Deep bradycardia and heart block caused by inducible cardiac-specific knockout of the pacemaker channel gene Hcn4

Abstract

Cardiac pacemaking generation and modulation rely on the coordinated activity of several processes. Although a wealth of evidence indicates a relevant role of the I f ("funny," or pacemaker) current, whose molecular constituents are the hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels and particularly HCN4, work with mice where Hcn genes were knocked out, or functionally modified, has challenged this view. However, no previous studies used a cardiac-specific promoter to induce HCN4 ablation in adult mice. We report here that, in an inducible and cardiac-specific HCN4 knockout (ciHCN4-KO) mouse model, ablation of HCN4 consistently leads to progressive development of severe bradycardia (∼50% reduction of original rate) and AV block, eventually leading to heart arrest and death in about 5 d. In vitro analysis of sinoatrial node (SAN) myocytes isolated from ciHCN4-KO mice at the mean time of death revealed a strong reduction of both the I f current (by ∼70%) and of the spontaneous rate (by ∼60%). In agreement with functional results, immunofluorescence and Western blot analysis showed reduced expression of HCN4 protein in SAN tissue and cells. In ciHCN4-KO animals, the residual I f was normally sensitive to β-adrenergic receptor (β-AR) modulation, and the permanence of rate response to β-AR stimulation was observed both in vivo and in vitro. Our data show that cardiac HCN4 channels are essential for normal heart impulse generation and conduction in adult mice and support the notion that dysfunctional HCN4 channels can be a direct cause of rhythm disorders. This work contributes to identifying the molecular mechanism responsible for cardiac pacemaking.funny current | heart rate | sinoatrial node | atrioventricular node | chronotropism T he degree of complexity of the processes involved in pacemaking and their individual contributions is still a hotly debated issue (1, 2). Despite this complexity, and granting the fact that perturbation of any participating mechanism can affect rate, there is evidence for a functional specificity of "funny" (f)-channels in mediating generation and physiological control of pacemaker activity.Native f-channels are encoded by four hyperpolarizationactivated, cyclic nucleotide-gated channel genes (Hcn1-4), of which Hcn4 is by far the most highly expressed in the cardiac pacemaker regions of different species (3-6). HCN4 contributes 80% of total HCN mRNA in rabbit and mouse sinoatrial node (SAN), the remaining 20% being a combination of HCN2 and HCN1, with species-dependent relative abundance (4,5,(7)(8)(9). Evidence for the role of f/HCN4 channels in pacemaking relies on several experimental data (10):i) The "funny" (I f ) current and pacemaker activity are correlated; i.e., functional expression of f-channels is restricted to pacemaking regions [SAN, atrioventricular node (AVN), and the ventricular conduction system], demonstrating a commitment to regulation of pacemaker activity. This correlation exists not only in the adult but also throughout development, such that ...

Popeye domain containing proteins are essential for stress-mediated modulation of cardiac pacemaking in mice

“…Several ion channels also directly bind cyclic nucleotides, which modulates the activation characteristics or gating properties of these channels; therefore, cAMP regulates membrane potential and excitability of cells. Although HCN4 plays an important role in cardiac pacemaker function (51,52), despite its ability to directly bind cAMP, its role in stress adaptation of the cardiac pacemaker remains a matter of debate (51,(53)(54)(55). Therefore, our present identification of Popdc proteins as a class of membrane-localized cAMP binding proteins in sinus node cells and our finding that genetic loss resulted in stress-induced bradycardia suggest that Popdc proteins are part of the molecular network involved in cardiac pacemaker regulation in response to adrenergic stimulation.…”

Embryonic Stem Cell–Derived CD166 + Precursors Develop Into Fully Functional Sinoatrial-Like Cells

“…11,12 Here, we have shown that CD166-expressing cells also express HCN4 (Figure 1), a protein that specifically delineates the SAN region during embryogenesis 14,15,41 and in adulthood. [42][43][44][45] HCN4 is also fundamental for sinus node function because its absence is incompatible with life 29,30 and mutations in its sequence can cause inherited rhythm disturbances. 46 Here, we show that freshly selected CD166 + cells, but not CD166 − cells, express high levels of HCN4 mRNA; at 1 day after sorting, most of the cells express the HCN4 proteins (Online Figure IV).…”

“…Because HCN channels are critical for pacemaker activity in both embryonic and adult SAN, 29,30 and because their ectopic expression induces repetitive spontaneous activity in the ventricle, 31 we evaluated the expression of various HCN subunits at the protein level after 1 or 3 weeks in culture ( Figure 5). In agreement with qRT-PCR data, we found that after 1 week in culture, CD166 + cells expressed both HCN1 ( Figure 5B) and HCN4 ( Figure 5E) together with the cardiac markers α-actinin (α-act; Figure 5A and 5D), whereas HCN2, the main ventricular isoform, 32 could not be detected in α-act-positive cells ( Figure 5G and 5H).…”

New Therapeutic Targets in Cardiology

“…49 In some nonconstitutive knockout models, only mild effects on cardiac automaticity were observed with no alterations of HR control. 50,51 Ablation of HCN4 in an inducible and cardiac-specific HCN4 knockout mouse model 52 led to progressive development of severe bradycardia (≈50% reduction of the original HR) and atrioventricular block, accompanied by cardiac arrest and death. In vitro analysis of SAN myocytes isolated from these mice revealed strong reductions of both the I f current (by ≈70%) and spontaneous HR generation (by ≈60%).…”