Dipeptidyl peptidase-4 inhibitors and GLP-1 reduce myocardial infarct size in a glucose-dependent manner.

BACKGROUND: The dipeptidyl peptidase-4 (DPP-4) inhibitors Sitagliptin and Vildagliptin lower blood glucose by augmenting endogenous levels of glucagon-like peptide-1 (GLP-1), an incretin which also confers cardioprotection. As such, we hypothesized that treatment with DPP-4 inhibitors are also cardioprotective. METHODS: In ex vivo experiments: Male Sprague-Dawley rats were randomized to receive by oral gavage either Vildagliptin (20 mg/kg/day), Sitagliptin (100 mg/kg/day), or water for 2 weeks. Excised hearts were Langendorff-perfused with buffer containing either 5 mmol/L or 11 mmol/L glucose and subjected to 35 minutes ischaemia/120 minutes reperfusion. In in vivo experiments: Male young Wistar and Sprague-Dawley rats, middle aged Wistar and Goto-Kakizaki diabetic rats were randomized to receive by oral gavage either Sitagliptin (100 mg/kg/day), or water for 2 weeks. Rats were then subjected to 30 minutes ischaemia/120 minutes reperfusion and infarct size ascertained. RESULTS: Two weeks pre-treatment with either Vildagliptin or Sitagliptin reduced ex vivo myocardial infarction (MI) size in hearts perfused with buffer containing 11 mmol/L glucose but not 5 mmol/L glucose. This effect was abolished by Exendin 9-39 (GLP-1 receptor antagonist) and H-89 (PKA antagonist). Treatment of perfused hearts with native GLP-1 was also glucose-sensitive, reducing MI size, at glucose concentrations 7, 9, and 11 mmol/L but not at 5 mmol/L. Finally, Sitagliptin reduced in vivo MI size in middle aged Wistar (7-8 mmol/L glucose) and Goto-Kakizaki (9-10 mmol/L glucose) rats where blood glucose was elevated, but not in young Wistar (5 mmol/L glucose) or Sprague-Dawley (5 mmol/L glucose) rats, where blood glucose was normal. CONCLUSIONS: We find that chronic treatment with DPP-4 inhibitors reduced MI size, via the GLP-1 receptor-PKA pathway, in a glucose-dependent manner. Glucose-sensitive cardioprotection of endogenous GLP-1 in diabetic patients may in part explain why intensive control of serum glucose levels has been associated with increased cardiovascular risk.

Hypoxic regulation of hand1 controls the fetal-neonatal switch in cardiac metabolism.

Cardiomyocytes are vulnerable to hypoxia in the adult, but adapted to hypoxia in utero. Current understanding of endogenous cardiac oxygen sensing pathways is limited. Myocardial oxygen consumption is determined by regulation of energy metabolism, which shifts from glycolysis to lipid oxidation soon after birth, and is reversed in failing adult hearts, accompanying re-expression of several "fetal" genes whose role in disease phenotypes remains unknown. Here we show that hypoxia-controlled expression of the transcription factor Hand1 determines oxygen consumption by inhibition of lipid metabolism in the fetal and adult cardiomyocyte, leading to downregulation of mitochondrial energy generation. Hand1 is under direct transcriptional control by HIF1α. Transgenic mice prolonging cardiac Hand1 expression die immediately following birth, failing to activate the neonatal lipid metabolising gene expression programme. Deletion of Hand1 in embryonic cardiomyocytes results in premature expression of these genes. Using metabolic flux analysis, we show that Hand1 expression controls cardiomyocyte oxygen consumption by direct transcriptional repression of lipid metabolising genes. This leads, in turn, to increased production of lactate from glucose, decreased lipid oxidation, reduced inner mitochondrial membrane potential, and mitochondrial ATP generation. We found that this pathway is active in adult cardiomyocytes. Up-regulation of Hand1 is protective in a mouse model of myocardial ischaemia. We propose that Hand1 is part of a novel regulatory pathway linking cardiac oxygen levels with oxygen consumption. Understanding hypoxia adaptation in the fetal heart may allow development of strategies to protect cardiomyocytes vulnerable to ischaemia, for example during cardiac ischaemia or surgery.

Cardioprotection in the aging, diabetic heart: the loss of protective Akt signalling.

AIMS: Old age and diabetes are risk factors that often coexist increasing the vulnerability of the heart to the lethal effects of ischaemia-reperfusion injury (IRI). However, to our knowledge, no investigations have examined IRI and cardioprotective signalling in animal models bearing these co-morbidities concomitantly. The ability of the heart to recover following IRI is greatly dependent on its innate cardioprotective potential, in which a central role is played by Akt. We aimed to investigate in an aging diabetic rat model, the susceptibility of the heart to IRI, the achievability of ischaemic preconditioning (IPC) against this lethal event, and the changes in Akt signalling, as the main prosurvival intracellular pathway. METHODS AND RESULTS: Our data showed that the isolated hearts of aged, diabetic Goto-Kakizaki rats were more susceptible to sub-lethal injury and not amenable to cardioprotection via IPC, compared with younger diabetic rat hearts. Western blot analysis of the heart tissue suggested a chronic up-regulation of Akt phosphorylation, and reduced expression of the mitochondrial regulator PGC-1α and of the anti-oxidant enzyme catalase, potentially due to the Akt up-regulation. Moreover, no further activation of Akt could be achieved following IPC. CONCLUSION: An increased susceptibility to IRI in the aged, diabetic heart could be a consequence of impaired Akt signalling due to chronic Akt phosphorylation. Additional Akt phosphorylation required for IPC protection may therefore not be possible in the aged, diabetic rat heart and may explain why this cardioprotective manoeuvre cannot be achieved in these hearts.

Glimepiride treatment facilitates ischemic preconditioning in the diabetic heart.

AIMS: The diabetic heart is resistant to the myocardial infarct-limiting effects of ischemic preconditioning (IPC). This may be in part due to the downregulation of the phosphatidylinositol 3'-kinase-Akt pathway, an essential component of IPC protection. We hypothesized that treating the diabetic heart with the sulfonylurea, glimepiride, which has been reported to activate Akt, may lower the threshold required to protect the diabetic heart by IPC. METHODS: Goto-Kakizaki rats (a type II lean model of diabetes) received glimepiride (20 mg/kg per d, by oral gavage) or vehicle for (a) 3 months (chronic treatment) or (b) 24 hours (subacute treatment). In the third group, glimepiride (10 µmol/L) was administered only to the isolated hearts on the Langendorff apparatus (acute treatment). All hearts were subjected to 35 minutes ischemia and 120 minutes reperfusion ex vivo, at the end of which infarct size was determined by tetrazolium staining. Preconditioning treatment comprised 1 (IPC-1) or 3 (IPC-3) cycles of 5 minutes global ischemia and 10 minutes reperfusion. RESULTS: The diabetic heart was found to be resistant to IPC such that 3-IPC cycles, instead of the usual 1-IPC cycle, were required for cardioprotection. However, pretreatment with glimepiride lowered the threshold for IPC such that both 1 and 3 cycles of IPC elicited cardioprotection: chronic glimepiride treatment (IPC-1 31.9% ± 3.8% and IPC-3 33.5% ± 2.4% vs 43.9% ± 1.4% control, P < .05; N > 6 per group); subacute glimepiride treatment (IPC-1 31.1% ± 3.0% and IPC-3 29.3% ± 3.3% vs 42.2% ± 2.3% control, P < .05 N > 6 per group); and acute glimepiride treatment (IPC-1 28.2% ± 3.7% and IPC-3 24.6% ± 5.4% vs 41.9% ± 5.4% control, P < .05; N > 6 per group). This effect of glimepiride was independent of changes in blood glucose. CONCLUSIONS: We report for the first time that glimepiride treatment facilitates the cardioprotective effect elicited by IPC in the diabetic heart.

The Akt1 isoform is an essential mediator of ischaemic preconditioning.

Phosphatidyl-inositol-3-kinase (PI3K)-Akt pathway is essential for conferring cardioprotection in response to ischaemic preconditioning (IPC) stimulus. However, the role of the individual Akt isoforms expressed in the heart in mediating the protective response to IPC is unknown. In this study, we investigated the specific contribution of Akt1 and Akt2 in cardioprotection against ischaemia-reperfusion (I-R) injury. Mice deficient in Akt1 or Akt2 were subjected to in vivo regional myocardial ischaemia for 30 min. followed by reperfusion for 2 hrs with or without a prior IPC stimulus. Our results show that mice deficient in Akt1 were resistant to protection with either one or three cycles of IPC stimulus (42.7 ± 6.5% control versus 38.5 ± 1.9% 1 χ IPC, N = 6, NS; 41.4 ± 6.3% control versus 32.4 ± 3.2% 3 χ IPC, N = 10, NS). Western blot analysis, performed on heart samples taken from Akt1(-/-) mice subjected to IPC, revealed an impaired phosphorylation of GSK-3ß, a downstream effector of Akt, as well as Erk1/2, the parallel component of the reperfusion injury salvage kinase pathway. Akt2(-/-) mice, which exhibit a diabetic phenotype, however, were amenable to protection with three but not one cycle of IPC (46.4 ± 5.6% control versus 35.9 ± 5.0% in 1 χ IPC, N = 6, NS; 47.0 ± 6.0% control versus 30.8 ± 3.3% in 3 χ IPC, N = 6; *P = 0.039). Akt1 but not Akt2 is essential for mediating a protective response to an IPC stimulus. Impaired activation of GSK-3ß and Erk1/2 might be responsible for the lack of protective response to IPC in Akt1(-/-) mice. The rise in threshold for protection in Akt2(-/-) mice might be due to their diabetic phenotype.

Failure of the adipocytokine, resistin, to protect the heart from ischemia-reperfusion injury.

Experimental studies have linked the adipocytokines with acute cardioprotection. Whether the adipocytokine, resistin, confers protection is, however, debatable. In the current study, the actions of resistin, administered at reperfusion, were investigated in in vivo and in vitro rodent and in vitro human models of myocardial ischemia-reperfusion (I/R) injury. Resistin did not reduce infarct size in Langendorff-perfused rat hearts or murine hearts perfused in vivo. Resistin also did not protect human atrial muscle subjected to hypoxia-reoxygenation. Although cyclosporin A delayed mitochondrial permeability transition pore (MPTP) opening in murine cardiomyocytes, resistin was ineffective. Western blot analysis revealed that resistin treatment was associated with enhanced phosphorylation of Akt, at both the serine-473 (+ 51.9%, P = .01) and threonine-308 (+107%, P < .01) phosphorylation sites, although not to the extent seen with ischemic preconditioning (+132.5%, P = .002 and +389.1%, P < .01, respectively). We conclude that resistin administered at reperfusion at concentrations/doses equivalent to normal (upper end) and pathological serum levels does not protect against I/R injury or inhibit MPTP opening.

Metformin prevents myocardial reperfusion injury by activating the adenosine receptor.

Metformin improves cardiovascular outcomes in patients with type 2 diabetes compared with other glucose-lowering drugs. Experimental studies have shown that metformin can increase the intracellular concentration of adenosine monophosphate, which is a major determinant of the intracellular formation of adenosine. We hypothesize that metformin, given at reperfusion, can limit myocardial infarct size due to increased adenosine receptor stimulation. Isolated perfused hearts from Sprague-Dawley rats were subjected to 35 minutes of regional ischemia and 120 minutes of reperfusion. Perfusion with metformin (50 microM) for the first 15 minutes of reperfusion reduced infarct size (percent area at risk) from 42% +/- 2% to 19% +/- 4% (n >or= 6; P < 0.01), which was blocked by a concomitant perfusion with the adenosine receptor antagonist 8-p-sulfophenyltheophylline (100 microM; 43% +/- 3%) or nitrobenzylthioinosine (a blocker of transmembranous adenosine transport; 1 microM; 45% +/- 5%). In addition, intravenous administration of metformin (5 mg/kg) reduced infarct size in a rat in situ model of myocardial infarction (34% +/- 6% vs. 62% +/- 5%; P < 0.01), which was completely abolished by 8-p-sulfophenyltheophylline (61% +/- 3%). We conclude that metformin, given at reperfusion, reduces infarct size in a rat model of myocardial infarction, which is critically dependent on adenosine receptor stimulation, probably via increased intracellular formation of adenosine.

Ischemia-reperfusion injury and cardioprotection: investigating PTEN, the phosphatase that negatively regulates PI3K, using a congenital model of PTEN haploinsufficiency.

Activation of the PI3K/Akt pathway protects the heart from ischemia-reperfusion injury (IRI). The phosphatase PTEN is the main negative regulator of this pathway. We hypothesized that reduced PTEN levels could protect against IRI. Isolated perfused mouse hearts from PTEN(+/-) and their littermates PTEN(+/+) (WT), were subjected to 35 min global ischemia and 30 min reperfusion, with and without 2, 4 or 6 cycles ischemic preconditioning (IPC). The end point was infarct size, expressed as a percentage of the myocardium at risk (I/R%). PTEN and Akt levels were determined using Western blot analysis. Unexpectedly, there were no significant differences in infarction between PTEN(+/-) and WT (42.1 +/- 5.0% Vs. 45.6 +/- 3.3%). However, the preconditioning threshold was significantly reduced in the PTEN(+/-) Vs. WT, with 4 cycles of IPC being sufficient to reduce I/R%, compared to 6 cycles in the WT (4 cycles IPC: 29.8. +/- 3.69% in PTEN(+/-) Vs. 45.5. +/- 5.08% in WT, P < 0.01). In addition, the ratio between the phospho/total Akt (Ser473 and Thr308) was slightly but significantly increased in the PTEN(+/-) indicating an upregulation of PI3K/Akt pathway. Interestingly, the levels of the other phosphatases that may negatively regulate the PI3K/Akt pathway (PP2A, SHIP2 and PHLPP) were not significantly different between littermates and PTEN(+/-). In conclusion, PTEN haploinsufficiency alone does not induce cardioprotection in this model; however, it reduces the threshold of protection induced by IPC.

Metformin protects the ischemic heart by the Akt-mediated inhibition of mitochondrial permeability transition pore opening.

BACKGROUND: In the majority of studies, metformin has been demonstrated to cardioprotect diabetic patients, the mechanism of which is unclear. We hypothesized that metformin cardioprotects the ischemic heart through the Akt-mediated inhibition of mitochondrial permeability transition pore (mPTP) opening. MATERIALS AND METHODS: Isolated perfused hearts from normoglycemic Wistar or from diabetic Goto-Kakizaki (GK) rats (N > or = 6/group) were subjected to 35 min ischemia and 120 min of reperfusion. Metformin (50 micromol/l) was added for 15 min at reperfusion, alone or with LY294002 (15 micromol/l), a PI3K inhibitor. Infarct size and Akt phosphorylation were measured. Furthermore, the effect of metformin on mPTP opening in adult cardiomyocytes isolated from both strains was determined. RESULTS: Metformin reduced infarct size in both Wistar (35 +/- 2.7% metformin vs. 62 +/- 3.0% control: P < 0.05) and GK hearts (43 +/- 4.7% metformin vs. 60 +/- 3.8% control: P < 0.05). This protection was accompanied by a significant increase in Akt phosphorylation. LY294002 abolished the metformin-induced Akt phosphorylation and the infarct-limiting effect of metformin in Wistar (61 +/- 6.7% metformin + LY294002 vs. 35 +/- 2.7% metformin: P < 0.05) and GK rats (56 +/- 5.7% metformin + LY294002 vs. 43 +/- 4.7% metformin: P < 0.05). In addition, metformin significantly inhibited mPTP opening and subsequent rigor contracture in both Wistar and GK cardiomyocytes subjected to oxidative stress, in a LY-sensitive manner. CONCLUSIONS: We report that metformin given at the time of reperfusion reduces myocardial infarct size in both the non-diabetic and diabetic heart and this protective effect is mediated through PI3K and is associated with Akt phosphorylation. Furthermore, cardioprotection appears to be executed through a PI3K-mediated inhibition of mPTP opening. These findings may explain in part the cardioprotective properties of metformin observed in clinical studies of diabetic patients.

Preconditioning the diabetic heart: the importance of Akt phosphorylation.

Conflicting evidence exists whether diabetic myocardium can be protected by ischemic preconditioning (IPC). The phosphatidylinositol 3-kinase (PI3K)-Akt pathway is important in IPC. However, components of this cascade have been found to be defective in diabetes. We hypothesize that IPC in diabetic hearts depends on intact signaling through the PI3K-Akt pathway to reduce myocardial injury. Isolated perfused Wistar (normal) and Goto-Kakizaki (diabetic) rat hearts were subjected to 1) 35 min of regional ischemia and 120 min of reperfusion with infarct size determined; 2) preconditioning (IPC) using 5 min of global ischemia followed by 10 min of reperfusion performed one, two, or three times before prolonged ischemia; or 3) determination of Akt phosphorylation after stabilization or after one and three cycles of IPC. In Wistar rats, one, two, and three cycles of IPC reduced infarct size 44.7 +/- 3.8% (P < 0.05), 31.4 +/- 4.9% (P < 0.01), and 34.3 +/- 6.1% (P < 0.01), respectively, compared with controls (60.7 +/- 4.5%). However, in diabetic rats only three cycles of IPC significantly reduced infarction to 20.8 +/- 2.6% from 46.6 +/- 5.2% in controls (P < 0.01), commensurate with significant Akt phosphorylation after three cycles of IPC. To protect the diabetic myocardium, it appears necessary to increase the IPC stimulus to achieve the threshold for cardioprotection and a critical level of Akt phosphorylation to mediate myocardial protection.

Failure to protect the myocardium against ischemia/reperfusion injury after chronic atorvastatin treatment is recaptured by acute atorvastatin treatment: a potential role for phosphatase and tensin homolog deleted on chromosome ten?

OBJECTIVES: We sought to ascertain whether chronic oral therapy with atorvastatin protects against ischemia/reperfusion (I/R) injury. BACKGROUND: We have recently shown that acute atorvastatin treatment protects against reperfusion-induced injury by activating the PI3K/Akt/eNOS pathway. However, many patients are on chronic statin therapy, and it is necessary to investigate whether this, in itself, provides a therapeutic advantage. METHODS: Sprague-Dawley rats were orally treated for one day, three days, one week, or two weeks with 20 mg/kg of atorvastatin or vehicle, after which the hearts underwent 35 min of ischemia and 120 min reperfusion (IR). Two additional groups were treated for one or two weeks with atorvastatin and then received a supplementary dose of 40 mg/kg before IR. The risk zone was determined using Evans blue and infarct size (IR%) using triphenyltetrazolium chloride staining. RESULTS: Treatment with atorvastatin for one and three days significantly reduced infarct size versus controls (38.9 +/- 3.1% vs. 56.4 +/- 2.3%; 39.3 +/- 2.4% vs. 61.3 +/- 3.8%, respectively). However, after one or two weeks of treatment, no protection was observed (52.6 +/- 3.8% vs. 58.6 +/- 4.3%; 58.3 +/- 2.7% vs. 52.4 +/- 5.7%, respectively). Surprisingly, a supplementary dose of atorvastatin recaptured the protection in the groups treated chronically (36.2 +/- 2.8% vs. 58.6 +/- 4.3%; 26.8 +/- 1.5% vs. 51.2 +/- 6.7%, at one and two weeks, respectively). Interestingly, we observed an increased level of phosphatase and tensin homolog deleted on chromosome ten (PTEN), the phosphatidylinositol-3 kinase inhibitor, in the chronic treated hearts. CONCLUSIONS: In conclusion, atorvastatin appears to have an acute protective effect that wanes with time associated with an increase in PTEN levels. This waning protection can be recaptured by an acute high dose given immediately before IR. These results may have protential clinical relevance.

Atorvastatin and myocardial reperfusion injury: new pleiotropic effect implicating multiple prosurvival signaling.

We investigated the potential role of atorvastatin, given at reperfusion, to improve survival of the ischemic/reperfused myocardium by activation of p44/42 MAPK and p38 MAPK with its downstream effector, HSP27. We have previously shown that atorvastatin attenuates lethal reperfusion-induced injury via activation of the phosphatidyl inositol 3-kinase (PI3K) prosurvival signaling pathway. In this study we hypothesize that other prosurvival kinases may also be implicated in this protection. Langendorff-perfused mouse hearts were subjected to 35 minutes of global ischemia followed by 30 minutes of reperfusion, and either infarct size or the levels of phosphorylated AKT, p44/42 MAPK, p38 MAPK, and HSP27 were analyzed. Atorvastatin was administered during reperfusion only. We used wortmannin to block PI3K/AKT, U0126 to block p44/42 MAPK, and SB203580 to prevent the phosphorylation of p38 MAPK and HSP27. Atorvastatin significantly reduced infarct size (32.96 +/- 3.4% versus 51.27 +/- 2.79% in controls, P < 0.05). This protection was abrogated by wortmannin (48.38 +/- 4.28%), U0126 (52.58 +/- 7.58), and SB203580 (49.37 +/- 4.16%). Western blot analysis confirmed significant phosphorylation of AKT, p44/42 MAPK, p38 MAPK, and HSP27 following administration of atorvastatin during reperfusion and abrogation of the respective phosphorylation in the presence of their specific inhibitors. Atorvastatin given at reperfusion attenuates lethal reperfusion-induced injury by the phosphorylation of multiple prosurvival pathways involving not only PI3K/AKT but also p44/42 MAPK, p38 MAPK, and HSP27.

Ischemic preconditioning protects by activating prosurvival kinases at reperfusion.

Pharmacological activation of the prosurvival kinases Akt and ERK-1/2 at reperfusion, after a period of lethal ischemia, protects the heart against ischemia-reperfusion injury. We hypothesized that ischemic preconditioning (IPC) protects the heart by phosphorylating the prosurvival kinases Akt and ERK-1/2 at reperfusion. In isolated perfused Sprague-Dawley rat hearts subjected to 35 min of lethal ischemia, the phosphorylation states of Akt, ERK-1/2, and p70 S6 kinase (p70S6K) were determined after 15 min of reperfusion, and infarct size was measured after 120 min of reperfusion. IPC induced a biphasic response in Akt and ERK-1/2 phosphorylation during the preconditioning and reperfusion phases after the period of lethal ischemia. IPC induced a fourfold increase in Akt, ERK-1/2, and p70S6K phosphorylation at reperfusion and reduced the infarct risk-to-volume ratio (56.9 +/- 5.7 and 20.9 +/- 3.6% for control and IPC, respectively, P < 0.01). Inhibiting the IPC-induced phosphorylation of Akt, ERK-1/2, and p70S6K at reperfusion with the phosphatidylinositol 3-kinase (PI3K) inhibitor LY-294002 or the MEK-1/2 inhibitor PD-98059 abrogated IPC-induced protection (46.3 +/- 5.8, 49.2 +/- 4.0, and 20.9 +/- 3.6% for IPC + LY-294002, IPC + PD-98059, and IPC, respectively, P < 0.01), demonstrating that the phosphorylation of these kinases at reperfusion is required for IPC-induced protection. In conclusion, we demonstrate that the reperfusion phase following sustained ischemia plays an essential role in mediating IPC-induced protection. Specifically, we demonstrate that IPC protects the heart by phosphorylating the prosurvival kinases Akt and ERK-1/2 at reperfusion.

Postconditioning: a form of "modified reperfusion" protects the myocardium by activating the phosphatidylinositol 3-kinase-Akt pathway.

Brief intermittent episodes of ischemia and reperfusion, at the onset of reperfusion after a prolonged period of ischemia, confer cardioprotection, a phenomenon termed "ischemic postconditioning" (Postcond). We hypothesized that this phenomenon may just represent a modified form of reperfusion that activates the reperfusion injury salvage kinase (RISK) pathway. Isolated perfused rat hearts were subjected to: (a) 35 minutes of ischemia and 120 minutes of reperfusion, and infarct size was determined by tetrazolium staining; or (b) 35 minutes of ischemia and 7 minutes of reperfusion, and the phosphorylation states of Akt, endothelial NO synthase (eNOS), and p70S6K were determined. Postcond reduced infarct size from 51.2+/-3.4% to 31.5+/-4.1% (P<0.01), an effect comparable with ischemic preconditioning (IPC; 27.5+/-2.3%; P<0.01). Of interest, the combined protective effects of IPC and Postcond were not additive (30.1+/-4.8% with IPC+Postcond; P=NS). Inhibiting phosphatidylinositol 3-kinase (PI3K) at reperfusion using LY or Wortmannin (Wort) during the first 15 minutes of reperfusion completely abolished Postcond-induced protection (31.5+/-4.1% with Postcond versus 51.7+/-4.5% with Postcond+LY, P<0.01; 56.2+/-10.1% with Postcond+ Wort; P<0.01), suggesting that Postcond protects the heart by activating PI3K-Akt. Western blot analysis demonstrated that Postcond induced a significant increase in phosphorylation of Akt, eNOS, and p70S6K in an LY- and Wort-sensitive manner. In conclusion, we show for the first time that ischemic Postcond protects the myocardium by activating the prosurvival kinases PI3K-Akt, eNOS, and p70S6K in accordance with the RISK pathway.

p53 down-regulation: a new molecular mechanism involved in ischaemic preconditioning.

Ischaemic preconditioning is associated with the activation of prosurvival mechanisms. Here we demonstrate that following a preconditioning protocol, the proapoptotic p53 is inactivated possibly via phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt)-murine double minute 2 (Mdm2) phosphorylation. Our data show that in preconditioned hearts Mdm2 was significantly phosphorylated, and wortmannin (a PI3K inhibitor) abrogated this effect (Western blotting). Also in preconditioned hearts p53 was shown to be bound to phospho-Mdm2 (co-immunoprecipitation). Furthermore, pifithrin alpha (a p53 inhibitor), administered to isolated perfused hearts prior to ischaemia, significantly attenuated the infarction. In conclusion our results suggest that p53 is implicated in ischaemia/reperfusion injury and that preconditioning counterbalances this effect via PI3K-Akt-Mdm2 phosphorylation.

Adenosine A(3) receptor activation protects the myocardium from reperfusion/reoxygenation injury.

Ischemia-reperfusion induces both necrotic and apoptotic cell death. The ability of adenosine to attenuate reperfusion-induced injury (RI) and the role played by adenosine receptors are unclear. We therefore studied the role of the A(3) receptor (A(3)R) in ameliorating RI using the specific A(3)R agonist 1-[2-chloro-6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-1-deoxi-N-methyl-b-D-ribofuranuronamide (2-Cl-IB-MECA). Isolated rat hearts and cardiomyocytes were subjected to ischemia or simulated ischemia, followed by reperfusion/reoxygenation. The end points were percent infarction/risk zone and annexin-V (apoptosis) and/or propidium iodide positivity (necrosis), respectively. In isolated hearts, 2-Cl-IB-MECA significantly limited infarct size (44.2 +/- 2.7% in control vs. 21.9 +/- 2.4% at 1 nM and 35.8 +/- 3.3% at 0.1 nM, P < 0.05). In isolated myocytes, apoptosis and necrosis were significantly reduced compared with controls (5.7 +/- 2.6% vs. 17.1 +/- 1.3% and 13.7 +/- 2.0% vs. 23.1 +/- 1.5%, respectively, P < 0.0001). In both models, the beneficial effects were abrogated using the A(3)R antagonist MRS-1191. The involvement of A(2a) receptor activation was also examined. This is the first study to demonstrate that A(3)R activation at reperfusion limits myocardial injury in the isolated rat heart and improves survival in isolated myocytes, possibly by antiapoptotic and antinecrotic mechanisms.