., Memon, M. A., Braidy, N.and Guillemin, G. J. 2012.

Neuroprotective effect of natural products against Alzheimer's disease. Neurochem Res. 37(9):

1829-1842. doi: 10.1007/s11064-012-0799-9. PMID: 22614926.

Frey, N.and Olson, E. N. 2003. Cardiac hypertrophy: the good, the bad, and the ugly. Annu Rev Physiol. 65:

45-79. doi: 10.1146/annurev.physiol.65.092101.142243. PMID: 12524460.

Gao, Y., Zhang, K., Zhu, F., Wu, Z., Chu, X., Zhang, X., et al. 2014. Salvia miltiorrhiza (Danshen) inhibits

L-type calcium current and attenuates calcium transient and contractility in rat ventricular myocytes. J

Ethnopharmacol. 158PA: 397-403. doi: 10.1016/j.jep.2014.10.049. PMID: 25446591.

Goyal, S. N., Arora, S., Sharma, A. K., Joshi, S., Ray, R., Bhatia, J., et al. 2010. Preventive effect of crocin of

Crocus sativus on hemodynamic, biochemical, histopathological and ultrastuctural alterations in

isoproterenol-induced cardiotoxicity in rats. Phytomedicine. 17(3-4): 227-232. doi:

10.1016/j.phymed.2009.08.009. PMID: 19747807.

Graham, T. P., Jr., Covell, J. W., Sonnenblick, E. H., Ross, J., Jr.and Braunwald, E. 1968. Control of myocardial

oxygen consumption: relative influence of contractile state and tension development. J Clin Invest.

47(2): 375-385. doi: 10.1172/JCI105734. PMID: 12066781.

Guan Bingcai, Z. H., Li Zhiwang. (2013). Basic Principles of Cellular Electrophysiology and Patch Clamp

Techniques: science press.

He, S. Y., Qian, Z. Y., Tang, F. T., Wen, N., Xu, G. L.and Sheng, L. 2005. Effect of crocin on experimental

atherosclerosis in quails and its mechanisms. Life Sci. 77(8): 907-921. doi: 10.1016/j.lfs.2005.02.006.

PMID: 15964309.

Hockberger, P. E.and Nam, S. C. 1994. High-voltage-activated calcium current in developing neurons is

insensitive to nifedipine. Pflugers Arch. 426(5): 402-411. doi. PMID: 8015890.

Page 14 of 25



Draft

15

Hosseinzadeh, H.and Ghenaati, J. 2006. Evaluation of the antitussive effect of stigma and petals of saffron

(Crocus sativus) and its components, safranal and crocin in guinea pigs. Fitoterapia. 77(6): 446-448.

doi: 10.1016/j.fitote.2006.04.012. PMID: 16814486.

Hosseinzadeh, H.and Sadeghnia, H. R. 2007. Effect of safranal, a constituent of Crocus sativus (saffron), on

methyl methanesulfonate (MMS)-induced DNA damage in mouse organs: an alkaline single-cell gel

electrophoresis (comet) assay. DNA Cell Biol. 26(12): 841-846. doi: 10.1089/dna.2007.0631. PMID:

17854266.

Melnyk, J. P., Wang, S.and Marcone, M. F. 2010. Chemical and biological properties of the world's most

expensive spice: Saffron. Food Research International. 43(8): 1981-1989. doi:

http://dx.doi.org/10.1016/j.foodres.2010.07.033.

Mousavi, S. H., Moallem, S. A., Mehri, S., Shahsavand, S., Nassirli, H.and Malaekeh-Nikouei, B. 2011.

Improvement of cytotoxic and apoptogenic properties of crocin in cancer cell lines by its nanoliposomal

form. Pharm Biol. 49(10): 1039-1045. doi: 10.3109/13880209.2011.563315. PMID: 21936628.

Ochiai, T., Ohno, S., Soeda, S., Tanaka, H., Shoyama, Y.and Shimeno, H. 2004. Crocin prevents the death of rat

pheochromyctoma (PC-12) cells by its antioxidant effects stronger than those of alpha-tocopherol.

Neurosci Lett. 362(1): 61-64. doi: 10.1016/j.neulet.2004.02.067. PMID: 15147781.

Pfander, H.and Schurtenberger, H. 1982. Biosynthesis of C20-carotenoids in Crocus sativus. Phytochemistry.

21(5): 1039-1042. doi: http://dx.doi.org/10.1016/S0031-9422(00)82412-7.

Piper, H. M., Garcia-Dorado, D.and Ovize, M. 1998. A fresh look at reperfusion injury. Cardiovasc Res. 38(2):

291-300. doi. PMID: 9709390.

Ren, Z., Ma, J., Zhang, P., Luo, A., Zhang, S., Kong, L., et al. 2012. The effect of ligustrazine on L-type calcium

current, calcium transient and contractility in rabbit ventricular myocytes. J Ethnopharmacol. 144(3):

555-561. doi: 10.1016/j.jep.2012.09.037. PMID: 23058991.

Saida, K., Umeda, M.and Itakura, A. 1994. Improvement of ischemic myocardial dysfunction by nisoldipine.

Cardiovasc Drugs Ther. 8 Suppl 2: 365-370. doi. PMID: 7947379.

Salem, K. A., Qureshi, A., Ljubisavijevic, M., Oz, M., Isaev, D., Hussain, M., et al. 2010. Alloxan reduces

amplitude of ventricular myocyte shortening and intracellular Ca2+ without altering L-type Ca2+

current, sarcoplasmic reticulum Ca2+ content or myofilament sensitivity to Ca2+ in Wistar rats. Mol

Cell Biochem. 340(1-2): 115-123. doi: 10.1007/s11010-010-0408-7. PMID: 20174963.

Shi, J. S., Li, D., Li, N., Lin, L., Yang, Y. J., Tang, Y., et al. 2010. Inhibition of L-type calcium currents by

salusin-beta in rat cardiac ventricular myocytes. Peptides. 31(6): 1146-1149. doi:

10.1016/j.peptides.2010.03.016. PMID: 20307603.

Shintani-Ishida, K., Inui, M.and Yoshida, K.-i. 2012. Ischemia–reperfusion induces myocardial infarction

through mitochondrial Ca2+ overload. J. Mol. Cell. Cardiol. 53(2): 233-239. doi:

10.1016/j.yjmcc.2012.05.012.

Shutt, R. H., Ferrier, G. R.and Howlett, S. E. 2006. Increases in diastolic [Ca2+] can contribute to positive

inotropy in guinea pig ventricular myocytes in the absence of changes in amplitudes of Ca2+ transients.

Am J Physiol. 291(4): H1623-1634. doi: 10.1152/ajpheart.01245.2005. PMID: 16699070.

Soler, F., Lax, A., Carmen Asensio, M., Pascual-Figal, D.and Fernandez-Belda, F. 2011. Passive Ca(2+) overload

in H9c2 cardiac myoblasts: assessment of cellular damage and cytosolic Ca(2+) transients. Arch

Biochem Biophys. 512(2): 175-182. doi: 10.1016/j.abb.2011.05.019. PMID: 21683055.

Sonnenblick, E. H.and Skelton, C. L. 1971. Myocardial energetics: basic principles and clinical implications. N

Engl J Med. 285(12): 668-675. doi: 10.1056/NEJM197109162851208. PMID: 4935226.

Tao, J., Wang, H., Chen, J., Xu, H.and Li, S. 2005. Effects of saponin monomer 13 of dwarf lilyturf tuber on

L-type calcium currents in adult rat ventricular myocytes. Am J Chin Med. 33(5): 797-806. doi:

Page 15 of 25



Draft

16

10.1142/s0192415x05003417. PMID: 16265992.

Tao, J., Xu, H., Yang, C., Liu, C. N.and Li, S. 2004. Effect of urocortin on L-type calcium currents in adult rat

ventricular myocytes. Pharmacol Res. 50(5): 471-476. doi: 10.1016/j.phrs.2004.05.006. PMID:

15458766.

Wang, Y., Han, T., Zhu, Y., Zheng, C. J., Ming, Q. L., Rahman, K., et al. 2010. Antidepressant properties of

bioactive fractions from the extract of Crocus sativus L. J Nat Med. 64(1): 24-30. doi:

10.1007/s11418-009-0360-6. PMID: 19787421.

Zhen, X. G., Xie, C., Yamada, Y., Zhang, Y., Doyle, C.and Yang, J. 2006. A single amino acid mutation attenuates

rundown of voltage-gated calcium channels. FEBS Lett. 580(24): 5733-5738. doi:

10.1016/j.febslet.2006.09.027. PMID: 17010345.

Zheng, Y. Q., Liu, J. X., Wang, J. N.and Xu, L. 2007. Effects of crocin on reperfusion-induced oxidative/nitrative

injury to cerebral microvessels after global cerebral ischemia. Brain Res. 1138: 86-94. doi:

10.1016/j.brainres.2006.12.064. PMID: 17274961.

Zhou, Q., Xiao, J., Jiang, D., Wang, R., Vembaiyan, K., Wang, A., et al. 2011. Carvedilol and its new analogs

suppress arrhythmogenic store overload-induced Ca2+ release. Nat Med. 17(8): 1003-1009. doi:

10.1038/nm.2406. PMID: 21743453.

Page 16 of 25



Draft

Figure legends

Fig. 1. Structural formula of crocin

Fig. 2. Confirmation of ICa-L

Representative ICa-L recordings with the steady-state activation protocol before and after

application of Nic (0.01 mM) (A), NiCl2 (0.1mM) (B), Ver (0.01 mM) (C). The ICa-L in rat

ventricular myocytes was completely blocked by Ver and Nic. Data are presented as means ±

S.E.M. (n = 6 cells). **P < 0.01, compared with control.

Fig. 3. Effects of crocin on ICa-L of ventricular myocytes

(A-C) Exemplary traces, pooled data and time course of ICa-L recorded under control

conditions, during application of crocin (300 µM) and during washout. Data are presented as

means ± S.E.M. (n = 6 cells). **P < 0.01, compared with control. (D) Time course and (E)

exemplary traces of ICa-L were recorded under control conditions during exposure to 1, 3, 10,

30, 100, 300 µM crocin or 0.01 mM Ver. (F) Concentration-response curves representing the

percent inhibitory effects of crocin. Data are presented as means ± S.E.M. (n = 9-12 cells).

Fig. 4. Effects of crocin on current-voltage relationship of ICa-L

(A) Representative ICa-L in absence or presence of crocin (3, 30 and 300 µM) or 0.01 mM Ver

was recorded according to the steady-state activation protocol. (B) The relationship of ICa-L in

cardiomyocytes in absence () or presence of 3 µM crocin (), 30 µM crocin (), 300 µM

crocin () or 0.01 mM Ver ( ). Data are presented as means ± S.E.M. (n = 9-12 cells).

Fig. 5. Effects of crocin on steady-state activation and inactivation of ICa-L

(A) Steady-state activation curves calculated from normalized conductance values from the

I-V curves in the absence () or presence of 3 µM crocin () or 30 µM crocin (). (B)

Page 17 of 25



Draft

Normalized steady-state inactivation of ICa-L in the absence () or presence of 3 µM crocin ()

or 30 µM crocin (). Data are presented as means ± S.E.M. (n = 6–9 cells).

Fig. 6. Effects of crocin on cell shortening and Ca2+ transients.

(A) Tracings demonstrating effects of 10-6 M crocin in a myocyte. (B) Representative signals

of cell shortening and fura-2 ratio (upper tracings). (C) Summary effects of 10-6 M crocin on

cell shortening and fura-2 ratio. Data are presented as means ± S.E.M. (**P < 0.01, n = 6 cells

compared with control).

Fig. 7. Effects of crocin on time parameters of cell shortening.

Summary results of Tp and Tr for cell shortening before and after application of 10-6 M

crocin. Data are presented as means ± S.E.M. (n = 6 cells). **P < 0.01,*P < 0.05, compared

with control.

Page 18 of 25



Draft

Figure 1.

84x51mm (300 x 300 DPI)

Page 19 of 25



Draft

Figure 2.

147x100mm (300 x 300 DPI)

Page 20 of 25



Draft

Figure 3.

168x129mm (300 x 300 DPI)

Page 21 of 25



Draft

Figure 4.

147x138mm (300 x 300 DPI)

Page 22 of 25



Draft

Figure 5.

81x137mm (300 x 300 DPI)

Page 23 of 25



Draft

Figure 6.

148x173mm (300 x 300 DPI)

Page 24 of 25



Draft

Figure 7.

56x129mm (300 x 300 DPI)

Page 25 of 25



draft - university of toronto t-space · draft crocin, a carotenoide component of crocus sativus l,...

Documents