Einar Bugge

Inhibition of sodium-hydrogen exchange reduces infarct size in the isolated rat heart — a protective additive to ischaemic preconditioning

OBJECTIVES Inhibition of Na+/H+ exchange with amiloride analogues has been shown to protect the ischaemic and reperfused heart. The aim of this study was to examine if preischaemic or postischaemic treatment with the selective Na+/H+ exchange inhibitor ethyl-isopropyl amiloride (EIPA, 1 microM) influenced infarct size in an isolated rat heart model of regional ischaemia and reperfusion, and if any such protection was additive to the protection afforded by ischaemic preconditioning. METHODS Langendorff perfused rat hearts were subjected to 30 or 45 min of regional ischaemia and 120 min of reperfusion. The risk zone was determined by fluorescent particles and infarct size was determined by staining with triphenyltetrazolium chloride. RESULTS Treatment with EIPA for 20 min before 30 min regional ischaemia significantly reduced infarct size (in % of the risk zone) compared to untreated controls [3.1 (SEM 1.0)% v 38.1(5.8)%, P < 0.001], a protection similar to that afforded by ischaemic preconditioning [6.1(2.5)%]. Combination of preischaemic EIPA treatment and ischaemic preconditioning also reduced infarct size [5.2(2.0)%, P < 0.01 v control group]. When EIPA was added to the buffer only during the first 30 min of reperfusion, no protection was observed [infarct size = 37.8(5.8)%, NS v control group]. In order to clarify if the protection observed with EIPA treatment was additive to protection by ischaemic preconditioning, another set of experiments was performed. In these experiments regional ischaemia was extended to 45 min. Preischaemic EIPA treatment reduced infarct size also in this model compared to controls [15.3(2.9)% v 64.3(2.9)%, P < 0.001], as did ischaemic preconditioning [23.5(4.2)%, P < 0.001 v controls, NS v EIPA treated hearts]. Combination of ischaemic preconditioning and preischaemic EIPA treatment further reduced infarct size significantly [3.9(0.6)%, P < 0.05 v all other groups with 45 min regional ischaemia]. CONCLUSIONS Inhibition of Na+/H+ exchange reduces infarct size in the isolated rat heart infarct model if the exchanger is inhibited during the ischaemic period, and this protection is additive to the protection afforded by ischaemic preconditioning.

Ischaemic preconditioning is protein kinase C dependent but not through stimulation of α adrenergic or adenosine receptors in the isolated rat heart

OBJECTIVE The aim was (1) to clarify whether alpha adrenoceptor and adenosine receptor stimulation is involved in the anti-infarct effect of ischaemic preconditioning in the rat heart, and (2) to test the hypothesis that signal transduction through membrane bound protein kinase C is essential for the protection. METHODS Isolated, buffer perfused rat hearts were subjected to 30 min of regional ischaemia and 120 min of reperfusion. The risk zone was determined by fluorescent particles, and infarct size was determined by staining with triphenyltetrazolium chloride. RESULTS Ischaemic preconditioning with three cycles of 5 min ischaemia plus 5 min of reperfusion significantly reduced infarct size as compared to non-preconditioned group [4.5(SEM 0.6)% of the risk zone v 45.5(5.7)%, P < 0.001]. Blockade of alpha adrenoceptors alone and simultaneous blockade of alpha adrenoceptors with phenoxybenzamine (10 microM) and adenosine receptors with sulphophenyltheophylline (100 microM) did not prevent the protective effect of ischaemic preconditioning [infarct size = 2.4(0.4) and 5.6(1.9)% respectively, NS v the non-treated preconditioned group]. Blocking either the membrane binding of protein kinase C with polymyxin B (1 microM) or direct inhibition of protein kinase C activity with chelerythrine (2 microM) completely abolished the infarct size reducing effect of ischaemic preconditioning [32.4(3.3)% and 48.2(4.0)% respectively, P < 0.005 v non-treated preconditioned group: NS v the non-preconditioned group]. CONCLUSIONS In the rat heart infarct model the protective effect of ischaemic preconditioning is not mediated through stimulation of alpha adrenoceptors alone or the combined stimulation of alpha adrenergic and adenosine receptors, and it is dependent on activation of membrane bound protein kinase C.

Endothelin-1 can reduce infarct size through protein kinase C and KATP channels in the isolated rat heart

OBJECTIVE Protection from ischaemic preconditioning (IP) is dependent on activation of protein kinase C (PKC), and preconditionings protection can be mimicked by stimulation of various membrane receptors which are known to activate PKC. It is well known that KATP channel activation is cardioprotective. We tested the hypothesis that preischaemic treatment with endothelin-1 (ET-1) can protect against infarction by a PKC-dependent mechanism and by activating KATP channels. METHODS Buffer-perfused isolated rat hearts were subjected to 30 min regional ischaemia and 120 min reperfusion. Risk zone was determined by fluorescent particles, and infarct size by TTC staining. RESULTS Treatment with ET-1 in a dose of 1 nM prior to ischaemia significantly reduced infarct size in % of the risk zone compared to the control group (infarct size: 14.1 +/- 2.6 vs. 41.9 +/- 3.4%), while ET-1 0.1 nM did not protect (infarct size: 40.9 +/- 3%). AS the protective dose of ET-1 resulted in a significant reduction of coronary flow, a control group with a similar preischaemic flow-reduction was included (infarct size: 48.1 +/- 4.2%). Both the nonselective ETA/ETB receptor antagonist bosentan (1 microM) and the ET(A)-receptor-selective antagonist BQ 123 (2 microM) abolished protection from ET-1 (infarct size: 43.3 +/- 3.5 and 41.3 +/- 3.3%, respectively), as did the PKC inhibitor chelerythrine (2 microM) (infarct size: 41.1 +/- 5.2%) and the KATP blocker 5-hydroxydecanoate (infarct size: 41.7 +/- 2.9%). None of the ET receptor antagonists bosentan and BQ-123 influenced infarct size alone (infarct size: 42.7 +/- 2.5 and 41.3 +/- 3.3%, respectively). IP, similarly to ET-1, reduced infarct size (infarct size: 6.1 +/- 1.4%), but the nonselective ET receptor antagonist bosentan did not interfere with preconditionings protection (infarct size: 13.2 +/- 4.3%). CONCLUSIONS ET-1 treatment prior to ischaemia can protect against infarction via ETA receptors by a PKC-dependent mechanism and by activating KATP channels, but ET does not mediate IP in the isolated rat heart.

Lipid peroxidation, arachidonic acid and products of the lipoxygenase pathway in ischaemic preconditioning of rat heart

OBJECTIVE Preconditioning with brief intermittent periods of ischaemia is known to provide protection against ischaemic injury. It has been suggested that myocardial ischaemia also activates phospholipase A2, which releases arachidonic acid from phospholipids. In the present study the possible role of phospholipid peroxidation, arachidonic acid and products of the lipoxygenase pathway in cellular mechanisms of ischaemic preconditioning was examined. METHODS Isolated, buffer-perfused rat hearts were freeze-clamped at the end of preconditioning (a cycle of 5 min global ischaemia +5 min reperfusion) and at the end of 30 min global ischaemia and analysed for non-esterified fatty acids and fatty acids in the 2-position of phospholipid. In a separate set of experiments, hearts pretreated with a lipoxygenase inhibitor, nordihydroguaiaretic acid (NDGA), were subjected to 30 min regional ischaemia and 120 min reperfusion. Infarct size was determined by tetrazolium staining and the ischaemic risk zone with fluorescent particles. RESULTS Myocardial levels of arachidonic as well as of linoleic and docosahexaenoic acid were significantly elevated by preconditioning. Also, the level of peroxidized polyunsaturated fatty acids (measured as hydroxy conjugated dienes) in myocardial phospholipid was significantly increased: 101.4 +/- 16.8 nmol/g versus 51.2 +/- 7.3 nmol/g tissue dw in the control group, p < 0.05. Pre-treatment of hearts with 5 microM NDGA blocked the infarct limiting effects of preconditioning: infarct size was 37.4 +/- 6.4% of risk zone in control, 9.0 +/- 0.9% in the preconditioning group and 27.7 +/- 3.8% in the preconditioning + NDGA group (p < 0.05 vs. i.p., n.s. vs. control). CONCLUSION Our findings provide evidence for the involvement of phospholipase A2 and lipoxygenase derived lipid second messengers in ischaemic preconditioning of the isolated rat heart.

Reduced infarct size in the rabbit heartin vivo by ethylisopropyl-amiloride. A role for Na+/H+ exchange

Inhibition of Na+/H+ exchange with amiloride analogues has been shown to offer functional protection during ischemia and reperfusion and reduce infarct size in isolated rat hearts and intact pigs. The aim of the present study was to examine if pre- or postischemic treatment with ethylisopropylamiloride (EIPA), a selective Na+/H+ exchange inhibitor, could reduce infarct size in anin situ rabbit model of regional ischemia and reperfusion. Anesthetized, open-chest rabbits were subjected to 30 min of regional ischemia and 180 min of reperfusion. The risk zone was determined by fluorescent particles, and infarct size was determined by TTC staining. Preischemic treatment with EIPA (0.65 mg/kg) significantly reduced infarct size from 45.8±3.5% of the risk zone in the control group to 10.6±3.1% (p<0.01). EIPA-treatment during the first part of the reperfusion period did not reduced infarct size compared to controls (41.9±3.5%). We conclude that EIPA, when administered prior to ischemia, reduces infarct size in the rabbit heartin situ, a protection most likely due to inhibition of Na+/H+ exchange.

Preischaemic bradykinin and ischaemic preconditioning in functional recovery of the globally ischaemic rat heart

OBJECTIVES Substantial release of bradykinin has been demonstrated to occur during short periods of myocardial ischaemia in various species. The aim of the present study was to investigate the protective effect of bradykinin in ischaemia and whether bradykinin could be involved in ischaemic preconditioning in the rat heart. METHODS Isolated, buffer-perfused hearts were subjected to 30 min of global ischaemia, followed by 30 min of reperfusion. Postischaemic functional recovery was recorded in the following groups: (1) control; (2) treatment with 0.1 microM bradykinin for 10 min before ischaemia (BK); (3) bradykinin treatment combined with pretreatment with the specific bradykinin B2-receptor antagonist, HOE 140; (4) ischaemic preconditioning by 5 min ischaemia +5 min reperfusion prior to sustained ischaemia (i.p.); and (5) ischaemic preconditioning combined with HOE 140 administration. RESULTS Postischaemic myocardial function was significantly improved in both BK and i.p. groups (developed pressure 66.9 +/- 6.8 and 67.6 +/- 7.1 mmHg, respectively, vs. 43.1 +/- 5.9 mmHg in controls, P < 0.05). Pretreatment with 1 microM HOE 140 completely abolished the effect of bradykinin, while protection achieved by i.p. was unaltered by this drug. None of the protective interventions was associated with any significant improvement in myocardial adenosine triphosphate, creatine phosphate, glycogen, lactate or glucose tissue levels, detected either at the end of ischaemia or after 30 min of reperfusion. CONCLUSIONS Bradykinin, acting via B2-receptors, can protect against postischaemic contractile dysfunction to a similar extent as i.p.. An involvement of B2-receptors in the ischaemic preconditioning phenomenon could, however, not be demonstrated.

Blockade of the KATP-channel by glibenclamide aggravates ischemic injury, and counteracts ischemic preconditioning

Blocking of the KATP-channel with glibenclamide has been shown to abolish the infarct-reducing effect of ischemic preconditioning in dog and swine. In the rabbit the results have been divergent purportedly related to anaesthesia. The aim of this study was to investigate the importance of the KATP-channel in a rabbit model where anaesthesia was not a condounding factor. Isolated rabbit hearts perfused with a Krebs-Henseleit bicarbonate buffer were subjected to 30 min regional ischemia by ligating a coronary artery, followed by 120 min reperfusion. The preconditioning protocol was 5 min global ischemia and 10 min reperfusion. Glibenclamide (100μM) was added to the perfusion solution before the preconditioning ischemia and stopped after 5 min regional ischemia. Infarcts were measured with tetrazolium staining and risk zones with fluorescent microspheres. The main results expressed as percent infarction of the risk zone±SEM for the different groups are as follows: control (n=12) 26.8±3.2, ischemic preconditioning (IP) (n=9) 7.3+1.5 (p<0.05 vs. control), control+ glibenclamide (n=9) 46.9±7.3 (p<0.05 vs. control), IP+ glibenclamide (n=10). 38.3±6.9 (p<0.05 vs. IP). These results show that glibenclamide treatment aggravates ischemia. Also, under the influence of glibenclamide ischemic preconditioning was no longer effective in reducing infarct size in the isolated perfused rabbit heart.

5-HD abolishes ischemic preconditioning independently of monophasic action potential duration in the heart.

AbstractObjective: Blocking of the KATP channel with either glibenclamide or 5-hydroxydecanoate (5-HD) has been shown to abolish the infarct reducing effect of ischemic preconditioning (IPC) in hearts from several species, but the results in rat and rabbit have been equivocal. In this study we investigated if 5-HD could abolish IPC in rat and rabbit and further if IPC or IPV + 5-HD were affecting action potential duration in the rabbit heart. Methods: The rat hearts were isolated and retrogradely perfused on a Langendorff perfusion apparatus with Krebs-Henseleit buffer. The rabbit experiments were performed in an in situ model. Rat and rabbit hearts were subjected to 30 min regional ischemia by ligating a coronary artery followed by 120 min (rat) or 150 min (rabbit) of reperfusion. The preconditioning protocol was one or three cycles of 5 min ischemia plus 5 min reperfusion in the rat and one cycle of 5 min ischemia plus 10 min reperfusion in the rabbit. In the rat 5-HD was added to the reservoir before ischemic preconditioning in different concentrations, and in the rabbit 5-HD was given as a bolus 5 mg/kg intraventricularly 2 min before the preconditioning ischemia. In the rabbit epicardial monophasic action potential duration at 50% repolarization (MAPD50) was measured at 1, 2 and 5 min in each of the ischemic periods using a contact pressure electrode. Infarcts were measured with tetrazolium staining and risk zone volumes with fluorescent microspheres. Results: All data are presented as infarct size in % of risk zone volume (mean ± SEM). In the rat 200 μM of 5-HD abolished the protective effect of one cycle of IPC (28.6 ± 4.7 versus 8.4 ± 0.8) and 500M of 5-HD abolished three cycles of IPC (50.7 ± 7.8 versus 8.4 ± 2.0). Control was 40.9 ± 2.8.In the rabbit 5-HD abolished IPC (41.2 ± 7.2 versus 8.1 ± 3.2). Control was 53.5 ± 12.4. MAPD50 were significantly more shortened compared to control at 1 and 2 min into the 30 min ischemia for the IPC and IPC+5-HD. Conclusions: We conclude that 5-HD abolishes ischemic preconditioning when given before the preconditioning ischemia in both rat and rabbit but does not abolish into ischemia induced shortening of the action potential duration in the rabbit; thus, a role for the mitochondrial KATP channel and not the sarcolemmal KATP channel in the protective mechanism behind IPC is probable.

Mepacrine protects the isolated rat heart during hypoxia and reoxygenation —but not by inhibition of phospholipase A2

Mepacrine (quinacrine) has in a number of studies been shown to protect the heart from ischemic injury, a protection commonly claimed to be due to inhibition of phospholipase A2. The aim of the present study was to investigate the effect of mepacrine 1 μM on isolated, buffer perfused rat hearts subjected to 60 min hypoxia and 30 min reoxygenation. We also wanted to clarify whether any cardioprotective effect was due to inhibition of phospholipase A2 or to other effects of the drug. Mepacrine led to a substantial fall in left ventricular developed pressure (LVDP) and coronary flow (CF) during normoxic perfusion. Treated hearts showed less increase in LVEDP and less fall in LVDP during the hypoxic period, and significantly fewer hearts stopped beating compared to untreated controls. Release of CK during hypoxia and reoxygenation was reduced in treated hearts compared to controls (19.9±3.8 vs. 73.1±13.3 IU, p<0.05). Lipid analyses of the myocardium showed a significant increase in the total amount of non esterified fatty acids (NEFA) in both untreated and mepacrine treated hypoxic hearts compared to normoxic controls, but to a significantly lower level in the mepacrine treated hearts. However contribution of polyunsaturated NEFAs to total NEFAs did not differ between the groups. Also, neither total amount of fatty acids nor amount of polyunsaturated fatty acids obtained from the 2-position of the phospholipid fraction differed between the treated and untreated groups. In an enzyme assay, mepacrine 1 μM did not inhibit phospholipase A2 activity. We conclude that in our model mepacrine protects the heart from hypoxic injury, but probably by another mechanism than inhibition of phospholipase A2 induced membrane damage.

Curative ablation for atrial fibrillation: a systematic review.

Objective. To perform a systematic review of randomized controlled trials (RCTs) on catheter ablation for atrial fibrillation (AF). Background. Radiofrequency catheter (RF)-ablation around pulmonary vein ostia and in left atrium may reduce or prevent recurrence of AF, as documented in observational studies and registry reports; however, few RCTs are available. Methods. Using relevant search phrases, Cochrane Library, MEDLINE and EMBASE were searched for RCTs, last time in May 2007. Titles and abstracts were screened. When entry criteria were fulfilled, full-text papers were read and graded according to quality and relevance. Results. One thousand and ninety four abstracts were evaluated, and five RCTs included (578 randomized patients). The studies had moderate quality and relevance, but the results were consistent: ablation is better than drug treatment in preventing AF recurrence; the relative risk (95% CI)) one year after ablation ranged from 0.20 (0.08–0.51) to 0.62 (0.39–0.99). Conclusions. Results from observational and registry studies are confirmed: RF-ablation reduces recurrence rate of AF, and can be done with few serious complications. Limitations are few patients >70 years, and only one year follow-up.