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ABSTRACT: This study aimed at characterizing expression and the functional role of the Gjb6 gene, encoding for connexin 30 (Cx30) protein, in the adult mouse heart.
The expression of the Gjb6 gene in the mouse heart was investigated by RT-PCR and sequencing of amplified cDNA fragments. The sites of Gjb6 expression were identified in the adult heart using transgenic mice with reporter genes (Cx30(LacZ/LacZ) and Cx30(LacZ/LacZ)/Cx40(EGFP/EGFP) mice), as well as anti-HCN4 (hyperpolarization activated cyclic nucleotide-gated potassium channel 4) or anti-connexin antibodies. Cine-magnetic resonance imaging and telemetric ECG recordings were used to evaluate the impact of Cx30 deficiency on cardiac physiology. Gjb6 was shown to be expressed in the sinoatrial (SA) node of the adult mouse heart. Eighty from 100 nuclei on average were LacZ-positive in the SA node of Cx30(LacZ/LacZ) mice. No significant LacZ expression was seen in other cardiac tissues. Cx30 protein was identified in low abundance in the SA node of wild-type mice, as indicated by immunofluorescence experiments. Telemetric ECG recordings indicated that Cx30-deficient mice displayed a mean daily heart rate (HR) that was 9% faster than that measured in control mice (572 +/- 38 b.p.m. vs. 524 +/- 23, P < 0.05). This moderate tachycardia was still observed after inhibition of the autonomic nervous system, demonstrating that Cx30 deficiency resulted in changes in the intrinsic electrical properties of the SA node. Consistent with this hypothesis, Cx30(LacZ/LacZ) displayed a significant reduction of SDNN (standard deviation of the interbeat interval) compared with control mice. Increase of both the cardiac index (20%) and the end-diastolic volume to body weight ratio (16%) with no deficiency in ejection fraction or stroke volume were observed in mutant mice. An increase in cardiac index was interpreted as being a direct consequence of high HR, whereas large end-diastolic volume may be an indirect consequence of prolonged high HR.
Cx30 is functionally expressed, in low abundance, in the SA node of the adult mouse heart where it participates in HR regulation.
Cardiovascular research 09/2009; 85(1):45-55. · 5.80 Impact Factor
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ABSTRACT: During both cardiac maturation and myopathy, elevated levels of circulating catecholamines coincide with alterations in impulse propagation. An in vitro model of cultured cardiomyocytes was used to study the effects of adrenergic stimulation on the conduction characteristics of immature heart cells.
Neonatal rat cardiomyocytes were cultured on preparations designed to measure conduction velocity (CV). CV was measured on the same preparation twice at t=0 and at t=24 h. Under control conditions (n=7), CV at t=0 (30.9+/-1.9 cm/s) and t=24 (32.4+/-4.4 cm/s) was similar (p=0.70). Immunohistochemistry revealed expression of the gap junction proteins connexin (Cx) 40, Cx43 and Cx45, with Cx43 being highly predominant. Stimulation for 24 h with the beta-adrenergic agonist isoproterenol (ISO) significantly increased CV from 28.0 +/-2.0 cm/s at t=0 to 34.8+/-2.2 cm/s at t=24 (p=0.002, n=5). Microelectrode recordings showed a faster upstroke of the action potential (AP) of ISO-treated cells. Reverse transcribed-polymerase chain reactions (RT-PCR) showed that ISO increased expression of SCN5A and alpha(1c) (alpha-subunit of the cardiac sodium and L-type calcium channel, respectively). Stimulation of cells with ISO did not induce alterations in distribution or expression of Cx40, Cx43 and Cx45 (both mRNA and protein), but slightly increased the phosphorylation of Cx43. Stimulation for 24 h with the alpha-adrenergic agonist phenylephrine did neither affect CV nor the expression of the connexin isoforms, SCN5A and alpha(1c).
Alpha- and beta-adrenergic stimulation differently affect propagation of the electric impulse, which is primarily not caused by a differential effect on intercellular coupling. RT-PCR analysis and an enhanced AP upstroke velocity indicate a higher functional expression level of alpha(1c) and SCN5A in beta-adrenergic stimulated cells, which may explain the observed increase in CV.
Circulation Journal 07/2007; 71(6):973-81. · 3.77 Impact Factor
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ABSTRACT: Expression of the tissue-specific gap junction protein connexin(Cx)40 is regulated by the interaction of ubiquitous and tissue-specific factors such as Sp1 and GATA4. Cardiac Cx40 expression is altered under pathological conditions such as atrial fibrillation. A human promoter polymorphism, a G-->A change at position -44 that has been associated with atrial-specific arrhythmias, is located between the TBE-NKE-Sp and GATA consensus transcription factor binding sites important for the regulation of the mouse Cx40 gene. The presence of the A-allele at position -44 in promoter-reporter constructs significantly reduces promoter activity. Using electrophoretic mobility shift assays and luciferase reporter assays in various cell types, we show that Sp1 and GATA4 are important regulators of human Cx40 gene transcription and that the -44 G-->A polymorphism negatively affects the promoter regulation by the transcription factors Sp1 and GATA4.
Biochimica et Biophysica Acta 11/2006; 1759(10):491-6. · 4.66 Impact Factor
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Mehran Firouzi,
Bart Kok,
Wilko Spiering,
Andreas Busjahn,
Connie R Bezzina,
Jan M Ruijter,
Bobby P C Koeleman,
Maria Schipper,
W Antoinette Groenewegen, Habo J Jongsma,
Peter W de Leeuw
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ABSTRACT: Gap junctions, formed by connexins (Cx), are important in the regulation of vascular tone. Previously, we reported two closely linked polymorphisms (-44G --> A and +71A --> G) within regulatory regions of the gene for Cx40, a major connexin in the vascular wall and the kidney. In the present study, we examined the hypothesis that these polymorphic variants are associated with hypertension and that they interact with blood pressure in healthy individuals.
Cx40 genotypes were determined in 191 subjects with essential hypertension, 198 normotensive individuals, and a healthy control population (178 twin pairs, 108 monozygotic, 70 dizygotic).
We found a significant contribution of the minor Cx40 allele or genotype (-44AA/+71GG) to the risk of hypertension in men (P = 0.013 or P = 0.035; odds ratio, 1.87 or 2.10, respectively), but not in women. Moreover, in the healthy control population a significant effect of Cx40 genotype and sex on systolic blood pressure was found (P < 0.05 and P < 0.0001, respectively). Women carrying the minor Cx40 genotype had significantly higher systolic blood pressure compared with non-carriers (P < 0.05). In men, systolic blood pressure in carriers of the minor Cx40 genotype was not significantly different from the other two genotypes, possibly because of the small number of men in this group. However, men carrying the -44GA/+71AG genotype had higher standing systolic blood pressure compared with the more common Cx40 genotype (-44GG; P = 0.033).
These findings suggest that the Cx40 polymorphisms may form a genetic susceptibility factor for essential hypertension in men.
Journal of Hypertension 03/2006; 24(2):325-30. · 4.02 Impact Factor
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ABSTRACT: Cardiac gap junction channels are crucial for conduction of the electric impulse. Between cardiomyocytes there exist gap junctions constructed from connexin40 (Cx40), Cx43 and Cx45. A fourth isoform, Cx37, is expressed in the endothelial lining. Each of these channel types possesses specific properties and their functioning is regulated by various mechanisms. In this chapter we compare the physiological differences between these channels and discuss the factors involved in modulation of channel properties. Next, we evaluate how alterations in expression and differential regulation of channel properties affect cardiac impulse propagation.
Advances in cardiology 02/2006; 42:18-40.
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ABSTRACT: Previous studies have shown that two linked polymorphisms within regulatory regions of the gene for connexin40 (Cx40), at nucleotides -44 (G --> A) and +71 (A --> G) occur in about 7% of the general population. Cx40 is abundant in the atrium, and homozygosity for the linked polymorphisms combined with an SCN5A mutation appeared to be responsible for familial atrial standstill. We hypothesized that these polymorphisms are associated with the atrial electrophysiologic substrate favoring reentrant mechanisms for initiation of atrial fibrillation (AF). Reentry is promoted by spatial dispersion of refractoriness that can be expressed as a coefficient of dispersion (CD).
CD was calculated from the standard deviation of 12 local mean fibrillatory intervals recorded at right atrial sites during induced AF in 30 patients without structural heart disease (14 sporadic AF episodes, 16 no AF history). CD <or= 3.0 was considered normal. Cx40 genotypes were determined by DNA sequencing.
Mean CD in AF patients was 5.96 +/- 0.70 and without AF 1.59 +/- 0.18 (p < 0.001). Thirteen of fourteen patients with AF had enhanced CD. Carriers of -44 AA genotype had higher CD compared with those with -44 GG genotype (6.37 +/- 1.21 vs. 2.38 +/- 0.39, p = 0.018), whereas heterozygotes showed intermediate values (3.95 +/- 1.38, NS).
The rare linked Cx40 polymorphisms are associated with enhanced CD and thus with the substrate for reentry in AF.
Advances in cardiology 02/2006; 42:284-91.
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ABSTRACT: Recordings of the electrical activity of mouse bundle branches (BBs) suggest reduced conduction velocity (CV) in the midseptal compared with the proximal part of the BB. The present study was performed to elucidate the mechanism responsible for this slowing of conduction.
Hearts of 16 mice were isolated and Langendorff perfused. After the right and left ventricular free walls were removed, the extracellular activity of the BB was mapped with a 247-point electrode. Premature stimulation was used to estimate CV restitution in the BBs. Expression/distribution of connexin40 (Cx40), Cx43, and Cx45 was determined. Morphology of the conduction system was assessed by whole-mount acetylcholine esterase staining and in Cx40(+/KI-GFP) hearts. Effective CV in the midseptal part of the left and right BBs was reduced by 50% compared with the proximal BB. CV restitution in the proximal and midseptal parts of the BBs was similar. Myocytes labeled positive for Cx40 and Cx45 in the entire BB. Cx43 colocalized with Cx40 and Cx45 only in the very distal BB. Subcellular distribution of gap junctions differed between proximal and distal BBs. Geometry of the midseptal and distal BBs revealed on both sides a profuse network of interlacing fibers, whereas the proximal BB consisted of a single (right BB) or multiple (left BB) parallel fibers.
Comparison of connexin expression/distribution, geometry of the BBs, and CV characteristics suggests that increased path length for activation resulting from BB geometry is responsible for the apparently reduced CV in the midseptal BB of the mouse heart.
Circulation 11/2005; 112(15):2235-44. · 14.74 Impact Factor
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Teun P De Boer,
Bart Kok,
Kirsten I E Neuteboom,
Nicole Spieker,
Jochum De Graaf,
Olivier H J Destrée,
Martin B Rook,
Toon A B Van Veen, Habo J Jongsma,
Marc A Vos,
Jacques M T De Bakker,
Marcel A G Van Der Heyden
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ABSTRACT: Connexin-containing gap junctions play an essential role in vertebrate development. More than 20 connexin isoforms have been identified in mammals. However, the number identified in Xenopus trails with only six isoforms described. Here, identification of a new connexin isoform from Xenopus laevis is described. Connexin40.4 was found by screening expressed sequence tag databases and carrying out polymerase chain reaction on genomic DNA. This new connexin has limited amino acid identity with mammalian (<50%) connexins, but conservation is higher (approximately 62%) with fish. During Xenopus laevis development, connexin40.4 was first expressed after the mid-blastula transition. There was prominent expression in the presomitic paraxial mesoderm and later in the developing somites. In adult frogs, expression was detected in kidney and stomach as well as in brain, heart, and skeletal muscle. Ectopic expression of connexin40.4 in HEK293 cells, resulted in formation of gap junction like structures at the cell interfaces. Similar ectopic expression in neural N2A cells resulted in functional electrical coupling, displaying mild, asymmetric voltage dependence. We thus cloned a novel connexin from Xenopus laevis, strongly expressed in developing somites, with no apparent orthologue in mammals.
Developmental Dynamics 08/2005; 233(3):864-71. · 2.54 Impact Factor
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Teun P. De Boer,
Bart Kok,
Kirsten I.E. Neuteboom,
Nicole Spieker,
Jochum De Graaf,
Olivier H.J. Destrée,
Martin B. Rook,
Toon A.B. Van Veen, Habo J. Jongsma,
Marc A. Vos,
Jacques M.T. De Bakker,
Marcel A.G. Van Der Heyden
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ABSTRACT: Connexin-containing gap junctions play an essential role in vertebrate development. More than 20 connexin isoforms have been identified in mammals. However, the number identified in Xenopus trails with only six isoforms described. Here, identification of a new connexin isoform from Xenopus laevis is described. Connexin40.4 was found by screening expressed sequence tag databases and carrying out polymerase chain reaction on genomic DNA. This new connexin has limited amino acid identity with mammalian (<50%) connexins, but conservation is higher (∼62%) with fish. During Xenopus laevis development, connexin40.4 was first expressed after the mid-blastula transition. There was prominent expression in the presomitic paraxial mesoderm and later in the developing somites. In adult frogs, expression was detected in kidney and stomach as well as in brain, heart, and skeletal muscle. Ectopic expression of connexin40.4 in HEK293 cells, resulted in formation of gap junction like structures at the cell interfaces. Similar ectopic expression in neural N2A cells resulted in functional electrical coupling, displaying mild, asymmetric voltage dependence. We thus cloned a novel connexin from Xenopus laevis, strongly expressed in developing somites, with no apparent orthologue in mammals. Developmental Dynamics 233:864–871, 2005. © 2005 Wiley-Liss, Inc.
Developmental Dynamics 06/2005; 233(3):864 - 871. · 2.54 Impact Factor
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ABSTRACT: Cardiac hypertrophic remodelling, initiated by signalling cascades in response to increased workload, injury or intrinsic disease, is initially adaptive. However, prolonged hypertrophy as a consequence of pathological stress leads to maladaptive changes that increase the risk for fatal ventricular arrhythmias. One of these changes is the remodelling of myocardial gap junctions, which provide for electrical coupling of adjacent cardiomyocytes. Myocardial gap junctions are composed of three connexin isotypes, connexin40 (Cx40), -43 (Cx43), and -45 (Cx45) and each display a characteristic developmental and regional expression pattern. Alterations in the distribution and expression of Cx43, the predominant isoform in the adult ventricles, has been the main focus of examination in humans, experimental animal models and cultured cardiomyocytes in response to hypertrophy. The molecular mechanisms and signalling pathways underlying these changes have been studied less thoroughly. In this review we summarize what is known about the remodelling of myocardial gap junctions during hypertrophy, the putative underlying mechanisms and functional consequences thereof.
European Heart Journal 12/2004; 25(22):1979-89. · 10.48 Impact Factor
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The Journal of Physiology 09/2004; 516(3):679 - 685. · 4.72 Impact Factor
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ABSTRACT: Alterations in distribution, density, and properties of cardiac gap junctions, which mediate electrical coupling of cardiomyocytes, are considered potentially arrhythmogenic. We recently reported 2 linked polymorphisms within regulatory regions of the gene for the atrial gap junction protein connexin40 (Cx40) at nucleotides -44 (G-->A) and +71 (A-->G), which were associated with familial atrial standstill. The present study examined whether these Cx40 polymorphisms were associated with increased atrial vulnerability in vivo and arrhythmia susceptibility. In 30 subjects without structural heart disease, of whom 14 had documented sporadic paroxysmal atrial fibrillation (AF) and 16 had no AF history, inducibility of AF was assessed using an increasingly aggressive atrial stimulation protocol. Coefficient of spatial dispersion of refractoriness (CD) was calculated. CD was defined as the SD of 12 local mean fibrillatory intervals recorded at right atrial sites, expressed as a percentage of the overall mean fibrillatory interval. Cx40 genotypes were determined by direct DNA sequencing. Subjects were stratified according to normal or increased CD with a cutoff value of 3.0, because CD >3.0 was previously shown to be strongly associated with enhanced atrial vulnerability. The prevalence of the minor Cx40 allele (-44A) and -44AA genotype was significantly higher in subjects with increased dispersion (n=13) compared with those with CD < or =3.0 (n=17; P=0.00046 and P=0.025; odds ratios of 6.7 and 7.4) and a control population (n=253; P=0.00002 and P=3.90x10(-7)). Carriers of -44AA genotype had a significantly higher CD compared with those with -44GG genotype (6.37+/-1.21 versus 2.38+/-0.39, P=0.018), whereas heterozygotes had intermediate values (3.95+/-1.38, NS). All subjects with increased CD had a history of idiopathic AF compared with only 1 subject with normal CD. The -44A allele and -44AA genotype were significantly more frequent in subjects with prior AF than in those without (P=0.0019 and P=0.031; odds ratios 5.3 and 6.2). This study provides strong evidence linking Cx40 polymorphisms to enhanced atrial vulnerability and increased risk of AF. The full text of this article is available online at http://circres.ahajournals.org.
Circulation Research 08/2004; 95(4):e29-33. · 9.49 Impact Factor
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Cardiovascular Research 06/2004; 62(2):225-7. · 6.06 Impact Factor
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ABSTRACT: Connexin 43 (Cx43) is a major determinant of conduction in the ventricular working myocardium of mammals. We investigated the effect of decreased Cx43 expression on conduction velocity and arrhythmogenesis using adult mice with inducible deletion of Cx43.
Cx43Cre-ER(T)/+ mice, in which 1 coding region of the Cx43 gene was replaced by Cre-ER(T), were mated to Cx43fl/fl mice, generating Cx43Cre-ER(T)/fl mice. Application of 4-hydroxytamoxifen (4-OHT) induced Cre-ER(T)-mediated deletion of the floxed Cx43 allele. Epicardial ventricular mapping using a 13x19 multiterminal electrode grid (300-microm spacing) was performed on Langendorff-perfused hearts from Cx43fl/fl plus carrier (n=10), Cx43fl/fl plus 4-OHT (n=10), Cx43 Cre-ER(T)/fl plus carrier (n=9), and Cx43Cre-ER(T)/fl plus 4-OHT (n=10). Cx43 protein amount in group 3 hearts was decreased by 50% compared with group 1. 4-OHT did not affect cardiac protein amounts in group 2 but decreased Cx43 expression up to 95% in group 4 compared with group 3. Epicardial activation of both left ventricle (LV) and right ventricle (RV) during sinus rhythm was similar in all groups. Conduction velocity (CV) changed only in group 4 animals. For RV (LV), longitudinal CV decreased from 38 (35) to 31.6 (33.6) and transverse CV from 24.4 (16.8) to 10.1 (11.3) cm/s. Dispersion of conduction in RV (LV) was increased by 91% (38%). Programmed stimulation resulted in ventricular arrhythmias in group 4 (7 of 10 mice) but never in groups 1 through 3.
Heterozygous expression of Cx43 did not affect ventricular conduction velocity. Up to 95% decrease of Cx43 protein in 4-OHT-treated Cx43(Cre-ER(T)/fl) mice reduced conduction velocity and increased dispersion of conduction and propensity for ventricular arrhythmias.
Circulation 04/2004; 109(8):1048-55. · 14.74 Impact Factor
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ABSTRACT: Voltage-gated Na(+)channels are essential for the amplitude and upstroke velocity of the cardiac action potential, which are important determinants for impulse propagation and impulse conduction velocity of throughout the working myocardium. Mutations in the major cardiac Na(+)channel gene SCN5A have been implicated in rare, familial forms of cardiac arrhythmias, namely LQT3, Brugada syndrome, progressive cardiac conduction disorder and sudden infant death syndrome. It is increasingly recognized that such mutations--apart from changing channel gating characteristics--may also be related to changes in channel protein trafficking and expression. Regulation of ion channel protein expression depends on a fine-tuned balance among various processes, such as gene transcription, RNA processing, protein synthesis, assembly and post-translational modification, the transport to the cell surface, the anchoring to the cytoskeleton, and regulation of endocytosis and controlled degradation of the protein. During the last decade, interest in factors that control the expression level of ion channels and mechanisms that are involved in targeting of channel proteins to specific sub-cellular and membrane domains is increasing. This review focuses on the current knowledge of mechanisms of cardiac Na(+) channel protein trafficking and expression in cardiomyocytes and its relation to Na(+)-channelopathies.
Journal of Molecular and Cellular Cardiology 03/2004; 36(2):185-93. · 5.17 Impact Factor
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ABSTRACT: Altered transcriptional control is likely to contribute to the down-regulation of connexin 43 (Cx43) expression observed in many forms of heart disease. However, little is known about the factors regulating Cx43 transcription in the heart under (patho)physiological conditions. Therefore, a systematic study of rat Cx43 (rCx43) proximal promoter regulation in rat primary neonatal ventricular cardiomyocytes (NCM) and, for comparison, different cell types was initiated. Luciferase assays revealed that, in NCM, the proximal promoter is preserved in a conserved region extending from 148 nucleotides upstream towards 281 nucleotides downstream relative to the transcription initiation site (TIS). Further deletional analysis suggested the involvement of four putative Sp- and two AP1-binding sites. The binding of both Sp1 and Sp3 to the Sp-binding elements and AP1 to the AP1-binding elements was demonstrated by electrophoretic mobility shift assays (EMSA). Promoter-luciferase assays using the natural rCx43 proximal promoter and mutated derivatives in NCM, HL-1 and A7r5 cells revealed that all sites contribute to basal promoter activity. Trans-activation of the Cx43 proximal promoter with Sp1 and Sp3 in Drosophila Schneider line 2 (SL2) cells demonstrated that Sp1 and, to a lesser extent, Sp3 determine rCx43 promoter activation. Thus Sp1, Sp3 and AP1 determine basal Cx43 expression. In addition, we studied the effect of the cardiac transcription factor Nkx2.5 on Cx43 regulation. NCM were infected with adenovirus encoding either beta-galactosidase (control) or Nkx2.5. Cx43 protein and mRNA were significantly decreased after Nkx2.5 infection as shown by Western and Northern blot analyses. Promoter-reporter assays demonstrated that the rCx43 promoter was down-regulated approximately twofold upon Nkx2.5 overexpression. Therefore, in NCM, Nkx2.5 appears to play a role in the regulation of Cx43 expression.
Gene 01/2004; 322:123-36. · 2.34 Impact Factor
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Lucas J Herfst,
Franck Potet,
Connie R Bezzina,
W Antoinette Groenewegen,
Hervé Le Marec,
Theo M Hoorntje,
Sophie Demolombe,
Isabelle Baró,
Denis Escande, Habo J Jongsma,
Arthur A M Wilde,
Martin B Rook
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ABSTRACT: We previously described a Dutch family in which congenital cardiac conduction disorder has clinically been identified. The ECG of the index patient showed a first-degree AV block associated with extensive ventricular conduction delay. Sequencing of the SCN5A locus coding for the human cardiac Na+ channel revealed a single nucleotide deletion at position 5280, resulting in a frame-shift in the sequence coding for the pore region of domain IV and a premature stop codon at the C-terminus.
Wild type and mutant Na+ channel proteins were expressed in Xenopus laevis oocytes and in mammalian cells. Voltage clamp experiments demonstrated the presence of fast activating and inactivating inward currents in cells expressing the wild type channel alone or in combination with the beta1 subinut (SCN1B). In contrast, cells expressing the mutant channels did not show any activation of inward current with or without the beta1 subunit. Culturing transfected cells at 25 degrees C did not restore the Na+ channel activity of the mutant protein. Transient expression of WT and mutant Na+ channels in the form of GFP fusion proteins in COS-7 cells indicated protein expression in the cytosol. But in contrast to WT channels were not associated with the plasma membrane.
The SCN5A/5280delG mutation results in the translation into non-function channel proteins that do not reach the plasma membrane. This could explain the cardiac conduction defects in patients carrying the mutation.
Journal of Molecular and Cellular Cardiology 06/2003; 35(5):549-57. · 5.17 Impact Factor
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ABSTRACT: Murine P19 embryonal carcinoma (EC) cells can differentiate into spontaneously beating cardiomyocytes in vitro and have revealed important insight into the early molecular processes of cardiomyocyte differentiation. We assessed the suitability of the P19 cell model for studying cardiac ion channel regulation at the molecular and functional level.
P19 cells were induced to differentiate towards cardiomyocytes. mRNAs for cardiac markers and ion channels were determined by RT-PCR at six timepoints during the differentiation process. Action potentials and individual ion currents were measured by whole cell patch clamp.
Ion channel mRNA expression of several channels is temporally regulated during differentiation, while others show little or no regulation. L-type calcium and transient outward channels are expressed from very early on, while sodium and delayed and inward rectifier channels are upregulated at somewhat later stages during differentiation, which mirrors the in vivo murine cardiomyocyte differentiation during embryogenesis. Spontaneous cardiomyocyte action potentials exhibit a low upstroke velocity, which often can be enhanced by hyperpolarizing the cells, hence activating thusfar dormant ion channels to contribute to the action potential upstroke. Action potential duration decreases considerably during the differentiation of spontaneously beating cells. In late stages, non-beating myocytes can be found which only generate action potentials upon electrical stimulation. Their shape is comparable to neonatal/juvenile ventricular mouse myocytes in culture. Finally, we show that P19-derived cardiomyocytes display a very complete set of functional ion channels.
P19 cells represent a powerful model to study the regulation of myocardial electrophysiological differentiation at the molecular and functional level.
Cardiovascular Research 06/2003; 58(2):410-22. · 6.06 Impact Factor
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ABSTRACT: Prolonged atrial fibrillation (AF) results in electrical, structural, and gap-junctional remodeling. We examined the reversibility of the changes in (ultra)structure and gap junctions.
Four groups of goats were used: (1) sinus rhythm (SR), (2) 4 months' AF (4 mo AF), (3) 2 months' SR after 4 mo AF (2 mo post-AF), and (4) 4 months' SR after 4 mo AF (4 mo post-AF). Atria were characterized electrophysiologically, (ultra)structure was studied by light and electron microscopy, and structural and gap-junctional protein expression was studied by immunohistochemistry or Western blotting. The atrial effective refractory period had completely returned to normal values 2 mo post-AF. Induced AF episodes still lasted for minutes at 2 and 4 mo post-AF, compared with seconds in the SR group. Structural abnormalities were still present at 2 and 4 mo post-AF, although to a lesser extent. The increased atrial myocyte diameter was back to normal at 4 mo post-AF. The number of myocytes with severe myolysis had almost normalized 4 mo post-AF, whereas myocytes with mild myolysis remained significantly increased. Extracellular matrix area fraction after 4 mo AF was similar to SR. However, the extracellular matrix fraction per myocyte had increased after 4 mo AF and remained higher post-AF. Changes in expression of structural proteins were partially restored post-AF. The reduction of connexin 40 that was observed during AF was completely reversed at 4 mo post-AF.
Recovery from structural remodeling after 4 mo AF is a slow process and is still incomplete 4 mo post-AF. Several months post-AF, the duration of AF episodes is still prolonged (minutes).
Circulation 05/2003; 107(15):2051-8. · 14.74 Impact Factor
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ABSTRACT: Cardiac conduction defects associate with mutations in SCN5A, the gene encoding the cardiac Na+ channel. In the present study, we characterized a family in which the proband was born in severe distress with irregular wide complex tachycardia. His older sister died at 1 year of age from severe conduction disease with similarly widened QRS-complexes. Mutational analysis of SCN5A in the proband demonstrated compound heterozygosity for a nonsense mutation (W156X), inherited from the father, and a missense mutation (R225W), inherited from the mother. Genotyping on DNA extracted from tissue from the deceased sibling revealed the same SCN5A genotype. Injection of cRNA encoding the W156X mutation in Xenopus oocytes did not produce any current. The R225W substitution neutralizes the third Arg residue within the voltage-sensing segment of domain I. Expression studies showed that this mutation leads to a severe reduction in I(Na) and is also associated with gating changes. Histological examination of the heart from the deceased sibling revealed changes consistent with a dilated type of cardiomyopathy and severe degenerative abnormalities of the specialized conduction system. The occurrence of compound heterozygosity for these two mutations implies that the proband carries solely severely dysfunctional cardiac Na+ channels. This explains his severe phenotype and that of his deceased sister who had been a carrier of the same genotype. The morphological changes within the heart of the deceased sibling may have occurred secondary to the Na+ channel abnormality and contributed to the severity of the disorder in this individual.
Circulation Research 03/2003; 92(2):159-68. · 9.49 Impact Factor
