Inhibition of glycogen synthase kinase 3beta prevents peroxide-induced collapse of mitochondrial membrane potential in rat ventricular myocytes.

1. Preconditioning has been proposed to protect the myocardium by inhibiting glycogen-synthase kinase (GSK) 3beta. The aim of the present study was to test whether transfection of ventricular myocytes with inactive GSK3 beta would mimic preconditioning and whether a constitutively active form of GSK3 beta would prevent protection by an opioid receptor agonist. 2. Isolated ventricular myocytes from adult rats were infected with live adenovirus containing either a wild-type (wtGSK), constitutively active (caGSK) or dominant-negative (dnGSK) GSK3 beta plasmid. Cells were loaded with tetramethylrhodamine ethyl ester (TMRE) and exposed to H(2)O(2) (100 micromol/L) for 40 min before mitochondrial membrane potential (Delta Psi(m)) was assessed using flow cytometric analysis. 3. Fluorescence intensity was reduced in H(2)O(2)-treated cells compared with untreated cells, presumably because oxidant injury opened mitochondrial permeability transition pores, causing mitochondria to lose TMRE. The selective GSK3 beta inhibitor SB216763, as well as the delta-opioid receptor agonist [d-Ala(2)-D-Leu(5)]-enkephalin (DADLE) (1 micromol/L), protected cells against peroxide-induced loss of Delta Psi(m). 4. Cells transfected with dnGSK (1 micromol/L) were equally protected against peroxide stress, when given throughout the TMRE and H(2)O(2) treatment, confirming a protective effect of GSK3 beta with a highly selective inhibition. Cells transfected with wtGSK did not show any difference in responses to H(2)O(2), SB216763 or DADLE compared with untransfected cells, suggesting that adenovirus infection itself had no effect. In contrast, caGSK-transfected myocytes could no longer be protected with DADLE, suggesting a role for GSK3 beta between the surface receptor and the mitochondria. 5. These experiments confirm that inhibition of GSK3 beta protects the myocytes, but also that the preconditioning mimetic DADLE loses its protective effect when a constitutively active GSK3 beta is present.

AMP579 is revealed to be a potent A2b-adenosine receptor agonist in human 293 cells and rabbit hearts.

The mixed A1/A2a-adenosine agonist AMP579 given at reperfusion is protective in animal models of myocardial infarction. Receptor-blocking studies have indicated that the protection came from an adenosine receptor (AR), but neither A1- nor A2a-selective agonists could duplicate its protection. We recently found that A2b-selective agonists given at reperfusion are protective, and, therefore, tested whether AMP579 might also be an A2b agonist. We used human embryonic kidney cells overexpressing human A2b receptors as an assay system. In these cells, A2b receptor occupancy causes phosphorylation of ERK. AMP579 induced ERK phosphorylation with an EC50 of 250 nM and this phosphorylation could be blocked by MRS1754 or PSB1115, two highly selective blockers of human A2b receptors. We attempted to confirm our A2b hypothesis in a rabbit heart model of ischemia-reperfusion. AMP579 (500 nM) for 1 h starting at reperfusion reduced infarct size in isolated rabbit hearts exposed to 30 min of regional ischemia and 2 h of reperfusion (12.9 +/- 2.2% infarction of risk zone vs. 32.0 +/- 1.9% in untreated hearts). PSB1115 (500 nM) given for the first 15 min of reperfusion blocked AMP579's protection (32.2 +/- 3.1% infarction) which is consistent with an A2b mechanism. We conclude that AMP579 is a non-selective, but potent A2b-AR agonist, and that its protection against infarction is through that receptor.

The delta-opioid receptor agonist DADLE at reperfusion protects the heart through activation of pro-survival kinases via EGF receptor transactivation.

The specific delta-opioid receptor agonist [D-Ala(2)-D-Leu(5)]enkephalin (DADLE) protects against infarction in the heart when given before ischemia. In rabbit, this protection leads to phosphorylation of the pro-survival kinases Akt and extracellular signal-regulated kinase (ERK) and is dependent on transactivation of the epidermal growth factor receptor (EGFR). DADLE reportedly protects rat hearts at reperfusion. We therefore tested whether DADLE at reperfusion could protect isolated rabbit hearts subjected to 30 min of regional ischemia and 120 min of reperfusion and whether this protection is dependent on Akt, ERK, and EGFR. DADLE (40 nM) was infused for 1 h starting 5 min before reperfusion and reduced infarct size from 31.0 +/- 2.3% in the control group to 14.6 +/- 1.6% (P = 0.01). This protection was abolished by cotreatment of the metalloproteinase inhibitor (MPI) and the EGFR inhibitor AG1478. In contrast, 20 nM DADLE, although known to be protective before ischemia, failed to protect. Western blotting revealed that DADLE's protection was correlated to increase in phosphorylation of the kinases Akt and ERK1 and -2 in reperfused hearts (2.5 +/- 0.5, 1.6 +/- 0.2, and 2.3 +/- 0.7-fold of baseline levels, P < 0.05 vs. control). The DADLE-dependent increases in Akt and ERK1/2 phosphorylation were abolished by either MPI or AG1478, confirming a signaling through the EGFR pathway. Additionally, DADLE treatment increased phosphorylation of EGFR (1.4 +/- 0.2-fold, P = 0.03 vs. control). Thus the delta-opioid agonist DADLE protects rabbit hearts at reperfusion through activation of the pro-survival kinases Akt and ERK and is dependent on the transactivation of the EGFR.

NECA at reperfusion limits infarction and inhibits formation of the mitochondrial permeability transition pore by activating p70S6 kinase.

The A1/A2 adenosine agonist 5'-(N-ethylcarboxamido) adenosine (NECA) limits infarction when administered at reperfusion. The present study investigated whether p70S6 kinase is involved in this anti-infarct effect. Adult rat ventricular myocytes were isolated and incubated in tetramethylrhodamine ethyl ester (TMRE, 100 nM), which causes cells to fluoresce in proportion to their mitochondrial membrane potential. A reduction in TMRE fluorescence serves as an indicator of collapse of the mitochondrial transmembrane potential. Cells were subjected to H2O2 (200 microM), which like ischemia induces loss of mitochondrial membrane potential. Fluorescence was measured every 3 min and to facilitate quantification membrane potential was arbitrarily considered as collapsed when fluorescence reached less than 60% of the starting value. Adding NECA (1 mM) to the cells prolonged the time to fluorescence loss (48.0+/-3.2 min in the NECA group versus 29.5+/-2.2 min in untreated cells, P<0.001) and the mTOR/p70S6 kinase inhibitor rapamycin (5 nM) abolished this protection (31.3+/-3.4 min). Since cyclosporine A offered similar protection, mitochondrial permeability transition pore formation is a likely cause of the H2O2-induced loss of potential. The direct GSK-3beta inhibitor SB216763 (3 microM) also prolonged the time to fluorescence loss (49.2+/-2.1 min, P<0.001 versus control), and its protection could not be blocked by rapamycin (42.2+/-2.3 min, P<0.001 versus control). NECA treatment (100 nM) of intact isolated rabbit hearts at reperfusion after 30 min of regional ischemia decreased infarct size from 33.0+/-3.8% of the risk zone in control hearts to 11.8+/-2.0% (P<0.001), and rapamycin blocked this NECA-induced protection (38.3+/-3.7%). A comparable protective effect was seen for SB216763 (1 microM) with infarct size reduction to 13.5+/-2.3% (P<0.001). NECA treatment (200 nM) of intact rabbit hearts at reperfusion also resulted in phosphorylation of p70S6 kinase more than that seen in untreated hearts. This NECA-induced phosphorylation was blocked by rapamycin. These experiments reveal a critical role for p70S6 kinase in the signaling pathway of NECA's cardioprotection at reperfusion.