Inhibition of HCN1 currents by norquetiapine, an active metabolite of the atypical anti-psychotic drug quetiapine.

Quetiapine is a second-generation atypical antipsychotic drug that has been commonly prescribed for the treatment of schizophrenia, major depressive disorder (depression), and other psychological disorders. Targeted inhibition of hyperpolarization-activated cyclic-nucleotide gated (HCN) channels, which generate Ih, may provide effective resistance against schizophrenia and depression. We investigated if HCN channels could contribute to the therapeutic effect of quetiapine, and its major active metabolite norquetiapine. Two-electrode voltage clamp recordings were used to assess the effects of quetiapine and its active metabolites 7-hydroxyquetiapine and norquetiapine on currents from HCN1 channels expressed in Xenopus laevis oocytes. Norquetiapine, but not quetiapine nor 7-hydroxyquetiapine, has an inhibitory effect on HCN1 channels. Norquetiapine selectively inhibited HCN1 currents by shifting the voltage-dependence of activation to more hyperpolarized potentials in a concentration-dependent manner with an IC50 of 13.9 ± 0.8 µM for HCN1 and slowing channel opening, without changing the kinetics of closing. Inhibition by norquetiapine primarily occurs from in the closed state. Norquetiapine inhibition is not sensitive to the external potassium concentration, and therefore, likely does not block the pore. Norquetiapine inhibition also does not dependent on the cyclic-nucleotide binding domain. Norquetiapine also inhibited HCN4 channels with reduced efficacy than HCN1 and had no effect on HCN2 channels. Therefore, HCN channels are key targets of norquetiapine, the primary active metabolite of quetiapine. These data help to explain the therapeutic mechanisms by which quetiapine aids in the treatment of anxiety, major depressive disorder, bipolar disorder, and schizophrenia, and may represent a novel structure for future drug design of HCN inhibitors.

HCN1 hyperpolarization-activated cyclic nucleotide-gated channels enhance evoked GABA release from parvalbumin-positive interneurons.

Hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels generate the cationic Ih current in neurons and regulate the excitability of neuronal networks. The function of HCN channels depends, in part, on their subcellular localization. Of the four HCN isoforms (HCN1-4), HCN1 is strongly expressed in the dendrites of pyramidal neurons (PNs) in hippocampal area CA1 but also in presynaptic terminals of parvalbumin-positive interneurons (PV+ INs), which provide strong inhibitory control over hippocampal activity. Yet, little is known about how HCN1 channels in these cells regulate the evoked release of the inhibitory transmitter GABA from their axon terminals. Here, we used genetic, optogenetic, electrophysiological, and imaging techniques to investigate how the electrophysiological properties of PV+ INs are regulated by HCN1, including how HCN1 activity at presynaptic terminals regulates the release of GABA onto PNs in CA1. We found that application of HCN1 pharmacological blockers reduced the amplitude of the inhibitory postsynaptic potential recorded from CA1 PNs in response to selective optogenetic stimulation of PV+ INs. Homozygous HCN1 knockout mice also show reduced IPSCs in postsynaptic cells. Finally, two-photon imaging using genetically encoded fluorescent calcium indicators revealed that HCN1 blockers reduced the probability that an extracellular electrical stimulating pulse evoked a Ca2+ response in individual PV+ IN presynaptic boutons. Taken together, our results show that HCN1 channels in the axon terminals of PV+ interneurons facilitate GABAergic transmission in the hippocampal CA1 region.

Upregulation of HCN2 in ventral tegmental area is involved in morphine-induced conditioned place preference in rats.

Morphine is an opioid commonly used to treat pain in clinic, but it also has the potential to be highly addictive, which can lead to abuse. Despite these known risks, the cellular and molecular mechanism of morphine conditioned place preference (CPP) is still unclear. In this study, using a rat model of chronic morphine administration, we found that compared with the control group, the mRNA and protein expression of HCN2 channel in the ventral tegmental area (VTA) were upregulated. Further immunofluorescence analysis showed that the fluorescence intensity of HCN2 channel of VTA dopaminergic neurons in morphine group was significantly enhanced, while the patch clamp recording of brain slices showed that both the magnitude and the density of Ih (HCN channel current) of VTA neurons were significantly increased. Moreover, intra-VTA infusion of ZD7288, a selective inhibitor of HCN channel, into rats of the morphine group decreased morphine CPP. Taken together, our results show that chronic morphine administration induces an upregulation of HCN2 in VTA dopamine neurons, while HCN inhibition reduces morphine CPP, suggesting that HCN channel may be a potential target for the treatment of morphine addiction.

Screening effects of HCN channel blockers on sleep/wake behavior in zebrafish.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels generate electrical rhythmicity in various tissues although primarily heart, retina and brain. The HCN channel blocker compound, Ivabradine (Corlanor), is approved by the US Food and Drug Administration (FDA) as a medication to lower heart rate by blocking hyperpolarization activated inward current in the sinoatrial node. In addition, a growing body of evidence suggests a role for HCN channels in regulation of sleep/wake behavior. Zebrafish larvae are ideal model organisms for high throughput drug screening, drug repurposing and behavioral phenotyping studies. We leveraged this model system to investigate effects of three HCN channel blockers (Ivabradine, Zatebradine Hydrochloride and ZD7288) at multiple doses on sleep/wake behavior in wild type zebrafish. Results of interest included shorter latency to daytime sleep at 0.1 µM dose of Ivabradine (ANOVA, p: 0.02), moderate reduction in average activity at 30 µM dose of Zatebradine Hydrochloride (ANOVA, p: 0.024) in daytime, and increased nighttime sleep at 4.5 µM dose of ZD7288 (ANOVA, p: 0.036). Taken together, shorter latency to daytime sleep, decrease in daytime activity and increased nighttime sleep indicate that different HCN channel antagonists affected different parameters of sleep and activity.

Role of HCN channels in the functions of basal ganglia and Parkinson's disease.

Parkinson's disease (PD) is a motor disorder resulting from dopaminergic neuron degeneration in the substantia nigra caused by age, genetics, and environment. The disease severely impacts a patient's quality of life and can even be life-threatening. The hyperpolarization-activated cyclic nucleotide-gated (HCN) channel is a member of the HCN1-4 gene family and is widely expressed in basal ganglia nuclei. The hyperpolarization-activated current mediated by the HCN channel has a distinct impact on neuronal excitability and rhythmic activity associated with PD pathogenesis, as it affects the firing activity, including both firing rate and firing pattern, of neurons in the basal ganglia nuclei. This review aims to comprehensively understand the characteristics of HCN channels by summarizing their regulatory role in neuronal firing activity of the basal ganglia nuclei. Furthermore, the distribution and characteristics of HCN channels in each nucleus of the basal ganglia group and their effect on PD symptoms through modulating neuronal electrical activity are discussed. Since the roles of the substantia nigra pars compacta and reticulata, as well as globus pallidus externus and internus, are distinct in the basal ganglia circuit, they are individually described. Lastly, this investigation briefly highlights that the HCN channel expressed on microglia plays a role in the pathological process of PD by affecting the neuroinflammatory response.

Ivabradine Alleviates Experimental Autoimmune Myocarditis-Mediated Myocardial Injury.

Ivabradine (IVA) reduces heart rate by inhibiting hyperpolarization-activated cyclic nucleotide-gated channels (HCNs), which play a role in the promotion of pacemaker activity in cardiac sinoatrial node cells. HCNs are highly expressed in neural and myocardial tissues and are involved in the modulation of inflammatory neuropathic pain. However, whether IVA exerts any effect on myocardial inflammation in the pathogenesis of heart failure is unclear. We employed single-cell RNA sequencing (scRNA-seq) in porcine cardiac myosin-induced experimental autoimmune myocarditis rat model to determine the effects and mechanisms of IVA. Lewis rats (n = 32) were randomly divided into the normal, control, high-dose-IVA, and low-dose-IVA groups. Heart rate and blood pressure were measured on days 0 and 21, respectively. Echocardiography was performed on day 22, and inflammation of the myocardium was evaluated via histopathological examination. Western blot was employed to detect the expression of HCN1-4, MinK-related protein 1 (MiRP1), matrix metalloproteinase 2 (MMP-2), MMP-9, and transforming growth factor-ß (TGF-ß). Furthermore, enzyme-linked immunosorbent assay was performed to measure serum IL-1, IL-6, and TNF-α. The relative mRNA levels of collagen I, collagen III, and α-smooth muscle actin (α-SMA) were determined via qRT-PCR. We found that IVA reduced the total number of cells infiltrated into the myocardium, particularly in the subset of fibroblasts, endocardia, and monocytes. IVA administration ameliorated cardiac inflammation and reduced collagen production. Results of the echocardiography indicated that left ventricular internal diameter at end-systole LVIDs increased whereas left ventricular ejection fraction and left ventricular fractional shortening decreased in the control group. IVA improved cardiac performance. The expression of HCN4 and MiRP1 protein and the level of serum IL-1, IL-6, and TNF-α were decreased by IVA treatment. In conclusion, HCNs and the helper proteins were increased in the profile of myocardial inflammation. HCNs may be involved in the regulation of myocardial inflammation by inhibiting immune cell infiltration. Our findings can contribute to the development of IVA-based combination therapies for the future treatment of cardiac inflammation and heart failure.

Interaction Between HCN and Slack Channels Regulates mPFC Pyramidal Cell Excitability in Working Memory Circuits.

The ability of monkeys and rats to carry out spatial working memory tasks has been shown to depend on the persistent firing of pyramidal cells in the prefrontal cortex (PFC), arising from recurrent excitatory connections on dendritic spines. These spines express hyperpolarization-activated cyclic nucleotide-gated (HCN) channels whose open state is increased by cAMP signaling, and which markedly alter PFC network connectivity and neuronal firing. In traditional neural circuits, activation of these non-selective cation channels leads to neuronal depolarization and increased firing rate. Paradoxically, cAMP activation of HCN channels in PFC pyramidal cells reduces working memory-related neuronal firing. This suggests that activation of HCN channels may hyperpolarize rather than depolarize these neurons. The current study tested the hypothesis that Na+ influx through HCN channels activates Slack Na+-activated K+ (KNa) channels to hyperpolarize the membrane. We have found that HCN and Slack KNa channels co-immunoprecipitate in cortical extracts and that, by immunoelectron microscopy, they colocalize at postsynaptic spines of PFC pyramidal neurons. A specific blocker of HCN channels, ZD7288, reduces KNa current in pyramidal cells that express both HCN and Slack channels, but has no effect on KNa currents in an HEK cell line expressing Slack without HCN channels, indicating that blockade of HCN channels in neurons reduces K+ current indirectly by lowering Na+ influx. Activation of HCN channels by cAMP in a cell line expressing a Ca2+ reporter results in elevation of cytoplasmic Ca2+, but the effect of cAMP is reversed if the HCN channels are co-expressed with Slack channels. Finally, we used a novel pharmacological blocker of Slack channels to show that inhibition of Slack in rat PFC improves working memory performance, an effect previously demonstrated for blockers of HCN channels. Our results suggest that the regulation of working memory by HCN channels in PFC pyramidal neurons is mediated by an HCN-Slack channel complex that links activation HCN channels to suppression of neuronal excitability.

Assessing neuroprotective effects of diroximel fumarate and siponimod via modulation of pacemaker channels in an experimental model of remyelination.

Cuprizone (CPZ)-induced alterations in axonal myelination are associated with a period of neuronal hyperexcitability and increased activity of hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels in the thalamocortical (TC) system. Substances used for the treatment of multiple sclerosis (MS) have been shown to normalize neuronal excitability in CPZ-treated mice. Therefore, we aimed to examine the effects of diroximel fumarate (DRF) and the sphingosine 1-phospate receptor (S1PR) modulator siponimod on action potential firing and the inward current (Ih) carried by HCN ion channels in naive conditions and during different stages of de- and remyelination. Here, DRF application reduced Ih current density in ex vivo patch clamp recordings from TC neurons of the ventrobasal thalamic complex (VB), thereby counteracting the increase of Ih during early remyelination. Siponimod reduced Ih in VB neurons under control conditions but had no effect in neurons of the auditory cortex (AU). Furthermore, siponimod increased and decreased AP firing properties of neurons in VB and AU, respectively. Computational modeling revealed that both DRF and siponimod influenced thalamic bursting during early remyelination by delaying the onset and decreasing the interburst frequency. Thus, substances used in MS treatment normalize excitability in the TC system by influencing AP firing and Ih.

Ivabradine ameliorates cardiomyopathy progression in a Duchenne muscular dystrophy model rat.

Duchenne muscular dystrophy (DMD) is an X-linked recessive myopathy caused by dystrophin mutations. Inevitable progressive cardiomyopathy is a current leading cause of premature death although respiratory management has improved the prognosis of patients with DMD. Recent evidence shows that reducing the heart rate is expected as one of the promising strategies for heart failure treatment, but administering a sufficient dose of ß-blocker for patients with DMD with tachycardia is difficult because of their low blood pressure (BP). Thus, this study aimed to clarify the role of ivabradine, which suppresses cardiac sinus node pacemakers without decreasing BP, in ameliorating cardiomyopathy progression in a rat model with DMD. A trans-oral single ivabradine administration demonstrated a declined dose-dependent heart rate without any significant BP reduction. Trans-gastric repeated administrations of 5 mg/kg of ivabradine twice a day for 3 months showed ameliorated cardiomyopathy in DMD rats based on echocardiography and histopathological observations (left ventricular dysfunction, right ventricular dysfunction, and myocardial fibrosis) as compared with vehicle administration. Our finding indicates that ivabradine is expected as another treatment choice for patients with DMD having tachycardia.

Inhibition of hyperpolarization-activated cyclic nucleotide-gated cation channel attenuates cerebral ischemia reperfusion-induced impairment of learning and memory by regulating apoptotic pathway.

Stroke is the second leading cause of death globally. Cognitive dysfunction is a common complication of stroke, which seriously affects the patient's quality of life. Previous studies have shown that the expression of hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channel is closely related to ischemia-reperfusion (IR) injury and subsequent cognitive impairment. We also found that ZD7288, a specific inhibitor of the HCN channel, attenuated IR injury during short-term reperfusion. Since apoptosis can induce cell necrosis and aggravate cognitive impairment after IR, the purpose of this study is to define whether ZD7288 could improve cognitive impairment after prolonged cerebral reperfusion in rats by regulating apoptotic pathways. Our data indicated that ZD7288 can ameliorate spatial cognitive behavior and synaptic plasticity, protect the morphology of hippocampal neurons, and alleviate hippocampal apoptotic cells in IR rats. This effect may be related to down-regulating the expressions of pro-apoptotic proteins such as AIF, p53, Bax, and Caspase-3, and increasing the ratio of Bcl-2/Bax. Taken together, it suggested that inhibition of the HCN channel improves cognitive impairment after IR correlated with its regulation of apoptotic pathways.

Volatile anesthetics inhibit presynaptic cGMP signaling to depress presynaptic excitability in rat hippocampal neurons.

Volatile anesthetics alter presynaptic function through effects on Ca2+ influx and neurotransmitter release. These actions are proposed to play important roles in their pleiotropic neurophysiological effects including immobility, unconsciousness and amnesia. Nitric oxide and cyclic guanosine monophosphate (NO/cGMP) signaling has been implicated in presynaptic mechanisms, and disruption of NO/cGMP signaling has been shown to alter sensitivity to volatile anesthetics in vivo. We investigated volatile anesthetic actions NO/cGMP signaling in relation to presynaptic function in cultured rat hippocampal neurons using pharmacological tools and genetically encoded biosensors and sequestering probes of cGMP levels. Using the fluorescent cGMP biosensor cGull, we found that electrical stimulation-evoked NMDA-type glutamate receptor-independent presynaptic cGMP transients were inhibited 33.2% by isoflurane (0.51 mM) and 26.4% by sevoflurane (0.57 mM) (p < 0.0001) compared to control stimulation without anesthetic. Stimulation-evoked cGMP transients were blocked by the nonselective inhibitor of nitric oxide synthase N-ω-nitro-l-arginine, but not by the selective neuronal nitric oxide synthase inhibitor N5-(1-imino-3-butenyl)-l-ornithine. Isoflurane and sevoflurane inhibition of stimulation-evoked increases in presynaptic Ca2+ concentration, measured with synaptophysin-GCaMP6f, and of synaptic vesicle exocytosis, measured with synaptophysin-pHlourin, was attenuated in neurons expressing the cGMP scavenger protein sponge (inhibition of exocytosis reduced by 54% for isoflurane and by 53% for sevoflurane). The anesthetic-induced reduction in presynaptic excitability was partially occluded by inhibition of HCN channels, a cGMP-modulated excitatory ion channel that can facilitate glutamate release. We propose that volatile anesthetics depress presynaptic cGMP signaling and downstream effectors like HCN channels that are essential to presynaptic function and excitability. These findings identify novel mechanisms by which volatile anesthetics depress synaptic transmission via second messenger signaling involving the NO/cGMP pathway in hippocampal neurons.

HCN channel inhibitor induces ketamine-like rapid and sustained antidepressant effects in chronic social defeat stress model.

Repeated, long-term (weeks to months) exposure to standard antidepressant medications is required to achieve treatment efficacy. In contrast, acute ketamine quickly improves mood for an extended time. Recent work implicates that hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are involved in mediating ketamine's antidepressant effects. In this study, we directly targeted HCN channels and achieved ketamine-like rapid and sustained antidepressant efficacy. Our in vitro electrophysiological recordings first showed that HCN inhibitor DK-AH 269 (also called cilobradine) decreased the pathological HCN-mediated current (Ih) and abnormal hyperactivity of ventral tegmental area (VTA) dopamine (DA) neurons in a depressive-like model produced by chronic social defeat stress (CSDS). Our in vivo studies further showed that acute intra-VTA or acute systemic administration of DK-AH 269 normalized social behavior and rescued sucrose preference in CSDS-susceptible mice. The single-dose of DK-AH 269, both by intra-VTA microinfusion and intraperitoneal (ip) approaches, could produce an extended 13-day duration of antidepressant-like efficacy. Animals treated with acute DK-AH 269 spent less time immobile than vehicle-treated mice during forced swim test. A social behavioral reversal lasted up to 13 days following the acute DK-AH 269 ip injection, and this rapid and sustained antidepressant-like response is paralleled with a single-dose treatment of ketamine. This study provides a novel ion channel target for acutely acting, long-lasting antidepressant-like effects.

Investigation of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in vitro inflammation model at molecular level.

In our study, we aimed to create an inflammation model in endothelial and macrophage cell lines and to examine the changes in the expression of hyperpolarization activated cyclic nucleotide gated (HCN) channels at the molecular level. HUVEC and RAW cell lines were used in our study. 1 µg/mL LPS was applied to the cells. Cell media were taken 6 h later. TNF-α, IL-1, IL-2, IL-4, IL-10 concentrations were measured by ELISA method. Cell media were cross-applied to cells for 24 h after LPS. HCN1/HCN2 protein levels were determined by Western-Blot method. HCN-1/HCN-2 gene expressions were determined by qRT-PCR method. In the inflammation model, a significant increase in TNF-α, IL-1, and IL-2 levels was observed in RAW cell media compared to the control. While no significant difference was observed in IL-4 level, a significant decrease was observed in IL-10 level. While a significant increase in TNF-α level was observed in HUVEC cell medium, no difference was observed in other cytokines. In our inflammation model, an 8.44-fold increase in HCN1 gene expression was observed in HUVEC cells compared to the control group. No significant change was observed in HCN2 gene expression. 6.71-fold increase in HCN1 gene expression was observed in RAW cells compared to the control. The change in HCN2 expression was not statistically significant. In the Western-Blot analysis, a statistically significant increase in HCN1 level was observed in the LPS group in HUVEC cells compared to the control; no significant increase in HCN2 level was observed. While a statistically significant increase in HCN1 level was observed in the LPS group in RAW cells compared to the control; no significant increase in HCN2 level was observed. In immunofluorescence examination, it was observed that the level of HCN1 and HCN2 proteins in the cell membrane of HUVEC and RAW cells increased in the LPS group compared to the control group. While HCN1 gene/protein levels were increased in RAW and HUVEC cells in the inflammation model, no significant change was observed in HCN2 gene/protein levels. Our data suggest that the HCN1 subtype is dominant in endothelium and macrophages and may play a critical role in inflammation.

Antidepressant-like activity of a brain penetrant HCN channel inhibitor in mice.

Changes in Hyperpolarization-Activated Cyclic Nucleotide-Gated (HCN) channel function have been linked to depressive-like traits, making them potential drug targets. However, there is currently no peer-reviewed data supporting the use of a small molecule modulator of HCN channels in depression treatment. Org 34167, a benzisoxazole derivative, has been patented for the treatment of depression and progressed to Phase I trials. In the current study, we analysed the biophysical effects of Org 34167 on HCN channels in stably transfected human embryonic kidney 293 (HEK293) cells and mouse layer V neurons using patch-clamp electrophysiology, and we utilised three high-throughput screens for depressive-like behaviour to assess the activity of Org 34167 in mice. The impact of Org 34167 on locomotion and coordination were measured by performing rotarod and ledged beam tests. Org 34167 is a broad-spectrum inhibitor of HCN channels, slowing activation and causing a hyperpolarising shift in voltage-dependence of activation. It also reduced I h-mediated sag in mouse neurons. Org 34167 (0.5 mg/kg) reduced marble burying and increased the time spent mobile in the Porsolt swim and tail suspension tests in both male and female BALB/c mice, suggesting reduced depressive-like behaviour. Although no adverse effects were seen at 0.5 mg/kg, an increase in dose to 1 mg/kg resulted in visible tremors and impaired locomotion and coordination. These data support the premise that HCN channels are valid targets for anti-depressive drugs albeit with a narrow therapeutic index. Drugs with higher HCN subtype selectivity are needed to establish if a wider therapeutic window can be obtained.

Interaction Between HCN and Slack Channels Regulates mPFC Pyramidal Cell Excitability and Working Memory.

The ability of monkeys and rats to carry out spatial working memory tasks has been shown to depend on the persistent firing of pyramidal cells in the prefrontal cortex (PFC), arising from recurrent excitatory connections on dendritic spines. These spines express hyperpolarization-activated cyclic nucleotide-gated (HCN) channels whose open state is increased by cAMP signaling, and which markedly alter PFC network connectivity and neuronal firing. In traditional neural circuits, activation of these non-selective cation channels leads to neuronal depolarization and increased firing rate. Paradoxically, cAMP activation of HCN channels in PFC pyramidal cells reduces working memory-related neuronal firing. This suggests that activation of HCN channels may hyperpolarize rather than depolarize these neurons. The current study tested the hypothesis that Na+ influx through HCN channels activates Slack Na+-activated K+ (KNa) channels to hyperpolarize the membrane. We have found that HCN and Slack KNa channels coimmunoprecipitate in cortical extracts and that, by immunoelectron microscopy, they colocalize at postsynaptic spines of PFC pyramidal neurons. A specific blocker of HCN channels, ZD7288, reduces KNa current in pyramidal cells that express both HCN and Slack channels, but has no effect on KNa currents in an HEK cell line expressing Slack without HCN channels, indicating that blockade of HCN channels in neurons reduces K+ +current indirectly by lowering Na+ influx. Activation of HCN channels by cAMP in a cell line expressing a Ca2+ reporter results in elevation of cytoplasmic Ca2+, but the effect of cAMP is reversed if the HCN channels are co-expressed with Slack channels. Finally, we used a novel pharmacological blocker of Slack channels to show that inhibition of Slack in rat PFC improves working memory performance, an effect previously demonstrated for blockers of HCN channels. Our results suggest that the regulation of working memory by HCN channels in PFC pyramidal neurons is mediated by an HCN-Slack channel complex that links activation HCN channels to suppression of neuronal excitability.

The role of P21-activated kinase (Pak1) in sinus node function.

Sinoatrial node (SAN) dysfunction (SND) and atrial arrhythmia frequently occur simultaneously with a hazard ratio of 4.2 for new onset atrial fibrillation (AF) in SND patients. In the atrial muscle attenuated activity of p21-activated kinase 1 (Pak1) increases the risk for AF by enhancing NADPH oxidase 2 dependent production of reactive oxygen species (ROS). However, the role of Pak1 dependent ROS regulation in SAN function has not yet been determined. We hypothesize that Pak1 activity maintains SAN activity by regulating the expression of the hyperpolarization activated cyclic nucleotide gated cation channel (HCN). To determine Pak1 dependent changes in heart rate (HR) regulation we quantified the intrinsic sinus rhythm in wild type (WT) and Pak1 deficient (Pak1-/-) mice of both sexes in vivo and in isolated Langendorff perfused hearts. Pak1-/- hearts displayed an attenuated HR in vivo after autonomic blockage and in isolated hearts. The contribution of the Ca2+ clock to pacemaker activity remained unchanged, but Ivabradine (3 µM), a blocker of HCN channels that are a membrane clock component, eliminated the differences in SAN activity between WT and Pak1-/- hearts. Reduced HCN4 expression was confirmed in Pak1-/- right atria. The reduced HCN activity in Pak1-/- could be rescued by class II HDAC inhibition (LMK235), ROS scavenging (TEMPOL) or attenuation of Extracellular Signal-Regulated Kinase (ERK) 1/2 activity (SCH772984). No sex specific differences in Pak1 dependent SAN regulation were determined. Our results establish Pak1 as a class II HDAC regulator and a potential therapeutic target to attenuate SAN bradycardia and AF susceptibility.

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.

Histamine H2 receptor deficit in glutamatergic neurons contributes to the pathogenesis of schizophrenia.

Schizophrenia is a serious mental disorder, and existing antipsychotic drugs show limited efficacy and cause unwanted side effects. The development of glutamatergic drugs for schizophrenia is currently challenging. Most functions of histamine in the brain are mediated by the histamine H1 receptor; however, the role of the H2 receptor (H2R) is not quite clear, especially in schizophrenia. Here, we found that expression of H2R in glutamatergic neurons of the frontal cortex was decreased in schizophrenia patients. Selective knockout of the H2R gene (Hrh2) in glutamatergic neurons (CaMKIIα-Cre; Hrh2 fl/fl) induced schizophrenia-like phenotypes including sensorimotor gating deficits, increased susceptibility to hyperactivity, social withdrawal, anhedonia, and impaired working memory, as well as decreased firing of glutamatergic neurons in the medial prefrontal cortex (mPFC) in in vivo electrophysiological tests. Selective knockdown of H2R in glutamatergic neurons in the mPFC but not those in the hippocampus also mimicked these schizophrenia-like phenotypes. Furthermore, electrophysiology experiments established that H2R deficiency decreased the firing of glutamatergic neurons by enhancing the current through hyperpolarization-activated cyclic nucleotide-gated channels. In addition, either H2R overexpression in glutamatergic neurons or H2R agonism in the mPFC counteracted schizophrenia-like phenotypes in an MK-801-induced mouse model of schizophrenia. Taken together, our results suggest that deficit of H2R in mPFC glutamatergic neurons may be pivotal to the pathogenesis of schizophrenia and that H2R agonists can be regarded as potentially efficacious medications for schizophrenia therapy. The findings also provide evidence for enriching the conventional glutamate hypothesis for the pathogenesis of schizophrenia and improve the understanding of the functional role of H2R in the brain, especially in glutamatergic neurons.

Chronic Cerebral Hypoperfusion-induced Dysregulations of Hyperpolarization- activated Cyclic Nucleotide-gated, KCNQ and G Protein-coupled Inwardly Rectifying Potassium Channels Correlated with Susceptibility and Unsusceptibility to Anxiety Behaviors.

BACKGROUND: Cerebrovascular lesions could induce affective disorders; however, the depression- and anxiety-related symptoms caused by Chronic Cerebral Hypoperfusion (CCH) and the roles of different Hyperpolarization-activated Cyclic Nucleotide-gated (HCN), KCNQ and G protein-coupled inwardly rectifying potassium (GirK) channel subunits in these pathological processes have been poorly elucidated so far. OBJECTIVE: To investigate the behavioral change and the alteration of HCN, KCNQ, and GirK subunits in amygdale rats suffering from CCH. METHODS: Permanent bilateral occlusion of the common carotid arteries was used to induce CCH. Anxiety and depression levels were assessed by the elevated plus maze test, sucrose preference test and forced swimming test to classify rats as highly anxious or depressive 'susceptibility' vs. 'unsusceptibility'. The expression of brain-derived neurotrophic factor (BDNF), tyrosine kinase receptor B (TrKB), HCN1/2, KCNQ2/3, and GirK1/2/3 were quantified by Western blotting. RESULTS: The main emotional change caused by 4 weeks of CCH is likely to be anxiety-like behavior (50%), accompanied by a down-regulation of BDNF and TrKB expression in amygdale. The increase of HCN1 and decrease of KCNQ3 expression in amygdale may be factors to blame for anxiety- like symptom caused by CCH, and the increase of KCNQ2 and Girk1 expression in amygdale may play a role in resilience to the anxiety induced by CCH. CONCLUSION: The different subunits of HCN, KCNQ and GirK channels in amygdale may contribute to distinct response to aversive stimuli or stress induced by CCH that evokes divergent influences on anxiety-like behavior in rats.