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.

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.

Role of hyperpolarization-activated cyclic nucleotide-gated cation nonselective channel in spontaneous contrac-tion of smooth muscles in human isolated ureters / 局解手术学杂志

Objective To investigate effects of hyperpolarization-activated cyclic nucleotide-gated cation nonselective channel in the hu-man ureter on the spontaneous contraction of smooth muscles. Methods Four HCN subtypes were detected in human ureteral tissue using reverse transcription polymerase chain reaction,Western blotting and immunohistochemical. ZD7288,the HCN blocker, was used to observe the changes of ureteral muscle contraction amplitude and frequency by applying the ureteral smooth muscle strip test in vitro. Results HCN1-4 isoforms were all identified in human ureter using reverse transcription-polymerase chain reaction and Western blotting. Through the immunohistochemical,HCN channel was found mostly in the urothelium layer and muscular layer of human ureteral wall. ZD7288 significantly decreased the bladder excitation. Conclusion All 4 HCN channel hypotypes exist in the human ureter, and affect the ureteral excitation.

Histology of canine pulmonary veins and expression of HCN channel / 重庆医学

Objective To observe the anisotropy of myocardial fiber orientation and conduction cells in canine pulmonary vein , and to investigate the expression of funny current (If ) channel subunit HCN1 ,HCN2 ,HCN4 mRNA in cadiocytes of pulmonary veins and left atrial in acute atrial fibrillation (AF) modle of caine .Methods Fourteen adult mongrel canines were randomly divided into atrial fibrillation group(n=7) and control group(n=7) .A model of acute AF has been developed in rapid atrial pacing (RAP) . The cardiocytes in control group were detect using hematoxylin hematoxylin and eosin (HE) and Periodic acid-Schiff(PAS) stain in the pulmonary vein .The mRNA expressions of HCN1 ,HCN2 ,HCN4 in two group were quantified by semiquantitative reverse transcription transcription-polymerase chain reaction(RT-PCR) .Results There were complex myocardial fiber orientation in pul-monary veins especially in PV-LA junction .PAS-positive cells could be seen in the endomembrane of pulmonary veins muscle sleeve .There were mRNA expression of HCN2 and HCN4 in pulmonary veins and left arial in two group ,but there was no mRNA expression of HCN1 .The mRNA levels of HCN4 from high to low were:pulmonary veins of atrial fibrillation group ,left atrial of atrial fibrillation group ,pulmonary veins of control group ,left atrial of control group(P0 .05) .Conclusion There are anisotro-py ,conduction cells in pulmonary veins .Up-regulation of pulmonary veins and left atrial HCN 2 ,HCN4 mRNA in atrial fibrillation caine ,may be correlated with the atrial fibrillation and maintenance .

Effect of Non-specific HCN1 Blocker CsCl on Spatial Learning and Memory in Mouse / 华中科技大学学报（医学）（英德文版）

It has been suggested that HCN1 is primarily expressed in hippocampus, however little is known about its effects on spatial learning and memory. In the present study, we investigated the effects of non-specific HCN1 blocker CsCl on spatial learning and memory by using Morris water maze and in situ hybridization in mice. The results showed CsCl 160 mg/kg ip for 4 days, and the mean escape latency was 34 s longer than that of normal control (P＜0.01). In hippocampal tissues, staining for the HCN1 mRNA was stronger in the DG and CA1 region of the hippocampus (P ＜0.05, P＜0.05, when CsCl-administration group was compared with normal group). Our results suggested that CsCl could significantly affect the spatial learning and memory in mice, and HCN channel is involved in the process of learning and memory.