SK4 Ca ²⁺ activated K ⁺ channel is a critical player in cardiac pacemaker derived from human embryonic stem cells

Abstract

Proper expression and function of the cardiac pacemaker is a critical feature of heart physiology. Two main mechanisms have been proposed: (i) the "voltage-clock," where the hyperpolarizationactivated funny current I f causes diastolic depolarization that triggers action potential cycling; and (ii) the "Ca 2+ clock," where cyclical release of Ca 2+ from Ca 2+ stores depolarizes the membrane during diastole via activation of the Na + -Ca 2+ exchanger. Nonetheless, these mechanisms remain controversial. Here, we used human embryonic stem cell-derived cardiomyocytes (hESC-CMs) to study their autonomous beating mechanisms. Combined current-and voltage-clamp recordings from the same cell showed the so-called "voltage and Ca 2+ clock" pacemaker mechanisms to operate in a mutually exclusive fashion in different cell populations, but also to coexist in other cells. Blocking the "voltage or Ca 2+ clock" produced a similar depolarization of the maximal diastolic potential (MDP) that culminated by cessation of action potentials, suggesting that they converge to a common pacemaker component. Using patch-clamp recording, realtime PCR, Western blotting, and immunocytochemistry, we identified a previously unrecognized Ca 2+ -activated intermediate K + conductance (IK Ca , KCa3.1, or SK4) in young and old stage-derived hESCCMs. IK Ca inhibition produced MDP depolarization and pacemaker suppression. By shaping the MDP driving force and exquisitely balancing inward currents during diastolic depolarization, IK Ca appears to play a crucial role in human embryonic cardiac automaticity.

Mechanisms underlying the cardiac pacemaker: the role of SK4 calcium-activated potassium channels

“…Interestingly, the depolarizing effect of KB-R7943 on the cell automaticity was previously reported in cardiomyocytes isolated from 8.5 days rodent embryos [167] and in guinea pig SAN cells [179] but the mechanism was not explained. This "I f -independent" pacemaker mechanism was observed in a subset of cells that represented 41% of the hESC-CMs [173] . In the two cell subpopulations, the "membrane clock" and "calcium clock" were operating in a mutually exclusive manner, although the two conductances coexisted in the same cell and the I NCX current density was higher than that of I f at MDP [173] .…”

“…This "I f -independent" pacemaker mechanism was observed in a subset of cells that represented 41% of the hESC-CMs [173] . In the two cell subpopulations, the "membrane clock" and "calcium clock" were operating in a mutually exclusive manner, although the two conductances coexisted in the same cell and the I NCX current density was higher than that of I f at MDP [173] . The ubiquitous presence of I f in all the tested hESC-CMs is also observed in early cardiogenesis [2,3] and confirms the primi-Weisbrod D et al Acta Pharmacologica Sinica npg tive embryonic phenotype of these cells.…”

“…By alternating the current and voltage clamp configurations of the patch clamp technique with a pharmacological approach on the same cell, we found that 32% of the hESC-CM population exhibited a prominent I f pacemaker mechanism. In this subset of cells, the I f blockers ZD7288 or zatebradine strongly decreased the funny current [174] , as monitored under voltage clamp, and led to a bradycardia and a depolarization of the maximum diastolic potential (MDP), which is frequently associated with a cessation of the pacemaker activity [173] . Similar concentrations of zatebradine were previously reported in hESC-CMs [175] or rabbit SAN, and, interestingly, a suppression of the MDP notch could already be observed in those studies but without a clear explanation [176] .…”

“…As hESCCMs usually start to beat spontaneously between the 9th and 12th day after differentiation begins, we focused our work on young cells (d11 to 21), which parallels the early beating cells of the cardiac tube [173] . By alternating the current and voltage clamp configurations of the patch clamp technique with a pharmacological approach on the same cell, we found that 32% of the hESC-CM population exhibited a prominent I f pacemaker mechanism.…”

“…The ubiquitous presence of I f in all the tested hESC-CMs is also observed in early cardiogenesis [2,3] and confirms the primi-Weisbrod D et al Acta Pharmacologica Sinica npg tive embryonic phenotype of these cells. The selection of a privileged pacemaker mechanism does not correlate with the relative current densities of I f and I NCX and remains completely unknown [173] . In the remaining 26% of the hESC-CMs tested, both I f and I NCX blockers decreased the firing rate by less than 50%.…”