Functional reclassification of variants of uncertain significance in the HCN4 gene identified in sudden unexpected death

The HCN4 gene encodes a subunit of the hyperpolarization‐activated cyclic nucleotide‐gated channel, type 4 that is essential for the proper generation of pacemaker potentials in the sinoatrial node. The HCN4 gene is often present in targeted genetic testing panels for various cardiac conduction system disorders and there are several reports of HCN4 variants associated with conduction disorders. Here, we report the in vitro functional characterization of four rare variants of uncertain significance (VUS) in HCN4, identified through testing a cohort of 296 sudden unexpected natural deaths. The variants are all missense alterations, leading to single amino acid changes: p.E66Q in the N‐terminus, p.D546N in the C‐linker domain, and both p.S935Y and p.R1044Q in the C‐terminus distal to the CNBD. We also identified a likely benign variant, p. P1063T, which has a high minor allele frequency in the gnomAD, which is utilized here as a negative control. Three of the HCN4 VUS (p.E66Q, p.S935Y, and p.R1044Q) had electrophysiological characteristics similar to the wild‐type channel, suggesting that these variants are benign. In contrast, the p.D546N variant in the C‐linker domain exhibited a larger current density, slower activation, and was unresponsive to cyclic adenosine monophosphate (cAMP) compared to wild‐type. With functional assays, we reclassified three rare HCN4 VUS to likely benign variants, eliminating the necessity for costly and time‐consuming further study. Our studies also provide a new lead to investigate how a VUS located in the C‐linker connecting the pore to the cAMP binding domain may affect the channel open state probability and cAMP response.     

Physiology and pharmacology of the cardiac pacemaker ("funny") current

First described over a quarter of a century ago, the cardiac pacemaker "funny" (I(f)) current has been extensively characterized since, and its role in cardiac pacemaking has been thoroughly demonstrated. A similar current, termed I(h), was later described in different types of neurons, where it has a variety of functions and contributes to the control of cell excitability and plasticity. I(f) is an inward current activated by both voltage hyperpolarization and intracellular cAMP. In the heart, as well as generating spontaneous activity, f-channels mediate autonomic-dependent modulation of heart rate: beta-adrenergic stimulation accelerates, and vagal stimulation slows, cardiac rate by increasing and decreasing, respectively, the intracellular cAMP concentration and, consequently, the f-channel degree of activation. Four isoforms of hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels have been cloned more recently and shown to be the molecular correlates of native f-channels in the heart and h-channels in the brain. Individual HCN isoforms have kinetic and modulatory properties which differ quantitatively. A comparison of their biophysical properties with those of native pacemaker channels provides insight into the molecular basis of the pacemaker current properties and, together with immunolabelling and other detection techniques, gives information on the pattern of HCN isoform distribution in different tissues. Because of their relevance to cardiac pacemaker activity, f-channels are a natural target of drugs aimed at the pharmacological control of heart rate. Several agents developed for their ability to selectively reduce heart rate act by a specific inhibition of f-channel function; these substances have a potential for the treatment of diseases such as angina and heart failure. In the near future, devices based on the delivery of f-channels in situ, or of a cellular source of f-channels (biological pacemakers), will likely be developed for use in therapies for diseases of heart rhythm with the aim of replacing electronic pacemakers.     

Pacemaker channel dysfunction in a patient with sinus node disease

The cardiac pacemaker current I(f) is a major determinant of diastolic depolarization in sinus nodal cells and has a key role in heartbeat generation. Therefore, we hypothesized that some forms of "idiopathic" sinus node dysfunction (SND) are related to inherited dysfunctions of cardiac pacemaker ion channels. In a candidate gene approach, a heterozygous 1-bp deletion (1631delC) in exon 5 of the human HCN4 gene was detected in a patient with idiopathic SND. The mutant HCN4 protein (HCN4-573X) had a truncated C-terminus and lacked the cyclic nucleotide-binding domain. COS-7 cells transiently transfected with HCN4-573X cDNA indicated normal intracellular trafficking and membrane integration of HCN4-573X subunits. Patch-clamp experiments showed that HCN4-573X channels mediated I(f)-like currents that were insensitive to increased cellular cAMP levels. Coexpression experiments showed a dominant-negative effect of HCN4-573X subunits on wild-type subunits. These data indicate that the cardiac I(f) channels are functionally expressed but with altered biophysical properties. Taken together, the clinical, genetic, and in vitro data provide a likely explanation for the patient's sinus bradycardia and the chronotropic incompetence.     

Transgenic characterization of two testis‐specific promoters in the silkworm,Bombyx mori

Sex‐specific regulatory elements are key components for developing insect genetic sexing systems. The current insect genetic sexing system mainly uses a female‐specific modification system whereas little success was reported on male‐specific genetic modification. In the silkworm Bombyx mori, a lepidopteran model insect with economic importance, a transgene‐based, female‐specific lethality system has been established based on sex‐specific alternative splicing factors and a female‐specific promoter BmVgp (vitellogenin promoter) has been identified. However, no male‐specific regulatory elements have yet been identified. Here we report the transgenic identification of two promoters that drive reporter gene expression in a testis‐specific manner in B. mori. Putative promoter sequences from the B. mori Radial spoke head 1 gene (BmR1) and beta‐tubulin 4 gene (Bmβ4) were introduced using piggybac‐based germline transformation. In transgenic silkworms, expression of the reporter gene enhanced green fluorescent protein (EGFP) directed by either BmR1 promoter (BmR1p) or Bmβ4p showed precisely testis‐specific manners from the larval to adult stage. Furthermore, EGFP expression of these two transgenic lines showed different localization in the testis, indicating that BmR1p or Bmβ4p might be used as distinct regulatory elements in directing testis‐specific gene expression. Identification of these testis‐specific promoters not only contributes to a better understanding of testis‐specific gene function in insects, but also has potential applications in sterile insect techniques for pest management.     

Stem cells as biological heart pacemakers

Abnormalities in the pacemaker function of the heart or in cardiac impulse conduction may result in the appearance of a slow heart rate, traditionally requiring the implantation of a permanent electronic pacemaker. In recent years, a number of experimental approaches have been developed in an attempt to generate biological alternatives to implantable electronic devices. These strategies include, initially, a number of gene therapy approaches (aiming to manipulate the expression of ionic currents or their modulators and thereby convert quiescent cardiomyocytes into pacemaking cells) and, more recently, the use of cell therapy and tissue engineering. The latter approach explored the possibility of grafting pacemaking cells, either derived directly during the differentiation of human embryonic stem cells or engineered from mesenchymal stem cells, into the myocardium. This review will describe each of these approaches, focusing mainly on the stem cell strategies, their possible advantages and shortcomings, as well as the avenues required to make biological pacemaking a clinical reality.     

Stem cells as biological heart pacemakers

Abnormalities in the pacemaker function of the heart or in cardiac impulse conduction may result in the appearance of a slow heart rate, traditionally requiring the implantation of a permanent electronic pacemaker. In recent years, a number of experimental approaches have been developed in an attempt to generate biological alternatives to implantable electronic devices. These strategies include, initially, a number of gene therapy approaches (aiming to manipulate the expression of ionic currents or their modulators and thereby convert quiescent cardiomyocytes into pacemaking cells) and, more recently, the use of cell therapy and tissue engineering. The latter approach explored the possibility of grafting pacemaking cells, either derived directly during the differentiation of human embryonic stem cells or engineered from mesenchymal stem cells, into the myocardium. This review will describe each of these approaches, focusing mainly on the stem cell strategies, their possible advantages and shortcomings, as well as the avenues required to make biological pacemaking a clinical reality.     

Mesenchymal stem cells as a gene delivery system to create biological pacemaker cells in vitro

Pacemaker cells differ from common cardiomyocytes due to the presence of a spontaneous depolarization process during the diastolic phase of the cardiac cycle. This is due to hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, which are responsible for providing an inward current. Genetically engineered mesenchymal stem cells (MSCs) were transfected with hHCN4 genes using lentiviral transfection, and their potential use as biological pacemaker cells was investigated. In addition to expressing an anticipated high level of the hHCN4 gene, MSCs transfected with hHCN4 genes also expressed characteristic hHCN4 protein, a cardiac pacemaker-like current and were capable of increasing the spontaneous beating rate of co-cultured cardiac myocytes. Control MSCs did not exert these effects. It is hypothesized that genetically engineered MSCs transfected with hHCN4 genes by lentiviral transfection can be modified to be cardiac pacemaker cells in vitro.     

心脏电子起搏器时代与生物起搏替代的前沿话题

植入电子起搏器是目前治疗症状性缓慢心律失常的主要方法,然而它存在许多缺点.能否利用分子生物学原理发展生物起搏器成为大家关注的热点.通过转染编码If电流的超极化激活环核苷酸门控通道基因,过度表达超极化激活环核苷酸门控通道,增加心脏舒张期内向电流,从而在窦房结被抑制时提供起搏作用,这种利用基因治疗和细胞治疗构建的生物起搏在不久的将来可能会成为电子起搏器最为理想的替代方法.目的:总结超极化激活环核苷酸门控通道基因构建生物起搏的研究进展.检索策略:由该论文的研究人员应用计算机检索.Pubmed数据库1979-01/2007-06的相关文献.检索词"hyperpolarization-activated cyclic nucleotide-gated nnel;biological pacemaker",并限定文章语言种类为English.共检索到157篇文献,对资料进行初审,纳入标准:①与生物起搏及超极化激活环核苷酸门控通道基因密切相关.②同一领域选择近期发表或在权威杂志上发表的文章.排除标准:重复性研究.文献评价:文献的来源主要是超极化激活环核苷酸门控通道基因的基础实验.所选剧的36篇文献中,10篇为综述,其余均为临床或基础实验研究.资料综合:①在4种异构体中超极化激活环核苷酸门控通道1,2,4是心脏中的主要部分,超极化激活环核苷酸门控通道3只在胚胎起搏细胞中有低水平表达.起搏活性小的区域(如心室肌),超极化激活环核苷酸门控通道2的表达占优势;而起搏活性高的区域超极化激活环核苷酸门控通道4的表达占优势.此外,超极化激活环核苷酸门控通道2在整个发育阶段,是心室的主要异构体,超极化激活环核苷酸门控通道2:超极化激活环核苷酸门控通道4的相当表达量在乳鼠为5:1,成年鼠为13:1.②超极化激活环核苷酸门控通道通道缺陷可导致病窦综合征.③到目前为止.转染编码If电流的超极化激活环核苷酸门控通道基因被认为是最有可能实现生物起搏的.结论:基因治疗和细胞治疗必将成为改善生物"起搏"功能最理想的方法,以超极化激活环核苷酸门控通道基因和细胞为主的生物起搏在缓慢性心律失常治疗中必将占有一席之地.     

Simple suspension culture system of human iPS cells maintaining their pluripotency for cardiac cell sheet engineering

In this study, a simple three‐dimensional (3D) suspension culture method for the expansion and cardiac differentiation of human induced pluripotent stem cells (hiPSCs) is reported. The culture methods were easily adapted from two‐dimensional (2D) to 3D culture without any additional manipulations. When hiPSCs were directly applied to 3D culture from 2D in a single‐cell suspension, only a few aggregated cells were observed. However, after 3 days, culture of the small hiPSC aggregates in a spinner flask at the optimal agitation rate created aggregates which were capable of cell passages from the single‐cell suspension. Cell numbers increased to approximately 10‐fold after 12 days of culture. The undifferentiated state of expanded hiPSCs was confirmed by flow cytometry, immunocytochemistry and quantitative RT–PCR, and the hiPSCs differentiated into three germ layers. When the hiPSCs were subsequently cultured in a flask using cardiac differentiation medium, expression of cardiac cell‐specific genes and beating cardiomyocytes were observed. Furthermore, the culture of hiPSCs on Matrigel‐coated dishes with serum‐free medium containing activin A, BMP4 and FGF‐2 enabled it to generate robust spontaneous beating cardiomyocytes and these cells expressed several cardiac cell‐related genes, including HCN4, MLC‐2a and MLC‐2v. This suggests that the expanded hiPSCs might maintain the potential to differentiate into several types of cardiomyocytes, including pacemakers. Moreover, when cardiac cell sheets were fabricated using differentiated cardiomyocytes, they beat spontaneously and synchronously, indicating electrically communicative tissue. This simple culture system might enable the generation of sufficient amounts of beating cardiomyocytes for use in cardiac regenerative medicine and tissue engineering. Copyright © 2013 John Wiley & Sons, Ltd.     

An efficient transfection method for mouse embryonic stem cells

Embryonic stem (ES) cells are considered to have potentials for tissue regeneration and treatment of diverse human diseases. ES cells are capable of indefinite renewal and proliferation, which can be induced to differentiate into tissues of all three germ lines. Despite these exciting potential, it remains unclear as to how the renewal and differentiation programs are operated and regulated at the genetic level. Genetic manipulation such as delivery of exogenous gene expression or knockdown with small interfering RNA (siRNA) is commonly used in most of cancer or transformed cells but relatively rare in ES cells. In this study, we compare the transfection efficacies of several liposome-based transfection methods by introduction of a plasmid encoding enhanced green fluorescent protein (EGFP) into mouse ES (mES) cells. Our results show that transfection by Effectene achieves the efficiency of >98% in CCE and >80% in D3 cells. The optimal ratio of DNA:Effectene for EGFP transfection is between 1:4 and 1:8. Transient-expressed EGFP or endogenous protein kinase A (PKA) were significantly knocked down by Effectene transfection of specific siRNA. High EGFP level expression and accumulation in mES cells induces minor cytotoxicity but can be reduced by introducing siRNA of EGFP. Further, this transfection method did not significantly affect mES properties of proliferation or differentiation. Our results provide an optimal protocol to achieve an efficient transfection for mES cells.     

Sheep embryonic stem‐like cells transplanted in full‐thickness cartilage defects

Articular cartilage regeneration is limited. Embryonic stem (ES) cell lines provide a source of totipotent cells for regenerating cartilage. Anatomical, biomechanical, physiological and immunological similarities between humans and sheep make this animal an optimal experimental model. This study examines the repair process of articular cartilage in sheep after transplantation of ES‐like cells isolated from inner cell masses (ICMs) derived from in vitro‐produced (IVP) vitrified embryos. Thirty‐five ES‐like colonies from 40 IVP embryos, positive for stage‐specific embryonic antigens (SSEAs), were pooled in groups of two or three, embedded in fibrin glue and transplanted into osteochondral defects in the medial femoral condyles of 14 ewes. Empty defect (ED) and cell‐free glue (G) in the controlateral stifle joint served as controls. The Y gene sequence was used to detect ES‐like cells in the repair tissue by in situ hybridization (ISH). Two ewes were euthanized at 1 month post‐operatively, three each at 2 and 6 months and four at 12 months. Repairing tissue was examined by biomechanical, macroscopic, histological, immunohistochemical (collagen type II) and ISH assays. Scores of all treatments showed no statistical significant differences among treatment groups at a given time period, although ES‐like grafts showed a tendency toward a better healing process. ISH was positive in all ES‐like specimens. This study demonstrates that ES‐like cells transplanted into cartilage defects stimulate the repair process to promote better organization and tissue bulk. However, the small number of cells applied and the short interval between surgery and euthanasia might have negatively affected the results. Copyright © 2009 John Wiley & Sons, Ltd.     

X‐ray Radiation Causes Electromagnetic Interference in Implantable Cardiac Pacemakers

Background: X‐rays are not thought to cause electromagnetic interference (EMI) in implantable cardiac pacemakers. However, x‐ray radiation during computed tomography (CT) scanning has been reported to cause EMI in some implantable cardiac pacemakers. The objectives of this study were to identify the location within the pacemakers where x‐ray radiation causes EMI and to investigate the association of EMI with the x‐ray radiation conditions. Methods: We verified the location where x‐ray radiation caused EMI using a CT scanner and conventional radiographic x‐ray equipment. An inhibition test and an asynchronous test were performed using five types of implantable cardiac pacemakers. Results: X‐ray radiation inhibited the pacing pulses of four types of implantable cardiac pacemakers when the body of each implantable cardiac pacemaker, containing a complementary metal‐oxide semiconductor (CMOS), was scanned using a CT scanner. We confirmed that x‐ray‐induced EMI depends on the x‐ray radiation conditions, that is, the tube voltage, tube current, x‐ray dose, and direction of x‐ray radiation, as well as the sensing thresholds of the implantable cardiac pacemakers. Conclusions: X‐ray radiation caused EMI in some implantable cardiac pacemakers, probably because the CMOS component was irradiated. The occurrence of EMI depended on the pacemaker model, sensing threshold of the pacemaker, and x‐ray radiation conditions. (PACE 2010; 33:1174–1181)     

Immune response to green fluorescent protein: implications for gene therapy.

Green fluorescent protein (GFP) is a widely used intracellular reporter molecule to assess gene transfer and expression. A potential use for GFP is as a co-expressed marker, to select and enrich gene-modified cells by flow cytometry. Processed peptides derived from GFP and presented by the major histocompatibility complex on the cell surface could potentially induce T cell immune responses against GFP+ cells. Thus, clinical application of GFP is premature, since in vivo studies on its immunogenicity are lacking. Therefore, we investigated immune responses against EGFP (enhanced-GFP) in two transplantable murine models: the BALB/c (H-2d) BM185 pre-B leukemia and the C57BL/6 (H-2b) EL-4 T cell lymphoma. BM185 and EL-4 cell lines modified to express high levels of EGFP showed drastic reduction of disease development when transplanted into immunocompetent mice. BM185/ EGFP did lead to rapid development of disease in immunodeficient Nu/Nu mice. Mice surviving BM185/EGFP leukemia challenge developed high cytotoxic T lymphocyte (CTL) responses against EGFP-expressing cells. Furthermore, immune stimulation against BM185/EGFP cells could also be induced by immunization with EGFP+ transduced dendritic cells. The effects of the co-expression of EGFP and immunomodulators (CD80 plus GM-CSF) were also investigated as an irradiated leukemia vaccine. EGFP co-expression by the vaccine did not interfere with the development of CTLs against the parental leukemia or with the anti-leukemia response in vivo. These results indicate that the immune response against EGFP may interfere with its applicability in gene insertion/replacement strategies but could potentially be employed for leukemia cell vaccines.     

MR tracking of SPIO‐labeled mesenchymal stem cells in rats with liver fibrosis could not monitor the cells accurately

Our previous study showed that in vivo magnetic resonance (MR) imaging is effective in tracking superparamagnetic iron oxide (SPIO)‐labeled bone marrow mesenchymal stem cells (BMSCs) in rats with liver fibrosis. SPIO‐labeling‐induced signal reduction on MR images was completely reversed within 15 days after transplantation. It is still unclear whether the signal changes in MR imaging could reflect the number of transplanted cells in the liver. In the present study, BMSCs of male rats were doubly labeled with enhanced green fluorescent protein (EGFP) and SPIO and injected intravascularly into female rats with liver fibrosis. At different time points after injection, MR imaging was performed. The distribution of SPIO particles and EGFP‐positive cells was determined by Prussian blue staining and EGFP immunohistochemistry, respectively. The distribution of transplanted BMSCs in various organs was assessed by detection of the SRY gene using real‐time quantitative PCR. At 15 days post transplantation, the numbers of transplanted cells were significantly decreased in the lung, kidney, spleen and muscle, but not liver and heart, in comparison with those at 7 days after transplantation. EGFP staining‐positive cells were observed in the liver intralobular parenchyma, while Prussian blue staining was negative at 42 days after transplantation. Taken together, SPIO particles and EGFP‐labeled BMSCs show a different tissue distribution pattern in rats with liver fibrosis after a long‐term period of monitoring. SPIO‐based MR imaging may not be suitable for long‐term tracking of transplanted BMSCs in vivo. Copyright © 2015 John Wiley & Sons, Ltd.     

The Role of Pacemaker Currents in Neuropathic Pain

Abstract:   Hyperpolarization-activated cation nonselective cyclic nucleotide-gated (HCN) channels mediate pacemaker currents that control basic rhythmic processes including heartbeat. Alterations in HCN channel expression or function have been described in both epilepsy and cardiac arrhythmias. Recent evidence suggests that pacemaker currents may also play an important role in ectopic neuronal activity that manifests as neuropathic pain. Pacemaker currents are subject to endogenous regulation by cyclic nucleotides, pH and perhaps phosphorylation. In addition, a number of neuromodulators with known roles in pain affect current density and kinetics. The pharmacology of a number of drugs that are commonly used to treat neuropathic pain includes effects on pacemaker currents. Altered pacemaker currents in injured tissues may be an important mechanism underlying neuropathic pain, and drugs that modulate these currents may offer new therapeutic options.     

ΔNp63 Is Essential for Epidermal Commitment of Embryonic Stem Cells

In vivo studies have demonstrated that p63 plays complex and pivotal roles in pluristratified squamous epithelial development, but its precise function and the nature of the isoform involved remain controversial. Here, we investigate the role of p63 in epithelial differentiation, using an in vitro ES cell model that mimics the early embryonic steps of epidermal development. We show that the ΔNp63 isoform is activated soon after treatment with BMP-4, a morphogen required to commit differentiating ES cells from a neuroectodermal to an ectodermal cell fate. ΔNp63 gene expression remains high during epithelial development. P63 loss of function drastically prevents ectodermal cells to commit to the K5/K14-positive stratified epithelial pathway while gain of function experiments show that ΔNp63 allows this commitment. Interestingly, other epithelial cell fates are not affected, allowing the production of K5/K18-positive epithelial cells. Therefore, our results demonstrate that ΔNp63 may be dispensable for some epithelial differentiation, but is necessary for the commitment of ES cells into K5/K14-positive squamous stratified epithelial cells.     

Sleep Disordered Breathing and Arrhythmia Burden in Pacemaker Recipients

Background: Sleep disordered breathing (SDB), a common condition among patients with permanent pacemaker (PM), is associated with greater incidence of cardiac arrhythmias. Scarce availability of sleep laboratories and the high costs of nocturnal‐attended polysomnography limit the routine screening of patients with PM for SDB. We investigated whether a novel PM that utilizes variations in transthoracic impedance to record the fluctuations in breathing pattern and minute ventilation could be used to screen patients for SDB. Methods: Twenty patients who underwent dual‐chamber PM implantation were studied. The Talent 3 DR PM (SORIN Group Italy S.r.l., Milan, Italy) calculates apnea‐hypopnea index (AHI) by computing minute ventilation signal derived from transthoracic impedance measurements. Within a month after PM implantation, an in‐home respiratory monitoring was performed to evaluate the accuracy of PM‐derived AHI. Patients were followed for mean ± standard deviation, 487 ± 166 days. The PM was checked at each follow‐up visit to retrieve the information about recurrent arrhythmias. Results: Eleven patients were diagnosed with SDB by an in‐home respiratory monitoring. An AHI derived from an in‐home respiratory monitoring was similar to pacemaker‐derived AHI (27 ± 14 vs 16 ± 13 events/hour, P = 0.15). The cumulative incidence of cardiac arrhythmias, including atrial fibrillation, extrasystolic beats, sustained and nonsustained ventricular tachycardia, and supraventricular tachycardia was similar in patients with and without SDB. Conclusion: SDB is highly prevalent in patients with permanent pacemaker. Screening for SDB with Talent 3 DR PM may facilitate diagnosis and treatment of SDB. (PACE 2010; 33:1462–1466)     

Genetic transformation of the housefly Musca domestica with the lepidopteran derived transposon piggyBac

The piggyBac transposable element was successfully used for stable genetic transformation of the housefly Musca domestica. The construct contains the EGFP marker under the control of Pax‐6 binding sites, which can drive eye‐specific expression in insect species as distantly related as Drosophila melanogaster and Tribolium castaneum[ Berghammer, A.J., Klingler, M. and Wimmer, E.A. (1999) Nature 402: 370–371]. We obtained seven independent integration events among 41 fertile G₀Musca flies. Most of the transformed lines contained two or more chromosomal insertions of the EGFP marker which were stably inherited over more than 15 generations. piggyBac‐mediated transposition was verified by identifying the characteristic TTAA duplication at the insertion sites. This first report of stable transmission of a genetic marker in Musca confirms the use of this vector‐marker system for effective gene transfer in a broad range of insect species.