Studies Of The Effects And Mechanisms Of17-methoxyl-7-hydroxyl-benzofuran Chalcone On Ion Channel Of Myocardial Membrane

YLS is the root of Millettia pulchra Kurz var(Papilionaceae).The investigation of YLS in recent years had confirmed flavonoid extracts of YLS has the protective effect on myocardial ischemia reperfusion (MIRI),17-methoxyl-7-hydroxyl-benzofuran chalcone (YLSC) is a Monomer extracted from flavonoid extracts of YLS,which belongs to Chalcones and has high activity of antioxidation.The studies in vitro and in vivo had showed that YLSC is the mainly bioactive of YLS,which can efficiently improvement blood flow dynamics, reduce myocardial enzyme exuding during the period of myocardial ischemia reperfusion, the mechanisms including antioxidation, anti-apoptosis and inhibition of myocardial calcium overloading.calcium overloading is the final pathway to death in ischemia-reperfusion injury, abnormal transport of calcium channel of myocardial membrane plays a key role in the formation of calcium overloading.Although previous studies had explored the mechanisms concerned with transporting of calcium, such as Na+-K+-ATPase, Ca2+-Mg2+-ATPase, as well as sodium-calcium exchanger (NCX), however its effects on L-type calcium current of ventricular myocytes have no been reported. In addition, there are also have sodium channel and potassium channel, the ion flow changes of which with calcium channel together, commonly constitute myocardial cell electric activities, However MIRI can cause various blood changes of flow dynamics, its pathological base is the abnormal heart electric activities that mainly for sodium, and potassium, and calcium ion flow exception, correcting electric activities exception or reducing its consequences would become treatment means.In this research, we investigated the effects of YLSC on L-type calcium current, sodium current, transient outward potassium current and inward rectifier potassium current of ventricular myocytes from KM mouse by patch clamp technique, meanwhile applying the laser scanning confocal microscope (LSCM) to observe the protective effect of YLSC on intracellular calcium overload in cultured cardiac myocytes of neonatal rats injured by H2O2Part I.The study of acute isolation methods and the electrophysiological characteristics of calcium-tolerant ventricular myocytes of KM mouseObjective:To explore and establish simple and reliable method of isolating single calcium-tolerant cardiomyocytes from KM mouse for patch clamping and recording of Action potential and the Currents of L-type Calcium Channels. Methods:single calcium-tolerant ventricular myocytes of KM mice were obtained enzymatically by Langendorff perfusion apparatus, The protocol containing two major steps, firstly perfused retrogradely with Ca2+free Tyrode’s solution, and then with digestive enzyme. During the dissolve,10μl20mM of CaCl2was added in experiment group, but add nothing to the control group. The efffect of Ca2+in disgestion on the yield of calcium-tolerant ventricular myocytes was investigated by calculating the Rod-shaped cells under microscope. The terminal of digestion was judged by observing the existence of single myocyte in the efflux solution. Whole-cell patch clamp recording technique was used to record the action potential and L-type calcium channels. Results:The immediately isolation survival rates of ventricular myocytes of experiment group is above seventy percent which equal to control group. But the yield of calcium-tolerant myocytes of experiment group were significantly higher than control group (P<0.05). Giga-ohm seal could be easily achieved and AP and ICa-L currents were successfully recorded. Conclusion:Adding Ca2+gradually in digestive enzyme in an applicable range is helpful for isolationg sigle calcium-tolerant cardiomyocytes. The isolation method can obtain large numbers and night activity of calcium-tolerant cardiomyocytes with normal electrophysiological properties. Part Ⅱ. Effects of YLSC on L-type calcium current in isolated ventricular myocytes of mouseObjective:To explore the effects of YLSC on L-type calcium current of ventricular myocytes. Methods:Acute isolation was used to obtain single myocytes, ICaL was recorded by the wholecell patch clamp,RT-PCR was used to detect the gene expression of Cav1.2. Results:①The inhibition percentage of YLSC under the does of200,400,600,800μmol.L-1respectively were (95.2±1.4)%,(67.8±2.3)%,(29.1±1.8)%,(8.2±1.1)%(n=5), and the Median effective concentration (EC50) was508.4∴mol.L-1.②YLSC can reduce the peak of ICaL and up-shifted the current-voltage(I-V) curves, It can also markedly shift the steadystate activation and inactivation curve of ICaL to the left under the does of500μmol.L-1,decreasing the half-activate voltage and the half-deactivate voltage.③The gene expression of Cavl.2was decreased when administrated by YLSC.Conclusion:YLSC can block ICaL reversibly in does-dependent manner, accelerate the process of activation and deactivation, reduce the gene expression of Cav1.2, and result to internal flow of ICaL reduction. The effects of YLSC on ICaL may be involved in the electrophysiological mechanism of calcium overload protection and negative inotropic effect. Part Ⅲ. Effects of YLSC on Sodium current in Isolated Ventricular Myocytes of mouseObjective:To explore the effects of YLSC on Sodium current of ventricular myocytes. Methods:Acute isolation was used to obtain single myocytes, INa was recorded by the wholecell patch clamp. Results:①The inhibition percentage of YLSC under the does of200,400,600μmol.L"1 respectively were (86.2±1.2)%,(52.4±3.6)%,(35.2±2.6)%(n=5), and the Median effective concentration (EC50) was326.6μmol.L-1.②YLSC could reduce the peak of INa and up-shifted the current-voltage relationship curves(I-V), but it can not change the reversal potential and maximum activate potential.③400μmol.L-1YLSC could decrese half-deactivate voltage.Conclusion:YLSC can block INa reversibly in does-dependent manner and accelerate the process of deactivation. The effects of YLSC on INa maybe involve in the electrophysiological mechanism of inhibition of Na/Ca exchange and Arrhythmia. PartⅣ. Effects of YLSC on transient outward potassium current in Isolated Ventricular Myocytes of mouseObjective:To explore the effects of YLSC on transient outward potassium current of ventricular myocytes. Methods:Acute isolation was used to obtain single myocytes, Ito was recorded by the wholecell patch clamp. Results:①YLSC under the does of200,400,800μmol.L-1can increase faintly the peak of Ito,but meaningless in statistics (n=5,P>0.05).②YLSC has no evident effects on Ⅰ-Ⅴ relationship and steadystate activation curve of Ito, but can increase the half-deactivate voltage.Conclusion:YLSC has no effect on Ito. Part V. Effects of YLSC on inward rectifier potassium current in isolated ventricular myocytes of mouseObjective:To explore the effects of YLSC on inward rectifier potassium current of ventricular myocytes. Methods:Acute isolation was used to obtain single myocytes, IK1was recorded by the wholecell patch clamp,RT-PCR was used to detect the gene expression of kir2.1. Results:(DYLSC under the does of200,400,800μmol.L-1could obviously block the inward currents of IK1on a does-dependent manner, It could also inhibit the outward currents of IK1but had no statistically significant (n=5,P>0.05).②LSC could not change the characteristics of inward rectifier.③The gene expression of Kir2.1was decreased when administrated by YLSC. Conclusion: YLSC can inhibit the inward currents component of IK1, reduce the gene expression of Kir2.1,so it mainly affect the resting membrane potential of cells, reducing cardiac muscle cell excitability and automaticity which maybe one of the electrophysiological mechanisms of its negative chronotropic effect. Part VI. Effects of YLSC on intracellular calcium overload in cultured cardiac myocytes of neonatal rats injured by H2O2 Objective:To investigate the effects of YLSC on intracellular calcium overload in cultured cardiac myocytes of neonatal rats injured by H2O2. Methods:The cells of primary cultured cardiac myocytes of neonatal rats were divided into groups:(1)control group;(2) model group:administrated with0.3mmol·L-1H2O2;(3)YLSC treated groups:incubation respectively with100,200,400μmol·L-1YLSC for24h, and add0.3mmol·L-1H2O2. Laser confocal microscopy was used with Fluo-3/Am as specific fluorescent indicator to detect changes of liber calcium concentration in myocardial cells immediately and15mintues after H2O2intervention. RT-PCR was used to detect the gene expression of RyR2. Results:The average fluorescence intensity values of the model group treated by H2O2higher significantly than control group and the fluorescence enhancement ratio was60.43%±7.75%. The YLSC could decline the intensity values in a does-dependant manner, the fluorescence enhancement ratios of low, middle and high does respectively were38.39%±13.87%,14.49%±2.94%,-28.1%±1.52%, significantly lower than H2O2group(P<0.01). The geneexpression of RyR2was decreased when administrated by YLSC. Conclusion:Hydrogen peroxide induces intracellular calcium overload in cardiomyocytes, and YLSC can decrease calcium overload induced by H2O2.