Examination of the Changes in Calcium Homeostasis in the Delayed Antiarrhythmic Effect of Sodium Nitrite.

We have evidence that the intravenous infusion of sodium nitrite (NaNO2) results in an antiarrhythmic effect when given 24 h prior to an ischemia and reperfusion (I/R) insult in anaesthetized dogs. This protection was associated with the reduction of reactive oxygen species resulting from I/R through the attenuation of mitochondrial respiration. Here, we examined whether the changes in calcium, which also contributes to arrhythmia generation, play a role in the NaNO2-induced effect. On the first day, 30 anaesthetized dogs were treated either with saline or NaNO2 (0.2 µmol/kg/min) for 20 min. Some animals were subjected to a 25 min LAD (anterior descending branch of the left coronary artery) occlusion and 2 min reperfusion (I/R = 4; NaNO2-I/R = 6), or the heart was removed 24 h later. We have shown that nitrite prevented the I/R-induced increase in cellular and mitochondrial calcium deposits. During simulated I/R, the amplitude of the calcium transient and the diastolic calcium level were significantly lower in the nitrite-treated hearts and the ERP (effective refractory period) fraction of the action potential was significantly increased. Furthermore, nitrite also enhanced the mitochondrial respiratory response and prevented the MPTPT opening during calcium overload. These results suggest that nitrite can reduce the harmful consequences of calcium overload, perhaps directly by modulating ion channels or indirectly by reducing the mitochondrial ROS (reactive oxygen species) production.

Is there a role of inducible nitric oxide synthase activation in the delayed antiarrhythmic effect of sodium nitrite?

This study aimed to examine whether inducible nitric oxide synthase (iNOS) plays a role in the delayed antiarrhythmic effect of sodium nitrite. Twenty-one dogs were infused intravenously with sodium nitrite (0.2 µmol·kg-1·min-1) for 20 min, either in the absence (n = 12) or in the presence of the iNOS inhibitor S-(2-aminoethyl)-isothiourea (AEST) (total dose 2.0 mg·kg-1 i.v., n = 9). Control dogs (n = 12) were given saline. Twenty-four hours later, all of the dogs were subjected to a 25 min period occlusion of the left anterior descending coronary artery followed by rapid reperfusion. Dogs treated with AEST and nitrite received again AEST prior to the occlusion. Compared with the controls, sodium nitrite markedly reduced the number of ectopic beats, the number and incidence of ventricular tachycardia, and the incidence of ventricular fibrillation during occlusion and increased survival (0% versus 50%) from the combined ischaemia and reperfusion insult. Although AEST completely inhibited iNOS activity, the nitrite-induced increase in NO bioavailability during occlusion was not substantially modified. Furthermore, AEST attenuated but did not completely abolish the antiarrhythmic effect of nitrite. The marked delayed antiarrhythmic effect of sodium nitrite is not entirely due to the activation of iNOS; other mechanisms may certainly play a role.

Examination of the effect of sodium nitrite on gap junction function during ischaemia and reperfusion in anaesthetized dogs.

It has previously been proved that sodium nitrite, infused prior to coronary artery occlusion or before reperfusion, results in marked antiarrhythmic effect in anaesthetized dogs. We have now examined whether this protection involves the modulation of gap junction (GJ) function by nitric oxide (NO), derived from nitrite administration under ischaemic conditions. Two groups of chloralose and urethane anaesthetized dogs, each containing 13 animals, were subjected to a 25 min period occlusion of the left anterior descending (LAD) coronary artery, followed by reperfusion. One group was infused with sodium nitrite (0.2 µmol/kg/min, i.v.), the other group with saline 10 min prior to reperfusion. The severities of arrhythmias and of ischaemia (epicardial ST-segment, total activation time), parallel with changes in myocardial tissue impedance, a measure of electrical coupling of gap junctions, were assessed during the experiments. Compared to the controls, nitrite infusion administered prior to reperfusion significantly attenuated the severity of ischaemia, the ischaemia-induced impedance changes and, consequently, the severity of arrhythmias, occurring during the 1B phase of the occlusion, and increase survival following reperfusion (0% vs. 85%). It is concluded that the marked antiarrhythmic effect of sodium nitrite is partly due, to the preservation of the electrical coupling of GJs by NO.

Further evidence for the role of gap junctions in the delayed antiarrhythmic effect of cardiac pacing.

UNLABELLED: 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.

Time course analysis of cardiac pacing-induced gene expression changes in the canine heart.

Rapid right ventricular pacing in anesthetized dogs results in marked protection against ischemia and reperfusion-induced ventricular arrhythmias, 24 h later. We have previous evidence that this protection associates with altered expression of genes, encoding proteins involved in the delayed cardioprotection. However, the sequence of transcriptional changes occurring between the pacing stimulus and the test ischemia has not yet been elucidated. Thus, we designed studies in which the expression of 29 genes was examined by real-time PCR at various time intervals, i.e., immediately (0 h), 6, 12, and 24 h after short periods (4 times 5 min) of rapid (240 beats min(-1)) right ventricular pacing in the canine. Sham-operated dogs (the pacing electrode was introduced but the dogs were not paced) served as controls. Compared with these dogs, pacing induced an early up-regulation of genes which encode, for example, HSP90, MnSOD, ERK1, PKCε, Bcl2, and sGC; all these somehow relate to the early phase of the protection. These genes remained either up-regulated or, after a transient lower expression (around 6 h), were up-regulated again, suggesting their involvement in the delayed protection. There were also some genes which down-regulated soon after the pacing stimulus (e.g., Bax, Casp3, Casp9, MMP9, GSK3ß), and showed also low expression 24 h later. Genes encoding eNOS and iNOS, as well as Cx43 were only up-regulated 12 h after pacing. We conclude that cardiac pacing induces time-dependent changes in gene expression, and the sequence of these changes is important in the development of the delayed protection.

The involvement of gap junctions in the delayed phase of the protection induced by cardiac pacing in dogs.

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.

Changes in gene expression following cardiac pacing-induced delayed cardioprotection in the canine heart.

The aim of the present study was to identify gene expression changes in the rapid cardiac pacing-induced delayed antiarrhythmic protection in the canine, using cDNA microarrays and quantitative real-time PCR (QRT -PCR) techniques. In all dogs under light pentobarbitone anaesthesia, a pacing electrode was introduced into the right ventricle, and then the animals were divided into three groups: (1) sham-operated and sham-paced group (SP, n = 3) (2) ischaemic control group (IC; n = 3); these were without cardiac pacing and subjected only to a 25 min occlusion of the left anterior descending coronary artery (LAD), and (3) paced group (PC, n = 3); these animals were paced at a rate of 220-240 beats min-1 24 h prior to ischaemia. With cDNA chip 23 genes were found with altered expression in response to rapid cardiac pacing and 10 genes in the IC group when compared to SP dogs. These genes encode transcription factors (MEF2); members of signaling pathways (TGFß2, PDE4D9), hormone related proteins (e.g. vasopressin V1 and V2 receptors). RT-QPCR was used either to confirm the results of the microarray analysis and also to study 46 genes which are already known to have a role in the late phase of PC. By this method 17 genes were up-regulated and 6 genes down-regulated in the IC group; their expression ratios changed either to the opposite or showed no alteration after cardiac pacing. This study would add some new information about those transcriptional changes that are involved in the delayed phase of cardiac protection.

A pharmacological analysis of the possible role of vasoactive mediators in compensatory coronary blood flow.

BACKGROUND: Coronary blood flow in one (circumflex) branch of the left coronary artery increases when an adjacent (left anterior descending [LAD]) branch is occluded for periods of between 1 min and 25 min. OBJECTIVE: To examine the possible role of the myocardial release of vasoactive substances in such 'compensatory blood flow' increase by the classical pharmacological approach of inhibition of synthesis, or blockade at the receptor level, of the most likely mediators. METHODS: In pentobarbitone anesthetized, thoracotomized dogs, coronary blood flow changes were measured in both the LAD (using a Doppler flow probe) and the left circumflex (using an electromagnetic flow probe) coronary arteries. RESULTS: The flow increase during 5 min occlusions of the LAD coronary artery was unaffected by blockade of adenosine receptors by 8-sulfophenyltheophylline, inhibition of prostanoid synthesis by sodium meclofenamate or celecoxib, blockade of bradykinin B(1) receptors by icatibant, inhibition of nitric oxide synthesis by N(omega)-nitro-L-arginine methyl ester (L-NAME), inhibition of guanylyl cyclase by methylene blue, or opening (using diazoxide) and closing (using glibenclamide or 5-hydroxydecanote) of ATP-dependent K(+) channels. Neither dual blockade with L-NAME and glibenclamide, or meclofenamate and 5-hydroxydecanote, nor triple blockade with L-NAME, glibenclamide and 8-sulfophenyltheophylline, modified the blood flow response. However, it was greatly reduced (60%) by metoprolol. CONCLUSIONS: These results suggest that coronary vascular beta(1)-adrenoceptors are involved in 'compensatory' vasodilation, whereas bradykinin, nitric oxide, prostanoids and ATP-dependent K(+) channels are seemingly not required for this flow increase.

Gap junctional uncoupling plays a trigger role in the antiarrhythmic effect of ischaemic preconditioning.

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 microM 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<0.05) during the prolonged occlusion. This protection was, however, attenuated when CBX was infused in PC dogs (VPBs: 203+/-32). Changes in electrical impedance, GJ permeability and Cx43 dephosphorylation were significantly less in the PC and CBX groups than in the controls but these were again increased in the CBX+PC group. CONCLUSIONS: Uncoupling of GJs prior to ischaemia either by PC or CBX preserves the electrical coupling of cells and results in an antiarrhythmic effect during a subsequent ischaemic insult, indicating that a partial closure of gap junctions may play a trigger role in the protection. In contrast, when CBX is administered in PC dogs the protection both against GJ uncoupling and arrhythmias is markedly attenuated, suggesting that the antiarrhythmic protection, at least in part, is mediated through GJs.

Examination of the Role of Mitochondrial Morphology and Function in the Cardioprotective Effect of Sodium Nitrite Administered 24 h Before Ischemia/Reperfusion Injury.

Background: We have previous evidence that in anesthetized dogs the inorganic sodium nitrite protects against the severe ventricular arrhythmias, resulting from coronary artery occlusion and reperfusion, when administered 24 h before. The present study aimed to examine, whether in this effect changes in mitochondrial morphology and function would play a role. Methods: Thirty dogs were infused intravenously either with saline (n = 15) or sodium nitrite (0.2 µmol/kg/min; n = 15) for 20 min, and 24 h later, 10 dogs from each group were subjected to a 25 min period of occlusion and then reperfusion of the left anterior descending coronary artery. The severity of ischaemia and ventricular arrhythmias were examined in situ. Left ventricular tissue samples were collected either before the occlusion (5 saline and 5 nitrite treated dogs) or, in dogs subjected to occlusion, 2 min after reperfusion. Changes in mitochondrial morphology, in complex I and complex II-dependent oxidative phosphorylation (OXPHOS), in ATP, superoxide, and peroxynitrite productions were determined. Results: The administration of sodium nitrite 24 h before ischemia/reperfusion significantly attenuated the severity of ischaemia, and markedly reduced the number and incidence of ventricular arrhythmias. Nitrite also attenuated the ischaemia and reperfusion (I/R)-induced structural alterations, such as reductions in mitochondrial area, perimeter, and Feret diameter, as well as the increase in mitochondrial roundness. The administration of nitrite, however, enhanced the I/R-induced reduction in the mitochondrial respiratory parameters; compared to the controls, 24 h after the infusion of nitrite, there were further significant decreases, e.g., in the complex I-dependent OXPHOS (by -20 vs. -53%), respiratory control ratio (by -14 vs. -61%) and in the P/E control coupling ratio (by 2 vs. -36%). Nitrite also significantly reduced the I/R-induced generation of superoxide, without substantially influencing the ATP production. Conclusions: The results suggest that sodium nitrite may have an effect on the mitochondria; it preserves the mitochondrial structure and modifies the mitochondrial function, when administered 24 h prior to I/R. We propose that nitrite affects primary the phosphorylation system (indicated by the decreased P/E ratio), and the reduction in superoxide production would result from the subsequent suppression of the ROS producing complexes; an effect which may certainly contribute to the antiarrhythmic effect of nitrite.

Metoprolol reduces 'compensatory' coronary blood flow following occlusion of an adjacent branch without altering post-occlusion hyperaemia.

In pentobarbitone anaesthetised, thoracotomised dogs, blood flow in one (circumflex; LCX) branch of the left coronary artery increases when an adjacent (anterior descending; LAD) branch is occluded. We show that this 'compensatory blood flow' increase results from an enhanced regional myocardial contractility, as assessed using piezoelectric crystals, and that this is to compensate for a marked decrease in segmental shortening (SS) in the region supplied by the occluded vessel. These changes in regional contractility are relatively unaffected by the intravenous administration of metoprolol whereas the LCX flow change is markedly reduced, suggesting a major contribution of coronary vascular beta(1)-adrenoceptors to such 'compensatory' flow changes.

Effect of levosimendan and milrinone on regional myocardial ischemia/reperfusion-induced arrhythmias in dogs.

Phosphodiesterase inhibitors as inodilators in heart failure are associated with promotion of arrhythmias. Calcium sensitizers have been proposed for the treatment of severe decompensated heart failure. The effect of levosimendan, a calcium sensitizer, and milrinone, a phosphodiesterase inhibitor, on ventricular arrhythmias was compared in a model of acute regional myocardial ischemia and reperfusion. The left anterior descending coronary artery in dogs was occluded for 25 minutes, followed by reperfusion. The 2 drugs were administered in a hemodynamically equieffective dose (0.1 micromol/kg) 10 minutes before coronary occlusion. Levosimendan, but not milrinone, significantly attenuated the pronounced increase in the number of ventricular premature beats (-63%), tachycardia (-50%), fibrillation (-70%), and inhomogeneity of ventricular electrical activation. Levosimendan significantly improved the overall survival rate. Levosimendan has a more beneficial profile than milrinone regarding the development of ventricular arrhythmias during and after regional myocardial ischemia.

The effect of levosimendan during long-term amiodarone treatment in dogs.

The haemodynamic and electrophysiological effects of levosimendan were studied in conscious dogs receiving long-term oral amiodarone treatment. Instrumented dogs were administered increasing doses of levosimendan (up to 0.9 microg/kg/min. intravenously) in three successive 30 min. infusions. This schedule was repeated on the 21st day of treatment with oral amiodarone 100 mg/kg/day. The extent of increase in left ventricular systolic pressure (LVSP) and the decrease in left ventricular end-diastolic pressure (LVEDP) seen with levosimendan were similar before and after long-term treatment with amiodarone. The levosimendan-induced increases in isovolumic contraction (+dP/dt) and in left ventricular contractility (dP/dt/P) seen prior to amiodarone administration were augmented during amiodarone treatment, an effect that was statistically significant (P<0.05) at the highest doses of levosimendan. A tendency towards a shortening of the QT interval and a rise in heart rate was observed for levosimendan alone but they did not exceed the physiological range when the drug was given in combination with amiodarone. QTc value was unaffected by levosimendan either alone or with amiodarone. These effects were apparent in animals with therapeutically meaningful plasma levels of levosimendan, amiodarone and desethylamiodarone levels. The results of this study show that the improvement in ventricular contractile performance usually associated with administration of levosimendan was somewhat enhanced by chronic oral treatment with amiodarone. It seems reasonable to infer that the inotropic potency and electrophysiological safety of parenteral levosimendan will be maintained in patients with heart failure during long-term treatment with oral amiodarone.

ORM-3819 promotes cardiac contractility through Ca(2+) sensitization in combination with selective PDE III inhibition, a novel approach to inotropy.

This study is the first pharmacological characterization of the novel chemical entity, ORM-3819 (L-6-{4-[N'-(4-Hydroxi-3-methoxy-2-nitro-benzylidene)-hydrazino]-phenyl}-5-methyl-4,5-dihydro-2H-pyridazin-3-one), focusing primarily on its cardiotonic effects. ORM-3819 binding to cardiac troponin C (cTnC) was confirmed by nuclear magnetic resonance spectroscopy, and a selective inhibition of the phosphodiesterase III (PDE III) isozyme (IC50=3.88±0.3 nM) was revealed during in vitro enzyme assays. The Ca(2+)-sensitizing effect of ORM-3819 was demonstrated in vitro in permeabilized myocyte-sized preparations from left ventricles (LV) of guinea pig hearts (ΔpCa50=0.12±0.01; EC50=2.88±0.14 µM). ORM-3819 increased the maximal rate of LV pressure development (+dP/dtmax) (EC50=8.9±1.7 nM) and LV systolic pressure (EC50=7.63±1.74 nM) in Langendorff-perfused guinea pig hearts. Intravenous administration of ORM-3819 increased LV+dP/dtmax (EC50=0.13±0.05 µM/kg) and improved the rate of LV pressure decrease (-dP/dtmax); (EC50=0.03±0.02 µM/kg) in healthy guinea pigs. In an in vivo dog model of myocardial stunning, ORM-3819 restored the depressed LV+dP/dtmax and improved % segmental shortening (%SS) in the ischemic area (to 18.8±3), which was reduced after the ischaemia-reperfusion insult (from 24.1±2.1 to 11.0±2.4). Our data demonstrate ORM-3819 as a potent positive inotropic agent exerting its cardiotonic effect by a cTnC-dependent Ca(2+)-sensitizing mechanism in combination with the selective inhibition of the PDE III isozyme. This dual mechanism of action results in the concentration-dependent augmentation of the contractile performance under control conditions and in the postischemic failing myocardium.

Nitric oxide involvement in the delayed antiarrhythmic effect of treadmill exercise in dogs.

We have shown previously that a single period of treadmill exercise in dogs protects the heart against the severe ventricular arrhythmias that arise when a major (anterior descending) branch of the left coronary artery is occluded following anaesthesia 24 h later. This protection is aminoguanidine sensitive, suggesting a role for nitric oxide (NO) in this exercise-induced delayed antiarrhythmic effect. The present study has further examined the possible role of NO as a mediator and/or as a trigger using the selective induced (iNOS) inhibitor S-(2-aminoethyl)-methyl-isothiourea (AEST) and the specific but not selective nitric oxide synthase inhibitor N(omega)-nitro-L-arginine-methyl-ester (L-NAME). Exercise markedly reduced the severity of ischaemia and reperfusion-induced ventricular arrhythmias 24 h later. Thus, only one of the dogs (8%) so exercised fibrillated on occlusion (contrast 46% in the control, non-exercised dogs; P<0.05) and the marked changes in the inhomogeneity of electrical activation that occur in the ischaemic region following occlusion were much reduced (P<0.05 compared to controls). This delayed exercise-induced cardioprotection was significantly attenuated by the nitric oxide synthase (NOS) inhibitors L-NAME, given prior to the exercise protocol and by AEST given prior to the coronary artery occlusion. For example, survival from the ischaemia-reperfusion insult was 54% in the exercise dogs, 0% in the controls and 14% in those dogs given a NOS inhibitor. We conclude that nitric oxide (NO) is both the trigger and the mediator of this delayed protection against ischaemia and reperfusion-induced arrhythmias.

A common mechanism in the protective effects of preconditioning, cardiac pacing and physical exercise against ischemia and reperfusion-induced arrhythmias.

Ischemic preconditioning results not only in a reduction in myocardial ischemic damage, but also in a marked suppression of those ventricular arrhythmias that result from a more prolonged period of ischemia and reperfusion insult. This protection is time-dependent and occurs in two distinct phases. There is an 'early phase' which is apparent immediately after the preconditioning stimulus but fades quickly (within 1 h to 2 h), and a 'delayed protection phase' in which the antiarrhythmic protection reappears 20 h to 24 h later. In both phases, the intensity of protection largely depends on the nature of the preconditioning stimulus. This can be ischemia resulting from brief coronary artery occlusions, cardiac pacing or vigorous physical exercise. Both cardiac pacing and exercise results in a marked reduction in the incidence and severity of ischemia and reperfusion-induced ventricular arrhythmias 24 h later. Although the precise mechanisms of the delayed protection that results from cardiac pacing and exercise are not yet fully understood, there is some evidence that similar endogenous protective substances (such as bradykinin, prostanoids and nitric oxide), as with ischemic preconditioning, play a pivotal trigger and mediator role in this anti-arrhythmic protection.

Noradrenaline, infused locally, reduces arrhythmia severity during coronary artery occlusion in anaesthetised dogs.

OBJECTIVE: To contribute to the debate, discussed in an earlier issue, regarding the role of adrenoceptors in the genesis of early, coronary artery occlusion-induced ventricular arrhythmias. METHODS: Mongrel dogs anaesthetised with chloralose and urethane were given either noradrenaline (NA, 100 ng kg(-1) min(-1)), phenylephrine (PHE, 200 ng kg(-1) min(-1)) or isoprenaline (ISO 12.5 ng kg(-1) min(-1)) by intracoronary infusion into a side branch of the left anterior descending coronary artery (LAD), commencing 10 min prior to the occlusion and then throughout the 25-min occlusion period. Control dogs were infused for the same period with saline. In another group of dogs noradrenaline was infused intravenously in a dose of 1 and then 2 microg kg(-1) min(-1) over a period of 60 min, 24 h prior to coronary artery occlusion. Haemodynamic and coronary blood flow changes, as well as changes in the epicardial ST-segment and in the degree of inhomogeneity were continuously recorded. Ventricular arrhythmias were evaluated as ventricular premature beats (VPBs), tachycardiac (VT) episodes and the incidences of VT and ventricular fibrillation (VF) during occlusion and following reperfusion. RESULTS: Compared to the controls, NA markedly reduced the severity of ventricular arrhythmias resulted from coronary artery occlusion and increased survival (to 40%) following reperfusion; there were no survivors in the control group. Noradrenaline released endogenously following guanethidine administration was also protective. Protection was also seen, although to a lesser extent with intracoronary PHE (occlusion VF 20% cp 80% in controls; survival 42%). In contrast, ISO enhanced arrhythmia severity; five out of seven dogs infused with ISO fibrillated within 10 min of the commencement of occlusion and no dog survived reperfusion. Other indices of ischaemia severity (epicardial ST-segment and inhomogeneity) were also reduced by NA and by PHE. NA, infused 24 h prior to occlusion was also protective against ischaemia and reperfusion-induced arrhythmias and ischaemia-induced changes in inhomogeneity. CONCLUSION: We conclude that exogenously administered NA, or released endogenously by chemical means, reduces the severity of ischaemia and reperfusion-induced ventricular arrhythmias and that this is mediated by alpha-adrenoceptors, perhaps through presynaptic inhibition of local NA release or by a 'preconditioning' effect presumably mediated by PKC.

The activation of PI 3-kinase/Akt pathway is involved in the acute effects of simvastatin against ischaemia and reperfusion-induced arrhythmias in anaesthetised dogs.

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.

Effect of sodium nitrite on ischaemia and reperfusion-induced arrhythmias in anaesthetized dogs: is protein S-nitrosylation involved?

BACKGROUND AND PURPOSE: To provide evidence for the protective role of inorganic nitrite against acute ischaemia and reperfusion-induced ventricular arrhythmias in a large animal model. EXPERIMENTAL APPROACH: Dogs, anaesthetized with chloralose and urethane, were administered intravenously with sodium nitrite (0.2 µmol kg(-1) min(-1)) in two protocols. In protocol 1 nitrite was infused 10 min prior to and during a 25 min occlusion of the left anterior descending (LAD) coronary artery (NaNO2-PO; n = 14), whereas in protocol 2 the infusion was started 10 min prior to reperfusion of the occluded vessel (NaNO2-PR; n = 12). Control dogs (n = 15) were infused with saline and subjected to the same period of ischaemia and reperfusion. Severities of ischaemia and ventricular arrhythmias, as well as changes in plasma nitrate/nitrite (NOx) levels in the coronary sinus blood, were assessed throughout the experiment. Myocardial superoxide and nitrotyrosine (NT) levels were determined during reperfusion. Changes in protein S-nitrosylation (SNO) and S-glutathionylation were also examined. KEY RESULTS: Compared with controls, sodium nitrite administered either pre-occlusion or pre-reperfusion markedly suppressed the number and severity of ventricular arrhythmias during occlusion and increased survival (0% vs. 50 and 92%) upon reperfusion. There were also significant decreases in superoxide and NT levels in the nitrite treated dogs. Compared with controls, increased SNO was found only in NaNO2-PR dogs, whereas S-glutathionylation occurred primarily in NaNO2-PO dogs. CONCLUSIONS: Intravenous infusion of nitrite profoundly reduced the severity of ventricular arrhythmias resulting from acute ischaemia and reperfusion in anaesthetized dogs. This effect, among several others, may result from an NO-mediated reduction in oxidative stress, perhaps through protein SNO and/or S-glutathionylation.