Bart Kok

Association of Human Connexin40 Gene Polymorphisms With Atrial Vulnerability as a Risk Factor for Idiopathic Atrial Fibrillation

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 ≤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=3.90×10−7). 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.

Polymorphisms in human connexin40 gene promoter are associated with increased risk of hypertension in men.

Objective 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. Methods 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). Results 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). Conclusion These findings suggest that the Cx40 polymorphisms may form a genetic susceptibility factor for essential hypertension in men.

Human cardiomyocyte progenitor cell-derived cardiomyocytes display a maturated electrical phenotype.

Cardiomyocyte progenitor cells (CMPCs) can be isolated from the human heart and differentiated into cardiomyocytes in vitro. A comprehensive assessment of their electrical phenotype upon differentiation is essential to predict potential future applications of this cell source. CMPCs isolated from human fetal heart were differentiated in vitro and examined using immunohistochemistry, Western blotting, RT-PCR, voltage clamp and current clamp techniques. Differentiated cultures presented up to 95% alpha-actinin positive cardiomyocytes. Adherens junction and desmosomal proteins beta-catenin, N-cadherin, desmin and plakophilin2 were upregulated. Expression levels of cardiac connexins were not affected by differentiation, however Cx43 phosphorylation was increased upon differentiation, accompanied by translocation of connexins to the cell border. RT-PCR analysis demonstrated upregulation of all major cardiac ion channel constituents during differentiation. Patch clamp experiments showed that cardiomyocytes had a stable resting membrane potential of -73.4+/-1.8 mV. Infusion of 1 mM BaCl(2) resulted in depolarization to -59.9+/-2.8 mV, indicating I(K1) channel activity. Subsequent voltage clamp experiments confirmed presence of near mature I(Na), I(Ca,L) and I(K1) current densities. Infusion of the I(Kr) blocker Almokalant caused prolongation of the action potential by 40%. Differentiated monolayers were not spontaneously contracting in the absence of serum, but responded to field stimulation, displaying adult ventricular-like action potentials. Human fetal CMPC-derived cardiomyocytes have a homogenous and rather mature electrical phenotype that benefits to in vitro physiology and pharmacology. In the context of cardiac repair, their properties may translate into a reduced pro-arrhythmic risk and enhanced electrical integration upon transplantation.

CTGF knockout does not affect cardiac hypertrophy and fibrosis formation upon chronic pressure overload

BACKGROUND One of the main contributors to maladaptive cardiac remodeling is fibrosis. Connective tissue growth factor (CTGF), a matricellular protein that is secreted into the cardiac extracellular matrix by both cardiomyocytes and fibroblasts, is often associated with development of fibrosis. However, recent studies have questioned the role of CTGF as a pro-fibrotic factor. Therefore, we aimed to investigate the effect of CTGF on cardiac fibrosis, and on functional, structural, and electrophysiological parameters in a mouse model of CTGF knockout (KO) and chronic pressure overload. METHODS AND RESULTS A new mouse model of global conditional CTGF KO induced by tamoxifen-driven deletion of CTGF, was subjected to 16weeks of chronic pressure overload via transverse aortic constriction (TAC, control was sham surgery). CTGF KO TAC mice presented with hypertrophic hearts, and echocardiography revealed a decrease in contractility on a similar level as control TAC mice. Ex vivo epicardial mapping showed a low incidence of pacing-induced ventricular arrhythmias (2/12 in control TAC vs. 0/10 in CTGF KO TAC, n.s.) and a tendency towards recovery of the longitudinal conduction velocity of CTGF KO TAC hearts. Picrosirius Red staining on these hearts unveiled increased fibrosis at a similar level as control TAC hearts. Furthermore, genes related to fibrogenesis were also similarly upregulated in both TAC groups. Histological analysis revealed an increase in fibronectin and vimentin protein expression, a significant reduction in connexin43 (Cx43) protein expression, and no difference in NaV1.5 expression of CTGF KO ventricles as compared with sham treated animals. CONCLUSION Conditional CTGF inhibition failed to prevent TAC-induced cardiac fibrosis and hypertrophy. Additionally, no large differences were found in other parameters between CTGF KO and control TAC mice. With no profound effect of CTGF on fibrosis formation, other factors or pathways are likely responsible for fibrosis development.

A systematic evaluation of protein kinase a-a-kinase anchoring protein interaction motifs

Protein kinase A (PKA) in vertebrates is localized to specific locations in the cell via A-kinase anchoring proteins (AKAPs). The regulatory subunits of the four PKA isoforms (RIα, RIβ, RIIα, and RIIβ) each form a homodimer, and their dimerization domain interacts with a small helical region present in each of the more than 40 AKAPs reported so far. This allows for tight anchoring of PKA and efficient communication with other signaling proteins that interact with the AKAP scaffold in a spatial and temporal manner. The hydrophobic interaction surfaces of the PKA-R dimer and several AKAP helices have been investigated in great detail. Despite this knowledge, not every suggested AKAP has had its binding motif specified. Here we created an efficient bioinformatic tool, termed THAHIT, to accurately map the PKA binding motif and/or additional motifs of all previously reported AKAPs. Moreover, THAHIT predicts its specificity toward PKA-RIα and/or PKA-RIIα binding. To verify the validity of these newly predicted anchoring sites and their putative specificities, we used computational modeling approaches (HADDOCK), biochemical affinity studies (fluorescence anisotropy), and cellular colocalization studies. We further demonstrate the potential of THAHIT to identify novel AKAPs in cAMP-based chemical proteomics discovery data sets, and the human proteome. We retrieved numerous novel AKAP candidates, including a never reported 330 kDa AKAP observed in heart tissue, which we further characterized biochemically as a PKA-RIIα binder. Altogether, THAHIT provides a comprehensive overview of known and novel PKA-AKAP interaction domains and their PKA-R specificities.

Changes in Cx43 and NaV1.5 Expression Precede the Occurrence of Substantial Fibrosis in Calcineurin-Induced Murine Cardiac Hypertrophy

In mice, the calcium-dependent phosphatase calcineurin A (CnA) induces a transcriptional pathway leading to pathological cardiac hypertrophy. Interestingly, induction of CnA has been frequently noticed in human hypertrophic and failing hearts. Independently, the arrhythmia vulnerability of such hearts has been regularly associated with remodeling of parameters determining electrical conduction (expression level of connexin43 (Cx43) and NaV1.5, connective tissue architecture), for which the precise molecular basis and sequence of events is still unknown. Recently, we observed reduced Cx43 and NaV1.5 expression in 4-week old mouse hearts, overexpressing a constitutively active form of CnA (MHC-CnA model), but the order of events is still unknown. Therefore, three key parameters of conduction (Cx43, NaV1.5 and connective tissue expression) were characterized in MHC-CnA ventricles versus wild-type (WT) during postnatal development on a weekly basis. At postnatal week 1, CnA overexpression induced cardiac hypertrophy in MHC-CnA. Moreover, protein and RNA levels of both Cx43 and NaV1.5 were reduced by at least 50% as compared to WT. Cx43 immunoreactive signal was reduced at week 2 in MHC-CnA. At postnatal week 3, Cx43 was less phosphorylated and RNA level of Cx43 normalized to WT values, although the protein level was still reduced. Additionally, MHC-CnA hearts displayed substantial fibrosis relative to WT, which was accompanied by increased RNA levels for genes previously associated with fibrosis such as Col1a1, Col1a2, Col3a1, Tgfb1, Ctgf, Timp1 and microRNA miR-21. In MHC-CnA, reduction in Cx43 and NaV1.5 expression thus coincided with overexpression of CnA and hypertrophy development and preceded significant presence of fibrosis. At postnatal week 4 the alterations in conductional parameters observed in the MHC-CnA model lead to abnormal conduction and arrhythmias, similar to those observed in cardiac remodeling in heart failure patients. The MHC-CnA model, therefore, provides for a unique model to resolve the molecular origin of conductional remodeling in detail.

Experimental Mapping of the Canine KCNJ2 and KCNJ12 Gene Structures and Functional Analysis of the Canine KIR2.2 ion Channel

For many model organisms traditionally in use for cardiac electrophysiological studies, characterization of ion channel genes is lacking. We focused here on two genes encoding the inward rectifier current, KCNJ2 and KCNJ12, in the dog heart. A combination of RT-PCR, 5′-RACE, and 3′-RACE demonstrated the status of KCNJ2 as a two exon gene. The complete open reading frame (ORF) was located on the second exon. One transcription initiation site was mapped. Four differential transcription termination sites were found downstream of two consensus polyadenylation signals. The canine KCNJ12 gene was found to consist of three exons, with its ORF located on the third exon. One transcription initiation and one termination site were found. No alternative splicing was observed in right ventricle or brain cortex. The gene structure of canine KCNJ2 and KCNJ12 was conserved amongst other vertebrates, while current GenBank gene annotation was determined as incomplete. In silico translation of KCN12 revealed a non-conserved glycine rich stretch located near the carboxy-terminus of the KIR2.2 protein. However, no differences were observed when comparing dog with human KIR2.2 protein upon ectopic expression in COS-7 or HEK293 cells with respect to subcellular localization or electrophysiological properties.

Calmodulin/CaMKII inhibition improves intercellular communication and impulse propagation in the heart and is antiarrhythmic under conditions when fibrosis is absent

AIM In healthy hearts, ventricular gap junctions are mainly composed by connexin43 (Cx43) and localize in the intercalated disc, enabling appropriate electrical coupling. In diseased hearts, Cx43 is heterogeneously down-regulated, whereas activity of calmodulin/calcium-calmodulin protein kinase II (CaM/CaMKII) signalling increases. It is unclear if CaM/CaMKII affects Cx43 expression/localization or impulse propagation. We analysed different models to assess this. METHODS AND RESULTS AC3-I mice with CaMKII genetically inhibited were subjected to pressure overload (16 weeks, TAC vs. sham). Optical and epicardial mapping was performed on Langendorff-perfused rabbit and AC3-I hearts, respectively. Cx43 subcellular distribution from rabbit/mouse ventricles was evaluated by immunoblot after Triton X-100-based fractionation. In mice with constitutively reduced CaMKII activity (AC3-I), conduction velocity (CV) was augmented (n = 11, P < 0.01 vs. WT); in AC3-I, CV was preserved after TAC, in contrast to a reduction seen in TAC-WT mice (-20%). Cx43 expression was preserved after TAC in AC3-I mice, though arrhythmias and fibrosis were still present. In rabbits, W7 (CaM inhibitor, 10 µM) increased CV (6-13%, n= 6, P< 0.05), while susceptibility to arrhythmias decreased. Immunoconfocal microscopy revealed enlarged Cx43 cluster sizes at intercalated discs of those hearts. Total Cx43 did not change by W7 (n= 4), whereas Triton X-100 insoluble Cx43 increased (+21%, n= 4, P< 0.01). Similar findings were obtained in AC3-I mouse hearts when compared with control, and in cultured dog cardiomyocytes. Functional implication was shown through increased intercellular coupling in cultured neonatal rat cardiomyocytes. CONCLUSION Both acute and chronic CaM/CaMKII inhibition improves conduction characteristics and enhances localization of Cx43 in the intercalated disc. In the absence of fibrosis, this reduced the susceptibility for arrhythmias.

Cloning and functional characterization of a novel connexin expressed in somites of Xenopus laevis.

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.

Alternative Promoter Usage and Splicing of the Human SCN5A Gene Contribute to Transcript Heterogeneity

The sodium channel isoform Na(v)1.5 mediates sodium current, excitability, and electrical conduction in the human heart. Recent studies have indicated alternative splicing within the protein-coding portion of its gene, SCN5A, as a mechanism to generate diversity in Na(v)1.5 protein structure and function. In the present study we identified several novel SCN5A transcripts in human heart, displaying distinct 5′-untranslated regions but identical protein-coding sequences. These transcripts originated from the splicing of alternative exons 1 (designated 1A, 1B, 1C, and 1D) to the translational start codon-containing exon 2, and were preferentially expressed in the heart as compared to other tissues. Comparison of their expression level between adult and fetal heart demonstrated that exon 1C- and 1D-derived sequences were more prominent in adult than in fetal heart. Two new promoters (designated P2 and P3) for the SCN5A gene were identified and functionally characterized in myocardial- and nonmyocardial-derived cell lines. Translation of the transcript containing exon 1D-derived sequences proved to be significantly impaired in these cell lines, which could be restored by mutation of an upstream translational start codon. These results implicate the usage of alternative promoters and 5′-untranslated regions as new mechanisms in the regulation of human Na(v)1.5 expression.