Background— The pacemaker current I_f contributes to spontaneous diastolic depolarization of cardiac autonomic cells. In heterologous expression, HCN channels exhibit a hyperpolarization-activated inward current similar to I_f. However, the links between HCN genes and native I_f are largely inferential, and it remains unknown whether I_f is essential for cardiac pacing.

Methods and Results— To clarify this situation, we generated a GYG_402–404AYA pore mutation of HCN2, which rendered the channel nonfunctional and suppressed wild-type HCN2 in a dominant-negative manner in Chinese hamster ovary cells. In addition, HCN2-AYA suppressed I_HCN4 in a dominant-negative manner when coexpressed with wild-type HCN4, indicating that the 2 isoforms HCN2 and HCN4 are able to coassemble to form heteromultimeric complexes. Given that HCN2 and HCN4 are the dominant HCN mRNA transcripts in neonatal rat ventricle, we expressed HCN2-AYA in neonatal cardiocytes using adenoviral gene transfer to test the effect of HCN suppression on native I_f. I_f density was indeed reduced markedly, from 7.8±1.6 pA/pF (n=13) in control cells to 0.3±0.2 pA/pF (n=11) in HCN2-AYA–infected cells when measured at −130 mV (P<0.001). To probe the effect of HCN on cardiac pacing, we infected spontaneously beating neonatal monolayers with adenoviral vectors expressing wild-type and mutant HCN channels. Infection with HCN2 and HCN4 accelerated the beating rate significantly, to 230.5±8.6 bpm (n=12) and 223.5±12.3 bpm (n=10), respectively, compared with control cultures (83.4±4.5 bpm, n=13, P<0.001). Conversely, HCN2-AYA completely undermined spontaneous pacing of neonatal cardiocytes.

Conclusions— HCN channels are the major molecular component of native I_f and are critical for spontaneous beating of neonatal cardiomyocytes.