Effects And Mechanism Of Dioscin On Rat Cardiac Contractility

Objective: To investigate the effects of Dioscin(Dio) on sodium current(INa), L-type calcium current(ICa,L) and intracellular free calcium concentration in isolated rat ventricular myocytes and to study its effects on rat myocardial contractility as well as explore its mechanism preliminarily.Methods: 1. Single rat ventricular myocyte was isolated from adult rat heart by enzymatic dissociation. Effects of dioscin on sodium current(INa), L-type calcium current(ICa,L) were observed and recorded with whole-cell patch clamp. 2. Left ventricle contractile function was measured using the Langendorff non-recirculating mode of isolated rat heart perfusion. Effects of dioscin and dioscin in presence of SEA0400, sodium-calcium exchanger(NCX) inhibitor, were investigated by measuring left ventricular systolic pressure(LVSP) and left ventricular end diastolic pressure(LVEDP). Also, heart rate(HR), peak rate of rise/fall of left ventricular pressure(±dp/dtmax) of isolated rat heart were calculated. After perfusion, isolated rat hearts were collected to analyze the content of superoxide dismutase(SOD) using SOD kit. 3. Effects of dioscin and SEA0400 on intracellular free calcium concentration in rat H9c2 cells were measured by labeling intracellular free calcium ion with Fluo3-AM and then detected the fluorescence intensity with confocal microscopy. Mitochondrial membrane potential was detected with multifunctional microplate reader in order to determine whether dioscin may cause cellular injuries.Results: 1. Dioscin shifted downward the I-V curve with increased current density of sodium current compared with those in the control group. With the 0.1, 1, 10 μmol·L-1 dioscin, the peak current density were dose-dependently changed from-52.91±1.58 p A·p F-1 to-57.93±2.28(n=8, p<0.05),-76.14±2.76 and-82.40±4.71 p A·p F-1(n=8, p<0.01) respectively. Dioscin facilitated the activation and recovery process of sodium current, but not the inactivation process.2. Compared with the control group, dioscin shifted upward the I-V curve with decreased current density of calcium current. With the 0.1, 1, 10 μmol·L-1 dioscin, the peak current density were dose-dependently changed from-3.46±0.15 p A·p F-1 to-3.31±0.12(n=8,p<0.05),-3.03±0.13 and-2.69±0.18 p A·p F-1(n=8, p<0.01). Dioscin retarded the activation and recovery process of calcium current, but had no significant effects on the inactivation process. 3. With 0.1, 1, 10 μmol·L-1 dioscin, the relative fluorescence intensity of intracellular free calcium concentrations were increased significantly from 16.62±0.89 to 21.48±0.80, 25.68±0.69 and 19.84±0.66(n=8, p<0.01) respectively. While in presence of SEA0400, the relative fluorescence intensity was changed by 0.1,1,10 μmol·L-1 dioscin to 11.20±0.82, 17.09±0.63 and 14.80±0.47(n=8, p<0.01). With 1 μmol·L-1 dioscin, the relative fluorescence intensity was changed to 8.17±0.50 and 12.73±0.39(n=8, p<0.01) without calcium or sodium in the extracellular fluid. 4. According to the results from multifunctional microplate reader, 0.1,1 μmol·L-1 dioscin, SEA0400 as well as 1 μmol·L-1 dioscin with SEA0400 all showed no significant effects on the mitochondrial membrane potential of rat H9c2 cells, while with effects of 10 μmol·L-1 dioscin, the ratio of JC-1 monomer and J-aggregates was changed from 1.14±0.03 to 1.35±0.06(n=6, p<0.01), causing a decrease in the mitochondrial membrane potential. 5. With 0.1, 1 μmol·L-1 dioscin, LVSP and dp/dtmax were significantly enhanced by 13%, 18% and 11%, 9% respectively. With the 10 μmol·L-1 dioscin, LVSP and dp/dtmax were both decreased. 6. 1 μmol·L-1 SEA0400 alone showed no significant effect but can partly inhibited the effects of dioscin on cardiac performances in isolated Langendorff-perfused rat hearts. 7. With 0.1, 1, 10 μmol·L-1 dioscin, the content of SOD in isolated Langendorff-perfused rat hearts was changed from 2.67±0.23 μg·m L-1 to 3.16±0.10, 3.32±0.24(n=6,p<0.01) and 2.95±0.16 μg·m L-1(n=6,p<0.05) respectively.Conclusions: 1. Dioscin increases the sodium current(INa) by facilitating the activation process and recovery process of the channel.2. Dioscin decreases the L-type calcium current(ICa,L) by inhibiting the activation process and recovery process of the channel. 3. Dioscin increases the intracellular concentration of Ca2+ in the cardiac myocytes and 0.1, 1 μmol·L-1 dioscin show no effect on the mitochondrial membrane potential. 4. Dioscin shows positive inotropic effect on isolated rat heart, enhancing the LVSP and +dp/dtmax by increasing Na+ influx and facilitating the reverse mode of the sodium-calcium exchanger.