Inward rectifier K channel subfamily 2 (Kir2) channels primarily maintain the normal resting membrane potential of cardiomyocytes. At subphysiological extracellular K concentrations or pathological hypokalaemia, human cardiomyocytes show both hyperpolarized and depolarized resting membrane potentials; these depolarized potentials cause cardiac arrhythmia; however, the underlying mechanism is unknown. In the present study, we show that inward rectifier K channel subfamily 2 isoform 1 (Kir2.1) currents non-linearly counterbalance hypokalaemia-induced two pore-domain K channel isoform 1 (K2P1) leak cation currents, reconstituting two levels of resting membrane potential in cardiomyocytes. Under hypokalaemic conditions, both human cardiomyocytes derived from induced pluripotent stem cells with enhanced Kir2.1 expression and mouse HL-1 cardiomyocytes with ectopic expression of K2P1 channels recapitulate two levels of resting membrane potential. These cardiomyocytes display N-shaped current-voltage relationships that cross the voltage axis three times and the first and third zero-current potentials match the two levels of resting membrane potential. Inhibition of K2P1 expression eliminates the phenomenon, indicating contributions of Kir2.1 and K2P1 channels to two levels of resting membrane potential. Second, in Chinese hamster ovary cells that heterologously express the channels, Kir2.1 currents non-linearly counterbalance hypokalaemia-induced K2P1 leak cation currents, yielding the N-shaped current-voltage relationships, causing the resting membrane potential to spontaneously jump from hyperpolarization at the first zero-current potential to depolarization at the third zero-current potential, again recapitulating two levels of resting membrane potential. These findings reveal ionic mechanisms of the two levels of resting membrane potential, demonstrating a previously unknown mechanism for the regulation of excitability, and support the hypothesis that Kir2 currents non-linearly balance inward background cation currents, accounting for two levels of resting membrane potential of human cardiomyocytes.

Background: Biological pacemakers derived from pluripotent stem cell (PSC) have been considered as a potential therapeutic surrogate for sick sinus syndrome. So it is essential to develop highly efficient strategies for enrichment of sinoatrial node-like cells (SANLCs) as seed cells for biological pacemakers. It has been reported that BMP, FGF, and RA signaling pathways are involved in specification of different cardiomyocyte subtypes, pacemaker, ventricular, and atrial cells. We aimed to investigate whether combined modulation of BMP, FGF, and RA signaling pathways could enrich the differentiation of SANLC from human pluripotent stem cell (hiPSC). Methods: During the differentiation process from human induced pluripotent stem cell to cardiomyocyte through small molecule-based temporal modulation of the Wnt signaling pathway, signaling of BMP, FGF, and RA was manipulated at cardiac mesoderm stage. qRT-PCR, immunofluorescence, flow cytometry, and whole cell patch clamp were used to identify the SANLC.

Spontaneous rhythmic action potential or pacemaking activity of pacemaker cells controls rhythmic signaling such as heartbeat. The mechanism underlying the origin of pacemaking activity is not well understood. In this study, we created human embryonic kidney (HEK) 293 cells that show pacemaking activity through heterologous expression of strong inward rectifier K subfamily 2 isoform 1 (Kir2.1) channels, hyperpolarization-activated cyclic nucleotide-gated isoform 2 (HCN2) nonselective cation channels, and voltage-gated Na subfamily 1 isoform 5 or Ca subfamily 3 isoform 1 (Na1.5 or Ca3.1) channels. A range of relative levels of Kir2.1 and HCN2 currents dynamically counterbalance, generating spontaneous rhythmic oscillation of resting membrane potential between -64 and -34 mV and determining oscillation rates. Each oscillation cycle begins with an autodepolarization phase, which slowly proceeds to the threshold potential that activates Na1.5 or Ca3.1 channels and triggers action potential, causing engineered HEK293 cells to exhibit pacemaking activity at a rate of ≤67 beats/min. Engineered HEK293 cells with Kir2.1 and either HCN3 or HCN4 also show the oscillation. Engineered HEK293 cells expressing HCN2 and other Kir2 channels, which lack Kir2.1-like complete inward rectification, do not show the oscillation. Therefore, Kir2.1-like inward rectification-controlled precise and dynamic balances between Kir2 and HCN currents initiate spontaneous rhythmic action potential and form an origin of pacemaking activity; Kir2 and HCN channels play essential roles in pacemaking activity.-Chen, K., Zuo, D., Wang, S.-Y. Chen, H. Kir2 inward rectification-controlled precise and dynamic balances between Kir2 and HCN currents initiate pacemaking activity.