The Effects of Puerarin on Rat Ventricular Myocytes and the Potential Mechanism

Figure 1. Puerarin prolonged APD of rat ventricular myocytes. (a) Puerarin at the concentrations of 100 μM significantly prolonged APD of rat ventricles; whereas isoproterenol at the concentrations of 10 nM significantly shortened APD of rat ventricles, in comparing with control. (b) Puerarin (100 μM) significantly offseted the reduced effect of isoproterenol (10 nM) and extended APD of rat ventricles at the 50% and 90% measurement points by respectively. *P < 0.01, **P < 0.001 versus control. n = 5 each point. (c) Puerarin at the concentrations of 10 μM, 100 μM, 1 mM, 10 mM, 100 mM and 200 mM extended APD of rat ventricles, in comparing with control. The statistical results were shown in table1. (d) The molecular structural formula of puerarin.

Figure 2. Puerarin inhibited Kir2.1 and Kir2.3 channels in a dosage dependent manner. (a) Puerarin at the concentrations of 10 μM, 100 μM, 10 mM, 50 mM, 100 mM, 200 mM significantly inhibited Kir2.1 currents illustrated by the ramp voltage protocol, respectively. This effect could be partly reversed by simple compound wash-off. (b) Concentration dependent inhibition by puerarin upon Kir2.1. The maximal inhibitory effect was (53.11 ± 1.84) % and saturation dosage was 50 mM. Dosage curve was fitted with non-linear Hill. IC50 = 1.27 mM, Hill coefficient is 0.990. n = 5 each point. (c) Puerarin at the concentrations of 10 μM, 100 μM, 1 mM, 10 mM, 50 mM, 100 mM, 200 mM significantly inhibited Kir2.3 currents illustrated by the ramp voltage protocol, respectively. This effect could be partly reversed by simple compound wash-off. (d) Concentration dependent inhibition by puerarin upon Kir2.3. Dosage curve was fitted with non-linear Hill. IC50 = 129.4 mM, Hill coefficient is 0.984. n = 5 each point.

Figure 3. Puerarin inhibited Kv7.1 channels in a dosage dependent manner. (a) Puerarin at the concentrations of 10 μM, 100 μM, 1 mM and10 mM significantly inhibited Kv7.1 currents illustrated by the protocol of voltage steps, respectively. (b) I-V curves summarized inhibitory effects of puerarin at concentrations of 10 μM, 100 μM, 1 mM, 10 mM and recovery after compound wash off. Different concentrations of puerarin decreased the Kv7.1 from (24.3 ± 1.1) pA/pF to (19.1 ± 1.0) pA/pF, (7.9 ± 0.8) pA/pF, (6.1 ± 0.4) pA/pF, (4.8 ± 0.8) pA/pF, and decreased the Kv7.1-tail current from (10.4 ± 0.6) pA/pF to (8.8 ± 0.7) pA/pF, (3.8 ± 0.6) pA/pF, (2.5 ± 0.9) pA/pF, (2.0 ± 0.5) pA/pF at 40 mV, respectively. (c) Concentration dependent inhibition by puerarin upon Kv7.1 at the peak currents and tail currents. Dosage curve was fitted with non-linear Hill. IC50 = 36.01 μM at the peak currents and IC50 = 39.77 μM at the tail currents, Hill coefficient is 0.997. n = 6 each point.

Figure 4. Puerarin inhibited IKs channels in a dosage dependent manner. (a) Puerarin at the concentrations of 10 μM, 100 μM, 1 mM and10 mM significantly inhibited IKs currents illustrated by the protocol of voltage steps, respectively. (b) I-V curves summarized inhibitory effects of puerarin at concentrations of 10 μM, 100 μM, 1 mM, 10 mM and recovery after compound wash off. Different concentrations of puerarin decreased the IKs from (33.8 ± 3.2) pA/pF to (25.6 ± 2.4) pA/pF, (15.7 ± 1.9) pA/pF, (6.0 ± 1.3) pA/pF, (5.4 ± 1.7) pA/pF, and decreased the IKs-tail current from (23.5 ± 1.2) pA/pF to (17.8 ± 1.5) pA/pF, (9.7 ± 1.1) pA/pF, (6.4 ± 1.2) pA/pF, (5.2 ± 1.3) pA/pF at 40 mV, respectively. (c) Concentration dependent inhibition by puerarin upon IKs at the peak currents and tail currents. Dosage curve was fitted with non-linear Hill. IC50 = 55.63 μM at the peak currents and IC50 = 59.75 μM at the tail currents, Hill coefficient is 0.989. n = 6 each point.

Figure 5. Puerarin might inhibit the current of Kv7.1/IKs in a PKA-independent pathway. (a) Isoproterenol significantly enhanced Kv7.1 current whereas puerarin (100 μM) antagonized such activation effect. (b) Forskolin significantly enhanced Kv7.1 current whereas puerarin (100 μM) antagonized such activation effect. (c) 8-Br-cAMP significantly enhanced Kv7.1 current whereas puerarin (100 μM) antagonized such activation effect. (d) 1 mM puerarin had no effect on intracellular cAMP concentration and no effect upon the increase of cAMP induced by isoproterenol, forskolin in rat cardiac myocytes. (e) forskolin significantly enhanced IKs current whereas puerarin (100 μM) antagonized such activation effect in rat cardiac myocytes. (f ) Statistical analysis of Fig. 5e.

Figure 6. Puerarin might directly inhibited Kv7.1 and IKs channels. (a) Single channel currents with the conductance of 4.4 pS elicited at the different voltages. (b) The open probability of Kv7.1 channel was reduced by puerarin at the concentration of 100 μM significantly, which was further abolished by the specific antagonist of Kv7.1, 293B. (c) Single channel currents with the conductance of 15.9 pS elicited at the different voltages. (d) The open probability of IKs channel was reduced by puerarin at the concentration of 100 μM significantly, which was further abolished by the specific antagonist of IKs, 293B.

Figure 7. Baicalin inhibited Kir 2.1 channels and enhanced IKs channels in a dosage dependent manner. (a) Baicalin at the concentrations of 1 mM significantly inhibited Kir2.1 currents illustrated by the ramp voltage protocol. This effect could be partly reversed by simple compound wash-off. (b) Concentration dependent inhibition by baicalin upon Kir 2.1. The maximal inhibitory effect was (40.95 ± 2.05) % and saturation dosage was 1 mM. Dosage curve was fitted with non-linear Hill. IC50 = 8.02 μM, Hill coefficient is 0.983. n = 5 each point. (c) Baicalin at the concentrations of 100 μM significantly enhanced IKs currents illustrated by the protocol of voltage steps, respectively. (d) Concentration dependent enhancement by baicalin upon IKs currents. The maximal increased effect was (57.32 ± 3.07) % and saturation dosage was 1 mM. Dosage curve was fitted with non-linear Hill. EC50 = 137.95 μM at the peak currents, Hill coefficient is 0.993. n = 6 each point. (e) The molecular structural formula of baicalin.