Gating movements and ion permeation in HCN4 pacemaker channels.

The HCN1-4 channel family is responsible for the hyperpolarization-activated cation current If/Ih that controls automaticity in cardiac and neuronal pacemaker cells. We present cryoelectron microscopy (cryo-EM) structures of HCN4 in the presence or absence of bound cAMP, displaying the pore domain in closed and open conformations. Analysis of cAMP-bound and -unbound structures sheds light on how ligand-induced transitions in the channel cytosolic portion mediate the effect of cAMP on channel gating and highlights the regulatory role of a Mg2+ coordination site formed between the C-linker and the S4-S5 linker. Comparison of open/closed pore states shows that the cytosolic gate opens through concerted movements of the S5 and S6 transmembrane helices. Furthermore, in combination with molecular dynamics analyses, the open pore structures provide insights into the mechanisms of K+/Na+ permeation. Our results contribute mechanistic understanding on HCN channel gating, cyclic nucleotide-dependent modulation, and ion permeation.

A gain-of-function HCN4 mutant in the HCN domain is responsible for inappropriate sinus tachycardia in a Spanish family.

In a family with inappropriate sinus tachycardia (IST), we identified a mutation (p.V240M) of the hyperpolarization-activated cyclic nucleotide-gated type 4 (HCN4) channel, which contributes to the pacemaker current (If) in human sinoatrial node cells. Here, we clinically study fifteen family members and functionally analyze the p.V240M variant. Macroscopic (IHCN4) and single-channel currents were recorded using patch-clamp in cells expressing human native (WT) and/or p.V240M HCN4 channels. All p.V240M mutation carriers exhibited IST that was accompanied by cardiomyopathy in adults. IHCN4 generated by p.V240M channels either alone or in combination with WT was significantly greater than that generated by WT channels alone. The variant, which lies in the N-terminal HCN domain, increased the single-channel conductance and opening frequency and probability of HCN4 channels. Conversely, it did not modify the channel sensitivity for cAMP and ivabradine or the level of expression at the membrane. Treatment with ivabradine based on functional data reversed the IST and the cardiomyopathy of the carriers. In computer simulations, the p.V240M gain-of-function variant increases If and beating rate and thus explains the IST of the carriers. The results demonstrate the importance of the unique HCN domain in HCN4, which stabilizes the channels in the closed state.

Caveolin-3 and Caveolin-1 Interaction Decreases Channel Dysfunction Due to Caveolin-3 Mutations.

Caveolae constitute membrane microdomains where receptors and ion channels functionally interact. Caveolin-3 (cav-3) is the key structural component of muscular caveolae. Mutations in CAV3 lead to caveolinopathies, which result in both muscular dystrophies and cardiac diseases. In cardiomyocytes, cav-1 participates with cav-3 to form caveolae; skeletal myotubes and adult skeletal fibers do not express cav-1. In the heart, the absence of cardiac alterations in the majority of cases may depend on a conserved organization of caveolae thanks to the expression of cav-1. We decided to focus on three specific cav-3 mutations (Δ62-64YTT; T78K and W101C) found in heterozygosis in patients suffering from skeletal muscle disorders. We overexpressed both the WT and mutated cav-3 together with ion channels interacting with and modulated by cav-3. Patch-clamp analysis conducted in caveolin-free cells (MEF-KO), revealed that the T78K mutant is dominant negative, causing its intracellular retention together with cav-3 WT, and inducing a significant reduction in current densities of all three ion channels tested. The other cav-3 mutations did not cause significant alterations. Mathematical modelling of the effects of cav-3 T78K would impair repolarization to levels incompatible with life. For this reason, we decided to compare the effects of this mutation in other cell lines that endogenously express cav-1 (MEF-STO and CHO cells) and to modulate cav-1 expression with an shRNA approach. In these systems, the membrane localization of cav-3 T78K was rescued in the presence of cav-1, and the current densities of hHCN4, hKv1.5 and hKir2.1 were also rescued. These results constitute the first evidence of a compensatory role of cav-1 in the heart, justifying the reduced susceptibility of this organ to caveolinopathies.

Channel HCN4 mutation R666Q associated with sporadic arrhythmia decreases channel electrophysiological function and increases protein degradation.

Mutations in the hyperpolarization-activated nucleotide-gated channel 4 (HCN4) are known to be associated with arrhythmias in which QT prolongation (delayed ventricular repolarization) is rare. Here, we identified a HCN4 mutation, HCN4-R666Q, in two sporadic arrhythmia patients with sinus bradycardia, QT prolongation, and short bursts of ventricular tachycardia. To determine the functional effect of the mutation, we conducted clinical, genetic, and functional analyses using whole-cell voltage-clamp, qPCR, Western blot, confocal microscopy, and co-immunoprecipitation. The mean current density of HEK293T cells transfected with HCN4-R666Q was lower in 24 to 36 h after transfection and was much lower in 36 to 48 h after transfection relative to cells transfected with wildtype HCN4. Additionally, we determined that the HCN4-R666Q mutant was more susceptible to ubiquitin-proteasome system-mediated protein degradation than wildtype HCN4. This decreased current density for HCN4-R666Q could be partly rescued by treatment with a proteasome inhibitor. Therefore, we conclude that HCN4-R666Q had an effect on HCN4 function in two aspects, including decreasing the current density of the channel as a biophysical effect and weakening its protein stability. Our findings provide new insights into the pathogenesis of the HCN4-R666Q mutation.

Optimized synthesis and pharmacological evaluation of HCN channel inhibitor EC18.

HCN4 channels are considered to be a promising target for cardiac pathologies, epilepsy, and multiple sclerosis. However, there are no subtype-selective HCN channel blockers available, and only a few compounds are reported to display subtype preferences, one of which is EC18 (cis-1). Herein, we report the optimized synthetic route for the preparation of EC18 and its evaluation in three different pharmacological models, allowing us to assess its activity on cardiac function, thalamocortical neurons, and immune cells.

Paradigm shift: new concepts for HCN4 function in cardiac pacemaking.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are the molecular correlate of the If current and are critically involved in controlling neuronal excitability and the autonomous rhythm of the heart. The HCN4 isoform is the main HCN channel subtype expressed in the sinoatrial node (SAN), a tissue composed of specialized pacemaker cells responsible for generating the intrinsic heartbeat. More than 40 years ago, the If current was first discovered in rabbit SAN tissue. Along with this discovery, a theory was proposed that cyclic adenosine monophosphate-dependent modulation of If mediates heart rate regulation by the autonomic nervous system-a process called chronotropic effect. However, up to the present day, this classical theory could not be reliably validated. Recently, new concepts emerged confirming that HCN4 channels indeed play an important role in heart rate regulation. However, the cellular mechanism by which HCN4 controls heart rate turned out to be completely different than originally postulated. Here, we review the latest findings regarding the physiological role of HCN4 in the SAN. We describe a newly discovered mechanism underlying heart rate regulation by HCN4 at the tissue and single cell levels, and we discuss these observations in the context of results from previously studied HCN4 mouse models.

Glucocorticoid Receptor/HCN4 Channels Interaction in Spinal Dorsal Horn Participates in the Regulation of Neuropathic Pain after Peripheral Nerve Injury in Rats.

We studied the interaction between glucocorticoid receptor (GR) and HCN4 channels in the rat model of spared nerve injury (SNI) in Sprague-Dawley rats (n=124). The animals were randomly divided into 6 groups: sham-operated (SO; n=24), SNI (reference group; n=20), and 4 experimental SNI groups intrathecally treated with dexamethasone (DEX; GR agonist; n=20), RU38486 (GR antagonist; n=20), ZD7288 (HCN channels blocker; n=20), and ZD7288+DEX (n=20). The paw mechanical withdrawal threshold (PWT) was measured one day before surgery (SO group) and on days 1, 3, 7, 14, and 21 after surgery. Behavioral results showed that mechanical hyperalgesia appeared on day 1 after SNI, while PWT decreased gradually with time. The expression of GR and HCN4 channels in L4-L6 dorsal horn of the spinal cord was detected by Western blotting and immunohistochemistry. In the reference group, SNI significantly increased GR expression up to day 14 after surgery in comparison with the SO group. The expression of GR showed a tendency to increase in the DEX group (with the maximum expression on days 14 and 21), significantly increased in the RU38486 group (maximum on day 7). In the ZD7288 group, GR expression was lower than in the SNI group and did not change throughout the experiment, suggesting that ZD7288 could block the expression of GR. In the DEX group, the expression of HCN4 channels was significantly higher on day 1 after SNI, but there were no differences in this parameter between the RU38486 and ZD7288 groups. In the ZD7288+DEX group, the expression of HCN4 channels significantly increased on days 14 and 21 after SNI. Thus, GR and HCN4 have the same linkage in the formation of central sensitization after SNI, but antagonists have no significant effect on the improvement of pain behavior.

Dual role of miR-1 in the development and function of sinoatrial cells.

miR-1, the most abundant miRNA in the heart, modulates expression of several transcription factors and ion channels. Conditions affecting the heart rate, such as endurance training and cardiac diseases, show a concomitant miR-1 up- or down-regulation. Here, we investigated the role of miR-1 overexpression in the development and function of sinoatrial (SAN) cells using murine embryonic stem cells (mESC). We generated mESCs either overexpressing miR-1 and EGFP (miR1OE) or EGFP only (EM). SAN-like cells were selected from differentiating mESC using the CD166 marker. Gene expression and electrophysiological analysis were carried out on both early mES-derived cardiac progenitors and SAN-like cells and on beating neonatal rat ventricular cardiomyocytes (NRVC) over-expressing miR-1. miR1OE cells increased significantly the proportion of CD166+ SAN precursors compared to EM cells (23% vs 12%) and the levels of the transcription factors TBX5 and TBX18, both involved in SAN development. miR1OE SAN-like cells were bradycardic (1,3 vs 2 Hz) compared to EM cells. In agreement with data on native SAN cells, EM SAN-like cardiomyocytes show two populations of cells expressing either slow- or fast-activating If currents; miR1OE SAN-like cells instead have only fast-activating If with a significantly reduced conductance. Western Blot and immunofluorescence analysis showed a reduced HCN4 signal in miR-1OE vs EM CD166+ precursors. Together these data point out to a specific down-regulation of the slow-activating HCN4 subunit by miR-1. Importantly, the rate and If alterations were independent of the developmental effects of miR-1, being similar in NRVC transiently overexpressing miR-1. In conclusion, we demonstrated a dual role of miR-1, during development it controls the proper development of sinoatrial-precursor, while in mature SAN-like cells it modulates the HCN4 pacemaker channel translation and thus the beating rate.

Structural and functional approaches to studying cAMP regulation of HCN channels.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are primarily activated by voltage and further modulated by cAMP. While cAMP binding alone does not open the channel, its presence facilitates the action of voltage, increasing channel open probability. Functional results indicate that the membrane-based voltage sensor domain (VSD) communicates with the cytosolic cyclic nucleotide-binding domain (CNBD), and vice-versa. Yet, a mechanistic explanation on how this could occur in structural terms is still lacking. In this review, we will discuss the recent advancement in understanding the molecular mechanisms connecting the VSD with the CNBD in the tetrameric organization of HCN channels unveiled by the 3D structures of HCN1 and HCN4. Data show that the HCN domain transmits cAMP signal to the VSD by bridging the cytosolic to the membrane domains. Furthermore, a metal ion coordination site connects the C-linker to the S4-S5 linker in HCN4, further facilitating cAMP signal transmission to the VSD in this isoform.

Mitochondrial thioredoxin-2 maintains HCN4 expression and prevents oxidative stress-mediated sick sinus syndrome.

OBJECTIVE: Sick sinus syndrome (SSS) is associated with loss of HCN4 (hyperpolarization-activated cyclic nucleotide-gated potassium channel 4) function in the cardiac conduction system. The underlying mechanism for SSS remains elusive. This study is to investigate how mitochondrial oxidative stress induces HCN4 downregulation associated with in sick sinus syndrome. METHODS AND RESULTS: Trx2lox/lox mice were crossed with α-myosin heavy chain (α-Mhc)-Cre and Hcn4-CreERT2 deleter mice to generate Trx2 deletion mice in the whole heart (Trx2cKO) and in the conduction system (Trx2ccsKO), respectively. Echocardiography was applied to measure hemodynamics and heart rhythm. Histological analyses, gene profiling and chromatin immunoprecipitation were performed to define the mechanism by which thioredoxin-2 (Trx2) regulates HCN4 expression and cardiac function. Trx2cKO mice displayed dilated cardiomyopathy, low heart rate, and atrial ventricular block (AVB) phenotypes. Immunofluorescence revealed that HCN4 expression was specifically reduced within the sinoatrial node in Trx2cKO mice. Interestingly, Trx2ccsKO mice displayed low heart rate and AVB without dilated cardiomyopathy. Both mRNA and protein levels of HCN4 were reduced in the sinoatrial node, suggesting transcriptional HCN4 regulation upon Trx2 deletion. ChIP indicated that the binding of MEF2 to the HCN4 enhancer was not altered by Trx2 deletion; however, histone 3 acetylation at the MEF2 binding site was decreased, and expression of histone deacetylase 4 (HDAC4) was elevated following Trx2 deletion. Moreover, HDAC4 binding to the HCN4 enhancer was mediated by MEF2. Mitochondrial ROS were increased by Trx2 deletion and importantly, mitochondria-specific ROS scavenger MitoTEMPO suppressed HDAC4 elevation, HCN4 reduction, and sinus bradycardia in Trx2ccsKO mice. CONCLUSION: In the conduction system, Trx2 is critical for maintaining HCN4-mediated normal heart rate. Loss of Trx2 reduces HCN4 expression via a mitochondrial ROS-HDAC4-MEF2C pathway and subsequently induces sick sinus syndrome in mice.

The Hyperpolarization-Activated HCN4 Channel is Important for Proper Maintenance of Oscillatory Activity in the Thalamocortical System.

Hyperpolarization-activated cation channels are involved, among other functions, in learning and memory, control of synaptic transmission and epileptogenesis. The importance of the HCN1 and HCN2 isoforms for brain function has been demonstrated, while the role of HCN4, the third major neuronal HCN subunit, is not known. Here we show that HCN4 is essential for oscillatory activity in the thalamocortical (TC) network. HCN4 is selectively expressed in various thalamic nuclei, excluding the thalamic reticular nucleus. HCN4-deficient TC neurons revealed a massive reduction of Ih and strongly reduced intrinsic burst firing, whereas the current was normal in cortical pyramidal neurons. In addition, evoked bursting in a thalamic slice preparation was strongly reduced in the mutant mice probes. HCN4-deficiency also significantly slowed down thalamic and cortical oscillations during active wakefulness. Taken together, these results establish that thalamic HCN4 channels are essential for the production of rhythmic intrathalamic oscillations and determine regular TC oscillatory activity during alert states.

[The effects of PDK1-Akt signaling pathway intervention on cardiomyocyte HCN4 ion channels].

Objective: To explore the effects of 3-phosphate dependent protein kinase 1-protein kinase B (PDK1-Akt) signaling pathway on the transcription, expression and function of cardiac hyperpolarized activated cyclic nucleotide gated 4 (HCN4) ion channels. Methods: Atrial myocytes were obtained from healthy male wild-type C57 mice and heart-specific PDK1 knockout mice (PDK1-KO) by enzymolysis. Then the atrial myocytes were divided into blank control group and PDK1-KO group. In further studies, the isolated atrial myocytes were cultured and further divided into drug control group (treated with dimethyl sulfoxide (DMSO)) and PDK1 knockdown group (treated with 1 µg/ml PDK1 short hairpin RNA (shRNA) interference plasmid), SC79 group (treated with 8 µmol/ml SC79), GSK2334470 group (treated with 10 nmol/L GSK2334470) and PDK1 knockdown+SC79 group (8 µmol/ml SC79 and 1 µg/ml PDK1 shRNA interference plasmid). Real time quantitative PCR (qRT-PCR) was used to detect the mRNA expression levels of PDK1 and HCN4, Western blot was used to detect the protein expression levels of PDK1, Akt and HCN4, the whole cell patch clamp was used to detecte the current density of HCN, and immunofluorescence was used to detecte the expression of HCN4 protein on atrial cells. Results: (1) the expression levels of HCN4 mRNA (1.46±0.03 vs. 0.99±0.01, P<0.001) and protein (1.14±0.02 vs. 1.00±0.06, P=0.017) in PDK1-KO group were higher than those in blank control group. The HCN current density in PDK1-KO group was higher than that in blank control group((-17.47±2.00) pA/pF vs. (-12.15±2.25) pA/pF, P=0.038). (2) The functions of PDK1 shRNA and specific Akt agonist SC79 were verified by comparing the PDK1 knockdown group and SC79 group with the drug control group. The results showed that the expression levels of PDK1 mRNA and protein in PDK1 knockdown group were lower than those in drug control group, and the expression level of phosphorylated Akt (Thr 308) protein in SC79 group was higher than that in drug control group. (3) The expression levels of HCN4 mRNA (3.61±0.46 vs. 1.00±0.08, P<0.001) and protein (2.33±0.11 vs. 1.00±0.05, P<0.001) in GSK2334470 group were higher than those in drug control group. (4) To reduce the effect of drug-miss target, the cultured atrial myocytes were transfected with shRNA plasmid of PDK1 and intervened with SC79. The results showed that the expression of HCN4 mRNA in PDK1 knockdown group was higher than that in the drug control group (1.76±0.11 vs. 1.00±0.06, P<0.001), and PDK1 knockdown+SC79 group (1.76±0.11 vs. 1.33±0.07, P=0.003). In PDK1 knockdown+SC79 group, the mRNA expression level was also higher than that in the drug control group (1.33±0.07 vs. 1.00±0.06, P<0.001). The expression level of HCN4 protein in PDK1 knockdown group was higher than that in drug control group (1.15±0.04 vs. 1.00±0.05, P=0.003). As for the The expression level of HCN4 protein, there was no significantly statistical difference between the PDK1 knockdown+SC79 group and the drug control group (P>0.05), but PDK1 knockdown+SC79 group was lower than PDK1 knockdown group (0.95±0.01 vs. 1.15±0.04, P<0.001). In patch clamp experiments, the results showed that the HCN current density was (-13.27±1.28) pA/pF in the drug control group, (-18.76±2.03) pA/pF in the PDK1 knockdown group, (-13.50±2.58) pA/pF in the PDK1 knockdown+SC79 group; the HCN current density of PDK1 knockdown group was higher than that of drug control group (P<0.001), but there was no significant difference between PDK1 knockdown+SC79 group and drug control group (P>0.05). (5) The results of immunofluorescence showed that the brightness of green fluorescence of PDK1 knockdown group was higher than that of drug control group, indicating that the expression of HCN4 localized on cell membrane was increased. However, the green fluorescence of PDK1 knockdown+SC79 group was lighter than that of PDK1 knockdown group, suggesting that the expression of HCN4 in PDK1-knockdown cell membrane decreased after further activating Akt. Conclusion: PDK1-Akt signaling pathway is involved in the regulation of HCN4 ion channel transcription, expression and function.

The C-terminal HCN4 variant P883R alters channel properties and acts as genetic modifier of atrial fibrillation and structural heart disease.

Atrial fibrillation (AF) is the most frequent sustained arrhythmia and can lead to structural cardiac changes, known as tachycardia-induced cardiomyopathy (TIC). HCN4 is implicated in spontaneous excitation of the sinoatrial node, while channel dysfunction has been associated with sinus bradycardia, AF and structural heart disease. We here asked whether HCN4 mutations may contribute to the development of TIC, as well. Mutation scanning of HCN4 in 60 independent patients with AF and suspected TIC followed by panel sequencing in carriers of HCN4 variants identified the HCN4 variant P883R [minor allele frequency (MAF): 0,88%], together with the KCNE1 variant S38G (MAF: 65%) in three unrelated patients. Family histories revealed additional cases of AF, sudden cardiac death and cardiomyopathy. Patch-clamp recordings of HCN4-P883R channels expressed in HEK293 cells showed remarkable alterations of channel properties shifting the half-maximal activation voltage to more depolarized potentials, while channel deactivation was faster compared to wild-type (WT). Co-transfection of WT and mutant subunits, resembling the heterozygous cellular situation of our patients, revealed significantly higher current densities compared to WT. In conclusion HCN4-P883R may increase ectopic trigger and maintenance of AF by shifting the activation voltage of If to more positive potentials and producing higher current density. Together with the common KCNE1 variant S38G, previously proposed as a genetic modifier of AF, HCN4-P883R may provide a substrate for the development of AF and TIC.

Functional reclassification of variants of uncertain significance in the HCN4 gene identified in sudden unexpected death.

The HCN4 gene encodes a subunit of the hyperpolarization-activated cyclic nucleotide-gated channel, type 4 that is essential for the proper generation of pacemaker potentials in the sinoatrial node. The HCN4 gene is often present in targeted genetic testing panels for various cardiac conduction system disorders and there are several reports of HCN4 variants associated with conduction disorders. Here, we report the in vitro functional characterization of four rare variants of uncertain significance (VUS) in HCN4, identified through testing a cohort of 296 sudden unexpected natural deaths. The variants are all missense alterations, leading to single amino acid changes: p.E66Q in the N-terminus, p.D546N in the C-linker domain, and both p.S935Y and p.R1044Q in the C-terminus distal to the CNBD. We also identified a likely benign variant, p. P1063T, which has a high minor allele frequency in the gnomAD, which is utilized here as a negative control. Three of the HCN4 VUS (p.E66Q, p.S935Y, and p.R1044Q) had electrophysiological characteristics similar to the wild-type channel, suggesting that these variants are benign. In contrast, the p.D546N variant in the C-linker domain exhibited a larger current density, slower activation, and was unresponsive to cyclic adenosine monophosphate (cAMP) compared to wild-type. With functional assays, we reclassified three rare HCN4 VUS to likely benign variants, eliminating the necessity for costly and time-consuming further study. Our studies also provide a new lead to investigate how a VUS located in the C-linker connecting the pore to the cAMP binding domain may affect the channel open state probability and cAMP response.

HCN4 pacemaker channels attenuate the parasympathetic response and stabilize the spontaneous firing of the sinoatrial node.

KEY POINTS: The contribution of HCN4 pacemaker channels in the autonomic regulation of the sino-atrial node (SAN) has been a matter of debate. The transgenic overexpression of HCN4 did not induce tachycardia, but reduced heart rate variability, while the conditional knockdown of HCN4 gave rise to sinus arrhythmia. The response of the SAN to ß-adrenergic stimulation was not affected by overexpression or knockdown of HCN4 channels. When HCN4 channels were knocked down, the parasympathetic response examined by cervical vagus nerve stimulation (CVNS) was enhanced; the CVNS induced complete sinus pause. The overexpression of HCN4 attenuated bradycardia induced by CVNS only during ß-adrenergic stimulation. We concluded that HCN4 pacemaker channels stabilize the spontaneous firing by attenuating the parasympathetic response of the SAN. ABSTRACT: The heart rate is dynamically controlled by the sympathetic and parasympathetic nervous systems that regulate the sinoatrial node (SAN). HCN4 pacemaker channels are the well-known causative molecule of congenital sick sinus syndrome. Although HCN4 channels are activated by cAMP, the sympathetic response of the SAN was preserved in patients carrying loss-of-function mutations of the HCN4 gene. In order to clarify the contribution of HCN4 channels in the autonomic regulation of the SAN, we developed novel gain-of-function mutant mice in which the expression level of HCN4 channels could be reversibly changed from zero to ∼3 times that in wild-type mice, using tetracycline transactivator and the tetracycline responsive element. We recorded telemetric ECGs in freely moving conscious mice and analysed the heart rate variability. We also evaluated the response of the SAN to cervical vagus nerve stimulation (CVNS). The conditional overexpression of HCN4 did not induce tachycardia, but reduced heart rate variability. The HCN4 overexpression also attenuated bradycardia induced by the CVNS only during the ß-adrenergic stimulation. In contrast, the knockdown of HCN4 gave rise to sinus arrhythmia, and enhanced the parasympathetic response; complete sinus pause was induced by the CVNS. In vitro, we compared the effects of acetylcholine on the spontaneous action potentials of single pacemaker cells, and found that similar phenotypic changes were induced by genetic manipulation of HCN4 expression both in the presence and absence of ß-adrenergic stimulation. Our study suggests that HCN4 channels attenuate the vagal response of the SAN, and thereby stabilize the spontaneous firing of the SAN.

RNA sequencing of mouse sinoatrial node reveals an upstream regulatory role for Islet-1 in cardiac pacemaker cells.

RATIONALE: Treatment of sinus node disease with regenerative or cell-based therapies will require a detailed understanding of gene regulatory networks in cardiac pacemaker cells (PCs). OBJECTIVE: To characterize the transcriptome of PCs using RNA sequencing and to identify transcriptional networks responsible for PC gene expression. METHODS AND RESULTS: We used laser capture microdissection on a sinus node reporter mouse line to isolate RNA from PCs for RNA sequencing. Differential expression and network analysis identified novel sinoatrial node-enriched genes and predicted that the transcription factor Islet-1 is active in developing PCs. RNA sequencing on sinoatrial node tissue lacking Islet-1 established that Islet-1 is an important transcriptional regulator within the developing sinoatrial node. CONCLUSIONS: (1) The PC transcriptome diverges sharply from other cardiomyocytes; (2) Islet-1 is a positive transcriptional regulator of the PC gene expression program.

Regulation of HCN Ion Channels by Non-canonical Cyclic Nucleotides.

The hyperpolarization-activated cyclic-nucleotide-modulated (HCN) proteins are cAMP-regulated ion channels that play a key role in nerve impulse transmission and heart rate modulation in neuronal and cardiac cells, respectively. Although they are regulated primarily by cAMP, other cyclic nucleotides such as cGMP, cCMP, and cUMP serve as partial agonists for the HCN2 and HCN4 isoforms. By competing with cAMP for binding, these non-canonical ligands alter ion channel gating, and in turn, modulate the cAMP-dependent activation profiles. The partial activation of non-canonical cyclic nucleotides can be rationalized by either a partial reversal of a two-state inactive/active conformational equilibrium, or by sampling of a third conformational state with partial activity. Furthermore, different mechanisms and degrees of activation have been observed upon binding of non-canonical cyclic nucleotides to HCN2 versus HCN4, suggesting that these ligands control HCN ion channels in an isoform-specific manner. While more work remains to be done to achieve a complete understanding of ion channel modulation by non-canonical cyclic nucleotides, it is already clear that such knowledge will ultimately prove invaluable in achieving a more complete understanding of ion channel signaling in vivo, as well as in the development of therapeutics designed to selectively modulate ion channel gating.

Rescuing cardiac automaticity in L-type Cav1.3 channelopathies and beyond.

Pacemaker activity of the sino-atrial node generates the heart rate. Disease of the sinus node and impairment of atrioventricular conduction induce an excessively low ventricular rate (bradycardia), which cannot meet the needs of the organism. Bradycardia accounts for about half of the total workload of clinical cardiologists. The 'sick sinus' syndrome (SSS) is characterized by sinus bradycardia and periods of intermittent atrial fibrillation. Several genetic or acquired risk factors or pathologies can lead to SSS. Implantation of an electronic pacemaker constitutes the only available therapy for SSS. The incidence of SSS is forecast to double over the next 50 years, with ageing of the general population thus urging the development of complementary or alternative therapeutic strategies. In recent years an increasing number of mutations affecting ion channels involved in sino-atrial automaticity have been reported to underlie inheritable SSS. L-type Cav 1.3 channels play a major role in the generation and regulation of sino-atrial pacemaker activity and atrioventricular conduction. Mutation in the CACNA1D gene encoding Cav 1.3 channels induces loss-of-function in channel activity and underlies the sino-atrial node dysfunction and deafness syndrome (SANDD). Mice lacking Cav 1.3 channels (Cav 1.3-/- ) fairly recapitulate SSS and constitute a precious model to test new therapeutic approaches to handle this disease. Work in our laboratory shows that targeting G protein-gated K+ (IKACh ) channels effectively rescues SSS of Cav 1.3-/- mice. This new concept of 'compensatory' ion channel targeting shines new light on the principles underlying the pacemaker mechanism and may open the way to new therapies for SSS.

HCN4 dynamically marks the first heart field and conduction system precursors.

RATIONALE: To date, there has been no specific marker of the first heart field to facilitate understanding of contributions of the first heart field to cardiac lineages. Cardiac arrhythmia is a leading cause of death, often resulting from abnormalities in the cardiac conduction system (CCS). Understanding origins and identifying markers of CCS lineages are essential steps toward modeling diseases of the CCS and for development of biological pacemakers. OBJECTIVE: To investigate HCN4 as a marker for the first heart field and for precursors of distinct components of the CCS, and to gain insight into contributions of first and second heart lineages to the CCS. METHODS AND RESULTS: HCN4CreERT2, -nuclear LacZ, and -H2BGFP mouse lines were generated. HCN4 expression was examined by means of immunostaining with HCN4 antibody and reporter gene expression. Lineage studies were performed using HCN4CreERT2, Isl1Cre, Nkx2.5Cre, and Tbx18Cre, coupled to coimmunostaining with CCS markers. Results demonstrated that, at cardiac crescent stages, HCN4 marks the first heart field, with HCN4CreERT2 allowing assessment of cell fates adopted by first heart field myocytes. Throughout embryonic development, HCN4 expression marked distinct CCS precursors at distinct stages, marking the entire CCS by late fetal stages. We also noted expression of HCN4 in distinct subsets of endothelium at specific developmental stages. CONCLUSIONS: This study provides insight into contributions of first and second heart lineages to the CCS and highlights the potential use of HCN4 in conjunction with other markers for optimization of protocols for generation and isolation of specific conduction system precursors.

Inducible gene deletion in the entire cardiac conduction system using Hcn4-CreERT2 BAC transgenic mice.

Developmental defects and disruption of molecular pathways of the cardiac conduction system (CCS) can cause life-threatening cardiac arrhythmias. Despite decades of effort, knowledge about the development and molecular control of the CCS remains primitive. Mouse genetics, complementary to other approaches such as human genetics, has become a key tool for exploring the developmental processes of various organs and associated diseases. Genetic analysis using mouse models will likely provide great insights about the development of the CCS, which can facilitate the development of novel therapeutic strategies to treat arrhythmias. To enable genetic studies of the CCS, CCS-associated Cre mouse models are essential. However, existing mouse models with Cre activity reported in the CCS have various limitations such as Cre leak, haploinsufficiency, and inadequate specificity of the Cre activity. To circumvent those limitations, we successfully generated Hcn4-CreERT2 bacterial artificial chromosome (BAC) transgenic mice using BAC recombineering in which Cre activity was specifically detected in the entire CCS after tamoxifen induction. Our Hcn4-CreERT2 BAC transgenic line will be an invaluable genetic tool with which to dissect the developmental control of CCS and arrhythmias.