HCN4 Protein Research Articles

Enhancement of Dorsal Hippocampal Activity by Knockdown of HCN1 Channels Leads to Anxiolytic- and Antidepressant-like Behaviors

The hippocampus is an integral brain region for affective disorders. TRIP8b knockout mice lacking functional HCN channels as well as both HCN1 and HCN2 knockout mice have been shown to display antidepressant-like behaviors. The mechanisms or brain regions involved in these alterations in behavior, however, are not clear. We developed a lentiviral shRNA system to examine whether knockdown of HCN1 protein in the dorsal hippocampal CA1 region is sufficient to produce antidepressant-like effects. We found that knockdown of HCN1 channels increased cellular excitability and resulted in physiological changes consistent with a reduction of I(h). Rats infused with lentiviral shRNA-HCN1 in the dorsal hippocampal CA1 region displayed antidepressant- and anxiolytic-like behaviors associated with widespread enhancement of hippocampal activity and upregulation of BDNF-mTOR signaling pathways. Our results suggest that HCN1 protein could be a potential target for treatment of anxiety and depression disorders.

Distinct perinatal features of the hyperpolarization-activated non-selective cation current Ih in the rat cortical plate

BackgroundDuring neocortical development, multiple voltage- and ligand-gated ion channels are differentially expressed in neurons thereby shaping their intrinsic electrical properties. One of these voltage-gated ion channels, the hyperpolarization-activated cyclic nucleotide-gated (HCN) channel and its current Ih, is an important regulator of neuronal excitability. Thus far, studies on an early Ih appearance in rodent neocortex are missing or conflicting. Therefore, we focused our study on perinatal neocortical Ih and its properties.ResultsIn the perinatal rat neocortex we observed a rapid increase in the number of neurons exhibiting Ih. Perinatal Ih had unique properties: first, a pronounced cAMP sensitivity resulting in a marked shift of the voltage sufficient for half-maximum activation of the current towards depolarized voltages and second, an up to 10 times slower deactivation at physiological membrane potentials when compared to the one at postnatal day 30. The combination of these features was sufficient to suppress membrane resonance in our in silico and in vitro experiments. Although all four HCN subunits were present on the mRNA level we only detected HCN4, HCN3 and HCN1 on the protein level at P0. HCN1 protein at P0, however, appeared incompletely processed. At P30 glycosilated HCN1 and HCN2 dominated. By in silico simulations and heterologous co-expression experiments of a ‘slow’ and a ‘fast’ Ih conducting HCN channel subunit in HEK293 cells, we mimicked most characteristics of the native current, pointing to a functional combination of subunit homo- or heteromeres.ConclusionTaken together, these data indicate a HCN subunit shift initiated in the first 24 hours after birth and implicate a prominent perinatal role of the phylogenetically older HCN3 and/or HCN4 subunits in the developing neocortex.

Expression of HCNl subunits is age-dependently regulated in developing hippocampus of rats

Hyperpolarization-activated, cyclic nucleotide-gated cation channels (HCN) encode hyperþolarization-activated cation current (Ih), playing regulatory roles in resting membrane properties, synaptic integration, and intrinsic exctabilities of hippocampal neurons. Expression patterns of HCN subtypes seem to be quietty variable to excitatory conditions of neurons, even though it has been previously observed that expression levels of HCN1, 2 and 4 are differentially regulated during developmental period. In the present study, we investigated expression patterns of HCN1 during an early developmental period within postnatal 3 weeks by using Western blot analysis. Inconsistent with previous reports, HCN1 expression was highest in neonatal and initial postnatal perlods, while neurons in postnatal third week revealed lower levels. This result indicates that HCN1 proteins are age dependently decreased during first postnatal 3 weeks. This finding suggests that CA1 neurons of early developmental hippocampi may have specific neuroprotective mechanisms correlated with HCN1 subunit against hyperexcitability or excitotoxic damages.

Bone marrow‐derived multipotent adult progenitor cells are capable of functional sinus node myocytes differentiation

Although recent studies of biological pacemaker are focused on overexpression of bioengineered ion channels and genetically modified stem cells. However, short‐term expression of gene and unkown bio‐safety limit the application of current biological pacemakers. A logical approach to recreate an ideal cellular microenvironment in Petri dishes is to hand the task over to differentiated cells. The aim of the present study is to investigate if multipotent adult progenitor cells(MAPCs) can be induced into pacemaker‐like cells by co‐culture assays. After 1 week co‐culture with neonatal sinus node myocytes, MAPCs from bone marrow of rat expressed HCN2 and HCN4 mRNA and proteins until 4 week. HCN4 mRNA was more abundant than HCN2. The Kir2.1 and Kir2.2 that are responsible for inward‐rectifier K+ channels were decreased with culture time. The co‐cultured MAPCs showed sinus node myocytes‐like action potentials and drived quiescence cardiomyocytes to beat. These results suggest that MAPCs could differentiate towards functional sinus node myocytes when co‐cultured with neonatal sinus node myocytes.

Chronic inflammatory pain is associated with increased excitability and hyperpolarization-activated current (Ih) in C- but not Aδ-nociceptors

Inflammatory pain hypersensitivity results partly from hyperexcitability of nociceptive (damage-sensing) dorsal root ganglion (DRG) neurons innervating inflamed tissue. However, most of the evidence for this is derived from experiments using acute inflammatory states. Herein, we used several approaches to examine the impact of chronic or persistent inflammation on the excitability of nociceptive DRG neurons and on their expression of Ih and the underlying hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, which regulate neuronal excitability. Using in vivo intracellular recordings of somatic action potentials from L4/L5 DRG neurons in normal rats and rats with hindlimb inflammation induced by complete Freund’s adjuvant (CFA), we demonstrate increased excitability of C- but not Aδ-nociceptors, 5 to 7days after CFA. This included an afterdischarge response to noxious pinch, which may contribute to inflammatory mechanohyperalgesia, and increased incidence of spontaneous activity (SA) and decreased electrical thresholds, which are likely to contribute to spontaneous pain and nociceptor sensitization, respectively. We also show, using voltage clamp in vivo, immunohistochemistry and behavioral assays that (1) the inflammation-induced nociceptor hyperexcitability is associated, in C- but not Aδ-nociceptors, with increases in the mean Ih amplitude/density and in the proportion of Ih expressing neurons, (2) increased proportion of small DRG neurons (mainly IB4-negative) expressing HCN2 but not HCN1 or HCN3 channel protein, (3) increased HCN2- immunoreactivity in the spinal dorsal horn, and (4) attenuation of inflammatory mechanoallodynia with the selective Ih antagonist, ZD7288. Taken together, the findings suggest that C- but not Aδ-nociceptors sustain chronic inflammatory pain and that Ih/HCN2 channels contribute to inflammation-induced C-nociceptor hyperexcitability.

Enhanced Embryonic Stem Cell Differentiation to Cardiac Pacemaker Cells by Transduction with a Single Transcription Factor

Background: Embryonic stem cells (ESCs) can spontaneously differentiate into heterogeneous aggregates of cardiomyocytes with atrial, ventricular, and pacemaker properties. We sought to bias the random cardiogenesis of ESCs toward pacemaker cardiomyocytes. Shox2 is an embryonic transcription factor essential for the patterning of pacemaker cells in the sinoatrial node (SAN). We hypothesized that Shox2 overexpression may tilt and dominate ESCs’ developmental program toward pacemaker myocytes.Methods: Mouse ESCs were transduced with an adenoviral vector expressing Shox2 or a control gene. Standard embryoid body (EB) method was used for differentiation.Pooled data are n≥3 with p 6-fold in Shox2-EBs compared to control at the early and late stages of differentiation. HCN4 protein level was up-regulated at all differentiation stages, with heightened cell surface expression in Shox2-EBs. Action potentials are mainly propagated by connexin(Cx)45 but not Cx43 in the SAN, which is mirrored in the atrial/ventricular myocardium. Shox2-overexpression led to an increase in the transcript (∼1.5-fold) and protein (∼3-fold) levels of Cx45. In contrast, Cx43 transcript and protein levels were reduced in Shox2-EBs by 33±5% and 30±10%, respectively, compared to control.Conclusion: Shox2 overexpression singularly biased ESC differentiation toward more pacemaker cells. Increased expression of NCX1, HCN4, Cx45, and down-regulation of Cx43 are all hallmarks of SA nodal cell biology. The data provide a novel and efficient platform to develop biological pacemakers from pluripotent cells.

The hyperpolarization-activated cyclic nucleotide-gated HCN2 channel transports ammonium in the distal nephron

Recent studies have identified Rhesus proteins as important molecules for ammonia transport in acid-secreting intercalated cells in the distal nephron. Here, we provide evidence for an additional molecule that can mediate NH3/NH4 excretion, the subtype 2 of the hyperpolarization-activated cyclic nucleotide-gated channel family (HCN2), in collecting ducts in rat renal cortex and medulla. Chronic metabolic acidosis in rats did not alter HCN2 protein expression but downregulated the relative abundance of HCN2 mRNA. Its cDNA was identical to the homolog from the brain and the protein was post-translationally modified by N-type glycosylation. Electrophysiological recordings in Xenopus oocytes injected with HCN2 cRNA found that potassium was transported better than ammonium, each of which was transported significantly better than sodium, criteria that are compatible with a role for HCN2 in ammonium transport. In microperfused rat outer medullary collecting duct segments, the initial rate of acidification, upon exposure to a basolateral ammonium chloride pulse, was higher in intercalated than in principal cells. A specific inhibitor of HCN2 (ZD7288) decreased acidification only in intercalated cells from control rats. In rats with chronic metabolic acidosis, the rate of acidification doubled in both intercalated and principal cells; however, ZD7288 had no significant inhibitory effect. Thus, HCN2 is a basolateral ammonium transport pathway of intercalated cells and may contribute to the renal regulation of body pH under basal conditions.

Colocalization of hyperpolarization-activated, cyclic nucleotide-gated channel subunits in rat retinal ganglion cells.

The current-passing pore of mammalian hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels is formed by subunit isoforms denoted HCN1-4. In various brain areas, antibodies directed against multiple isoforms bind to single neurons, and the current (I(h)) passed during hyperpolarizations differs from that of heterologously expressed homomeric channels. By contrast, retinal rod, cone, and bipolar cells appear to use homomeric HCN channels. Here, we assess the generality of this pattern by examining HCN1 and HCN4 immunoreactivity in rat retinal ganglion cells, measuring I(h) in dissociated cells, and testing whether HCN1 and HCN4 proteins coimmunoprecipitate. Nearly half of the ganglion cells in whole-mounted retinae bound antibodies against both isoforms. Consistent with colocalization and physical association, 8-bromo-cAMP shifted the voltage sensitivity of I(h) less than that of HCN4 channels and more than that of HCN1 channels, and HCN1 coimmunoprecipitated with HCN4 from membrane fraction proteins. Finally, the immunopositive somata ranged in diameter from the smallest to the largest in rat retina, the dendrites of immunopositive cells arborized at various levels of the inner plexiform layer and over fields of different diameters, and I(h) activated with similar kinetics and proportions of fast and slow components in small, medium, and large somata. These results show that different HCN subunits colocalize in single retinal ganglion cells, identify a subunit that can reconcile native I(h) properties with the previously reported presence of HCN4 in these cells, and indicate that I(h) is biophysically similar in morphologically diverse retinal ganglion cells and differs from I(h) in rods, cones, and bipolar cells.

Spironolactone diminishes spontaneous ventricular premature beats by reducing HCN4 protein expression in rats with myocardial infarction

Hyperpolarization-activated current (If) is the major ionic current contributing to the spontaneous diastolic depolarization of cardiac sinus node pacemaker cells. It is mediated by hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels. However, several observations support a potential role of HCN channels in the arrhythmogenesis of working myocardium under pathological conditions. Spironolactone, a classic aldosterone blocker, has been proved to prevent spontaneous ventricular arrhythmias after myocardial infarction (MI). Here, we examined the effect of spironolactone on the expression of HCN channels and ventricular premature beats (VPBs) using a rat MI model. Sprague-Dawley rats were divided into a sham-operated group and MI groups treated with intragastric administration of saline or spironolactone (80 µg/kg/day) for 7 days immediately after ligation of the left coronary artery. Compared to the sham group, HCN2 and HCN4 protein levels were increased in MI rats. Treatment with spironolactone prevented the MI-induced increase of HCN4 protein levels (1.47 ± 0.16 vs. 1.81 ± 0.21, P<0.05). MI rats exhibited a marked increase of VPBs compared to the sham group (104 ± 17 vs. 3 ± 1 VPBs/h, P < 0.05). This the increase was reduced by spironolactone (55 ± 14 vs. 104 ± 17 VPBs/h, P < 0.05). Moreover, If current inhibitor (ivabradine, 0.5 mg/kg) further decreased the occurrence of VPBs in the control and spironolactone groups to the same level (54 ± 13 vs. 49 ± 8 VPBs/h, P > 0.05). In conclusion, spironolactone may prevent ischemia-induced VPBs by reducing HCN4 protein expression to basal levels.

CSPE Young Investigator Award Session

CSPE Young Investigator Award Session

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

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.

E0097 Canine marrow mesenchymal stem cells with lentiviral mHCN4 gene transfer create cardiac pacemakers

The purpose Research on biological pacemakers for the heart has so far mainly focused on short-term gene and cell therapies. To develop a clinically relevant biological pacemaker, long-term function is...

Dynamic changes in HCN2, HCN4, KCNE1, and KCNE2 expression in ventricular cells from acute myocardial infarction rat hearts

Aims: To investigate dynamic changes in the expression of HCN2, HCN4, as well as KCNE1, and KCNE2 mRNA and protein levels in ventricular cells from acute myocardial infarction (AMI) rat hearts. Main methods: An AMI model was induced by ligating the left anterior descending coronary artery (LAD) of Sprague–Dawley rats. The rats were randomly divided into four experimental groups: 24-hour (24 h) post-AMI, 1-week (1w) post-AMI, 2-week (2w) post-AMI, and 4-week (4w) post-AMI; sham-operated control rat groups were established in parallel for each time point. HCN2, HCN4, KCNE1, and KCNE2 mRNA and protein levels were measured by reverse transcription-polymerase chain reaction (RT-PCR) and by immunohistochemistry and Western blot, respectively. Key findings: Ventricular arrhythmias occurred in all the post-AMI groups, particularly in the 1w and 2w post-AMI groups. Although HCN2, HCN4, KCNE1, and KCNE2 genes were expressed in the left ventricular myocardium of sham-operated control rats, their expression increased in rat ischemic left ventricular myocardium, with dynamic changes in expression observed 4 weeks after AMI. HCN2, HCN4, and KCNE2 protein levels were highest at 1w and KCNE2 protein levels peaked at 2w post-AMI. Significance: The expression of the HCN2, HCN4, as well as KCNE1, and KCNE2 genes in ventricular cells from AMI rat hearts underwent dynamic changes, reaching peak levels at 1 or 2 weeks post-AMI. The increased expression maybe related to ventricular arrhythmogenesis after AMI.

Isoform- and Species-Specific Proteolysis of Cardiac Pacemaker Channels

Proteolysis of cardiac pacemaker channels affects biophysical properties of functional channels. Hyperpolarization-activated channels HCN2 and HCN4 can form homomeric or heteromeric functional pacemaker channels in cardiac ventricles. Employing Western blot and immunoprecipitation techniques with antibodies against N- or C- terminus of HCN2 or HCN4, respectively, we investigated protein expression patterns of endogenous HCN2 and HCN4 in cardiac ventricles of small (mouse, rat) and large (sheep, canine) animals and human. Using an antibody against N-terminus of HCN2, more full length protein at 100kD and less cleaved bands around 50kD were detected in small than in large animals. An additional cleaved band around 60kD was exclusively expressed in human. HCN2 C-terminal antibody could not detect any full length protein in all species tested. A 75kD cleaved band was detected in mice, rat, canine and substantially higher in sheep heart ventricles. A 60kD band was observed in human only. Using an N-terminal HCN4 antibody, the full length protein signals (at 160kD) were present in sheep and canine only. The cleaved bands near 100kD predominated in small animals but absent in large animals. With a C-terminal HCN4 antibody, the full length protein was observed in mice, barely detectable in rat, and clearly present in sheep, canine and human. A cleaved band around 100kD predominated in all animals. A minor cleaved band around 50kD appeared in all tested species except human. Overall, there was less HCN2 and more HCN4 proteolysis in small than in large animal cardiac ventricles. Endogenous myocardial HCN2 and HCN4 underwent intensive proteolysis at both N- and C- termini in an isoform- and species-specific pattern. In conclusion, results obtained from HCN2 and HCN4 protein expression in small animals may not be directly applied to large ones including human.

Proteolytic Processing of HCN2 and Co-assembly with HCN4 in the Generation of Cardiac Pacemaker Channels

In sino-atrial and atrio-ventricular nodal cells, hyperpolarization-activated cyclic nucleotide-gated (HCN) inward current carrying cationic channels, I(f), are expressed that contribute importantly to the diastolic depolarization critical for cardiac pacemaker activity. Although previous studies have demonstrated myocardial expression of both the HCN2 and HCN4 subunits, the specific roles of these subunits in the generation of functional myocardial I(f) channels remain unclear. To explore the molecular compositions of functional cardiac I(f) channels, antibodies targeted against specific C- and N-terminal sequences in HCN2 and HCN4 were exploited to examine HCN2 and HCN4 subunit expression in adult (mouse) heart and to immunoprecipitate endogenous HCN-encoded cardiac I(f) channel complexes. Western blot experiments revealed that although the full-length HCN2 (105 kDa) and HCN4 (160 kDa) proteins are readily detected in transiently transfected HEK-293 cells and in adult (mouse) brain, the molecular mass of the HCN2 protein in the myocardium is approximately 60 kDa. In addition, the myocardial 60-kDa HCN2 protein lacks the C terminus, which contains the cAMP binding domain. In heterologous cells, the C-terminal-truncated HCN2 protein co-assembles with HCN4 to form functional heteromeric HCN channels, which activate faster than homomeric HCN2 or homomeric HCN4 channels, and display properties similar to endogenous myocardial I(f) channels Taken together, these results suggest that functional myocardial I(f) channels reflect the heteromeric assembly of HCN2 and HCN4 subunits and further that the HCN4 subunit underlies the cAMP-mediated regulation of cardiac I(f) channels.

Distinct regulation of cardiac If current via thyroid receptors alpha1 and beta1

Thyroid hormone (TH) markedly modulates cardiovascular function and heart rate. The pacemaker current I(f) and encoding hyperpolarization-activated cation (HCN) genes have been identified as TH targets. To analyze the specific contribution and functional significance of thyroid receptor isoforms responsible for HCN gene transactivation, we generated transgenic neonatal rat cardiomyocytes with adenovirus-mediated overexpression of the thyroid receptors alpha1 (TR alpha 1) and beta1 (TR beta 1), and analyzed native I(f) current and expression levels of the underlying molecular components HCN2 and HCN4. Initial results revealed that spontaneous beating activity was higher in TR alpha 1- and lower in TR beta 1-expressing cardiomyocytes. This was associated with accelerated depolarization velocity and abbreviated action potential duration in cells overexpressing TR alpha 1, while TR beta 1 suppressed phase 4 depolarization and prolonged action potentials. Consistently, TR alpha 1-infected myocytes exhibited larger I(f) current densities along with increased HCN2 and HCN4 mRNA and protein levels. In contrast, HCN2 gene expression was not significantly affected by TR beta 1. TR beta 1 exclusively suppressed HCN4 transcription. T3 application led to significant effects only in controls and TR alpha 1-infected cardiomyocytes; whereas, no ligand-dependent actions were observed in TR beta 1-expressing neonatal cardiomyocytes. Our results demonstrate that TR alpha 1 and TR beta 1 divergently regulate cardiac pacing activity. TH-induced positive chronotropic effects are likely to be mediated by TR alpha 1 through enhanced expression of I(f) pacemaker current and its underlying genes.

Activity-dependent regulation of HCN1 protein in cortical neurons

Homeostasis of neuronal activity is crucial to neuronal physiology. In dendrites, hyperpolarization-activated cyclic nucleotide-gated channel (HCN) 1 is considered to play critical roles in this process. While electrophysiological studies have demonstrated the dynamic modulation of I h current mediated by HCN1 proteins, little is known about the underlying molecular and cellular mechanisms. In this study, we utilized cortical cultured neurons and biochemical methods to identify molecular and cellular mechanisms that mediate the physiological regulation of HCN1 channel functions in cortical neurons. Pharmacological manipulations of neuronal activity resulted in changes in the expression level of HCN1. In addition, the surface expression of HCN1 was dynamically regulated by neuronal activity. Both of these changes led to functional modulations of HCN1 channels. Our study suggests that coordinated changes in protein expression and surface expression of HCN1 serve as the key regulatory mechanisms controlling the function of endogenous HCN1 protein in cortical neurons.

Association with the Auxiliary Subunit PEX5R/Trip8b Controls Responsiveness of HCN Channels to cAMP and Adrenergic Stimulation

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are key modulators of neuronal activity by providing the depolarizing cation current I(h) involved in rhythmogenesis, dendritic integration, and synaptic transmission. These tasks critically depend on the availability of HCN channels, which is dynamically regulated by intracellular cAMP; the range of this regulation, however, largely differs among neurons in the mammalian brain. Using affinity purification and high-resolution mass spectrometry, we identify the PEX5R/Trip8b protein as the beta subunit of HCN channels in the mammalian brain. Coassembly of PEX5R/Trip8b affects HCN channel gating in a subtype-dependent and mode-specific way: activation of HCN2 and HCN4 by cAMP is largely impaired, while gating by phosphoinositides and basal voltage-dependence remain unaffected. De novo expression of PEX5R/Trip8b in cardiomyocytes abolishes beta-adrenergic stimulation of HCN channels. These results demonstrate that PEX5R/Trip8b is an intrinsic auxiliary subunit of brain HCN channels and establish HCN-PEX5R/Trip8b coassembly as a mechanism to control the channels' responsiveness to cyclic nucleotide signaling.

TRIP8b Splice Variants Form a Family of Auxiliary Subunits that Regulate Gating and Trafficking of HCN Channels in the Brain

Hyperpolarization-activated cyclic nucleotide-regulated (HCN) channels, which generate the I(h) current, mediate a number of important brain functions. The HCN1 isoform regulates dendritic integration in cortical pyramidal neurons and provides an inhibitory constraint on both working memory in prefrontal cortex and spatial learning and memory in the hippocampus. Altered expression of HCN1 following seizures may contribute to the development of temporal lobe epilepsy. Yet the regulatory networks and pathways governing HCN channel expression and function in the brain are largely unknown. Here, we report the presence of nine alternative N-terminal splice forms of the brain-specific cytoplasmic protein TRIP8b and demonstrate the differential effects of six isoforms to downregulate or upregulate HCN1 surface expression. Furthermore, we find that all TRIP8b isoforms inhibit channel opening by shifting activation to more negative potentials. TRIP8b thus functions as an auxiliary subunit that provides a mechanism for the dynamic regulation of HCN1 channel expression and function.

Distribution of the pacemaker HCN4 channel mRNA and protein in the rabbit sinoatrial node

Several studies of the pacemaker mechanisms in mammalian cells, most of which were carried out in cells isolated from the rabbit sinoatrial node (SAN), have highlighted the role of the I f current. While the distribution of Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) channels, the molecular correlates of f-channels, is known at the mRNA level, the identification of f-channel proteins in this tissue is still undetermined. Here we investigate HCN protein expression in the rabbit pacemaker region. We found that HCN4 is the main isoform, and set therefore to analyze its distribution within the SAN and surrounding areas with the aim of correlating protein expression and pacemaking function. The analysis was carried out in tissue slices and single cells of the intercaval area, which includes the crista terminalis (CT), the SAN, and the septum interatrialis (SI). Immunolabeling, in situ hybridization, qRT-PCR analysis, and electrophysiological recordings identified the SAN as a region characterized by high HCN4 signal and current levels, while the expression in the CT and in the SI was either negligible or absent. Detailed analysis of the central SAN area showed that cells are predominantly distributed in islets interconnected by cell prolongations, and single-cell HCN4 labeling suggested sites of channel clustering. Our data indicate that in the rabbit SAN, HCN4 proteins are major constituents of native f-channels, and their distribution matches closely the SAN as defined morphologically and electrophysiologically. Until recently, the SAN was identified as the region where Cx43 and atrial natriuretic peptide are not expressed; we propose here that expression of HCN4 is an appropriate tool to map and identify the cardiac SAN pacemaker region.