Role of hyperpolarization-activated cyclic nucleotide-gated channels in aging bladder phenotype.

OBJECTIVE: To assess the functional role of Hyperpolarization-activated cyclic nucleotide-gated gated channel (HCN) subtypes in the aging bladder phenotype characterized by diminished bladder volume sensation (BVS) with or without the detrusor instability (DI). METHODS: Expression of HCN subtypes was examined by quantitative RT-PCR and Western blot in aged male Fisher 344 rats (n = 15) and young rats (n = 15). Nocturnal urination and awake cystometry (CMG) were assessed in presence and absence of a steady state HCN channel blockade achieved with daily oral gavage of vehicle or Ivabradine (HCN blocker) 6 mg/kg for 7 days. RESULTS: The association of BVS with the age-related downregulation (~30%) of cAMP sensitive HCN1, HCN2 subtypes, and (~50%) upregulation of cAMP insensitive HCN3 subtype is evinced by the doubling in the mean urine volume of nocturnal voids (0.82 ± 0.22 mL vs 0.41 ± 0.12 mL; n = 10; p < 0.05) predicting an age-related rise in the micturition volume threshold (p < 0.0001) in CMG, which is raised further by Ivabradine treatment (p < 0.0005). Ivabradine also doubled non-voiding contractions (NVC) and maximum voiding pressure (MVP) in young and aged rats, respectively (p < 0.0001) to abolish the age-related, innate two -fold elevation in NVC not accompanied with MVP rise in untreated aged rats (p < 0.005). CONCLUSION: The age-related HCN downregulation is mechanistically linked to the exhibition of aging bladder phenotype with the manifestation of DI following steady state blockade of HCN channels in Ivabradine treated young rats. The amplification of MVP in aged rats mediated by FDA approved Ivabradine hints at potential repurposing opportunity in detrusor underactivity.

Deficiency of CXXC finger protein 1 leads to small changes in heart rate but moderate epigenetic alterations and significant protein downregulation of hyperpolarization-activated cyclic nucleotide-gated 4 (HCN4) ion channels in mice.

BACKGROUND: The normal cardiac rhythm is generated in the sinoatrial node (SAN). Changes in ionic currents of the SAN may cause sinus arrhythmia. CXXC finger protein 1 (Cfp1) is an epigenetic regulator that is involved in transcriptional regulation of multiple genes. OBJECTIVE: The purpose of this study was to explore whether Cfp1 controls SAN function through regulation of ion channel-related genes. METHODS: Electrophysiological study, patch clamp recording, reverse transcriptase polymerase chain reaction, optical mapping, chromatin immunoprecipitation, and immunofluorescence staining were performed to evaluate the function of SAN and underlying mechanism on Cfp1 heterozygous knockout (Cfp1+/-) mice. RESULTS: Heart rate was slower slightly and SAN recovery time was longer in Cfp1+/- mice than controls. Whole-cell patch-clamp recording showed that the firing rate of action potentials was reduced in Cfp1+/- mice. The density of If current was reduced by 66% in SAN cells of Cfp1+/- mice but the densities of ICa, ICa-L, and ICa-T were not changed. The hyperpolarization-activated cyclic nucleotide-gated 4 (HCN4) mRNA level in SAN tissue of Cfp1+/- mice was reduced. The HCN4 protein was significantly decreased in SAN cells and tissues after heterozygous deletion of Cfp1. Chromatin immunoprecipitation assay on cultured HL-1 cells demonstrated that Cfp1 was enriched in the promoter regions of HCN4. Knockdown of Cfp1 reduced H3K4 trimethylation, H3K9 acetylation, and H3K27 acetylation of HCN4 promoter region. CONCLUSION: Deficiency of Cfp1 leads to small changes in heart rate by moderate epigenetic modification alterations and significant protein downregulation of HCN4 ion channels in mice.

A circadian clock in the sinus node mediates day-night rhythms in Hcn4 and heart rate.

BACKGROUND: Heart rate follows a diurnal variation, and slow heart rhythms occur primarily at night. OBJECTIVE: The lower heart rate during sleep is assumed to be neural in origin, but here we tested whether a day-night difference in intrinsic pacemaking is involved. METHODS: In vivo and in vitro electrocardiographic recordings, vagotomy, transgenics, quantitative polymerase chain reaction, Western blotting, immunohistochemistry, patch clamp, reporter bioluminescence recordings, and chromatin immunoprecipitation were used. RESULTS: The day-night difference in the average heart rate of mice was independent of fluctuations in average locomotor activity and persisted under pharmacological, surgical, and transgenic interruption of autonomic input to the heart. Spontaneous beating rate of isolated (ie, denervated) sinus node (SN) preparations exhibited a day-night rhythm concomitant with rhythmic messenger RNA expression of ion channels including hyperpolarization-activated cyclic nucleotide-gated potassium channel 4 (HCN4). In vitro studies demonstrated 24-hour rhythms in the human HCN4 promoter and the corresponding funny current. The day-night heart rate difference in mice was abolished by HCN block, both in vivo and in the isolated SN. Rhythmic expression of canonical circadian clock transcription factors, for example, Brain and muscle ARNT-Like 1 (BMAL1) and Cryptochrome (CRY) was identified in the SN and disruption of the local clock (by cardiomyocyte-specific knockout of Bmal1) abolished the day-night difference in Hcn4 and intrinsic heart rate. Chromatin immunoprecipitation revealed specific BMAL1 binding sites on Hcn4, linking the local clock with intrinsic rate control. CONCLUSION: The circadian variation in heart rate involves SN local clock-dependent Hcn4 rhythmicity. Data reveal a novel regulator of heart rate and mechanistic insight into bradycardia during sleep.

HCN channels in the mammalian cochlea: Expression pattern, subcellular location, and age-dependent changes.

Neuronal diversity in the cochlea is largely determined by ion channels. Among voltage-gated channels, hyperpolarization-activated cyclic nucleotide-gated (HCN) channels open with hyperpolarization and depolarize the cell until the resting membrane potential. The functions for hearing are not well elucidated and knowledge about localization is controversial. We created a detailed map of subcellular location and co-expression of all four HCN subunits across different mammalian species including CBA/J, C57Bl/6N, Ly5.1 mice, guinea pigs, cats, and human subjects. We correlated age-related hearing deterioration in CBA/J and C57Bl/6N with expression levels of HCN1, -2, and -4 in individual auditory neurons from the same cohort. Spatiotemporal expression during murine postnatal development exposed HCN2 and HCN4 involvement in a critical phase of hair cell innervation. The huge diversity of subunit composition, but lack of relevant heteromeric pairing along the perisomatic membrane and axon initial segments, highlighted an active role for auditory neurons. Neuron clusters were found to be the hot spots of HCN1, -2, and -4 immunostaining. HCN channels were also located in afferent and efferent fibers of the sensory epithelium. Age-related changes on HCN subtype expression were not uniform among mice and could not be directly correlated with audiometric data. The oldest mice groups revealed HCN channel up- or downregulation, depending on the mouse strain. The unexpected involvement of HCN channels in outer hair cell function where HCN3 overlaps prestin location emphasized the importance for auditory function. A better understanding may open up new possibilities to tune neuronal responses evoked through electrical stimulation by cochlear implants.

HCN2 contributes to oxaliplatin-induced neuropathic pain by inducing spinal long-term potentiation via activation of NMDA receptor-mediated CaMKII signaling.

Our previous findings indicate that HCN2 contributes to oxaliplatin-induced neuropathic pain, but the mechanisms underlying the development of neuropathic pain are still unclear. Here, we found that the rat HCN2 levels significantly increased after high-frequency stimulation-induced long-term potentiation (LTP). Spinal local application of ZD7288 (a cyclic-nucleotide-gated-channel-specific inhibitor) prevented LTP induction after intraperitoneal injection of oxaliplatin. In addition, oxaliplatin administration induced spinal LTP via activation of the CaMKII-CREB cascade in the rat spinal dorsal horn. Moreover, we found that administration of oxaliplatin significantly increased the amplitude of excitatory postsynaptic currents and the number of action potentials, but these effects were attenuated by pretreatment with either CaMKII inhibitor KN-93 or NR2B antagonist Ro 25-6981. An increase in the phosphorylation of spinal N-methyl-d-aspartate (NMDA) receptor subunit 1 (NR1) after oxaliplatin administration was weakened by ZD7288 pretreatment. Administration of noncompetitive NMDA receptor antagonist MK-801 blocked oxaliplatin-evoked CaMKII-CREB cascade activation and prevented HCN2-mediated spinal-LTP induction in vitro and suppressed neuropathic-pain behaviors of rats. All these data suggest that HCN2 contributes to the development of neuropathic pain by inducing spinal LTP via activation of NMDA receptor-mediated CaMKII signaling.

Protein and surface expression of HCN2 and HCN4 subunits in mesocorticolimbic areas after cocaine sensitization.

The Ih is a mixed depolarizing current present in neurons which, upon activation by hyperpolarization, modulates neuronal excitability in the mesocorticolimbic (MCL) system, an area which regulates emotions such as pleasure, reward, and motivation. Its biophysical properties are determined by HCN protein expression profiles, specifically HCN subunits 1-4. Previously, we reported that cocaine-induced behavioral sensitization increases HCN2 protein expression in all MCL areas with the Ventral Tegmental Area (VTA) showing the most significant increase. Recent evidence suggests that HCN4 also has an important expression in the MCL system. Although there is a significant expression of HCN channels in the MCL system their role in addictive processes is largely unknown. Thus, in this study we aim to compare HCN2 and HCN4 expression profiles and their cellular compartmental distribution in the MCL system, before and after cocaine sensitization. Surface/intracellular (S/I) ratio analysis indicates that VTA HCN2 subunits are mostly expressed in the cell surface in contrast to other areas tested. Our findings demonstrate that after cocaine sensitization, the HCN2 S/I ratio in the VTA was decreased whereas in the Prefrontal Cortex it was increased. In addition, HCN4 total expression in the VTA was decreased after cocaine sensitization, although the S/I ratio was not altered. Together, these results demonstrate differential cocaine effects on HCN2 and HCN4 protein expression profiles and therefore suggest a diverse Ih modulation of cellular activity during cocaine addictive processes.

Downregulation of HCN1 channels in hippocampus and prefrontal cortex in methamphetamine re-exposed mice with enhanced working memory.

The hyperpolarization-activated cyclic-nucleotide-gated non-selective cation (HCN) channels play a potential role in the neurological basis underlying drug addiction. However, little is known about the role of HCN channels in methamphetamine (METH) abuse. In the present study, we examined the changes in working memory functions of METH re-exposed mice through Morris water maze test, and investigated the protein expression of HCN1 channels and potential mechanisms underlying the modulation of HCN channels by Western blotting analysis. Mice were injected with METH (1 mg/kg, i.p.) once per day for 6 consecutive days. After 5 days without METH, mice were re-exposed to METH at the same concentration. We found that METH re-exposure caused an enhancement of working memory, and a decrease in the HCN1 channels protein expression in both hippocampus and prefrontal cortex. The phosphorylated extracellular regulated protein kinase 1/2 (p-ERK1/2), an important regulator of HCN channels, was also obviously reduced in hippocampus and prefrontal cortex of mice with METH re-exposure. Meanwhile, acute METH exposure did not affect the working memory function and the protein expressions of HCN1 channels and p-ERK1/2. Overall, our data firstly showed the aberrant protein expression of HCN1 channels in METH re-exposed mice with enhanced working memory, which was probably related to the down-regulation of p-ERK1/2 protein expression.

Non-CpG methylation by DNMT3B facilitates REST binding and gene silencing in developing mouse hearts.

The dynamic interaction of DNA methylation and transcription factor binding in regulating spatiotemporal gene expression is essential for embryogenesis, but the underlying mechanisms remain understudied. In this study, using mouse models and integration of in vitro and in vivo genetic and epigenetic analyses, we show that the binding of REST (repressor element 1 (RE1) silencing transcription factor; also known as NRSF) to its cognate RE1 sequences is temporally regulated by non-CpG methylation. This process is dependent on DNA methyltransferase 3B (DNMT3B) and leads to suppression of adult cardiac genes in developing hearts. We demonstrate that DNMT3B preferentially mediates non-CpG methylation of REST-targeted genes in the developing heart. Downregulation of DNMT3B results in decreased non-CpG methylation of RE1 sequences, reduced REST occupancy, and consequently release of the transcription suppression during later cardiac development. Together, these findings reveal a critical gene silencing mechanism in developing mammalian hearts that is regulated by the dynamic interaction of DNMT3B-mediated non-CpG methylation and REST binding.

Hyperpolarization-activated cation and T-type calcium ion channel expression in porcine and human renal pacemaker tissues.

Renal pacemaker activity triggers peristaltic upper urinary tract contractions that propel waste from the kidney to the bladder, a process prone to congenital defects that are the leading cause of pediatric kidney failure. Recently, studies have discovered that hyperpolarization-activated cation (HCN) and T-type calcium (TTC) channel conductances underlie murine renal pacemaker activity, setting the origin and frequency and coordinating upper urinary tract peristalsis. Here, we determined whether this ion channel expression is conserved in the porcine and human urinary tracts, which share a distinct multicalyceal anatomy with multiple pacemaker sites. Double chromagenic immunohistochemistry revealed that HCN isoform 3 is highly expressed at the porcine minor calyces, the renal pacemaker tissues, whereas the kidney and urinary tract smooth muscle lacked this HCN expression. Immunofluorescent staining demonstrated that HCN(+) cells are integrated within the porcine calyx smooth muscle, and that they co-express TTC channel isoform Cav3.2. In humans, the anatomic structure of the minor calyx pacemaker was assayed via hematoxylin and eosin analyses, and enabled the visualization of the calyx smooth muscle surrounding adjacent papillae. Strikingly, immunofluorescence revealed that HCN3(+) /Cav3.2(+) cells are also localized to the human minor calyx smooth muscle. Collectively, these data have elucidated a conserved molecular signature of HCN and TTC channel expression in porcine and human calyx pacemaker tissues. These findings provide evidence for the mechanisms that can drive renal pacemaker activity in the multi-calyceal urinary tract, and potential causes of obstructive uropathies.

Therapeutic effect of astragaloside-IV on bradycardia is involved in up-regulating klotho expression.

AIMS: In order to determine whether klotho is involved in the therapeutic effects of Astragaloside-IV on bradycardia, we evaluated the effect of ASG-IV on klotho and the effect of klotho on HCN4 and If. MAIN METHODS: Administrating isoproterenol (5 mg/kg) for 15 days to establish a rat bradycardia model randomized SD rats into control, model (ISO) and ASG-IV (5 mg/kg/day) groups to explore the effect of ASG-IV on klotho. Rats were sacrificed on day 15 after heart rate and heart function were measured; SAN tissues were collected to measure the expression of klotho and HCN4. In vitro, neonatal rat myocardial cells were incubated with LPS for 24 h to inhibit the expression of HCN4 and incubated with LPS+ klotho to explore the effect of klotho on HCN4 expression. We also adopted full-patch-clamp technique to explore the effect of klotho on If. KEY FINDINGS: Heart rate in model group was significantly decreased (356.6±19.7 vs. 428.9±19.9 in control group, P<0.01) and ASG-IV can increase heart rate (401.4±12.0 vs. 356.6±19.7 in model group, P<0.01). The expression of klotho was also up-regulated (P<0.05). In vitro, after incubation with LPS for 24h, HCN4 expression was significantly decreased in neonatal rat myocardial cells (0.6±0.07 vs. 1.0, P<0.01) and If was significantly declined. Exogenous klotho showed protective effect on HCN4 expression (1.58±0.16 in ASG-IV group vs. 0.6±0.07 in LPS group, P<0.05) and If. SIGNIFICANCE: Klotho is involved in the treatment mechanism of ASG-IV.

Fluoxetine ameliorates cognitive impairments induced by chronic cerebral hypoperfusion via down-regulation of HCN2 surface expression in the hippocampal CA1 area in rats.

Chronic cerebral hypoperfusion (CCH) causes cognitive impairments and increases the risk of Alzheimer's disease (AD) and vascular dementia (VD) through several biologically plausible pathways, yet the underlying neurobiological mechanisms are still poorly understood. In this study, we investigated whether fluoxetine, a selective serotonin reuptake inhibitor (SSRI), could play a neuroprotective role against chronic cerebral hypoperfusion injury and to clarify underlying mechanisms of its efficacy. Rats were subjected to permanent bilateral occlusion of the common carotid arteries (two-vessel occlusion, 2VO). Two weeks later, rats were treated with 30 mg/kg fluoxetine (intragastric injection, i.g.) for 6 weeks. Cognitive function was evaluated by Morris water maze (MWM) and novel objects recognition (NOR) test. Long-term potentiation (LTP) was used to address the underlying synaptic mechanisms. Western blotting was used to quantify the protein levels. Our results showed that fluoxetine treatment significantly improved the cognitive impairments caused by 2VO, accompanied with a reversion of 2VO-induced inhibitory of LTP. Furthermore, 2VO caused an up-regulation of hyperpolarization-activated cyclic nucleotide-gated channel 2 (HCN2) surface expressions in the hippocampal CA1 area and fluoxetine also effectively recovered the disorder of HCN2 surface expressions, which may be a possible mechanism that fluoxetine treatment ameliorates cognitive impairments in rats with CCH.

Age-associated expression of HCN channel isoforms in rat sinoatrial node.

The expression of hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channel isoforms varies among species, cardiac tissues, developmental stages, and disease generation. However, alterations in the HCN channels during aging remain unclear. We investigated the protein expressions of HCN channel isoforms, HCN1-HCN4, in the sinoatrial nodes (SANs) from young (1-month-old), adult (4-month-old), and aged (30-month-old) rats. We found that HCN2 and HCN4 proteins were present in rat SAN using immunohistochemistry; therefore, we quantitatively analyzed their expression by Western blot. Aim to correlate protein expression and pacemaking function, specific blockade of HCN channels with 3 µmol/L ivabradine prolonged the cycle length in the intact rat heart. During the senescent process, the HCN2 and HCN4 protein levels declined, which was accompanied with a decreased effect of ivabradine on rat SAN automaticity. These results indicated the age-associated expression and relative function of HCN channel isoforms.

Development of a universal RNA beacon for exogenous gene detection.

Stem cell therapy requires a nontoxic and high-throughput method to achieve a pure cell population to prevent teratomas that can occur if even one cell in the implant has not been transformed. A promising method to detect and separate cells expressing a particular gene is RNA beacon technology. However, developing a successful, specific beacon to a particular transfected gene can take months to develop and in some cases is impossible. Here, we report on an off-the-shelf universal beacon that decreases the time and cost of applying beacon technology to select any living cell population transfected with an exogenous gene.

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

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. SIGNIFICANCE: 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.

PP2 prevents ß-adrenergic stimulation of cardiac pacemaker activity.

One of the main strategies for cancer therapy is to use tyrosine kinase inhibitors for inhibiting tumor proliferation. Increasing evidence has demonstrated the potential risks of cardiac arrhythmias (such as prolonged QT interval) of these drugs. We report here that a widely used selective inhibitor of Src tyrosine kinases, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2), can inhibit and prevent ß-adrenergic stimulation of cardiac pacemaker activity. First, in dissected rat sinus node, PP2 inhibited and prevented the isoproterenol-induced increase of spontaneous beating rate. Second, in isolated rat sinus node myocytes, PP2 suppressed the hyperpolarization-activated "funny" current (traditionally called cardiac pacemaker current, I(f)) by negatively shifting the activation curve and decelerating activation kinetics. Third, in isolated rat sinus node myocytes, PP2 decreased the Src kinase activity, the cell surface expression, and tyrosine phosphorylation of hyperpolarization-activated, cyclic nucleotide-modulated channel 4 (HCN4) channel proteins. Finally, in human embryonic kidney 293 cells overexpressing recombinant human HCN4 channels, PP2 reversed the enhancement of HCN4 channels by isoproterenol and inhibited 573x, a cyclic adenosine momophosphate-insensitive human HCN4 mutant. These results demonstrated that inhibition of Src kinase activity in heart by PP2 decreased and prevented ß-adrenergic stimulation of cardiac pacemaker activity. These effects are mediated, at least partially, by a cAMP-independent attenuation of channel activity and cell surface expression of HCN4, the main channel protein that controls the heart rate.

Overexpression of hyperpolarization-activated cyclic nucleotide-gated channels into the ventral tegmental area increases the rewarding effects of ethanol in UChB drinking rats.

BACKGROUND: A number of studies have shown that ethanol (EtOH) activates dopamine neurocircuitries and is self-administered into the ventral tegmental area (VTA) of the rat brain. In vitro and in silico studies have showed that hyperpolarization-activated cyclic nucleotide-gated (HCN) ionic channels on VTA dopamine neurons may constitute a molecular target of EtOH; however, there is no in vivo evidence supporting this assumption. METHODS: Wistar-derived University of Chile Drinking (UChB) rats were microinjected into the VTA with a lentiviral vector coding for rat HCN-2 ionic channel or a control vector. Four days after vector administration, daily voluntary EtOH intake was assessed for 30 days under a free-access paradigm to 5% EtOH and water. After EtOH consumption studies, the effect of HCN-2 overexpression was also assessed on EtOH-induced conditioned place preference (CPP); EtOH-induced locomotion, and EtOH-induced dopamine release in the nucleus accumbens (NAcc). RESULTS: Rats microinjected with the HCN-2 coding vector into the VTA showed (i) a ~2-fold increase in their voluntary EtOH intake compared to control animals, (ii) lentiviral-HCN-2-treated animals also showed an increased CPP to EtOH (~3-fold), (iii) a significant higher locomotor activity (~2-fold), and (iv) increased dopamine release in NAcc upon systemic administration of EtOH (~2-fold). CONCLUSIONS: Overexpression of HCN-2 ionic channel in the VTA of rats results in an increase in voluntary EtOH intake, EtOH-induced CPP, locomotor activity, and dopamine release in NAcc, suggesting that HCN levels in the VTA are relevant for the rewarding properties of EtOH.

Increased expression of HCN channels in the ventricular myocardium contributes to enhanced arrhythmicity in mouse failing hearts.

BACKGROUND: The efficacy of pharmacological interventions to prevent sudden arrhythmic death in patients with chronic heart failure remains limited. Evidence now suggests increased ventricular expression of hyperpolarization-activated cation (HCN) channels in hypertrophied and failing hearts contributes to their arrythmicity. Still, the role of induced HCN channel expression in the enhanced arrhythmicity associated with heart failure and the capacity of HCN channel blockade to prevent lethal arrhythmias remains undetermined. METHODS AND RESULTS: We examined the effects of ivabradine, a specific HCN channel blocker, on survival and arrhythmicity in transgenic mice (dnNRSF-Tg) expressing a cardiac-specific dominant-negative form of neuron-restrictive silencer factor, a useful mouse model of dilated cardiomyopathy leading to sudden death. Ivabradine (7 mg/kg per day orally) significantly reduced ventricular tachyarrhythmias and improved survival among dnNRSF-Tg mice while having no significant effect on heart rate or cardiac structure or function. Ivabradine most likely prevented the increase in automaticity otherwise seen in dnNRSF-Tg ventricular myocytes. Moreover, cardiac-specific overexpression of HCN2 in mice (HCN2-Tg) made hearts highly susceptible to arrhythmias induced by chronic ß-adrenergic stimulation. Indeed, ventricular myocytes isolated from HCN2-Tg mice were highly susceptible to ß-adrenergic stimulation-induced abnormal automaticity, which was inhibited by ivabradine. CONCLUSIONS: HCN channel blockade by ivabradine reduces lethal arrhythmias associated with dilated cardiomyopathy in mice. Conversely, cardiac-specific overexpression of HCN2 channels increases arrhythmogenicity of ß-adrenergic stimulation. Our findings demonstrate the contribution of HCN channels to the increased arrhythmicity seen in failing hearts and suggest HCN channel blockade is a potentially useful approach to preventing sudden death in patients with heart failure.

Testosterone enhances cardiomyogenesis in stem cells and recruits the androgen receptor to the MEF2C and HCN4 genes.

Since a previous study (Goldman-Johnson et al., 2008 [4]) has shown that androgens can stimulate increased differentiation of mouse embryonic stem (mES) cells into cardiomyocytes using a genomic pathway, the aim of our study is to elucidate the molecular mechanisms regulating testosterone-enhanced cardiomyogenesis. Testosterone upregulated cardiomyogenic transcription factors, including GATA4, MEF2C, and Nkx2.5, muscle structural proteins, and the pacemaker ion channel HCN4 in a dose-dependent manner, in mES cells and P19 embryonal carcinoma cells. Knock-down of the androgen receptor (AR) or treatment with anti-androgenic compounds inhibited cardiomyogenesis, supporting the requirement of the genomic pathway. Chromatin immunoprecipitation (ChIP) studies showed that testosterone enhanced recruitment of AR to the regulatory regions of MEF2C and HCN4 genes, which was associated with increased histone acetylation. In summary, testosterone upregulated cardiomyogenic transcription factor and HCN4 expression in stem cells. Further, testosterone induced cardiomyogenesis, at least in part, by recruiting the AR receptor to the regulatory regions of the MEF2C and HCN4 genes. These results provide a detailed molecular analysis of the function of testosterone in stem cells and may offer molecular insight into the role of steroids in the heart.