Hyperthermia Influences the Effects of Sodium Channel Blocking Drugs in Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes

<div><p>Introduction</p><p>Fever can increase the susceptibility to supraventricular and ventricular arrhythmias, in which sodium channel dysfunction has been implicated. Whether fever influences the efficacy of sodium channel blocking drugs is unknown. The current study was designed to investigate the temperature dependent effects of distinct sodium channel blocking drugs on the sodium currents in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs).</p><p>Methods and Results</p><p>hiPSC-CMs were generated from human skin fibroblasts of a healthy donor. The peak and late sodium currents (I_Na), steady-state activation, inactivation and recovery from inactivation of I_Na in hiPSC-CMs were analyzed using the whole-cell patch clamp technique. The effects of different concentrations of the antiarrhythmic drugs flecainide, lidocaine, ajmaline and the antianginal drug ranolazine on I_Na were tested at 36°C and 40°C. Increasing the temperature of the bath solution from 36°C to 40°C enhanced the inhibition of peak I_Na but reduced the inhibition of late I_Na by flecainide and lidocaine. By contrast, increasing the temperature reduced the effect of ajmaline and ranolazine on the peak I_Na but not late I_Na. None of the tested drugs showed temperature-dependent effects on the steady-state activation and inactivation as well as on the recovery from inactivation of I_Na in hiPSC-CMs.</p><p>Conclusions</p><p>Temperature variation from the physiological to the febrile range apparently influences the effects of sodium channel blockers on the sodium currents. This may influence their antiarrhythmic efficacy in patients suffering from fever.</p></div

Characterizations of hiPSC-CMs.

<p>qPCR analysis was carried out to assess the expression of the pluripotency gene POU5F (A) and the cardiac genes TNNT2 (B), MYH6 (C), NKX2.5 (D) and ACTN2 (E) as well as the sodium channel gene SCN5A (F) at different times after onset of differentiation. Spontaneous APs were recorded in current-clamp mode without injection of current. Three forms of AP were observed (G-H). (I) Representative cardiac I_Na, resistant to 20 nM TTX but sensitive to 20 μM TTX, was measured with the protocol shown as an inset at the bottom. The late I_Na was shown from 200 ms to 250 ms (indicated by dotted line) with different scale (inset). Values given are mean ± SEM. n = 4/2 (technical replicates/biological replicates). *, p<0.05 vs. d0.</p

Hyperthermia enhanced the effect of flecainide and lidocaine but reduced the effect of ajmaline and ranolazine on the peak I_Na.

<p>I_Na was recorded with the same protocol as that shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166143#pone.0166143.g001" target="_blank">Fig 1A</a> in absence and presence of different concentrations of the tested drugs at 36°C and 40°C. Representative traces (left panels) and mean values (right panels) of percent inhibition of peak I_Na recorded at -35 mv in the presence of flecainide (A), lidocaine (B), ajmaline (C) and ranolazine (D) at 36°C and 40°C are shown by the bar graphs. Values given are mean ± SEM. n, number of cells. ns, P>0.05.</p

Hyperthermia diminished the effect of flecainide and lidocaine on the late I_Na.

<p>The late I_Na was recorded by a depolarizing pulse from the holding potential of -100 mV to -45 mV with the duration of 400 ms. The area under the current curve was measured between 50 ms and 350 ms of the depolarizing pulse to illustrate the quantity of the late I_Na. Mean values of the percent inhibition of late I_Na in the presence of flecainide (A), lidocaine (B), ajmaline (C) and ranolazine (D) at 36°C and 40°C as well as 36°C plus 20 μM TTX are shown by the bar graphs. Representative traces of late I_Na inhibited by flecainide and lidocaine at 36°C and 40°C are also shown. Values given are mean ± SEM. n, number of cells. ns, P>0.05.</p

Effects of the Na channel blocking drugs on the recovery from inactivation of peak I_Na.

<p>(A)-(B) Representative recovery curves in the presence of different concentrations of ranolazine at 36°C and 40°C. Mean values of the time constants of the recovery from inactivation of peak I_Na in the presence of ranolazine (C), flecainide (D), lidocaine (E) and ajmaline (F) are shown by the bar graphs. Values given are mean ± SEM. n, number of cells. ns, P>0.05.</p

Effects of the Na channel blocking drugs on the inactivation of peak I_Na.

<p>The normalized peak I_Na (I_m/I_max) was plotted against the membrane potentials to obtain the inactivation curves. The curves are fitted by Boltzmann equation to obtain the half maximal voltage (V0.5) of inactivation. (A)-(B) Representative inactivation curves in the presence of different concentrations of flecainide at 36°C and 40°C. Mean values of V0.5 of the inactivation curves in the presence of flecainide (C), lidocaine (D), ajmaline (E) and ranolazine (F) are shown by the bar graphs. Values given are mean ± SEM. n, number of cells. ns, P>0.05.</p

Effects of the Na channel blocking drugs on the activation of peak I_Na.

<p>Peak I_Na was recorded with the same protocol as that shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166143#pone.0166143.g001" target="_blank">Fig 1A</a> and converted to membrane conductance (G_m) and normalized to the maximum (G_max). The normalized G_m was plotted against the membrane potentials to obtain the activation curves. The curves are fitted by Boltzmann equation to obtain the half maximal voltage (V0.5) of activation. (A)-(B) Representative activation curves in the presence of different concentrations of ajmaline at 36°C and 40°C. Mean values of V0.5 of the activation curves in the presence of ajmaline (C), flecainide (D), lidocaine (E) and ranolazine (F) at 36°C and 40°C are shown by the bar graphs. Values given are mean ± SEM. n, number of cells. ns, P>0.05.</p

Effects of temperature variation on sodium channel currents and current kinetics.

<p>(A) Representative current-voltage relationships for peak I_Na acquired with indicated voltage protocols (the inset on the left) at 36°C and 40°C. (B) Normalized membrane conductance (G_m/G_max) versus membrane potentials demonstrating the steady-state activation of I_Na measured at 36°C and 40°C. (C) Normalized peak I_Na (I_m/I_max) measured at -30 mV with the indicated protocol (inset) versus membrane potentials demonstrating the steady-state inactivation of I_Na measured at 36°C and 40°C. (D) Recovery of I_Na from inactivation measured with the indicated protocol (inset) at 36°C and 40°C. (E) Late I_Na illustrated as the area under the current curve (pA*ms) between 50 and 350 ms of the pulse at -45 mV. (F) Examples of Na⁺ current traces showing the peak I_Na and late I_Na (at -40 mV) recorded at 36°C and 40°C. Values given are mean ± SEM. n, number of cells. ns, P>0.05.</p