Basal late sodium current is a significant contributor to the duration of action potential of guinea pig ventricular myocytes

Abstract

In cardiac myocytes, an enhancement of late sodium current (I_NaL) under pathological conditions is known to cause prolongation of action potential duration (APD). This study investigated the contribution of I_NaL under basal, physiological conditions to the APD Whole-cell I_NaL and the APD of ventricular myocytes isolated from healthy adult guinea pigs were measured at 36°C. The I_NaL inhibitor GS967 or TTX was applied to block I_NaL The amplitude of basal I_NaL and the APD at 50% repolarization in myocytes stimulated at a frequency of 0.17 Hz were -0.24 ± 0.02 pA/pF and 229 ± 6 msec, respectively. GS967 (0.01-1 μmol/L) concentration dependently reduced the basal I_NaL by 18 ± 3-82 ± 4%. At the same concentrations, GS967 shortened the APD by 9 ± 2 to 25 ± 1%. Similarly, TTX at 0.1-10 μmol/L decreased the basal I_NaL by 13 ± 1-94 ± 1% and APD by 8 ± 1-31 ± 2%. There was a close correlation (R² = 0.958) between the percentage inhibition of I_NaL and the percentage shortening of APD caused by either GS967 or TTX MTSEA (methanethiosulfonate ethylammonium, 2 mmol/L), a Na_V1.5 channel blocker, reduced the I_NaL by 90 ± 5%, suggesting that the Na_V1.5 channel isoform is the major contributor to the basal I_NaL KN-93 (10 μmol/L) and AIP (2 μmol/L), blockers of CaMKII, moderately reduced the basal I_NaL Thus, this study provides strong evidence that basal endogenous I_NaL is a significant contributor to the APD of cardiac myocytes. In addition, the basal I_NaL of guinea pig ventricular myocytes is mainly generated from Na_V1.5 channel isoform and is regulated by CaMKII.

Keywords: Action potential duration; Ca2+/calmodulin‐dependent protein kinase II; NaV1.5 channel; late sodium current; ventricular myocytes.

Figure 1

Concentration‐dependent inhibition by GS967 of ATX‐II (5 nmol/L)‐induced I_{N aL}. Inward currents were activated by depolarizing pulses from −90 to −50 mV. Panel A, superimposed currents recorded in the order of a–e from a single myocyte before (control) and after drug treatments. Panel B, summary of the average amplitude of I_{N aL} recorded before (A) and after (B–E) drug treatments, as shown in panel A (n = 12/5). *P < 0.001 versus control; ^† P < 0.001 versus ATX‐II alone.

Figure 2

Concentration‐dependent inhibition by GS967 or TTX of basal I_{N aL}. I_{N aL} was elicited by voltage‐clamp pulses from −90 to −30 mV. Panel A, example of current traces recorded from a single myocyte in the absence of drugs (control) and in the presence of 0.3 and 1 μmol/L GS967 (GS). Panel B, concentration–response relationship of the inhibitory effect of GS967 on I_{N aL}. Each data point represents an average inhibition observed from 10 myocytes isolated from 3 to 5 hearts. Data points are fitted with a four‐parameter logistic curve. Panel C, current traces recorded before (A) and after (B) application of TTX, and after washing out TTX (C). Panel D, bars show an average inhibition of I_{N aL} by 0.1 (n = 13/4), 1 (n = 13/4), and 10 (n = 18/6) μmol/L TTX, respectively.

Figure 3

Concentration‐dependent shortening by GS967 or TTX of the action potential duration (APD). Panel A, example of action potential traces recorded from a single myocyte before (control) and after applications of 0.01, 0.1, and 1 μmol/L GS967 (GS). Panel B, summary of APD shortening caused by GS967 at concentrations of 0.01 (n = 10/4), 0.1 (n = 25/10), and 1 (n = 19/7) μmol/L, respectively. Panel C, action potentials recorded from a myocyte in the absence of drug (control) and in the presence of 0.1, 1, and 10 μmol/L TTX. Panel D, average shortening of APD caused by TTX at concentrations of 0.1 (n = 9/2), 1 (n = 15/4), and 10 (n = 15/3) μmol/L.

Figure 4

Correlation of APD shortening and I_{N aL} inhibition in the presence of GS967 (●) and TTX (○). The percentage shortenings of APD are plotted against the percentage inhibition of I_{N aL} caused by GS967 and TTX at the same concentrations. Coefficient of determination (R ²) is calculated using SigmaPlot linear regression curve analysis.

Figure 5

Inhibition of I_{N aL} by the selective Na_V1.5 channel blocker MTSEA (2 mmol/L). Panel A, currents recorded from a myocyte before (control) and after application of MTSEA. Panel B, summary of the results obtained from experiments shown in panel A. Bars represent the average amplitude of I_{N aL} determined from 12 myocytes isolated from six hearts. *P < 0.001 versus control.

Figure 6

Decrease in I_{N aL} in the presence of CaMKII inhibitors. Panel A, representative current traces recorded from four myocytes treated with no drug (control), KN‐93 (10 μmol/L), KN‐92 (10 μmol/L), and AIP (2 μmol/L), respectively. Panel B, summary of the results obtained from experiments shown in panel A. Bars represent the average amplitude of I_{N aL} in control (n = 40/17) and in the presence of KN‐93 (n = 11/3), KN‐92 (n = 12/4), and AIP (n = 11/5). *P < 0.05 versus control.