Hyperpolarization-activated Cyclic Nucleotide-gated Expression Research Articles

Interaction between miRNA-155 targeting neuronal pacemaker ion channels and release of amino acid transmitters during cerebral ischemia.

Cerebral hemorrhage can cause hemorrhagic stroke, leading to severe brain damage. MiRNA-155 is closely related to the development of stroke. However, the regulatory mechanism of miRNA-155 during cerebral ischemia has not yet been elucidated. SD rats were separated to sham operation group, model group, miRNA-155 inhibitor group and miRNA-155 agonist group followed by analysis of neural function, miRNA-155 and pacemaker ion channel hyperpolarization-activated, cyclic nucleotide-gated (HCN) expression by Real Time PCR. Meanwhile, Caspase 3 activity, glutamic acid (Glu) and γ-aminobutyric acid (GABA) content were also measured along with analysis of IL-6 and IL-1β secretion by ELISA as well as reactive oxygen species (ROS) content and superoxide dismutase (SOD) activity. Compared to sham operation group, model group presented significantly elevated miRNA-155 level and Longa score, decreased HCN expression, elevated Caspase 3 activity and Glu content, and reduced GABA and SOD activity. Meanwhile, model group also had elevated IL-6 and IL-1β secretion, and ROS content (p<0.05). The miRNA-155 agonist group further exacerbated these changes (p<0.01). The miRNA-155 inhibitor group could inhibit miRNA-155 expression and significantly reverse the above pathological changes (p<0.05). miRNA-155 is increased in cerebral ischemia. Regulation of miRNA-155 expression can target neuronal pacing ion channel HCN channel to regulate the release of amino acid transmitters, thereby alleviating the progress of cerebral ischemia.

Aging Changes in Bladder Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels Are Associated With Increasing Heterogeneity of Adrenergic/Mucosal Influence on Detrusor Control in the Mouse.

A geroscience-informed approach to the increasing prevalence of bladder control problems in older adults requires understanding the impact of aging on dynamic mechanisms that ensure resilience in response to stressors challenging asymptomatic voluntary control over urine storage and voiding. Bladder control is predicated on sensory neural information about bladder volume. Modulation of volume-induced bladder wall tensions by autonomic and mucosal factors controls neural sensitivity to bladder volume. We hypothesized that hyperpolarization-activated cyclic nucleotide-gated (HCN) channels integrate these factors and thereby mediate adrenergic detrusor tension control. Furthermore, loss of HCN expression compromises that integration and could result in loss of precision of detrusor control. Using a life-span mouse model, reverse transcription quantitative real-time PCR and pharmacologic studies in pretensioned intact and mucosa-denuded bladder strips were made. The dominant hcn1 expression declines with maturation and aging; however, aging is also associated with increased variance around mean values. In strips from Mature animals, isoproterenol had less effect in denuded muscle strips than in intact strips, and HCN blockade diminished isoproterenol responsiveness. With aging, variances about mean response values significantly increased, paralleling hcn1 expression. Our findings support a role for HCN in providing neuroendocrine/paracrine integration and suggest an association of increased heterogeneity of HCN expression in aging with reductions in response precision to neuroendocrine control. The functional implication is an increased risk of dysfunction of brainstem/bladder regulation of neuronal sensitivity to bladder volume. This supports the clinical model of the aging bladder phenotype as an expression of loss of resilience, and not as emerging bladder pathology with aging.

Alleviation of trigeminal neuropathic pain by electroacupuncture: the role of hyperpolarization-activated cyclic nucleotide-gated channel protein expression in the Gasserian ganglion.

The aim of this study was to examine the effect of electroacupuncture (EA) on trigeminal neuropathic pain in rats and explore the potential mechanism underlying the putative therapeutic effect of EA. Trigeminal neuropathic pain behavior was induced in rats by unilateral chronic constriction injury of the distal infraorbital nerve (dIoN-CCI). EA was administered at ST2 (Sibai) and Jiachengjiang. A total of 60 Sprague Dawley rats were divided into the following four groups (n = 15 per group) to examine the behavioral outcomes after surgery and/or EA treatment: sham (no ligation); dIoN-CCI (received isoflurane only, without EA treatment); dIoN-CCI+EA-7d (received EA treatment for 7 days); and dIoN-CCI+EA-14d (received EA treatment for 14 days). Both evoked and spontaneous nociceptive behaviors were measured. Of these, 12 rats (n = 4 from sham, dIoN-CCI, and dIoN-CCI+EA-14d groups, respectively) were used to analyze protein expression of hyperpolarization-activated cyclic nucleotide-gated (HCN) channel in the Gasserian ganglion (GG) by immunohistochemistry. dIoN-CCI rats exhibited mechanical allodynia and increased face-grooming activity that lasted at least 35 days. EA treatment reduced mechanical allodynia and face-grooming in dIoN-CCI rats. Overall, 14 days of EA treatment produced a prolonged anti-nociceptive effect as compared to 7-day EA treatment. The counts of HCN1 and HCN2 immunopositive puncta were increased in the ipsilateral GG in dIoN-CCI rats and were reduced by 14 days of EA treatment. EA treatment relieved trigeminal neuropathic pain in dIoN-CCI rats, and this effect was dependent on the duration of EA treatment. The downregulation of HCN expression may contribute to the anti-nociceptive effect of EA in this rat model of trigeminal neuropathic pain.

The function of hyperpolarization-activated cyclic nucleotide-gated channel in diabetic cystopathy.

We aimed at investigating changes in the expression and physiological function of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in interstitial cells of Cajal (ICC) in diabetic state. Twenty adult female Sprague-Dawley (SD) rats were randomly assigned to control and Zucker diabetic fatty (ZDF) group. The protein and mRNA expression of HCN isoforms and C-kit in the rat bladders were detected using Western blotting and reverse transcription-polymerase chain reaction (RT-PCR). The bladder contraction was evaluated using a bladder smooth muscle strip test. Whole cell patch-clamp techniques were used to detect the activity of HCN channels. Immunofluorescent staining was used to the positive expression of HCN and C-kit in ICC. cAMP, as HCN channel-specific stimulant, could increase the frequency and amplitude of spontaneous contractions in both group, while cAMP inducing contraction of ZDF rats, was still significantly lower compared with the control group. Acute bladder ICCs were isolated by collagenase digestion. Classic Ih current pattern was recorded on ICCs while Ih current amplitude of ICCs from ZDF diabetic rats was significantly lower than the control group. The expression and mRNA of HCN1-4 isoforms in ZDF diabetic rats were both significantly lower compared with the control group. Meanwhile, the number of c-kit positive cells in ZDF diabetic rats showed no significant differences compared with controls. The morphological structure of ICC in the bladder of ZDF rats was relatively loose and the number of their cell process was apparently decreased. The structure of ICCs in ZDF rats was relatively loose, their connection to each other was also diminished. The expression of HCN was down-regulated and its response to cAMP was also decreased. HCN channels in bladder ICCs might regulate detrusor contraction. Changes in HCN expression and activity in bladder ICCs might be one of the most important mechanisms of diabetic cystopathy.

HCN channelopathy and cardiac electrophysiologic dysfunction in genetic and acquired rat epilepsy models

Evidence from animal and human studies indicates that epilepsy can affect cardiac function, although the molecular basis of this remains poorly understood. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels generate pacemaker activity and modulate cellular excitability in the brain and heart, with altered expression and function associated with epilepsy and cardiomyopathies. Whether HCN expression is altered in the heart in association with epilepsy has not been investigated previously. We studied cardiac electrophysiologic properties and HCN channel subunit expression in rat models of genetic generalized epilepsy (Genetic Absence Epilepsy Rats from Strasbourg, GAERS) and acquired temporal lobe epilepsy (post-status epilepticus SE). We hypothesized that the development of epilepsy is associated with altered cardiac electrophysiologic function and altered cardiac HCN channel expression. Electrocardiography studies were recorded in vivo in rats and in vitro in isolated hearts. Cardiac HCN channel messenger RNA (mRNA) and protein expression were measured using quantitative PCR and Western blotting respectively. Cardiac electrophysiology was significantly altered in adult GAERS, with slower heart rate, shorter QRS duration, longer QTc interval, and greater standard deviation of RR intervals compared to control rats. In the post-SE model, we observed similar interictal changes in several of these parameters, and we also observed consistent and striking bradycardia associated with the onset of ictal activity. Molecular analysis demonstrated significant reductions in cardiac HCN2 mRNA and protein expression in both models, providing a molecular correlate of these electrophysiologic abnormalities. These results demonstrate that ion channelopathies and cardiac dysfunction can develop as a secondary consequence of chronic epilepsy, which may have relevance for the pathophysiology of cardiac dysfunction in patients with epilepsy.

Phylogeny and effects of anoxia on hyperpolarization-activated, cyclic nucleotide-gated channel gene expression in the heart of a primitive chordate, the Pacific Hagfish (eptatretus stoutii)

The aneural heart of the Pacific hagfish, Eptatretus stoutii, varies heart rate fourfold during recovery from anoxia. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, which play an important role in establishing the pacemaker rate of vertebrate hearts, were postulated to be present in this ancestral vertebrate heart, and it was also theorized that changes in hagfish heart rate with oxygen availability involved altered HCN expression. Partial gene cloning revealed six HCN isoforms in the hagfish heart. Hagfish representatives of HCN2, HCN3 and HCN4 were discovered, with HCN2 and HCN3 existing as isoforms designated as HCN2a, HCN2b, HCN3a, two paralogs of HCN3b, and HCN3c. Phylogenetic analysis revealed HCN3b and HCN3c to be ancestral, followed by HCN3a, HCN4 and HCN2. Moreover, HCN3a expression was dominant in both the atrial and ventricular chambers, suggesting that the HCN4 dominance in adult mammalian hearts appeared after hagfish divergence. HCN expression was higher in the atrium than in the ventricle, as might be expected given that atrial beating rate is known to be faster than the ventricular rate. In addition, mRNA expression under normoxic conditions was compared with that following 24 h of anoxia, and either a 2-h or 36-h recovery in normoxic water. In the ventricle, anoxia decreased HCN3a but not HCN4 expression. In contrast, atrial HCN3a expression significantly increased following 2 h of recovery, before returning to control levels following 36 h of recovery, possibly contributing to heart rate changes previously observed under these conditions.

TRIP8b-Independent Trafficking and Plasticity of Adult Cortical Presynaptic HCN1 Channels

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are subthreshold activated voltage-gated ion channels. In the cortex, these channels are predominantly expressed in dendrites where they significantly modify dendritic intrinsic excitability as well synaptic potential shapes and integration. HCN channel trafficking to dendrites is regulated by the protein, TRIP8b. Additionally, altered TRIP8b expression may be one mechanism underlying seizure-induced dendritic HCN channel plasticity. HCN channels, though, are also located in certain mature cortical synaptic terminals, where they play a vital role in modulating synaptic transmission. In this study, using electrophysiological recordings as well as electron microscopy we show that presynaptic, but not dendritic, cortical HCN channel expression and function is comparable in adult TRIP8b-null mice and wild-type littermates. We further investigated whether presynaptic HCN channels undergo seizure-dependent plasticity. We found that, like dendritic channels, wild-type presynaptic HCN channel function was persistently decreased following induction of kainic acid-induced seizures. Since TRIP8b does not affect presynaptic HCN subunit trafficking, seizure-dependent plasticity of these cortical HCN channels is not conditional upon TRIP8b. Our results, thus, suggest that the molecular mechanisms underlying HCN subunit targeting, expression and plasticity in adult neurons is compartment selective, providing a means by which pre- and postsynaptic processes that are critically dependent upon HCN channel function may be distinctly influenced.

Characterization of HCN and cardiac function in a colonial ascidian

Hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels generate the rhythmic beating of mammalian hearts. We identified an HCN homolog in the colonial ascidian Botryllus schlosseri, a nonvertebrate chordate which possesses a tubular heart that beats bidirectionally. Contractions initiate at one end of the heart and travel across the length of the organ, and these periodically reverse, suggesting the presence of two pacemakers, one on each side. We find that HCN expression is highly enriched in cells scattered throughout the myocardium. We functionally analyzed the role of HCN channels in heartbeat using the antagonists Cilobradine and Zatebradine, which decreased the heartbeat in a reversible manner. We also assessed the role of β-adrenoreceptors in regulating HCN function using the antagonist Metoprolol, which lowered heartbeat rate (HR), as well as the agonist Isoproterenol, which did not alter HR, but caused simultaneous beating, analogous to a fibrillation. Measurements of direction and velocity of blood flow by making use of a novel system to study heart function in model systems amenable to live imaging revealed a significant correlation between heartbeat arrhythmia and drug treatment, similar to that observed with the same drugs in vertebrates. These results suggest that the heart pacemaker in tunicates may be homologous to that in their vertebrate counterparts in both development and function.

Direct evidence for calcium conductance of hyperpolarization-activated cyclic nucleotide-gated channels and human native If at physiological calcium concentrations

The hyperpolarization-activated cyclic nucleotide-gated (HCN) current I(f)/I(HCN) is generally thought to be carried by Na(+) and K(+) under physiological conditions. Recently, Ca(2+) influx through HCN channels has indirectly been postulated. However, direct functional evidence of Ca(2+) permeation through I(f)/I(HCN) is still lacking. To possibly provide direct evidence of Ca(2+) influx through I(HCN)/I(f), we performed inside-out and cell-attached single-channel recordings of heterologously expressed HCN channels and native rat and human I(f), since Ca(2+)-mediated I(f)/I(HCN) currents may not readily be recorded using the whole-cell technique. Original current traces demonstrated HCN2 Ca(2+) inward currents upon hyperpolarization with a single-channel amplitude of -0.87+/-0.06 pA, a low open probability of 3.02+/-0.48% (at -110 mV, n=6, Ca(2+) 2 mmol/L), and a Ca(2+) conductance of 8.9+/-1.2 pS. I(HCN2-Ca2+) was significantly activated by the addition of cAMP with an increase in the open probability and suppressed by the specific I(f) inhibitor ivabradine, clearly confirming that Ca(2+) influx indeed was conducted by HCN2 channels. Changing [Na(+)] (10 vs. 100 mmol/L) in the presence or absence of 2 mmol/L Ca(2+) caused a simple shift of the reversal potential along the voltage axis without significantly affecting Na(+)/Ca(2+) conductance, whereas the K(+) conductance of HCN2 increased significantly in the absence of external Ca(2+) with increasing K(+) concentrations. The mixed K(+)-Ca(2+) conductance, however, was unaffected by the external K(+) concentration. Notably, we could also record hyperpolarization-activated Ca(2+) permeation of single native I(f) channels in neonatal rat ventriculocytes and human atrial myocytes in the presence of blockers for all known cardiac calcium conduction pores (Ca(2+) conductance of human I(f), 9.19+/-0.34 pS; amplitude, -0.81+/-0.01 pA; open probability, 1.05+/-0.61% at -90 mV). We directly show Ca(2+) permeability of native rat and, more importantly, human I(f) at physiological extracellular Ca(2+) concentrations at the physiological resting membrane potential. This might have particular implications in diseased states with increased I(f) density and HCN expression.

Sinus node dysfunction and hyperpolarization-activated (HCN) channel subunit remodeling in a canine heart failure model

The hyperpolarization-activated cation current I(f) contributes significantly to sinoatrial node pacemaker function and possibly to ectopic arrhythmogenesis. Little is known about the expression of corresponding hyperpolarization-activated cyclic nucleotide-gated (HCN) channel subunits in normal hearts and HCN remodeling by diseases, like congestive heart failure (CHF), associated with disturbances of cardiac rhythm. We assessed expression of HCN1, 2 and 4 in normal mongrel dogs and dogs subjected to 2-week ventricular tachypacing-induced CHF. Competitive RT-PCR, Western blot and immunohistochemistry were used to quantify HCN subunit mRNA and protein expression in the right atrium (RA) and sinoatrial node. CHF approximately doubled sinus node recovery time, indicating suppressed sinus node pacemaker function. HCN expression under control conditions was HCN4 > HCN2 >> HCN1. HCN2 and HCN4 expression was greater at both protein and mRNA levels in sinoatrial node than RA. CHF significantly decreased sinus node HCN expression at both mRNA and protein levels (HCN2 by 78% and 82%; HCN4 by 42% and 77%, respectively). RA HCN2 expression was unaltered by CHF, but HCN4 was significantly upregulated (by 209%). HCN4 is the dominant subunit in canine sinoatrial node and RA; strong sinus node HCN expression likely contributes to its pacemaker function; downregulation of HCN4 and HCN2 expression contribute to CHF-induced sinus node dysfunction; and upregulation of atrial HCN4 may help to promote atrial arrhythmia formation. These findings provide novel information about the molecular basis of normal and disease-related impairments of cardiac impulse formation.

MiRP1 Modulates HCN2 Channel Expression and Gating in Cardiac Myocytes

MinK-related protein (MiRP1 or KCNE2) interacts with the hyperpolarization-activated, cyclic nucleotide-gated (HCN) family of pacemaker channels to alter channel gating in heterologous expression systems. Given the high expression levels of MiRP1 and HCN subunits in the cardiac sinoatrial node and the contribution of pacemaker channel function to impulse initiation in that tissue, such an interaction could be of considerable physiological significance. However, the functional evidence for MiRP1/HCN interactions in heterologous expression studies has been accompanied by inconsistencies between studies in terms of the specific effects on channel function. To evaluate the effect of MiRP1 on HCN expression and function in a physiological context, we used an adenovirus approach to overexpress a hemagglutinin (HA)-tagged MiRP1 (HAMiRP1) and HCN2 in neonatal rat ventricular myocytes, a cell type that expresses both MiRP1 and HCN2 message at low levels. HA-MiRP1 co-expression with HCN2 resulted in a 4-fold increase in maximal conductance of pacemaker currents compared with HCN2 expression alone. HCN2 activation and deactivation kinetics also changed, being significantly more rapid for voltages between -60 and -95 mV when HA-MiRP1 was co-expressed with HCN2. However, the voltage dependence of activation was not affected. Co-immunoprecipitation experiments demonstrated that expressed HA-MiRP1 and HCN2, as well as endogenous MiRP1 and HCN2, co-assemble in ventricular myocytes. The results indicate that MiRP1 acts as a beta subunit for HCN2 pacemaker channel subunits and alters channel gating at physiologically relevant voltages in cardiac cells.