A multi-modal composition of the late Na+ current in human ventricular cardiomyocytes

Abstract

Objective: We reported an ultraslow late Na+ current (INaL) in ventricular cardiomyocytes of human hearts. INaL has been implicated in regulation of action potential duration in normal hearts and repolarization abnormalities in failing hearts. We have also identified sodium channel (NaCh) gating modes including bursts (BM) and late scattered openings (LSM) that together comprise INaL; however, the contribution of these gating modes to Na+ current (INa) remains unknown. In the present study, the late NaCh activity was recorded, analyzed, and modeled for heterologously expressed NaCh, Nav1.5, and for the native NaCh of ventricular mid-myocardial cardiomyocytes from normal and failing hearts.

Methods and results: We found that LSM gating was significantly slower in failing compared to normal myocytes and Nav1.5 (tau=474+/-10 vs. 299+/-9, and 229+/-12 ms, m+/-SEM; P<0.05, n=5-6). Total burst length of BM decreased with depolarization and was larger in failing compared to normal myocytes and Nav1.5. A complete INa decay was then numerically approximated as composed of NaCh populations operating in three gating modes described by separate Markov kinetic schemes: transient mode (TM), LSM, and BM. The populations of NaCh operating in each gating mode were estimated as 79.8% for TM, 20% for LSM, and 0.2% for BM, yielding an apparent four-exponential INa decay at -30 mV (maximum INa) (tau i approximately 0.4, 4, 50, and 500 ms). Whole-cell recordings confirmed the existence of all four predicted components. The model also predicted voltage and temperature dependence of INaL as well as INaL increase and slower decay in failing hearts and acceleration by amiodarone.

Conclusions: The early phase of Na+ current decay (<40 ms) involves all three NaCh gating modes, the intermediate phase (from 40 to 300 ms) is produced by BM+LSM, although the contribution of BM decreases with depolarization, and ultra-late decay (>300 ms) is determined solely by LSM. The concept of multi-mode composition for INaL provides a new rationale for INaL modulation by factors such as voltage, temperature, pharmacological agents, and pathological conditions.

Fig. 1.

Single-channel data from human ventricular myocytes. A-C. Three major types of single Na⁺ channel modal activity: transient (A), late scattered openings (B) and bursts (C), including total burst length and envelopes. D. Ensemble current was from 160 original traces containing no bursts. A double-exponential function (solid line) was fitted to the current decay (dots). E. Time constant of the latency of the late scattered openings in heterologously expressed Na_v1.5 in HEK293 cells and human normal and failing ventricular myocytes; m±SEM, n=number of patches, *P<0.05 failing cells, vs. normal or Na_v1.5. F. The total burst length evaluated at different membrane potentials. Data were collected from 5-8 patches; m±SEM, *P<0.05, heart failure vs. normal heart or clone. Cell-attached configuration: V_h = -140 mV, 24°C.

Fig. 2.

Kinetic models describing the three major modes of NaCh gating: transient mode (A), late scattered openings (B) and bursts (C), together with simulated traces and transition rates. Rates a and b (†) for the activation process were taken from [8]. All other rates were calculated from single-channel data obtained at -30 mV (see text for details). Traces in A, B, and C were generated from 3, 5, and 1 channel(s), respectively. Total simulation times are indicated above the traces. Simulated traces had a noise amplitude and bandwidth similar to actual single-channel recordings.

Fig. 3.

Simulated total Na⁺ current (I_total) from 100,000 channels described by a composite multi-modal stochastic model (Fig. 2): 79,807 channels were operating in transient mode (TM), 20,000 in late scattered mode (LSM) and 193 in burst mode (BM). I_total and the contributions of the different gating modes (I_total=I_TM+I_LSM+I_BM) are shown at different time scales increasing from A to C. The early phase (A) is derived mainly from TM, the intermediate phase (B) from BM+LSM, and the late phase (C) entirely from LSM. Note that in C, I_total and I_LSM almost overlap. Simulated I_total and experimental whole-cell currents appear identical in time frames 20 and 2000 ms (patch-clamp recording insets in A and C, respectively). D. Instantaneous percent contributions to total I_Na predicted by the model for the three different gating modes during 300 ms of membrane depolarization to -30 mV.

Fig. 4.

Multi-exponential decay function closely fits whole-cell I_Na recordings in human ventricular myocytes. A and B. Four-exponential fit for a total I_Na (in gray) recorded at a low bath Na⁺ (5 mM), showing the same traces for transient (I_NaT) and late (I_NaL) currents, respectively, at two different time scales. C. Simplified two-exponential fit for I_NaL decay recorded with 140 mM Na⁺ in the bath. The respective fitting functions are shown as solid black lines, with a boxed numerical representation indicated by the arrows.

Fig. 5.

Voltage dependence of Na⁺ channel bursts. A. Experimentally measured activity reproduced by the model simulations. B and C. Model predictions for time course of P_open (B) and average current of a single channel operating in burst mode at various membrane voltages (C). Predictions were obtained by averaging the activity from 1000 simulations of the bursting channel. Calibration bars are 1 pA and 10 ms.

Fig. 6.

Model predicts acceleration of I_NaL decay and increase in I_NaL amplitude as temperature increases from 24°C (A, B) to 37°C (C, D). The figure shows simulated cumulative activity of 20,000 late scattered mode channels (LSM, Panels A and C) and 193 burst mode channels (BM, panels B and D) at different voltages indicated at the traces. All channels were available and activated upon depolarization. Channel numbers were chosen to correspond to a human ventricular myocyte (same as in Fig. 3). Gating schemes for LSM and BM are shown in Fig. 2B and C, respectively. Temperature dependence was explored using Q10 factors as discussed in the text. Single-channel currents for 24°C were calculated with a conductance of 11 pS. Equilibrium Na⁺ potential was calculated as E_Na=(RT/F)*ln([Na]_o/[Na]_i). The LSM currents were low-pass (100 Hz) filtered.

Fig. 7.

Model predicts augmented and slower I_NaL at various membrane potentials in a human ventricular myocyte in heart failure vs. a normal myocyte and Na_v1.5 expressed in HEK293 cells. A. Late scattered mode (LSM). B. Burst mode (BM). The model parameters were changed based on the single-channel data (Fig. 1E and F) as follows: rate e in Fig. 2B (LSM) was 0.0043554, 0.00334, and 0.00211 ms^-1 and rate e in Fig. 2C (BM) was 3, 0.2, and 0.086 ms^-1 for Nav1.5, normal and heart failure VM, respectively. Activity is shown at -30 mV from 20,000 LSM channels and 193 BM channels with a conductance of 11 pS. LSM currents were low-pass (100 Hz) filtered. Inset shows a larger integral of the LSM current in a heart failure myocyte vs. a normal myocyte; the box size is 50 pC x 1990 ms.

Fig. 8.

Experimental effect of amiodarone (AMIO, 5 μM) on I_NaL (A) in human ventricular myocytes as predicted by the new Markov model of late Na⁺ channel gating (B). Model simulations using the kinetic schemes shown in Fig. 2 were performed for 170,000 Na⁺ channels, 34,000 operating in LSM mode and 328 in burst mode. Rate constants e and d for LSM mode were 0.00145 and 0.01 ms^-1 in control cells and 0.0028 and 0.0062 ms^-1 in the presence of AMIO.