by Xianming Lin, Mark Crye, and Richard D. Veenstra Regulation of Connexin43 Gap Junctional Conductance by Ventricular Action Potentials by Xianming Lin, Mark Crye, and Richard D. Veenstra Circulation Research Volume 93(6):e63-e73 September 19, 2003 Copyright © American Heart Association, Inc. All rights reserved.

Figure 2. A, Junctional current recordings from a single experiment at a CL of 1000 ms. Figure 2. A, Junctional current recordings from a single experiment at a CL of 1000 ms. The Ij signals from the first and ensemble average of the last 100 action potentials (SS) from a train of 200 action potentials are illustrated. All Ij recordings have the same characteristic action potential shape. The APD95 for the CL=1000-ms action potential is shown. B, Junctional conductance (gj) calculations from the same cell pair as in panel A. Peak gj was 4.14 nS initially and declined to a steady-state value of 1.61 nS during constant pacing at a CL of 1000 ms. An increase in gj toward initial values is evident during repolarization of cardiac action potential. C, Average normalized junctional conductance (Gj) from 6 experiments at a CL of 1000 ms for the first and ensemble average of the final 100 action potentials (SS) from a train of 200 action potentials. All gj values were normalized to the peak gj of the first action potential in each experiment. The average steady-state Gj declined by 55% during the action potential before beginning its recovery to maximum resting values during phase 3 repolarization. Xianming Lin et al. Circ Res. 2003;93:e63-e73 Copyright © American Heart Association, Inc. All rights reserved.

Figure 3. A, Ten individual Ij traces from a low gj (≤0 Figure 3. A, Ten individual Ij traces from a low gj (≤0.8 nS) experiment demonstrating the high incidence of open channels at the beginning of the CL=1000-ms action potential and lower incidence of the same during the plateau phase. Figure 3. A, Ten individual Ij traces from a low gj (≤0.8 nS) experiment demonstrating the high incidence of open channels at the beginning of the CL=1000-ms action potential and lower incidence of the same during the plateau phase. The ensemble average of the 10 displayed and all 68 Ij traces are also displayed to demonstrate that the temporal sum of several Cx43 gap junction channels reproduces the shape of the Ij responses from macroscopic recordings (Figure 2A). B, gj calculations for the same traces shown in panel A. A maximum of 8 and an average of 5 100-pS channels were initially open with an average of only 2 equivalent channels remaining open during the action potential plateau channel until final repolarization commences. Xianming Lin et al. Circ Res. 2003;93:e63-e73 Copyright © American Heart Association, Inc. All rights reserved.

Figure 4. A, Ensemble-averaged Gj over the last 100 action potentials from 5 to 6 experiments was calculated relative to the APD95 at each CL. Gj declined to a minimum plateau value of 0.37 to 0.50 and recovered to 0.70 to 1.00 of its initial value at the ADP95 for each CL. B, Steady-state Gj−Vj curve for Cx43 obtained from continuous Vj ramps from 0 to 100 mV in 200-ms/mV increments. Figure 4. A, Ensemble-averaged Gj over the last 100 action potentials from 5 to 6 experiments was calculated relative to the APD95 at each CL. Gj declined to a minimum plateau value of 0.37 to 0.50 and recovered to 0.70 to 1.00 of its initial value at the ADP95 for each CL. B, Steady-state Gj−Vj curve for Cx43 obtained from continuous Vj ramps from 0 to 100 mV in 200-ms/mV increments. The solid curved line is the best fit of a Boltzmann distribution (Equation 2, see text) to the data from 4 experiments. Each Vj ramp was repeated 5 times per experiment and the ensemble-averaged Gj from each experiment was pooled to calculate the mean Gj data points (symbols). Xianming Lin et al. Circ Res. 2003;93:e63-e73 Copyright © American Heart Association, Inc. All rights reserved.

Figure 7. A, Average Gj and Vj for the CL=1000-ms action potential in time. Figure 7. A, Average Gj and Vj for the CL=1000-ms action potential in time. The vertically striped bar indicates the first 25 ms of the action potential during which conduction delays and complete conduction block develop. This critical time period for propagation is characterized by a rapid inactivation of Cx43 Gj to 60% of its peak value. Gj declines another 15% to a minimum of 0.45 during the plateau phase. The horizontally striped bar indicates the initial phase of Gj recovery that produces a 20% increase in Gj. This early recovery phase occurs during phase 3 repolarization and closely resembles the relative refractory period of the cardiac ventricular action potential. The late phase of Gj recovery (diagonally striped bar) occurs during final repolarization and is temporally associated with the occurrence of the supernormal period of cardiac excitability. B, Same average Gj values plotted relative to Vj to indicate the regions of Vj where the fast inactivation, initial recovery, and final recovery phases of Cx43 Gj occur. It becomes readily apparent that inactivation is driven by high Vj>100 mV and that complete recovery requires repolarization to Vj <10 mV. Xianming Lin et al. Circ Res. 2003;93:e63-e73 Copyright © American Heart Association, Inc. All rights reserved.

Figure 8. A through F, Thin solid line in each panel indicates the average Gj curve for the indicated CL. The thick solid black line is the modeled Gj curve for each CL according to Equation 13. Figure 8. A through F, Thin solid line in each panel indicates the average Gj curve for the indicated CL. The thick solid black line is the modeled Gj curve for each CL according to Equation 13. The computed output of each Vj-dependent component is illustrated as the long dashed line (Equation 4, G1t+1), the short dashed line (Equation 5, G2t+1), the long dotted line (Equation 9, R1t), and the short dotted line (Equation 10, R2t). The slow inactivation and final recovery components of Gj increase in prominence with decreasing frequencies of stimulation. Recovery of Gj is most rapid at high rates of stimulation (CL=250 ms, panel A). Xianming Lin et al. Circ Res. 2003;93:e63-e73 Copyright © American Heart Association, Inc. All rights reserved.

Figure 1. Voltage command signals applied to cell 1 (V1) and cell 2 (V2) of Cx43-transfected N2A cells to examine the effects of a train of 200 cardiac action potentials on the flow of gap junction current (Ij) and conductance (gj). Figure 1. Voltage command signals applied to cell 1 (V1) and cell 2 (V2) of Cx43-transfected N2A cells to examine the effects of a train of 200 cardiac action potentials on the flow of gap junction current (Ij) and conductance (gj). The transjunctional voltage, Vj=V1−V2. Stimulus cycle lengths were 250, 500, 750, 1000, 1500, and 2000 ms, respectively. Xianming Lin et al. Circ Res. 2003;93:e63-e73 Copyright © American Heart Association, Inc. All rights reserved.

Figure 5. A, Whole-cell 2 currents (I2) in response to a Vj pulse (=ΔV1) applied to cell 1 as indicated. Figure 5. A, Whole-cell 2 currents (I2) in response to a Vj pulse (=ΔV1) applied to cell 1 as indicated. The common holding potential (Vj=0 mV) was −40 mV and the pulse duration was 2.5 seconds. The I2 traces represent unsubtracted values. Ij=−ΔI2 where ΔI2 is the baseline (V2=−40 mV) subtracted value. All I2 recordings are ensemble averages of 5 pulses obtained from the same experiment. The Vj-dependent decay time constants were obtained from the exponential fit (solid curved lines) of each ensemble-averaged trace. B, Reciprocal of the average (mean±SD) Vj-dependent decay time constants from 3 to 12 experiments were well described by an exponential function with a voltage constant of 22.1 mV. 1/τ was assumed to decline to zero with decreasing Vj from an estimated rate of 0.00046 ms−1 at 40 mV (Equation 3, see text). Xianming Lin et al. Circ Res. 2003;93:e63-e73 Copyright © American Heart Association, Inc. All rights reserved.

Figure 6. A, Recovery of Gj was examined using premature stimuli. Figure 6. A, Recovery of Gj was examined using premature stimuli. The first 30 ms of the CL=1000-ms action potential was applied with increasing 10-ms delays from 120 to 190 ms after the onset of the preceding normal CL=1000-ms action potential. Each Ij trace is the ensemble average of 20 sweeps per stimulus delay. B, Average Gj (±SD) calculated from the peak Ij from 6 different delayed extrasystole experiments. The dashed line represents the time course of Gj for the CL=1000-ms action potential (Figures 2C and 4A). C, Time-dependent relaxation of Ij during a fixed Vj step to 10 to 70 mV from Vj≅80 mV achieved during a CL=1000-ms action potential. Again, each Ij trace is the ensemble average of 20 sweeps. Exponential time constants ranged from 30 to 96 ms (see Table 1). D, Average Gj values (n=5) from the end of the action potential waveform in panel C (AP), to the start (Ginst), and the end (Gss) of the indicated 125 ms Vj pulses are compared with the steady-state Gj−Vj curve (dot-dash line) from Figure 4B (Cx43). Xianming Lin et al. Circ Res. 2003;93:e63-e73 Copyright © American Heart Association, Inc. All rights reserved.