A novel Na+ channel agonist, dimethyl lithospermate B, slows Na+ current inactivation and increases action potential duration in isolated rat ventricular myocytes

Abstract

Voltage-gated Na(+) channel blockers have been widely used as local anaesthetics and antiarrhythmic agents. It has recently been proposed that Na(+) channel agonists can be used as inotropic agents. Here, we report the identification of a natural substance that acts as a Na(+) channel agonist. Using the patch-clamp technique in isolated rat ventricular myocytes, we investigated the electrophysiological effects of the substances isolated from the root extract of Salvia miltiorrhiza, which is known as 'Danshen' in Asian traditional medicine. By the intensive activity-guided fractionation, we identified dimethyl lithospermate B (dmLSB) as the most active component, while LSB, which is the major component of the extract, showed negligible electrophysiological effect. Action potential duration (APD(90)) was increased by 20 microM dmLSB from 58.8 +/- 12.1 to 202.3 +/- 9.5 ms. In spite of the prolonged APD, no early after-depolarization (EAD) was observed. dmLSB had no noticeable effect on K(+) or Ca(2+) currents, but selectively affected Na(+) currents (I(Na)). dmLSB slowed the inactivation kinetics of I(Na) by increasing the proportion of slowly inactivating component without inducing any persistent I(Na). The relative amplitude of slow component compared to the peak fast I(Na) was increased dose dependently by dmLSB (EC(50) = 20 microM). Voltage dependence of inactivation was not affected by dmLSB, while voltage dependence of activation shifted by 5 mV to the depolarised direction. Since the APD prolongation by dmLSB did not provoke EAD, which is thought as a possible mechanism for the proarrhythmia seen in other Na(+) channel agonists, dmLSB might be an excellent candidate for a Na(+) channel agonist.

Figure 1

Chemical structure of dmLSB.

Figure 2

Effects of dmLSB on cardiac AP. (a) Change in AP duration during the bath application of dmLSB. All data points were obtained from the same cell. Horizontal bars above the plot indicate the duration of the bath application of dmLSB. APs were evoked by depolarizing current pulse (55 pA, 5 ms, 2 Hz). APD₉₀ (AP duration at 90% repolarization) was obtained from the average of five sequential APs selected every 12 s. (b) Left, exemplary AP records at various concentration of dmLSB (a: control; b: 4 μM; c: 10 μM; d: 20 μM; e: wash out of dmLSB). The time point of each AP record was indicated by a triangle in (a). Right, APD₉₀ as a function of the concentration of dmLSB. APD values obtained from seven different cells were superimposed (smaller open symbols). At each concentration of dmLSB, a mean value for APD₉₀ was also superimposed (larger closed circles; error bars, s.e.m.). Asterisks indicate statistical significance (paired t-test; P<0.01).

Figure 3

Effects of dmLSB on the whole-cell current. (a) Current responses to step voltage pulses (inset, −120 to +80 mV) from V_h of −80 mV were recorded in whole-cell mode using K⁺-rich pipette solution. Left: control condition; right: in the presence of 10 μM dmLSB. (b) Voltage dependence of current amplitude measured at 20 ms (left) and at 950 ms (right) of the step pulses. The I–V relationships before (open circle) and after (closed circle) the dmLSB application were superimposed in each plot.

Figure 4

Effects of dmLSB on cardiac inward current (a). Current in response to the double step depolarizing pulses (inset). Sets of current traces before (left) and after (middle) the application of 10 μM dmLSB. Notice the slowing of the fast inward current in the presence of dmLSB (arrow heads). Right, voltage dependence of peak amplitude of the current evoked by the second step pulses. The I–V relationships before (open circle) and after (closed circle) the dmLSB application were superimposed (n=4; error bars, s.e.m.). (b) Effects of dmLSB on the inward current in the presence of 100 μM TTX. Sets of current traces in response to the depolarization pulses (inset) before and after bath-applying dmLSB (10 μM).

Figure 5

Effects of dmLSB on cardiac I_Na in the condition of low [Na⁺] (a). Whole-cell I_Na was elicited from a V_h of −120 mV to test potentials ranging from −80 to +10 mV in 10 mV step increments before (left) and after (right) the application of 10 μM dmLSB to the ventricular myocyte from the young rat (3 weeks old). (b) A pair of I_Na was superimposed in control (grey line) and in dmLSB (black line) at the same test potential for comparison. Inset, the same I_Na at −20 mV in the expanded vertical scale. Notice the absence of persistent I_Na in dmLSB. (c) Left, shows the I–V relationships, which represent the peak I_Na values before (open circle) and after (closed circle) the application of dmLSB. Right, the activation curves were estimated from I–V curves of four different cells. The half-activation voltage was significantly shifted to the positive direction by dmLSB (n=4, paired t-test; P<0.05). (d) Left, time constants estimated from fitting inactivation phases with mono- or biexponential function. Only a single time constant for each test potential was plotted when monoexponential function was sufficient for fitting (for V_T=−60, −50 and −40 mV). Right, the plot of relative amplitude of slow component induced by dmLSB as a function of test potential.

Figure 6

No effect of dmLSB on the steady-state inactivation of I_Na. (a) I_Na elicited by step pulses to −20 mV after 500 ms prepulses of various levels in control condition (left) and in the presence of 20 μM dmLSB (right). (b) Summary of mean availability of Na⁺ channel as a function of prepulse voltages in the absence (open circle, n=28) and in the presence (closed circle, n=32) of 20 μM dmLSB. Data were fitted by 1−(1/(1+exp((V_1/2−V_m)/k))). V_1/2 and k were −75.7 and 10.20 for control, and −77.8 and 9.99 for dmLSB, respectively. Error bars, s.e.m.

Figure 7

Dose–response relationship. (a) Upper, the change in the relative amplitude of I_Slow to the peak I_Na as a function of WCR time. A pair of step pulse to −20 mV from the prepulse of −130 mV and from that of −40 mV were applied every 10 s to obtain I_Na (see text for details). The dmLSB-induced I_Slow was obtained by subtracting I_Na in control condition from I_Na in the presence of dmLSB. Lower, I_Na (grey) traces in control condition and in the presence of dmLSB and I_Slow (black) were superimposed. The time when each set of current records was obtained is marked in the upper plot with ‘a' and ‘b'. (b) Relative amplitude of slow components induced by dmLSB to the peak I_Na was plotted as a function of the concentration of dmLSB. The relationship was fitted to a Hill equation. The half-maximum effective concentration and Hill coefficient were 21.1 μM and 1.02, respectively.

Figure 8

Effects of intra- and extracellular dmLSB on I_Na. The whole-cell mode was obtained with the pipette solution containing 20 μM dmLSB. I_Na was evoked by test pulse to −20 mV from V_h of −80 mV. (a) The time course of peak I_Slow (I_{Na, dmLSB}–I_{Na, control}) during bath application of 5 μM dmLSB. The arrow heads indicate the start and the end of the solution changes. (b) Current traces obtained from before (left) and after (middle) the bath application of dmLSB to the same cell. Extracellular dmLSB slowed the I_Na inactivation. The dmLSB effect was completely reversed after the wash out of external dmLSB (right). In each plot, the initial I_Na recorded just after break-in was superimposed for comparison (grey traces).