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Background and purpose The reliable assessment of proarrhythmic risk of compounds under development remains an elusive goal. Current safety guidelines focus on the effects of blocking the KCNH2/HERG ion channel-in tissues and animals with intact repolarization. Novel models with better predictive value are needed that more closely reflect the conditions in patients with cardiac remodelling and reduced repolarization reserve. Experimental approach We have developed a model for the long QT syndrome type-5 in rabbits (LQT5 ) with cardiac-specific overexpression of a mutant (G52R) KCNE1 β-subunit of the channel that carries the slow delayed-rectifier K(+) -current (IKs ). ECG parameters, including short-term variability of the QT interval (STVQT ), a biomarker for proarrhythmic risk, and arrhythmia development were recorded. In vivo, arrhythmia susceptibility was evaluated by i.v. administration of the IKr blocker dofetilide. K(+) currents were measured with the patch-clamp technique. Key results Patch-clamp studies in ventricular myocytes isolated from LQT5 rabbits revealed accelerated IKs and IKr deactivation kinetics. At baseline, LQT5 animals exhibited slightly but significantly prolonged heart-rate corrected QT index (QTi) and increased STVQT . Dofetilide provoked Torsade-de-Pointes arrhythmia in a greater proportion of LQT5 rabbits, paralleled by a further increase in STVQT . Conclusion and implications We have created a novel transgenic LQT5 rabbit model with increased susceptibility to drug-induced arrhythmias that may represent a useful model for testing proarrhythmic potential and for investigations of the mechanisms underlying arrhythmias and sudden cardiac death due to repolarization disturbances.

Hypertriglyceridemia is not only a serious risk factor in the development of cardiovascular diseases, but it is linked to neurodegeneration, too. Previously, we generated transgenic mice overexpressing the human APOB-100 protein, a mouse model of human atherosclerosis. In this model we observed high plasma levels of triglycerides, oxidative stress, tau hyperphosphorylation, synaptic dysfunction, cognitive impairment, increased neural apoptosis and neurodegeneration. Neurovascular dysfunction is recognized as a key factor in the development of neurodegenerative diseases, but the cellular and molecular events linking cerebrovascular pathology and neurodegeneration are not fully understood. Our aim was to study cerebrovascular changes in APOB-100 transgenic mice. We described the kinetics of the development of chronic hypertriglyceridemia in the transgenic animals. Increased blood-brain barrier permeability was found in the hippocampus of APOB-100 transgenic mice which was accompanied by structural changes. Using transmission electron microscopy, we detected changes in the brain capillary endothelial tight junction structure and edematous swelling of astrocyte endfeet. In brain microvessels isolated from APOB-100 transgenic animals increased Lox-1, Aqp4, and decreased Meox-2, Mfsd2a, Abcb1a, Lrp2, Glut-1, Nos2, Nos3, Vim, and in transgenic brains reduced Cdh2 and Gfap-σ gene expressions were measured using quantitative real-time PCR. We confirmed the decreased P-glycoprotein (ABCB1) and vimentin expression related to the neurovascular unit by immunostaining in transgenic brain sections using confocal microscopy. We conclude that in chronic hypertriglyceridemic APOB-100 transgenic mice both functional and morphological cerebrovascular pathology can be observed, and this animal model could be a useful tool to study the link between cerebrovascular pathology and neurodegeneration.

The present study has examined the role of GJ (gap junctions) in the delayed anti-arrhythmic effect of cardiac pacing, with particular reference to the time-course changes in Cx43 (connexin43) expression both after pacing (4×5 min, at a rate of 240 beats/min) and 24 h later, when the dogs were subjected to a 25 min occlusion and reperfusion of the LAD (left anterior descending coronary artery). Compared with the SP (sham-paced) controls (n=20), in dogs paced 24 h previously (n=16) there were reductions in arrhythmia severity [e.g. number of VPB (ventricular premature beats) during occlusion 294±78 compared with 63±25; survival from the combined ischaemia/reperfusion insult 20% compared with 78%], and in other ischaemic changes [epicardial ST-segment, TAT (total activation time) and tissue impedance]. Pacing also prevented the ischaemia-induced structural impairment of the intercalated discs, and preserved GJ permeability and Cx43 phosphorylation, without modifying Cx43 protein content. Following cardiac pacing the membrane and total Cx43 protein contents were unchanged up to 6 h, but were significantly reduced 12 h later (preceded by a down-regulation of Cx43 mRNA at 6 h), and returned to normal by 24 h. Interestingly, dogs that were subjected to ischaemia 12 h after cardiac pacing showed increased arrhythmia generation. We conclude that cardiac pacing results in time-dependent changes in Cx43 expression, which may alter GJ function and influence arrhythmia generation during a subsequent ischaemia/reperfusion insult. This effect is manifested in protection 24 h after pacing, but of potential clinical interest is the finding that there is a time interval after pacing during which an ischaemic event may generate severe ventricular arrhythmias.

Background and purpose: Both ischaemia preconditioning (PC) and the intracoronary infusion of peroxynitrite (PN) suppress ischaemia and reperfusion (I/R)-induced arrhythmias and the generation of nitrotyrosine (NT, a marker of PN). However, it is still unclear whether this latter effect is due to a reduction in nitric oxide (NO) or superoxide (O2-) production. Experimental approach: Dogs anaesthetized with chloralose and urethane were infused, twice for 5 min, with either saline (control) or 100 nM PN, or subjected to similar periods of occlusion (PC), 5 min prior to a 25 min occlusion and reperfusion of the left anterior descending coronary artery. Severities of ischaemia and ventricular arrhythmias, as well as changes in the coronary sinus nitrate/nitrite (NOx) levels were assessed throughout the experiment. The production of myocardial NOx, O2- and NT was determined following reperfusion. Key results: Both PC and PN markedly suppressed the I/R-induced ventricular arrhythmias, compared to the controls, and increased NOx levels during coronary artery occlusion. Reperfusion induced almost the same increases in NOx levels in all groups, but superoxide production and, consequently, the generation of NT were significantly less in PC- and PN-treated dogs than in controls. Conclusions and implications: Since both PC and the administration of PN enhanced NOx levels during I/R, the attenuation of endogenous PN formation in these dogs is primarily due to a reduction in the amount of O2 produced. Thus, the anti-arrhythmic effect of PC and PN can almost certainly be attributed to the preservation of NO availability during myocardial ischaemia.

Background and purpose: Nitric oxide (NO) donors provide a preconditioning-like anti-arrhythmic protection in the anaesthetized dog. As NO may modulate gap junction (GJ) function, the present study investigated whether this anti-arrhythmic effect is due to a modification of GJs by NO, derived from the NO donor sodium nitroprusside (SNP). Experimental approach: In chloralose-urethane-anaesthetized, open-chest dogs, either saline (controls; n= 11) or SNP (0.2 µg·kg−1·min−1; n= 10) was infused at a rate of 0.5 mL·min−1 by the intracoronary route. The infusions were started 20 min prior to and maintained throughout the entire 60 min occlusion period of the left anterior descending coronary artery. The severity of ischaemia and of arrhythmias, tissue electrical impedance and permeability, as well as the phosphorylation of connexin43, were assessed. Key results: Compared with the controls, SNP infusion markedly suppressed the total number of ventricular premature beats (666 ± 202 vs. 49 ± 18; P < 0.05), and the number of ventricular tachycardiac episodes (8.1 ± 2.3 vs. 0.2 ± 0.1; P < 0.05) without significantly modifying the incidence of ventricular tachycardia or ventricular fibrillation. The severity of ischaemia (epicardial ST-segment changes, inhomogeneity of electrical activation) and tissue electrical impedance changes were significantly less in the SNP-treated dogs. SNP improved GJ permeability and preserved the phosphorylated form of connexin43. Conclusion and implications: The anti-arrhythmic protection resulting from SNP infusion in the anaesthethized dog may, in part, be associated with the modulation of gap junctional function by NO.

The objective of this study was to examine whether the PI3-kinase/Akt pathway is involved in the activation of endothelial nitric oxide synthase (eNOS) and in the subsequent increase of nitric oxide (NO) production that has been proved to play a role in the antiarrhythmic effect of acute simvastatin treatment in anaesthetised dogs, subjected to a 25min occlusion and reperfusion of the left anterior descending coronary artery. Using the same model, 12 dogs out of the 26 controls (given the solvent of simvastatin) and 11 dogs out of the 23 animals treated with intracoronary administered simvastatin (0.1mg/kg), were now received wortmannin (1.5mg/kg, ic.), a selective inhibitor of PI3-kinase. In another 13 dogs the effects of DMSO (0.1%), the vehicle of wortmannin, were examined. Compared to the controls, simvastatin markedly reduced the severity of ischaemia (epicardial ST-segment, inhomogeneity) and ventricular arrhythmias that were reversed (except the occlusion-induced ventricular fibrillation [VF; 50%, 0%, 0%]) by the administration of wortmannin. Thus in these groups there were 310±45, 62±14, 307±59 ectopic beats, 7.1±1.4, 0.3± 0.2, 4.3±1.3 tachycardiac episodes that occurred 93%, 17% and 73% of the dogs during occlusion, whereas survival following reperfusion was 0%, 67% and 0%, respectively. Simvastatin also increased the phosphorylation of eNOS and the plasma nitrate/nitrite levels, but reduced myocardial superoxide production on reperfusion. These effects of simvastatin were also abolished in the presence of wortmannin. We conclude that the NO-dependent antiarrhythmic effect of simvastatin involves the rapid activation of eNOS through the stimulation of the PI3-kinase/Akt pathway.

Loss-of-function mutations of the KCNJ2 gene encoding for the inward rectifier potassium channel subunit Kir2.1 cause Andersen–Tawil Syndrome (ATS), a rare genetic disorder characterised by periodic paralysis, ventricular arrhythmias, and dysmorphic features. Clinical manifestations of the disease appear to vary greatly with the nature of mutation, therefore, functional characterisation of ATS-causing mutations is of clinical importance. In this study, we describe the identification and functional analysis of a novel KCNJ2 mutation, Val302del, identified in a patient with ATS. Heterologously expressed wild type (WT) and Val302del mutant alleles showed similar subcellular distribution of the Kir2.1 protein with high intensity labelling from the membrane region, demonstrating normal membrane trafficking of the Val302del Kir2.1 variant. Cells transfected with the WT allele displayed a robust current with strong inward rectification, while no current above background was detected in cells expressing the Val302del Kir2.1 subunit. Co-transfection of CHO cells with the WT and the Val302del Kir2.1 revealed a dose-dependent inhibitory effect of the Val302del Kir2.1 mutant subunit on WT Kir2.1 currents. These observations indicate that the WT and the Val302del mutant subunits co-assemble in the cell membrane and that the mutation affects potassium conductivity and (or) gating of the WT/Val302del heteromeric Kir2.1 channels.

The objective of this study was to provide evidence that gap junctions are involved in the delayed antiarrhythmic effect of cardiac pacing. Twenty-four dogs were paced through the right ventricle (4 × 5 min, rate of 240 beats/min) 24 h prior to a 25 min occlusion of the left anterior descending coronary artery. Some of these paced dogs were infused with 50 (n = 7) or 100 μmol/L (n = 7) of the gap junction uncoupler carbenoxolone (CBX), prior to and during the occlusion. Ten sham-paced dogs, subjected only to occlusion, served as the controls. Cardiac pacing markedly reduced the number of ectopic beats and episodes of ventricular tachycardia (VT), as well the incidence of VT and ventricular fibrillation during occlusion. The changes in severity of ischaemia and tissue electrical resistance were also less marked compared with the unpaced controls. Pacing also preserved the permeability of gap junctions, the phosphorylation of connexin43, and the structural integrity of the intercalated discs. The closing of gap junctions with CBX prior to and during ischaemia markedly attenuated or even abolished these protective effects of pacing. Conclusion: Our results support the previous findings that gap junctions play a role in the delayed antiarrhythmic effect of cardiac pacing.

obJeCtives: To examine whether changes in nitric oxide (NO) bioavailability occurring during a 25 min occlusion of the left anterior descending coronary artery and following ischemic preconditioning (PC) in anesthetized dogs (chloralose and urethane, intravenous) are associated with changes in the activation of NO synthase (NOS) enzymes. Methods: In 12 control and 16 preconditioned dogs, total NOS activity (measured using radioimmunoassay), superoxide (measured using confocal microscopy) and nitrotyrosine (NT; measured using Western blot) production were assessed in tissue samples obtained from the left ventricular wall both after the PC (two 5 min occlusions) procedure and at two time points (5 min and 25 min) during prolonged ischemia. Plasma nitrate/nitrite concentrations in the blood of the coronary sinus were also determined. ResuLts: Compared with the sham control group that was not subjected to ischemia, occlusion of the left anterior descending coronary artery resulted in an initial increase, followed by a significant decrease in NOS activity and nitrate/nitrite levels. There were parallel increases in superoxide and NT production by the end of the ischemic period. PC itself enhanced NOS activity and the concentration of NO metabolites, which were maintained during the prolonged occlusion. Furthermore, PC prevented prolonged ischemia-induced superoxide and NT formation. ConCLusions: The present study demonstrated that changes in NO bioavailability during ischemia are closely associated with changes in NOS activation. PC stimulates and maintains the activation of NOS, thereby providing better NO bioavailability and suppressed free radical formation during sustained ischemia. All of these effects may have importance in the previously observed marked antiarrhythmic effect of PC.

The present study has examined the effects and the possible mechanisms of a single dose of simvastatin on the severity of arrhythmias resulting from a 25min occlusion and reperfusion of the left anterior descending coronary artery in anaesthetized (chloralose and urethane) dogs. The control animals (n=16) were given the solvent of simvastatin by slow (over 5min) intracoronary (ic.) injection just prior to the occlusion. Twenty-six dogs were treated with simvastatin (0.1mg/kg) by the same route, both in the absence (n=15) and in the presence (n=11) of l-NAME. This latter was administered (5mg/kg, ic.) either alone (n=12) or 10min before the simvastatin treatment. The severity of ischaemia (epicardial ST-segment, inhomogeneity) and ventricular arrhythmias (ventricular premature beats [VPBs], ventricular tachycardia [VT] and fibrillation [VF]), plasma nitrite/nitrate levels, myocardial superoxide production and eNOS activity were assessed. Compared with controls simvastatin significantly reduced the number of VPBs (289+/-34vs. 94+/-25) and the episodes of VT (5.6+/-1.3vs. 0.3+/-0.2), the incidence of VT (88% vs. 20%) and VF (56% vs. 0%) during occlusion and increased survival (0% vs. 33%) on reperfusion. There were also less marked ischaemic changes in the simvastatin-treated dogs than in the controls. Simvastatin preserved eNOS activity and nitric oxide (NO) bioavailability during occlusion and attenuated superoxide production following reperfusion. All these effects of simvastatin (except for the protection against VF) were reversed by l-NAME. We conclude that simvastatin given just prior to ischaemia/reperfusion reduces the severity of arrhythmias. This effect involves both NO-dependent and NO-independent mechanisms.

This study has examined whether peroxynitrite (PN), generated during the preconditioning (PC) procedure or administered by brief intracoronary infusions, plays a trigger role in the anti-arrhythmic effects of preconditioning and peroxynitrite in anaesthetized dogs. To achieve this we infused the peroxynitrite scavenger uric acid (UA; 0.2 mg/kg/min, i.v.) over a 30 min period, just prior to a 25 min occlusion of the left anterior descending coronary artery, in preconditioned (UA + PC, n = 8), peroxynitrite-treated (UA + PN, n = 8) and in control (UAC; n = 9) dogs. The effects were compared to those obtained from groups (PC, n = 10; PN, n = 10; C1, n = 14) without uric acid administration. Severities of ischaemia (ST-segment elevation, inhomogeneity of electrical activation) and ventricular arrhythmias (VPBs, VT, VF), plasma nitrate/nitrite levels, as well as myocardial superoxide and nitrotyrosine productions were determined. Both preconditioning and the infusion of peroxynitrite increased nitrotyrosine formation which was abolished by the simultaneous administration of urate. Despite this, the protective effects of preconditioning (i.e. reductions in arrhythmias, superoxide and nitrotyrosine productions, as well as the increase in nitric oxide availability), occurring during the prolonged period of occlusion and reperfusion were still present. In contrast, urate completely abolished the protection resulted from peroxynitrite administration. This effect is most probably due to the fact that urate has already scavenged peroxynitrite during the infusion. Interestingly, urate itself, given prior to ischaemia and reperfusion, was also protective. We conclude that peroxynitrite in nanomolar concentrations can induce an anti-arrhythmic effect but peroxynitrite, generated during the preconditioning stimulus, is not necessary for the preconditioning-induced anti-arrhythmic protection.

Objective : The aim of this study was to determine whether uncoupling of gap junctions (GJ) prior to ischaemia would modify the antiarrhythmic effect of ischaemic preconditioning (PC) in a canine model of ischaemia/reperfusion. Methods : Twenty control dogs, anaesthetised with chloralose and urethane, were thoracotomised and subjected either to a 25 or a 60 min occlusion of the left anterior descending (LAD) coronary artery. This prolonged ischaemia was preceded 20 min earlier by a single 5 min LAD occlusion in preconditioned dogs (PC group; n =14) or by a 20 min intracoronary infusion of 50 μM carbenoxolone (CBX group; n =15), a relatively selective uncoupler of gap junctions. CBX was also infused in PC dogs (CBX+PC group; n =11). The severity of ischaemia (epicardial ST-segment changes, inhomogeneity of electrical activation) and of ventricular arrhythmias, such as ventricular premature beats (VPBs), ventricular tachycardiac (VT) episodes and ventricular fibrillation (VF), as well as changes in electrical impedance was assessed throughout the experiments. Connexin 43 (Cx43) phosphorylation and GJ permeability were determined at the end of the occlusion periods. Results : Compared to the controls PC and, interestingly, CBX markedly reduced, e.g. the total number of VPBs (440±104 vs 47±11 and 60±15; P

Introduction: We have evidence that rapid cardiac pacing preserves gap junction (GJ) function by preventing the structural alterations that occur during ischaemia, 24h later; a mechanism which play a role the pacing-induced delayed protection against arrhythmias. Purpose: The present study has now examined whether closing of GJs prior to ischaemia with carbenoxolone (CBX) would attenuate the beneficial effects of pacing. Methods: Under light pentobarbitone anaesthesia 22 dogs were subjected to rapid right ventricular pacing (4x5 min, at a rate of 240 beats/min) and, 24 hours later, to a 30 min occlusion of the left anterior descending coronary artery (LAD). In 7 and 5 of these paced dogs CBX was infused locally into a small branch of the LAD in doses of 50 and 100 μM, respectively. The infusion was started 20 min prior to and maintained over the entire occlusion period. Another group of dogs (n=10) were instrumented but not paced and only subjected to occlusion. These dogs served as controls. At the end of the occlusion period, myocardial tissue samples were taken for the measurement of Cx43 phosphorylation, gap junction permeability, and immunofluorescence analysis of the intercalated discs (ID). Results: Compared with controls, cardiac pacing prevented the ischaemia-induced reduction in GJ permeability and the marked dephosphorylation of Cx43. Pacing also preserved the structural integrity of ID. CBX in both doses reversed the pacing-induced protection; i.e. the permeability of GJs was again decreased and the disruption of ID increased. CBX, however, did not affect the phosphorylation of Cx43. Conclusion: Our results that the inhibition of GJs with CBX prior to an ischaemic insult attenuates the protective effects of cardiac pacing confirm the role of GJs in the delayed phase of the pacing-induced protection.

In a previous study we have shown that a single dose of simvastatin markedly reduced the severity of ventricular arrhythmias that resulted from a 25 min ischaemia and reperfusion (I/R) in anaesthetized dogs. This protection seems to involve the generation of nitric oxide (NO) via the activation of NO synthase (NOS), since the antiarrhythmic effect of simvastatin was abolished by L-NAME. The signaling pathways, which are involved in the rapid activation of NOS by simvastatin, are however, not fully elucidated; some studies have suggested that the stimulation of the PI3/Akt pathway may play a role. Thus, the present study has now examined whether this signaling pathway has an importance in the acute antiarrhythmic effect of simvastatin. Mongrel dogs, anaesthetized with chloralose and urethane (i.v.) were subjected to a 25 min occlusion of the left anterior descending (LAD) coronary artery, which was followed in some cases by reperfusion. The control animals were infused either with the solvent of simvastatin (C1; n=16) or DMSO (the solvent of wortmannin; C2; n=11). In three other groups simvastatin (S; 0.1 mg/kg; n=15) and wortmannin a selective inhibitor of phosphatidyl-inositol-3-kinase (W; 1.5 mg/kg; n=10) were administered alone or together (W+S; n=9) in slow intracoronary injection just prior to the occlusion. The severity of ischaemia (degree of inhomogeneity of electrical activation, epicardial ST-segment) and of arrhythmias, as well as the plasma NOx levels in blood samples taken from the coronary sinus were assessed. The activity of eNOS was determined by Western blot, the tissue superoxide production was evaluated by dihydroethidium fluorescence staining using confocal microscopy. Compared with controls (C1+C2), simvastatin significantly decreased the number of VPBs (272±73 vs. 94±25) and episodes of VT (4.7±1.6 vs. 0.3±0.2), the incidence of VT (76% vs. 20%) and VF (28% vs. 0%) during the occlusion, and increased survival following reperfusion (17% vs 38%). This protective effect of simvastatin was abolished in the presence of wortmannin (VPBs: 257±113, VT episodes: 4.6±2.2, VT and VF%: 67% and 83%, survival: 17%). Simvastatin also enhanced eNOS activity and preserved NO bioavailability during occlusion and reduced superoxide production following reperfusion. We conclude from these results that the antiarrhythmic effect of simvastatin is due to an increased NO bioavailability resulting from the rapid activation of eNOS via the activation of the PI3K/Akt signaling pathway.

Purpose: We have now examined whether the ischaemia-induced structural alterations of the main gap junction (GJ) protein connexin43 (Cx43) are modified by rapid cardiac pacing, 24h previously. Methods: Under light pentobarbital anesthesia, in 8 dogs a pacing electrode was introduced into the right ventricle through which, in half of these dogs, the heart was paced four times for 5 min at a rate of 240 beats/min. Twenty-four hours later, both the non-paced controls (SPO; n=4) and the paced (PO; n=4) animals were subjected to a 30 min occlusion of the left anterior descending (LAD) coronary artery. Another 3 dogs were also instrumented, but these animals were not subjected to either pacing or coronary artery occlusion (SP, n=3). In each experimental group left ventricular tissue samples from the LAD region were taken for immunohistochemical, western blot and co-immunoprecipitation analyses. Results: The immunohistochemical images showed that a 30 min of ischaemia resulted in increased intensity of Cx43 and obvious structural alterations of the intercalated discs (ID); the connections became disoriented and ‘blurred’ particularly at the longitudinal sections. In contrast, in the paced dogs the structural integrity of GJs was well-preserved and the intensity of Cx43was reduced to the same level as has been observed in the SP group. Under normal conditions, the GJs are mainly located at the periphery of the ID, the intensity and size of GJs is less pronounced at the center region of the disc. Ischaemia altered this distribution pattern resulting in enhanced intensity in both the peripheral and central regions of the ID. These ischaemia-induced changes were, however, almost completely abolished by pacing, commenced 24h previously. Compared with the SP dogs, ischaemia reduced the phosphorylation and increased the dephosphorylation of Cx43 (phospho/dephospho ratio was 40/60%). Pacing reversed this phospho/dephospho ratio of Cx43 (55/45%). Conclusions: These results suggest that rapid cardiac pacing favorably modifies the ischaemia-induced structural changes of GJs occurring within the ID. The preliminary results of the co-immunoprecipitation studies indicate that these changes may associate with alterations in the connection of Cx43 and ZO1 proteins.