Christopher C. T. Smith

Necrostatin: A Potentially Novel Cardioprotective Agent?

BackgroundNecrostatin-1 (Nec-1), a small tryptophan-based molecule, was recently reported to protect the cerebral cortex against ischemia-reperfusion (I/R) injury. We investigated the actions of Nec-1 and its so-called inactive analog, Nec-1i, in the setting of myocardial I/R injury.Materials and methodsThe actions of Nec-1 and Nec-1i were examined in cultured C2C12 and H9c2 myocytes, cardiomyocytes isolated from male Sprague–Dawley rats, Langendorff isolated perfused C57Bl/6J mouse hearts and an in vivo open-chest C57Bl/6J mouse heart model.ResultsNec-1 at 30xa0μM and 100xa0μM (but not 100xa0μM Nec-1i) reduced peroxide-induced cell death in C2C12 cells from 51.2u2009±u20091.1% (control) to 26.3u2009±u20092.9% (pu2009<u20090.01 vs control) and 17.8u2009±u20090.9% (pu2009<u20090.001), respectively. With H9c2 cells cell death was also reduced from 73.0u2009±u20090.4% (control) to 56.7u2009±u20090% (30xa0μM Nec-1, pu2009<u20090.05) and 45.4u2009±u20093.3% (100xa0μM Nec-1, pu2009<u20090.01). In the isolated perfused heart Nec-1 (30xa0μM) reduced infarct size (calculated as a percentage of the risk area) from 48.0u2009±u20092.0% (control) to 32.1u2009±u20095.4% (pu2009<u20090.05). Nec-1i (30xa0μM) also reduced infarct size (32.9u2009±u20095.1%, pu2009<u20090.05). In anesthetized C57Bl/6J mice Nec-1 (1.65xa0mg/kg), given intraperitoneally to coincide with reperfusion following left anterior descending artery ligation (30xa0min), also reduced infarct size from 45.3u2009±u20095.1% (control) to 26.6u2009±u20094.0% (pu2009<u20090.05), whilst Nec-1i (1.74xa0mg/kg) was ineffective (37.8u2009±u20096.0%). Stimulus-induced opening of the mitochondrial permeability transition pore (MPTP) in rat cardiomyocytes, as reflected by the time until mitochondrial depolarisation, was unaffected by Nec-1 or Nec-1i at 30xa0μM but increased at 100xa0μM i.e. 91% (pu2009<u20090.05 vs control) and 152% (pu2009<u20090.001) for Nec-1 and Nec-1i, respectively.ConclusionThis is the first study to demonstrate that necrostatins inhibit myocardial cell death and reduce infarct size, possibly via a mechanism independent of the MPTP.

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.

The Cardioprotective Effect of Necrostatin Requires the Cyclophilin-D Component of the Mitochondrial Permeability Transition Pore

BackgroundNecrostatin (Nec-1) protects against ischemia–reperfusion (IR) injury in both brain and heart. We have previously reported in this journal that necrostatin can delay opening of the mitochondrial permeability transition pore (MPTP) in isolated cardiomyocytes.AimThe aim of the present study was to investigate in more detail the role played by the MPTP in necrostatin-mediated cardioprotection employing mice lacking a key component of the MPTP, namely cyclophilin-D.MethodAnaesthetized wild type (WT) and cyclophilin-D knockout (Cyp-D−/−) mice underwent an open-chest procedure involving 30xa0min of myocardial ischemia and 2xa0h of reperfusion, with subsequent infarct size assessed by triphenyltetrazolium staining. Nec-1, given at reperfusion, significantly limited infarct size in WT mice (17.7u2009±u20093% vs. 54.3u2009±u20093%, Pu2009<u20090.05) but not in Cyp-D−/− mice (28.3u2009±u20097% vs. 30.8u2009±u20096%, Pu2009>u20090.05).ConclusionThe data obtained in Cyp-D−/− mice provide further evidence that Nec-1 protects against myocardial IR injury by modulating MPTP opening at reperfusion.

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.

The novel adipocytokine visfatin exerts direct cardioprotective effects.

Visfatin is an adipocytokine capable of mimicking the glucose‐lowering effects of insulin and activating the pro‐survival kinases phosphatidylinositol‐3‐OH kinase (PI3K)‐protein kinase B (Akt) and mitogen‐activated protein kinase kinase 1 and 2 (MEK1/2)‐extracellular signal‐regulated kinase 1 and 2 (Erk 1/2). Experimental studies have demonstrated that the activation of these kinases confers cardioprotection through the inhibition of the mitochondrial permeability transition pore (mPTP). Whether visfatin is capable of exerting direct cardioprotective effects through these mechanisms is unknown and is the subject of the current study. Anaesthetized C57BL/6 male mice were subjected to in situ 30 min. of regional myocardial ischaemia and 120 min. of reperfusion. The administration of an intravenous bolus of visfatin (5 × 10−6μmol) at the time of myocardial reperfusion reduced the myocardial infarct size from 46.1 ± 4.1% in control hearts to 27.3 ± 4.0% (n≥ 6/group, P < 0.05), an effect that was blocked by the PI3K inhibitor, wortmannin, and the MEK1/2 inhibitor, U0126 (48.8 ± 5.5% and 45.9 ± 8.4%, respectively, versus 27.3 ± 4.0% with visfatin; n≥ 6/group, P < 0.05). In murine ventricular cardiomyocytes subjected to 30 min. of hypoxia followed by 30 min. of reoxygenation, visfatin (100 ng/ml), administered at the time of reoxygenation, reduced the cell death from 65.2 ± 4.6% in control to 49.2 ± 3.7%(n > 200 cells/group, P < 0.05), an effect that was abrogated by wortmannin and U0126 (68.1 ± 5.2% and 59.7 ± 6.2%, respectively; n > 200 cells/group, P > 0.05). Finally, the treatment of murine ventricular cardiomyocytes with visfatin (100 ng/ml) delayed the opening of the mPTP induced by oxidative stress from 81.2 ± 4 sec. in control to 120 ± 7 sec. (n > 20 cells/group, P < 0.05) in a PI3K‐ and MEK1/2‐dependent manner. We report that the adipocytokine, visfatin, is capable of reducing myocardial injury when administered at the time of myocardial reperfusion in both the in situ murine heart and the isolated murine cardiomyocytes. The mechanism appears to involve the PI3K and MEK1/2 pathways and the mPTP.

Adipocytokines, cardiovascular pathophysiology and myocardial protection.

Reducing myocardial damage resulting from ischaemia-reperfusion (I/R) is vital in ensuring patient recovery and survival. It relies upon the activation of the so-called Reperfusion Injury Salvage Kinase (RISK) pathway. Experimentally various treatments, both mechanical and chemical, have been shown to protect the myocardium against I/R injury. Chemical facilitators of myocardial preservation include endogenous factors such as insulin, erythropoietin and glucagon-like peptide 1. The adipocytokines, products of white adipose tissue, are important peptide hormones with respect to metabolic control and satiety, and were formerly considered in the context of obesity and metabolic disease. More recently, however, evidence has been presented indicating that the adipocytokines play significant roles in cardiac function and, as we have suggested, in myocardial protection. To date leptin, adiponectin, apelin and visfatin have all been shown to protect against I/R injury. Significantly, the protection afforded by these peptides involves the activation of kinases which are key elements of the mechanisms underlying tissue preservation, including the RISK pathway components PI3K-Akt and p44/42, and inhibition of the mitochondrial permeability transition pore (MPTP). In this article we examine the roles played by the adipocytokines in cardiovascular function and disease. In particular, we focus on the evidence that these peptides promote myocardial survival, much of it having been obtained in this laboratory. To conclude, we discuss some future directions in the field, including the prospects for some of the adipocytokines finding application as therapeutic agents in myocardial infarction.

Necroptosis, necrostatins and tissue injury.

•u2002 Introduction ‐u2002 Cell death and pathophysiology ‐u2002 Apoptosis ‐u2002 Autophagy ‐u2002 Necrosis ‐u2002 Necroptosis ‐u2002 Necrostatin and inhibition of cell death ‐u2002 Necroptosis, necrostatin and RIP‐1 kinase ‐u2002 Necrostatin and tissue injury ‐u2002 Necrostatin and cerebrocortical injury ‐u2002 Necrostatin and myocardial injury ‐u2002 Necrostatin and retinal injury •u2002 Concluding remarks

Temporal Changes in Myocardial Salvage Kinases During Reperfusion Following Ischemia: Studies Involving the Cardioprotective Adipocytokine Apelin

IntroductionActivation 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 methodsLangendorff perfused C57Bl/6J mouse hearts were subjected to 35xa0min global ischemia followed by 0, 2.5, 5 or 10xa0min reperfusion with or without 1xa0μM 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).ResultsBasal 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 5xa0min (NS) and 10xa0min (p<0.05) reperfusion.DiscussionWe 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.

The cannabinoid CB1 receptor antagonist, rimonabant, protects against acute myocardial infarction.

CB1 antagonism is associated with reduced doxorubicin-induced cardiotoxicity and decreased cerebrocortical infarction. Rimonabant, a selective CB1 receptor antagonist, was, before it was withdrawn, proposed as a treatment for obesity and reported to reduce cardiovascular risk by improving glucose and lipid profiles and raising adiponectin levels. The cardioprotective actions of rimonabant in 6-week-old C57BL/6J mice fed either high-fat (HFD) or standard diets (STD) for 8xa0weeks were investigated. At 14xa0weeks, mice received rimonabant (10xa0mg/kg/day, i.p.) or vehicle for 1xa0week and were then subjected to an in vivo acute myocardial infarction. The influence of rimonabant on infarct size (IS) in CB1 knockout (CB1−/−) and wild-type (CB1+/+) mice was also examined. C57BL/6J mice that had been maintained on STD or HFD exhibited 4.3 and 21.4% reductions in body weight following 7xa0days rimonabant treatment. Rimonabant reduced IS in both STD (29.6xa0±xa03.5% vs. 49.8xa0±xa06.9% in control, Pxa0<xa00.05) and HFD (26.9xa0±xa01.5% vs. 48.7xa0±xa07% in control, Pxa0<xa00.05) mice. In CB1−/− mice rimonabant failed to reduce body weight or IS (51.0xa0±xa05.3% vs. 49.7xa0±xa04.7% in control, Pxa0>xa00.05), although significant reductions were seen in CB1+/+ mice (IS, 48.9xa0±xa04.6% control vs. 30.5xa0±xa03.1% rimonabant, Pxa0<xa00.05). To exclude the possibility that weight loss alone induced cardioprotection, HFD mice were switched to STD for 7xa0days (HFD–STD), resulting in an 11.3xa0±xa01.0% decrease in body weight compared to control (+2.1xa0±xa01.1% in HFD). This, however, was not associated with IS reduction (39.1xa0±xa03.9% HFD–STD vs. 40.0xa0±xa05.3% HFD, Pxa0>xa00.05). Serum and cardiac adiponectin levels were unaltered by rimonabant treatment. HL-1 cell death was not prevented by 1 or 7xa0days treatment with rimonabant. We conclude that rimonabant-induced infarct limitation may involve the CB1 receptor, although not necessarily cardiac CB1 receptors, and is unrelated to weight loss or altered adiponectin synthesis.

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.