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(Circulation. 2003;107:485.)
© 2003 American Heart Association, Inc.

Basic Science Reports

Dominant-Negative Suppression of HCN Channels Markedly Reduces the Native Pacemaker Current I_f and Undermines Spontaneous Beating of Neonatal Cardiomyocytes

Fikret Er, MD; Robert Larbig; Andreas Ludwig, MD; Martin Biel, MD; Franz Hofmann, MD; Dirk J. Beuckelmann, MD; Uta C. Hoppe, MD

From the Department of Medicine III, University of Cologne (F.E., R.L., D.J.B., U.C.H.); the Institut für Pharmakologie und Toxikologie, Technische Universität München (A.L., F.H.); and the Department of Pharmacy, Zentrum für Pharmaforschung, Ludwig-Maximilians-Universität München (M.B.), Germany.

Correspondence to Uta C. Hoppe, MD, Department of Medicine III, University of Cologne, Joseph-Stelzmann-Straße 9, 50924 Cologne, Germany. E-mail uta.hoppe{at}uni-koeln.de

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.

Key Words: ion channels • pacemakers • viruses • gene therapy • electrophysiology

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