Characterisation of the role of TTX-sensitive sodium channels (TTX-S Nav) and calcium activated potassium channels (KCa1.1) in cardiac automaticity using huwentoxin-IV and iberiotoxin

Introduction

Cardiac automaticity is defined by heart capacity to spontaneously generate action potentials (APs) in sino-atrial node mouse cells (SANCs). This capacity is due to diastolic depolarization phase (DD) which is a slow depolarizing phase developing during the diastole of the cardiac contraction cycle. DD is the results of a complex and not yet totally unraveled, interaction between intracellular calcium dynamic and sarcolemma ion channels. Among these ion channels, TTX-sensitive sodium channels (TTX-S Nav) and calcium activated potassium channels (KCa1.1) have been associated with intranodal conduction failure.

Objective

To investigate the implication of TTX-S Nav and KCa1.1 ion channels in cardiac automaticity, by using two different natural peptides [huwentoxin-IV (HwTx-IV) and iberiotoxin (IbTx)] inhibiting respectively and selectively these ion channels.

Method

SANCs isolated from wild-type mouse were used for ion current and AP recordings using patch-clamp technique. 100 nM HWTX-IV was perfused on SANCs alone or in combination with 50 nM TTX (as positive control). ECG intervals were recorded in anesthetised rats in the same conditions to confirm the results in more integrated model. In order to define the right condition to study the effect of KCa1.1 channels in SANCs, HEK cells were transfected with KCa1.1 channels. KCa1.1 current was recorded before and after application of NS 19504 (a KCa1.1 activator) 10 μM, IbTx 300 nM and Paxillin 1 μM.

Results

Inhibition of TTX-S Nav channels by 100 nM HwTx-IV induced a significant reduction of the sodium current which was not enhanced by 50 nM TTX, a dose knowing to block 100% of TTX-S Nav channels. Moreover, 100 nM HwTx-IV showed a significant 13% reduction of spontaneous firing frequency in SANCs whereas 50 nM TTX did not further reduce the SANCs APs rate validating a complete inhibition of TTX-S Nav channels by 100 nM HwTx-IV. In anesthetised rats, injection of 50 μg/kg HwTx-IV induced a significant reduction in heart rate without affecting cardiac conduction. In transfected HEK cells, 10 μM NS doubled KCa1.1 current while 300 nM IbTx and 1 μM Paxillin blocked 100% of KCa1.1 current.

Conclusion

Inhibition of TTX-S Nav channels significantly reduced spontaneous APs frequency in SANCs, suggesting their important role in cardiac automaticity. IbTx 300 nM totally blocked KCa1.1 current in HEK transfected cells. Experiments to clarify the role of KCa1.1 channels in cardiac automaticity and crosstalk with TTX-S channels are currently performed.