Solute exchange in the rabbit myocardium: Ischaemia, reflow, and myocardial necrosis

Objective: Coronary occlusion in the rabbit reduces the delivery of particulate tracers to close to zero, but exchange of diffusible solutes, derived from non-arterial sources, continues at a significant level. We investigated the relationships between the exchange of diffusible solutes during coronary occlusion and the extent of myocardial necrosis and between duration of ischaemia and the extent of recovery of solute exchange during reflow. Methods: In an anaesthetised rabbit model of regional ischaemia and reflow, solute exchange is measured using the voltammetric hydrogen clearance technique. The area at risk and infarct size are determined ex vivo with monastral blue and nitroblue tetrazolium staining, respectively. Three groups are studied: control perfusion for 130 minutes (group A); 30 minutes coronary ligation followed by 90 minutes reflow (group B) and 40 minutes coronary ligation followed by 90 minutes reflow (group C). Results: There was no significant difference in area at risk between the groups B and C (50±2% and 45±5%; p=ns) or in infarct size when expressed relative to the area at risk (42±7% and 55±5%; p=ns). During coronary ligation hydrogen clearance remained constant at 22±4% of the control region in group B and 32±4% in group C, at the same time period in group A it was 87±2% (ANOVA=p<0.05, with a significant non-linear trend). Although the duration of ischaemia and the level of solute exchange during ischaemia did not correlate individually with the extent of myocardial necrosis, together they showed a significant correlation (ANOVA; p<0.05). Following coronary occlusion, hydrogen clearance recovered to 72±9% after 30 minutes ischaemia but only to 57±5% following 40 minutes ischaemia and was 95±2% in the control group (ANOVA between the three groups p<0.05 with a significant linear trend). Myocardial hydration fell in the apical region following coronary ligation by 27±5% in group B and by 25±5% in group C, and rose on reperfusion but only to 80±3% in group B and 83±3% in group C of their preligation values. Conclusion: In collateral deficient myocardium, the extent of myocardial necrosis is dependent on the level of solute exchange occurring during ischaemia. The level of solute exchange during reflow is dependent on the duration of ischaemia. Received: 4 December 1997, Returned for 1. revision: 12 January 1998, 1. Revision received: 16 April 1998, Accepted: 13 May 1998     

Mild Hypothermia reduces infarct size in the beating rabbit heart: A practical intervention for acute myocardial infarction?

The present study describes a method for rapidly cooling the whole body via its blood pool and tests whether cooling instituted after ischemia has begun can sill limit infarction. We also evaluated whether the cardiac protection seen with cooling could be added to that from ischemic preconditioning. Recently it was reported that lowering myocardial temperature by only several degrees greatly slows the extent of myocardial infarction in the beating heart experiencing regional ischemia. To further explore the potential of hypothermia for myocardial protection, rabbits underwent either a 30-, 45- or 60-min coronary artery occlusion and 3-h reperfusion. Blood from a carotid artery was allowed to circulate through a heat exchanger immersed in ice water and return to a jugular vein until the blood temperature in the left atrium reached the target temperature of 35 or 32°C. Furthermore, to elucidate the mechanism of hypothermia's protection, we also examined its effect on isolated cardiomyocytes. Rewarming began upon reperfusion in all protocols. Cooling to 32°C before a 30-min ischemia reduced infarct size from 37.3±2.5% (n=6) of the risk zone in normothermic controls to 3.6±0.3% (n=6). When cooling was begun 10 or 20 min after the onset of ischemia infarct size was still significantly smaller [8.1±1.2% and 22.8±1.8%, respectively (n=6 in each group)]. Less but significant protection was also seen with cooling to 35°:C. Cooling caused only mild bradycardia and hypotension and no apparent arrhythmias. Forty-five min of regional ischemia caused 50.7±3.3% (n=6) of risk zone to infarct in untreated hearts. Preconditioning with 5-min ischemia/10-min reperfusion reduced infarct size to 27.5±2.5% (n=6). Cooling to 32°C starting 20 min after the onset of ischemia protected the heart (28.7±2.6% infarction, n=8), and this protection could be added to the effect from ischemic preconditioning delayed the progressive increase in osmotic fragility that occurs during simulated ischemia in an additive way, but only hypothermia delayed the appearance of contracture suggesting that different mechanisms are involved. Hence blood pool cooling was easily induced and well tolerated and protected the beating heart against infarction even when hypothermia was started after the onset of coronary occlusion. We conclude that hypothermia might be a simple and useful therapy for patients presenting with acute myocardial infarction. Received: 5 January 1998, Returned for 1. revision: 3 February 1998, 1. Revision received: 24 February 1998, Returned for 2. revision: 1 April 1998, 2. Revision received: 7 April 1998, Accepted: 14 July 1998     

Failure of N-2-mercaptopropionyl glycine to reduce myocardial infarction after 3 days of reperfusion in rabbits

Recent studies have repoarted that prolonged infusion of N-2-mercaptopropionyl glycine (MPG), a diffusible antioxidant, could limit infarct size in dogs. However, there are no comparable studies testing this agent in other species. We examined the efficacy of MPG in a rabbit model of infarction. Rabbit hearts were subjected to a 30-min coronary artery occlusion. Infarct size expressed as a percentage of risk zone was determined by either triphenyltetrazolium chloride (TTC) staining after 3 h of reperfusion (study 1) or by histology after 72 h of reperfusion (study 2). In study 1, 37 ± 2.6 % of the risk zone infarcted in the control group. Intravenous MPG at a rate of 100 mg/kg/h starting 15 min after the onset of ischemia and continuing until 1 h after reperfusion had no effect on infarct size (35.4 ± 3.4 % infarction). However, infusion of MPG until the end of reperfusion significantly reduced infarct size as measured with TTC to 17.2 ± 2.5 % (p < 0.01 vs. control group). In study 2, 48.6 ± 4.0 % of the risk zone infarcted in the control group. In the treatment group MPG was started as above and was continued for 4 h of reperfusion followed by an intramuscular injection at the termination of the intravenous infusion. No protection was seen after 72 h of reperfusion (43.8 ± 2.1 % infarction). These findings reveal that MPG at a dose and schedule that appeared to protect the dog heart could not effect sustained protection in the rabbit heart. TTC staining revealed that MPG appeared to have preserved viability for up to 3 h of reperfusion suggesting that failure may have been due to early withdrawal of the drug. Alternatively, early TTC staining may yield spurious results under conditions in which protection is dependent upon antioxidant or free radical scavenger treatment as has previously been suggested. It is concluded that MPG as administered in the previous canine studies does not limit infarct size in all species, thus raising a concern about MPG's potential efficacy in man. Received: 6 July 1998, Returned for revision: 29 July 1998, Revision received: 8 December 1998, Accepted: 9 December 1998     

Preconditioning-induced cardioprotection and release of the second messenger inositol (1,4,5)-trisphosphate are both abolished by neomycin in rabbit heart

The mechanisms responsible for infarct size reduction with preconditioning remain controversial. Our aim was to determine whether release of the second messenger inositol (1,4,5)-trisphosphate (Ins(1,4,5)P₃) during the preconditioning stimulus may play a role. To test this concept, Langendorff-perfused rabbit hearts underwent sham perfusion, 5 min of coronary artery occlusion (CO), or 5 min of CO+infusion of neomycin, an agent which inhibits formation of Ins(1,4,5)P₃. Direct quantitation (by competitive binding assay) revealed a 2-fold increase in Ins(1,4,5)P₃ content with brief ischemia vs shams (0.69±0.14 vs 0.34±0.05 pmol/mg tissue; p<.05) that was blocked by neomycin (0.15±0.04 pmol/mg). Infarct size (by tetrazolium staining) was assessed in additional hearts that underwent 30 min of sustained CO and 2 h of reperfusion. As expected, two 5-min episodes of preconditioning ischemia reduced infarct size versus controls (30±6% versus 63±3% of the myocardium at risk; p<.01). In contrast, infarct size was comparable (54–56% of the risk region) in neomycin-treated control and preconditioned hearts. These results demonstrate that myocardial Ins(1,4,5)P₃ content is increased in response to brief preconditioning ischemia and are consistent with the concept that Ins(1,4,5)P₃ may be a potential mediator of infarct size reduction with preconditioning in isolated rabbit heart. Received: 12 February 1998, Returned for revision: 1 April 1998, Revision received: 1 June 1998, Accepted: 12 August 1998     

Influence of the angiotensin II AT1 receptor antagonist irbesartan on ischemia/reperfusion injury in the dog heart

The aim of the present study was to investigate whether the non-peptide angiotensin II type 1 (AT₁) receptor antagonist irbesartan (SR 47436, BMS 186295, 2-n-butyl-3[2‘-(1H-tetrazol-5-yl)-biphenyl-4-yl)methyl]-1,3-diaza-spiro[4,4]non-1-en-4-one) has myocardial protective effects during regional myocardial ischemia/reperfusion in vivo. Eighteen anesthetized open-chest dogs were instrumented for measurement of left ventricular and aortic pressure (tip manometer and pressure transducer, respectively), and coronary flow (ultrasonic flowprobes). Regional myocardial function was assessed by Doppler displacement transducers as systolic wall thickening (sWT) in the antero-apical and the postero-basal wall. The animals underwent 1 h of left anterior descending coronary artery (LAD) occlusion and subsequent reperfusion for 3 hours. Irbesartan (10 mg kg⁻¹, n=9) or the vehicle (KOH, control, n=9) was injected intravenously 30 min before LAD occlusion. Regional myocardial blood flow (RMBF) was measured after irbesartan injection and at 30 min LAD occlusion using colored microspheres. Infarct size was determined by triphenyltetrazolium chloride staining after 3 h of reperfusion. There was no recovery of sWT in the LAD perfused area in both groups at the end of the experiments (systolic bulging, −15.1±6.1% of baseline (irbesartan) vs. −12.3±3.0% (control), mean±SEM). Irbesartan led to an increase in RMBF in normal myocardium (2.47±0.40 vs. 1.35±0.28 ml min⁻¹ g⁻¹, P<0.05), and also to an increase in collateral blood flow to the ischemic area (0.27±0.04 vs. 0.17±0.02 ml min⁻¹ g⁻¹, P=<0.05). Infarct size (percent of area at risk) was 24.8±3.2% in the treatment group compared with 26.9±4.8% in the control group (P=0.72). These results indicate that a blockade of angiotensin II AT₁ receptors with irbesartan before coronary artery occlusion led to an increase in RMBF, but did not result in a significant reduction of myocardial infarct size. Received: 6 October 1999 Returned for revision: 16 November 1999 Revision received: 7 February 2000 Accepted: 29 March 2000     

Limitation of infarct size and attenuation of myeloperoxidase activity by an endothelin A receptor antagonist following ischaemia and reperfusion

It has previously been shown that endothelin (ET) receptor antagonists limit myocardial ischaemia/reperfusion (I/R) injury. The mechanism behind this effect is still unclear. The aim of this study was to elucidate the possible relationship between cardioprotection by an ET_A receptor antagonist and inhibition of neutrophil accumulation or activation in the myocardium determined as myeloperoxidase (MPO) activity during I/R. Anaesthetised pigs were subjected to 45 min ischaemia by ligation of the left anterior descending coronary artery (LAD) followed by 4 h of reperfusion. Infiltration of MPO-containing cells, presumably neutrophils, into the ischaemic area was confirmed with an immunohistochemical technique using antibodies against porcine MPO. Vehicle (n = 7) or the selective ETA receptor antagonist LU 135252 (LU; n = 7) were given into the LAD during the last 10 min of ischaemia and the first 5 min of reperfusion. There were no significant differences in LAD flow, mean arterial pressure, heart rate, or rate pressure product between the groups during I/R. The area at risk was similar in the two groups. LU reduced the final infarct size to 40 ± 6 % of the area at risk compared to 80 ± 6 % in the vehicle group (P < 0.001). Endothelin-like immunoreactivity increased 2-fold in the ischaemic area in the vehicle group (P < 0.01), but not in the group given LU. MPO activity was higher (2.5x) in the ischaemic than in the non-ischaemic myocardium of the vehicle group. The MPO activity in the ischaemic myocardium was significantly lower in the group given LU (7.0 ± 1.2 units g⁻¹) than in the vehicle group (14.2 ± 1.9 units g⁻¹; P < 0.01). There was a significant correlation between the infarct size and MPO activity (P < 0.01, r = 0.68). In conclusion, local administration of the selective ET_A receptor antagonist LU during the last period of ischaemia and early reperfusion reduces the extent of myocardial necrosis and MPO activity. This suggests that LU may exert its cardioprotective effect by inhibiting neutrophil-mediated injury. Received: 20 November 2000, Returned for revision: 11 December 2000, Revision received: 9 February 2001, Accepted: 13 February 2001     

Effects of BIIB513 on ischemia-induced arrhythmias and myocardial infarction in anesthetized rats

Na⁺/H⁺ exchange (NHE) plays an important role in the regulation of the intracellular pH (pH_i) and in cardiac cell injury induced by ischemia and reperfusion. In the present study, we investigated the effects of BIIB513, a selective NHE-1 inhibitor on myocardial ischemia induced arrhythmias and myocardial infarction, provoked by 30 minutes of left main coronary artery occlusion followed by 2 hours of reperfusion in an anesthetized rat model. Intravenous administration of BIIB513 (0.01–3.0 mg/kg) did not induce changes in blood pressure or heart rate. BIIB513 (0.01, 0.1, 0.3, 1.0, 3.0 mg/kg) given prior to the coronary artery occlusion dose-dependently reduced ventricular premature beats, ventricular tachycardia, and a complete suppression of ventricular fibrillation down to the dose of 0.1 mg/kg. BIIB513 (0.01, 0.1, 0.3, 1.0, 3.0 mg/kg) given prior to the coronary artery occlusion dose-dependently reduced the infarct size with an ED50 value of 0.16 mg/kg. BIIB513 (1.0 mg/kg) given prior to reperfusion also reduced infarct size by 47.3 ± 13.1%. The reduction in infarct size was accompanied by a decrease in circulating levels of creatine phosphokinase (CPK). In conclusion, the present study demonstrates the cardioprotective ability of NHE-1 inhibition during myocardial ischemia and reperfusion by reducing serious ventricular arrhythmias and myocardial infarct size in anesthetized rats. Received: 18 November 1999, Returned for 1.revision: 9 December 1999, 1.Revision received: 2 May 2000, Returned for 2.revision: 24 May 2000, 2.Revision received: 5 June 2000, Accepted: 7 June 2000     

Evidence for a role of platelet activating factor in the pathogenesis of irreversible but not reversible myocardial injury after reperfusion in dogs

The role of platelet activating factor (PAF) in myocardial injury after either brief (15 minutes, stunned myocardium) or prolonged (90 minutes, infarcted myocardium) coronary artery occlusion and 3 hours of reperfusion of the left anterior descending coronary artery was investigated in barbital-anesthetized dogs. Regional myocardial blood flow was measured by radioactive microspheres, regional segment shortening by sonomicrometry, and infarct size by the triphenyltetrazolium chloride stain. Infarct size expressed as a percentage of the area at risk was significantly reduced by the intravenous administration of two structurally unrelated PAF antagonists, BN 52021 (10 mg/kg and 1 mg/kg/hr) and CV-3988 (3 mg/kg and 0.3 mg/kg/hr). Infarct size was 38% +/- 5% in the saline (control) group, (n = 7), 22% +/- 5% in the BN 52021 group (n = 7), and 19% +/- 5% in the CV-3988 group (n = 8). However, the intravenous administration of BN 52021 (5 and 10 mg/kg) and CV-3988 (5 mg/kg) had no effect on functional recovery (regional segment shortening) in the stunned myocardium after brief occlusion and reperfusion. Regional myocardial blood flow, hemodynamic data, and the incidence of cardiac arrhythmias were not significantly affected by PAF antagonists in both series of experiments at any time. These data suggest that PAF may play an important role in the pathogenesis of an evolving myocardial infarction that follows a prolonged coronary artery occlusion and reperfusion. Furthermore, PAF antagonists may have a beneficial role in reduction of the injury produced during an acute infarction. Finally, these data indicate that PAF does not appear to be an important mediator of myocardial stunning.     

SB 203580, an inhibitor of p38 MAPK, abolishes infarct-limiting effect of ischemic preconditioning in isolated rabbit hearts

There is debate concerning the involvement of p38 mitogen-activated protein kinase (MAPK) in ischemic preconditioning (PC). At the center of the controversy are data obtained after administration of SB 203580, a specific inhibitor of p38 MAPK. Whereas several studies have reported that SB 203580 abolishes the cardioprotective effect of PC, others claim that this compound is actually cardioprotective against ischemia. Many of these latter observations have been made in isolated myocardial cells. Accordingly the present study was designed to test the effect of SB 203580 in a model of preconditioning in intact rabbit hearts in which infarct size was the end-point. Isolated hearts experienced 30 min of regional ischemia followed by 120 min of reperfusion. Infarct size was measured with triphenyltetrazolium chloride. In control hearts infarction was 30.2 ± 3.3% of the risk zone. PC with 5 min of global ischemia and 10 min of reperfusion before the 30-min period of ischemia significantly reduced infarct size to 10.2 ± 2.4% (P < 0.05 vs. control). SB 203580 (2 μ M) added to the perfusate for 20 min starting 5 min before the index ischemia totally blocked the protection from PC (27.4 ± 3.3% infarction). SB 203580 alone had no effect on infarct size (28.6 ± 4.6% infarction). These results reveal that SB 203580 does not affect infarct size on its own, but selectively blocks preconditioning's anti-infarct effect in the intact rabbit heart. Received: 21 August 2000, Returned for revision: 30 August 2000, Revision received: 2000, Accepted: 6 September 2000     

Nifedipine limits infarct size via NO-dependent mechanisms in dogs

Objectives Amlodipine increases NO levels in coronary vessels and aorta via bradykinin-dependent mechanisms in vitro. We have previously reported that nifedipine increases cardiac NO levels in the ischemic canine hearts, suggesting that nifedipine may also have protective effects against ischemia and reperfusion injury, because the enhancement of NO production limits infarct size. We tested whether nifedipine limits infarct size via NO-dependent mechanisms. Methods In open chest dogs, the left anterior descending coronary artery was perfused with blood through a bypass tube and occluded for 90 min followed by 6 hours of reperfusion. Infarct size was assessed at 6 hours of reperfusion. Nifedipine of 3 or 6 μg/kg/min was infused into the bypass tube between 10 min prior to the onset of ischemia and 60 min of reperfusion. Results Neither systemic blood pressure nor heart rate changed during infusion of nifedipine. Infarct size was reduced by the administration of nifedipine (3 or 6 μg/kg/min) compared with the untreated condition (25.6 plusmn; 2.6 and 19.1 ± 3.5 vs. 43.4 ± 5.6 %, respectively), which was completely blunted by L-NAME (45.0 ± 3.6 and 45.4 ± 4.2 vs. 47.9 ± 3.9 % in the nifedipine (3 or 6 μg/kg/min) with L-NAME groups vs. the L-NAME group). Myeloperoxidase activity of the myocardium increased after 6 hours of reperfusion, which was attenuated by nifedipine. The limitation of infarct size and the attenuation in myeloperoxidase activity were completely blunted by L-NAME. There were no significant differences in collateral blood flow at 45 min of ischemia between each group. Conclusions We conclude that the Ca channel blocker, nifedipine, limits infarct size via NO-dependent mechanisms. Received: 21 September 2000, Returned for 1. revision: 9 October 2000, 1. Revision received: 17 January 2001, Returned for 2. revision: 5 February 2001, 2. Revision received: 13 February 2001, Accepted: 14 February 2001     

Evidence for an essential role of cyclooxygenase-2 as a mediator of the late phase of ischemic preconditioning in mice

Recent studies have demonstrated that cyclooxygenase-2 (COX-2) is an essential mediator of the cardioprotective effects of the late phase of ischemic preconditioning (PC) in rabbits. The goal of this study was to determine whether COX-2 also plays an essential role in late PC in the mouse. B6129F₂/J mice underwent a 30-min coronary occlusion followed by 24 h of reperfusion. Administration of the COX-2 selective inhibitor, NS-398, 30 min prior to the 30-min occlusion (5 mg/kg i.p.) had no appreciable effect on infarct size compared with untreated controls (58.8 ± 2.1%, vs. 58.8 ± 4.3% of the risk region, respectively). When mice were preconditioned with six cycles of 4-min coronary occlusion/4-min reperfusion 24 h prior to the 30-min occlusion, infarct size was markedly reduced (19.3 ± 3.4%), indicating a late PC effect. The protective effect of late PC was completely abrogated by administration of NS-398 30 min before the 30-min coronary occlusion (67.7 ± 3.0%), but not by administration of vehicle alone (23.6 ± 3.7%). These results indicate that COX-2 mediates the late phase of ischemic PC in the mouse and imply that the role of this enzyme in cardioprotection is not species-specific. Received: 1 September 2000, Returned for revision: 13 September 2000, Revision received: 18 September 2000, Accepted: 20 September 2000     

The influence of maturation and gender on the anti-arrhythnmic effect of ischaemic preconditioning in rats

The aim of this study was to investigate the influence of maturation and gender on the anti-arrhythmic effect of myocardial ischaemic preconditioning in rats. Coronary artery occlusion was carried out in either rats anaesthetised with sodium pentobarbitone or in rat isolated hearts. Cardiac arrhythmias occurring in the 30 min post-occlusion period were assessed. In anaesthetised 3 month (m) old male rats ischaemic preconditioning, with a 3 min temporary coronary artery occlusion, significantly reduced the total number of ventricular ectopic beats (VEBs) from 2074±206 to 490±139 and the incidence of ventricular fibrillation (VF) from 40 to 0% during a subsequent 30 min occlusion (P<0.05). In middle-aged male rats (16 m) the anti-arrhythmic effect of preconditioning was unaltered (VEBs were reduced from 1958±121 to 245±66 and VF from 70 to 0%). In 3 m old anaesthetised female rats the effect of ischaemic preconditioning was also evident (VEBs reduced from 961±170 to 154±48; P<0.05). In non-preconditioned age-matched female animals the total number of VEBs (961±170), VF (0%) and mortality (0%) were significantly (P<0.05) lower than in respective male animals. In female rats, attenuation of ischaemia-induced arrhythmic severity was most pronounced in the oestrus state. In hearts isolated from weight-matched male and female rats the incidence of ventricular tachycardia (81 vs 25%) and the total number of VEBs (351±73 vs 81±50) were significantly (P<0.05) different. It is concluded that in rats neither maturation nor gender influence the anti-arrhythmic effect of ischaemic preconditioning. However, female rats exhibit a lower level of arrhythmic activity during sustained coronary artery occlusion than male rats both in vivo and in vitro. Received: 8 April 1998, Returned for revision: 2 June 1998, Revision received: 9 July 1998, Accepted: 21 July 1998     

Acute coronary vascular and myocardial perfusion effects of conjugated equine estrogen in the anesthetized dog

Women generally exhibit angina rather than myocardial infarction as the first manifestation of heart disease. Postmenopausal use of hormone replacement therapy, specifically estrogens, is associated with reduced incidence of major cardiac events suggesting estrogen may protect the heart during ischemia. We recently showed that acute administration of conjugated equine estrogens prior to ischemia attenuated the ventricular arrhythmias of ischemia as well as those of reperfusion. This study looks at basal effects of estrogen on coronary blood flow and the effects of estrogen on regional blood flow during ischemia to determine if estrogen exerts its antiarrhythmic effects during ischemia by altering blood flow. Under conditions of natural blood flow, estrogen caused cyclic changes in blood flow. When coronary blood flow was controlled and limited, estrogen increased coronary perfusion pressure (118±8 mmHg vs. 85±10 mmHg in non-treated dogs, P<0.05) demonstrating an overall vasoconstrictor effect. Coronary blood flow and regional myocardial perfusion were determined before and during ischemia in anesthetized dogs with and without acutely-administered estrogen. Colored microspheres were injected at steady state prior to ischemia, and during steady state myocardial ischemia. Conjugated equine estrogen (10 μg/kg), administered about 6 min before ischemia, had no effect on regional perfusion under steady state conditions, nor in the non-ischemic zone during ischemia. Perfusion in the subepicardial and subendocardial ischemic zones in estrogen-treated dogs was significantly lower than in non-treated dogs [0.14±0.01 ml/min/g vs. 0.23±0.02 ml/min/g (P<0.05) in the epicardial ischemic zone; and, 0.15±0.02 ml/min/g vs. 0.22±0.03 ml/min/g (P<0.05) in the endocardial ischemic zone]. We conclude that the acute, systemic administration of estrogen in the anesthetized dog decreases regional perfusion in the ischemic myocardium and causes significant coronary vasoconstriction when flow is controlled and limited. Received: 3 March 1998, Returned for 1. revision: 28 April 1998, 1. Revision received: 29 May 1998, Accepted: 18 June 1998     

Myocardial protection by insulin is dependent on phospatidylinositol 3-kinase but not protein kinase C or KATP channels in the isolated rabbit heart

Because tyrosine kinase blockade prevents protection by ischemic preconditioning (PC) in several species, activation of tyrosine kinase appears to be critical for cardioprotection. The tyrosine kinase's identity, however, is unknown. The present study tested whether activation of a receptor tyrosine kinase, the insulin receptor, could mimic PC and if the mechanism of protection was similar to that of PC. Isolated rabbit hearts were subjected to 30 min of regional ischemia and 2 h of reperfusion. Infarct size was determined by triphenyltetrazolium staining and expressed as a percentage of the area at risk. Infarct size in control hearts was 32.6 ± 2.3 %. A 5-min infusion of insulin (5 mU/ml) followed by a 10-min washout period prior to ischemia significantly reduced infarction to 14.7 ± 2.1 % (P < 0.05). The tyrosine kinase inhibitor genistein (50 μM) given around the insulin infusion blocked protection (28.9 ± 2.8 %). However, when present during the onset of ischemia, genistein had no effect on protection triggered by insulin (14.0 ± 2.4 %; P < 0.05). Inhibition of either PKC by polymyxin B (50 μM) or K_ATP channels by 5-hydroxydecanoate (100 μM) also failed to prevent protection by insulin (17.5 ± 3.2 % and 17.6 ± 3.0 %, respectively). However, the reduction in infarct size by insulin was significantly attenuated by wortmannin (100 nM), a selective inhibitor of phosphatidylinositol 3-kinase (P13K, 28.3 ± 2.2 %). Insulin was still able to protect the heart when given only during the reperfusion period (13.2 ± 3.4 %). PC reduced infarction to 12.8 ± 2.0 % (P < 0.05). In conclusion, activation of the insulin receptor reduces infarct size in the rabbit heart even when instituted upon reperfusion. However, the mechanism of protection is quite different from that of PC and involves activation of P13K but not PKC or K_ATP channels. Received: 12 November 1998, Returned for revision: 25 November 1998, Revision received: 8 December 1998, Accepted: 10 December 1998     

Evidence that cytosolic and ecto 5'-nucleotidases contribute equally to increased interstitial adenosine concentration during porcine myocardial ischemia

The purpose of this study was to determine the roles of cytosolic and ecto 5'-nucleotidase in myocardial ischemia-induced increases in interstitial fluid (ISF) adenosine. Pentobarbital anesthetized, open chest pigs were instrumented with two microdialysis fibers in the distally perfused bed of the left anterior descending (LAD) coronary artery to estimate ISF metabolites. Fibers in control hearts were perfused with standard Krebs buffer. In two additional groups, after collecting one dialysate sample with normal Krebs, fibers were perfused with buffer supplemented with either L-homocysteine thiolactone (5 mM) or the ecto 5'-nucleotidase inhibitor α, β-methylene adenosine 5'-diphosphate (AOPCP, 5 mM). Hearts were then submitted to 60 minutes LAD occlusion and two hours reperfusion. Dialysate nucleosides and AMP were measured by high performance liquid chromatography. The local delivery of homocysteine did not alter preischemic dialysate adenosine concentration (0.30 ± 0.04 μM) compared to pre-homocysteine infusion (0.39 ± 0.04 μM) or control hearts (0.36 ± 0.04 μM), but AOPCP significantly decreased preischemic dialysate adenosine levels (from 0.36 ± 0.02 to 0.14 ± 0.03 μM). During LAD occlusion both homocysteine and AOPCP reduced dialysate levels by approximately 50 %. At 30 minutes ischemia dialysate adenosine concentrations were 19.47 ± 2.72, 11.41 ± 2.44, and 7.93 ± 1.01 μ:M in control, homocysteine, and AOPCP hearts, respectively. AOPCP significantly increased dialysate AMP levels; at 60 minutes ischemia AMP levels were 6.22 ± 2.97 μM in control hearts and 38.60 ± 5.69 μM in AOPCP treated hearts. These results suggest that both cytosolic and ecto 5'-nucleotidase contribute to ischemia-induced increases in ISF adenosine in porcine myocardium. Received: 13 October 1998, Returned for revision: 5 November 1998, Revision received: 17 December 1998, Accepted: 4 January 1999     

Blockade of ATP-sensitive potassium channels prevents myocardial preconditioning in dogs.

Single or multiple brief periods of ischemia (preconditioning) have been shown to protect the myocardium from infarction after a subsequent more prolonged ischemic insult. To test the hypothesis that preconditioning is the result of opening ATP-sensitive potassium (KATP) channels, a selective KATP channel antagonist, glibenclamide, was administered before or immediately after preconditioning in barbital-anesthetized open-chest dogs subjected to 60 minutes of left circumflex coronary artery (LCX) occlusion followed by 5 hours of reperfusion. Preconditioning was elicited by 5 minutes of LCX occlusion followed by 10 minutes of reperfusion before the 60-minute occlusion period. Glibenclamide (0.3 mg/kg i.v.) or vehicle was given 10 minutes before the initial ischemic insult in each of four groups. In a fifth group, glibenclamide was administered immediately after preconditioning. In a final series (group 6), a selective potassium channel opener, RP 52891 (10 micrograms/kg bolus and 0.1 micrograms/mg/min i.v.) was started 10 minutes before occlusion and continued throughout reperfusion. Transmural myocardial blood flow was measured at 30 minutes of occlusion, and infarct size was determined by triphenyltetrazolium staining and expressed as a percent of the area at risk. There were no significant differences in hemodynamics, collateral blood flow, or area at risk between groups. The ratio of infarct size to area at risk in the control group (28 +/- 6%) was not different from the group pretreated with glibenclamide in the absence of preconditioning (31 +/- 6%). Preconditioning produced a marked reduction (p less than 0.002) in infarct size (28 +/- 6% to 6 +/- 2%), whereas glibenclamide administered before or immediately after preconditioning completely abolished the protective effect (28 +/- 6% and 30 +/- 8%, respectively). RP 52891 also produced a significant (p less than 0.03) reduction (28 +/- 6% to 13 +/- 3%) in infarct size. These results suggest that myocardial preconditioning in the canine heart is mediated by activation of KATP channels and that these channels may serve an endogenous myocardial protective role.     

Beneficial actions of amlodipine in the multiple-stunned canine myocardium

Summary The effects of the long-acting dihydropyridine calcium-entry blocker, amlodipine, on subendocardial segment shortening (%SS), regional myocardial blood flow (radioactive microspheres), and tissue high-energy phosphate levels were compared with those of a saline-treated group of barbital-anesthetized dogs subjected to nine 5-minute coronary artery occlusions interspersed with 15 minutes of reperfusion and finally by 1 hour of reperfusion (multiple stunned myocardium). Saline or amlodipine (200 μg/kg, IV) were administered 15 minutes prior to the first coronary occlusion. There were no major differences between groups in ischemic bed size or hemoydnamics throughout the experiment. Subendocardial collateral blood flow was significantly increased in the amlodipine-treated group during coronary occlusion 1; however, tissue blood flow in the ischemic region was not significantly different between groups during occlusion 9. Following each occlusion, %SS in the ischemic region was equally reduced in both groups and passive systolic lengthening resulted. In spite of similar decreases in %SS during occlusion, the amlodipine-treated dogs showed a marked improvement in myocardial segment function (%SS) of the ischemic-reperfused region at 15 minutes following each occlusion (1–9) and at 15, 30, and 60 minutes of reperfusion following occlusion 9, as compared to saline-treated animals. In addition, amlodipine attenuated the loss of adenine nucleotides in the ischemic-reperfused area at 1 hour of reperfusion. These results suggest that amlodipine has a favorable effect on the functional and metabolic recovery of the multiple-stunned myocardium and may have potential as a cardioprotective agent for the treatment of myocardial reperfusion injury.     

Establishment of a long-surviving murine model of myocardial infarction: Qualitative and quantitative conventional microscopic findings during pathological evolution

Summary Ongoing basic molecular analyses are being performed in mice, and a simple long-surviving murine model of myocardial infarction (MI) would be very useful in this regard. Although a few studies have included MI in mice by coronary artery ligation, the induction involves a complex technique and has a relatively high mortality rate. In addition, the identification of the basic pathological sequence is essential to the interpretation of experimental results. We developed a simple technique for the induction of MI in mice and examined qualitative and quantitative conventional microscopic findings during the pathological evolution over a 28-day observation period. Male BALB/c mice weighing approximately 25 – 30 g were anesthetized and then ventilated with a positive pressure ventilator. The heart was exposed by thoracotomy. Left coronary artery occlusion was performed by thermocoagulation using a thermocoagulation knife at the level of the tip of the left atrium. After establishing this surgical method, we used it to induce MI in 71 mice. The operative and postoperative mortality rates of this model were 5.6 % (4/71) and 12.6 % (9/71), respectively. In 3 (5.2%) of the 58 surviving mice, the area of infarct was not sufficient. The infarct area in the remaining 55 mice was 40 ± 9 % of the entire perimeter of the left ventricle. Conventional microscopic examinations with hematoxylin-eosin and Masson-trichrome staining disclosed that all of the characteristic histopathological features of MI occurred 1 – 2 days earlier than those in rats. Our surgical technique provides a sufficient infarct area, with an acceptable mortality rate. The present study clarified the histopathological sequence in this long surviving murine MI model. Received: 2 July 1998, Returned for revision: 19 August 1998, Revision received: 7 October 1998, Accepted: 7 October 1998     

Effect of WEB 2086 on myocardial infarct size and regional blood flow in the dog.

OBJECTIVE: Systemic administration of platelet activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-phosphocholine) produces hypotension and decreased cardiac output; in isolated heart preparations PAF increases coronary vascular resistance and depresses inotropic state. A precursor of PAF bioactivity has been found early in myocardial ischaemia and other reports have suggested that PAF antagonists can reduce myocardial damage and ventricular arrhythmia. This study concerns the effects of WEB 2086, a PAF antagonist, on myocardial infarct size and coronary blood flow after total coronary artery occlusion. METHODS: Open chest anaesthetised dogs (n = 26) were pretreated with either WEB 2086 (5 mg.kg-1) or saline before proximal occlusion of the circumflex artery and constant infusion of WEB 2086 (1 mg.kg-1.h-1) or saline was maintained for 5 h. Cardiac output and regional myocardial flow were measured with radiolabelled microspheres (46Sc, 57Co, and 113Sn) before and immediately after occlusion and 5 h later. In the 22 dogs surviving occlusion, infarct size was determined by planimetry of cross sectional slices after exposure to triphenyltetrazolium chloride. RESULTS: Infarct size was not different between treated and control groups, at 23.6(SEM 2.3)% v 24.8(3.7)% of left ventricle, and was not different between groups when related to vasculature at risk and to collateral blood flow determined with microspheres. CONCLUSIONS: No beneficial effect of a relatively large dose of the potent PAF antagonist, WEB 2086, on myocardial infarct size or collateral blood flow was found after relatively short duration of myocardial ischaemia in the dog.     

Salvage of ischemic myocardium by ibuprofen during infarction in the conscious dog

Because nonsteroidal anti-inflammatory drugs differ in potency and degree of prostaglandin inhibition, they may have different effects on ischemic myocardium. The effect of ibuprofen, an agent of this type, on myocardial infarct size was measured 2 days after occlusion of the left circumflex coronary artery in conscious dogs. Treatment was randomized in dogs after occlusion: Intravenous infusions of ibuprofen (6.25 mg/kg per hour) were administered to 13 dogs and saline solution (0.9 percent) to 13 control dogs over a period of 6 hours. The boundary of the occluded coronary bed, or anatomic risk region, was defined by postmortem coronary arteriography. Masses of infarct and occluded bed were measured by planimetry of weighed transverse sections of the left ventricle. Ibuprofen decreased infarct size compared with that in control dogs, both as percent of the left ventricle (mean ± standard error of the mean 7.5 ± 1.4 versus 15.2 ± 3.1, p < 0.05) and as percent of the occluded bed (16.3 ± 2.3 versus 38.6 ± 5.7, p < 0.005). Ibuprofen also altered (p < 0.001) the relation between the size of the infarct and the size of the occluded bed, so that hearts with occluded beds of similar size had smaller infarcts than those of control dogs. Morphologically, ibuprofen salvaged myocardium in both lateral and epicardial regions of the occluded bed. Changes in arterial pressure, left atrial pressure and heart rate were similar in the two groups. Changes in regional myocardial blood flow measured with 7 to 9 μm radioactive microspheres were also similar in both groups, with an increase in flow to infarcted regions and borders between 20 seconds to 15 minutes after occlusion, but no further change from 15 minutes to 6 hours. Thus, protection of ischemic myocardium by ibuprofen was not due to changes in collateral flow or myocardial oxygen demands, suggesting that cellular and metabolic effects might be important.