Bi-directional flow of the funny current (I_f) during the pacemaking cycle in murine sinoatrial node myocytes

Abstract

Sinoatrial node myocytes (SAMs) act as cardiac pacemaker cells by firing spontaneous action potentials (APs) that initiate each heartbeat. The funny current, If, is critical for the generation of these spontaneous APs; however, its precise role during the pacemaking cycle remains unresolved. We used the AP-clamp technique to quantify If during the cardiac cycle in mouse SAMs. We found that If is persistently active throughout the sinoatrial AP, with surprisingly little voltage-dependent gating. As a consequence, it carries both inward and outward current around its reversal potential of -30 mV. Despite operating at only 2-5% of its maximal conductance, If carries a substantial fraction of both depolarizing and repolarizing net charge movement during the firing cycle. We also show that β-adrenergic receptor stimulation increases the percentage of net depolarizing charge moved by If, consistent with a contribution of If to the fight-or-flight increase in heart rate. These properties were confirmed by heterologously-expressed HCN4 channels and by mathematical models of If. Modelling further suggested that the slow activation and deactivation of the HCN4 isoform underlie the persistent activity of If during the sinoatrial AP. These results establish a new conceptual framework for the role of If in pacemaking, in which it operates at a very small fraction of maximal activation but nevertheless drives membrane potential oscillations in SAMs by providing substantial driving force in both inward and outward directions.

Intracellular Na+ Modulates Pacemaking Activity in Murine Sinoatrial Node Myocytes: An In Silico Analysis

“…We simulated the murine SAM AP using the published model developed by Kharche et al [ 34 ], updated to more closely recapitulate our experimental datasets. First, we adjusted the formulation of several ionic currents based on our measurements of I CaL [ 35 ], transient and steady-state outward K + currents (I to and I sus ) [ 36 ], and I f [ 37 ] ( Figure 1 A). Then, we applied a global optimization method [ 38 ] to scale selected model parameters ( Table A1 ) to match measured AP characteristics ( Figure 1 B and Table A2 ) [ 39 , 40 ].…”

“…Experimental data previously collected by the Proenza laboratory [ 35 , 36 , 37 , 39 ] were used to constrain model reparameterization. Briefly, SAMs were isolated from 2–3 months old male C58BL/6 J mice [ 73 , 74 ], and membrane currents and spontaneous APs were recorded at 35 °C in whole-cell and amphotericin perforated patch configurations, respectively [ 35 , 36 , 39 ].…”

“… (iii) I f . The original I f the formulation of the Kharche et al model was replaced with our recently updated version [ 37 ]. Briefly, we modified and extended the Hodgkin-Huxley type I f model originally present in the Kharche et al framework based on our novel data in murine SAMs describing voltage-dependence of I f availability and activation/deactivation kinetics.…”

“…Briefly, we modified and extended the Hodgkin-Huxley type I f model originally present in the Kharche et al framework based on our novel data in murine SAMs describing voltage-dependence of I f availability and activation/deactivation kinetics. Notably, in our novel formulation, activation and deactivation of I f exhibit both fast and slow kinetics [ 37 ].…”

Speeding Up the Heart? Traditional and New Perspectives on HCN4 Function

“…However, until now this concept could not be validated in vivo and therefore remained controversially discussed. Moreover, it has been shown that, especially in the mouse SAN, the gating kinetics of HCN channels are much too slow to open and close the channels during successive action potentials, because the very high sinus rhythm and thus the firing rate of murine SAN pacemaker cells are markedly faster than the channel kinetics ( Fenske et al, 2011 , 2020 ; Hennis et al, 2021 ; Peters et al, 2021 ). As a result, HCN4-mediated current will manifest mainly as a nearly time-independent but bidirectionally flowing background current during the pacing cycle ( Figure 2E ), strongly arguing against a role for HCN4 in adjusting the slope of SDD and altering HR.…”