Hyperpolarization-activated Cyclic Nucleotide-gated Gene Research Articles

Mutants of the Zebrafish K+ Channel Hcn2b Exhibit Epileptic-like Behaviors.

Epilepsy is a chronic neurological disorder that affects 50 million people worldwide. The most common form of epilepsy is idiopathic, where most of the genetic defects of this type of epilepsy occur in ion channels. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are activated by membrane hyperpolarization, and are mainly expressed in the heart and central and peripheral nervous systems. In humans, four HCN genes have been described, and emergent clinical data shows that dysfunctional HCN channels are involved in epilepsy. Danio rerio has become a versatile organism to model a wide variety of diseases. In this work, we used CRISPR/Cas9 to generate hcn2b mutants in zebrafish, and characterized them molecularly and behaviorally. We obtained an hcn2b mutant allele with an 89 bp deletion that produced a premature stop codon. The mutant exhibited a high mortality rate in its life span, probably due to its sudden death. We did not detect heart malformations or important heart rate alterations. Absence seizures and moderate seizures were observed in response to light. These seizures rarely caused instant death. The results show that mutations in the Hcn2b channel are involved in epilepsy and provide evidence of the advantages of zebrafish to further our understanding of the pathogenesis of epilepsy.

HCN ion channels and accessory proteins in epilepsy: genetic analysis of a large cohort of patients and review of the literature

The Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) channels are highly expressed in the Central Nervous Systems, where they are responsible for the Ih current. Together with specific accessory proteins, these channels finely regulate neuronal excitability and discharge activity. In the last few years, a substantial body of evidence has been gathered showing that modifications of Ih can play an important role in the pathogenesis of epilepsy. However, the extent to which HCN dysfunction is spread among the epileptic population is still unknown. The aim of this work is to evaluate the impact of genetic mutations potentially affecting the HCN channels’ activity, using a NGS approach. We screened a large cohort of patients with epilepsy of unknown etiology for mutations in HCN1, HCN2 and HCN4 and in genes coding for accessory proteins (MiRP1, Filamin A, Caveolin-3, TRIP8b, Tamalin, S-SCAM and Mint2). We confirmed the presence of specific mutations of HCN genes affecting channel function and predisposing to the development of the disease. We also found several previously unreported additional genetic variants, whose contribution to the phenotype remains to be clarified. According to these results and data from literature, alteration of HCN1 channel function seems to play a major role in epilepsy, but also dysfunctional HCN2 and HCN4 channels can predispose to the development of the disease. Our findings suggest that inclusion of the genetic screening of HCN channels in diagnostic procedures of epileptic patients should be recommended. This would help pave the way for a better understanding of the role played by Ih dysfunction in the pathogenesis of epilepsy.

Ivabradine Improves Cognitive and Behavioral Responses Following Traumatic Stress

BackgroundAlpha‐2 adrenergic receptor (α2AR) agonists, such as clonidine or dexmedetomidine, can decrease the working memory acquisition of a traumatic event, and also, blocking the post‐synaptic action of the catecholamines with a β‐adrenergic receptor (β‐AR) antagonist, propranolol, can reduce the emotional response to memories of the traumatic event. α2ARs and β‐ARs divergently regulate neuronal intracellular cyclic adenosine monophosphate (cAMP) concentrations. It is suggested that other pharmacologic mechanisms that similarly lead to reductions in neuronal cAMP could also be salutary in the treatment of post‐traumatic stress (PTSD). In the central nervous system, hyperpolarization‐activated, cyclic nucleotide‐gated (HCN) “funny” channels are important for dendritic integration, synaptic transmission, setting membrane potential, and neuronal firing rate. These funny channels (HCN1 and HCN2) are expressed in the forebrain and pre‐frontal cortex where they are co‐located and cross talk with A2ARs that mediate intracellular cAMP concentrations and drive working memory and the emotional valence associated with memories. Also, targeted disruption of HCN gene expression, or inhibition of cyclic nucleotide binding to HCN channels in the brain, has been shown to result in antidepressant‐like behavior with improved coping in animal models of stress.HypothesisWe postulated that pharmacological inhibition of HCN channels would diminish the anxiolytic and cognitive decline that occurs in an animal model of PTSD.MethodsWe infused an HCN antagonist, ivabradine (10 mg/kg/day), subcutaneously or performed sham surgery in a mouse model of PTSD. After six days of random cued and contextual fear conditioning, mouse anxiety and cognition was assessed by their performance over five minutes on three standard behavioral challenges—Open Field Test (OFT), Elevated Plus Maze (EPM), and Object Recognition Test (ORT).ResultsDuring the OFT, ivabradine did not significantly affected the total exploratory distance traveled. However, ivabradine significantly increased the time the mice spent in the center of the field (all values expressed in sec ± SEM), from 19.0 ± 2.2 to 27.7 ± 3.2, p = 0.029. During EPM, ivabradine increased the time mice spent traversing the open arm from 66.8 ± 8.5 to 116 ± 11.5, p = 0.002, During the ORT, social isolation during PTSD reduced total exploratory time from 24.7 ± 4.9 to 13.1 ± 2.2, and the ivabradine group increased exploratory time to 30.0 ± 3.3 compared to the mice living in isolation with PTSD (p = 0.0004). Furthermore, when compared to the untreated PTSD mice living in social isolation, ivabradine significantly increased the exploratory times of both familiar (14.3 ± 1.9 vs. 5.6 ± 1.1, p=0.0008) and novel objects (15.8 ± 2.4 vs. 7.5 ± 1.6, p = 0.01).ConclusionIvabradine may offer salutary advantage to men and women who experience PTSD.Support or Funding InformationDepartment of DefenseThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Hyperpolarization-activated cyclic nucleotide-gated gene signatures and poor clinical outcome of cancer patient

Background: we investigated the mRNA expression of hyperpolarization-activated cyclic nucleotide-gated genes (HCN1-4) in multiple types and subtypes of cancers. Methods: We performed a meta-analysis of public microarray data from Oncomine and NextBio Research databases to discover the mRNA expression level of HCN1-4 in cancers. Survival analysis was also used to investigate the correlation between overexpression of HCN gene family with overall survival rate of cancer patients using Kaplan-Meier Plotter database and PROGgene V2 database. Results: HCN genes (HCN1-4) over-expression and under-expression in multiples types of cancers such as CNS and brain cancer, breast cancer, colorectal cancer, melanoma, and lymphoma were found. HCN1 was signi cantly correlated with low overall survival of breast cancer [hazard ratio (HR) =7.42, P=0.0019] and colorectal cancer (HR =1.66, P=0.0071) patients. The lower survival rates of lung cancer (HR =2.5, P= 0.0107), kidney cancer (HR =1.1, P=0.004) and gastric cancer (HR =1.33, P=0.0037) patients were significantly correlated with the expression of HCN2. HCN3 was signi cantly correlated to lower survival rates of breast cancer (HR =1.65, P=0.0016), and kidney cancer (HR =1.17, P=0.0049). HCN4 was highly correlated with the lower survival rates of breast cancer of gastric cancer (HR =1.25, P=0.022), lung cancer (HR =5.37, P=0.0433) and ovarian cancer (HR =13.58, P=0.0426). Conclusions: These data suggested that HCN genes (HCN1-4) are likely to be potential candidates for cancer diagnosis and prognosis.

Hyperpolarization-activated cyclic nucleotide-gated channels in peripheral diaphragmatic lymphatics.

Diaphragmatic lymphatic function is mainly sustained by pressure changes in the tissue and serosal cavities during cardiorespiratory cycles. The most peripheral diaphragmatic lymphatics are equipped with muscle cells (LMCs), which exhibit spontaneous contraction, whose molecular machinery is still undetermined. Hypothesizing that spontaneous contraction might involve hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in lymphatic LMCs, diaphragmatic specimens, including spontaneously contracting lymphatics, were excised from 33 anesthetized rats, moved to a perfusion chamber containing HEPES-Tyrode's solution, and treated with HCN channels inhibitors cesium chloride (CsCl), ivabradine, and ZD-7288. Compared with control, exposure to 10 mM CsCl reduced (-65%, n = 13, P < 0.01) the contraction frequency (FL) and increased end-diastolic diameter (DL-d, +7.3%, P < 0.01) without changes in end-systolic diameter (DL-s). Ivabradine (300 μM) abolished contraction and increased DL-d (-14%, n = 10, P < 0.01) or caused an incomplete inhibition of FL (n = 3, P < 0.01), leaving DL-d and DL-s unaltered. ZD-7288 (200 μM) completely (n = 12, P < 0.01) abolished FL, while DL-d decreased to 90.9 ± 2.7% of control. HCN gene expression and immunostaining confirmed the presence of HCN1-4 channel isoforms, likely arranged in different configurations, in LMCs. Hence, all together, data suggest that HCN channels might play an important role in affecting contraction frequency of LMCs.

In situ investigation of allografted mouse HCN4 gene–transfected rat bone marrow mesenchymal stromal cells with the use of patch-clamp recording of ventricular slices

BackgroundRecently, proof-of-concept experiments have shown that genetically modified bone marrow mesenchymal stromal cells (MSCs) carrying hyperpolarization-activated cyclic nucleotide-gated (HCN) channels were able to express the funny current (If) in vitro, which played a key role in the process of pacemaker generation for heart rate, and were capable of pacemaker function after transplantation into the host heart. Nevertheless, because of the lack of direct experimental access to the implanted cells in situ, the changes in electrophysiological characteristics and the mechanisms underlying the pacemaker function of engrafted HCN gene–transfected MSCs in vivo remain unclear. Methods and ResultsOn the basis of the improved preparation of ventricular slices, we successfully performed an in situ investigation of allografted mouse HCN4 gene (mHCN4)-transfected rat MSCs (rMSCs) with the use of patch-clamp recording in ventricular slices. We demonstrate that allografted mHCN4-transfected rMSCs survived in the host heart for >4 weeks; that they expressed If, which is generated by the mHCN4 channel, with a similar amplitude but greater negative activation compared with parallel cells cultured in vitro; that they did not express optical action potentials or depolarization-activated inward sodium or calcium currents; and that they exhibited a low incidence of gap-junctional coupling with host cardiomyocytes. ConclusionsThis study provides direct experimental access to investigate MSCs after transplantation into the host heart. We propose that mHCN4-transfected rMSCs survived in the host heart with altered electrophysiological characteristics of If and were accompanied by a low efficiency of connexin 43 expression at 4 weeks after transplantation, which may affect its pacemaker function in vivo.

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.

Abstract 4626: Optimal Dose of Adv-hHCN4 Creates Stable and Long-lasting Biopacing Activity in Cultured Ventricular Cardiomyocytes

Purpose: Hyperpolarization-activated cyclic nucleotide-gated (HCN) genes have been successfully used as a strategy for recreating cardiac biological pacemakers in animal models. However, optimal dose of HCN and toxicity from HCN overexpression have not been investigated. Therefore, we assessed the effects of various titers of adenoviral human HCN4-GFP vector (Adv-hHCN4) on cardiomyocytes. Methods: Neonatal rat ventricular myocytes (NRVMs) were isolated, selected and cultured on microelectrode arrays to assess their automaticities. Morphology and apoptosis with and without HCN or Ca 2+ channel inhibitor were also assessed. Results: Beating rates significantly increased in NRVMs after hHCN4 infection (Fig. 1 ). For example, the rates were gradually increased to 235±11 beat/min on day 7 after hHCN4 infection with 1×10 5 PFU/array. In contrast, control cells showed low rates. NRVMs with ≥10 6 PFU/array Adv-hHCN4 reached faster rates early and subsequently stopped beating (Fig. 1 ). In addition, myocytes with ≥10 6 PFU/array Adv-hHCN4 underwent significant apoptosis (&gt;50%) which potentially resulted from hHCN4 overexpression and was blocked by the HCN channel blocker Cs + (1 mM), but not by the Ca 2+ channel inhibitor nifedipine. In addition, myocytes infected with ≥10 6 PFU/array Adv-GFP maintained normal morphology and rate. Our data demonstrate that hHCN4 transfer significantly and dose-dependently increased beating rates of NRVMs. However, overexpression of HCN could cause apoptosis. Therefore, an optimal dose of HCN gene is important for reducing toxicity and creating stable and long-lasting biopacing activity in cardiomyocytes in vitro, and probably also in vivo. Figure 1. Effects of hHCN4 infection on automaticities of neonatal rat ventricular myocytes. Each data point represents an averaged beating rate (mean ± SE) from 8 to 10 arrays. Various titers (1×10 5 to 1×10 7 PFU/500,000 cells per array) of Adv-Hhcn4 (expect control) were added to the arrays after measurements on day 0 (see the arrow)

Hyperpolarization-activated cyclic nucleotide-gated channel gene: The most possible therapeutic applications in the field of cardiac biological pacemakers

The implantation of electronic devices has become the preferred treatment for symptomatic bradyarrhythmias. However, there are many shortcomings in electronic pacemakers. More recently, the data has suggested that application of hyperpolarization-activated cyclic nucleotide-gated (HCN) channel gene may hold great promise in development of biological pacemaker. The evidence for the hypotheses included that: (1) The expression of four HCN genes varies somewhat among cardiac tissue. And also, the main difference between pacemaker cells and common cardiomyocytes depends upon which HCN genes are fully expressed; (2) the animals lacking HCN channels show a pronounced cardiac phenotype, which is due to dysfunction of the sinus node; (3) it is suggested that primary defects in HCN channels might underlie certain cardiac arrhythmias in several families with hereditary sinus node dysfunction; (4) recently, several studies demonstrated that overexpression of HCN gene in myocytes via gene delivery or implantation of stem cells with overexpression of I(f) successfully induced automaticity of a biological pacemaker in arrhythmic animal models.

Pacemaking by HCN Channels Requires Interaction with Phosphoinositides

Hyperpolarization-activated, cyclic-nucleotide-gated (HCN) channels mediate the depolarizing cation current (termed I(h) or I(f)) that initiates spontaneous rhythmic activity in heart and brain. This function critically depends on the reliable opening of HCN channels in the subthreshold voltage-range. Here we show that activation of HCN channels at physiologically relevant voltages requires interaction with phosphoinositides such as phosphatidylinositol-4,5-bisphosphate (PIP(2)). PIP(2) acts as a ligand that allosterically opens HCN channels by shifting voltage-dependent channel activation approximately 20 mV toward depolarized potentials. Allosteric gating by PIP(2) occurs in all HCN subtypes and is independent of the action of cyclic nucleotides. In CNS neurons and cardiomyocytes, enzymatic degradation of phospholipids results in reduced channel activation and slowing of the spontaneous firing rate. These results demonstrate that gating by phospholipids is essential for the pacemaking activity of HCN channels in cardiac and neuronal rhythmogenesis.