We investigated the effect of sildenafil in protection against necrosis or apoptosis in cardiomyocytes. Adult mouse ventricular myocytes were treated with sildenafil (1 or 10 M) for 1 h before 40 min of simulated ischemia (SI). Necrosis was determined by trypan blue exclusion and lactate dehydrogenase release following SI alone or plus 1 or 18 h of reoxygenation (RO). Apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated nick end labeling assay and mitochondrial membrane potential measured using a fluorescent probe 5,5 ,6,6 -tetrachloro-1,1 ,3,3 -tetraethylbenzimidazolyl-carbocyanine iodide (JC-1). Sildenafil reduced necrosis as indicated by decrease in trypan blue-positive myocytes and leakage of lactate dehydrogenase compared with untreated cells after either SI or SI-RO. The number of terminal deoxynucleotidyl transferase-mediated nick end labeling-positive myocytes or loss of JC-1 fluorescence following SI and 18 h of RO was attenuated in the sildenafil-treated group with concomitant inhibition of caspase 3 activity. An early increase in Bcl-2 to Bax ratio with sildenafil treatment was also observed in myocytes after SI-RO. The increase of Bcl-2 expression by sildenafil was inhibited by nitric-oxide synthase (NOS) inhibitor, L-nitro-amino-methyl-ester. The drug also enhanced mRNA and protein content of inducible NOS (iNOS) and endothelial NOS (eNOS) in the myocytes. Sildenafil-induced protection against necrosis and apoptosis was absent in the myocytes derived from iNOS knock-out mice and was attenuated in eNOS knock-out myocytes. The up-regulation of Bcl-2 expression by sildenafil was also absent in iNOS-deficient myocytes. Reverse transcription-PCR, Western blots, and immunohistochemical assay confirmed the expression of phosphodiesterase-5 in mouse cardiomyocytes. These data provide strong evidence for a direct protective effect of sildenafil against necrosis and apoptosis through NO signaling pathway. The results may have possible therapeutic potential in preventing myocyte cell death following ischemia/reperfusion.

Background-Experimental interleukin-1 receptor antagonist gene overexpression has shown that interleukin-1 receptor antagonist is cardioprotective during global cardiac ischemia. The aim of the present study was to test the impact of an exogenous recombinant human interleukin-1 receptor antagonist (anakinra) in experimental acute myocardial infarction. Methods and Results-Two animal studies were conducted: one of immediate anakinra administration during ischemia in the mouse and one of delayed anakinra administration 24 hours after ischemia in the rat. Seventy-eight Institute of Cancer Research mice and 20 Wistar rats underwent surgical coronary artery ligation (or sham operation) and were treated with either anakinra 1 mg/kg or NaCl 0.9% (saline). Treatment was administered during surgery and then daily for 6 doses in the mice and starting on day 2 daily for 5 doses in the rats. Twenty-eight mice underwent infarct size assessment 24 hours after surgery, 6 saline-treated mice and 22 mice treated with increasing doses of anakinra (1 mg/kg [nϭ6], 10 mg/kg [nϭ6], and 100 mg/kg [nϭ10]); 6 mice were euthanized at 7 days for protein expression analysis. The remaining animals underwent transthoracic echocardiography before surgery and 7 days later just before death. Cardiomyocyte apoptosis was measured in the peri-infarct regions. The antiapoptotic effect of anakinra was tested in a primary rat cardiomyocyte culture during simulated ischemia and in vitro on caspase-1 and -9 activities. At 7 days, 15 of the 16 mice (94%) treated with anakinra were alive versus 11 of the 20 mice (55%) treated with saline (Pϭ0.013).No differences in infarct size at 24 hours compared with saline were observed with the 1-and 10-mg/kg doses, whereas a 13% reduction in infarct size was found with the 100-mg/kg dose (Pϭ0.015). Treatment with anakinra was associated with a significant reduction in cardiomyocyte apoptosis in both the immediate and delayed treatment groups (3.1Ϯ0.2% versus 0.5Ϯ0.3% [PϽ0.001] and 4.2Ϯ0.4% versus 1.1Ϯ0.2% [PϽ0.001], respectively). Compared with saline-treated animals, anakinra-treated mice and rats showed signs of more favorable ventricular remodeling. In vitro, anakinra significantly prevented apoptosis induced by simulated ischemia and inhibited caspase-1 and -9 activities. Conclusions-Administration of anakinra within 24 hours of acute myocardial infarction significantly ameliorates the remodeling process by inhibiting cardiomyocyte apoptosis in 2 different experimental animal models of AMI. This may open the door for using anakinra to prevent postischemic cardiac remodeling and heart failure.

Sildenafil citrate (Viagra) is the pharmacological agent used to treat erectile dysfunction in men. Because this drug has a vasodilatory effect, we hypothesized that such an action may induce a preconditioning-like cardioprotective effect via opening of mitochondrial ATP-sensitive K (K(ATP)) channels. Rabbits were treated with sildenafil citrate (0.7 mg/kg iv) either 30 min (acute phase) or 24 h (delayed phase) before 30 min of ischemia and 3 h of reperfusion. Mitochondrial K(ATP) channel blocker 5-hydroxydecanoate (5-HD, 5 mg/kg iv) was given 10 min before ischemia-reperfusion. Infarct size was measured by tetrazolium staining. Sildenafil caused reduction in arterial blood pressure within 2 min of treatment, which returned to nearly baseline levels 3 min later. The infarct size (% risk area, means +/- SE) reduced from 33.8 +/- 1.7 in control rabbits to 10.8 +/- 0.9 during the acute phase (68% reduction, P < 0.05) and 19.9 +/- 2.0 during the delayed phase (41% reduction, P < 0.05). 5-HD abolished protection with an increase in infarct size to 35.6 +/- 0.4% and 36.8 +/- 1.6% during the acute and delayed phase, respectively (P < 0.05). Similar acute and delayed cardioprotective effects were observed when sildenafil was administered orally. Systemic hemodynamics also decreased after oral administration of the drug. However, these changes were mild and occurred slowly. For the first time, we demonstrate that sildenafil induces acute and delayed protective effects against ischemia-reperfusion injury, which are mediated by opening of mitochondrial K(ATP) channels.

Background-Sildenafil, a phosphodiesterase-5 inhibitor, induces cardioprotection against ischemia/reperfusion injury via opening of mitochondrial K ATP channels. It is unclear whether sildenafil would provide similar protection from doxorubicin-induced cardiotoxicity. Methods and Results-Male ICR mice were randomized to 1 of 4 treatments: saline, sildenafil, doxorubicin (5 mg/kg IP), and sildenafil (0.7 mg/kg IP) plus doxorubicin (nϭ6 per group). Apoptosis was assessed with the use of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling and in situ oligo ligation methods. Desmin distribution was determined via immunofluorescence. Bcl-2 expression was analyzed by Western blot. Left ventricular function was assessed by measuring developed pressure and rate pressure product in Langendorff mode. ECG changes indicative of doxorubicin cardiotoxicity were also measured. For in vitro studies, adult ventricular cardiomyocytes were exposed to doxorubicin (1 mol/L), sildenafil (1 mol/L) with or without N G -nitro-L-arginine methyl ester (L-NAME) (100 mol/L), or 5-hydroxydecanoate (100 mol/L) 1 hour before doxorubicin and incubated for 18 hours. Doxorubicintreated mice demonstrated increased apoptosis and desmin disruption, which was attenuated in the sildenafilϩdoxorubicin group. Bcl-2 was decreased in the doxorubicin group but was maintained at basal levels in the sildenafilϩdoxorubicin group. Left ventricular developed pressure and rate pressure product were significantly depressed in the doxorubicin group but were attenuated in the sildenafilϩdoxorubicin group. ST interval was significantly increased in the doxorubicin group over 8 weeks. In the sildenafilϩdoxorubicin group, ST interval remained unchanged from baseline. Doxorubicin caused a significant increase in apoptosis, caspase-3 activation, and disruption of mitochondrial membrane potential in vitro. In contrast, sildenafil significantly protected against doxorubicin cardiotoxicity; however, this protection was abolished by both L-NAME and 5-hydroxydecanoate. Conclusions-Prophylactic

Expression of the STAT3 transcription factor in the heart is cardioprotective and decreases the levels of reactive oxygen species. Recent studies indicate that a pool of STAT3 resides in the mitochondria where it is necessary for the maximal activity of complexes I and II of the electron transport chain. However, it has not been explored whether mitochondrial STAT3 modulates cardiac function under conditions of stress. Transgenic mice with cardiomyocyte-specific overexpression of mitochondria-targeted STAT3 with a mutation in the DNA-binding domain (MLS-STAT3E) were generated. We evaluated the role of mitochondrial STAT3 in the preservation of mitochondrial function during ischemia. Under conditions of ischemia heart mitochondria expressing MLS-STAT3E exhibited modest decreases in basal activities of complexes I and II of the electron transport chain. In contrast to WT hearts, complex I-dependent respiratory rates were protected against ischemic damage in MLS-STAT3E hearts. MLS-STAT3E prevented the release of cytochrome c into the cytosol during ischemia. In contrast to WT mitochondria, ischemia did not augment reactive oxygen species production in MLS-STAT3E mitochondria likely due to an MLS-STAT3E-mediated partial blockade of electron transport through complex I. Given the caveat of STAT3 overexpression, these results suggest a novel protective mechanism mediated by mitochondrial STAT3 that is independent of its canonical activity as a nuclear transcription factor. STAT3 was originally identified as an IL-6-induced transcriptional activator of acute phase genes (1). However, other members of the IL-6 family, which utilize gp-130 receptor, as well as leptin, IL-12, IFN␣/␤, IL-10, GM-CSF, several growth factors, oncogenes, and stress such as hypoxia, also activate STAT3 (1). STAT3 is vital to embryonic development and STAT3-null mice are embryonic lethal (2). Analysis of tissuespecific conditional STAT3 knock-out mice has provided strong evidence that transcriptional activity of STAT3 plays a central role in the control of cell growth and host responses to inflammation and cellular stress (1). STAT3 positively regulates expression of anti-apoptotic (Bcl-2 and Bcl-xL) (1) and antioxidative proteins (MnSOD and metallothionein-1 and -2) (3, 4).Expression of STAT3 in the heart is associated with cardiac survival (5). When STAT3 is selectively deleted in cardiomyocytes, mice develop enhanced cardiac inflammation with fibrosis, dilated cardiomyopathy, and die prematurely due to congestive heart failure (5). Female mice, where STAT3 is not expressed in cardiomyocytes, develop post-partum cardiomyopathy, which is also seen in humans with reduced STAT3 expression in the myocardium (6). Ventricles from STAT3-null hearts show elevated levels of reactive oxygen species (ROS) 2 (6). Ischemic and pharmacologic preconditioning protected the viability of wild type but not STAT3 Ϫ/Ϫ cardiomyocytes (5). When STAT3 is overexpressed in cardiomyocytes, mice are less sensitive to the cardiotoxic effects of doxorubicin, which exerts i...

Previous studies have argued that enhanced activity of the epidermal growth factor receptor (EGFR) and the mitogen-activated protein kinase (MAPK) pathway can promote tumor cell survival in response to cytotoxic insults. In this study, we examined the impact of MAPK signaling on the survival of primary hepatocytes exposed to low concentrations of deoxycholic acid (DCA, 50 microM). Treatment of hepatocytes with DCA caused MAPK activation, which was dependent upon ligand independent activation of EGFR, and downstream signaling through Ras and PI(3) kinase. Neither inhibition of MAPK signaling alone by MEK1/2 inhibitors, nor exposure to DCA alone, enhanced basal hepatocyte apoptosis, whereas inhibition of DCA-induced MAPK activation caused approximately 25% apoptosis within 6 h. Similar data were also obtained when either dominant negative EGFR-CD533 or dominant negative Ras N17 were used to block MAPK activation. DCA-induced apoptosis correlated with sequential cleavage of procaspase 8, BID, procaspase 9, and procaspase 3. Inhibition of MAPK potentiated bile acid-induced apoptosis in hepatocytes with mutant FAS-ligand, but did not enhance in hepatocytes that were null for FAS receptor expression. These data argues that DCA is causing ligand independent activation of the FAS receptor to stimulate an apoptotic response, which is counteracted by enhanced ligand-independent EGFR/MAPK signaling. In agreement with FAS-mediated cell killing, inhibition of caspase function with the use of dominant negative Fas-associated protein with death domain, a caspase 8 inhibitor (Ile-Glu-Thr-Asp-p-nitroanilide [IETD]) or dominant negative procaspase 8 blocked the potentiation of bile acid-induced apoptosis. Inhibition of bile acid-induced MAPK signaling enhanced the cleavage of BID and release of cytochrome c from mitochondria, which were all blocked by IETD. Despite activation of caspase 8, expression of dominant negative procaspase 9 blocked procaspase 3 cleavage and the potentiation of DCA-induced apoptosis. Treatment of hepatocytes with DCA transiently increased expression of the caspase 8 inhibitor proteins c-FLIP-(S) and c-FLIP-(L) that were reduced by inhibition of MAPK or PI(3) kinase. Constitutive overexpression of c-FLIP-(s) abolished the potentiation of bile acid-induced apoptosis. Collectively, our data argue that loss of DCA-induced EGFR/Ras/MAPK pathway function potentiates DCA-stimulated FAS-induced hepatocyte cell death via a reduction in the expression of c-FLIP isoforms.

Purified PGH synthase when acting on arachidonic acid in the presence of reduced nicotinamideadenine dinucleotide or reduced nicotinamide-adenine dinucleotide 3'-phosphate generated superoxide in burst-like fashion. In eight experiments using different batches of enzyme, the mean ± SE rate of superoxide generation from 100 U of enzyme measured as the superoxide dismutase-inhibitable reduction of cytochrome c was 5.06 ± 0.19 nmol/min in the first minute and 0.35 ± 0.03 nmol/min subsequently. Optimum rates of superoxide were seen at concentrations of reduced nicotinamideadenine dinucleotide in excess of 80 /xM and reduced nicotinamide-adenine dinucleotide 3-phosphate in excess of 100 juM. Using prostaglandin G 2 or linoleic acid as substrate rather than arachidonate also resulted in superoxide generation. When prostaglandin H 2 was used as substrate, no superoxide was generated. The rate of superoxide generation was markedly inhibited by cyclooxygenase inhibitors. Superoxide generation was also observed during the action of lipoxygenase on either linoleic or arachidonic acid in the presence of reduced nicotinamide-adenine dinucleotide or reduced nicotinamide-adenine dinucleotide 3-phosphate but not in their absence. Indomethacin had no effect on superoxide generation from lipoxygenase. We conclude that PGH synthase and lipoxygenase produce superoxide via a side-chain reaction dependent on the presence of suitable reducing cosubstrate. This mechanism is analogous to that described for peroxidases in general. and topical application of arachidonate 89 cause a spectrum of functional, morphological, and biochemical cerebral arteriolar abnormalities. These consist of sustained dilation, reduced responsiveness to vasoconstrictor and vasodilator influences, focal destructive lesions of the endothelium and vascular smooth muscle, and reduced oxygen consumption of the vessel wall. Superoxide anion and other reactive agents derived from it, such as hydrogen peroxide and the hydroxyl radical, are the likely mediators of the cerebral arteriolar abnormalities seen in these conditions, since the latter are inhibited by scavengers of superoxide anion, hydrogen peroxide, and of the hydroxyl radical. 6 Received January 21, 1986; accepted August 25, 1986. ioles to these agents for more than a few minutes causes vascular injury. The enzymatic source of superoxide anion in the experimental conditions noted above is not firmly established. The following evidence suggests strongly that superoxide is produced via the action of the prostaglandin hydroperoxidase. First, the cerebral arteriolar abnormalities noted above can be reproduced by topical application of prostaglandin G 2 (PGG 2 ) 8 or 15-hydroperoxy-eicosatetraenoic acid (15-HPETE)," which are good substrates of the prostaglandin hydroperoxidase. The cerebral arteriolar abnormalities are not produced by prostaglandin H 2 (PGH 2 ), the product of the reaction catalyzed by prostaglandin hydroperoxidase.8 Second, the vasodilation and cerebral arteriolar abnormalities due to hype...

Sildenafil citrate (Viagra) is the most widely used drug for treating erectile dysfunction in men. We recently demonstrated that it induces potent protective effects against ischemia-reperfusion (I-R) injury in rabbit hearts through the opening of mitochondrial ATP-dependent K ؉ channels. In the present study, we investigated the role of the NO-dependent signaling pathway in delayed cardioprotection by sildenafil. Adult male ICR mice were treated with saline or sildenafil (0.7 mg/kg IP) 24 hours before global I-R in the Langendorff mode. Infarct size was reduced from 27.6؎3.3% in saline-treated control mice to 6.9؎1.2% in sildenafil-treated mice (mean؎SEM, P<0.05) without compromising cardiac function. Reverse transcription-polymerase chain reaction revealed a transient increase in endothelial and inducible NO synthase (eNOS and iNOS, respectively) mRNA in sildenafil-treated mice, peaking at 45 minutes (eNOS) and 2 hours (iNOS) after sildenafil injection. The magnitude of mRNA increase was more pronounced for iNOS than for eNOS. In addition, a significant increase in both iNOS and eNOS protein was detected 24 hours after sildenafil treatment. A selective inhibitor of iNOS, 1400W (10 mg/kg IP given 30 minutes before I-R), abolished sildenafil-induced protection (23.7؎2.8%, P<0.05 versus sildenafil). These data suggest that the induction of NO synthase isoforms is an essential component of the signaling mechanism for sildenafil-induced delayed preconditioning. However, iNOS appears to be the primary isoform that mediates the robust cardioprotection. S ildenafil citrate (Viagra) is a selective inhibitor of phosphodiesterase-5, which catalyzes the breakdown of cGMP, one of the primary factors involved in smooth muscle relaxation. It enhances NO-driven cGMP accumulation, which, in turn, causes vasodilatation in the corpus cavernosum. Sildenafil has become the most widely used drug for treating erectile dysfunction in men since its market debut in 1998. 1 Interestingly, we recently discovered a powerful preconditioning-like effect of sildenafil in rabbit hearts. 2 Both intravenous and oral administration of sildenafil caused significant reduction of infarct size after ischemia-reperfusion (I-R). The protection was abolished by 5-hydroxydecanoate, a selective blocker of mitochondrial ATP-dependent K ϩ (mitoK ATP ) channels. 2 However, the mechanism by which sildenafil triggers the signaling cascade leading to the opening of mitoK ATP remains speculative. 2 There is mounting evidence suggesting a role of NO in modulating mitoK ATP . [3][4][5][6] The synthesis of NO is catalyzed by 3 isoforms of NO synthase (NOS), namely, neuronal NOS, inducible NOS (iNOS), and endothelial NOS (eNOS), among which iNOS has been identified as the essential mediator of delayed preconditioning induced by divergent pathophysiological stimuli or pharmacological agents, such as brief episodes of I-R, 7 endotoxin derivatives, 8,9 G-protein-coupled membrane receptor agonists, 10,11 whole-body hyperthermia, 12 and systemic hypoxia. 13 However, no ...