Expression Of Cardiac Ion Channels Research Articles

Deciphering the roles of triiodothyronine (T3) and thyroid-stimulating hormone (TSH) on cardiac electrical remodeling in clinical and experimental hypothyroidism.

Hypothyroidism is the most frequent endocrine pathology. Although clinical or overt hypothyroidism has been traditionally associated to low T3 / T4 and high thyrotropin (TSH) circulating levels, other forms exist such as subclinical hypothyroidism, characterized by normal blood T3 / T4 and high TSH. In its different forms is estimated to affect approximately 10% of the population, especially women, in a 5:1 ratio with respect to men. Among its consequences are alterations in cardiac electrical activity, especially in the repolarization phase, which is accompanied by an increased susceptibility to cardiac arrhythmias. Although these alterations have traditionally been attributed to thyroid hormone deficiency, recent studies, both clinical trials and experimental models, demonstrate a fundamental role of TSH in cardiac electrical remodeling. Thus, both metabolic thyroid hormones and TSH regulate cardiac ion channel expression in many and varied ways. This means that the different combinations of hormones that predominate in different types of hypothyroidism (overt, subclinic, primary, central) can generate different forms of cardiac electrical remodeling. These new findings are raising the relevant question of whether serum TSH reference ranges should be redefined.

Impact of stress on cardiac phenotypes in mice harboring an ankyrin-B disease variant

Encoded by ANK2, ankyrin-B (AnkB) is a multifunctional adapter protein critical for the expression and targeting of key cardiac ion channels, transporters, cytoskeletal-associated proteins, and signaling molecules. Mice deficient for AnkB expression are neonatal lethal, and mice heterozygous for AnkB expression display cardiac structural and electrical phenotypes. Human ANK2 loss-of-function variants are associated with diverse cardiac manifestations; however, human clinical 'AnkB syndrome' displays incomplete penetrance. To date, animal models for human arrhythmias have generally been knock-out or transgenic overexpression models and thus the direct impact of ANK2 variants on cardiac structure and function invivo is not clearly defined. Here, we directly tested the relationship of a single human ANK2 disease-associated variant with cardiac phenotypes utilizing a novel invivo animal model. At baseline, young AnkBp.E1458G+/+ mice lacked significant structural or electrical abnormalities. However, aged AnkBp.E1458G+/+ mice displayed both electrical and structural phenotypes at baseline including bradycardia and aberrant heart rate variability, structural remodeling, and fibrosis. Young and old AnkBp.E1458G+/+ mice displayed ventricular arrhythmias following acute (adrenergic) stress. In addition, young AnkBp.E1458G+/+ mice displayed structural remodeling following chronic (transverse aortic constriction) stress. Finally, AnkBp.E1458G+/+ myocytes harbored alterations in expression and/or localization of key AnkB-associated partners, consistent with the underlying disease mechanism. In summary, our findings illustrate the critical role of AnkB in invivo cardiac function as well as the impact of single AnkB loss-of-function variants invivo. However, our findings illustrate the contribution and in fact necessity of secondary factors (aging, adrenergic challenge, pressure-overload) to phenotype penetrance and severity.

Abstract P1102: Rna-seq Analyses Reveal Roles Of The Hvcn1 Proton Channel In Cardiac Ph Homeostasis

Introduction: The voltage-gated proton channel Hv1 is a member of the voltage-gated ion channel family. Hv1 channel controls acid extrusion and regulates pH homeostasis in various cell types. Recent evidence showed that the Hv1 were expressed in the heart. Hypothesis: We propose the Hv1 channel is associated with cardiac function. Methods: To investigate the role of Hv1 in cardiac myocytes, we performed a transcriptomic analysis of wild-type (WT) and Hv1-/- mouse hearts. Results: Hv1 null hearts exhibited a differential transcriptome profile compared with wild-type hearts. The RNA-seq data indicating impaired pH homeostasis in Hv1 null hearts were the downregulated NADPH oxidoreductases (NOXs) and decreased expression of Cl-/HCO3- exchanger. Additionally, Hv1 null hearts exhibited differential expression of cardiac ion channels, suggesting a potential role of Hv1 in cardiac electrophysiological remodeling. Conclusions: The data indicated that Hv1 was involved in cardiac pH homeostasis and showed that Hv1 was a regulator of gene transcriptional networks controlling NOX signaling and CO2 homeostasis in the heart. The study highlights the importance of Hv1 in cardiac function and may present a new target associated with heart diseases.

Cardiac ion channel expression in the equine model ‐ In‐silico prediction utilising RNA sequencing data from mixed tissue samples

Understanding cardiomyocyte ion channel expression is crucial to understanding normal cardiac electrophysiology and underlying mechanisms of cardiac pathologies particularly arrhythmias. Hitherto, equine cardiac ion channel expression has rarely been investigated. Therefore, we aim to predict equine cardiac ion channel gene expression. Raw RNAseq data from normal horses from 9 datasets was retrieved from ArrayExpress and European Nucleotide Archive and reanalysed. The normalised (FPKM) read counts for a gene in a mix of tissue were hypothesised to be the average of the expected expression in each tissue weighted by the proportion of the tissue in the mix. The cardiac‐specific expression was predicted by estimating the mean expression in each other tissues. To evaluate the performance of the model, predicted gene expression values were compared to the human cardiac gene expression. Cardiac‐specific expression could be predicted for 91 ion channels including most expressed Na+ channels, K+ channels and Ca2+‐handling proteins. These revealed interesting differences from what would be expected based on human studies. These differences included predominance of NaV1.4 rather than NaV1.5 channel, and RYR1, SERCA1 and CASQ1 rather than RYR2, SERCA2, CASQ2 Ca2+‐handling proteins. Differences in channel expression not only implicate potentially different regulatory mechanisms but also pathological mechanisms of arrhythmogenesis.

Cardiac ion channels associated with unexplained stillbirth- an immunohistochemical study.

Despite the use of post-mortem investigations, approximately 20% of stillbirths remain unexplained. Cardiac ion channelopathies have been identified as a cause of death in Sudden Infant Death Syndrome (SIDS) and could be associated with unexplained stillbirths. This study aimed to understand if the expression or localisation of cardiac ion channels associated with channelopathies were altered in cases of unexplained stillbirths. A case control study was conducted using formalin-fixed cardiac tissue from 20 cases of unexplained stillbirth and a control group of 20 cases of stillbirths from intrapartum hypoxia. 4µm tissue sections were stained using haematoxylin and eosin, Masson's trichrome (MT) and Elastic van Gieson (EVG). Immunohistochemistry (IHC) was performed using antibodies against CACNA1G, KCNJ2, KCNQ1, KCNH2 and KCNE1. The cardiac conduction system in samples stained with MT and EVG could not be identified. Therefore, the levels of immunoperoxidase staining were quantified using QuPath software. The nuclear-cytoplasmic ratio of sections stained with haematoxylin and eosin was higher for the hypoxia group (hypoxia median 0.13 vs. 0.04 unexplained, p<0.001). CACNA1G (unexplained median 0.26 vs. hypoxia 0.30, p=0.009) and KCNJ2 (unexplained median 0.35 vs. hypoxia 0.41, p=0.001) had lower staining intensity in the unexplained stillbirth group. There were no statistically significant differences in the staining intensity of KCNQ1, KCNH2 and KCNE1. Two ion channels associated with channelopathies demonstrated lower levels of expression in cases of unexplained stillbirth. Further genetic studies using human tissue should be performed to understand the association between channelopathies and otherwise unexplained stillbirths.

The role of the cardiomyocyte circadian clocks in ion channel regulation and cardiac electrophysiology.

Daily variations in cardiac electrophysiology and the incidence for different types of arrhythmias reflect ≈24h changes in the environment, behaviour and internal circadian rhythms. This article focuses on studies that use animal models to separate the impact that circadian rhythms, as well as changes in the environment and behaviour, have on 24h rhythms in heart rate and ventricular repolarization. Circadian rhythms are initiated at the cellular level by circadian clocks, transcription-translation feedback loops that cycle with a periodicity of 24h. Several studies now show that the circadian clock in cardiomyocytes regulates the expression of cardiac ion channels by multiple mechanisms; underlies time-of-day changes in sinoatrial node excitability/intrinsic heart rate; and limits the duration of the ventricular action potential waveform. However, the 24h rhythms in heart rate and ventricular repolarization are primarily driven by autonomic signalling. A functional role for the cardiomyocyte circadian clock appears to buffer the heart against perturbations. For example, the cardiomyocyte circadian clock limits QT-interval prolongation (especially at slower heart rates), and it may facilitate the realignment of the 24h rhythm in heart rate to abrupt changes in the light cycle. Additional studies show that modifying rhythmic behaviours (including feeding behaviour) can dramatically impact the 24h rhythms in heart rate and ventricular repolarization. If these mechanisms are conserved, these studies suggest that targeting endogenous circadian mechanisms in the heart, as well as modifying the timing of certain rhythmic behaviours, could emerge as therapeutic strategies to support heart function against perturbations and regulate 24h rhythms in cardiac electrophysiology.

Progesterone Changes the Pregnancy-Induced Adaptation of Cardiomyocyte Kv2.1 Channels via MicroRNA-29b.

The cardiovascular system adaptation occurs during pregnancy to ensure adequate maternal circulation. Progesterone (P4) is widely used in hormone therapy to support pregnancy, but little is known about its effects on maternal cardiac function. In this study, we investigated the cardiac repolarization and ion channel expression in pregnant subjects and mice models and studied the effects of P4 administrations on these pregnancy-mediated adaptations. P4 administrations shortened the prolongation of QTC intervals and action potential duration (APD) that occurred during pregnancy, which was mainly attributable to the reduction in the voltage-gated potassium (Kv) current under basal conditions. In vitro studies indicated that P4 regulated the Kv2.1 channel in a bidirectional manner. At a low dose (1 μM), P4 induced upregulation of Kv2.1 through P4 receptor, while at a higher dose (5 μM), P4 downregulated Kv2.1 by targeting microRNA-29b (miR-29b). Our data showed that P4 modulated maternal cardiac repolarization by regulating Kv2.1 channel activity during pregnancy. Kv2.1, as well as miR-29b, might be used as potential therapeutic targets for adaptations of the maternal cardiovascular system or evaluation of progesterone medication during pregnancy.

RNA-Seq Analyses Reveal Roles of the HVCN1 Proton Channel in Cardiac pH Homeostasis.

The voltage-gated proton channel HVCN1 is a member of the voltage-gated ion channel family. HVCN1 channel controls acid extrusion and regulates pH homeostasis in various cell types. Recent evidence indicated that the HVCN1 channel was associated with cardiac function. To investigate the role of HVCN1 in cardiac myocytes, we performed an RNA sequencing analysis of murine hearts and showed that HVCN1 null hearts exhibited a differential transcriptome profile compared with wild-type hearts. The RNA-seq data indicating impaired pH homeostasis in HVCN1 null hearts were the downregulated NADPH oxidoreductases (NOXs) and decreased expression of Cl−/HCO3 − exchanger, indicating HVCN1 is a regulator of gene transcriptional networks controlling NOX signaling and CO2 homeostasis in the heart. Additionally, HVCN1 null hearts exhibited differential expression of cardiac ion channels, suggesting a potential role of HVCN1 in cardiac electrophysiological remodeling. The study highlights the importance of HVCN1 in cardiac function and may present a novel target associated with heart diseases.

Phenotypes of mutations related to voltage-dependent sodium channels on children and adolescents.

Cardiac channelopathies are a heterogeneous group of inherited cardiac diseases that are associated with mutations in the genes that encode the expression of cardiac ion channels. In view of this, it can be mentioned that the main hereditary arrhythmias in children and adolescents, caused by dysfunction of the ion channels, are Brugada Syndrome (BrS) and Long QT Syndrome (LQTS). However, few studies address the physiological effects of these conditions on children and adolescents. Thus, the aim of this study is to describe the mutation phenotype related to voltage-gated sodium channels in children and adolescents. A search was performed in the literature of PubMed, Scielo, and Google scholar. The search was limited to articles written in the last 5 years, so articles published between 2014 and 2019 were included. Among 2196 studies identified through a systematic literature review, 30 studies related to the theme were identified for a complete review and after applying exclusion criteria, 4 articles were included in the results of this study. As the most frequently observed channelopathy, BrS was also more identified in children and adolescents, characterized by episodes of syncope or sudden cardiac death. LQTS shows clinical manifestations with a mild phenotype and good prognosis, although it is necessary to monitor and correct serum electrolyte disturbances to prevent ventricular arrhythmias and, consequently, sudden death in patients with the pathology. The aim of this study is to find the general phenotypes related to genetic mutations of voltage-gated sodium channels, in a population aged from 7- to 14-year-old.

Changes in ion channel expression and function associated with cardiac arrhythmogenic remodeling by Sorbs2

The Sorbin and SH3 domain-containing protein 2 (Sorbs2) is an important component of cardiomyocyte sarcomere. It has been recently reported that loss of Sorbs2 is causally associated with arrhythmogenic cardiomyopathy in human. However, the ionic mechanisms leading to cardiac arrhythmogenesis by Sorbs2 deficiency are unknown. In this study, we hypothesized that Sorbs2 plays an important role in regulating cardiac ion channel expression and function. Using electrophysiological and molecular biological approaches, we found that the Sorbs2 knockout (KO) mice progressively developed cardiac structural and electrical remodeling as early as 1 to 2 months of age and died prematurely at 5 to 7 months of age. Electrocardiographic recordings showed that Sorbs2 KO mice had conduction delays, spontaneous ventricular extrasystoles and polymorphic ventricular tachyarrhythmia. Intracellular recordings revealed abnormal action potentials with depolarized resting potential, reduced upstroke velocity, prolonged repolarization, and effective refractory period in the ventricular preparations of Sorbs2 KO mice. Patch clamp experiments demonstrated that Sorbs2 KO mice displayed distinct abnormalities in the expression and function of cardiac ion channels, including those of the voltage-gated Na+ channels, L-type Ca2+ channels, the voltage-gated K+ channels and the inward-rectifier K+ channels. Moreover, Sorbs2 physically interacted with the RNAs and/or proteins of important cardiac ion channels and directly regulated their expression in vitro. Our results indicate that Sorbs2 plays a pivotal role in the regulation of cardiac channel physiology. Loss of Sorbs2 promotes cardiac ion channelopathies and life-threatening arrhythmias.

Electrical Features of the Diabetic Myocardium. Arrhythmic and Cardiovascular Safety Considerations in Diabetes.

Diabetes is a chronic metabolic disease characterized by hyperglycemia in the absence of treatment. Among the diabetes-associated complications, cardiovascular disease is the major cause of mortality and morbidity in diabetic patients. Diabetes causes a complex myocardial dysfunction, referred as diabetic cardiomyopathy, which even in the absence of other cardiac risk factors results in abnormal diastolic and systolic function. Besides mechanical abnormalities, altered electrical function is another major feature of the diabetic myocardium. Both type 1 and type 2 diabetic patients often show cardiac electrical remodeling, mainly a prolonged ventricular repolarization visible in the electrocardiogram as a lengthening of the QT interval duration. The underlying mechanisms at the cellular level involve alterations on the expression and activity of several cardiac ion channels and their associated regulatory proteins. Consequent changes in sodium, calcium and potassium currents collectively lead to a delay in repolarization that can increase the risk of developing life-threatening ventricular arrhythmias and sudden death. QT duration correlates strongly with the risk of developing torsade de pointes, a form of ventricular tachycardia that can degenerate into ventricular fibrillation. Therefore, QT prolongation is a qualitative marker of proarrhythmic risk, and analysis of ventricular repolarization is therefore required for the approval of new drugs. To that end, the Thorough QT/QTc analysis evaluates QT interval prolongation to assess potential proarrhythmic effects. In addition, since diabetic patients have a higher risk to die from cardiovascular causes than individuals without diabetes, cardiovascular safety of the new antidiabetic drugs must be carefully evaluated in type 2 diabetic patients. These cardiovascular outcome trials reveal that some glucose-lowering drugs actually reduce cardiovascular risk. The mechanism of cardioprotection might involve a reduction of the risk of developing arrhythmia.

The mineralocorticoid receptor leads to increased expression of EGFR and T-type calcium channels that support HL-1 cell hypertrophy

The EGF receptor (EGFR) has been extensively studied in tumor biology and recently a role in cardiovascular pathophysiology was suggested. The mineralocorticoid receptor (MR) is an important effector of the renin–angiotensin–aldosterone-system and elicits pathophysiological effects in the cardiovascular system; however, the underlying molecular mechanisms are unclear. Our aim was to investigate the importance of EGFR for MR-mediated cardiovascular pathophysiology because MR is known to induce EGFR expression. We identified a SNP within the EGFR promoter that modulates MR-induced EGFR expression. In RNA-sequencing and qPCR experiments in heart tissue of EGFR KO and WT mice, changes in EGFR abundance led to differential expression of cardiac ion channels, especially of the T-type calcium channel CACNA1H. Accordingly, CACNA1H expression was increased in WT mice after in vivo MR activation by aldosterone but not in respective EGFR KO mice. Aldosterone- and EGF-responsiveness of CACNA1H expression was confirmed in HL-1 cells by Western blot and by measuring peak current density of T-type calcium channels. Aldosterone-induced CACNA1H protein expression could be abrogated by the EGFR inhibitor AG1478. Furthermore, inhibition of T-type calcium channels with mibefradil or ML218 reduced diameter, volume and BNP levels in HL-1 cells. In conclusion the MR regulates EGFR and CACNA1H expression, which has an effect on HL-1 cell diameter, and the extent of this regulation seems to depend on the SNP-216 (G/T) genotype. This suggests that the EGFR may be an intermediate for MR-mediated cardiovascular changes and that SNP analysis can help identify subgroups of patients that will benefit most from MR antagonists.

Cardiomyocyte Deletion of Bmal1 Exacerbates QT- and RR-Interval Prolongation in Scn5a+/ΔKPQ Mice

Circadian rhythms are generated by cell autonomous circadian clocks that perform a ubiquitous cellular time-keeping function and cell type-specific functions important for normal physiology. Studies show inducing the deletion of the core circadian clock transcription factor Bmal1 in adult mouse cardiomyocytes disrupts cardiac circadian clock function, cardiac ion channel expression, slows heart rate, and prolongs the QT-interval at slow heart rates. This study determined how inducing the deletion of Bmal1 in adult cardiomyocytes impacted the in vivo electrophysiological phenotype of a knock-in mouse model for the arrhythmogenic long QT syndrome (Scn5a+/ΔKPQ). Electrocardiographic telemetry showed inducing the deletion of Bmal1 in the cardiomyocytes of mice with or without the ΔKPQ-Scn5a mutation increased the QT-interval at RR-intervals that were ≥130 ms. Inducing the deletion of Bmal1 in the cardiomyocytes of mice with or without the ΔKPQ-Scn5a mutation also increased the day/night rhythm-adjusted mean in the RR-interval, but it did not change the period, phase or amplitude. Compared to mice without the ΔKPQ-Scn5a mutation, mice with the ΔKPQ-Scn5a mutation had reduced heart rate variability (HRV) during the peak of the day/night rhythm in the RR-interval. Inducing the deletion of Bmal1 in cardiomyocytes did not affect HRV in mice without the ΔKPQ-Scn5a mutation, but it did increase HRV in mice with the ΔKPQ-Scn5a mutation. The data demonstrate that deleting Bmal1 in cardiomyocytes exacerbates QT- and RR-interval prolongation in mice with the ΔKPQ-Scn5a mutation.

Electrophysiological and pharmacological analysis with LQT2 patient (453delC‐KNCH2)‐derived induced pluripotent stem cell‐derived cardiomyocytes

Objective Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are promising options for cell-based cardiac repair and in vitro models of human cardiomyocytes. The hiPSC-CMs are used as a diagnostic disease modeling and drug toxicity screening platform. Because hiPSCs retain the genetic information of the cells from which they are derived, cells differentiated from iPSCs can potentially recapitulate the disease phenotype. Methods In this study, we investigated the electrophysiological properties of hiPSC-CMs derived from a LQT2 patient with mutation in the KNCH2 gene (453delC-KNCH2) for hERG channel (LQT2 453delC-iPSC-CM). Results The genomic DNA analysis of LQT2 453delC-iPSC-CM confirmed the heterozygous mutation of 453delC KCNH2. The mRNA expression of cardiac ion channels and ventricular markers were analyzed to verify cardiomyocyte maturity. In the whole-cell patch clamp study, LQT2 453delC-iPSC-CMs showed smaller amplitude of hERG-type K+ current (IKr) and prolonged action potential duration compare to the control hiPSC-CMs. The results were consistent with the loss-of-function mutation of KCNH2 and LQT2 phenotype. In a multi-electrode array (MEA) study, TdP risk was compared between the control hiPSC-CMs and LQT2 453delC-iPSC-CMs. High, intermediate, and low risk TdP drugs were applied, and the drug-induced changes of field potential were analyzed to compare TdP risk. Conclusion The data set might be helpful for the drug investigation in the LQT syndrome and the development of patient-specific antiarrhythmic strategies.

Sex-Related Differences in Cardiac Channelopathies: Implications for Clinical Practice.

Sex-related differences in prevalence, clinical presentation, and outcome of cardiac channelopathies are increasingly recognized, despite their autosomal transmission and hence equal genetic predisposition among sexes. In congenital long-QT syndrome, adult women carry a greater risk for Torsades de pointes and sudden cardiac death than do men. In contrast, Brugada syndrome is observed predominantly in adult men, with a considerably higher risk of arrhythmic sudden cardiac death in adult men than in women. In both conditions, the risk for arrhythmias varies with age. Sex-associated differences appear less evident in other cardiac channelopathies, likely a reflection of their rare(r) occurrence and our limited knowledge. In several cardiac channelopathies, sex-specific predictors of outcome have been identified. Together with genetic and environmental factors, sex hormones contribute to the sex-related disparities in cardiac channelopathies through modulation of the expression and function of cardiac ion channels. Despite these insights, essential knowledge gaps exist in the mechanistic understanding of these differences, warranting further investigation. Precise application of the available knowledge may improve the individualized care of patients with cardiac channelopathies. Promoting the reporting of sex-related phenotype and outcome parameters in clinical and experimental studies and advancing research on cardiac channelopathy animal models should translate into improved patient outcomes. This review provides a critical digest of the current evidence for sex-related differences in cardiac channelopathies and emphasizes their clinical implications and remaining gaps requiring further research.

Genome-wide association study identifies 18 new susceptibility variants loci associated with Brugada Syndrome

Abstract The Brugada Syndrome (BrS) is characterized by ST-segment elevation in the right precordial leads and is associated with an increased risk of sudden cardiac death. The disorder was initially described as a monogenic primary cardiac electrical disease. However, mutations in SCN5A, encoding the cardiac sodium channel (NaV1.5), which is the major gene associated with the disorder are found in only around 20% of cases and are associated with low penetrance. Furthermore many cases did not display familial aggregation. Based on a previous GWAS conducted on 312 BrS patients and the discovery of the unexpected strong effect of 3 common variants, we proposed that the BrS may comprise a more complex inheritance model. We conducted a genome-wide association study on 2820 individuals with BrS and 10001 ancestry-matched controls to uncover additional genetic loci that modulate susceptibility to BrS, to characterize further the BrS genetic architecture and to uncover new molecular mechanisms. We identified 21 susceptibility variants that passed the genome-wide statistical significance threshold (P&amp;lt;5.10–8), of which 18 were novel. Eight were located at the SCN5A-SCN10A locus, illustrating the central role of NaV1.5 in the disease. Interestingly, 9 occur in the vicinity of genes known to play a crucial role in cardiac development (HEY2, TBX20, GATA4, ZFPM2, WT1, TBX5, IRX3, IRX5) and / or control cardiac ion channel expression. Of note, 2 others signals occurred in the vicinity of microtubule / cytoskeleton associated proteins (MAPRE2 and MYO18B). Through studies in zebrafish and in human iPSC-derived cardiomyocytes, we demonstrate a role of MAPRE2 on NaV1.5 function. We identified 18 new susceptibility variants associated with BrS and uncovered a new pathophysiological molecular mechanism underlying BrS susceptibility. We provided further support for a complex genetic architecture underlying susceptibility for the disorder. Funding Acknowledgement Type of funding source: Public Institution(s). Main funding source(s): H2020 - Marie Sklodowska Curie IF grant, Rising star grant from the Pays de la Loire regional council

Co-expression of calcium and hERG potassium channels reduces the incidence of proarrhythmic events.

Cardiac electrical activity is extraordinarily robust. However, when it goes wrong it can have fatal consequences. Electrical activity in the heart is controlled by the carefully orchestrated activity of more than a dozen different ion conductances. While there is considerable variability in cardiac ion channel expression levels between individuals, studies in rodents have indicated that there are modules of ion channels whose expression co-vary. The aim of this study was to investigate whether meta-analytic co-expression analysis of large-scale gene expression datasets could identify modules of co-expressed cardiac ion channel genes in human hearts that are of functional importance. Meta-analysis of 3653 public human RNA-seq datasets identified a strong correlation between expression of CACNA1C (L-type calcium current, ICaL) and KCNH2 (rapid delayed rectifier K+ current, IKr), which was also observed in human adult heart tissue samples. In silico modelling suggested that co-expression of CACNA1C and KCNH2 would limit the variability in action potential duration seen with variations in expression of ion channel genes and reduce susceptibility to early afterdepolarizations, a surrogate marker for proarrhythmia. We also found that levels of KCNH2 and CACNA1C expression are correlated in human-induced pluripotent stem cell-derived cardiac myocytes and the levels of CACNA1C and KCNH2 expression were inversely correlated with the magnitude of changes in repolarization duration following inhibition of IKr. Meta-analytic approaches of multiple independent human gene expression datasets can be used to identify gene modules that are important for regulating heart function. Specifically, we have verified that there is co-expression of CACNA1C and KCNH2 ion channel genes in human heart tissue, and in silico analyses suggest that CACNA1C-KCNH2 co-expression increases the robustness of cardiac electrical activity.

Mitogen-activated protein kinase p38 modulates pacemaker ion channels differentiation in P19-derived pluripotent cells

Signal regulators during early cardiogenetic differentiation for the cellular automaticity are largely unknown. Our investigations were designed to clarify the role of transcription factors and their modulators in P19-derived cardiomyocytes to the expression of cardiac pacemaker ion channels. Transcription factors Csx/Nkx2.5 and GATA4 but not MEF2C were markedly inhibited by p38 MAP kinase inhibition in a distinct manner; expression but not phosphorylation of GATA4 was reduced by inhibition of p38 MAP kinase actions. In the presence of an ERK1/2,5 inhibitor PD98059 or a JNK MAP kinase inhibitor SP600125, P19 cells successfully differentiated into cardiomyocytes displaying spontaneous beatings with expression of three types of pacemaker ion channels. We demonstrate that acquisition of cellular automaticity and the expression of pacemaker ion channels are regulated by the transcription factors, Csx/Nkx2.5 and GATA4, through intracellular signals including p38 MAP kinase in the process of P19-derived pluripotent cells differentiation into cardiomyocytes.

Effect of Cell Labeling on the Function of Human Pluripotent Stem Cell-Derived Cardiomyocytes.

Cell labeling technologies are required to monitor the fate of transplanted cells in vivo and to select target cells for the observation of certain changes in vitro. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have been transplanted for the treatment of heart injuries or used in vitro for preclinical cardiac safety assessments. Cardiomyocyte (CM) labeling has been used in these processes to facilitate target cell monitoring. However, the functional effect of the labeling agent on hiPSC-CMs has not been studied. Therefore, we investigated the effects of labeling agents on CM cellular functions. 3’-Dioctadecyloxacarbocyanine perchlorate (DiO), quantum dots (QDs), and a DNA plasmid expressing EGFP using Lipo2K were used to label hiPSC-CMs. We conclude that the hiPSC-CM labeling with DiO and QDs does not induce arrhythmogenic effects but rather improves the mRNA expression of cardiac ion channels and Ca2+ influx by L-type Ca2+ channels. Thus, DiO and QD labeling agents may be useful tools to monitor transplanted CMs, and further in vivo influences of the labeling agents should be investigated in the future.

Cardiac specific transcription factor Csx/Nkx2.5 regulates transient-outward K+ channel expression in pluripotent P19 cell-derived cardiomyocytes

The homeobox-containing gene Csx/Nkx2.5 codes several cardiac transcription factors and plays a critical role in early cardiogenesis. We investigated the effect of Csx/Nkx2.5 on the expression of cardiac ion channels using P19-derived cardiomyocytes. P19CL6 cells and P19CL6 cells with Csx/Nkx2.5 overexpression (P19CL6-Csx cells) were induced to differentiate into cardiomyocytes by treatment with dimethyl sulfoxide. Action potentials and membrane currents were measured by whole cell patch clamp at different differentiation stage: the early stage (1–5 days after beating had begun) and the late stage (10–15 days after beating). Expression of Csx/Nkx2.5 mRNA was increased as the differentiation stages advanced in both P19CL6 and P19CL6-Csx cells. In action potential configuration, maximal diastolic potentials in P19CL6-Csx cells exhibited more hyperpolarized potential (‒ 64.2 mV) than those in P19CL6 cells (‒ 54.8 mV, p < 0.01) in the early stage. In P19CL6 cells, among 6 different voltage-gated and ligand-operated K+ channels expressed during the early stage, the transient-outward K+ channel was most predominant. By overexpression of Csx/Nkx2.5, developmental decrease in the transient-outward K+ channel was suppressed. Homeobox-containing gene Csx/Nkx2.5 modifies the amount of distinct ionic channels, during differentiation periods, predominantly changing the expression of the transient-outward K+ channel.