Dynamic model for ventricular junctional conductance during the cardiac action potential

Abstract

The ventricular action potential was applied to paired neonatal murine ventricular myocytes in the dual whole cell configuration. During peak action potential voltages >100 mV, junctional conductance (g(j)) declined by 50%. This transjunctional voltage (V(j))-dependent inactivation exhibited two time constants that became progressively faster with increasing V(j). G(j) returned to initial peak values during action potential repolarization and even exceeded peak g(j) values during the final 5% of repolarization. This facilitation of g(j) was observed <30 mV during linearly decreasing V(j) ramps. The same behavior was observed in ensemble averages of individual gap junction channels with unitary conductances of 100 pS or lower. Immunohistochemical fluorescent micrographs and immunoblots detect prominent amounts of connexin (Cx)43 and lesser amounts of Cx40 and Cx45 proteins in cultured ventricular myocytes. The time dependence of the g(j) curves and channel conductances are consistent with the properties of predominantly homomeric Cx43 gap junction channels. A mathematical model depicting two inactivation and two recovery phases accurately predicts the ventricular g(j) curves at different rates of stimulation and repolarization. Functional differences are apparent between ventricular myocytes and Cx43-transfected N2a cell gap junctions that may result from posttranslational modification. These observations suggest that gap junctions may play a role in the development of conduction block and the genesis and propagation of triggered arrhythmias under conditions of slowed conduction (<10 cm/s).

Fig. 1

Voltage-clamp protocol for steady-state inactivation and recovery curves. The whole cell currents representing the negative junctional current signal (top) and the whole cell currents and command voltages for the partner cell (middle and bottom) during one run of the 200 ms/mV, ± 120-mV voltage ramp applied to ventricular myocytes are shown. The linear slope of the current (I)-voltage (V) relationship at low voltages was used to normalize the data from different experiments. The linear slope conductances from 0 to ±20 mV were 6.20 and 6.95 nS for traces 1 and 2 during the rising phase of the transjunctional voltage (V_j) ramp and were 10.50 and 10.30 nS during the returning phase, indicative of a facilitation of junctional conductance (g_j) during the recovery process.

Fig. 2

Fluorescent immunolocalization of endogenous murine connexin (Cx) expression. Immunohistochemical localization of endogenous Cx40 (A), Cx45 (D), or Cx43 (B and E) expression and colocalization of Cx40 with Cx43 (C) or Cx45 (F) in paired ventricular myocytes after 2 days in culture are shown. Only trace amounts of Cx40 and Cx45 are evident in neonatal murine ventricular myocytes cultured at low density for 48 h to mimic experimental electrophysiological conditions. Negative controls without overnight incubation with the specific Cx antibody revealed negligible background fluorescence (data not shown). G: immunoblot analysis of Cx43, Cx40, and Cx45 in cultured cells or fresh frozen ventricular tissue from neonatal mice. Whole cell lysates were loaded in the following order: lane 1, N2a untransfected cells (for Cx43 detection 10 μg protein, for Cx40 or Cx45 detection 100 μg protein); lane 2, Cx-expressing cells (for Cx43 detection 10 μg protein from N2a cells stably transfected with rat Cx43; for Cx40 detection 100 μg protein from N2a cells stably transfected with rat Cx40; for Cx45 detection 200 μg protein from HEK293 cells); lane 3, cultured ventricular myocytes (for Cx43 detection 10 μg protein, for Cx40 or Cx45 detection 50 μg protein); lane 4, ventricular tissue from neonatal mice (for Cx43 detection 10 μg protein; for Cx40 or Cx45 detection 100 μg protein). Samples were resolved by SDS-PAGE, transferred to membranes, and blotted with anticonnexin antibodies as described in Materials and Methods.

Fig. 3

Junctional currents and conductance during the ventricular action potential. A: junctional current (I_j) recorded as − ΔI₂ during application of the ventricular action potential for the first beat (AP #1) and the average of the last 100 beats (AP #SS) at a rate of 1 beat/s for 200 s. B: calculated g_j during this same experiment using the actual applied V_j (Eq. 1, see Materials and Methods). C: normalized g_j from 7 experiments. The g_j was normalized to the peak value of g_j for each ventricular myocyte cell pair during the train of 200 action potentials. D: G_j values of ventricular cardiac gap junctions after pacing at 6 different cycle lengths (CL) of stimulation. On average, g_j inactivates by 40–50% and recovers completely during phase 3 and phase 4 of the action potential. Some facilitation (increase in g_j above peak values) of g_j is evident in ventricular myocytes during the late phases of repolarization. E: single sweep depicting single gap junction channel responses to the ventricular action potential. The averaged I_j trace from all 200 sweeps closely resembles the macroscopic recordings shown in C. F: corresponding g_j calculations for the I_j traces shown in E. This indicates that the basis for the time-dependent changes in g_j during the action potential are the result of the V_j-dependent gating of individual gap junction channels.

Fig. 4

Steady-state V_j-dependent inactivation and recovery conductance curves. A and D: average I_j in response to a ±120 mV, 200 ms/mV V_j ramp during the increasing and decreasing V_j phases for ventricular myocyte (A) and Cx43-N2a (D) cell gap junctions. B and E: steady-state G_j-V_j ventricular myocyte (B) and Cx43 (E) curves during the inactivation (increasing V_j) phase of the V_j ramp. C and F: steady-state G_j-V_j curves for ventricular myocytes (C) and Cx43 (F) gap junctions during the recovery (decreasing V_j) phase do not follow the same path as inactivation. The Boltzmann curves (solid line) for each were determined using Eq. 2 (see Materials and Methods). The parameter values are provided in Table 1.

Fig. 5

V_j-dependent kinetics of ventricular G_j inactivation. A: unsubtracted whole cell currents during a voltage clamp step to the indicated V_j value from a single ventricular myocyte cell pair. The displayed current traces are the ensemble average of 5 V_j pulses. The decay time constants (τ_decay) were determined from exponential fits of the first second of the 2.5-s duration pulse. B: inactivation rate (1/τ_decay) was plotted as a function of V_j for N experiments (indicated in parentheses). The exponential fit of the rate constants reveals an e-fold increase in the rate of inactivation for every 20.3-mV increase in V_j.

Fig. 6

Effect of prolonged repolarization on the recovery process of ventricular G_j. A: average V_j from 7 experiments indicating the extension of the repolarization phase of the CL = 1,000 ms ventricular action potential by increasing the time step (dt) from 1 to 5 ms, as indicated. B: average I_j from the same experiments in response to a train of 20 action potentials/experiment. C: time-dependent G_j curves for the same experiments. D: same G_j curves as in C plotted as a function of V_j, as depicted in A. G_j increases with increasing dt during phase 3 and 4 repolarization. There is no evidence for a V_j-dependent recovery time constant, since the different curves demonstrate convergence toward the dt = 5 ms curve at all voltages.

Fig. 7

Model of ventricular G_j at different cycle lengths of stimulation and rates of repolarization. The average time-dependent G_j curve at each CL, as shown in Fig. 3D, was modeled according to Eq. 11 (see Appendix) for 250 (A), 500 (B), 2,000 (C), and 1,000 (D) ms CL. Parameter values for Eqs. 4, 5, 9, and 10 used to define the 2 inactivation ( $G_1^{t+1}$ and $G_2^{t+1}$) and recovery ( ${\text{R}}_1^{\text{t}+1}$ and ${\text{R}}_2^{\text{t}+1}$) components (see Appendix) are provided in Table 2. The average time-dependent G_j curves shown in Fig. 6C were modeled using Eq. 11, and the results are displayed graphically for the 1.5-ms (E) and 5.0-ms (F) dt applied to phase 3 and early phase 4 of the CL = 1,000 ms ventricular action potential. The values of the individual inactivation and recovery components for all dt intervals are listed in Table 3.