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Diabetic patients suffer from both cardiac lipid accumulation and an increased risk of arrhythmias and sudden cardiac death. This correlation suggests a link between diabetes induced cardiac steatosis and electrical abnormalities, however, the underlying mechanism remains unknown. We previously showed that cardiac conduction velocity slows in Zucker diabetic fatty rats and in fructose-fat fed rats, models that both exhibit prominent cardiac steatosis. The aim of this study was to investigate whether acute cardiac lipid accumulation reduces conduction velocity per se. Cardiac lipid accumulation was induced acutely by perfusing isolated rat hearts with palmitate-glucose buffer, or subacutely by fasting rats overnight. Subsequently, longitudinal cardiac conduction velocity was measured in right ventricular tissue strips, and intramyocardial triglyceride and lipid droplet content was determined by thin layer chromatography and BODIPY staining, respectively. Perfusion with palmitate-glucose buffer significantly increased intramyocardial triglyceride levels compared to perfusion with glucose (2.16 ± 0.17 (n = 10) vs. 0.92 ± 0.33 nmol/mg WW (n = 9), P < 0.01), but the number of lipid droplets was very low in both groups. Fasting of rats, however, resulted in both significantly elevated intramyocardial triglyceride levels compared to fed rats (3.27 ± 0.43 (n = 10) vs. 1.45 ± 0.24 nmol/mg WW (n = 10)), as well as a larger volume of lipid droplets (0.60 ± 0.13 (n = 10) vs. 0.21 ± 0.06% (n = 10), P < 0.05). There was no significant difference in longitudinal conduction velocity between palmitate-glucose perfused and control hearts (0.77 ± 0.025 (n = 10) vs. 0.75 m/sec ± 0.029 (n = 9)), or between fed and fasted rats (0.75 ± 0.042 m/sec (n = 10) vs. 0.79 ± 0.047 (n = 10)). In conclusion, intramyocardial lipid accumulation does not slow cardiac longitudinal conduction velocity per se. This is true for both increased intramyocardial triglyceride content, induced by palmi

Astrocytes in the mammalian central nervous system are interconnected by gap junctions made from connexins of the subtypes Cx30 and Cx43. These proteins may exist as hemichannels in the plasma membrane in the absence of a ‘docked’ counterpart on the neighboring cell. A variety of stimuli are reported to open the hemichannels and thereby create a permeation pathway through the plasma membrane. Cx30 and Cx43 have, in their hemichannel configuration, been proposed to act as ion channels and membrane pathways for different molecules, such as fluorescent dyes, ATP, prostaglandins, and glutamate. Published studies about astrocyte hemichannel behavior, however, have been highly variable and/or contradictory. The field of connexin hemichannel research has been complicated by great variability in the experimental preparations employed, a lack of highly specific pharmacological inhibitors and by confounding changes associated with genetically modified animal models. This review attempts to critically assess the gating, inhibition and permeability of astrocytic connexin hemichannels and proposes that connexins in their hemichannel configuration act as gated pores with isoform-specific permeant selectivity. We expect that some, or all, of the controversies discussed here will be resolved by future research and sincerely hope that this review serves to motivate such clarifying investigations.

L-type voltage gated Ca(2+) channels are considered to be the primary source of calcium influx during the myogenic response. However, many vascular beds also express T-type voltage gated Ca(2+) channels. Recent studies suggest that these channels may also play a role in autoregulation. At low pressures (40-80 mm Hg) T-type channels affect myogenic responses in cerebral and mesenteric vascular beds. T-type channels also seem to be involved in skeletal muscle autoregulation. This review discusses the expression and role of T-type voltage gated Ca(2+) channels in the autoregulation of several different vascular beds. Lack of specific pharmacological inhibitors has been a huge challenge in the field. Now the research has been strengthened by genetically modified models such as mice lacking expression of T-type voltage gated Ca(2+) channels (CaV3.1 and CaV3.2). Hopefully, these new tools will help further elucidate the role of voltage gated T-type Ca(2+) channels in autoregulation and vascular function.

Connexins form gap junction channels made up of two connexons (hemichannels) from adjacent cells. Unopposed hemichannels may open toward the extracellular space upon stimulation by, e.g., removal of divalent cations from the extracellular solution and allow isoform-specific transmembrane flux of fluorescent dyes and physiologically relevant molecules, such as ATP and ions. Connexin (Cx)43 and Cx30 are the major astrocytic connexins. Protein kinase C (PKC) regulates Cx43 in its cell-cell gap junction configuration and may also act to keep Cx43 hemichannels closed. In contrast, the regulation of Cx30 hemichannels by PKC is unexplored. To determine phosphorylation-dependent regulation of Cx30 and Cx43 hemichannels, these were heterologously expressed in Xenopus laevis oocytes and opened with divalent cation-free solution. Inhibition of PKC activity did not affect hemichannel opening of either connexin. PKC activation had no effect on Cx43-mediated hemichannel activity, whereas both dye uptake and current through Cx30 hemichannels were reduced. We detected no PKC-induced connexin internalization from the plasma membrane, indicating that PKC reduced Cx30 hemichannel activity by channel closure. In an attempt to resolve the PKC phosphorylation site(s) on Cx30, alanine mutations of putative cytoplasmic PKC consensus sites were created to prevent phosphorylation (T5A, T8A, T102A, S222A, S225A, S239A, and S258A). These Cx30 mutants responded to PKC activation, suggesting that Cx30 hemichannels are not regulated by phosphorylation of a single site. In conclusion, Cx30, but not Cx43, hemichannels close upon PKC activation, illustrating that connexin hemichannels display not only isoform-specific permeability profiles but also isoform-specific regulation by PKC.

Connexin43 (Cx43) generates intercellular gap junction channels involved in, among others, cardiac and brain function. Gap junctions are formed by the docking of two hemichannels from neighbouring cells. Undocked Cx43 hemichannels can upon different stimuli open towards the extracellular matrix and allow transport of molecules such as fluorescent dyes and ATP. A range of phosphorylated amino acids have been detected in the C-terminus of Cx43 and their physiological role has been intensively studied both in the gap junctional form of Cx43 and in its hemichannel configuration. We present the current knowledge of protein kinase C (PKC)-dependent regulation of Cx43 and discuss the divergent results.

BACKGROUND: Type 2 diabetes is associated with abnormal electrical conduction and sudden cardiac death, but the pathogenic mechanism remains unknown. This study describes electrophysiological alterations in a diet-induced pre-diabetic rat model and examines the underlying mechanism.METHODS: Sprague-Dawley rats were fed either high-fat diet and fructose water or normal chow and water for 6 weeks. The electrophysiological properties of the whole heart was analyzed by in vivo surface ECG recordings, as wells as ex vivo in Langendorff perfused hearts during baseline, ischemia and reperfussion. Conduction velocity was examined in isolated tissue strips. Ion channel and gap junction conductances were analyzed by patch-clamp studies in isolated cardiomyocytes. Fibrosis was examined by Masson's Trichrome staining and thin-layer chromatography was used to analyze cardiac lipid content. Connexin43 (Cx43) expression and distribution was examined by western blotting and immunofluorescence respectively.RESULTS: Following 6 weeks of feeding, fructose-fat fed rats (FFFRs) showed QRS prolongation compared to controls (16.1 ± 0.51 (n = 6) vs. 14.7 ± 0.32 ms (n = 4), p < 0.05). Conduction velocity was slowed in FFFRs vs. controls (0.62 ± 0.02 (n = 13) vs. 0.79 ± 0.06 m/s (n = 11), p < 0.05) and Langendorff perfused FFFR hearts were more prone to ventricular fibrillation during reperfusion following ischemia (p < 0.05). The patch-clamp studies revealed no changes in Na(+) or K(+) currents, cell capacitance or gap junctional coupling. Cx43 expression was also unaltered in FFFRs, but immunofluorescence demonstrated an increased fraction of Cx43 localized at the intercalated discs in FFFRs compared to controls (78 ± 3.3 (n = 5) vs. 60 ± 4.2 % (n = 6), p < 0.01). No fibrosis was detected but FFFRs showed a significant increase in cardiac triglyceride content (1.93 ± 0.19 (n = 12) vs. 0.77 ± 0.13 nmol/mg (n = 12), p < 0.0001).CONCLUSION: Six weeks on

Cardiomyocytes are connected by mechanical and electrical junctions located at the intercalated discs (IDs). Although these structures have long been known, it is becoming increasingly clear that their components interact. This review describes the involvement of the ID in electrical disturbances of the heart and focuses on the role of the gap junctional protein connexin 43 (Cx43). Current evidence shows that Cx43 plays a crucial role in organizing microtubules at the intercalated disc and thereby regulating the trafficking of the cardiac sodium channel NaV1.5 to the membrane.

INTRODUCTION: SK channels have functional importance in the cardiac atrium of many species, including humans. Pharmacological blockage of SK channels has been reported to be antiarrhythmic in animal models of atrial fibrillation, however, the exact antiarrhythmic mechanism of SK channel inhibition remains unclear.OBJECTIVES: We speculated that together with a direct inhibition of repolarizing SK current, the previously observed depolarization of the atrial resting membrane potential (RMP) after SK channel inhibition reduces sodium channel availability thereby prolonging the effective refractory period (ERP) and slowing the conduction velocity. We therefore aimed at elucidating these properties of SK channel inhibition and the underlying antiarrhythmic mechanisms by using; microelectrode action potential recordings and conduction velocity measurements in isolated rat atrium. Automated patch-clamping and two-electrode voltage-clamp was used to access INa and IK,ACh respectively.RESULTS: The SK channel inhibitor N-(pyridin-2-yl)-4-(pyridin-2-yl)thiazol-2-amine (ICA) exhibited antiarrhythmic effects. ICA prevented electrically induced runs of atrial fibrillation in the isolated right atrium and induced atrial post-repolarization-refractoriness and depolarized the resting membrane potential (RMP). Moreover, ICA (1-10 µM) was found to slow conduction velocity, however, due to a marked prolongation of ERP the calculated wavelength was increased. Furthermore, at increased pacing frequencies SK channel inhibition by ICA (10-30 µM) demonstrated prominent depression of other sodium channel-dependent parameters. ICA did not inhibit IK,ACh, but at concentrations above 10 µM ICA use-dependently inhibited INa.CONCLUSION: SK channel inhibition modulates multiple parameters of the action potential. It prolongs the action potential duration, and shifts the RMP towards more depolarized potentials through direct I

Diabetes increases the risk of cardiovascular complications including arrhythmias, but the underlying mechanisms remain to be established. Decreased conduction velocity (CV), which is an independent risk factor for re-entry arrhythmias, is present in models with streptozotocin (STZ) induced type 1 diabetes. Whether CV is also disturbed in models of type 2 diabetes is currently unknown.

Udgivelsesdato: 2009-Nov

Udgivelsesdato: 2009-Aug, Intercellular communication via gap junction channels can be quantified by several methods based on diffusion of fluorescent dyes or metabolites. Given the variation in intercellular coupling of cells, even under untreated control conditions, it is of essence to quantify the coupling between numerous cells to obtain reliable estimates of metabolic coupling. Quantification is often based on manual counting of fluorescent cells, which is time consuming and may include some degree of subjectivity. In this report, we introduce a technique based on digital image analysis, and the software for the analysis is presented together with a detailed protocol in the online supplemental material (http://bmi.ku.dk/matlab_program/). Fluorescent dye was introduced in connexin 43-expressing C6 glioma cells by in situ electroporation, and fluorescence intensity was measured in the electroporated cells and in cells receiving dye by intercellular diffusion. The analysis performed is semiautomatic, and comparison with traditional cell counting shows that this method reliably determines the effect of uncoupling by several interventions. This new method of analysis yields a rapid and objective quantification process with a high degree of reproducibility.

Udgivelsesdato: 2008-Nov, Changes in the lipid composition of cardiac myocytes have been reported during cardiac hypertrophy, cardiomyopathy, and infarction. Because a recent study indicates a relation between low phosphatidylinositol-bisphosphate (PIP(2)) levels and reduced intercellular coupling, we tested the hypothesis that agonist-induced changes in PIP(2) can result in a reduction of the functional coupling of cardiomyocytes and, consequently, in changes in conduction velocity. Intercellular coupling was measured by Lucifer Yellow dye transfer in cultured neonatal rat cardiomyocytes. Conduction velocity was measured in cardiomyocytes grown on microelectrode arrays. Intercellular coupling was reduced by angiotensin II (43.7 +/- 9.3%, N = 11) and noradrenaline (58.0 +/- 10.7%, N = 11). To test if reduced intercellular coupling after agonist stimulation was caused by PIP(2)-depletion, myocytes were stimulated by angiotensin II (57.3 +/- 5.7%, N = 14) and then allowed to recover in medium with or without wortmannin (an inhibitor of PIP(2) synthesis). Intercellular coupling fully recovered in control medium (102.1 +/- 8.9%, N = 10), whereas no recovery occurred in the presence of wortmannin (69.3 +/- 7.8%, N = 12). Inhibition of PKC, calmodulin, or arachidonic acid production did not affect the response to either angiotensin II or noradrenaline. Furthermore, decreasing or increasing PIP(2) also decreased and increased intercellular coupling, respectively. This supports the role of PIP(2) in the regulation of intercellular coupling. In beating myocytes, conduction velocity was reduced by angiotensin II stimulation, and recovery after wash out was prevented by inhibition of PIP(2) production. Reductions in PIP(2) inhibit intercellular coupling in cardiomyocytes, and stimulation by physiologically relevant agonists reduces intercellular coupling by this mechanism. The reduction in intercellular coupling lowered conduction velocity.

Udgivelsesdato: null-null, This synopsis covers the main results and conclusions from the platform presentations during the International Gap Junction Conference. More detailed information is provided in the mini reviews on controversial scientific issues, short reports of research results and conference abstracts published in this issue of Cell Communication and Adhesion.

Udgivelsesdato: 2006-Feb, Treatment with non-selective drugs (eg, long-chain alcohols, halothane) that reduce gap junction intercellular communication (GJIC) is associated with reduced infarct size after myocardial infarction (MI). Therefore, it has been suggested that gap junction intercellular communication stimulating compounds may increase infarct size. The antiarrhythmic peptide analogue rotigaptide (ZP123) increases cardiac gap junction intercellular communication and the purpose of the present study was to examine the effects of rotigaptide treatment on infarct size. Myocardial infarction was induced in male rats by ligation of the left anterior descending artery (LAD). Rats (n = 156) were treated with rotigaptide at three dose levels or vehicle from the onset of ischemia and for 3 weeks following LAD occlusion. Infarct size was determined using histomorphometry after 3 weeks treatment. Rotigaptide treatment producing steady state plasma levels of 0.8 +/- 0.1, 5.5 +/- 0.5, and 86 +/- 8 nmol/L had no effect on mortality, but reduced infarct size to 90 +/- 10% (P = 0.41), 67 +/- 7% (P = 0.005), and 82 +/- 7% (P = 0.13), respectively relative to vehicle-treated myocardial infarction rats (100 +/- 12%). In contrast to what was predicted, our data demonstrates that rotigaptide treatment was associated with a significant infarct size reduction. We conclude that whereas treatment with non-selective inhibitors of gap junction intercellular communication cause a reduction in infarct size, this information cannot be extrapolated to the effects of compounds that selectively increase gap junction intercellular communication.