Dominant-negative suppression of HCN channels markedly reduces the pacemaker current If and undermines spontaneous beating of neonatal cardiomyocytes

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(402-404)AYA 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.