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.

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.

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.

Preconditioning the diabetic human myocardium.

Our objective was to determine whether human diabetic myocardium is amenable to the cardioprotective actions of ischaemic preconditioning. Human right atrial appendages were harvested from diabetic and non-diabetic patients undergoing elective coronary artery bypass graft surgery. The atrial trabeculae were isolated and subjected to 90 min. of hypoxia followed by 120 min. of reoxygenation, following which the percentage recovery of baseline contractile function was determined. The atrial trabeculae were randomized to: (i) controls (groups 1 and 3); (ii) standard hypoxic preconditioning (HPC) protocol consisting of 4 min. of hypoxia/16 min. of reoxygenation before the 90 min. index hypoxic period (groups 2 and 4); (iii) Prolonged HPC protocol consisting of: 7 min. of hypoxia /16 min. of reoxygenation before the index hypoxic period (group 5). In addition, basal levels of Akt phosphorylation were determined in right atrial appendages harvested from non-diabetic patients and diabetic patients to determine whether PI3K-Akt signalling is down-regulated in the diabetic heart. Standard HPC improved baseline contractile function in human atrial trabeculae harvested from non-diabetic patients (52.4 +/- 3.8% with HPC versus 30.0 +/- 3.2% in control: P = 0.001; N = 6/group), but not in atrial trabeculae isolated from diabetic patients (22.6 +/- 3.3% with HPC versus 28.5 +/- 1.9% in control: P > 0.05; N = 6/group). However, the prolonged HPC protocol did improve baseline contractile function in atrial trabeculae harvested from diabetic patients (42.0 +/- 2.4% with HPC versus 28.5 +/- 1.9% in control: P= 0.001; N > or = 6/group). Western blot analysis demonstrated lower levels of phosphorylated Akt in diabetic myocardium compared to non-diabetic myocardium (0.13 +/- 0.03 arbitrary units versus 0.39 +/- 0.11 arbitrary units: P= 0.047; N > or = 4/group). From the data obtained it appears that the threshold for preconditioning the diabetic myocardium is elevated which may be related to the down-regulation of the PI3K-Akt pathway.

The cardioprotective actions of leptin are lost in the Zucker obese (fa/fa) rat.

Protection against myocardial ischemia-reperfusion injury, including that induced by leptin, involves activation of the reperfusion injury salvage kinase pathway and inhibition of the mitochondrial permeability transition pore. In the current study, we explored the mechanisms underlying leptin-induced cardioprotection further with reference to the leptin receptor (OB-R) and obesity. We examined hearts from Wistar and Zucker lean rats that express functional OB-R and Zucker obese (fa/fa) rats with nonfunctional OB-R. In Langendorff experiments, leptin (10 nM) caused significant reductions in infarct size in hearts from Wistar (leptin treated, 32.4% +/- 3.9% vs. control, 53.2% +/- 3.2%, P < 0.01) and Zucker lean animals (leptin treated, 25.2% +/- 3.7% vs. control, 53.9% +/- 11.3%, P < 0.01). By contrast, hearts from (fa/fa) did not exhibit significant decreases in infarct size. Leptin increased p44 and p42 phosphorylation in Wistar rat hearts by 103.9% (P < 0.05) and 157.3% (P < 0.001), respectively, and by 97.0% (P < 0.05) and 158.1% (P < 0.05) in hearts from Zucker lean rats. Akt/serine-473 phosphorylation was increased in Wistar hearts by 96.7% (P < 0.05), whereas Akt/threonine-308 phosphorylation was elevated by 43.9% (P < 0.05) in Zucker lean rat hearts. Leptin did not influence Akt or p44/42 phosphorylation in (fa/fa) animals. On leptin treatment, mitochondrial permeability transition pore opening was delayed by 43% (P < 0.01) and 30.9% (P < 0.01), respectively, in cardiomyocytes from Wistar and Zucker lean rat hearts but not in cardiomyocytes from (fa/fa). This study provides the first evidence that myocardial sensitivity to the tissue preserving actions of leptin is influenced by adiposity and OB-R status.

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.

Temporal changes in myocardial salvage kinases during reperfusion following ischemia: studies involving the cardioprotective adipocytokine apelin.

INTRODUCTION: Activation of the Reperfusion Injury Salvage Kinase (RISK) pathway, which incorporates phosphatidylinositol-3-OH kinase (PI3K)-Akt/protein kinase B (PKB) and p44/42 mitogen-activated protein kinase (MAPK), underlies protection against ischemia-reperfusion (I/R) injury. The temporal nature of the activation of these RISK pathway components during reperfusion is, however, uncertain. We examined Akt and p44/42 phosphorylation in hearts subjected to ischemia and varying periods of reperfusion in the absence or presence of the putative cardioprotectant, apelin-13. Akt activity was also measured. MATERIALS AND METHODS: Langendorff perfused C57Bl/6J mouse hearts were subjected to 35 min global ischemia followed by 0, 2.5, 5 or 10 min reperfusion with or without 1 microM apelin-13. Basal and apelin-induced phosphorylation of Akt (at both the threonine 308 and serine 473 phosphorylation sites) and p44/42 during the reperfusion phase was determined by Western blotting and Akt activity measured using an Enzyme-Linked ImmunoSorbent Assay (ELISA). RESULTS: Basal phosphorylation of both Akt and p44/42 increased progressively with time of reperfusion. Apelin enhanced Akt and p44/42 phosphorylation at all reperfusion time points. Akt activity did not change under basal conditions but was increased by apelin at 5 min (NS) and 10 min (p<0.05) reperfusion. DISCUSSION: We conclude that under basal conditions Akt and p44/42 phosphorylation increases with time of reperfusion but that this is not accompanied by increased kinase (Akt) activity. On application of a cardioprotectant, however, kinase phosphorylation and activity are enhanced suggesting that it is the combination of these two mechanisms that may underly the tissue preserving actions of such agents.

Apelin-13 and apelin-36 exhibit direct cardioprotective activity against ischemia-reperfusion injury.

Protection against myocardial ischemia-reperfusion (I/R) injury involves activation of phosphatidylinositol-3-OH kinase (PI3K)- Akt/protein kinase B and p44/42 mitogen-activated protein kinase (MAPK), components of the reperfusion injury salvage kinase (RISK) pathway. The adipocytokine, apelin, activates PI3K-Akt and p44/42 in various tissues and we, therefore, hypothesised that it might demonstrate cardioprotective activity. Employing both in vivo (open-chest) and in vitro (Langendorff and cardiomyocytes) rodent (mouse and rat) models ofmyocardial I/R injury we investigated if apelin administered at reperfusion at concentrations akin to pharmacological doses possesses cardioprotective activity. Apelin-13 and the physiologically less potent peptide, apelin-36, decreased infarct size in vitro by 39.6% (p<0.01) and 26.1% (p<0.05) respectively. In vivo apelin-13 and apelin-36 reduced infarct size by 43.1% (p<0.01) and 32.7% (p<0.05). LY294002 and UO126, inhibitors of PI3K-Akt and p44/42 phosphorylation respectively, abolished the protective effects of apelin-13 in vitro.Western blot analysis provided further evidence for the involvement of PI3K-Akt and p44/42 in the cardioprotective actions of apelin. In addition,mitochondrial permeability transition pore (MPTP) opening was delayed by both apelin- 13 (127%, p<0.01) and apelin-36 (93%, p<0.01) which, in the case of apelin-13, was inhibited by LY294002 and mitogen-activated protein kinase kinase (MEK) inhibitor 1. This is the first study to yield evidence that the adipocytokine, apelin, produces direct cardioprotective actions involving the RISK pathway and the MPTP.

Ischemic preconditioning targets the reperfusion phase.

Emerging studies suggest that signaling during the myocardial reperfusion phase contributes to ischemic preconditioning (IPC). Whether the activation of PKC, the opening of the mKATP channel, redox signaling and transient acidosis specifically at the time of myocardial reperfusion are required to mediate IPC-induced protection is not known. Langendorff-perfused rat hearts were subjected to 35 min ischemia followed by 120 min reperfusion at the end of which infarct size was determined by tetrazolium staining. Control and IPC-treated hearts were randomized to receive for the first 15 min of reperfusion: (1) DMSO (0.02%) vehicle control; (2) chelerythrine (10 micromol/l), a PKC antagonist; (3) 5 hydroxydecanoate (5- HD,100 micromol/l), a mKATP channel blocker; (4) N-mercaptopropionylglycine (MPG,1 mmol/l), a reactive oxygen species scavenger; (5) NaHCO3 (pH 7.6), to counteract any acidosis. Interestingly, all four agents given at the time of myocardial reperfusion abolished the infarct reduction elicited by IPC (N>6/group): (1) DMSO at reperfusion: 49.3+/-3.6% in control versus 21.0+/-3.6% with IPC:P<0.05; (2) chelerythrine at reperfusion: 57.1+/-2.5% in control versus 60.1+/-3.3% with IPC:P=NS; (3) 5-HD at reperfusion: 53.4+/-6.5 % in control versus 42.6+/-4.4% with IPC:P=NS; (4) MPG at reperfusion: 55.3+/-4.6% in control versus 43.9+/-5.2% with IPC:P=NS; (5) NaHCO3 at reperfusion 53.4+/-2.5% in control versus 59.0+/-3.3% with IPC:P=NS. In conclusion, we report for the first time that PKC activation, mKATP channel opening, redox signaling and a low pH at the time of myocardial reperfusion are required to mediate the cardioprotection elicited by ischemic preconditioning.