Regulatory Effects Of On Ion Channels In Cardiomyocytes

Saffron has been used in clinical therapy in China for thousand years,especially as antispasmodic,anticatarrhal,nerve sedative,stimulant,etc.The saffron extracts contains more than 150 components as determined by chemical analysis,the major characteristic components of which are crocin,picrocrocin and safranal.As one of the major bioactive constituents of saffron,crocin has demonstrated various medicinal activities,such as antioxidant,antitumorigenic,memory enhancing,antidepressant,neuroprotective and cardiprotective effects.However,studies that have systematically demonstrated cellular mechanisms of cardioprotective effects of crocin are limited.Ca2+ regulates many physiological activities of cells as an important intracellular messenger,such as heart pace-making,excitation-contraction coupling.The opening of voltage dependent L-type calcium channel induced by excitation of cardiocyte contractility.And it makes Ca2+ fluxing into cardiomyocytes,subsequently leading to increase in intracellular Ca2+ concentration.The calcium influx is increasing,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 arrythmia and cellular damage occur.Therefore,the effects of calcium channel blocks are inhibiting the rise of diastolic calcium concentration to improve normal physiological functions of cardiomyocytes.The slowly activating delayed rectifier potassium current（I_Ks）and the rapidly activating delayed rectifier potassium current(I_Kr)are important to the repolarization of human cardiac action potential.Electrical remodeling and the genetic mutations in the genes in diseased heart maybe due to Loss or reduction of I_Ks and I_Kr channels function,and it will break down the balance of the channels,inducing a decrease in the K+ out flow and net repolarizing current.I_Ks and I_Kr blocker can cause long QT syndrome（LQTS）.In order to evaluate the risk of arrhythmia induced by new drugs,I_Ks and I_Kr are widely studied by the pharmaceutical industry.Some studies have shown that crocin has protective effects against cardiovascular conditions,such as atherosclerosis,cardiac arrhythmias and myocardial injury.Few studies on the mechanism of crocin at the cellular level or ion channels have been reported thus far.In this study,we examined the influences of crocin on ion channels,Ca2+ transient and contractility in rat ventricular myocytes under physiological conditions to expound the cellular mechanisms of its cardioprotective effects.Objectives: To examine the influences of crocin on ion channels,Ca2+ transient and contractility in rat ventricular myocytes under physiological conditions to expound the cellular mechanisms of its cardioprotective effects.Methods: In this study,we examined the influences of crocin on ion channels,Ca2+ transient and contractility in rat ventricular myocytes by using the whole-cell patch-clamp technique and video-based edge detection and dual excitation fluorescence photomultiplier systems.Results:1 Confirmation of I_Ca-LThe steady-state activation protocol was confirmed to elicit I_Ca-L in rat ventricular myocytes.Nicardipin（0.01 mM）,could almost abolished currents,which indicated that these currents were Ca2+ currents（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.Verapamil（10 μM）,a specific LTCC blocker,could nearly completely block the I_Ca-L,which indicated that these currents were L-type Ca2+ currents（P<0.01）.Peak calcium current measured at 0 mV in the presence of drugs was normalized to the peak current measured in the absence of drugs（n = 5-7）.2 Effects of crocin on I_Ca-L of ventricular myocytesThe peak of I_Ca-L was significantly reduced after exposure to 300 μM（P < 0.01）.After washing out crocin with the external solution,the I_Ca-L partially recovered,indicating that the effect of crocin on I_Ca-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.Representative current recordings according to the activation protocol at different concentrations of crocin（1,3,10,30,100,300 μM）are shown in result.Crocin obviously decreased the current density of I_Ca-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（n = 9-12）.3 Effects of crocin on current-voltage relationship of I_Ca-LTaking 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 result.It also shows the current generated at different test voltages in the range-60 to +60 mV.The amplitude of I_Ca-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 I_Ca-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（n = 8-11）.4 Effects of crocin on steady-state activation and inactivation of I_Ca-LIt shows the voltage dependence of steady-state activation and inactivation of I_Ca-L in the absence and presence of crocin（3 μM and 30 μM）.V1/2 is half-active voltage,and k is the slope in steady-state activation.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 m V for 30 μM.V1/2 is the half-inactive voltage,and k is the slope in inactivation.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,n = 8-10）.5 Effects of crocin on I_Kr and I_Ks300 μM crocin had no significant effects on the expressed I_Kr at all test potentials 2.70 ± 0.8% and 3.78 ± 0.4%,respectively（P > 0.05,n = 6）.6 Effects of crocin on cell shortening and Ca2+ transientIt shows that the representative cell shortening and Ca2+ transient recording before and after administration of crocin（1 μM）.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,n = 6-8）.7 Effects of crocin on time parameters of cell shorteningThe time to 10% of the peak（Tp）is an important parameter for the speed of cell contraction.In 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,n = 6-8）.Conclusions:The present study clearly demonstrated the significant inhibitory effects of crocin as a Ca2+ antagonist on I_Ca-L,[Ca2+]i and contraction of adult rat cardiomyocytes.By demonstrating that crocin could decrease the I_Ca-L and reduce the Ca2+ flow into cardiomyocytes.Meanwhile,crocin at 300 μM didn’t have any effects on the wxpressed I_Ks and I_Kr at all test potentials.It indicated that crocin may exert cardioprotective effects without causing drug-induced LQTS.This study provides new ionic evidence of a possible link between cardiac effects of crocin and ion channels and may help to expand clinical treatments for cardiovascular disease.