Ischaemic preconditioning and contractile function: studies with normothermic and hypothermic global ischaemia.

A significant reduction in the extent of cell necrosis or the incidence of reperfusion-induced arrhythmias can be achieved with ischaemic preconditioning. If preconditioning was also found to be effective in protecting against global ischaemia, then this may have significant implications for the preservation of the heart during cardiac surgery. We therefore investigated this phenomenon in relation to recovery of contractile function after global ischaemia in the isolated rat heart. Isolated working rat hearts (n = 6 per group) were perfused aerobically at 37 degrees C for 20 min and contractile function recorded. This was followed by 10 min of aerobic Langendorff perfusion (control hearts) or 5 min global ischaemia (37 degrees C) + 5 min Langendorff reperfusion (preconditioned hearts). The hearts were then subjected to 10, 15, 20 or 25 min of global ischaemia (37 degrees C) and reperfusion (15 min Langendorff + 20 min working) after which function was again assessed. Preconditioning improved functional recovery after all durations of ischaemia. Thus aortic flow after 10, 15, 20 and 25 min of ischaemia and 35 min of reperfusion recovered to 84, 58, 16 and 5%, respectively, in controls and 88, 74, 55 and 20%, respectively, in the preconditioned groups. To assess whether preconditioning was effective in a surgically relevant model of hypothermic ischaemia, the experiments were repeated with longer periods (45, 70, 90, 115, 135 and 160 min) of ischaemia at 20 degrees C. Under these conditions, normothermic preconditioning increase the post-ischaemic recovery of aortic flow after 115, 135 and 160 min of ischaemic (from 36, 20 and 10%, respectively, in controls to 57, 39 and 26%, respectively, in preconditioned hearts). There was no consistent correlation between tissue high energy phosphate content and enhanced post-ischaemic recovery. Thus, we have demonstrated that ischaemic preconditioning can improve contractile function after global ischaemia in the isolated rat heart, we have defined the duration of ischaemia for which it is operative, and we have shown that this protection is additive to that of hypothermia-induced protection during ischaemia. This may have clinical implications for cardiac surgery.     

Ischemic preconditioning: Infarct size is a more reliable endpoint than functional recovery

The search for the mechanism of preconditioning-induced cardioprotection has been hampered by controversial results obtained by workers using different animal species, experimental models, protocols and endpoints. The aim of this study was to evaluate the role of the perfusion model (retrograde vs working), the infarct size and severity of ischaemia (regional vs global) as well as the endpoint (functional recovery vs infarct size) in preconditioning. The isolated perfused rat heart was preconditioned by 3 × 5 min global ischaemia, followed by different periods of regional or global ischaemia and reperfusion. Ischaemic preconditioning of working hearts resulted in increased functional recovery after 25–35 min global ischaemia, while retrogradely perfused hearts showed no significant improvement (except after 30 min global ischaemia). In addition, the percentage reduction in functional performance during reperfusion observed in the latter group was signicantly less than in working hearts. Hearts were also subjected to regional ischaemia, perfused in either retrograde or working mode and infarct size determined. Regionally ischaemic working as well as retrogradely perfused hearts when preconditioned showed a signicant increase in functional recovery after 35 min ischaemia only. In contrast to global ischaemia, the percentage recovery in mechanical performance of regionally ischaemic hearts was not affected by the mode of perfusion. Preconditioning of working hearts caused a signicant reduction in infarct size after both 30 and 35 min ischaemia. However, preconditioned retrogradely perfused hearts showed a signicant decline in infarct size after 35 min regional ischaemia only. In conclusion, the effect of the perfusion mode on functional recovery is dependent on the size and severity of ischaemia. It also affects the ischaemic time at which infarct size reduction by prior preconditioning occurs in the retrogradely perfused heart.     

Protection of hearts from reperfusion injury by propofol is associated with inhibition of the mitochondrial permeability transition

OBJECTIVE: Diminishing oxidative stress may protect the heart against ischaemia-reperfusion injury by preventing opening of the mitochondrial permeability transition (MPT) pore. The general anaesthetic agent propofol, a free radical scavenger, has been investigated for its effect on the MPT and its cardioprotective action following global and cardioplegic ischaemic arrest. METHOD: Isolated perfused Wistar rat hearts were subjected to either warm global ischaemia (Langendorff) or cold St. Thomas' cardioplegia (working heart mode) in the presence or absence of propofol. MPT pore opening was determined using [3H]-2-deoxyglucose-6-phosphate ([3H]-DOG-6P) entrapment. The respiratory function of isolated mitochondria was also determined for evidence of oxidative stress. RESULTS: Propofol (2 micrograms/ml) significantly improved the functional recovery of Langendorff hearts on reperfusion (left ventricular developed pressure from 28.4 +/- 6.2 to 53.3 +/- 7.3 mmHg and left ventricular end diastolic pressure from 52.9 +/- 4.3 to 37.5 +/- 3.9 mmHg). Recovery was also improved in propofol (4 micrograms/ml) treated working hearts following cold cardioplegic arrest. External cardiac work on reperfusion improved from 0.42 +/- 0.05 to 0.60 +/- 0.03 J/s, representing 45-64% of baseline values, when compared to controls (P < 0.05). Propofol inhibited MPT pore opening during reperfusion, [3H]-DOG-6P entrapment being 16.7 vs. 22.5 ratio units in controls (P < 0.05). Mitochondria isolated from non-ischaemic, propofol-treated hearts exhibited increased respiratory chain activity and were less sensitive to calcium-induced MPT pore opening. CONCLUSION: Propofol confers significant protection against global normothermic ischaemia and during cold cardioplegic arrest. This effect is associated with less opening of mitochondrial MPT pores, probably as a result of diminished oxidative stress. Propofol may be a useful adjunct to cardioplegic solutions in heart surgery.     

Does ischemic preconditioning require reperfusion before index ischemia?

BACKGROUND: Ischemic preconditioning (IP) is initiated through one or several short bouts of ischemia and reperfusion which precede a prolonged ischemia. To test whether a reperfusion must precede the prolonged index ischemia, a series without reperfusion (intraischemic preconditioning: IIP) and a series with gradual onset of ischemia, i.e. ramp ischemia (RI), which is possibly related to the development of hibernation, was compared to conventional IP (CIP). METHOD: Experiments were performed an 27 blood-perfused rabbit hearts (Langendorff apparatus) that were randomized into one of four series: (1) control (n = 7): 60 min normal flow - 60 min low flow (10%) ischemia - 60 min reperfusion. (2) CIP (n = 7): 4 times 5 min zero flow with 10 min reperfusion each - 60 min low flow (10%) - ischemia 60 min reperfusion. (3) IIP (n = 7): 50 min normal flow - 10 min no flow - 60min low flow (10%) ischemia -4 60min reperfusion. (4) RI (n=6): gradual reduction to 10% flow during 60min - 60min low flow (10%) ischemia - 60min reperfusion. At the end of each protocol, the infarcted area was assessed. RESULTS: The infarct area in control hearts was 6.7+/-1.4% (means+/-SEM) of LV total area, in CIP hearts 2.6+/-0.8%, in IIP hearts 3.1+/-0.5%, and in RI hearts 3.0+/-0.3% (all p<0.05 vs. control). The differences between the three protection protocols were statistically not significant, and no protective protocol reduced post-ischemic myocardial dysfunction. CONCLUSION: The preconditioning effect (infarct size reduction) appears not to depend on intermittent reperfusion. Thus, the protective mechanism of IP develops during the initial ischemia that precedes the index ischemia. Alternatively, low-flow ischemia is effectively a sort of reperfusion.     

Endocannabinoids are implicated in the infarct size-reducing effect conferred by heat stress preconditioning in isolated rat hearts

OBJECTIVE: We have investigated the involvement of the endocannabinoid system in the delayed cardioprotection conferred by heat stress preconditioning in the isolated rat heart. METHODS: Rats were divided into eight groups (n=7 in each group), subjected to either heat stress (42 degrees C for 15 min, HS groups) or sham anaesthesia (Sham groups). Twenty-four hours later, their hearts were isolated, retrogradely perfused, and subjected to a 30-min occlusion of the left coronary artery followed by 120 min of reperfusion. Some hearts were perfused with either SR 141716 (a cannabinoid CB(1) receptor antagonist, 1 microM), SR 144528 (a CB(2) receptor antagonist, 1 microM) or L-NAME (a NOS inhibitor, 3 microM) 5 min before ischaemia and during the ischaemic period. RESULTS: The infarct size-reducing effect conferred by heat stress (35.7+/-1.8% in Sham to 14.1+/-0.6% in HS groups) was not altered by the perfusion of SR 141716 (11.2+/-1.5%) but was abolished by both SR 144528 (36.6+/-1.6%) and L-NAME (32.0+/-4.4%). In hearts from non-heat-stressed rats, perfusion with SR 141716 (32.8+/-1.6%), SR 144528 (33.4+/-2.2%) and L-NAME (31.6+/-2.9%) had no effect on infarct size. CONCLUSION: These results suggest an involvement of endocannabinoids, acting through CB(2) receptors, and NO in the cardioprotection conferred by heat stress against myocardial ischaemia. The possible interaction between both mediators of the heat stress response remains to be determined.     

Normothermic transfer times up to 3 min will not precondition the isolated rat heart

Isolated, perfused heart preparations suffer an inevitable peri-operative delay (POD) before retrograde perfusion restores coronary flow. By varying this ischaemic period we investigated the threshold of POD-induced inadvertent preconditioning (PC) in the rat heart. Hearts subjected to POD at 37 degrees C increasing from 1, 2, 3, 5, 10 up to 15 min prior to 20 min retrograde perfusion were further subjected to 30 min global, normothermic ischaemia and 30 min reperfusion (index I/R). The functional recovery was 32 +/- 4.1% in hearts subject to 1 min POD. After 3 min POD functional recovery started to improve and peaked at 10 min POD (78 +/- 7.1%, P < 0.001). At 4 degrees C functional recovery started to improve after 5 min POD and peaked at 10 min POD. To demonstrate that the POD-mediated protection was true PC, two conventional models of PC were established. In both models, hearts were retrogradely perfused within 1 min POD prior to a standard PC protocol (one episode of 10 min ischaemia, or four episodes of 5 min ischaemia). In the conventional PC models protection against the index I/R was abolished using 100 microM 5-hydroxydecanoate (5-HD), the mitrochondrial KATP channel inhibitor. Likewise, 10 min POD-mediated recovery at 37 degrees C (70 +/- 3.2%) was reversed by 100 microM 5-HD perfusion (36 +/- 5.9%; NS v.s. 2 min POD). We conclude: (1) the threshold for PC is greater than 3 min at 37 degrees C and greater than 5 min at 4 degrees C: (2) blockade of the mitochondrial KATP channel abolishes protection in three models of PC in the rat heart, including prolonged POD.     

Ischaemic preconditioning reduces infarct size following global ischaemia in the murine myocardium

Ischaemic preconditioning has not been demonstrated to reduce infarct size following global ischaemia in murine myocardium. Eighteen mouse hearts were isolated, perfused in the Langendorff mode, and randomised to control or preconditioning groups. Preconditioned hearts received four periods of five minutes global ischaemia with five minutes of intervening reperfusion. Control hearts were perfused normally during this period. Both groups were then subjected to 30 minutes global ischaemia followed by 30 minutes reperfusion. Infarct size, contractile recovery and LDH leakage were assessed. Mean infarct size was reduced from 57% of ventricular volume (controls) to 33% in preconditioned hearts (P=0.003). A small improvement in contractile recovery was also observed (6.3% of baseline in controls, 15.5% in preconditioned hearts; P=0.004). Release of LDH did not differ significantly between groups. This study confirms for the first time that ischaemic preconditioning can delay the onset of myocardial necrosis following global ischaemia in the isolated mouse heart. Received: 26 January 1998, Returned for 1. revision: 16 February 1998, 1. Revision received: 16 April 1998, Accepted: 14 May 1998     

Left ventricular unloading during reperfusion does not limit myocardial infarct size

To determine whether venting the left ventricle during coronary reperfusion limits myocardial infarct size, we studied paced (200 beats/min) Langendorff rabbit hearts, perfused with blood from a support rabbit. A left coronary artery was occluded for 60 minutes, followed by 2 hours of reperfusion. Four experimental conditions, as follows, were used: In group 1 (control), the hearts contracted isovolumetrically on a fluid-filled balloon in the left ventricle during both occlusion and reperfusion. In group 2, the balloon was present only during occlusion, and the heart was vented during reperfusion. Hearts in group 3 were vented during occlusion and developed pressure during reperfusion. In group 4, the left ventricle was vented during occlusion and reperfusion. Perfusion pressure (91.2 +/- 0.9 mm Hg) and coronary flow (0.88 +/- 0.03 ml/min/g) were not different between groups. Left ventricular pressures (mean of all groups) were 87.3 +/- 1.5 mm Hg systolic and 6.5 +/- 0.6 mm Hg diastolic. Infarcted myocardium was assessed by triphenyl tetrazolium staining and expressed as a percentage of the area at risk, as measured by fluorescent particles. Venting during both ischemia and reperfusion (n = 10) did result in significantly smaller infarcts than in the unvented controls (n = 10), that is, 13 +/- 5% vs. 41 +/- 6%, respectively. Venting only during reperfusion (n = 10) or occlusion (n = 11) did not significantly limit infarct size (57 +/- 6% and 32 +/- 5%, respectively), as compared with controls. Thus, the clinically feasible intervention of left ventricular venting during reperfusion was not cardioprotective.     

Cyclooxygenase inhibition converts the effect of nitric oxide synthase inhibition from infarct size reduction to expansion in isolated rabbit hearts.

Nitric oxide (NO) and prostacyclin (PGI2) are putative cardioprotective agents. Evidence indicates that there may be a reciprocal relationship involved in the synthesis of NO and PGI2, so that inhibiting the release of one mediator may promote the synthesis of the other. Therefore, we investigated the effects of concomitantly inhibiting NO and PGI2 synthesis, using NG-nitro-L-arginine (L-NOARG) or indomethacin, respectively, on infarct size. Langendorff-perfused rabbit hearts were assigned randomly to one of five treatment groups of n=6: control L-NOARG 100 micromol/l; indomethacin 3 micromol/l L-NOARG 100 micromol/l + indomethacin 3 micromol/l; or L-NOARG 100 micromol/l + L-arginine 1 mmol/l. After 30 min regional ischaemia and 120 min reperfusion, infarct size was assessed by tetrazolium staining. Infarct size was reduced significantly in hearts treated with L-NOARG (20.8+/-1.3%) compared to control hearts (34.7+/-0.4%). This reduction in infarct size was abolished by co-perfusing with a 10-fold excess of L-arginine (30.7+/-1.7%). While indomethacin alone had no effect (33.4+/-2.3%), perfusion with both L-NOARG and indomethacin resulted in a significant increase in infarct size (44.0+/-1.9%) compared to controls. Treatment with L-NOARG alone increased 6-keto PGF1alpha in coronary effluent prior to ischaemia (30.5+/-1.2 vs 16.6+/-1.3 pg/min/g in controls, P<0.05). This effect was reversed by co-perfusion with either L-arginine or indomethacin. These results indicate that the reduction in infarct size by L-NOARG may be due to increased PGI2 release. Concomitant administration of indomethacin negated this effect and revealed an adverse effect of NO synthase inhibition on infarct size.     

S-nitroso human serum albumin reduces ischaemia/reperfusion injury in the pig heart after unprotected warm ischaemia

AIMS: Uncoupled endothelial nitric oxide synthase (eNOS) is a major contributor to vascular reactive oxygen species generation in ischaemia/reperfusion (I/R) injury. Supplementation of NO by the novel NO donor S-nitroso human serum albumin (S-NO-HSA) may inhibit uncoupling of eNOS (feedback inhibition). METHODS AND RESULTS: Pigs (n = 14; 33.1 +/- 1.7 kg) were continuously monitored for heart rate (HR), mean arterial pressure (MAP), left ventricular systolic pressure (LVSP), and coronary flow (CF). Infusion of either human serum albumin (n = 8; controls) or S-NO-HSA (n = 6) lasted 60 min (0.1 micromol/kg/h) starting 15 min prior to ischaemia. After clamping the aorta under cardiopulmonary bypass (CPB), the hearts underwent 15 min of warm, unprotected ischaemia (37 degrees C). Reperfusion lasted 150 min (30 min under CPB; 15 min weaning; additional 105 min reperfusion). In biopsies from non-ischaemic hearts and myocardial biopsies taken after 150 min of reperfusion, high-energy phosphates were measured and the calcium ionophore-stimulated release of NO, superoxide, and peroxynitrite (ONOO(-)) were monitored with nanosensors. Compared with non-ischaemic hearts, the NO level decreased from 930 +/- 25 to 600 +/- 15 nmol/L (P < 0.001) while the superoxide level increased from 45 +/- 5 to 110 +/- 10 nmol/L (P < 0.001) after ischaemia. S-NO-HSA restored the NO level to 825 +/- 20 nmol/L, shifted favourably the [NO]/[ONOO(-)] balance (a marker of eNOS uncoupling) from 1.36 +/- 0.06 (ischaemia) to 3.59 +/- 0.18, significantly improved CF (65 +/- 10 vs. control, 43 +/- 5 mL/min, P < 0.05), MAP (57 +/- 5 vs. 39 +/- 3 mm Hg, P < 0.01), LVSP (106 +/- 5 vs. 81 +/- 4 mm Hg, P < 0.01) and phosphocreatine (PCr) content (41.5 +/- 7.3 vs. 18.0 +/- 5.6 micromol/g protein; P < 0.01) at 150 min of reperfusion. CONCLUSION: Long-lasting release of NO by S-NO-HSA prevented uncoupling of eNOS and thereby improved systolic and diastolic function, myocardial perfusion, and the energetic reserve of the heart after I/R injury.     

Inhibiting mitochondrial permeability transition pore opening at reperfusion protects against ischaemia-reperfusion injury

OBJECTIVE: The opening of the mitochondrial permeability transition pore (mPTP) in the first few minutes of post-ischaemic reperfusion is a critical determinant of reperfusion-induced cell death. We hypothesised that the novel immunosuppressant, sanglifehrin-A (SFA), given at the time of reperfusion, protects the myocardium from ischaemia-reperfusion injury, by suppressing mPTP opening. METHODS: Isolated perfused rat hearts were subjected to 35 min ischaemia/120 min reperfusion, and were treated with (1) SFA (1.0 microM) or (2) DMSO vehicle for the first 15 min of reperfusion or (3) SFA (1.0 microM) after the first 15 min of reperfusion. We examined the effect of SFA on mPTP opening directly, using a myocyte model of oxidative stress. Laser illumination of adult rat myocytes loaded with the fluorophore, TMRM, generates oxidative stress, which induces mPTP opening (represented by mitochondrial membrane depolarisation) followed by rigour contracture. RESULTS: In the isolated perfused heart model, SFA, given during the first 15 min of post-ischaemic reperfusion, reduced the infarct-risk volume ratio from 43.9+/-2.5% in the control group to 23.8+/-4.2% with SFA (p=0.001). However, when SFA was given after the first 15 min of reperfusion, there was no change in infarct size (43.8+/-5.7% with SFA vs. 43.9+/-2.5% in control; p=NS), suggesting that SFA has to be present during the first 15 min of reperfusion to induce protection. In the isolated adult myocyte model, SFA was shown to inhibit mPTP opening in the setting of oxidative stress, represented by an increase in the ROS threshold required to induce: mitochondrial membrane depolarisation (from 269+/-21 to 777+/-100 s; p<0.001) and rigour contracture (from 613+/-14 to 1329+/-129 s; p<0.001). CONCLUSIONS: Inhibiting mPTP opening during the first few minutes of reperfusion, using sanglifehrin-A, limits infarct size and protects myocytes from oxidative stress.     

Ischaemic postconditioning protects isolated mouse hearts against ischaemia/reperfusion injury via sphingosine kinase isoform-1 activation

AIMS: Sphingosine-1-phosphate (S1P) plays a vital role in cytoskeletal rearrangement, development, and apoptosis. Sphingosine kinase-1 (SphK1), the key enzyme catalyzing the formation of S1P, mediates ischaemic preconditioning. Ischaemic postconditioning (POST) has been shown to protect hearts against ischaemia/reperfusion injury (IR). To date, no studies have examined the role of SphK1 in POST. METHODS AND RESULTS: Wild-type (WT) and SphK1 null (KO) mouse hearts were subjected to IR (45 min of global ischaemia and 45 min of reperfusion) in a Langendorff apparatus. Left ventricular developed pressure (LVDP), maximum velocity of increase or decrease of LV pressure (+/-dP/dtmax), and LV end-diastolic pressure (LVEDP) were recorded. Infarction size was measured by 1% triphenyltetrazolium chloride staining. POST, consisting of 5 s of ischaemia and 5 s of reperfusion for three cycles after the index ischaemia, protected hearts against IR: recovery of LVDP and +/-dP/dtmax were elevated; LVEDP was decreased; infarction size (% of risk area) was reduced from 40 +/- 2% in the control group to 29 +/- 2% of the risk area in the POST group (P < 0.05, n = 4 per group). Phosphorylation of Akt and extracellular signal-regulated kinases detected by Western blotting was increased at 10 min of reperfusion. The protection induced by POST was abolished in KO hearts. Infarction size in KO hearts (57 +/- 5%) was not different from the KO control group (53 +/- 5% of risk area, n = 4, P = NS). CONCLUSIONS: A short period of ischaemic POST protected WT mouse hearts against IR. The cardiac protection induced by POST was abrogated in SphK1-KO mouse hearts. Thus, SphK1 is critical for successful ischaemic POST.     

Cariporide attenuates myocardial ischaemia, reperfusion injury and apoptosis in isolated rat hearts

OBJECTIVE: Inhibition of Na+/H+ exchanger (NHE) may protect the ischaemic reperfused myocardium by attenuating sodium overload. This study investigated the time-dependent beneficial effect of the NHEI inhibitor cariporide and the effect on early apoptosis in a rat model of ischaemia and reperfusion. METHODS AND RESULTS: The hearts of rats were perfused in a Langendorff apparatus. In the first part of the experiment, rats were divided into four groups: I/R (cariporide was not given), HOE-Pr+I/R (cariporide was given 15 min before ischaemia), HOE-Is+I/R (cariporide was given shortly before ischaemia), HOE-Re+I/R (cariporide was given during reperfusion). The left ventricular systolic pressure (LVSP), the left ventricular end-diastolic pressure (LVEDP), arrhythmia, coronary flow and myocardial enzymes were measured. In the second part of the experiment, hearts were bathed in the presence or absence of cariporide before ischaemia. Terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling (TUNEL) assays were used to quantify apoptotic cells. Improvement of recovery from ischaemia, defined as increased LVSP, decreased LVEDP, improvement in arrhythmia score, CK-MB and LDH was more significant in the HOE-Pr+I/R group than in the I/R group. Furthermore, cariporide given during ischaemia caused intermediate improvement of ischaemic recovery, while there was no difference between HOE-Re+I/R and I/R hearts. The number of apoptotic cells after ischaemia and reperfusion was 51.8 +/- 15.2 per 1000 cardiomyocytes compared to 11.5 +/- 5.7 (p < 0.05) in hearts pretreated before ischaemia with cariporide. CONCLUSIONS: In conclusion, cariporide decreases the degree of impaired cardiac recovery and early apoptosis after ischaemia/reperfusion injury. The cardioprotective effects of cariporide are more pronounced when the drug is given before ischaemia.     

Activation of kappa-opioid receptors at reperfusion affords cardioprotection in both rat and mouse hearts

The temporal properties of kappa-opioid receptor (kappa-OR) mediated cardioprotection are less well characterised than delta-opioid receptor (delta-OR) responses. This study was aimed at delineating the time course of kappa-OR-mediated protection in two experimental models: an in vivo rat model of regional myocardial infarction (30 min of left coronary artery occlusion with 120 min of reperfusion), and an in vitro perfused murine heart model (undergoing 25 min of global ischemia and 45 min of reperfusion). In the rat model, the selective kappa-OR agonist U50, 488 (0.1 mg/kg, IV bolus), administered either 10 min prior to ischemia or 5 min prior to reperfusion, significantly reduced infarct size (38 +/- 3% and 43 +/- 2% infarct size/area-at-risk (IS/AAR), respectively; P < 0.05) compared to untreated rats (56 +/- 1% IS/AAR). Administration of U50, 488 10 s after onset of reperfusion failed to elicit protection. Cardioprotection with U50,448 administered immediately prior to reperfusion was abolished by a kappa-OR antagonist, (0.1 mg/kg nor-BNI), given 10 min prior to reperfusion. In the in vitro murine model, untreated hearts exhibited 28 +/- 2% (IS/AAR) infarct size. Infusion of U50, 488 (at a final 100 nM concentration) significantly limited infarct size in mouse hearts when applied at the onset of reperfusion (15 +/- 2% IS/AAR; P < 0.05), yet failed to afford protection when infused prior to ischemia. Additionally, in both models studied, treatment with either wortmannin or 5-hydroxydecanoate (5-HD) abrogated the protective effects of U50,488 applied just prior to reperfusion. In summary, kappa-ORs afford cardioprotection primarily when activated prior to and not after reperfusion. This protection may involve activation of the PI3 kinase (PI3K) pathway and mitochondrial (mito) K (ATP) channels.     

Effects of diltiazem on the energy metabolism of the isolated rat heart submitted to ischaemia: a 31P NMR study

The possible direct attenuating modification by diltiazem (DZ) 10(-6) M of ischaemia-induced metabolic damage was studied by 31P NMR spectroscopy (at 101.3 MHz) on retrogradely perfused rat hearts submitted to a 24 min, normothermic (37 degrees C), global low-flow ischaemia (1% of the pre-ischaemic spontaneous coronary flow), followed by a 30 min reperfusion. The presence of DZ 10(-6) M altered neither the heart rate and the left intraventricular pressure under normoxic conditions, nor the extent of ATP and CP depletion during ischaemia, whilst the intramyocardial Pi accumulation during ischaemia was significantly reduced (by about 30%). The intracellular acidification induced by ischaemia was initially less in the presence of DZ, but the pH values reached by the end of ischaemia were somewhat lower than in control (albeit not significantly so): 5.85 +/- 0.07 v. 6.00 +/- 0.07 (Means +/- S.E.M.). On reperfusion, DZ-treated hearts exhibited a greater oxidative phosphorylation capacity than did control hearts. Indeed, NMR spectroscopy revealed a prompter, greater and durable rephosphorylation of creatine together with a simultaneous more rapid and furthermore sharp drop in Pi content in DZ-treated hearts. Moreover, although NMR spectroscopy did not reveal any significant difference in ATP alteration on reperfusion in DZ-treated hearts as compared with controls, biochemical measurements indicated slightly higher ATP content at the end of reperfusion and, more particularly, a better recovery of the adenylate charge: 0.81 +/- 0.03 v. 0.72 +/- 0.03, means +/- S.E.M. (Pre-ischaemic value 0.90-0.91). The intracellular pH differed insignificantly from its pre-ischaemic value at the end of reperfusion in DZ-treated hearts (7.08), while remaining below initial values in controls (7.00). From these results, it is inferred that, at relatively low concentration (10(-6) M), DZ exerts a direct beneficial effect on the energy metabolism of the ischaemic heart without preserving high-energy phosphate compounds during ischaemia and, most importantly, without reducing the extent of the concomitant intracellular acidification.     

Heat stress fails to protect myocardium of streptozotocin-induced diabetic rats against infarction

OBJECTIVE: Protection conferred by heat stress (HS) against ischaemia-reperfusion injury, in term of mechanical function and myocardial necrosis, has been extensively studied. In contrast, the effects of disease states on this HS-induced cytoprotective response are less known. Therefore, we investigated the effects of prior heat stress on the infarct size in the isolated heart and on the myocardial heat stress protein (HSP) 72 synthesis, in a model of insulin-dependent diabetic rats. METHODS: Three groups of animals were studied: D rats were rendered diabetic by 55 mg/kg streptozotocin i.v. injection. DI rats received the same treatment plus a daily injection of insulin started 2 weeks after and V rats received the vehicle of streptozotocin plus a daily injection of saline. Eight weeks later, D, DI and V rats were either heat-stressed (42 degrees C for 15 min) or sham-anaesthetised. Twenty-four hours later, their hearts were isolated, perfused using the Langendorff technique, and subjected to a 30 min occlusion of the left coronary artery followed by 120 min of reperfusion. Myocardial HSP72 content was measured 24 h after HS or sham treatment using an electrophoresis coupled with a Western blot analysis. RESULTS: Infarct-to-risk ratio (I/R) was significantly reduced in hearts from heat-stressed (11.7 +/- 2.0%) compared to sham (30.0 +/- 3.2%) V rats. This cardioprotection was not observed in hearts from D (I/R: 31.4 +/- 3.3 vs. 34.3 +/- 3.5%) and DI (I/R: 28.7 +/- 1.6 vs. 30.3 +/- 1.6%) rats. Risk zones were similar between all experimental groups. The incidence of ventricular arrhythmias during ischaemia and reperfusion periods was not different between the six experimental groups. Western blot analysis of the myocardial HSP72 content showed a comparable heat stress-induced increase of this protein, in V, D and DI animals. CONCLUSION: These results demonstrate that myocardial protective effect induced by heat stress could not extend to a pathological animal model like the diabetic rat and seems to be unrelated to the HSP72 level. Further investigations are required to elucidate the precise role of the heat stress proteins in this adaptive response.     

Developmental changes in tolerance to ischaemia in the rabbit heart: disparity between interpretations of structural, enzymatic and functional indices of injury.

The vulnerability of the heart to injury during ischaemia and reperfusion and its responsiveness to various protective and pharmacological interventions are age-dependent. Using three independent indices of tissue injury (cardiac structure, contractile function and creatine kinase leakage), we compared the response of adult (60-90 days old) and neonatal (7 days old) isolated perfused rabbit hearts to global ischaemia and reperfusion. Prior to ischaemia, heart rate was significantly higher in neonatal hearts, as were control values for coronary flow, aortic flow and cardiac output when expressed on a dry wt basis. In experiments in which adult and neonatal hearts (n = 8 per group) were subjected to 2 min of cardioplegia and 45 min of ischaemia, the post-ischaemic recovery of all indices of cardiac function (when expressed as a percentage of pre-ischaemic control) was significantly higher in neonatal than in adult hearts. Thus, cardiac output recovered to 82.9 +/- 3.6% in the neonate but to only 57.9 +/- 6.7% in the adult (P < 0.05). The functional evidence of a greater resistance to ischaemia in the neonate was, however, contradicted by the levels of creatine kinase leakage which tended to be greater in the neonatal than in the adult heart (32.0 +/- 4.7 vs 20.0 +/- 3.1 IU/15 min/g dry wt). Morphological studies indicated that injury was comparable (moderate-to-severe in degree) in both groups. To assess further the relationship between the three indices, additional experiments were undertaken in which the duration of ischaemia in the neonate was extended to 60 min so that the post-ischaemic recovery of function was reduced to a level similar to that seen in the adult after 45 min of ischaemia. Under these conditions cardiac output recovered to 55.6 +/- 4.8% in the neonatal heart (P = NS when compared with the adult) and creatine kinase leakage increased to 88.2 +/- 13.9 IU/15 min/g dry wt--a value over four times greater than that measured in adult hearts with a comparable degree of functional injury. Morphological examination of tissue obtained after 15 min of reperfusion revealed a remarkable recovery of structure in both age groups. In conclusion, in functional terms the neonatal heart was more resistant to ischaemia than the adult; enzymic leakage, however, indicated the opposite and structural assessment revealed no differences. Thus, in comparing injury during ischaemia and reperfusion between different age groups, it is clearly important to employ several independent indices.     

Infarct size limitation by adrenomedullin: protein kinase A but not PI3-kinase is linked to mitochondrial KCa channels

AIM: Adrenomedullin (ADM) has been shown to protect the heart against ischaemic injury, but little is known of the underlying mechanism. Mitochondrial Ca(2+)-activated K(+) (mitoK(Ca)) channels play a key role in cardioprotection. This study examined whether mitoK(Ca) channel is involved in the protection afforded by ADM. METHODS: Flavoprotein fluorescence in rabbit ventricular myocytes was measured to assay mitoK(Ca) channel activity. Infarct size in the isolated perfused rabbit hearts subjected to 30-min global ischaemia and 120-min reperfusion was determined by triphenyltetrazolium chloride staining. RESULTS: The mitoK(Ca) channel opener NS1619 (30 microM) partially oxidized flavoprotein. ADM (10 nM) augmented the NS1619-induced flavoprotein oxidation when applied after the effect of NS1619 had reached steady state. This potentiating effect of ADM was prevented by the protein kinase A (PKA) inhibitor KT5720 (200 nM), but not by the phosphatidylinositol 3-kinase (PI3-K) inhibitor LY294002 (5 microM). The mitoK(Ca) channel blocker paxilline (PX, 2 microM) completely blocked the oxidative effects of NS1619 in the presence of ADM. Treatment with ADM for 10 min before ischaemia significantly reduced infarct size after ischaemia/reperfusion from 63 +/- 3% in controls to 32 +/- 4% (P < 0.01). This infarct size-limiting effect of ADM was abolished by PX (61 +/- 2%), as well as by KT5720 (62 +/- 3%). ADM treatment for the first 10 min of reperfusion significantly reduced infarct size compared with controls (42 +/- 3%, P < 0.01). This cardioprotective effect of ADM was unaffected by PX (38 +/- 4%), but was abolished by LY294002 (60 +/- 4%). CONCLUSIONS: ADM augments the opening of mitoK(Ca) channels by PKA activation, but not by PI3-K activation. ADM treatment prior to ischaemia reduces infarct size via PKA-mediated activation of mitoK(Ca) channels. On the other hand, ADM treatment upon reperfusion reduces infarct size via a PI3-K-mediated pathway without activating mitoK(Ca) channels.     

Adiponectin protects against myocardial ischaemia-reperfusion injury via AMP-activated protein kinase, Akt, and nitric oxide

AIMS: Cardiovascular disease and type 2 diabetes mellitus are associated with low plasma concentration of adiponectin. The aim of this study was to investigate whether adiponectin exerts cardioprotective effects during myocardial ischaemia-reperfusion and whether this effect is related to the production of nitric oxide (NO). METHODS AND RESULTS: Isolated rat hearts were subjected to 30 min of either global or local ischaemia followed by 60 min of reperfusion. The hearts received vehicle, adiponectin (3 microg/mL), the NO-synthase inhibitor nitro-l-arginine (L-NNA) (0.1 mM), or a combination of adiponectin and L-NNA at the onset of ischaemia. Haemodynamics, infarct size, and expression of endothelial NO-synthase (eNOS), AMP-activated protein kinase (AMPK), and Akt were determined. Adiponectin significantly increased left ventricular function and coronary flow during reperfusion in comparison with the vehicle group. Co-administration of L-NNA abrogated the improvement in myocardial function induced by adiponectin. Infarct size following local ischaemia-reperfusion was 40 +/- 6% of the area at risk in the vehicle group. Adiponectin reduced infarct size to 19 +/- 2% (P < 0.01). L-NNA did not affect infarct size per se but abolished the protective effect of adiponectin (infarct size 40 +/- 5%). Phosphorylation of eNOS Ser1177, AMPK Thr172, and Akt Ser 473 was increased in the adiponectin group (P < 0.05). CONCLUSION: Adiponectin protects from myocardial contractile dysfunction and limits infarct size following ischaemia and reperfusion by a mechanism involving activation of AMPK and production of NO.     

Whole body heat stress fails to limit infarct size in the reperfused rabbit heart.

OBJECTIVE: It has recently been shown that induction of heat stress proteins by whole body heat stress confers myocardial protection in the isolated in vitro rat and rabbit heart. This study extends the above studies by examining the effects of stress protein synthesis on the limitation of infarct size in the in vivo rabbit heart model. METHODS: 30 male New Zealand white rabbits were used. Six rabbits were used for measurement of heat stress protein; 10 were used for infarct size determination in a heat stress group (HS); 14 were used for infarct size determination in a control group. There were 10 exclusions. Under anaesthesia, body temperature was raised to 42 degrees C for 15 min in the HS group. Following 24 hours of recovery rabbits were reanaesthetised and the hearts subjected to a 45 min period of regional ischaemia followed by 3 h reperfusion. The risk zone was defined with fluorescent particles and the infarct area determined by tetrazolium staining. Western blotting showed an increase in the 72 KD heat stress protein in hearts in the HS group. RESULTS: Infarct size as a percent of risk area was 61.4 (SEM 6.4)% (n = 14) in control hearts and 71.8(7.3)% (n = 10) in the HS hearts. These results were not statistically significant. CONCLUSIONS: No protective effect of heat stress could be seen when infarct size was used as the end point. Either the protection seen in earlier studies using the Krebs perfused isolated heart model does not accurately reflect protection against myocardial infarction, or heat stress itself may induce injurious factors in the blood which will negate any direct protective effect to the myocardium in this model.