Klaus Steinmeyer

Reprogramming of the Human Atrial Transcriptome in Permanent Atrial Fibrillation. Expression of a Ventricular-Like Genomic Signature

Atrial fibrillation is associated with increased expression of ventricular myosin isoforms in atrial myocardium, regarded as part of a dedifferentiation process. Whether reexpression of ventricular isoforms in atrial fibrillation is restricted to transcripts encoding for contractile proteins is unknown. Therefore, this study compares atrial mRNA expression in patients with permanent atrial fibrillation to atrial mRNA expression in patients with sinus rhythm and to ventricular gene expression using Affymetrix U133 arrays. In atrial myocardium, we identified 1434 genes deregulated in atrial fibrillation, the majority of which, including key elements of calcium-dependent signaling pathways, displayed downregulation. Functional classification based on Gene Ontology provided the specific gene sets of the interdependent processes of structural, contractile, and electrophysiological remodeling. In addition, we demonstrate for the first time a prominent upregulation of transcripts involved in metabolic activities, suggesting an adaptive response to increased metabolic demand in fibrillating atrial myocardium. Ventricular-predominant genes were 5 times more likely to be upregulated in atrial fibrillation (174 genes upregulated, 35 genes downregulated), whereas atrial-specific transcripts were predominantly downregulated (56 genes upregulated, 564 genes downregulated). Overall, in fibrillating atrial myocardium, functional classes of genes characteristic of ventricular myocardium were found to be upregulated (eg, metabolic processes), whereas functional classes predominantly expressed in atrial myocardium were downregulated (eg, signal transduction and cell communication). Therefore, dedifferentiation with adoption of a ventricular-like signature is a general feature of the fibrillating atrium.

Characterization of TASK-4, a novel member of the pH-sensitive, two-pore domain potassium channel family

We report the primary sequence of TASK‐4, a novel member of the acid‐sensitive subfamily of tandem pore K+ channels. TASK‐4 transcripts are widely expressed in humans, with highest levels in liver, lung, pancreas, placenta, aorta and heart. In Xenopus oocytes TASK‐4 generated K+ currents displaying a marked outward rectification which was lost by elevation of extracellular K+. TASK‐4 currents were efficiently blocked by barium (83% inhibition at 2 mM), only weakly inhibited by 1 mM concentrations of quinine, bupivacaine and lidocaine, but not blocked by tetraethylammonium, 4‐aminopyridine and Cs+. TASK‐4 was sensitive to extracellular pH, but in contrast to other TASK channels, pH sensitivity was shifted to more alkaline pH. Thus, TASK‐4 in concert with other TASK channels might regulate cellular membrane potential over a wide range of extracellular pH.

DCPIB is a novel selective blocker of ICl,swell and prevents swelling‐induced shortening of guinea‐pig atrial action potential duration

We identified the ethacrynic‐acid derivative DCPIB as a potent inhibitor of ICl,swell, which blocks native ICl,swell of calf bovine pulmonary artery endothelial (CPAE) cells with an IC50 of 4.1 μM. Similarly, 10 μM DCPIB almost completely inhibited the swelling‐induced chloride conductance in Xenopus oocytes and in guinea‐pig atrial cardiomyocytes. Block of ICl,swell by DCPIB was fully reversible and voltage independent. DCPIB (10 μM) showed selectivity for ICl,swell and had no significant inhibitory effects on ICl,Ca in CPAE cells, on chloride currents elicited by several members of the CLC‐chloride channel family or on the human cystic fibrosis transmembrane conductance regulator (hCFTR) after heterologous expression in Xenopus oocytes. DCPIB (10 μM) also showed no significant inhibition of several native anion and cation currents of guinea pig heart like ICl,PKA, IKr, IKs, IK1, INa and ICa. In all atrial cardiomyocytes (n=7), osmotic swelling produced an increase in chloride current and a strong shortening of the action potential duration (APD). Both swelling‐induced chloride conductance and AP shortening were inhibited by treatment of swollen cells with DCPIB (10 μM). In agreement with the selectivity for ICl,swell, DCPIB did not affect atrial APD under isoosmotic conditions. Preincubation of atrial cardiomyocytes with DCPIB (10 μM) completely prevented both the swelling‐induced chloride currents and the AP shortening but not the hypotonic cell swelling. We conclude that swelling‐induced AP shortening in isolated atrial cells is mainly caused by activation of ICl,swell. DCPIB therefore is a valuable pharmacological tool to study the role of ICl,swell in cardiac excitability under pathophysiological conditions leading to cell swelling.

Molecular mechanisms of early electrical remodeling: transcriptional downregulation of ion channel subunits reduces ICa,Land Itoin rapid atrial pacing in rabbits ☆

OBJECTIVES The purpose of the study was to characterize the ionic and molecular mechanisms in the very early phases of electrical remodeling in a rabbit model of rapid atrial pacing (RAP). BACKGROUND Long-term atrial fibrillation reduces L-type Ca(2+) (I(Ca,L)) and transient outward K(+) (I(to)) currents by transcriptional downregulation of the underlying ionic channels. However, electrical remodeling starts early after the onset of rapid atrial rates. The time course of ion current and channel modulation in these early phases of remodeling is currently unknown. METHODS Rapid (600 beats/min) right atrial pacing was performed in rabbits. Animals were divided into five groups with pacing durations between 0 and 96 h. Ionic currents were measured by patch clamp techniques; messenger ribonucleic acid (mRNA) and protein expression were measured by reverse transcription-polymerase chain reaction and Western blot, respectively. RESULTS L-type calcium current started to be reduced (by 47%) after 12 h of RAP and continued to decline as pacing continued. Current changes were preceded or paralleled by decreased mRNA expression of the Ca(2+) channel beta subunits CaB2a, CaB2b, and CaB3, whereas significant reductions in the alpha(1) subunit mRNA and protein expression began 24 h after pacing onset. Transient outward potassium current densities were not altered within the first 12 h, but after 24 h, currents were reduced by 48%. Longer pacing periods did not further decrease I(to). Current changes were paralleled by reduced Kv4.3 mRNA expression. Kv4.2, Kv1.4, and the auxiliary subunit KChIP2 were not affected. CONCLUSIONS L-type calcium current and I(to) are reduced in early phases of electrical remodeling. A major mechanism appears to be transcriptional downregulation of underlying ion channels, which partially preceded ion current changes.

hKChIP2 is a functional modifier of hKv4.3 potassium channels: cloning and expression of a short hKChIP2 splice variant.

OBJECTIVE The Ca(2+) independent transient outward K(+) current (I(to1)) in the heart is responsible for the initial phase of repolarization. The hKv4.3 K(+) channel alpha-subunit contributes to the I(to1) current in many regions of the human heart. Consistently, downregulation of hKv4.3 transcripts in heart failure and atrial fibrillation is linked to reduction in I(to1) conductance. The recently cloned KChIP family of calcium sensors has been shown to modulate A-type potassium channels of the Kv4 K(+) channel subfamily. METHODS AND RESULTS We describe the cloning and tissue distribution of hKChIP2, as well as its functional interaction with hKv4.3 after expression in Xenopus oocytes. Furthermore, we isolated a short splice variant of the hKChIP2 gene (hKCNIP2), which represents the major hKChIP2 transcript. Northern blot analyses revealed that hKChIP2 is expressed in the human heart and occurs in the adult atria and ventricles but not in the fetal heart. Upon coexpression with hKv4.3 both hKChIP2 isoforms increased the current amplitude, slowed the inactivation and increased the recovery from inactivation of hKv4.3 currents. For the first time we analyzed the influence of a KChIP protein on the voltage of half-maximal inactivation of Kv4 channels. We demonstrate that the hKChIP2 isoforms shifted the half-maximal inactivation to more positive potentials, but to a different extent. By elucidating the genomic structure, we provide important information for future analysis of the hKCNIP2 gene in candidate disorders. In the course of this work we mapped the hKCNIP2 gene to chromosome 10q24. CONCLUSIONS Heteromeric hKv4.3/hKChIP2 currents more closely resemble native epicardial I(to1), suggesting that hKChIP2 is a true beta-subunit of human cardiac I(to1). As a result hKChIP2 might play a role in cardiac diseases, where a contribution of I(to1) has been shown.

Global gene expression in human myocardium-oligonucleotide microarray analysis of regional diversity and transcriptional regulation in heart failure.

To obtain region- and disease-specific transcription profiles of human myocardial tissue, we explored mRNA expression from all four chambers of eight explanted failing [idiopathic dilated cardiomyopathy (DCM), n=5; ischemic cardiomyopathy (ICM), n=3], and five non-failing hearts using high-density oligonucleotide arrays (Affymetrix U95Av2). We performed pair-wise comparisons of gene expression in the categories (1) atria versus ventricles, (2) disease-regulated genes in atria and (3) disease-regulated genes in ventricles. In the 51 heart samples examined, 549 genes showed divergent distribution between atria and ventricles (272 genes with higher expression in atria, 277 genes with higher expression in ventricles). Two hundred and eighty-eight genes were differentially expressed in failing myocardium compared to non-failing hearts (19 genes regulated in atria and ventricles, 172 regulated in atria only, 97 genes regulated in ventricles only). For disease-regulated genes, down-regulation was 4.5-times more common than up-regulation. Functional classification according to Gene Ontology identified specific biological patterns for differentially expressed genes. Eleven genes were validated by RT-PCR showing a good correlation with the microarray data. Our goal was to determine a gene expression fingerprint of the heart, accounting for region- and disease-specific aspects. Recognizing common gene expression patterns in heart failure will significantly contribute to the understanding of heart failure and may eventually lead to the development of pathway-specific therapies.

KCNE2 modulates current amplitudes and activation kinetics of HCN4: influence of KCNE family members on HCN4 currents

AbstractThe HCN4 gene encodes a hyperpolarization-activated cation current contributing to the slow components of the pacemaking currents If in the sinoatrial node and Ih or Iq in the thalamus. Heterologous expression studies of individual HCN channels have, however, failed to reproduce fully the diversity of native If/h/q currents, suggesting the presence of modulating auxiliary subunits. Consistent with this is the recent description of KCNE2, which is highly expressed in the sinoatrial node, as a β-subunit of rapidly activating HCN1 and HCN2 channels. To determine whether KCNE2 can also modulate the slow component of native If/h/q currents, we co-expressed KCNE2 with HCN4 in Xenopus oocytes and in Chinese hamster ovary (CHO) cells and analysed the resulting currents using two-electrode voltage-clamp and patch-clamp techniques, respectively. In both cell types, co-expressed KCNE2 enhanced HCN4-generated current amplitudes, slowed the activation kinetics and shifted the voltage for half-maximal activation of currents to more negative voltages. In contrast, the related family members KCNE1, KCNE3 and KCNE4 did not change current characteristics of HCN4. Consistent with these electrophysiological results, the carboxy-terminal tail of KCNE2, but not of other KCNE subunits, interacted with the carboxy-terminal tail of HCN4 in yeast two-hybrid assays. KCNE2, by modulating If or Ih currents, might thus contribute to the electrophysiological diversity of known pacemaking currents in the heart and brain.

Functional profiling of human atrial and ventricular gene expression.

The purpose of our investigation was to identify the transcriptional basis for ultrastructural and functional specialization of human atria and ventricles. Using exploratory microarray analysis (Affymetrix U133A+B), we detected 11,740 transcripts expressed in human heart, representing the most comprehensive report of the human myocardial transcriptome to date. Variation in gene expression between atria and ventricles accounted for the largest differences in this data set, as 3.300 and 2.974 transcripts showed higher expression in atria and ventricles, respectively. Functional classification based on Gene Ontology identified chamber-specific patterns of gene expression and provided molecular insights into the regional specialization of cardiomyocytes, correlating important functional pathways to transcriptional activity: Ventricular myocytes preferentially express genes satisfying contractile and energetic requirements, while atrial myocytes exhibit specific transcriptional activities related to neurohumoral function. In addition, several pro-fibrotic and apoptotic pathways were concentrated in atrial myocardium, substantiating the higher susceptibility of atria to programmed cell death and extracellular matrix remodelling observed in human and experimental animal models of heart failure. Differences in transcriptional profiles of atrial and ventricular myocardium thus provide molecular insights into myocardial cell diversity and distinct region-specific adaptations to physiological and pathophysiological conditions. Moreover, as major functional classes of atrial- and ventricular-specific transcripts were common to human and murine myocardium, an evolutionarily conserved chamber-specific expression pattern in mammalian myocardium is suggested.

The antihistamine fexofenadine does not affect IKr currents in a case report of drug-induced cardiac arrhythmia

The human HERG gene encodes the cardiac repolarizing K+ current IKr and is genetically inactivated in inherited long QT syndrome 2 (LQTS2). The antihistamine terfenadine blocks HERG channels, and can cause QT prolongation and torsades de pointes, whereas its carboxylate fexofenadine lacks HERG blocking activity. In the present study the ability of fexofenadine to block the K897T HERG channel variant was investigated. The underlying single nucleotide polymorphism (SNP) A2960C was identified in a patient reported to develop fexofenadine‐associated LQTS. K897T HERG channels produced wild‐type‐like currents in Xenopus oocytes. Even at a concentration of 100 μM, fexofenadine did not inhibit wild‐type or K897T HERG channels. Coexpression of wild‐type and K897T HERG with the ß‐subunit MiRP1, slightly changed current kinetics but did not change sensitivity to terfenadine and fexofenadine. Western blot analysis and immunostaining of transiently transfected COS‐7 cells demonstrated that overall expression level, glycosylation pattern and subcellular localization of K897T HERG is indistinguishable from wild‐type HERG protein, and not altered in the presence of 1 μM fexofenadine. We provide the first functional characterization of the K897T HERG variant. We demonstrated that K897T HERG is similar to wild‐type HERG, and is insensitive to fexofenadine. Although the polymorphism changes PKA and PKC phosphorylation sites, regulation of K897T HERG by these kinases is not altered. Our results strongly indicate that QT lengthening and cardiac arrhythmia in the reported case of drug‐induced LQT are not due to the K897T exchange or to an inhibitory effect of fexofenadine on cardiac IKr currents.

Novel KChIP2 isoforms increase functional diversity of transient outward potassium currents

Kv4.3 channels conduct transient outward K+ currents in the human heart and brain where they mediate the early phase of action potential repolarization. KChIP2 proteins are members of a new class of calcium sensors that modulate the surface expression and biophysical properties of Kv4 K+ channels. Here we describe three novel isoforms of KChIP2 with an alternatively spliced C‐terminus (KChIP2e, KChIP2f) or N‐terminus (KChIP2g). KChIP2e and KChIP2f are expressed in the human atrium, whereas KChIP2g is predominantly expressed in the brain. The KChIP2 isoforms were coexpressed with Kv4.3 channels in Xenopus oocytes and currents recorded with two‐microelectrode voltage‐clamp techniques. KChIP2e caused a reduction in current amplitude, an acceleration of inactivation and a slowing of the recovery from inactivation of Kv4.3 currents. KChIP2f increased the current amplitude and slowed the rate of inactivation, but did not alter the recovery from inactivation or the voltage of half‐maximal inactivation of Kv4.3 channels. KChIP2g increased current amplitudes, slowed the rate of inactivation and shifted the voltage of half‐maximal inactivation to more negative potentials. The biophysical changes induced by these alternatively spliced KChIP2 proteins differ markedly from previously described KChIP2 proteins and would be expected to increase the diversity of native transient outward K+ currents.