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

Fig 1. Characterizations of hiPSC-CMs. 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 INa, 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 INa 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.

Fig 2. Effects of temperature variation on sodium channel currents and current kinetics. (A) Representative current-voltage relationships for peak INa acquired with indicated voltage protocols (the inset on the left) at 36°C and 40°C. (B) Normalized membrane conductance (Gm/Gmax) versus membrane potentials demonstrating the steady-state activation of INa measured at 36°C and 40°C. (C) Normalized peak INa (Im/Imax) measured at -30 mV with the indicated protocol (inset) versus membrane potentials demonstrating the steady-state inactivation of INa measured at 36°C and 40°C. (D) Recovery of INa from inactivation measured with the indicated protocol (inset) at 36°C and 40°C. (E) Late INa 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 INa and late INa (at -40 mV) recorded at 36°C and 40°C. Values given are mean ± SEM. n, number of cells. ns, P>0.05.

Fig 3. Hyperthermia enhanced the effect of flecainide and lidocaine but reduced the effect of ajmaline and ranolazine on the peak INa. INa was recorded with the same protocol as that shown in Fig 1A 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 INa 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.

Fig 4. Hyperthermia diminished the effect of flecainide and lidocaine on the late INa. The late INa 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 INa. Mean values of the percent inhibition of late INa 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 INa 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.

Fig 5. Effects of the Na channel blocking drugs on the activation of peak INa. Peak INa was recorded with the same protocol as that shown in Fig 1A and converted to membrane conductance (Gm) and normalized to the maximum (Gmax). The normalized Gm 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.

Fig 6. Effects of the Na channel blocking drugs on the inactivation of peak INa. The normalized peak INa (Im/Imax) 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.

Fig 7. Effects of the Na channel blocking drugs on the recovery from inactivation of peak INa. (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 INa 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.