The effects of nitroglycerin on coronary collaterals and myocardial contractility.

Nitroglycerin (TNG) causes a prolonged dilatation of coronary collaterals. To demonstrate a functional significance of this dilatation we measured the effect of TNG on myocardial contractile force in dogs 2(1/2)-4 wk after the left anterior descending coronary artery (LAD) had been embolized in closed-chest animals. Development of collaterals was documented by angiography. Via a left thoracotomy the main left coronary artery (LCA) and LAD distal to the embolized plug were cannulated. Coronary flow and perfusion pressure were recorded. Contractile force was measured with gauges sutured to epicardial areas supplied by the left circumflex coronary artery (LCf) and occluded LAD. Coronary perfusion pressure in the LCA was gradually decreased until the contractile force recorded by the LAD gauge diminished while the LCf gauge was unaffected. Under these conditions, with coronary perfusion pressure held constant with the aid of a Starling resistance, TNG (18 mug) injected into the LCA increased peripheral LAD pressure by 3-12 mm Hg and contractile force in the LAD region by 36% (range 20-90%), returning it to near-normal levels, while having minimal effect in the LCf area. These changes persisted for 5 min. When LCf and LAD areas were both ischemic, intracoronary TNG had minimal effect on peripheral LAD pressure and contractile force. Thus, TNG causes prolonged dilatation of coronary collaterals and presumed increased collateral flow with subsequent enhancement of myocardial contractile force in ischemic areas. This effect is seen only when ischemia is limited to an area supplied by the collaterals. When the whole heart is ischemic, collaterals are unresponsive to TNG, suggesting that these collaterals dilate fully when the regions from which they originate become ischemic.

Cardioprotection: spotlight on PKG.

Classical ischaemic preconditioning, delayed or second window preconditioning and postconditioning are forms of cardioprotection that are dependent on cell surface receptors, intracellular signalling molecules and kinases that ultimately block formation of the mitochondrial permeability transition. The latter is presumed to cause myocardial necrosis as well as apoptosis, so prevention of its formation upon resumption of perfusion after a prolonged coronary occlusion should be cardioprotective. In all of these forms of cardioprotection, formation of cGMP and activation of protein kinase G (PKG) are recognized to be key steps in the signal transduction pathway. Burley et al. highlight the roles of cGMP and PKG in their comprehensive review. They describe the basic biology of PKG and emphasize its compartmentalization, which may be responsible for the frustration induced by assays for PKG in whole cell lysates and for the spurious conclusions about the role of PKG in cardioprotection. This review will be useful to both the novice and the seasoned investigator.

Protecting the acutely ischemic myocardium beyond reperfusion therapies: are we any closer to realizing the dream of infarct size elimination?

Patients currently treated for acute myocardial infarction receive reperfusion therapy as their only anti-infarct intervention. Although pharmacologic agents have been evaluated in the past for their ability to salvage ischemic myocardium when administered at reperfusion, until very recently none has demonstrated clear efficacy in clinical trials. However, a new generation of interventions has emerged which protects the heart by activating the reperfusion-induced salvage kinase (RISK) pathway. Unlike the disappointing results documented with previously touted putative cardioprotective agents, the preclinical experience with these newer interventions is very consistent indicating that there is a high likelihood that they will be effective clinically. Ischemic postconditioning, which also acts by activating the RISK pathway, has shown marked reduction in infarct size in small-scale trials. Finally, if a strategy for rapidly cooling the heart can be devised so that the in-hospital normothermic ischemic time can be significantly reduced, then infarct size can be even further decreased. In our opinion it is well within our reach using existing technologies to see the day when infarction can be virtually eliminated in the patient with acute coronary occlusion.

Acetylcholine, bradykinin, opioids, and phenylephrine, but not adenosine, trigger preconditioning by generating free radicals and opening mitochondrial K(ATP) channels.

It has been assumed that all G(i)-coupled receptors trigger the protective action of preconditioning by means of an identical intracellular signaling pathway. To test this assumption, rabbit hearts were isolated and perfused with Krebs buffer. All hearts were subjected to a 30-minute coronary artery occlusion followed by 120 minutes of reperfusion. Risk area was measured with fluorescent particles and infarct size with triphenyltetrazolium chloride staining. Control hearts showed 29.1+/-2.8% infarction of the risk zone. A 5-minute infusion of acetylcholine (0.55 mmol/L) beginning 15 minutes before the 30-minute occlusion resulted in significant protection (9.2+/-2.7% infarction). This protection could be blocked by administration of 300 micromol/L N-2-mercaptopropionyl glycine (MPG), a free radical scavenger, or by 200 micromol/L 5-hydroxydecanoate (5-HD), a mitochondrial K(ATP) antagonist, for 15 minutes beginning 5 minutes before the acetylcholine infusion (35.2+/-3.9% and 27.8+/-2.4% infarction, respectively). Similar protection was observed with other known triggers, ie, bradykinin (0.4 micromol/L), morphine (0.3 micromol/L), and phenylephrine (0.1 micromol/L), and in each case protection was completely abrogated by either MPG or 5-HD. In contrast, protection by adenosine or its analog N(6)-(2-phenylisopropyl) adenosine could not be blocked by either MPG or 5-HD. Therefore, whereas most of the tested agonists trigger protection by a pathway that requires opening of mitochondrial K(ATP) channels and production of free radicals, the protective action of adenosine is not dependent on either of these steps. Hence, it cannot be assumed that all G(i)-coupled receptors use the same signal transduction pathways to trigger preconditioning.

Ischemic preconditioning activates MAPKAPK2 in the isolated rabbit heart: evidence for involvement of p38 MAPK.

Recent studies suggest that p38 mitogen-activated protein kinase (MAPK) may be involved in ischemic preconditioning (PC). To further test this possibility, the regulation of MAPK-activated protein kinase 2 (MAPKAPK2), a kinase immediately downstream from p38 MAPK, and the activity of c-Jun NH(2)-terminal kinase (JNK), a second MAPK, were examined in preconditioned hearts. Isolated, perfused rabbit hearts were subjected to 20 to 30 minutes of global ischemia. Ventricular biopsies before treatment and after 20 minutes of ischemia were homogenized, and the activities of MAPKAPK2 and JNK were evaluated. For the MAPKAPK2 experiments, 7 groups were studied, as follows: control hearts; preconditioned hearts; hearts treated with 500 nmol/L R(-) N(6)-(2-phenylisopropyl) adenosine (PIA), an A(1)-adenosine receptor agonist; preconditioned hearts pretreated with 100 micromol/L 8-(p-sulfophenyl) theophylline (SPT), an adenosine receptor antagonist; preconditioned hearts also treated with SB 203580, a potent inhibitor of p38 MAPK activation; hearts treated with 50 ng/mL anisomycin (a p38 MAPK/JNK activator); and hearts treated with both anisomycin (50 ng/mL) and the tyrosine kinase inhibitor genistein (50 micromol/L). MAPKAPK2 activity was not altered in control hearts after 20 minutes of global ischemia. By contrast, there was a 3.8-fold increase in activity during ischemia in preconditioned hearts. Activation of MAPKAPK2 in preconditioned hearts was blocked by both SPT and SB 203580. MAPKAPK2 activity during ischemia increased 3.5-fold and 3.3-fold in hearts pretreated with PIA or anisomycin, respectively. MAPKAPK2 activation during ischemia in hearts pretreated with anisomycin was blocked by genistein. In separate hearts, anisomycin mimicked the anti-infarct effect of PC, and that protection was abolished by genistein. JNK activity was measured in control and preconditioned hearts. There was a comparable, modest decline in activity during 30 minutes of global ischemia in both groups. As a positive control, a third group of hearts was treated with anisomycin before global ischemia, and in these, JNK activity increased by 290% above baseline. These results confirm that the p38 MAPK/MAPKAPK2 pathway is activated during ischemia only if the heart is in a preconditioned state. These data further support p38 MAPK as an important signaling component in ischemic PC.

Opening of mitochondrial K(ATP) channels triggers the preconditioned state by generating free radicals.

The critical time for opening mitochondrial (mito) K(ATP) channels, putative end effectors of ischemic preconditioning (PC), was examined. In isolated rabbit hearts 29+/-3% of risk zone infarcted after 30 minutes of regional ischemia. Ischemic PC or 5-minute exposure to 10 micromol/L diazoxide, a mito K(ATP) channel opener, reduced infarction to 3+/-1% and 8+/-1%, respectively. The mito K(ATP) channel closer 5-hydroxydecanoate (200 micromol/L), bracketing either 5-minute PC ischemia or diazoxide infusion, blocked protection (24+/-3 and 28+/-6% infarction, respectively). However, 5-hydroxydecanoate starting 5 minutes before long ischemia did not affect protection. Glibenclamide (5 micromol/L), another K(ATP) channel closer, blocked the protection by PC only when administered early. These data suggest that K(ATP) channel opening triggers protection but is not the final step. Five minutes of diazoxide followed by a 30-minute washout still reduced infarct size (8+/-3%), implying memory as seen with other PC triggers. The protection by diazoxide was not blocked by 5 micromol/L chelerythrine, a protein kinase C antagonist, given either to bracket diazoxide infusion or just before the index ischemia. Bracketing preischemic exposure to diazoxide with 50 micromol/L genistein, a tyrosine kinase antagonist, did not affect infarction, but genistein blocked the protection by diazoxide when administered shortly before the index ischemia. Thus, although it is not protein kinase C-dependent, the protection by diazoxide involves tyrosine kinase. Bracketing diazoxide perfusion with N:-(2-mercaptopropionyl) glycine (300 micromol/L) or Mn(III)tetrakis(4-benzoic acid) porphyrin chloride (7 micromol/L), each of which is a free radical scavenger, blocked protection, indicating that diazoxide triggers protection through free radicals. Therefore, mito K(ATP) channels are not the end effectors of protection, but rather their opening before ischemia generates free radicals that trigger entrance into a preconditioned state and activation of kinases.

Favorable remodeling enhances recovery of regional myocardial function in the weeks after infarction in ischemically preconditioned hearts.

BACKGROUND: In a previous study, we found that recovery of segment shortening in the ischemic zone of conscious, chronically instrumented rabbits was significantly better in ischemically preconditioned than control animals after 72 hours of reperfusion. However, although this period of reperfusion was felt to be sufficient to allow recovery from stunning, regional function was disproportionately low for the size of the infarcts. METHODS AND RESULTS: To further characterize the recovery of left ventricular regional function, rabbits were chronically instrumented with a balloon occluder around a branch of the left coronary artery and a pair of ultrasonic crystals to monitor segment shortening in the ischemic zone. The preconditioned group had 1 cycle of 5-minute occlusion/10-minute reperfusion before a 30-minute occlusion, whereas control rabbits experienced only the 30-minute occlusion. All monitored segments were either dyskinetic or akinetic during the 30-minute occlusion. There was no difference in function between the 2 groups until 24 hours of reperfusion. At 72 hours, systolic shortening in control hearts averaged only 5% of the preischemic value, whereas shortening was 29% of baseline in preconditioned hearts. By day 21, systolic shortening averaged 26% in control hearts and 65% in preconditioned hearts (P<0.02) and appeared to have reached a plateau. Infarct size was 31.4+/-2.8% and 15.5+/-2.1% in control and preconditioned hearts, respectively. Moreover, in ischemically preconditioned hearts, the recovery of regional function was better than in controls for any given amount of microinfarction in the myocardial segment between crystals (P=0.02). CONCLUSIONS: The progressive improvement in preconditioned hearts is most consistent with favorable remodeling in the ischemic zone, which the preconditioning process seems to accentuate.

Impaired coronary vasodilator responsiveness as a cause of lactate production during pacing-induced ischemia in patients with angina pectoris and normal coronary arteries.

Subgroups of patients with angina pectoris and normal coronary arteries are known to have pacing-induced lactate production and, therefore, myocardial ischemia. To examine the mechanism of this pacing-induced ischemia, the effect of incremental atrial pacing on coronary blood flow and metabolism was studied in 27 patients with angina and normal coronary arteries. Seventeen patients continued to exhibit normal lactate extraction even at heart rates up to 160 beats/min (Group 1), whereas in 10 patients (Group 2) lactate extraction changed to production at the highest pacing rate. Coronary blood flow increased in Group 1 patients by 18, 41 and 75%, respectively, as heart rate was increased by 20 beat/min increments from 100 to 160 beats/min. In contrast, coronary blood flow increased by only 8, 7 and 14%, at the three respective pacing rates in Group 2. Between the heart rates of 100 and 160 beats/min, coronary vascular resistance decreased 32% in Group 1 patients but was unchanged in Group 2 patients. There was no significant change in the ratio of myocardial O2 consumption/rate-pressure product in Group 1 patients, but this ratio decreased from 0.91 +/- 0.26 ml O2 X min-1 X (mm Hg X beats/min)-1 to 0.53 +/- 0.11 (p less than 0.05) in Group 2 patients as heart rate increased from baseline to 160 beats/min. Thus, patients with angina and normal coronary arteries who develop ischemia with pacing have a decreased coronary vasodilator response that interferes with their ability to increase myocardial oxygen supply to match the higher demand.

Acute ethanol exposure fails to elicit preconditioning-like protection in in situ rabbit hearts because of its continued presence during ischemia.

OBJECTIVES: Is the timing of exposure critical for ethanol's ability to induce cardioprotection? BACKGROUND: Acute ethanol exposure has been reported to mimic ischemic preconditioning in vitro, but it failed to protect in situ. We hypothesized that these conflicting findings were related to ethanol's presence during ischemia in situ. METHODS: The effect on infarct size (triphenyltetrazolium chloride) of acute ethanol exposure (0.35, 0.7, and 1.4 g/kg IV) 10 min before ischemia was measured in open-chest rabbits after 30 min of regional ischemia and reperfusion and was compared to ethanol's ability to reduce infarct size in isolated hearts in which the timing of ethanol exposure could be varied. RESULTS: Ethanol exposure in situ shortly before ischemia did not reduce infarct size. Moreover, ethanol abolished protection from both ischemic preconditioning and mitochondrial KATP channel activation. In contrast, in buffer-perfused hearts exposed to 10 to 50 mmol/liter ethanol for 5 min followed by washout before ischemia, infarct size was significantly reduced. When ethanol exposure was prolonged until the end of ischemia in isolated hearts, protection was abolished. Conversely, protection was seen when ethanol was infused in situ followed by removal of the heart and perfusion with ethanol-free buffer prior to ischemia in a Langendorff preparation. When 50 min were allowed to metabolize the ethanol prior to ischemia, protection could also be shown in situ. CONCLUSIONS: Ethanol exposure followed by washout or sufficient time to metabolize the alcohol prior to ischemia induces preconditioning-like myocardial protection. However, if present throughout ischemia, ethanol actually blocks all preconditioning-related protection.

Ischemic preconditioning: from basic mechanisms to clinical applications.

When the heart is subjected to a transient nonlethal period of ischemia, it quickly adapts itself to become resistant to infarction from a subsequent ischemic insult. This adaptation is called preconditioning. This cardioprotection has been shown to be mediated by stimulation of receptors linked to protein kinase C (PKC) (adenosine, bradykinin, opioids, etc.), and these receptors protect by activating PKC. PKC appears to be the first element of a complex kinase cascade that is activated during the prolonged ischemia in the preconditioned heart. Recent studies imply that p38 mitogen-activated protein kinase carries the signal from PKC to the mitochondrial K(ATP) channels, causing them to open and thus protect the heart. The cardioprotection of preconditioning occurs in all species tested to date, and possibly also humans. It is expected that as the mechanism of preconditioning is more thoroughly understood, pharmacological preconditioning will become practical for clinical use.

Coronary vascular reserve in the greyhound with left ventricular hypertrophy.

The effects of physiological left ventricular hypertrophy on coronary reactivity and reserve were examined by comparing greyhounds with their remarkable myocardial hypertrophy with mongrel dogs (left ventricular body weight ratios 7.7(0.1) and 4.4(0.1) g X kg-1 respectively). Peak reactive hyperaemia and flow debt repayment after 15 s coronary occlusions were virtually identical in both groups at baseline heart rates and paced atrial rates up to 270 beats X min-1. Furthermore, the changes in myocardial blood flow and its transmural distribution after release of a 45 s coronary occlusion both at baseline heart rate and during atrial pacing at 210 beats X min-1 were similar in both greyhounds and mongrel dogs. Collateral flow during a 1 min coronary occlusion in conscious animals and myocardial flows and distribution during treadmill exercise were also similar. To determine minimal coronary vascular resistance intracoronary adenosine (400-600 micrograms X min-1) was infused to dilate maximally the coronary vasculature without having appreciable systemic effects. In both groups of dogs adenosine increased coronary flow six to seven fold. Coronary vascular resistance per gram of left ventricle decreased equally, and the minimal resistance was 2200(147) kPa X litre-1 X min X g (RU X g-1) in greyhounds and 2360(453) RU X g-1 in mongrels, thus indicating similar maximal vascularity in each gram of the hypertrophied and mongrel left ventricles. Because of the larger greyhound heart, the minimal coronary vascular resistance for the entire left ventricle (11.7(0.7)RU) was 50% of that in mongrel dogs (24.1(0.7)RU). Thus all forms of left ventricular hypertrophy cannot be assumed to have similar effects on the coronary vasculature. Whereas the pathological hypertrophy associated with renovascular hypertension and aortic banding is accompanied by decreased coronary vascularity, the myocardial hypertrophy of the greyhound is accompanied by increased total vascularity and preserved coronary reserve.

Training in dogs with normal coronary arteries: lack of effect on collateral development.

STUDY OBJECTIVE - The aim of the study was to investigate the effect of exercise training on coronary collateral development in a normal heart animal model. DESIGN - Dogs with normal hearts were fitted with balloon occluders around the left circumflex coronary artery. Haemodynamic variables and myocardial blood flow (using radioactive microspheres) were measured during 1 min circumflex occlusions at rest and during exercise, before and after a 12 week exercise programme. Resting measurements were also made after 4 weeks. SUBJECTS - Trained subjects were 10 one year old beagles; eight beagles served as sedentary controls. Although measurements were made in all dogs at 4 weeks, four trained and three control animals died during the experiment because of complications, so the data reported at 12 weeks are derived from six trained and five sedentary animals. Except during the few occasions when the left circumflex artery was transiently occluded to allow measurement of collateral flow, all coronary arteries were patent and without impediment to normal flow. MEASUREMENTS and RESULTS - Measurements of haemodynamic variables and coronary blood flow were made at the start and repeated at 4 weeks (resting measurements only) and 12 weeks of exercise training. Initial resting circumflex collateral flow was 0.26(SEM 0.05) cm3.g-1.min-1 in the training group and 0.23(0.03) in the control group (NS). The ratio of ischaemic to normal blood flow was 0.16(0.02) in both groups. At 4 weeks there were no changes in either group. At 12 weeks the ischaemic to normal blood flow ratio had increased in both trained and sedentary dogs to 0.24(0.05) and 0.26(0.06) respectively, but the trend over the 12 week period was not significant. The decline in cardiac output and dramatic increase in left atrial pressure during combined coronary occlusion and exercise were comparable in both groups at weeks 1 and 12. CONCLUSIONS - Exercise does not accelerate the development of coronary collaterals in dogs with normal coronary arteries.

Lack of effect of propranolol on canine coronary collateral development during progressive coronary stenosis and occlusion.

OBJECTIVE: The aim was to test the effect of propranolol, a beta adrenergic receptor blocking drug which diminishes myocardial ischaemia, on coronary collateral development. METHODS: Dogs were chronically instrumented with a left circumflex coronary artery Doppler flow probe, ameroid constrictor, pneumatic occluder, and left atrial and aortic catheters. Haemodynamic studies and measurements of normal myocardial and collateral blood flows were performed at least weekly after surgery, or sooner if the left circumflex artery occluded spontaneously, in eight control dogs and seven animals treated with propranolol, 240 mg twice daily (a dose which successfully attenuated the chronotropic effect of a graded infusion of isoprenaline). In dogs in which the left circumflex artery occluded within the first four weeks after surgery, collateral blood flow was measured during exercise on a motorized treadmill. RESULTS: There was no difference between the groups in the rate or pattern of coronary collateral development, magnitude of collateral flow before or after spontaneous left circumflex occlusion, and collateral vascular reserve during exercise. At the time of coronary occlusion resting collateral flow was equivalent to normal perfusion levels in both groups and averaged 87-96% of normal flows during exercise. CONCLUSIONS: Propranolol has no observable effect on coronary collateral development despite its acknowledged ability to prevent or attenuate myocardial ischaemia.

Lack of effect of prior training on subsequent ischaemic and infarcting myocardium and collateral development in dogs with normal hearts.

To determine whether exercise training in animals with normal coronary arteries has a salutary effect on ischaemic myocardium, 24 dogs were randomly assigned to be either trained or confined to cages for three months. All dogs then underwent left thoracotomy for placement of indwelling right and left atrial and aortic catheters and a loose snare ligature around the proximal left circumflex coronary artery. Three days after operation control scintigrams were recorded after injection of thallous chloride-201 in animals running on a treadmill to achieve exercise heart rates of 220 beats.min-1. Four days later the snares were pulled to occlude the left circumflex artery and infarct size determined by measuring venous activity of creatine phosphokinase. Three days after infarction the first post-ligation scintigram was performed after thallium-201 injection in exercising animals. Exercise scans were repeated at 10 days and 2, 4, and 6 weeks after coronary ligation. During the final exercise study collateral blood flow was measured with radioactive microspheres. There was no difference in mean creatine phosphokinase appearance time, peak creatine phosphokinase activity, or measured infarct size between the trained and sedentary dogs. The ratio of thallium-201 activity in left circumflex artery or ischaemic area to left anterior descending artery or normally perfused myocardium fell from 100% before occlusion to 86.6% in the sedentary animals and 80.6% in the trained dogs three days after coronary ligation. Although these falls were significant (p less than 0.025 and p less than 0.005 respectively), there was no difference between groups. Over the next five and a half weeks the scintigraphic defect shrank as the thallium-201 ratio gradually increased, but changes were again similar in both groups. At six weeks there was little difference in exercise collateral flow to left circumflex artery myocardium and flow to normal myocardial tissue in cage confined and trained dogs. Therefore, no beneficial effect of exercise training in dogs with normal hearts could be seen on ischaemic or infarcting myocardium or coronary collateral development after coronary ligation.

Ventricular fibrillation threshold is influenced by left ventricular stretch and mass in the absence of ischaemia.

STUDY OBJECTIVE: Ischaemic heart disease is known to contribute to the development of ventricular arrhythmias. However, the role of non-ischaemic variables has been less well defined. We therefore studied the effect of myocardial stretch and ventricular mass on the vulnerability of the rat heart to ventricular fibrillation. DESIGN AND EXPERIMENTAL MATERIAL: Two groups of rat hearts were studied in an isolated buffer perfused apparatus: group I, mature female animals with an average dry left ventricular weight of 73 mg and group II, a group of retired breeders with an average left ventricular weight of 122 mg. Hearts performed isovolumetric work at either low (0 mm Hg) or high (20 mm Hg) left ventricular end diastolic pressures. MEASUREMENTS AND MAIN RESULTS: Ventricular fibrillation was provoked by trains of ventricular extrastimuli delivered at increasing current until development of the arrhythmia. The current required to provoke ventricular fibrillation decreased in both groups at the high left ventricular end diastolic pressure and the larger hearts in group II were more vulnerable to ventricular fibrillation than those in group I. The decrease in ventricular fibrillation threshold occurred in the absence of an increase in myocardial lactate production or a decrease in endocardial to epicardial flow ratios. CONCLUSIONS: This study thus identified two variables, myocardial stretch and ventricular mass, which influence the development of ventricular fibrillation and which are independent of myocardial ischaemia in this animal model. These observations may be relevant to an understanding of the increased incidence of lethal arrhythmias which occur in patients with dilated cardiomyopathies.

Cyclosporine A limits myocardial infarct size even when administered after onset of ischemia.

OBJECTIVE: The role of the immunosuppressant cyclosporine A as a preconditioning-mimetic in the rabbit heart was examined. METHODS: Cyclosporine A, a potent protein 2B or calcium/calmodulin-dependent phosphatase (PP) inhibitor, was administered isolated rabbit hearts starting either 15 min prior to or 10 or 20 min after the onset of a 30 min period of regional ischemia and continuing until the onset of reperfusion. The effect of pretreatment with a second PP2B antagonist, FK-506, was also examined. In an additional protocol L-NAME was perfused for 50 min starting 5 min before the 45-min infusion of cyclosporine A. After 2 h of reperfusion infarct size was measured with triphenyltetrazolium chloride. In a second study left ventricular biopsies of isolated rabbit hearts were obtained to measure the effect of cyclosporine A on dephosphorylation of [32P] phosphorylase kinase by calcium/calmodulin-dependent phosphatases. RESULTS: Pretreatment with cyclosporine A resulted in only 10.0%, infarction of the risk zone, significantly less than that in untreated control hearts (28.7%, p < 0.001) but comparable to the extent of infarction in ischemically preconditioned hearts (10.0% p < 0.001 vs. control). Equivalent protection was also observed in hearts with treatment delayed for 10 min following the onset of ischemia (10.4% infarction, p < 0.001 vs. control). However, protection waned when cyclosporine A was administered only during the last 10 min of the 30-min ischemic period (25.5% infarction, p = n.s. vs. control). Pretreatment with FK-506 also resulted in myocardial salvage (10.4% infarction, p < 0.001 vs. control). When hearts were exposed to a co-infusion of L-NAME and cyclosporine A, protection was still evident (18.1% infarction, p < 0.05 vs. L-NAME), although not as robust as that seen with the PP2B blocker alone. In hearts pretreated with cyclosporine A dephosphorylation of [32P] phosphorylase kinase by calcium/calmodulin-dependent phosphatases was inhibited by 67%. CONCLUSIONS: Cyclosporine A and FK-506, potent PP2B inhibitors, can protect the ischemic rabbit heart, and at least cyclosporine A continues to be effective when infusion is delayed until after the onset of ischemia. The mechanism of this protection may be related to inhibition of phosphatases and prolongation of the phosphorylation state of ischemic cells.

Acadesine extends the window of protection afforded by ischaemic preconditioning.

OBJECTIVE: The aim was to test whether acadesine (5-amino-4-imidazolecarboxamide riboside, AICAR), an adenosine regulating agent which increases tissue adenosine during ischaemia, could prolong the window of protection from ischaemic preconditioning. METHODS: A branch of the left coronary artery of a rabbit heart was occluded for 30 min and reperfused for 180 min to induce infarction. Infarct size was determined with triphenyl tetrazolium staining. Prior to the 30 min ischaemia, rabbits were subjected to one of the following seven protocols: (1) No treatment (controls). (2) Preconditioning with 5 min of regional ischaemia followed by 2 h of reperfusion. (3) Treatment with acadesine (2.5 mg.kg-1.min-1 intravenously for 5 min starting 155 min prior to 30 min ischaemia followed by 210 min infusion of 0.5 mg.kg-1.min-1. (4) Treatment with acadesine (same schedule as in group 3) plus preconditioning as in group 2. (5) Treatment with acadesine for a shorter period (acadesine 2.5 mg.kg-1.min-1 for 5 min starting 30 min prior to preconditioning followed by 0.5 mg.kg-1.min-1 for only 60 min) plus preconditioning as in group 2. (6) Treatment with preconditioning followed by adenosine receptor blockade with 8-(p-sulphophenyl)theophylline (SPT) 10 mg.kg-1 intravenously immediately after and again 15 min after preconditioning. (7) Treatment with short infusion of acadesine plus preconditioning plus SPT. RESULTS: Preconditioning followed by 2 h of reperfusion offered little protection against infarction [28.6(SEM 2.7)% of the ischaemic zone infarcted] as compared to control [38.7(3.1)% infarction]. Treatment with acadesine alone did not modify the infarct size [37.8(3.5)%], but both of the acadesine plus preconditioning groups showed a significant limitation of infarct size with 13.9(3.1)% infarction in group 4 and 12.7(2.2)% infarction in group 5 (both p < 0.01 v control). Although SPT alone did not modify the infarct size [26.8(3.3)%], SPT blocked the protective effect of acadesine [25.3(2.9)%, p < 0.05 v group 5]. CONCLUSION: Acadesine can delay the natural decay of preconditioning. This delay appeared to be mediated by adenosine and may have therapeutic potential.

A new animal model of controlled coronary artery occlusion in conscious rabbits.

OBJECTIVE: The aim was to develop a method for installing a pneumatic occluder on the coronary artery of a rabbit, and to see whether repetitive coronary occlusions could induce collateral development as in dogs. METHODS: A superficial branch of the left coronary artery was encircled with a balloon occluder, and a catheter was inserted into the left atrial appendage of New Zealand White rabbits, with a survival rate of 89.5%. Baseline collateral flow was measured 5-7 d after surgery with radioactive microspheres while the artery was occluded. According to a predetermined schedule animals were assigned to control and experimental groups. In the latter the balloon was inflated for 2 min once every 15 min for 8 h per d, 5 d per week. Collateral flow measurements were repeated after 100 and 300 occlusions. In control animals repeat flow measurements were made approximately 4 and 14 d after the first determination. RESULTS: Inflation of the balloon occluder reliably resulted in marked S-T segment elevation which quickly resolved after balloon deflation. These changes were nearly identical after two weeks of observation. Average baseline collateral flows in both groups were less than 5% of normal myocardial flow. There was no appreciable change during the two week period of observation in either control rabbits or those receiving at least 300 2 min coronary occlusions. CONCLUSIONS: The rabbit tolerates implantation of a pneumatic coronary occluder without noticeable problem and can successfully serve as a small animal model of repetitive or chronic myocardial ischaemia. The surgical preparation is not difficult, and ischaemia can be reproducibly created and relieved for at least three weeks following surgery. Coronary collaterals are sparse in the rabbit, and repetitive occlusions can be instituted without the complication of collateral development.

Acadesine extends the window of protection afforded by ischaemic preconditioning in conscious rabbits.

OBJECTIVE: Ischaemic preconditioning protects myocardium from infarction if the reperfusion interval between the brief and prolonged ischaemic intervals is less than 1 h. In anaesthetised rabbits acadesine (5-amino-4-imidazolecarboxamide riboside, AICAR), an adenosine enhancer which increases tissue adenosine during ischaemia, prolongs the window of protection to 2 h. The aim of this study was to try to determine the maximum extension of this window of protection, using chronically instrumented, unsedated rabbits. METHODS: Rabbits were instrumented with a balloon occluder around a major branch of the left coronary artery for reversible coronary occlusion. Five to seven days after surgery all animals underwent a 30 min coronary occlusion. Animals were randomised to one of seven groups: (1) No additional treatment (control); (2) Ischaemic preconditioning with 5 min regional ischaemia followed by 10 min reperfusion before the 30 min coronary occlusion; (3) and (4) Ischaemic preconditioning followed by 2 or 4 h of reperfusion before the 30 min occlusion, respectively; (5) Treatment with acadesine (2.5 mg.kg-1.min-1 intravenously for 5 min and then 0.5 mg.kg-1.min-1 beginning 45 min before and continuing until 30 min after release of the 30 min occlusion) without ischaemic preconditioning; (6) and (7) Treatment with the higher dose of acadesine for 5 min beginning 35 min before the 5 min ischaemic period, and then the lower dose continuing until 30 min after release of the 30 min coronary occlusion in rabbits with 4 or 6 h reperfusion intervals, respectively. RESULTS: Rabbits with ischaemic preconditioning with 10 min reperfusion preceding the 30 min coronary occlusion (group 2) had only 5.6(SEM 1.1)% infarction of the ischaemic zone. Ischaemic preconditioning followed by 2 h reperfusion (group 3) offered continued protection [18.2(2.2)% infarction] as compared to control animals [37.7(2.6)% infarction]. However, protection waned if ischaemic preconditioning was followed by 4 h reperfusion (group 4) [36.7(3.0)% infarction]. Additionally, treatment with acadesine alone did not modify infarct size (group 7) [39.5(4.0)%], but acadesine largely restored the protection of ischaemic preconditioning despite a 4 h reperfusion interval (group 5) [20.4(3.0)% infarction, P < 0.01 v control]. However, when reperfusion was extended to 6 h (group 6) acadesine could no longer restore protection [36.2(0.9)% infarction]. CONCLUSIONS: The protection afforded by a 5 min ischaemic preconditioning period lasts from 2 to 4 h in the awake, unsedated rabbit, and acadesine can extend the duration of this window of protection to at least 4 h but not to 6 h.