Viktoria Szuts

Regional and tissue specific transcript signatures of ion channel genes in the non‐diseased human heart

The various cardiac regions have specific action potential properties appropriate to their electrical specialization, resulting from a specific pattern of ion‐channel functional expression. The present study addressed regionally defined differential ion‐channel expression in the non‐diseased human heart with a genomic approach. High‐throughput real‐time RT‐PCR was used to quantify the expression patterns of 79 ion‐channel subunit transcripts and related genes in atria, ventricular epicardium and endocardium, and Purkinje fibres isolated from 15 non‐diseased human donor hearts. Two‐way non‐directed hierarchical clustering separated atria, Purkinje fibre and ventricular compartments, but did not show specific patterns for epicardium versus endocardium, nor left‐ versus right‐sided chambers. Genes that characterized the atria (versus ventricles) included Cx40, Kv1.5 and Kir3.1 as expected, but also Cav1.3, Cav3.1, Cavα2δ2, Navβ1, TWIK1, TASK1 and HCN4. Only Kir2.1, RyR2, phospholamban and Kv1.4 showed higher expression in the ventricles. The Purkinje fibre expression‐portrait (versus ventricle) included stronger expression of Cx40, Kv4.3, Kir3.1, TWIK1, HCN4, ClC6 and CALM1, along with weaker expression of mRNA encoding Cx43, Kir2.1, KChIP2, the pumps/exchangers Na+,K+‐ATPase, NCX1, SERCA2, and the Ca2+‐handling proteins RYR2 and CASQ2. Transcripts that were more strongly expressed in epicardium (versus endocardium) included Cav1.2, KChIP2, SERCA2, CALM3 and calcineurin‐α. Nav1.5 and Navβ1 were more strongly expressed in the endocardium. For selected genes, RT‐PCR data were confirmed at the protein level. This is the first report of the global portrait of regional ion‐channel subunit‐gene expression in the non‐diseased human heart. Our data point to significant regionally determined ion‐channel expression differences, with potentially important implications for understanding regional electrophysiology, arrhythmia mechanisms, and responses to ion‐channel blocking drugs. Concordance with previous functional studies suggests that regional regulation of cardiac ion‐current expression may be primarily transcriptional.

Gender-related differences in ion-channel and transporter subunit expression in non-diseased human hearts

Gender-related differences in ventricular electrophysiology are known to be important determinants of human arrhythmic risk, but the underlying molecular basis is poorly understood. The present work aims to provide the first detailed analysis of gender-related cardiac ion-channel gene-distribution, based on samples from non-diseased human hearts. By using a high-throughput quantitative approach, we investigated at a genome-scale the expression of 79 genes encoding ion-channel and transporter subunits in epicardial and endocardial tissue samples from non-diseased transplant donors (10 males, 10 females). Gender-related expression differences involved key genes implicated in conduction and repolarization. Female hearts showed reduced expression for a variety of K(+)-channel subunits with potentially important roles in cardiac repolarization, including HERG, minK, Kir2.3, Kv1.4, KChIP2, SUR2 and Kir6.2, as well as lower expression of connexin43 and phospholamban. In addition, they demonstrated an isoform switch in Na(+)/K(+)-ATPase, expressing more of the alpha1 and less of the alpha3 subunit than male hearts, along with increased expression of calmodulin-3. Iroquois transcription factors (IRX3, IRX5) were more strongly expressed in female than male epicardium, but the transmural gradient remained. Protein-expression paralleled transcript patterns for all subunits examined: HERG, minK, Kv1.4, KChIP2, IRX5, Nav1.5 and connexin43. Our results indicate that male and female human hearts have significant differences in ion-channel subunit composition, with female hearts showing decreased expression for a number of repolarizing ion-channels. These findings are important for understanding sex-related differences in the susceptibility to ventricular arrhythmias, particularly for conditions associated with repolarization abnormalities like Brugada and Long QT syndrome.

Structure, function and clinical relevance of the cardiac conduction system, including the atrioventricular ring and outflow tract tissues.

It is now over 100years since the discovery of the cardiac conduction system, consisting of three main parts, the sinus node, the atrioventricular node and the His-Purkinje system. The system is vital for the initiation and coordination of the heartbeat. Over the last decade, immense strides have been made in our understanding of the cardiac conduction system and these recent developments are reviewed here. It has been shown that the system has a unique embryological origin, distinct from that of the working myocardium, and is more extensive than originally thought with additional structures: atrioventricular rings, a third node (so called retroaortic node) and pulmonary and aortic sleeves. It has been shown that the expression of ion channels, intracellular Ca(2+)-handling proteins and gap junction channels in the system is specialised (different from that in the ordinary working myocardium), but appropriate to explain the functioning of the system, although there is continued debate concerning the ionic basis of pacemaking. We are beginning to understand the mechanisms (fibrosis and remodelling of ion channels and related proteins) responsible for dysfunction of the system (bradycardia, heart block and bundle branch block) associated with atrial fibrillation and heart failure and even athletic training. Equally, we are beginning to appreciate how naturally occurring mutations in ion channels cause congenital cardiac conduction system dysfunction. Finally, current therapies, the status of a new therapeutic strategy (use of a specific heart rate lowering drug) and a potential new therapeutic strategy (biopacemaking) are reviewed.

Ionic mechanisms limiting cardiac repolarization reserve in humans compared to dogs.

•  Cardiac repolarization, through which heart‐cells return to their resting state after having fired, is a delicate process, susceptible to disruption by common drugs and clinical conditions. •  Animal models, particularly the dog, are often used to study repolarization properties and responses to drugs, with the assumption that such findings are relevant to humans. However, little is known about the applicability of findings in animals to man. •  Here, we studied the contribution of various ion‐currents to cardiac repolarization in canine and human ventricle. •  Humans showed much greater repolarization‐impairing effects of drugs blocking the rapid delayed‐rectifier current IKr than dogs, because of lower repolarization‐reserve contributions from two other important repolarizing currents (the inward‐rectifier IK1 and slow delayed‐rectifier IKs). •  Our findings clarify differences in cardiac repolarization‐processes among species, highlighting the importance of caution when extrapolating results from animal models to man.

Terminal differentiation of chondrocytes is arrested at distinct stages identified by their expression repertoire of marker genes

During endochondral bone formation, cells in the emerging cartilaginous model transit through a cascade of several chondrocyte differentiation stages, each characterized by a specific expression repertoire of matrix macromolecules, until, as a final step, the hypertrophic cartilage is replaced by bone. In many permanent cartilage tissues, however, late differentiation of chondrocytes does not occur, due to negative regulation by the environment of the cells. Here, addressing the reason for the difference between chondrocyte fates in the chicken embryo sternum, cells from the caudal and cranial part were cultured separately in serum-free agarose gels with complements defined earlier that either permit or prevent hypertrophic development. Total RNA was extracted using a novel protocol adapted to agarose cultures, and the temporal changes in developmental stage-specific mRNA expression were monitored by Northern hybridization and phosphor image analysis. Kinetic studies of the mRNA accumulation not only showed significant differences between the expression patterns of cranial and caudal cultures after recovery, but also revealed two checkpoints of chondrocyte differentiation in keeping with cartilage development in vivo. Terminal differentiation of caudal chondrocytes is blocked at the late proliferative stage (stage Ib), while the cranial cells can undergo hypertrophic development spontaneously. The differentiation of cranial chondrocytes is reversible, since they can re-assume an early proliferative (stage Ia) phenotype under the influence of insulin, fibroblast growth factor-2 and transforming growth factor-beta in combination. Thus, the expression pattern in the latter culture resembles that of articular chondrocytes. We also provide evidence that the capacities of caudal and sternal chondrocytes to progress from the late proliferative (stage Ib) to hypertrophic stage (stage II) correlate with their differing abilities to express the Indian hedgehog gene.

Cytotoxic effects of a doxorubicin-transferrin conjugate in multidrug-resistant KB cells

Cancer chemotherapy is often limited by the emergence of multidrug-resistant tumor cells. Multidrug resistance (MDR) can be caused by amplification of the MDR genes and overexpression of the P-glycoprotein, which is capable of lowering intracellular drug concentrations. A doxorubicin-transferrin conjugate has been synthesized and exerts its cytotoxic effects through a transmembrane mechanism. We have examined the cytotoxicity of this conjugate and compared it with doxorubicin in sensitive (KB-3-1) and in multidrug-resistant KB cell lines (KB-8-5, KB-C1, and KB-V1). In the clonogenic assay, doxorubicin exhibited IC50 concentrations of 0.03 and 0.12 microM in the sensitive (KB-3-1) and resistant (KB-8-5) cell lines, respectively, whereas, doxorubicin-transferrin conjugate was more effective with IC50 concentrations of 0.006 and 0.028 microM, respectively. In highly multidrug-resistant KB-C1 and KB-V1 cells, doxorubicin up to 1 microM did not cause any cytotoxic effects, while the doxorubicin-transferrin conjugate inhibited colony formation of these cells with IC50 levels of 0.2 and 0.025 microM, respectively. These results demonstrate that doxorubicin-transferrin is effective against multidrug-resistant tumor cells.

Transcriptional profiling of ion channel genes in Brugada syndrome and other right ventricular arrhythmogenic diseases.

AIMS Brugada syndrome is an inherited sudden-death arrhythmia syndrome. Na(+)-current dysfunction is central, but mutations in the SCN5A gene (encoding the cardiac Na(+)-channel Nav1.5) are present in only 20% of probands. This study addressed the possibility that Brugada patients display specific expression patterns for ion-channels regulating cardiac conduction, excitability, and repolarization. METHODS AND RESULTS Transcriptional profiling was performed on right-ventricular endomyocardial biopsies from 10 unrelated Brugada probands, 11 non-diseased organ-donors, seven heart-transplant recipients, 10 with arrhythmogenic right-ventricular cardiomyopathy, and nine with idiopathic right-ventricular outflow-tract tachycardia. Brugada patients showed distinct clustering differences vs. the two control and two other ventricular-tachyarrhythmia groups, including 14 of 77 genes encoding important ion-channel/ion-transporter subunits. Nav1.5 and K(+)-channels Kv4.3 and Kir3.4 were more weakly expressed, whereas the Na(+)-channel Nav2.1 and the K(+)-channel TWIK1 were more strongly expressed, in Brugada syndrome. Differences were also seen in Ca(2+)-homeostasis transcripts, including stronger expression of RYR2 and NCX1. The molecular profile of Brugada patients with SCN5A mutations did not differ from Brugada patients without SCN5A mutations. CONCLUSION Brugada patients exhibit a common ion-channel molecular expression signature, irrespective of the culprit gene. This finding has potentially important implications for our understanding of the pathophysiology of Brugada syndrome, with possible therapeutic and diagnostic consequences.

Zerumbone exerts a beneficial effect on inflammatory parameters of cholecystokinin octapeptide-induced experimental pancreatitis but fails to improve histology.

Objective: Our experiments were designed to investigate the effects of zerumbone pretreatment on cholecystokinin octapeptide (CCK-8)-induced acute pancreatitis in rats. Methods: Male Wistar rats weighing 240 to 280 g were divided into a control group, a group treated with CCK-8, a group receiving 20 mg/kg zerumbone before CCK-8 administration, and a group treated with zerumbone only. Results: The serum amylase and lipase activities and the pancreatic weight-body weight ratio were significantly reduced by zerumbone pretreatment, but the drug failed to influence the histological parameters of pancreatitis. The anti-inflammatory effects of the drug were manifested in decreases in the cytosolic interleukin 6 and tumor necrosis factor &agr; concentrations and an elevation in the I-&kgr;B concentration, whereas the antioxidant ability of zerumbone was demonstrated by reductions in inducible nitric oxide synthase, Mn- and Cu/Zn-superoxide dismutase activities in the zerumbone-treated rats. Conclusion: Zerumbone ameliorated the changes of several parameters of acute pancreatitis probably by interfering with I-&kgr;B degradation, but in the applied dose, it failed to influence the histology of the disease.Abbreviations: CCK-8 - cholecystokinin octapeptide, DMSO - dimethyl sulfoxide, iNOS - inducible nitric oxide synthase, cNOS - constitutive nitric oxide synthase, TNF, tumor necrosis factor, IL-6 - interleukin 6, NF-&kgr;B - nuclear factor &kgr;B, SOD - superoxide dismutase, ASAT - aspartate aminotransferase

Altered expression of genes for kir ion channels in dilated cardiomyopathy

Dilated cardiomyopathy (DCM) is a multifactorial disease characterized by left ventricular dilation that is associated with systolic dysfunction and increased action potential duration. The Kir2.x K⁺ channels (encoded by KCNJ genes) regulate the inward rectifier current (IK1) contributing to the final repolarization in cardiac muscle. Here, we describe the transitions in the gene expression profiles of 4 KCNJ genes from healthy or dilated cardiomyopathic human hearts. In the healthy adult ventricles, KCNJ2, KCNJ12, and KCNJ4 (Kir2.1-2.3, respectively) genes were expressed at high levels, while expression of the KCNJ14 (Kir2.4) gene was low. In DCM ventricles, the levels of Kir2.1 and Kir2.3 were upregulated, but those of Kir2.2 channels were downregulated. Additionally, the expression of the DLG1 gene coding for the synapse-associated protein 97 (SAP97) anchoring molecule exhibited a 2-fold decline with increasing age in normal hearts, and it was robustly downregulated in young DCM patients. These adaptations could offer a new aspect for the explanation of the generally observed physiological and molecular alterations found in DCM.

Binding of doxorubicin-conjugated transferrin to u937 cells

Binding of transferrin (Trf) and its doxorubicin-conjugated forms (Conj) to U937 cells at 0 degrees C were compared using 125I-labelled Trf or Conj. The apparent binding affinity (Ka) of Conj to the surface of U937 cells was (1.9 +/- 0.4).10(8) l/mol; it is about 40% of that of Trf [(5.0 +/- 1.2).10(8) l/mol]. Binding of 125I-labelled ligands was blocked by the unlabelled ligands to the same degree, however, it was not blocked by a great excess of doxorubicin (Dox). N-ethylmaleimide caused about 10% inhibition while dithiothreitol was without effect. Dissociation of 125I-labelled ligands in the presence of different concentrations of unlabelled ligands (Trf and Conj in the all 4 variations) resulted in different R50 values (the concentration of the unlabelled ligand where 50% of the radiolabelled ligand was released). While Trf displaced Trf with an R50 value close to the binding affinity, Conj displacement by Conj occurred with much lower efficiency. The heterolog displacement experiments yielded R50 values in between the two extrema. These results suggest that 1) binding of Conj to the surface of cells is governed by the binding of the Trf part of Conj to the transferrin receptor, 2) -SH groups are not involved in the binding, and 3) a second interaction between the Conj and some constituent(s) of the plasma membrane may modify the binding of Conj in comparison to that of Trf.