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.

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.

Chronic metformin associated cardioprotection against infarction: not just a glucose lowering phenomenon.

PURPOSE: Clinical and experimental investigations demonstrated that metformin, a widely used anti-diabetic drug, exhibits cardioprotective properties against myocardial infarction. Interestingly, metformin was previously shown to increase the expression of PGC-1α a key controller of energy metabolism in skeletal muscle, which is down-regulated in diabetic conditions. We hypothesized that chronic treatment with metformin could protect the aged, diabetic heart against ischemia-reperfusion injury (IRI) by up-regulating PGC-1α and improving the impaired functionality of diabetic mitochondria. METHODS: Following 4 weeks of metformin (300 mg/kg) administered in the drinking water, 12 month-old diabetic Goto Kakizaki and non-diabetic Wistar rat hearts were assigned for infarct measurement following 35 min ischemia and 60 min reperfusion or for electron microscopy (EM) and Western blotting (WB) investigations. RESULTS: Metformin elicited a cardioprotective effect in both non-diabetic and diabetic hearts. In contrast with the diabetic non-treated hearts, the diabetic hearts treated with metformin showed more organized and elongated mitochondria and demonstrated a significant increase in phosphorylated AMPK and in PGC-1α expression. CONCLUSIONS: In summary these results show for the first time that chronic metformin treatment augments myocardial resistance to ischemia-reperfusion injury, by an alternative mechanism in addition to the lowering of blood glucose. This consisted of a positive effect on mitochondrial structure possibly via a pathway involving AMPK activation and PGC-1α. Thus, metformin prescribed chronically to patients may lead to a basal state of cardioprotection thereby potentially limiting the occurrence of myocardial damage by cardiovascular events.

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.

Retrograde heart perfusion: the Langendorff technique of isolated heart perfusion.

In the late 19th century, a number of investigators were working on perfecting isolated heart model, but it was Oscar Langendorff who, in 1895, pioneered the isolated perfused mammalian heart. Since that time, the Langendorff preparation has evolved and provided a wealth of data underpinning our understanding of the fundamental physiology of the heart: its contractile function, coronary blood flow regulation and cardiac metabolism. In more recent times, the procedure has been used to probe pathophysiology of ischaemia/reperfusion and disease states, and with the dawn of molecular biology and genetic manipulation, the Langendorff perfused heart has remained a stalwart tool in the study of the impact upon the physiology of the heart by pharmacological inhibitors and targeted deletion or up-regulation of genes and their impact upon intracellular signalling and adaption to clinically relevant stressful stimuli. We present here the basic structure of the Langendorff system and the fundamental experimental rules which warrant a viable heart preparation. In addition, we discuss the use of the isolated retrograde perfused heart in the model of ischaemia-reperfusion injury ex-vivo, and its applicability to other areas of study. The Langendorff perfusion apparatus is highly adaptable and this is reflected not only in the procedure's longevity but also in the number of different applications to which it has been turned.

Enhancing AMPK activation during ischemia protects the diabetic heart against reperfusion injury.

AMPK activation during ischemia helps the myocardium to cope with the deficit of energy production. As AMPK activity is considered to be impaired in diabetes, we hypothesized that enhancing AMPK activation during ischemia above physiological levels would protect the ischemic diabetic heart through AMPK activation and subsequent inhibition of mitochondrial permeability transition pore (mPTP) opening. Isolated perfused hearts from normoglycemic Wistar or diabetic Goto-Kakizaki (GK) rats (n ≥ 6/group) were subjected to 35 min of ischemia in the presence of 10, 20, and 40 µM of A-769662, a known activator of AMPK, followed by 120 min of reperfusion with normal buffer. Myocardial infarction and AMPK phosphorylation were assessed. The effect of A-769662 on mPTP opening in adult cardiomyocytes isolated from both strains was also determined. A-769662 at 20 µM reduced infarct size in both Wistar (30.5 ± 2.7 vs. 51.8 ± 3.9% vehicle; P < 0.001) and GK hearts (22.7 ± 3.0 vs. 48.5 ± 4.7% vehicle; P < 0.001). This protection was accompanied by a significant increase in AMPK and GSK-3ß phosphorylation. In addition, A-769662 significantly inhibited mPTP opening in both Wistar and GK cardiomyocytes subjected to oxidative stress. We demonstrate that AMPK activation during ischemia via A-769662 reduces myocardial infarct size in both the nondiabetic and diabetic rat heart. Furthermore, this cardioprotective effect appears to be mediated through inhibition of mPTP opening. Our findings suggest that improving AMPK activation during ischemia can be another mechanism for protecting the ischemic heart.

Translating novel strategies for cardioprotection: the Hatter Workshop Recommendations.

Ischemic heart disease (IHD) is the leading cause of death worldwide. Novel cardioprotective strategies are therefore required to improve clinical outcomes in patients with IHD. Although a large number of novel cardioprotective strategies have been discovered in the research laboratory, their translation to the clinical setting has been largely disappointing. The reason for this failure can be attributed to a number of factors including the inadequacy of the animal ischemia-reperfusion injury models used in the preclinical cardioprotection studies and the inappropriate design and execution of the clinical cardioprotection studies. This important issue was the main topic of discussion of the UCL-Hatter Cardiovascular Institute 6th International Cardioprotection Workshop, the outcome of which has been published in this article as the "Hatter Workshop Recommendations". These have been proposed to provide guidance on the design and execution of both preclinical and clinical cardioprotection studies in order to facilitate the translation of future novel cardioprotective strategies for patient benefit.

Transitory activation of AMPK at reperfusion protects the ischaemic-reperfused rat myocardium against infarction.

PURPOSE: AMPK plays a crucial role in the regulation of the energy metabolism of the heart. During ischaemia, AMPK activation is a known adaptative prosurvival mechanism that helps to maintain the energy levels of the myocardium. However, it still remains unclear if activation of AMPK during reperfusion is beneficial for the heart. Two known AMPK activators (metformin and AICAR) were used to verify the hypothesis that a transitory activation of AMPK at reperfusion may exert cardioprotection, as reflected in a reduction in myocardial infarct size. METHODS: Perfused rat hearts were subjected to 35 min ischaemia and 120 min reperfusion. Metformin (50 microM) or AICAR (0.5 mM) were added for 15 min at the onset of reperfusion alone or with Compound C (CC, 10 microM), an AMPK inhibitor. Infarct size and alpha-AMPK phosphorylation were measured. RESULTS: Metformin significantly reduced infarct size from 47.8 +/- 1.7% in control to 31.4 +/- 2.9%, an effect abolished by CC when the drugs were given concomitantly. Similarly, AICAR also induced a significant reduction in infarct size to 32.3 +/- 4.8%, an effect also abrogated by CC. However, metformin's protection was not abolished if CC was administered later in reperfusion. In addition, alpha-AMPK phosphorylation was significantly increased in the metformin treated group during the initial 30 min of reperfusion. CONCLUSIONS: Our data demonstrated that, in our ex vivo model of myocardial ischaemia-reperfusion injury, AMPK activation in early reperfusion is associated with a reduction in infarct size.

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.

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.

Glucagon-like peptide 1 can directly protect the heart against ischemia/reperfusion injury.

Glucagon-like peptide 1 (GLP-1), a gut incretin hormone that stimulates insulin secretion, also activates antiapoptotic signaling pathways such as phosphoinositide 3-kinase and mitogen-activated protein kinase in pancreatic and insulinoma cells. Since these kinases have been shown to protect against myocardial injury, we hypothesized that GLP-1 could directly protect the heart against such injury via these prosurvival signaling pathways. Both isolated perfused rat heart and whole animal models of ischemia/reperfusion were used, with infarct size measured as the end point of injury. In both studies, GLP-1 added before ischemia demonstrated a significant reduction in infarction compared with the valine pyrrolidide (an inhibitor of its breakdown) or saline groups. This protection was abolished in the in vitro hearts by the GLP-1 receptor antagonist exendin (9-39), the cAMP inhibitor Rp-cAMP, the PI3kinase inhibitor LY294002, and the p42/44 mitogen-activated protein kinase inhibitor UO126. Western blot analysis demonstrated the phosphorylation of the proapoptotic peptide BAD in the GLP-1-treated groups. We show for the first time that GLP-1 protects against myocardial infarction in the isolated and intact rat heart. This protection appears to involve activating multiple prosurvival kinases. This finding may represent a new therapeutic potential for this class of drug currently undergoing clinical trials in the treatment of type 2 diabetes.

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.

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.

Cross-talk between the survival kinases during early reperfusion: its contribution to ischemic preconditioning.

OBJECTIVES: Recruitment of the survival kinase cascades, PI3K-Akt and Raf-MEK1/2-Erk1/2, at the time of reperfusion, following a lethal ischemic insult, may mediate the protection associated with ischemic preconditioning (IPC). The exact interplay between these two kinase cascades in mediating this effect is not clear. We examine the 'cross-talk' between these kinase cascades in their contribution to IPC-induced protection. METHODS AND RESULTS: In isolated perfused rat hearts subjected to 35 min of lethal ischemia +/- ischemic preconditioning, the phosphorylation states of Akt, Erk1/2, p70S6K were determined after 15 min of reperfusion, and infarct size was measured after 120 min of reperfusion. IPC induced a threefold increase in Akt, Erk1/2, and p70S6K phosphorylation, at reperfusion. We found that inhibiting the PI3K-Akt (using LY294008) at reperfusion induced the phosphorylation of Erk1/2-p70S6K, and conversely, that inhibiting the MEK1/2-Erk1/2 pathway (using PD 98059) at reperfusion, induced the phosphorylation of Akt, suggesting 'cross-talk' between the two kinase pathways. However, this effect was not accompanied by a reduction in infarct size (43.1 +/- 7.2% with LY 294008 and 57.7 +/- 7.0% with PD 98059 vs. 46.3 +/- 5.8% in control; P = NS), suggesting that both the kinase cascades may need to be activated to mediate IPC-induced protection. IPC reduced the infarct-risk volume ratio to 17.8 +/- 2.3% from 46.3 +/- 5.8% in control (P < 0.01). Inhibiting p70S6K, a kinase situated downstream of both PI3K and Erk1/2, using rapamycin, abolished IPC-induced protection (46.0 +/- 7.7% with IPC+RAPA vs. 17.8 +/- 2.3% with IPC; P < 0.01). CONCLUSIONS: We report that, the survival kinase cascades PI3K-Akt and MEK1/2-Erk1/2, which are recruited at the time of reperfusion in response to ischemic preconditioning, exhibit 'cross-talk' such that inhibiting one cascade activates the other and vice versa. Furthermore, at the time of reperfusion, these kinase cascades mediate IPC-induced protection, by acting in concert via p70S6K.

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.

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.

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.