Roger A. Wolff

Met5-enkephalin protects isolated adult rabbit cardiomyocytes via δ-opioid receptors

In rats and rabbits, endogenous opioid peptides participate in ischemic preconditioning. However, it is not known which endogenous opioid(s) can trigger cardioprotection. We examined preconditioning-induced and opioid-induced limitation of cell death in isolated, calcium-tolerant, adult rabbit cardiomyocytes. Cells were subjected to simulated ischemia by pelleting and normothermic hypoxic incubation. Preconditioning was elicited with 15 min of simulated ischemia followed by 15 min of resuspension and reoxygenation. All cells underwent 180 min of simulated ischemia. Cell death was assessed by trypan blue permeability. Morphine protected cells, as did preconditioning; naloxone blocked the preconditioning-induced protection. Exogenous Met5-enkephalin (ME) induced protection, but exogenous β-endorphin did not. ME-induced protection was blocked by the δ-selective antagonist naltrindole. Additionally, two other proenkephalin products, Leu5-enkephalin and Met5-enkephalin-Arg-Phe, provided protection equipotent to ME. These data suggest that one or more proenkephalin products interact with δ-opioid receptors to endogenously trigger opioid-mediated protection.

Naloxone blockade of myocardial ischemic preconditioning does not require central nervous system participation

Objective The hypothesis that naloxone blockade of ischemic preconditioning (IP)-induced infarct limitation does not require central nervous system participation was evaluated using quaternary naloxone in anesthetized rabbits (Study I) and naloxone hydrochloride in isolated rabbit hearts (Study II). Methods In Study I, rabbits underwent 30 min coronary artery occlusion and 180 min reperfusion. IP was elicited with a 5 min coronary artery occlusion beginning 15 min before the 30 min occlusion. Intravenous naloxone methiodide, 12.9 mg/kg, was bolused 10 or 1 min before IP. In Study II, rabbit hearts underwent 45 min coronary artery occlusion and 120 min reperfusion. IP was elicited with 2 cycles of 5 min coronary artery occlusion plus 5 min reperfusion, beginning 20 min before the 45 min occlusion. Naloxone hydrochloride, 1µmol/L, was added to the buffer perfusate for 25 min preceding the long coronary artery occlusion. In both studies, infarct size was assessed with tetrazolium, normalized to risk volume, and analyzed using ANOVA. Results In both studies, IP reduced infarct size compared to control (6.3 ± 2,3 vs. 29.5 ± 4,4, P = 0.007, Study I; 11.8 ± 4.7 vs. 47.7 ± 6.7, P = 0.03, Study II). In Study I, IP was not blocked when naloxone methiodide was given 10 min before IP (13.8 ± 4.8 vs. 42.3 ± 5.4, P = 0.004) but was blocked when given 1 min before IP (25.3 ± 7.2 vs. 28.4 ± 5.0, P = ns). In Study II, infarct size was intermediate in the 1µmol/L naloxone hydrochloride+IP group (19.0 ± 6.5 vs. 48.9 ± 8.4, P = ns) but IP was blocked by 100 µmol/L naloxone hydrochloride (62.6 ± 4.5 vs. 56.2 ± 6.7, P = ns). Conclusion Naloxone blockade of IP-induced infarct limitation involves a cardiac mechanism.

Remifentanil limits infarct size but attenuates preconditioning-induced infarct limitation

ObjectivesWe investigated the influence of the narcotic anesthetic remifentanil on irreversible myocardial ischemic injury. MethodsNew Zealand White rabbits were anesthetized with propofol (0.7–1.8 mg·kg−1·min−1) and then subjected to 30 min regional myocardial ischemia and 3 h reperfusion (CON). Some animals also underwent ischemic preconditioning, elicited by either one (IP1) or two (IP2) cycles of 5 min ischemia and 5 min reperfusion, and/or remifentanil, administered either as a transient infusion mimicking the preconditioning protocol (RP2, 10 μg·kg−1·min−1) or as a continuous infusion (R, 3–10 μg·kg−1·min−1). Rabbits were randomly assigned to experimental groups. Infarct size was assessed with tetrazolium. Results are reported as mean±SD. ResultsNon-preconditioned infarct size was ∼50% of the area-at-risk (49.6±20.1% CON). Both one and two cycles of ischemic preconditioning markedly reduced infarct size (49.6±20.1% CON versus 18.6±8.6% IP and versus 7.5±7.6% IP2; both p<0.001). Preconditioning with remifentanil modestly reduced infarct size (49.6±20.1% CON versus 29.3±8.5% RP2; p<0.01). However, sustained administration of remifentanil did not provide protection (49.6±20.1% CON versus 43.9±16.2% R), and it attenuated the protection offered by preconditioning (49.6±20.1% CON versus 35.6±20.7% R+IP1, p=NS; and versus 14.5±14.5% R+IP2; p<0.05). ConclusionTransient pre-ischemic administration of remifentanil modestly reduces infarct size in propofol-anesthetized rabbits, but continuous administration of remifentanil increases the threshold for ischemic preconditioning-induced infarct limitation.

Dichloroacetate reduces plasma lactate levels but does not reduce infarct size in rabbit myocardium.

Dichloroacetate (DCA), an activator of pyruvate dehydrogenase (PDHC), enhances postischemic mechanical recovery of isolated hearts. It is not known whether this is secondary to reduced infarction or preservation of contractile function in viable cardiomyocytes. This study investigated the effect of DCA on myocardial infarct size. Anesthetized open chest rabbits underwent regional coronary occlusion and reperfusion. DCA (300 mg/kg plus 150 mg/kg 1 h later) was administered intravenously either before occlusion (DCA-O; n = 8) or at reperfusion (DCA-R; n = 7). Control rabbits (n = 8) received saline vehicle. Myocardial PDHC activity was measured after administration of 300 mg/kg i.v. DCA in 10 separate rabbits. DCA reduced plasma lactate levels and increased PDHC activity by 76%, from 2.79 +/- .30 mumol/min.g-1 to 4.92 +/- .44 mumol/min.g-1 (p < .005). However, infarct size in DCA-treated animals was not significantly different from Control (60 +/- 5% DCA-O, 57 +/- 6% DCA-R, 58 +/- 7% Control). We conclude that stimulation of pyruvate dehydrogenase does not limit infarct size.

Manipulations in glycogen metabolism and the failure to influence infarct size in the ischaemic rabbit heart

BACKGROUND AND OBJECTIVE Myocardial ischaemic preconditioning is characterized by a reduction in the rate of glycolysis. Brief myocardial ischaemia also reduces the glycogen content of the heart. The first objective was to determine whether augmenting glucose oxidation by activation of the pyruvate dehydrogenase complex would prevent the infarct limitation of ischaemic preconditioning. The second part of the study evaluates whether glycogen depletion before ischaemia mimics the infarct-limiting effect of ischaemic preconditioning. METHODS Dichloroacetate (300 + 150 mg kg(-1)), an activator of the pyruvate dehydrogenase complex, was administered intravenously in the anaesthetized open-chest rabbit. All animals underwent 45 min of regional ischaemia and 3 h of reperfusion. Ischaemic preconditiong was elicited by 5 min of coronary occlusion. Control rabbits, those with ischaemic preconditioning with no dichloroacetate, received a saline vehicle. An isolated perfused rabbit heart model was employed to test the second hypothesis. Hearts were depleted of glycogen by perfusing them with a substrate-free buffer. Infarction was assessed by triphenyl tetrazolium chloride and area at risk determined with fluorescent particles. RESULTS (a) Pyruvate dehydrogenase complex activation experiments. Treatment with dichloroacetate alone did not alter infarct size (58 +/- 7% control vs. 60 +/- 5% dichloroacetate). Addition of dichloroacetate did not attenuate the infarct-limiting effect of ischaemic preconditioning as evidenced by a similar reduction in infarct size in the ischaemic preconditioning group (22 +/- 5%) and in the ischaemic preconditioning + dichloroacetate group (27 +/- 7%). (b) Glycogen depletion experiments. Compared with control hearts with a normal glycogen content (4.84 +/- 0.15 mg g(-1) wet weight), glycogen depleted and ischaemic preconditioning hearts had reduced glycogen content before ischaemia (2.15 +/- 0.26, 1.62 +/- 0.17 mg g(-1) wet weight, respectively; P < 0.01). Glycogen depletion did not reduce infarct size: 25.0 +/- 4.5% cf. 27.9 +/- 3.4% in the control group. However, ischaemic preconditioning resulted in a significant reduction of infarct size (11.5 +/- 2.3% vs. 27.9 +/- 3.4% control; P < 0.01). CONCLUSIONS Augmentation of oxidative glycolysis by dichloroacetate in in situ rabbit hearts does not alter the effect of ischaemic preconditioning, and glycogen depletion in the isolated rabbit heart does not influence infarct size after subsequent coronary occlusion.

High Spinal Anesthesia Does Not Alter Experimental Myocardial Infarction Size or Ischemic Preconditioning

OBJECTIVE The role of the central nervous system in the development of myocardial infarction and ventricular fibrillation in virgin and ischemically preconditioned myocardium was investigated. DESIGN Infarct size and ventricular arrhythmias were assessed after regional ischemia-reperfusion. Animals were randomly assigned to four groups: (1) preconditioned, central nervous system intact; (2) nonpreconditioned, nervous system intact; (3) preconditioned, nervous system blocked; and (4) nonpreconditioned, nervous system blocked. Differences in hemodynamics and infarct size were assessed with analysis of variance, and differences in ventricular fibrillation were assessed with the Kruskal-Wallis test. SETTING Experiments were performed in the Anesthesiology Research Laboratory at a medical center. PARTICIPANTS Anesthetized open-chest New Zealand white rabbits were used for these studies. INTERVENTIONS Rabbits underwent 30 minutes of coronary artery occlusion and 3 hours of reperfusion. The central nervous system was blocked with total spinal anesthesia. Ischemic preconditioning was elicited with 5 minutes of coronary artery occlusion and 10 minutes of reperfusion. Infarction was assessed with tetrazolium and expressed as a percentage of the risk zone (mean +/- SEM). MEASUREMENTS AND MAIN RESULTS Preconditioning resulted in infarct size limitation compared with the control (8% +/- 4% v 43% +/- 5%; p < 0.001) and delayed the onset of fibrillation (15.5 minutes v 11 minutes; p = 0.001). Spinal blockade neither altered nonpreconditioned infarct size nor attenuated preconditioning (32% +/- 7% v 8% +/- 3%; p = 0.04), but it was associated with ventricular fibrillation in 24/25 rabbits as compared with 6/14 rabbits without blockade. In blocked animals, preconditioning resulted in a decreased duration of fibrillation (2.5 minutes v 12.5 minutes; p = 0.0004). However, spinal blockade eliminated the preconditioning-induced delay in fibrillation (10 minutes v 12 minutes; p = NS). CONCLUSIONS It is concluded that (1) activation of efferent sympathetic nerves is not necessary for ischemic preconditioning; (2) preconditioning delays the onset of ventricular arrhythmias; and (3) spinal blockade exacerbates ischemia-induced ventricular arrhythmias.