Arrhythmogenic and antiarrhythmic actions of late sustained sodium current in the adult human heart

Figure 1. Characterization of late INa in adult human primary ventricular myocytes. (a) Schematic representation of the voltage ramp protocol to measure late INa. (b) and (c) Typical current recordings from an adult human primary ventricular myocyte and average current–voltage relationships of normal physiological late INa in the presence of Vehicle control and after exposure to either ranolazine (60 µM, n = 6 cells) or ATX-II (0.06 µM, n = 6 cells). (d) and (e) Effects of ranolazine on ATX-II-enhanced late INa. Representative recordings (d) and average current–voltage relationships (e) of late INa in the presence of Vehicle control and in the presence of ranolazine (60 µM) before and after application of ATX-II (0.06 µM, n = 6 cells). Data in (c) and (e) are expressed as mean ± SEM. Statistical analysis was conducted by ANOVA with a Bonferroni posthoc test. *,*p < 0.05 versus vehicle control in (c) and (e), and *p < 0.05 versus ATX-II in (e). Fitmaster analysis software package (HEKA Elektronik, Germany, www.heka.com) and SigmaPlot v14.0 (Systat Software Inc., CA, USA, www.systatsoftware.com) were used to generate the representative late INa current traces.

Figure 2. Physiological late INa and normal human ventricular action potential. (a) Typical action potentials (APs) recorded from a ventricular cell at a pacing of 1 Hz in the presence of vehicle control and after exposure to 0.1 µM GS-967 or 0.06 µM ATX-II. (b) shows a typical example of a Poincaré plot of APD90 from a ventricular cell (same cell as in a) under vehicle and in the presence of GS-967 or ATX-II. Note the large complex polygons during the application of ATX-II. (c) and (d) show mean % changes in APD (measured at 20% (APD20), 50% (APD50) and 90% (APD90) of repolarization) and STV(APD90), short-term variability of APD90 (n = 5 cells). (e) Typical APs recorded from a ventricular cell at a pacing frequency of 1 Hz in the presence of vehicle control and after exposure to 10 µM ranolazine or 0.06 µM ATX-II. (f) and (g) show mean % changes in APD and STV(APD90) under vehicle and in the presence of ranolazine or ATX-II (n = 5 cells). *,*p < 0.05 versus values from vehicle control. Fitmaster analysis software package (HEKA Elektronik, Germany, www.heka.com) and SigmaPlot v14.0 (Systat Software Inc., CA, USA, www.systatsoftware.com) were used to generate the representative AP traces.

Figure 3. Effects of GS-967 and ranolazine on human ventricular action potential in condition mimicking LQTS3. (a) Typical action potentials (APs) recorded from a ventricular cell at a pacing of 1 Hz in the presence of vehicle control and after exposure to 0.06 µM ATX-II alone or in combination with 0.1 µM GS-967. (b) and (c) show mean % changes in APD (measured at 20% (APD20), 50% (APD50) and 90% (APD90) of repolarization) and STV(APD90), short-term variability of APD90 induced by addition of ATX-II alone or in the presence of GS-967 (n = 5 cells) at 1 Hz. (d) Typical APs recorded from a ventricular cell at a pacing frequency of 1 Hz in the presence of vehicle control and after exposure to 0.06 µM ATX-II alone or in combination with ranolazine 10 µM. (e) and (f) show mean % changes in APD and STV(APD90) induced by addition of ATX-II alone or in the presence of ranolazine (n = 5 cells) at 1 Hz. *,*p < 0.05 versus values from vehicle control. #p < 0.05 versus values from ATX-II. Fitmaster analysis software package (HEKA Elektronik, Germany, www.heka.com) and SigmaPlot v14.0 (Systat Software Inc., CA, USA, www.systatsoftware.com) were used to generate the representative AP traces.

Figure 4. Effects of GS-967 on human ventricular action potential in condition mimicking LQTS2. (a) Typical action potentials (APs) recorded from a ventricular cell at a pacing of 1 Hz in the presence of vehicle control and after exposure to 1 µM E-4031 alone or in combination with 0.3 µM GS-967. (b) and (c) show mean % changes in APD (measured at 20% (APD20), 50% (APD50) and 90% (APD90) of repolarization) and STV(APD90), short-term variability of APD90 induced by addition of E-4031 alone or in the presence of GS-967 (n = 5 cells) at 1 Hz. #p < 0.05 versus values from E-4031. Fitmaster analysis software package (HEKA Elektronik, Germany, www.heka.com) and SigmaPlot v14.0 (Systat Software Inc., CA, USA, www.systatsoftware.com) were used to generate the representative AP traces.

Figure 5. Effects of GS-967 and ranolazine on human ventricular Ca2+ transient in condition mimicking LQTS3. (a–c) and (d–f) show typical Ca2+ transients recorded from ventricular cells at a pacing of 1 Hz in the presence of vehicle control (a and d) and after exposure to 0.03 µM ATX-II alone (b and e) or in combination with 0.3 µM GS-967 (c) and 10 µM ranolazine (f). (g), (h) and (i) show mean % incidence in ATX-II-induced arrhythmia induced by addition of ATX-II alone or in the presence of GS-967 (g, n = 45 cells) or ranolazine (10 µM, h, n = 58 cells; 60 µM, i, n = 35 cells) at 1 Hz. *,*p < 0.05 versus values from vehicle control. #p < 0.05 versus values from ATX-II. MetaMorph analysis software (Molecular Devices, CA, USA, www.moleculardevices.com) and a validated custom written MatLab program (The MathWorks Inc., MA, USA, www.mathworks.com) were used to generate the representative Ca2+ transients.

Figure 6. Effects of ATX-II on human ventricular contractility. (a–e) show typical contractility transients recorded from a ventricular cell at a pacing of 1 Hz in the presence of vehicle control and after exposure to four ascending concentrations of ATX-II. (f) shows ATX-effect curve for sarcomere shortening as a function of concentrations tested (EC50 = 0.012 µM, n = 6 cells). (g) shows mean % incidence of aftercontraction and contraction failure when cardiomyocytes were incubated with ATX-II at 1 Hz pacing frequency. *,*p < 0.05 versus values from vehicle control. IonWizard software (v1.2.22, IonOptix LLC, MA, USA, www.ionoptix.com) and SigmaPlot v14.0 (Systat Software Inc., CA, USA, www.systatsoftware.com) were used to generate the representative contractility transients and fitted C-E curve, respectively.

Figure 7. Effects of GS-967 and ranolazine on human ventricular contractility in condition mimicking LQTS3. (a–c) show typical contractility transients recorded from a ventricular cell at a pacing of 1 Hz in the presence of vehicle control (a) and after exposure to 0.03 µM ATX-II alone (b) or in combination with 0.3 µM GS-967 (c). (d) and (e) show mean % change in sarcomere shortening (d) and mean % incidence of aftercontraction and contraction failure (e) when cardiomyocytes were treated with 0.03 µM ATX alone or in combination with 0.3 µM GS-967 (n = 8 cells) at 1 Hz. (f) and (g) show mean % change in sarcomere shortening (f) and mean % incidence of aftercontraction and contraction failure (g) when cardiomyocytes were treated with 0.03 µM ATX alone or in combination with 10 and 60 µM ranolazine (n = 6 cells) at 1 Hz pacing frequency. *,*,*p < 0.05 versus values from vehicle control. #,#,#p < 0.05 versus values from ATX-II. IonWizard software (v1.2.22, IonOptix LLC, MA, USA, www.ionoptix.com) was used to generate the representative contractility transients.

Figure 8. Effects of GS-967 on human ventricular contractility in condition mimicking LQTS2. (a–c) show typical contractility transients recorded from a ventricular cell at a pacing of 1 Hz in the presence of vehicle control (a) and after exposure to 1 µM E-4031 alone (b) or in combination with 0.3 µM GS-967 (c). (d) and (e) show mean % change in sarcomere shortening (d) and mean % incidence of aftercontraction and contraction failure (e) when cardiomyocytes were treated with 1 µM E-4031 alone or in combination with 0.3 µM GS-967 (n = 8 cells) at 1 Hz. *p < 0.05 versus values from vehicle control. #p < 0.05 versus values from E-4031. IonWizard software (v1.2.22, IonOptix LLC, MA, USA, www.ionoptix.com) was used to generate the representative contractility transients.

Figure 9. Effects of GS-967 on human atrial contractility in condition mimicking LQTS3. (a–c) show typical contractility transients recorded from a ventricular cell at a pacing of 1 Hz in the presence of vehicle control (a) and after exposure to 0.03 µM ATX-II alone (b) or in combination with 0.3 µM GS-967 (c). (d) and (e) show mean % change in sarcomere shortening (d) and mean % incidence of aftercontraction and contraction failure (e) when cardiomyocytes were treated with 0.03 µM ATX-II alone or in combination with 0.3 µM GS-967 (n = 5 cells) at 1 Hz. *,*p < 0.05 versus values from vehicle control. #,#p < 0.05 versus values from ATX-II. IonWizard software (v1.2.22, IonOptix LLC, MA, USA, www.ionoptix.com) was used to generate the representative contractility transients.

Figure 10. Effects of GS-967 on human atrial trabeculae contraction from donors with atrial fibrillation (AF) in condition of spontaneous arrhythmia. (a–b) and (c–d) show typical contraction/relaxation cycles recorded from two atrial trabecula of donors with AF at a pacing of 1 Hz in the presence of vehicle control (a and c with incidence of spontaneous arrhythmic markers: aftercontraction (a and c) and premature atrial contraction (c)) and after exposure to 0.3 µM GS-967 (b and d with no incidence of arrhythmic markers). (e–f) show mean % control in maximum amplitude of contraction (Max. ampl. Contraction) (e) and mean % incidence of aftercontraction (f) when atrial trabeculae were treated with vehicle or after exposure to 0.3 µM GS-967 (n = 4 trabeculae) at 1 Hz. *p < 0.05 versus values from vehicle. LabChart Software (v8.1.16, ADInstruments Inc., CO, USA, www.adinstruments.com) was used to generate the representative contraction/relaxation cycles.