Differential thermosensitivity in mixed syndrome cardiac sodium channel mutants

Abstract

Cardiac arrhythmias are often associated with mutations in SCN5A the gene that encodes the cardiac paralogue of the voltage-gated sodium channel, NaV 1.5. The NaV 1.5 mutants R1193Q and E1784K give rise to both long QT and Brugada syndromes. Various environmental factors, including temperature, may unmask arrhythmia. We sought to determine whether temperature might be an arrhythmogenic trigger in these two mixed syndrome mutants. Whole-cell patch clamp was used to measure the biophysical properties of NaV 1.5 WT, E1784K and R1193Q mutants. Recordings were performed using Chinese hamster ovary (CHOk1) cells transiently transfected with the NaV 1.5 α subunit (WT, E1784K, or R1193Q), β1 subunit, and eGFP. The channels' voltage-dependent and kinetic properties were measured at three different temperatures: 10ºC, 22ºC, and 34ºC. The E1784K mutant is more thermosensitive than either WT or R1193Q channels. When temperature is elevated from 22°C to 34°C, there is a greater increase in late INa and use-dependent inactivation in E1784K than in WT or R1193Q. However, when temperature is lowered to 10°C, the two mutants show a decrease in channel availability. Action potential modelling using Q10 fit values, extrapolated to physiological and febrile temperatures, show a larger transmural voltage gradient in E1784K compared to R1193Q and WT with hyperthermia. The E1784K mutant is more thermosensitive than WT or R1193Q channels. This enhanced thermosensitivity may be a mechanism for arrhythmogenesis in patients with E1784K sodium channels.

Figure 1

Na⁺ currents Current recordings of the channel variants are shown at three temperatures (A1-A3 (WT), B1-B3 (R1193Q), C1-C3 (E1784K)). Panel D shows the current density for the different channel variants as a function of temperature. Panel E shows the time to half peak of maximal Na current measured at 0 mV.

Figure 2

Activation Panels A–F show the voltage-dependence of activation as normalized conductance plotted against membrane potential plotted versus membrane potential. The insets show pulse protocols used to measure I_Na at different voltages. Panels A–C show the channel variant effect at 10°C, 22°C and 34°C. Panels D–F show the temperature effect for WT, R1193Q and E1784K.

Figure 3

Steady-state fast inactivation Panels A–F show the voltage-dependence of steady-state fast inactivation as normalized current plotted against membrane potential. The insets show pulse protocols used to measure I_Na at different voltages. Panels A–C show the channel variant effect at 10°C, 22°C and 34°C. Panels D–F show the temperature effect for WT, R1193Q and E1784K.

Figure 4

Activation and steady-state fast inactivation Q₁₀ curves Panels A and B show the conductance midpoint and conductance slope for the channel variants as a function of temperature fit with Q₁₀ curves. Panels C and D show the steady-state fast inactivation midpoint and slope Q₁₀ curves.

Figure 5

Fast inactivation (FI) time constants Panels A–F show the recovery from fast inactivation at −130 mV as normalized currents versus recovery time duration. The insets show the FI time constants plotted against the membrane potential. Panels A–C show the channel variant effects at 10°C, 22°C and 34°C. Panels D–F show the temperature effect for WT, R1193Q and E1784K. Pulse protocols of recovery and onset were not shown for clarity. Please refer to Methods.

Figure 6

Fast inactivation (FI) kinetics Q₁₀ fits Panels A–H show the rates of fast inactivation for WT, R1193Q and E1784K at different voltages plotted as a function of temperature.

Figure 7

Persistent sodium currents Panels A–F show normalized current plotted as a function of time with insets that focus on a narrower current window to show persistent I_Na. Panels A and B show the channel variant effect at both 22°C and 34°C. Panels C and D show the temperature effect for WT, R1193Q and E1784K. The pulse protocol inset is not shown in Panel E for clarity purposes. Panel F shows the Q₁₀ fit for persistent current of all the channel variants plotted as a function of temperature.

Figure 8

Use-dependent inactivation (1 Hz) Panels A–F show normalized current plotted as a function of time. Panels A–C show the channel variant effect for 10°C, 22°C and 34°C. Panels D–F show the temperature effect for WT, R1193Q and E1784K. Insets show the pulse protocols used to measure use-dependent inactivation (UDI) at 1 Hz.

Figure 9

Use-dependent inactivation (3 Hz) Panels A–F show normalized current plotted as a function of time. Panels A–C show the channel variant effect for 10°C, 22°C and 34°C. Panels D–F show the temperature effect for WT, R1193Q and E1784K. Insets show the pulse protocols used to measure use-dependent inactivation (UDI) at 3 Hz.

Figure 10

Use-dependent inactivation (UDI; 1 Hz and 3 Hz) parameters Q₁₀ fits Panels A and D show the initial rate of use-dependent inactivation for 1 Hz and 3 Hz, respectively. Panels B and E show the secondary rate of use-dependent inactivation for 1 Hz and 3 Hz, respectively. Panels C and F show the inverse of plateau of use-dependent inactivation for 1 Hz and 3 Hz, respectively.

Figure 11

AP model simulation