Long-QT syndrome-related sodium channel mutations probed by the dynamic action potential clamp technique

Abstract

Long-QT3 syndrome (LQT3) is linked to cardiac sodium channel gene (SCN5A) mutations. In this study, we used the 'dynamic action potential clamp' (dAPC) technique to effectively replace the native sodium current (I(Na)) of the Priebe-Beuckelmann human ventricular cell model with wild-type (WT) or mutant I(Na) generated in a human embryonic kidney (HEK)-293 cell that is voltage clamped by the free-running action potential of the ventricular cell. We recorded I(Na) from HEK cells expressing either WT or LQT3-associated Y1795C or A1330P SCN5A at 35 degrees C, and let this current generate and shape the action potential (AP) of subepicardial, mid-myocardial and subendocardial model cells. The HEK cell's endogenous background current was completely removed by a real-time digital subtraction procedure. With WT I(Na), AP duration (APD) was longer than with the original Priebe-Beuckelmann model I(Na), due to a late I(Na) component of approximately 30 pA that could not be revealed with conventional voltage-clamp protocols. With mutant I(Na), this late component was larger ( approximately 100 pA), producing a marked increase in APD ( approximately 70-80 ms at 1 Hz for the subepicardial model cell). The late I(Na) magnitude showed reverse frequency dependence, resulting in a significantly steeper APD-frequency relation in the mutant case. AP prolongation was more pronounced for the mid-myocardial cell type, resulting in increased APD dispersion for each of the mutants. For both mutants, a 2 s pause following rapid (2 Hz) pacing resulted in distorted AP morphology and beat-to-beat fluctuations of I(Na). Our dAPC data directly demonstrate the arrhythmogenic nature of LQT3-associated SCN5A mutations.

Figure 1. Dynamic action potential clamp (dAPC) technique

A, general experimental design. The cardiac ventricular cell is in current clamp. The SCN5A cDNA-transfected HEK cell is voltage clamped with V_m of the ventricular cell. I_Na from the HEK cell is continuously applied to the ventricular cell as an external current input, partly or entirely replacing I_Na in the myocyte. B, model cell mode of dAPC technique: after real-time digital subtraction of HEK cell I_bck, I_Na from the HEK cell is scaled by factor F₂; model cell I_Na density is reduced to 40% of the original value (scaling factor F₁). The I_Na of the PB model cell incorporates a ‘heterozygous’I_Na composed of reduced model cell I_Na and scaled HEK cell (input) I_Na(+). The V_m of the human ventricular cell model is computed in real time using the thus-obtained I_Na and applied as voltage-clamp command potential to the HEK cell, thus establishing dAPC.

Figure 2. Square wave-, step-ramp-, and AP-elicited currents in HEK-293 cells

A, I_bck in non-transfected HEK cells. Upper left panel depicts the voltage-step protocol; lower left panel shows the corresponding I_bck–V, determined from amplitudes at the end of the 500-ms voltage steps (arrow). The fit (continuous line) is a third-order polynomial equation (see text for details). A step-ramp waveform (upper right panel) was used to determine time-course of averaged I_bck (lower right panel). B, step-ramp and ‘subepicardial’-type AP-waveforms (top) to activate I_bck (bottom) in a WT SCN5A cDNA-transfected HEK cell (representative examples). Note the absence (○) and the presence (arrows) of fast I_Na, as a function of V_hold of −60 mV and −90 mV, respectively (see text for details). APs were generated using the PB model cell; broken line shows zero current level; peak I_Na is off scale.

Figure 3. Real-time HEK-293 cell I_bck subtraction in a dAPC experiment, using a WT SCN5A cDNA-transfected HEK-293 cell

A, step-ramp voltage protocol and the first AP elicited in a subepicardial model cell. The maximum diastolic potential (MDP) during dAPC was −90 mV (top); Middle: I_Na in the presence of I_bck (arrows) and after I_bck removal (bottom). Note the transient WT late I_Na during AP repolarization. Broken line shows zero current level. B, I–V relationship of I_bck in the experiment shown in A, fit with the scaled polynomial equation (P= 0.39) (see text for details).

Figure 4. Whole-cell currents in HEK-293 cells transfected with WT, Y1795C, or A1330P cDNAs

A, representative I_Na traces. The voltage protocol is shown as an inset. Interpulse interval was 2 s. B, typical, 250 ms step depolarization-evoked TTX-sensitive whole-cell I_Na traces obtained by subtraction and plotted as percentage of peak I_Na; inset: protocol. Note that late I_Na is hardly detectable with WT SCN5A channels upon a step depolarization. Peak currents are off scale; broken lines indicate zero current level. C, superimposed tracing of AP clamp-evoked currents: control (I_total, blue), TTX-resistant (I_bck, grey), and difference (TTX-sensitive, red). Currents were plotted as percentage of peak I_Na; to record WT and mutant I_Na traces, the same cells were used as in B; in all cases I_Na was ∼10 nA; peak currents are off scale; broken line shows zero current level. AP clamp waveform shown as inset.

Figure 5. Dynamic action potential clamp with WT or Y1795C I_Na

A, subepicardial cell APs elicited at 1 Hz before and after a 2 s pause. APs with mutant I_Na are prolonged compared to WT (see Table 1). B, boxed APs from A and associated I_Na in C, on expanded time scale. HEK cell I_Nas were scaled to identical peak amplitudes (21 nA), and applied to the ‘subepicardial’ PB model cell as an external current input. Note that late I_Na-increase after the pause is more pronounced with the mutant (*). Broken line shows zero current level; peak I_Na is off scale; note the slower time-course of peak I_Na-inactivation of the mutant compared to the WT (arrows). D, relationship between the APs from B and selected membrane current components of the PB model cell, showing the changes in the time-course of transient outward K⁺ current (I_to), slowly and rapidly activating components of the delayed rectifier K⁺ current (I_Ks and I_Kr, respectively), and L-type Ca²⁺ current (I_Ca), along with changes in mutant I_Na.

Figure 6. Prolonging effects of the A1330P mutation are reduced at fast stimulation rate

A, subepicardial cell APs generated with WT or A1330P I_Na, at 2 Hz. B, boxed APs from A and C, associated I_Nas. After a 2 s pause, late A1330P I_Na increases (*), resulting in a prolonged AP; note the slower time-course of peak A1330P I_Na inactivation compared to that of WT (arrows). D, selected associated individual membrane currents in the PB model cell.

Figure 7. Representative examples of the effects of pacing rate and of a pause on WT or mutant peak I_Nas

APs were recorded from a ‘subepicardial’ cell successively coupled to HEK cells transfected with WT (top), Y1795C (middle) or A1330P (bottom) channels. A normal (WT) response is shown in the top panels, while APs in LQT syndrome are prolonged compared to normal (middle and bottom). At 2 Hz, after the pause, the extent of AP prolongation is exaggerated and heterogeneous. Note the corresponding I_Nas.

Figure 8. AP duration at 90% repolarization (APD₉₀) with the subepicardial cell with WT or mutant I_Nas

See Table 1 for n at 1 and 2 Hz; at 0.5 Hz n was ≥ 6 in all cases. *Significant difference versus control (P < 0.05 for mutant versus WT).

Figure 9. AP prolongation caused by the Y1795C and A1330P mutations in ‘subendocardial’ (A) and ‘M’ (B) cells

Representative APs (top) at 1 Hz, the corresponding HEK cell I_Na (middle), and associated individual membrane currents in the PB model cell (bottom). AP prolongation was exaggerated in subendocardial and M cells compared to subepicardial cells. The same input (WT, Y1795C, or A1330P) I_Na peak amplitude was used in A and B, respectively. Arrows in bottom panels indicate plateau amplitude and the corresponding late I_Na amplitude.