Impact of Mitochondrial Ca2+-Sensitive Potassium (mBKCa) Channels in Sildenafil-Induced Cardioprotection in Rats

Background

Mitochondrial large-conductance Ca²⁺-sensitive potassium (mBK_Ca) channels are involved in myocardial ischemic preconditioning. Their role in sildenafil-induced cardioprotection is unknown. We investigated whether sildenafil-induced acute cardioprotection is mediated by activation of mBK_Ca channels in the rat heart in vitro.

Methods

Male Wistar rats (n = 8 per group) were randomized and anesthetized with pentobarbital (90 mg/kg). Hearts were isolated, mounted on a Langendorff system and perfused with Krebs-Henseleit buffer at a constant pressure of 80 mmHg. Hearts underwent 30 min of global ischemia followed by 60 min of reperfusion. At the end of the experiments infarct size was determined by TTC staining. In the control group rats were not further treated. Sildenafil (3 μM) was administered over 10 min before the beginning of ischemia. The mBK_Ca channel inhibitor paxilline (1 μM) was administered with and without sildenafil before the onset of ischemia. The pathway underlying sildenafil-induced cardioprotection was further investigated with the protein kinase G blocker KT5823 (1 μM). Myocardial cGMP concentration was measured by ELISA. Data (mean±SD) were analysed with a one and two-way analysis of variance as appropriate.

Results

In control animals infarct size was 52±8%. Sildenafil increased cGMP concentration and reduced infarct size to 35±6% (P<0.05 vs. control). Paxilline and KT5823 completely blocked sildenafil-induced cardioprotection (paxilline+sildenafil: 50±8%, KT5823+sildenafil: 45±8%; both P<0.05 vs. sildenafil). Functional heart parameters and coronary flow were not different between the study groups.

Conclusion

This study shows that in male rats protein kinase G-dependent opening of mBK_Ca channels plays a pivotal role in sildenafil-induced cardioprotection.     

Essential role of nitric oxide in acute ischemic preconditioning: S-Nitros(yl)ation versus sGC/cGMP/PKG signaling?

Nitric oxide (NO) plays an important role in acute ischemic preconditioning (IPC). In addition to activating soluble guanylyl cyclase (sGC)/cyclic guanosine monophosphate (cGMP)/protein kinase G (PKG) signaling pathways, NO-mediated protein S-nitros(yl)ation (SNO) has been recently shown to play an essential role in cardioprotection against ischemia–reperfusion (I/R) injury. In our previous studies, we have shown that IPC-induced cardioprotection could be blocked by treatment with either N-nitro-L-arginine methyl ester (L-NAME, a constitutive NO synthase inhibitor) or ascorbate (a reducing agent to decompose SNO). To clarify NO-mediated sGC/cGMP/PKG-dependent or -independent (i.e., SNO) signaling involved in IPC-induced cardioprotection, mouse hearts were Langendorff-perfused in the dark to prevent SNO decomposition by light exposure. Treatment with 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, a highly selective inhibitor of sGC) or KT5823 (a potent and selective inhibitor of PKG) did not abolish IPC-induced acute protection, suggesting that the sGC/cGMP/PKG signaling pathway does not play an important role in NO-mediated cardioprotective signaling during acute IPC. In addition, treatment with ODQ in IPC hearts provided an additional protective effect on functional recovery, in parallel with a higher SNO level in these ODQ+IPC hearts. In conclusion, these results suggest that the protective effect of NO is not related primarily to activation of the sGC/cGMP/PKG signaling pathway, but rather through SNO signaling in IPC-induced acute cardioprotection.     

Protein kinase C plays an essential role in sildenafil-induced cardioprotection in rabbits

Sildenafil citrate (Viagra) is the most widely used pharmacological drug for treating erectile dysfunction in men. It has potent cardioprotective effects against ischemia-reperfusion injury via nitric oxide and opening of mitochondrial ATP-sensitive K(+) channels. We further investigated the role of protein kinase C (PKC)-dependent signaling pathway in sildenafil-induced cardioprotection. Rabbits were treated (orally) with sildenafil citrate (1.4 mg/kg) 30 min before index ischemia for 30 min and reperfusion for 3 h. The PKC inhibitor chelerythrine (5 mg/kg i.v.) was given 5 min before sildenafil. Infarct size (% of risk area) reduced from 33.65 +/- 2.17 in the vehicle (saline) group to 15.07 +/- 0.63 in sildenafil-treated groups, a 45% reduction compared with vehicle (mean +/- SE, P < 0.05). Chelerythrine abolished sildenafil-induced protection, as demonstrated by increase in infarct size to 31.14 +/- 2.4 (P < 0.05). Chelerythrine alone had an infarct size of 33.5 +/- 2.5, which was not significantly different compared with DMSO-treated group (36.8 +/- 1.7, P > 0.05). Western blot analysis demonstrated translocation of PKC-alpha, -, and -delta isoforms from cytosol to membrane after treatment with sildenafil. However, no change in the PKC-beta and -epsilon isoforms was observed. These data provide direct evidence of an essential role of PKC, and potentially PKC-alpha, -, and -delta, in sildenafil-induced cardioprotection in the rabbit heart.     

Ouabain protects rat hearts against ischemia-reperfusion injury via pathway involving src kinase, mitoKATP, and ROS

We showed recently that mitochondrial ATP-dependent K(+) channel (mitoK(ATP)) opening is required for the inotropic response to ouabain. Because mitoK(ATP) opening is also required for most forms of cardioprotection, we investigated whether exposure to ouabain was cardioprotective. We also began to map the signaling pathways linking ouabain binding to Na(+)-K(+)-ATPase with the opening of mitoK(ATP). In Langendorff-perfused rat hearts, 10-80 microM ouabain given before the onset of ischemia resulted in cardioprotection against ischemia-reperfusion injury, as documented by an improved recovery of contractile function and a reduction of infarct size. In skinned cardiac fibers, a ouabain-induced protection of mitochondrial outer membrane integrity, adenine nucleotide compartmentation, and energy transfer efficiency was evidenced by a decreased release of cytochrome c and preserved half-saturation constant of respiration for ADP and adenine nucleotide translocase-mitochondrial creatine kinase coupling, respectively. Ouabain-induced positive inotropy was dose dependent over the range studied, whereas ouabain-induced cardioprotection was maximal at the lowest dose tested. Compared with bradykinin (BK)-induced preconditioning, ouabain was equally efficient. However, the two ligands clearly diverge in the intracellular steps leading to mitoK(ATP) opening from their respective receptors. Thus BK-induced cardioprotection was blocked by inhibitors of cGMP-dependent protein kinase (PKG) or guanylyl cyclase (GC), whereas ouabain-induced protection was not blocked by either agent. Interestingly, however, ouabain-induced inotropy appears to require PKG and GC. Thus 5-hydroxydecanoate (a selective mitoK(ATP) inhibitor), N-(2-mercaptopropionyl)glycine (MPG; a reactive oxygen species scavenger), ODQ (a GC inhibitor), PP2 (a src kinase inhibitor), and KT-5823 (a PKG inhibitor) abolished preconditioning by BK and blocked the inotropic response to ouabain. However, only PP2, 5-HD, and MPG blocked ouabain-induced cardioprotection.     

Phosphodiesterase 5 inhibitors prevent 3,4-methylenedioxymethamphetamine-induced 5-HT deficits in the rat

Phosphodiesterase 5 (PDE5) inhibitors are often used in combination with club drugs such as 3,4‐methylenedioxymethamphetamine (MDMA or ecstasy). We investigated the consequences of such combination in the serotonergic system of the rat. Oral administration of sildenafil citrate (1.5 or 8 mg/kg) increased brain cGMP levels and protected in a dose‐dependent manner against 5‐hydroxytryptamine depletions caused by MDMA (3 × 5 mg/kg, i.p., every 2 h) in the striatum, frontal cortex and hippocampus without altering the acute hyperthermic response to MDMA. Intrastriatal administration of the protein kinase G (PKG) inhibitor, KT5823 [(9S, 10R, 12R)‐2,3,9,10,11,12‐Hexahydro‐10‐methoxy‐2,9‐dimethyl‐1‐oxo‐9,12‐epoxy‐1H‐diindolo[1,2,3‐fg:3′,2′,1′‐kl]pyrrolo[3,4‐i][1,6]benzodiazocine‐10‐carboxylic acid, methyl ester)], suppressed sildenafil‐mediated protection. By contrast, the cell permeable cGMP analogue, 8‐bromoguanosine cyclic 3′,5′‐monophosphate, mimicked sildenafil effects further suggesting the involvement of the PKG pathway in mediating sildenafil protection. Because mitochondrial ATP‐sensitive K⁺ channels are a target for PKG, we next administered the specific mitochondrial ATP‐sensitive K⁺ channel blocker, 5‐hydroxydecanoic acid, 30 min before sildenafil. 5‐hydroxydecanoic acid completely reversed the protection afforded by sildenafil, thereby implicating the involvement of mitochondrial ATP‐sensitive K⁺ channels. Sildenafil also increased Akt phosphorylation, and so the possible involvement of the Akt/endothelial nitric oxide synthase (eNOS)/sGC signalling pathway was analysed. Neither the phosphatidylinositol 3‐kinase inhibitor, wortmannin, nor the selective eNOS inhibitor, l ‐N5‐(1‐iminoethyl)‐l ‐ornithine dihydrochloride, reversed the protection afforded by sildenafil, suggesting that Akt/eNOS/sGC cascade does not participate in the protective mechanisms. Our data also show that the protective effect of sildenafil can be extended to vardenafil, another PDE5 inhibitor. In conclusion, sildenafil protects against MDMA‐induced long‐term reduction of indoles by a mechanism involving increased production of cGMP and subsequent activation of PKG and mitochondrial ATP‐sensitive K⁺ channel opening.     

Estradiol-induced protection against ischemia-induced heart mitochondrial damage and caspase activation is mediated by protein kinase G

We have previously reported that estradiol can protect heart mitochondria from the ischemia-induced mitochondrial permeability transition pore-related release of cytochrome c and subsequent apoptosis. In this study we investigated whether the effect of 17-beta-estradiol on ischemia-induced mitochondrial dysfunctions and apoptosis is mediated by activation of signaling protein kinases in a Langendorff-perfused rat heart model of stop-flow ischemia. We found that pre-perfusion of non-ischemic hearts with 100 nM estradiol increased the resistance of subsequently isolated mitochondria to the calcium-induced opening of mitochondrial permeability transition pore and this was mediated by protein kinase G. Loading of the hearts with estradiol prevented ischemia-induced loss of cytochrome c from mitochondria and respiratory inhibition and these effects were reversed in the presence of the inhibitor of Akt kinase, NO synthase inhibitor L-NAME, guanylyl cyclase inhibitor ODQ and protein kinase G inhibitor KT5823. Estradiol prevented ischemia-induced activation of caspases and this was also reversed by KT5823. These findings suggest that estradiol may protect the heart against ischemia-induced injury activating the signaling cascade which involves Akt kinase, NO synthase, guanylyl cyclase and protein kinase G, and results in blockage of mitochondrial permeability transition pore-induced release of cytochrome c from mitochondria, respiratory inhibition and activation of caspases.     

PDE5 inhibitor promotes melanin synthesis through the PKG pathway in B16 melanoma cells

PDE inhibitors could increase cellular cGMP levels and are used to treat erectile dysfunction as well as pulmonary arterial hypertension. cGMP production was reported to be necessary for UVB-induced melanin synthesis, however, the effect of PDE5 inhibitor on melanin synthesis has not been examined. We found that PDE5 inhibitor (sildenafil or vardenafil) and the cGMP analog 8-CPT-cGMP stimulated CREB phosphorylation, leading to increased tyrosinase expression and melanin synthesis, which was counteracted by KT5823, a selective cGMP-dependent protein kinase (PKG) inhibitor. However, KT5823 did not affect cAMP-elevating agent-mediated melanin synthesis, indicating that KT5823 selectively inhibited cGMP-induced melanin synthesis. This is the first study to find that PDE5 inhibitor can promote melanin synthesis and reveal that PKG-dependent CREB phosphorylation and tyrosinase expression is involved in cGMP-induced melanin synthesis. Our results suggest that PDE5 inhibitor may be beneficial for the treatment of hypopigmentation diseases.     

Glucosamine cardioprotection in perfused rat hearts associated with increased O-linked N-acetylglucosamine protein modification and altered p38 activation

We have shown that, in the perfused heart, glucosamine improved functional recovery following ischemia and that this appeared to be mediated via an increase in O-linked N-acetylglucosamine (O-GlcNAc) levels on nucleocytoplasmic proteins. Several kinase pathways, specifically Akt and the mitogen-activated protein kinases (MAPKs) p38 and ERK1/2, which have been implicated in ischemic cardioprotection, have also been reported to be modified in response to increased O-GlcNAc levels. Therefore, the goals of this study were to determine the effect of ischemia on O-GlcNAc levels and to evaluate whether the cardioprotection resulting from glucosamine treatment could be attributed to changes in ERK1/2, Akt, and p38 phosphorylation. Isolated rat hearts were perfused with or without 5 mM glucosamine and were subjected to 5, 10, or 30 min of low-flow ischemia or 30 min of low-flow ischemia and 60 min of reperfusion. Glucosamine treatment attenuated ischemic contracture and improved functional recovery at the end of reperfusion. Glucosamine treatment increased flux through the hexosamine biosynthesis pathway and increased O-GlcNAc levels but had no effect on ATP levels. Glucosamine did not alter the response of either ERK1/2 or Akt to ischemia-reperfusion; however, it significantly attenuated the ischemia-induced increase in p38 phosphorylation and paradoxically increased p38 phosphorylation at the end of reperfusion. These data support the notion that O-GlcNAc may play an important role as an internal stress response and that glucosamine-induced cardioprotection may be mediated via the p38 MAPK pathway.     

Mitigation of the progression of heart failure with sildenafil involves inhibition of RhoA/Rho-kinase pathway

Chronic inhibition of phosphodiesterase-5 with sildenafil immediately after permanent occlusion of the left anterior descending coronary artery was shown to limit ischemic heart failure (HF) in mice. To mimic a more clinical scenario, we postulated that treatment with sildenafil beginning at 3 days post-myocardial infarction (MI) would also reduce HF progression through the inhibition of the RhoA/Rho-kinase pathway. Adult male ICR mice with fractional shortening < 25% at day 3 following permanent left anterior descending coronary artery ligation were continuously treated with either saline (volume matched, ip, 2 times/day) or sildenafil (21 mg/kg, ip, 2 times/day) for 25 days. Echocardiography showed fractional shortening preservation and less left ventricular end-diastolic dilatation with sildenafil treatment compared with saline treatment at 7 and 28 days post-MI (P < 0.05). Both fibrosis and apoptosis, determined by Masson's trichrome and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL), respectively, were attenuated in the sildenafil-treated mice (P < 0.05 vs. saline). Western blot analysis showed enchanced Bcl-2-to-Bax ratio with sildenafil treatment (P < 0.05 vs. saline). Activity assay showed sildenafil-mediated PKG activation 1 day after treatment (P < 0.05 vs. sham and saline). PKG activation was associated with sildenafil-mediated inhibition of Rho kinase (P < 0.05) compared with saline treatment, whereas PKG inhibition with KT-5823 abolished this inhibitory effect of sildenafil. In conclusion, for the first time, our findings show that chronic sildenafil treatment, initiated at 3 days post-MI, attenuates left ventricular dysfunction independent of its infarct-sparing effect, and this cardioprotection involves the inhibition of the RhoA/Rho-kinase pathway. Sildenafil may be a promising therapeutic tool for advanced HF in patients.     

The phosphodiesterase 5 inhibitor sildenafil stimulates angiogenesis through a protein kinase G/MAPK pathway

cGMP-degrading pathways have received little attention in the context of angiogenesis. In the present study we set out to determine whether cGMP-specific phosphodiesterase 5 (PDE5) inhibition affects new blood vessel growth. Incubation of chicken chorioallantoic membranes (CAMs) in vivo with sildenafil increased vascular length in a dose-dependent manner. Moreover, incubation of cultured endothelial cells (ECs) with the PDE5 inhibitor promoted proliferation, migration, and organization into tube-like structures. The effects of sildenafil on the angiogenesis-related properties of EC could be blocked by pre-treatment with the soluble guanylyl cyclase (sGC) inhibitor ODQ or the protein kinase G (PKG) I inhibitor DT-3. In addition, over-expression of sGC in EC led to an enhanced growth and migratory response to sildenafil. To study the signaling pathways implicated in the sildenafil-stimulated angiogenic responses we determined the phosphorylation status of mitogen-activated protein kinase (MAPK) members. Incubation of cells with sildenafil increased both extracellular signal regulated kinase 1/2 (ERK1/2) and p38 phosphorylation in a time-dependent manner. Inhibition of MEK by PD98059 and p38 with SB203580 blocked sildenafil-induced proliferation and migration, respectively, suggesting that these MAPK members are downstream of PDE5 and mediate the angiogenic effects of sildenafil. PDE5 inhibitors could, thus, be used in disease states where neo-vessel growth is desired.     

Attenuation of ischemia–reperfusion injury by sevoflurane postconditioning involves protein kinase B and glycogen synthase kinase 3 beta activation in isolated rat hearts

Volatile anesthetic ischemic postconditioning reduces infarct size following ischemia/reperfusion. Whether phosphorylation of protein kinase B (PKB/Akt) and glycogen synthase kinase 3 beta (GSK3β) is causal for cardioprotection by postconditioning is controversial. We therefore investigated the impact of PKB/Akt and GSK3β in isolated perfused rat hearts subjected to 40 min of ischemia followed by 1 h of reperfusion. 2.0% sevoflurane (1.0 minimum alveolar concentration) was administered at the onset of reperfusion in 15 min as postconditioning. Western blot analysis was used to determine phosphorylation of PKB/Akt and its downstream target GSK3β after 1 h of reperfusion. Mitochondrial and cytosolic content of cytochrome C checked by western blot served as a marker for mitochondrial permeability transition pore opening. Sevoflurane postconditioning significantly improved functional cardiac recovery and decreased infarct size in isolated rat hearts. Compared with unprotected hearts, sevoflurane postconditioning-induced phosphorylation of PKB/Akt and GSK3β were significantly increased. Increase of cytochrome C in mitochondria and decrease of it in cytosol is significant when compared with unprotected ones which have reversal effects on cytochrome C. The current study presents evidence that sevoflurane-induced cardioprotection at the onset of reperfusion are partly through activation of PKB/Akt and GSK3β.     

Exogenous nitric oxide generates ROS and induces cardioprotection: involvement of PKG, mitochondrial KATP channels, and ERK

We examined whether cGMP-dependent protein kinase (PKG) and mitochondrial ATP-sensitive potassium (K(ATP)) channels are involved in S-nitroso-N-acetyl penicillamine (SNAP)-induced reactive oxygen species (ROS) generation. SNAP significantly increased ROS generation in cardiomyocytes. This increase was suppressed by both 5-hydroxydecanoate (5-HD) and glibenclamide. Direct opening of mitochondrial K(ATP) channels with diazoxide led to ROS generation. The increased ROS generation was reversed by N-(2-mercaptopropionyl)glycine (MPG), a scavenger of ROS. Myxothiazol partially suppressed the ROS generation. KT-5823, an inhibitor of PKG, prevented ROS generation, indicating that PKG is required for ROS generation. In addition, 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP), an activator of PKG, induced ROS generation. The effect of 8-BrcGMP was reversed by either 5-HD or MPG. YC-1, an activator of guanylyl cyclase, also increased ROS production, which was reversed by 5-HD. Neither LY-294002 nor wortmannin, the inhibitors of phosphatidylinositol 3-kinase (PI3-kinase), affected SNAP's action. In a whole heart study, SNAP significantly reduced infarct size. The anti-infarct effect of SNAP was abrogated by either MPG or 5-HD. This effect was also blocked by PD-98059, an ERK inhibitor, but not by LY-294002. A Western blotting study showed that SNAP significantly enhanced phosphorylation of ERK, which was reversed by MPG. These results suggest that SNAP-induced ROS generation is mediated by activation of PKG and mitochondrial K(ATP) channels and that opening of mitochondrial K(ATP) channels is the downstream event of PKG activation. ROS and mitochondrial K(ATP) channels participate in the anti-infarct effect of SNAP. Moreover, phosphorylation of ERK is the downstream signaling event of ROS and plays a role in the cardioprotection of SNAP.     

Intermittent hypobaric hypoxia improves postischemic recovery of myocardial contractile function via redox signaling during early reperfusion

Intermittent hypobaric hypoxia (IHH) protects hearts against ischemia-reperfusion (I/R) injury, but the underlying mechanisms are far from clear. ROS are paradoxically regarded as a major cause of myocardial I/R injury and a trigger of cardioprotection. In the present study, we investigated whether the ROS generated during early reperfusion contribute to IHH-induced cardioprotection. Using isolated perfused rat hearts, we found that IHH significantly improved the postischemic recovery of left ventricular (LV) contractile function with a concurrent reduction of lactate dehydrogenase release and myocardial infarct size (20.5 ± 5.3% in IHH vs. 42.1 ± 3.8% in the normoxic control, P < 0.01) after I/R. Meanwhile, IHH enhanced the production of protein carbonyls and malondialdehyde, respective products of protein oxidation and lipid peroxidation, in the reperfused myocardium and ROS generation in reperfused cardiomyocytes. Such effects were blocked by the mitochondrial ATP-sensitive K(+) channel inhibitor 5-hydroxydecanoate. Moreover, the IHH-improved postischemic LV performance, enhanced phosphorylation of PKB (Akt), PKC-ε, and glycogen synthase kinase-3β, as well as translocation of PKC-ε were not affected by applying H(2)O(2) (20 μmol/l) during early reperfusion but were abolished by the ROS scavengers N-(2-mercaptopropionyl)glycine (MPG) and manganese (III) tetrakis (1-methyl-4-pyridyl)porphyrin. Furthermore, IHH-reduced lactate dehydrogenase release and infarct size were reversed by MPG. Consistently, inhibition of Akt with wortmannin and PKC-ε with εV1-2 abrogated the IHH-improved postischemic LV performance. These findings suggest that IHH-induced cardioprotection depends on elevated ROS production during early reperfusion.     

Nitric oxide inhibits spleen cell proliferative response after burn injury by inducing cytostasis,apoptosis, and necrosis of activated T lymphocytes: role of the guanylate cyclase

We previously showed that an overproduction of nitric oxide (NO) by macrophages was responsible for the collapse of lymphoproliferative responses after burn injury in rats. First, we demonstrate here that 10 days post-burn, the inhibition of splenocyte response to concanavalin-A results from cytostatic, apoptotic, and necrotic effects of NO on activated T cells. This was evidenced by various criteria at the levels of DNA, mitochondria, and plasma membrane. Inhibition of NO synthase by S-methylisothiourea (10 microM) normalized all the parameters. Second, we show that two soluble guanylate cyclase (sGC) inhibitors, LY83583 and ODQ, restored the proliferative response in a concentration-dependent manner. LY83583 (0.5 microM) rescued T cells from apoptosis. Similar results were obtained with KT5823 (5 microM) a specific inhibitor of protein kinase G (PKG). In contrast, neither LY83583 nor KT5823 inhibited NO-induced necrosis. These results suggest that NO blocked T cells in the G1 phase and induced apoptosis through a sGC-PKG-dependent pathway and necrosis through an independent one.     

Phosphodiesterase-5 inhibition mimics intermittent reoxygenation and improves cardioprotection in the hypoxic myocardium

Although chronic hypoxia is a claimed myocardial risk factor reducing tolerance to ischemia/reperfusion (I/R), intermittent reoxygenation has beneficial effects and enhances heart tolerance to I/R. Aim of the study: To test the hypothesis that, by mimicking intermittent reoxygenation, selective inhibition of phosphodiesterase-5 activity improves ischemia tolerance during hypoxia. Adult male Sprague-Dawley rats were exposed to hypoxia for 15 days (10% O₂) and treated with placebo, sildenafil (1.4 mg/kg/day, i. p.), intermittent reoxygenation (1 h/day exposure to room air) or both. Controls were normoxic hearts. To assess tolerance to I/R all hearts were subjected to 30-min regional ischemia by left anterior descending coronary artery ligation followed by 3 h-reperfusion. Whereas hypoxia depressed tolerance to I/R, both sildenafil and intermittent reoxygenation reduced the infarct size without exhibiting cumulative effects. The changes in myocardial cGMP, apoptosis (DNA fragmentation), caspase-3 activity (alternative marker for cardiomyocyte apoptosis), eNOS phosphorylation and Akt activity paralleled the changes in cardioprotection. However, the level of plasma nitrates and nitrites was higher in the sildenafil+intermittent reoxygenation than sildenafil and intermittent reoxygenation groups, whereas total eNOS and Akt proteins were unchanged throughout. Conclusions: Sildenafil administration has the potential to mimic the cardioprotective effects led by intermittent reoxygenation, thereby opening the possibility to treat patients unable to be reoxygenated through a pharmacological modulation of NO-dependent mechanisms.     

Akt activation induced by an antioxidant compound during ischemia-reperfusion

Molecular mechanisms of cardioprotection afforded by modified mexiletine compounds were investigated during ischemia-reperfusion (IR) in Langendorff perfused hearts. Rat hearts were subjected to a global 25 min ischemia followed by reperfusion, either untreated or treated with mexiletine, or three substituted mexiletine derivates (5 muM). A modified mexiletine derivative (H-2693) promoted best the recovery of myocardial energy metabolism (assessed by (31)P NMR spectroscopy) compared to untreated and mexiletine-treated hearts. H-2693 also preserved cardiac contractile function and attenuated the IR-induced lipid peroxidation (TBARS formation) and protein oxidation (carbonyl content). Western blot revealed that H-2693 propagated the phosphorylation of Akt (activation) and its downstream substrate glycogen synthase kinase-3beta (GSK-3beta, inactivation) compared to untreated IR. Parallel treatment with the phosphatidylinositol-3-kinase (upstream activator of Akt) inhibitor wortmannin (100 nM) abolished the beneficial effects of H-2693 on energetics and function, and reduced Akt and GSK-3beta phosphorylation. As a result of the antiapoptotic impacts of Akt activation, H-2693 decreased caspase-3 activity, which was neutralized by wortmannin. Here we first demonstrated that a free radical-entrapping compound could activate the prosurvival Akt pathway beyond its proven ability to scavenge reactive oxygen species. In conclusion, the favorable influence of H-2693 on signaling events during IR may have considerably contributed to its cardioprotective effect.     

Reduction of SIRT1 blunts the protective effects of ischemic post-conditioning in diabetic mice by impairing the Akt signaling pathway

Ischemic post-conditioning (IPO) activates Akt signaling to confer cardioprotection. The responsiveness of diabetic hearts to IPO is impaired. We hypothesized that decreased cardiac SIRT1, a positive regulator of Akt, may be responsible for the impaired responsiveness of diabetic hearts to IPO-mediated cardioprotection. High-fat diet and streptozotocin-induced diabetic mice were subjected to myocardial ischemia/reperfusion (MI/R, 30 min ischemia and 180 min reperfusion) or IPO (three cycles of 10 s of reperfusion and ischemia at the onset of reperfusion). Adenoviral vectors encoding GFP or SIRT1 (Ad-SIRT1) were administered by direct injection into the left ventricular. Our results showed that IPO activated the Akt signaling pathway and reduced MI/R injury in non-diabetic hearts but not in diabetic hearts, in which reduced expression of SIRT1 and increased Akt acetylation were observed. Delivery of Ad-SIRT1 into the diabetic hearts reduced Akt acetylation and restored the cardioprotective effects of IPO by modulating Akt signaling pathway. In contrast, cardiac-specific SIRT1 knockout increased Akt acetylation and blunted the cardioprotective effects of IPO. In in vitro study, transfection with wild-type SIRT1 but not inactive mutant SIRT1 reduced the expression of Akt acetylation and restored the protective effects of hypoxic post-conditioning in high glucose-incubated cardiomyocytes. Moreover, the cardiomyocytes transfected with constitutive Akt acetylation showed repressed Akt phosphorylation and blunted protective effects against hypoxia/reoxygenation injury. These findings demonstrate that the reduction of SIRT1 blunts the protective effects of IPO by impairing Akt signaling pathway and that SIRT1 up-regulation restores IPO-mediated cardioprotection in diabetic mice via deacetylation-dependent activation of Akt signaling pathway.     

Infarct limitation by a protein kinase G activator at reperfusion in rabbit hearts is dependent on sensitizing the heart to A2b agonists by protein kinase C

PKG activator 8-(4-chlorophenylthio)-guanosine 3',5'-cyclic monophosphate (CPT) at reperfusion protects ischemic hearts, but the mechanism is unknown. We recently proposed that in preconditioned hearts PKC lowers the threshold for adenosine to initiate signaling from low-affinity A2b receptors during early reperfusion thus allowing endogenous adenosine to activate survival kinases phosphatidylinositol 3-kinase (PI3K) and ERK. We tested whether CPT might also sensitize A2b receptors to adenosine. CPT (10 microM) during the first minutes of reperfusion markedly reduced infarction in isolated rabbit hearts undergoing 30-min regional ischemia/2-h reperfusion, and salvage was blocked by MRS 1754, an A2b-selective antagonist. Coadministration of wortmannin (PI3K inhibitor) or PD-98059 (MEK1/2 and therefore ERK1/2 inhibitor) also blocked protection. In nonischemic hearts, 10-min infusion of CPT did not change phosphorylation of Akt or ERK1/2. Neither did a subthreshold dose (2.5 nM) of the nonselective but A2b-potent receptor agonist 5'-(N-ethylcarboxamido)adenosine (NECA). However, when 2.5 nM NECA was combined with 10 microM CPT, both phospho-Akt and phospho-ERK1/2 significantly increased, indicating CPT had lowered the threshold for A2b-dependent signaling. The PKC antagonist chelerythrine blocked this phosphorylation induced by CPT + NECA. Chelerythrine also blocked the anti-infarct effect of CPT as did nonselective (glibenclamide) and mitochondrial-selective (5-hydroxydecanoate) K(ATP) channel blockers. A free radical scavenger, N-(2-mercaptopropionyl)glycine, also blocked CPT protection. We propose CPT targets PKG, which activates PKC through mitochondrial K(ATP) channel (mitoKATP)-dependent redox signaling, a sequence mimicking that already documented in preconditioning. Activated PKC then augments sensitivity of normally low-affinity cardiac adenosine A2b receptors so endogenous adenosine can protect by activating Akt and ERK.     

Cyclic GMP Reduces Ventricular Myocyte Stunning after Simulated Ischemia–Reperfusion

We tested the hypothesis that the second messenger activated by nitric oxide, cyclic GMP, would reduce the effects of myocyte stunning following simulated ischemia-reperfusion and that this was related to cyclic GMP protein kinase. Ventricular cardiac myocytes were isolated from New Zealand White rabbits (n = 8). Cell shortening was measured by a video edge detector and protein phosphorylation was determined autoradiographically after SDS gel electrophoresis. Cell shortening data were acquired at: (i) baseline followed by 8-Bromo-cGMP 10(-6) M (8-Br-cGMP) and then KT 5823 10(-6) M (cyclic GMP protein kinase inhibitor) and (ii) simulated ischemia (20 min of 95% N(2)-5% CO(2) at 37 degrees C) followed by simulated reperfusion (reoxygenation) with addition of 8-Br-cGMP 10(-6) M followed by KT 5823 10(-6) M, (iii) addition of 8-Br-cGMP prior to ischemia followed by the addition of KT 5823 10(-6) M after 30 min of reoxygenation. In the control group, 8-Br-cGMP 10(-6) M decreased percentage shortening (%short) (5.0 +/- 0.6 vs 3.8 +/- 0. 4) and the maximum velocity (V(max), microm/s) (48.6 +/- 6.9 vs 40.2 +/- 6.4). KT 5823 10(-6) M added after 8-Br-cGMP partially restored %short (4.6 +/- 0.5) and V(max) (46.6 +/- 8.0). After stunning, baseline myocytes had decreased %short (3.4 +/- 0.2) and V(max) (36. 0 +/- 4.2). After the addition of 8-Br-cGMP, the %short (2.7 +/- 0. 2) and V(max) (27.6 +/- 2.5) decreased further. The addition of KT 5823 did not change either the %short or the V(max). The myocytes with 8-Br-cGMP during ischemia had increased %short (4.2 +/- 0.2) and V(max) (37.2 +/- 3.4) when compared to the stunned group. The addition of KT 5823 did not significantly alter %short (3.3 +/- 0.4) or V(max) (29.2 +/- 5.0) in the myocytes pretreated with 8-Br-cGMP. Protein phosphorylation was increased by 8-Br-cGMP in control and stunned myocytes. KT 5823 blocked this effect in control but not stunned myocytes, suggesting some change in the cyclic GMP protein kinase. Ischemia-reperfusion produced myocyte stunning that was reduced when 8-Br-cGMP was added prior to but not after ischemia.