HCN4 Research Articles

Title: Dissociation-related behaviors in mice emerge from the inhibition of retrosplenial cortex parvalbumin interneurons

Dissociation, characterized by altered consciousness and perception, underlies multiple mental disorders, but the specific neuronal subtypes involved remain elusive. In mice, we find that dissociation-inducing doses of ketamine significantly inhibit retrosplenial cortex (RSC) parvalbumin interneurons (PV-INs), enhancing delta oscillations (1-3Hz) and delta-gamma phase-amplitude coupling (δ-γ PAC) and inducing dissociation-like behaviors. Optogenetic inhibition of RSC PV-INs triggers delta oscillations, δ-γ PAC, and some dissociation-like behaviors without ketamine. Furthermore, activation of RSC PV-INs or knockdown of the N-methyl-D-aspartate receptor subunit NR1 and the hyperpolarization-activated cyclic nucleotide-gated channel 1 (HCN1) in RSC PV-INs attenuates ketamine-induced delta oscillations, δ-γ PAC, and certain dissociation-like behaviors. These findings reveal that PV-INs regulate delta oscillations and δ-γ PAC and identify NR1 and HCN1 as ketamine targets in PV-INs that may cooperatively affect dissociation, possibly providing potential therapeutic targets for dissociative symptoms.

The Role of HCN4 Variants in Human Systolic Heart Failure and Protein Interaction Network

Introduction: An elevated heart rate (HR) results in adverse outcomes in human systolic heart failure with a sinus rhythm. Genetic variants may alter HCN4 interactions with regulatory proteins and increase HR. This study aimed to generate a protein interaction network (PIN) associated with elevated HR and to determine HCN4 gene variants (718G>A, 1571G>A, and 2648C>G) in patients with systolic heart failure, sinus rhythm, and elevated heart. Methods: STRING protein database was used to generate a PIN. Laboratory exploration was performed to identify HCN4 gene variants in patients with systolic heart failure using PCR and DNA sequencing. Results: PIN revealed eight nodes and 13 edges. STRING functional enrichment showed the essential proteins (ADRB1 and HCN channels) involved in HR regulation. GNAS and ADCY1 contributed to the regulation process. HCN4 gene variants (718G>A, 1571G>A, and 2648C>G) alter the properties of HCN4 channel. STRING scores of protein-protein interactions that involved HCN4, ADRB1, GNAS, and ADCY1 were high (in the range of 0.879-0.979). The three gene variants were evaluated in 49 study participants with HR≥70 bpm after 10 mg bisoprolol therapy. However, only 718G>A was identified in three of 49 patients. Conclusion: PIN revealed that the three essential proteins associated with HCN4 channels in elevating HR were ADRB1, GNAS, and ADCY1. Only HCN4 718G>A was found in three out of 49 patients with systolic heart failure, sinus rhythm, and increased HR, according to a laboratory investigation on HCN4 gene variants

A Novel Loss of Function Variant in HCN1 Gene Underlies Early Infantile Epileptic Encephalopathy 24 [EIEE24]

Background: Early infantile epileptic encephalopathy (EIEE) is a rare neurological condition characterized by frequent seizures in the early stages of life, resulting in severely impaired cognitive and motor development. Although the specific causes of EIEE remain unknown, one of the primary causes is gene pathogenicity (even in the absence of consanguinity). Hyperpolarization-activated cyclic nucleotide-gated channels (HCNs) are essential for proper brain function. They are regulated by multiple genes, and mutations in these genes induce channel malfunction, which can result in various severe conditions, including EIEE. Herein, we have reported a patient presenting hallmarks of EIEE. Methods: The patient underwent clinical, radiographic, and genetic analysis. A thorough clinical examination and molecular study were conducted utilizing whole exome sequencing and Sanger sequencing. Results: Whole exome sequencing of the proband revealed a novel de novo nonsynonymous/nonsense variant (c.1468A>T; (p.Lys490Ter) in exon 6 of the HCN1 gene. This variant may cause channel dysfunction via nonsynonymous/nonsense-mediated decay or aberrant protein, which may be associated with EIEE phenotypes. Conclusions: This evidence backs the idea that HCN1 has a vital role in brain development and lose of function can cause a range of debilitating conditions. Still, the functional characterization study of the HCN1 variants will be the fundamental tool for a better understanding of EIEE in the near future.

Atrial standstill in a young patient with ischemic stroke associated with inheritance of a novel HCN4 mutation.

We present a case of HCN4 gene mutation presenting with atrial standstill and stroke in the young.

Abstract P351: Obesity in Early Life Leads to Endothelial and Erectile Dysfunction in Young Adult Rats.

Background: Erectile dysfunction (ED) is largely associated with obesity, however, the consequences of early life obesity for ED in young adulthood are unknown. We hypothesize that obesity induced by preweaning overfeeding impairs vascular and erectile function and affects blood pressure in young adult male rats (5-month-old). Methods: At postnatal day (PND) 1, Wistar rats were divided into a normal litter (NL, 5 female + 5 male pups/dam) or small litter (SL, 2 male + 1 female pup/dam) during lactation. After weaning, SL pups had increased body weight. Next, rats from both groups returned to normal size colonies (4-5 rats per cage) and were fed standard chow. At PND160, we evaluated body weight and fat pad deposition, mean arterial pressure (MAP) by tail-cuff, and pudendal artery (PA) and corpus cavernosum (CC) reactivity. Concentration-response curves to phenylephrine (PE) or acetylcholine (ACh) were performed in both preparations. In the CC, contraction and relaxation to electrical field stimulation (EFS) were obtained. Concentration-response curves were analyzed using non-linear regression analysis. RNA sequencing (RNAseq) analyses were performed in both PA and CC. Data were analyzed by Student’s t-test and presented as mean ± S.E.M. Statistical significance was set at p<0.05. Results: At PND160 no differences in body weight were observed in SL (594±14g) compared to NL (559±13g), however, there was an increase in fat deposition in SL (retroperitoneal fat: NL 7.47±0.69g vs SL 11.25±1.23g; perigonadal fat: NL 10.05±0.55g vs SL 13.24±1.16g). MAP was higher in SL (120.5±1.20 mmHg vs 114.9±0.58 NL). In the PA, ACh-induced relaxation was reduced in SL (Emax: 28±6%) vs NL (Emax: 68±5.5%), with no changes in the contraction to PE. In the CC, EFS-induced relaxation was significantly decreased in SL rats compared to NL rats (16 Hz: NL 3.06 ± 0.28% vs SL 2.39 ± 0.53%). RNAseq revealed that, in PA from SL rats, 289 and 224 genes were down and upregulated, respectively, while in the CC from SL rats, 115 genes were downregulated, and 110 genes were upregulated. Among them, hcn2 gene (which encodes HCN2, the hyperpolarization-activated cyclic nucleotide-gated potassium and sodium channel 2) was strongly downregulated in both PA and CC, and this may affect mechanotransduction. Conclusion: Our data shows that early-life obesity causes long-lasting vascular and cavernous damage that might be associated with endothelial and erectile dysfunction in young adulthood.

(005) OBESITY IN EARLY-LIFE CAUSES LONG-TERM INFLAMMATION AND WNT SIGNALING PATHWAY DOWNREGULATION IN PUDENDAL ARTERIES OF YOUNG ADULT RATS: THE EARLY ONSET OF CELL SENESCENCE IN THE DEVELOPMENT OF ERECTILE DYSFUNCTION IN MALE RATS

Abstract Introduction Erectile dysfunction (ED) is largely associated with obesity, however, the consequences of early-life obesity for ED in young adulthood are unknown. Objective This study tested the hypothesis that obesity induced by preweaning overfeeding causes long-term metabolic damages that lead to inflammation, impaired vascular and erectile function, and affect blood pressure in young adult male rats (5-month-old). Methods At postnatal day (PND) 1, Wistar rats were divided into a normal litter (NL, 5 female +5 male pups/dam) or a small litter (SL, 2 male +1 female pup/dam) during lactation. After weaning, SL pups had increased body weight. Next, rats from both groups returned to normal size colonies (4-5 rats per cage) and were fed standard chow. At PND160, we evaluated body weight and fat pad deposition, mean arterial pressure (MAP) by tail-cuff, and pudendal artery (PA) and corpus cavernosum (CC) reactivity. Concentration-response curves to phenylephrine (PE) or acetylcholine (ACh) were performed in both preparations. In the CC both contraction and relaxation to electrical field stimulation (EFS) were obtained. Concentration-response curves were analyzed using non-linear regression analysis. A cytokine array was performed in plasma. RNA sequencing (RNAseq) analyses were performed in both PA and CC. Data was analyzed by Student’s t-test and presented as mean ± S.E.M. Statistical significance was set at p < 0.05. Results At PND160 no differences in body weight were observed in SL (594 ± 14 g) compared to NL (559 ± 13 g), however, there was an increase in fat deposition in SL (retroperitoneal fat: NL 7.47 ± 0.69 g vs SL 11.25 ± 1.23 g; perigonadal fat: NL 10.05 ± 0.55 g vs SL 13.24 ± 1.16 g). MAP was higher in SL (120.5 ± 1.20 mmHg vs 114.9 ± 0.58 NL). In the PA, ACh-induced relaxation was reduced in SL (Emax: 28 ± 6%) vs NL (Emax: 68 ± 5.5%), with no changes in the contraction to PE. In the CC, EFS-induced relaxation was significantly decreased in SL rats compared to NL rats (16 Hz: NL 3.06 ± 0.28% vs SL 2.39 ± 0.53%). The cytokine array demonstrated increased plasma levels of pro-inflammatory cytokines associated with metabolic dysfunction and aging, including CCL5, Lipocalin, MCP-1, CX3CL1, and MMP2. RNAseq revealed that in PA from SL rats, 289 and 224 genes were down and upregulated, respectively, while in the CC from SL rats, 115 genes were downregulated, and 110 genes were upregulated. Among them, the hcn2 gene (which encodes HCN2, the hyperpolarization-activated cyclic nucleotide-gated potassium and sodium channel 2) was strongly downregulated in both PA and CC, which may affect mechanotransduction. In addition, enriched gene ontology revealed the downregulation of the Wnt signaling pathway in PA, which is associated with the early onset of cell senescence. Conclusions Our data shows that, in rats, early-life obesity has long-term consequences leading to endothelial and cavernosal dysfunction observed in young adulthood and this might be linked to metabolic dysfunction, inflammation, and premature cell senescence. Disclosure No.

Genetic aspects associated with increased risk of developing atrial fibrillation

Abstract Atrial Fibrillation (AF) is the most common sustained arrhythmia in clinical practice with increasing prevalence and association with increased morbidity and mortality. Studies have suggested a greater contribution of the genetic factor to the etiopathogenesis of arrhythmia, which could indicate up to 40% more risk for the individual who has a family member with the arrhythmia, and later in the form of single nucleotide polymorphisms (SNPs). Objective To verify the association of polymorphisms in the PITX2, HCN4, CAV1 and KCNN3 genes with the occurrence of AF. Method The study is cross-sectional with group comparison, prospective with a quantitative approach. 505 randomly selected subjects were included (357 cases and 148 controls). The real-time PCR methodology was used, using the TAQMAN® system (Thermo scientific, CA, USA). Result The sample was matched by sex and age. The most common type of AF was permanent (69%) followed by paroxysmal (23%). When comparing the groups selected for CAV1, statistical significance was found for AA vs AG genotypes (p 0.003; OR= 2.2; CI 1.29-3.7). For the HCN4 gene allele and genotypes A vs G (p 0.024; OR= 0.66; CI 0.47-0.9) and AA vs AG (p 0.014, OR= 0.57; CI 0.37-0.88), respectively. Regarding the comparison of the dominant and recessive genetic model of each gene, the following models/genes presented statistical significance: in the genotypic model of the CAV1 gene (AA vs AG vs GG) p 0.013, as well as the same model applied to the HCN4 gene (AA vs AG vs GG) (p 0.03); when comparing the dominant models, the HCN4 gene (AA vs AG/GG) presented (p 0.01; OR=0.58; 95% CI 0.38 – 0.88), with the recessive model of the CAV1 gene (AA vs AG/GG) with (p 0.008; OR=2.0; 95% CI: 1.19 – 3.34). Regarding allelic associations between the two groups, a greater chance of susceptibility to developing AF was observed in patients who had the "G" allele (25.6%; p 0.024; OR= 0.66, CI 0.47-0.9) in the HCN4 gene polymorphism. Polymorphisms in the CAV1 (rs3807989) and HCN4 (rs7164883) genes were also included in the multivariate analysis with maintenance of the risk factor for AF. However, only HCN4 rs7164883 showed statistical significan, thus showing it to be an independent variable regarding the probability of the patient with this polymorphism presenting AF. The present study showed a significant association with a greater chance of susceptibility to developing atrial fibrillation in patients with a polymorphism in the CAV1 gene (rs3807989) in a univariate and multivariate model and for patients with the HCN4 gene SNP (rs7164883). Conclusion Therefore, the results revealed an association between the HCN4 gene polymorphism rs7164883 and susceptibility to the development of AF, suggesting that the presence of the G allele may be one of the genetic factors related to the arrhythmia. The CAV1 rs3807989 gene polymorphism also seemed to suggest a risk for the development of AF, also relating the G allele.

HCN2-based biological pacing in a modified rat AV-block model

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): Eurostars - Heartpace 114245 Background Electronic pacemakers have several important shortcomings which are difficult to overcome due to their hardware-based design. Gene therapy-based biological pacemakers may provide a different platform which can overcome these limitations. Such biological pacemaker strategies center around overexpression of the pacemaker ion channel HCN2, either alone or with other transgenes to potentiate pacemaker performance, such as the skeletal muscle sodium channel SkM1. In order to develop clinically applicable pacemaker gene therapies further engineering is needed, which would benefit from a reliable small animal model. At present it is unknown if the rat AV block model can be used to evaluate ion channel-based biological pacing. Aim In the present study, we aim to validate a modified rat AV-block model for the evaluation of HCN2 and SKM1-based biological pacing. Methods AV-block was generated in female rats by epicardially applied needle ablation of the AV-nodal region, as previously described (N.K. ). Complete AV-block was confirmed 7 days post-ablation by loss of P-wave and QRS-complex association on the body surface ECG. Seven days post-ablation, 3 groups of animals were injected into the left ventricular apex with Ad5-TNNT2-HCN2, Ad5-TNNT2-SkM1 or saline (control). 14 days post-ablation, ECG analysis was performed during anesthesia under baseline and after i.v. administration of isoprenaline. Upon completion of functional testing, tissue samples were harvested followed by gene expression analysis using qPCR. Results Injection of Ad5-TNNT2-HCN2, Ad5-TNNT2-SkM1 or saline did not result in ectopic activation patterns in baseline conditions 7 days after injection. Administration of isoprenaline results in reversal of the QRS complexes 90% of total measurement time in Ad5-TNNT2-HCN2 injected animals. Saline and Ad5-TNNT2-SkM1 showed no change in activation pattern upon isoprenaline administration. Gene expression analysis revealed expression of HCN2 in the injection site following Ad5-TNNT2-HCN2 injection, with minimal spread to non-target regions. However, this analysis also showed Ad5-TNNT2-SkM1 did not express well in this context. Conclusion Our modified rat AV-block model, when administered with isoprenaline, recapitulated expected biological pacemaker activity of HCN2 gene delivery. However, lack of effective expression of SkM1 renders direct comparison of these relevant transgenes difficult. Nevertheless, we expect this rat AV-block model can provide a valuable tool for the evaluation of novel ion channel-based biological pacemaker strategies.

TBX3 transfection and nodal signal pathway inhibition promote differentiation of adipose mesenchymal stem cell to cardiac pacemaker-like cells.

Mesenchymal stem cells (MSCs) are known as one of the best candidate cells to produce cardiac pacemaker-like cells (CPLCs). Upregulation of TBX3 transcription factor and inhibition of the nodal signal pathway have a significant role in the formation of cardiac pacemaker cells such as sinoatrial and atrioventricular nodes, which initiate the heartbeat and control the rhythm of heart contractions. This study aimed to confirm the effects of transfection of TBX3 transcription factor and inhibition of the nodal signal pathway on differentiating adipose-derived MSCs (AD-MSCs) to CPLCs. AD-MSCs were characterized using flow cytometry and three-lineage differentiation staining. The transfection of TBX3 plasmid was carried out using lipofectamine, and inhibition of the nodal signal pathway was done using the small-molecule SB431542. The morphology of the cells was observed using a light microscope. Pacemaker-specific markers, including TBX3, Cx30, HCN4, HCN1, HCN3, and KCNN4, were evaluated using the qRT-PCR method. For protein level, TBX3 and Cx30 were evaluated using ELISA and immunofluorescence staining. The electrophysiology of cells was evaluated using a patch clamp. The TBX3 expression in the TBX3, SM, and TBX + SM groups significantly higher (p < 0.05) compared to the control group and cardiomyocytes. The expression of Cx40 and Cx43 genes were lower in TBX3, SM, TBX + SM groups. In contrast, Cx30 gene showed higher expression in TBX3 group. The expression HCN1, HCN3, and HCN4 genes are higher in TBX3 group. The transfection of TBX3 and inhibition of the nodal signal pathway by small-molecule SB431542 enhanced differentiation of AD-MSCs to CPLCs.

The Perioperative Period of Heart Transplantation Is Affected by Thyroid Hormone Status.

Background: Orthotopic heart transplantation (HTx) is a long-term surgical therapeutic approach for patients with end-stage heart failure. The objective of the present study was to uncover associations between altered thyroid hormone (TH) status and adverse outcomes after HTx. Methods: In this prospective, single-center cohort study, 283 patients underwent HTx between 2013 and 2020 at the Heart and Vascular Center of Semmelweis University in Hungary. We measured serum free triiodothyronine (fT3), free thyroxine (fT4), and thyrotropin (TSH) pre- and postoperatively. TaqMan qPCR was used to measure type 2 deiodinase and type 3 deiodinase mRNA (Dio2 and Dio3, respectively) levels from the diseased heart bioptates. To assess the local TH action of the heart, mRNA levels of Hcn2 and Myh7 were measured in a subgroup of patients receiving extracorporeal membrane oxygenation (ECMO) postoperatively. Groups were compared using nonparametric tests. Cox regression analysis and logistic regression test were used to investigate the outcomes. The connection between serum TH parameters and cardiac gene expressions was assessed using linear regression. Results: Serum TSH (p = 0.009), fT3 (p < 0.001), and fT4 (p < 0.001) levels were lower after HTx than preoperatively. Levothyroxine (LT4) administered to donors was associated with better survival after 30 days (p = 0.049). LT4 replacement given to recipients after HTx was associated with better survival after 30 days (p = 0.018), 1 year (p = 0.002), and 2 years (p = 0.001). Dio3 mRNA level was significantly increased in patients who were treated with ECMO (p = 0.026), left ventricular assist device (LVAD) (p = 0.008), and biventricular assist device (BiVAD) (p = 0.013) preoperatively, and ECMO (p = 0.042) postoperatively, compared with those who did not require any type of mechanical circulatory support (MCS). We found no significant difference in the expression of the Hcn2 and Myh7 marker genes between patients on postoperative ECMO and those without MCS, and neither did they correlate with serum hormone levels (p = 0.519 and p = 0.056, respectively). Conclusions: We conclude that TH status plays an important role in HTx patients, and monitoring of TH status in the perioperative period may contribute to improved treatment outcomes. Our findings require independent confirmation in a randomized controlled clinical trial.

Characterization of HCN channel subtypes at hypoglossal motoneurons throughout postnatal maturation in mice

The inspiratory phase of breathing includes a stiffening of the upper airway to decrease airway resistance and is mediated by excitatory motor output from hypoglossal motoneurons (XII MNs). This excitation decreases from wakefulness to sleep. This decrease in excitation is in large part due to increasing acetylcholine levels, which activate muscarinic acetylcholine receptors (mAChRs) that have a net inhibitory effect in adult preparations. By contrast, activation of mAChRs in neonatal rodent preparations has a net effect of potentiating inspiratory burst amplitude. Therefore, characterizing changes in muscarinic effects at XII MNs across postnatal maturation will help elucidate the mechanism underlying the shift from muscarinic excitation to inhibition. mAChRs modulate several effector ion channels including the hyperpolarization activated cyclic nucleotide gated (HCN) channel. HCN channels give rise to I h, a mixed cation current activated at hyperpolarized membrane potentials. I h additionally shows upregulation at XII MNs with postnatal maturation. Since there are four HCN subtypes (HCN1-4), the reported changes in I h may be caused by changes in HCN channel subtype levels. HCN1 and HCN2 are most prominent in adult XII MNs, whereas the expression of HCN1-4 in neonates is unknown. Thus, the purpose of this research is to characterize changes in HCN gene and channel expression levels at XII MNs across postnatal maturation. We hypothesis that both HCN1 and HCN2 subtypes will increase with postnatal maturation, and that HCN2 has higher levels than HCN1. To test this hypothesis, we used neuroanatomical techniques to evaluate changes in expression patterns of the four HCN proteins at the XII nuclei across postnatal maturation in CD-1 mice. We performed double-labeled immunofluorescence experiments against HCN1 and HCN2 and co-labeled with choline acetyltransferase (ChAT) on 20μm transverse brainstem slices across six postnatal age groups under identical conditions: P0-P1, P4-P6, P9-P10, P12-P14, P17-P19, and adult. Images of the XII nuclei were collected using a confocal microscope. ImageJ was used to determine the average XII MN HCN subtype intensity across postnatal maturation. Preliminary data suggest that HCN1 (n=2) channels undergo a decrease in expression at P17-19 before a final increase in adulthood (P0-1= 97-100%, P4-6= 92-100%, P9-10= 85-95%, P12-14= 77-97%, P17-19= 61-67%, adult= 71-82%). By contrast, preliminary data suggest that HCN2 (n=2) expression intensity remained relatively consistent with a decrease in labeling intensity at P4-6 (P0-1= 97-100%, P4-6=62-84%, P9-10= 45-95%, P12-14= 67-90%, P17-19= 50-93%, adult= 75-100%). These modest changes in labeling intensity of HCN channel subtypes may not contribute to the observed shift of mAChR modulation at XII MNs from excitation to inhibition with postnatal maturation. Biomedical Sciences, College of Graduate Studies; Department of Physiology, College of Graduate Studies, Midwestern University; NIH/NHLBI Funding: R15HL148870. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Investigation of Some Ion Channel Expressions in Cochlear Nucleus of Tinnitus Induced Rats

Aim: The aim of this study is to gain a better understanding of how certain ion channels play a role in the molecular mechanisms of salicylate- and noise-induced tinnitus. Method: The present study was conducted on thirty-two, 4-month-old, male Wistar Albino rats. Rats were equally divided into four groups; two experimental groups and two control groups. The assessment of tinnitus was based on a behavioral test which was modified from the conditional suppression method. Tinnitus was induced by sodium salicylate administration and noise exposure in rats in which the suppression ratios were zero (0). All animals in both experimental and control groups were decapitated in deep anaesthesia for 2 h after salicylate or saline administration and noise exposure, consecutively. Tissues from the left and right cochlear nucleus were dissected immediately in ice-cold RNA later (Invitrogen). Before reverse transcription, the RNA pools were arranged. Quantitative changes in HCN1, HCN2, HCN4, SCN1A, SCN2A1, SCN3A, TRPM2, TRPM7 and GAPDH mRNA expressions in the cochlear nucleus in both experimental and control groups were examined by quantitative real-time PCR method. Statistical data were analysed using the SPSS 21 program (Version 21.0, SPSS Inc., Chicago, IL, USA) with the Kruskal-Wallis and Mann-Whitney U tests. Results: Fold changes in the expression levels of SCNA1, SCN2A1, SCN3A, TRPM2, TRPM7, CACNA1B, HCN1, HCN2 and HCN4 genes in both salicylate-induces tinnitus (SAT) and noise-induced tinnitus (NT) groups compared with the control group. According to these data, it is seen that the mRNA levels of all genes are lower in the cochlear nucleus area of the rats in both SAT and NT groups than in the control group. Considering each of these genes in NT group: SCNA1, SCN3A, TRPM7 genes slightly decreased; SCN2A1, TRPM2, HCN1 and HCN4 genes slightly increased compared with the SAT group. For HCN2 gene fold changes were nearly the same in the NT and SAT groups. Conclusion: The findings of this study suggest that tinnitus generation may be closely related to alterations in several key ion channel families activity including voltage-gated calcium channels, hyperpolarization-activated cyclic nucleotide–gated (HCN) channels, transient receptor potential (TRP) channels, voltage-gated sodium channels within the CN, specifically in response to salicylate-induced and noise-induced tinnitus models.

Characterization of hyperpolarization-activated cyclic nucleotide-gated channels in oligodendrocytes.

Mature oligodendrocytes (OLG) are the myelin-forming cells of the central nervous system. Recent work has shown a dynamic role for these cells in the plasticity of neural circuits, leading to a renewed interest in voltage-sensitive currents in OLG. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels and their respective current (Ih) were recently identified in mature OLG and shown to play a role in regulating myelin length. Here we provide a biochemical and electrophysiological characterization of HCN channels in cells of the oligodendrocyte lineage. We observed that mice with a nonsense mutation in the Hcn2 gene (Hcn2ap/ap) have less white matter than their wild type counterparts with fewer OLG and fewer oligodendrocyte progenitor cells (OPCs). Hcn2ap/ap mice have severe motor impairments, although these deficits were not observed in mice with HCN2 conditionally eliminated only in oligodendrocytes (Cnpcre/+; Hcn2F/F). However, Cnpcre/+; Hcn2F/F mice develop motor impairments more rapidly in response to experimental autoimmune encephalomyelitis (EAE). We conclude that HCN2 channels in OLG may play a role in regulating metabolism.

Identification and Analysis of Genes Related to Testicular Size in 14-Day-Old Piglets.

The RNA-Seq technology was used to screen the key genes that affect the early development of the testes of Duroc × Landrace × Yorkshire piglets, to determine the regulatory pathway and provide reference for subsequent reproductive performance research, breeding, and other production practices. This study selected 14-day-old Duroc × Landrace × Yorkshire piglets as the trial animals. Testes from piglets with similar weights and no pathological changes were divided into small testis (ST) and large testis (LT) groups, and the RNA-Seq screening of differentially expressed genes (DEGs) was performed to find candidate genes and regulatory pathways related to early testicular development. The results show that 570 DEGs were found in the ST and LT groups, with 281 upregulated and 289 downregulated. The DEGs were mainly enriched on 47 gene ontology (GO) functional items. The Kyoto encyclopedia of genes and genotypes (KEGG) enrichment analysis found that there were 44 significantly enriched KEGG signal pathways, and the regulation of testicular development mainly focused on the arachidonic acid metabolism, Wnt signaling pathway and GnRH secretion pathways. The PTGES, SFRP1, SPP1, PLA2G4E, KCNJ5, PTGS2, and HCN1 genes were found to be as closely related to the testicular development of these Duroc × Landrace × Yorkshire piglets, and the differential gene expression was consistent with the real-time quantitative reverse transcription PCR (real-time qRT-PCR) validation results. This study was validated by high-throughput sequencing analysis and real-time qRT-PCR, and showed that the PTGES, SFRP1, SPP1, PLA2G4E, KCNJ5, PTGS2, and HCN1 genes may be involved in the regulation of germ cell development, spermatogenesis and semen traits. These should be further studied as candidate genes for early testicular development and reproductive trait regulation in boars.

Brugada syndrome: variability of clinical and genetic characteristics

AIM: To evaluate the clinical characteristics of patients with diverse genetic variants of Brugada syndrome.&#x0D; MATERIALS AND METHODS: 24 patients (17 male and 7 female) aged 18 to 55 years (median age 32.5 [20; 42] years) with a pattern of Brugada syndrome on electrocardiogram were observed for 3 years. From their ECGs, a type 1 pattern was found in 9 (37.5%) of these patients, type 2 pattern in 14 (58.3%) and type 3 pattern only in 1 patient. The clinical and instrumental study included 12-lead electrocardiogram, 24-hour Holter electrocardiogram monitoring, provocative drug test with intravenous administration of sodium channel blockers (novocainamide), electrophysiologic study according to indications, genealogical history collection and family history of sudden cardiac death, transthoracic echocardiography and cardiac magnetic resonance imaging to detect structural myocardial changes. High-throughput sequencing was utilized to search for mutations in genes linked to the onset of channelopathies and other inherited rhythm disorders.&#x0D; RESULTS: In 15 (62.5%) of the 24 probands included in the study, variants of the nucleotide sequence of pathogenicity classes III–V according to The American College of Medical Genetics and Genomics criteria (2015) were found in genes encoding sodium (SCN5A, SCN10A) and potassium (KCNE3, KCNJ2, KCNJ8, KCNA5) channels, as well as in HCN4 and SNTA1 genes linked with these channels. Moreover, 3 variants were identified in ANK2 gene associated with ankyrinopathies, and 3 variants in DSP and DES genes connected with arrhythmogenic right ventricular cardiomyopathy. Four genetic variants in SCN5A gene were of pathogenicity classes IV and V, the rest were variants of uncertain clinical significance (class III). Six (40.0%) of the 15 genotype-positive patients had several genetic variants. The most severe form of the disease, manifested by the development of ventricular fibrillation with successful resuscitation and subsequent cardioverter-defibrillator implantation, was observed in patients with mutations in SCN5A, SCN10A genes. Recurrent syncope, polymorphic ventricular tachycardia induced by programmed ventricular stimulation during electrophysiologic study, followed by cardioverter-defibrillator implantation were observed in patients with variants KCNJ8 and HCN4, DES and MYH11. In 2 patients with clinical manifestations, no mutations were identified. 13 (54.2%) patients were asymptomatic, while 3 of them had pathogenic and likely pathogenic mutations in SCN5A gene, as well as variants of uncertain clinical significance.&#x0D; CONCLUSION: Thus, this study examined various genetic variants in patients with Brugada syndrome based on their clinical manifestation. The impact of the genotype on the Brugada syndrome phenotype is not unambiguous. The most severe form of the disease with the development of ventricular fibrillation and successful resuscitation with subsequent cardioverter-defibrillator implantation was observed mainly in patients with variants in several genes (SCN5A and JUP, KCNJ8 and HCN4, DES and MYH11). This substantiate the idea that Brugada syndrome, along with monogenic, may also have a polygenic nature of the disease, in which the clinical phenotype is determined by variants in respective genes linked to the onset of cardiovascular disorders.

The Action Potential Clamp Technique as a Tool for Risk Stratification of Sinus Bradycardia Due to Loss-of-Function Mutations in HCN4: An In Silico Exploration Based on In Vitro and In Vivo Data.

These days, in vitro functional analysis of gene variants is becoming increasingly important for risk stratification of cardiac ion channelopathies. So far, such risk stratification has been applied to SCN5A, KCNQ1, and KCNH2 gene variants associated with Brugada syndrome and long QT syndrome types 1 and 2, respectively, but risk stratification of HCN4 gene variants related to sick sinus syndrome has not yet been performed. HCN4 is the gene responsible for the hyperpolarization-activated 'funny' current If, which is an important modulator of the spontaneous diastolic depolarization underlying the sinus node pacemaker activity. In the present study, we carried out a risk classification assay on those loss-of-function mutations in HCN4 for which in vivo as well as in vitro data have been published. We used the in vitro data to compute the charge carried by If (Qf) during the diastolic depolarization phase of a prerecorded human sinus node action potential waveform and assessed the extent to which this Qf predicts (1) the beating rate of the comprehensive Fabbri-Severi model of a human sinus node cell with mutation-induced changes in If and (2) the heart rate observed in patients carrying the associated mutation in HCN4. The beating rate of the model cell showed a very strong correlation with Qf from the simulated action potential clamp experiments (R2 = 0.95 under vagal tone). The clinically observed minimum or resting heart rates showed a strong correlation with Qf (R2 = 0.73 and R2 = 0.71, respectively). While a translational perspective remains to be seen, we conclude that action potential clamp on transfected cells, without the need for further voltage clamp experiments and data analysis to determine individual biophysical parameters of If, is a promising tool for risk stratification of sinus bradycardia due to loss-of-function mutations in HCN4. In combination with an If blocker, this tool may also prove useful when applied to human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) obtained from mutation carriers and non-carriers.

Functional Characterization of the A414G Loss-of-Function Mutation in HCN4 Associated with Sinus Bradycardia

Patients carrying the heterozygous A414G mutation in the HCN4 gene, which encodes the HCN4 protein, demonstrate moderate to severe bradycardia of the heart. Tetramers of HCN4 subunits compose the ion channels in the sinus node that carry the hyperpolarization-activated ‘funny’ current (If), also named the ‘pacemaker current’. If plays an essential modulating role in sinus node pacemaker activity. To assess the mechanism by which the A414G mutation results in sinus bradycardia, we first performed voltage clamp measurements on wild-type (WT) and heterozygous mutant HCN4 channels expressed in Chinese hamster ovary (CHO) cells. These experiments were performed at physiological temperature using the amphotericin-perforated patch-clamp technique. Next, we applied the experimentally observed mutation-induced changes in the HCN4 current of the CHO cells to If of the single human sinus node cell model developed by Fabbri and coworkers. The half-maximal activation voltage V1/2 of the heterozygous mutant HCN4 current was 19.9 mV more negative than that of the WT HCN4 current (p &lt; 0.001). In addition, the voltage dependence of the heterozygous mutant HCN4 current (de)activation time constant showed a −11.9 mV shift (p &lt; 0.001) compared to the WT HCN4 current. The fully-activated current density, the slope factor of the activation curve, and the reversal potential were not significantly affected by the heterozygous A414G mutation. In the human sinus node computer model, the cycle length was substantially increased, almost entirely due to the shift in the voltage dependence of steady-state activation, and this increase was more prominent under vagal tone. The introduction of a passive atrial load into the model sinus node cell further reduced the beating rate, demonstrating that the bradycardia of the sinus node was even more pronounced by interactions between the sinus node and atria. In conclusion, the experimentally identified A414G-induced changes in If can explain the clinically observed sinus bradycardia in patients carrying the A414G HCN4 gene mutation.

The optimized method for mesenchymal stem cell differentiation into pacemaker-like cell

Link of Video Abstract: https://www.youtube.com/watch?v=HIbeTAOCFcABackground: Mesenchymal stem cells (MSCs) differentiate into pacemaker-like cells by activating TBX3 and downregulating Pitx2c by inhibiting the nodal signaling pathway. This optimization study aimed to determine the optimal conditions for differentiating MSCs into pacemaker-like cells. Methods: The differentiation of MSCs into pacemaker-like cells was carried out in three ways: transfecting TBX3 pcDNA into MSCs, inhibiting the nodal signaling pathway using the small molecule SB431542, and a combination of both methods. This optimization study was conducted in two batches. The first batch was an experiment with transfection carried out on the 5th day of differentiation and addition of 20 μM/mL of the small molecule SB431542. The second batch was an experiment with transfection carried out on the 3rd day of differentiation and the addition of 2 μM/mL of the small molecule SB431542. From the two batches, the expression patterns of TBX3, Cx30.2, Cx40, Cx43, HCN4, HCN1, HCN3, and KCNN4 genes were analyzed using the qRT-PCR method. Results: The results showed that the second batch had a better gene expression pattern in pacemaker-like cells than the first batch. These results were indicated by the increased expression of the TBX3, Cx30.2, HCN4, HCN1, HCN3, and KCNN4 genes. Furthermore, there was a decrease in Cx40 and Cx43 expression levels. Conclusion: The results showed that TBX3 transfection on the third day and a smaller dose (2 μM/mL) of small molecules and combination treatment induced differentiation from the initial MSC, leading to the expression of pacemaker-like cells.

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. 1µg/mL LPS was applied to the cells. Cell media were taken 6h 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 24h 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.

Fetal Heart Rate < 3rd Percentile for Gestational Age Can Be a Marker of Inherited Arrhythmia Syndromes.

Repeated fetal heart rates (FHR) < 3rd percentile for gestational age (GA) with 1:1 atrioventricular conduction (sinus bradycardia) can be a marker for long QT syndrome. We hypothesized that other inherited arrhythmia syndromes might present with fetal sinus bradycardia. We reviewed pregnancies referred with sinus bradycardia to the Colorado Fetal Care Center between 2013 and 2023. FHR/GA data, family history, medication exposure, normalized isovolumic contraction times (n-IVRT), postnatal genetic testing, and ECGs at 4-6 weeks after birth were reviewed. Twenty-nine bradycardic subjects were evaluated by fetal echocardiography. Five were lost to follow-up, one refused genetic testing, and one had negative genetic testing for any inherited arrhythmia. Six had non-genetic causes of fetal bradycardia with normal prenatal n-IVRT and postnatal QTc. Thirteen carried pathogenic variants in RYR2 (n = 2), HCN4 (n = 2), KCNQ1 (6), and other LQTS genes (n = 4). The postnatal QTc was <470 ms in subjects with RYR2, HCN4, and two of those with KCNQ1 mutations, and >470 ms in subjects with CALM 2, KCNH2, SCN5A, and four of those with KCNQ1 mutations. LQTS and RYR2 mutations were associated with prolonged n-IVRT, but HCN4 was not. Two fetuses died in utero with variants of uncertain significance (CACNA1 and KCNE1). Cascade testing uncovered six affected but undiagnosed parents and confirmed familial inheritance in five. In addition to heralding LQTS, repeated FHR < 3rd percentile for GA is a risk factor for other inherited arrhythmia syndromes. These findings suggest that genetic testing should be offered to infants with a history of FHR < 3rd percentile for GA even if the postnatal ECG demonstrates a normal QTc interval.