Abigail Wynne

Transient Mitochondrial Permeability Transition Pore Opening Mediates Preconditioning-Induced Protection

Background—Transient (low-conductance) opening of the mitochondrial permeability transition pore (mPTP) may limit mitochondrial calcium load and mediate mitochondrial reactive oxygen species (ROS) signaling. We hypothesize that transient mPTP opening and ROS mediate the protection associated with myocardial preconditioning and mitochondrial uncoupling. Methods and Results—Isolated perfused rat hearts were subjected to 35 minutes of ischemia/120 minutes of reperfusion, and the infarct-risk-volume ratio was determined by tetrazolium staining. Inhibiting mPTP opening during the preconditioning phase with cyclosporine-A (CsA, 0.2 μmol/L) or sanglifehrin-A (SfA, 1.0 μmol/L) abolished the protection associated with ischemic preconditioning (IPC) (20.2±3.6% versus 45.9±2.5% with CsA, 49.0±7.1% with SfA; P <0.001); and pharmacological preconditioning with diazoxide (Dzx, 30 μmol/L) (22.1±2.7% versus 46.3±3.0% with CsA, 48.4±5.5% with SfA; P <0.001), CCPA (the adenosine A1-receptor agonist, 200 nmol/L) (24.9±4.5% versus 54.4±6.6% with CsA, 42.6±9.0% with SfA; P <0.001), or 2,4-dinitrophenol (DNP, the mitochondrial uncoupler, 50 μmol/L) (15.7±2.7% versus 40.8±5.5% with CsA, 34.3±3.1% with SfA; P <0.001), suggesting that mPTP opening during the preconditioning phase is required to mediate protection in these settings. Inhibiting ROS during the preconditioning protocols with N-mercaptopropionylglycine (MPG, 1 mmol/L) also abolished the protection associated with IPC (20.2±3.6% versus 47.1±3.8% with MPG; P <0.001), diazoxide (22.1±2.7% versus 56.3±3.8% with MPG; P <0.001), and DNP (15.7±2.7% versus 50.7±6.6% with MPG; P <0.001) but not CCPA (24.9±4.5% versus 26.5±8.4% with MPG; P =NS). Further experiments in adult rat myocytes demonstrated that diazoxide induced CsA-sensitive, low-conductance transient mPTP opening (represented by a 28±3% reduction in mitochondrial calcein fluorescence compared with control; P <0.01). Conclusions—We report that the protection associated with IPC, diazoxide, and mitochondrial uncoupling requires transient mPTP opening and ROS.

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.

Leptin, the obesity‐associated hormone, exhibits direct cardioprotective effects

Background and purpose: Protection against ischaemia‐reperfusion (I/R) injury involves PI3K‐Akt and p44/42 MAPK activation. Leptin which regulates appetite and energy balance also promotes myocyte proliferation via PI3K‐Akt and p44/42 MAPK activation. We, therefore, hypothesized that leptin may also exhibit cardioprotective activity.

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 μmol/l), a PKC antagonist; (3) 5 hydroxydecanoate (5- HD,100 μmol/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.

Leptin-induced cardioprotection involves JAK/STAT signaling that may be linked to the mitochondrial permeability transition pore

Leptin-induced protection against myocardial ischemia-reperfusion (I/R) injury involves the activation of the reperfusion injury salvage kinase pathway, incorporating phosphatidylinositol 3-kinase-Akt/protein kinase B and p44/42 MAPK, and the inhibition of the mitochondrial permeability transition pore (MPTP). Recently published data indicate that the JAK/STAT signaling pathway, which mediates the metabolic actions of leptin, also plays a pivotal role in cardioprotection. Consequently, in the present study we considered the possibility that JAK/STAT signaling linked to the MPTP may be involved in modulating the cardioprotective actions of leptin. Employing rat in vitro models (Langendorff-perfused hearts and cardiomyocytes) of I/R injury, we investigated the actions of leptin (10 nM), administered at reperfusion, in the presence or absence of the JAK2 inhibitor, AG-490 (5 μM). Leptin reduced infarct size significantly (control, 60.05 ± 7.41% vs. leptin treated, 29.9 ± 3.24%, P < 0.05), protection being abolished by AG-490. Time course studies revealed that leptin caused a 171% (P < 0.001) increase in STAT3/tyrosine-705 phosphorylation at 2.5 min reperfusion; however, increases were not seen at 5, 10, 15, or 30 min reperfusion. Contrasting with STAT3, Akt/serine-473 phosphorylation was not significantly increased until 15 min into the reperfusion phase (140%, P < 0.05). AG-490 blocked the leptin-induced rise in STAT3 phosphorylation seen at 2.5 min reperfusion but did not influence Akt/serine-473 phosphorylation at 15 min. Leptin reduced the MPTP opening (P < 0.001), which was blocked by AG-490. This is the first study to yield evidence that JAK/STAT signaling linked to the MPTP plays a role in leptin-induced cardioprotection. Under the experimental conditions employed, STAT3 phosphorylation appears to have occurred earlier during reperfusion than that of Akt. Further research into the interactions between these two signaling pathways in the setting of I/R injury is, however, required.

Pioglitazone mimics preconditioning in the isolated perfused rat heart : A role for the prosurvival kinases PI3K and P42/44MAPK

Ischemic preconditioning, the most powerful protection against infarction, activates PI3Kinase (PI3K)/AKT and P42/44MAPK. Pioglitazone, a thiazolidinedione and PPARγ receptor agonist used in Type II diabetes treatment, has been shown to activate these kinase cascades. We therefore hypothesized that pioglitazone could protect the myocardium when given prior to myocardial ischemia/reperfusion injury. Langendorff perfused rat hearts underwent 40 minutes of stabilization then 35 minutes of regional ischemia and 120 minutes of reperfusion (control) or Pioglitazone (1, 2, 5, and 10 μM)-given before ischemia. Additional groups underwent the same protocol but with either PI3K inhibitors (15 μM LY294002 or 100 nM wortmannin) or P42/44MAPK inhibitors (10 μM U0126 or 10 μM PD98059) given either during stabilization or at reperfusion. Infarct size was determined as a percentage of risk zone (I/R%). Pioglitazone (2 μM) significantly reduced I/R% compared with control (25.4 ± 3.1 versus 47.3 ± 3.4; P < 0.05). This protection was abolished by PI3K inhibitors (pioglitazone+LY294002 46.5 ± 5.0, pioglitazone + wortmannin 48.8 ± 4.6 versus pioglitazone alone 25.4 ± 3.1; P ≤ 0.05) but not by P42/44MAPK inhibitors (pioglitazone+U0126 30.7 ± 5.7, pioglitazone + PD98059 28.5 ± 6.3 versus pioglitazone alone 25.4 ± 3.1; P ≤ 0.05) given in stabilization. However when the inhibitors were given at reperfusion, the protection was abrogated by blocking either pathway (pioglitazone+LY294002 49.8 ± 3.1, pioglitazone+U0126 48.7 ± 3.7 versus pioglitazone alone 25.4 ± 3.1; P ≤ 0.05). In conclusion pioglitazone induced significant protection against ischemia/reperfusion injury when administered prior to ischemia. This protection appears to involve PI3K and P42/44MAPK.

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 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.

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.