Funny Current and Cardiac Rhythm: Insights from HCN Knockout and Transgenic Mouse Models

Abstract

In the adult animal the sinoatrial node (SAN) rhythmically generates a depolarizing wave that propagates to the rest of the heart. However, the SAN is more than a simple clock; it is a clock that adjusts its pace according to the metabolic requirements of the organism. The Hyperpolarization-activated Cyclic Nucleotide-gated channels (HCN1-4) are the structural component of the funny (I(f)) channels; in the SAN the I(f) current is the main driving electrical force of the diastolic depolarization and the HCN4 is the most abundant isoform. The generation of HCN KO and transgenic mouse models has advanced the understanding of the role of these channels in cardiac excitability. The HCN4 KO models that were first developed allowed either global or cardiac-specific constitutive ablation of HCN4 channels, and resulted in embryonic lethality. A further progress was made with the development of three separate inducible HCN4 KO models; in one model KO was induced globally in the entire organism, in a second, ablation occurred only in HCN4-expressing cells, and finally in a third model KO was confined to cardiac cells. Unexpectedly, the three models yielded different results; similarities and differences among these models will be presented and discussed. The functional effects of HCN2 and HCN3 knockout models and transgenic HCN4 mouse models will also be discussed. In conclusion, HCN KO/transgenic models have allowed to evaluate the functional role of the I(f) currents in intact animals as well as in single SAN cells isolated from the same animals. This opportunity is therefore unique since it allows (1) to verify the contribution of specific HCN isoforms to cardiac activity in intact animals, and (2) to compare these results to those obtained in single cell experiments. These combined studies were not possible prior to the development of KO models. Finally, these models represent critical tools to improve our understanding of the molecular basis of some inheritable arrhythmic human pathologies.

Keywords: HCN KO mouse models; cardiac pacemaking; sinoatrial node.

Figure 1

(A) Top, left: structure of the floxed HCN4 gene carrying the loxP sites (triangles) in the introns flanking exon 2 of the inducible cardiac-specific HCN4 KO mouse model. Top, right: structure of the wild type HCN4 protein isoform. Bottom, left: HCN4 gene structure after the Cre-induced recombination; the gene is now lacking exon 2. Bottom, right: Removal of exon 2 eliminates the first four transemembrane segments and generates a frame-shift leading to an early stop-codon 3 residues downstream to the recombination site. The hypothetical resulting protein should therefore be formed by the N-terminus and by a small portion of the S1 segment. No data on the presence/absence of both transcript and protein are available (B) Bottom: Mean I_f current densities measured during hyperpolarization steps at the indicated potentials (holding potential −35 mV) from SAN cells isolated from cardiac-specific HCN4 KO mice before (dashed bars) and 5 days after (empty bars) KO induction (tamoxifen treatment). Top: Representative I_f traces recorded at the same potentials.

Figure 2

(A) Top: Representative telemetric ECG traces recorded from freely moving mice before (left) and after (right) cardiac-specific HCN4 ablation (five tamoxifen injections). Bottom: Sample action potentials recorded from SAN cells isolated before (left) and after (right) the knockout. (B) Bar-graph showing normalized telemetric heart rates of control (left) and KO (middle left) mice recorded before (day 0) and during the Tam-procedure (day 2 and day 5). The normalized spontaneous activity (middle right) and the I_f current conductance (right) of SAN cells isolated from KO mice in the same days. Clearly the induction of the knockout decreases the heart rates of freely moving animals, the spontaneous activity of isolated SAN cells, and the I_f current conductance (gf) in a similar manner. Only a minor heart rate reduction was observed in control animals undergoing the same treatment.