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 hyperpolarization-activated funny current If 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, real-time 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 hESC-CMs. 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.