Search

The inhibition of mammalian target of rapamycin (mTOR) with rapamycin (RAPA) provides protection against myocardial ischemia/reperfusion (I/R) injury in diabetes. Since interactions between transcripts, including long non-coding RNA (lncRNA), microRNA(miRNA) and mRNA, regulate the pathophysiology of disease, we performed unbiased miRarray profiling in the heart of diabetic rabbits following I/R injury with/without RAPA treatment to identify differentially expressed (DE) miRNAs and their predicted targets of lncRNAs/mRNAs. Results showed that among the total of 806 unique miRNAs targets, 194 miRNAs were DE after I/R in diabetic rabbits. Specifically, eight miRNAs, including miR-199a-5p, miR-154-5p, miR-543-3p, miR-379-3p, miR-379-5p, miR-299-5p, miR-140-3p, and miR-497-5p, were upregulated and 10 miRNAs, including miR-1-3p, miR-1b, miR-29b-3p, miR-29c-3p, miR-30e-3p, miR-133c, miR-196c-3p, miR-322-5p, miR-499-5p, and miR-672-5p, were significantly downregulated after I/R injury. Interestingly, RAPA treatment significantly reversed these changes in miRNAs. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis indicated the participation of miRNAs in the regulation of several signaling pathways related to I/R injury, including MAPK signaling and apoptosis. Furthermore, in diabetic hearts, the expression of lncRNAs, HOTAIR, and GAS5 were induced after I/R injury, but RAPA suppressed these lncRNAs. In contrast, MALAT1 was significantly reduced following I/R injury, with the increased expression of miR-199a-5p and suppression of its target, the anti-apoptotic protein Bcl-2. RAPA recovered MALAT1 expression with its sponging effect on miR-199-5p and restoration of Bcl-2 expression. The identification of novel targets from the transcriptome analysis in RAPA-treated diabetic hearts could potentially lead to the development of new therapeutic strategies for diabetic patients with myocardial infarction. [ABSTRACT FROM AUTHOR]

Robust activation of mTOR (mammalian target of rapamycin) signaling in diabetes exacerbates myocardial injury following lethal ischemia due to accelerated cardiomyocyte death with cardiac remodeling and inflammatory responses. We examined the effect of rapamycin (RAPA, mTOR inhibitor) on cardiac remodeling and inflammation following myocardial ischemia/reperfusion (I/R) injury in diabetic rabbits. Diabetic rabbits (DM) were subjected to 45 min of ischemia and 10 days of reperfusion by inflating/deflating a previously implanted hydraulic balloon occluder. RAPA (0.25 mg/kg, i.v.) or DMSO (vehicle) was infused 5 min before the onset of reperfusion. Post-I/R left ventricular (LV) function was assessed by echocardiography and fibrosis was evaluated by picrosirius red staining. Treatment with RAPA preserved LV ejection fraction and reduced fibrosis. Immunoblot and real-time PCR revealed that RAPA treatment inhibited several fibrosis markers (TGF-β, Galectin-3, MYH, p-SMAD). Furthermore, immunofluorescence staining revealed the attenuation of post-I/R NLRP3-inflammasome formation with RAPA treatment as shown by reduced aggregation of apoptosis speck-like protein with a caspase recruitment domain and active-form of caspase-1 in cardiomyocytes. In conclusion, our study suggests that acute reperfusion therapy with RAPA may be a viable strategy to preserve cardiac function with the alleviation of adverse post-infarct myocardial remodeling and inflammation in diabetic patients. [ABSTRACT FROM AUTHOR]

The CXC chemokine IL-8, which promotes adhesion, activation, and transmigration of polymorphonuclear neutrophils (PMN), has been associated with production of tissue injury in reperfused myocardium. Hypoxia-inducible factor-1 (HIF-1) is a heterodimeric peptide that is a key regulator of genes such as heine oxygenase (HO)-1 expressed under hypoxic conditions. We hypothesized that HO-1 plays an important role in regulating proinflammatory mediator production under conditions of ischemia-reperfusion. HIF-1 was activated in the human microvascular endothelial cell line (HMEC-1) with the prolyl hydroxylase inhibitor dimethyloxalylglycine (DMOG). DMOG significantly attenuated cytokine-induced IL-8 promoter activity and protein secretion and cytokine-induced PMN migration across human microvascular endothelial cell line HMEC-1 monolayers. In vivo studies in a rabbit model of myocardial ischemia-reperfusion showed that rabbits pretreated with a 20 mg/kg DMOG infusion (n = 6) 24 h before study exhibited a 21.58 [+ or -] 1.76% infarct size compared with 35.25 [+ or -] 2.06% in saline-treated ischemia-reperfusion animals (n = 6, change in reduction = 39%; P < 0.001). In DMOG-pretreated (20 mg/kg) animals, plasma IL-8 levels at 3 h after onset of reperfusion were 405 [+ or -] 40 pg/ml vs. 790 [+ or -] 40 pg/ml in saline-treated ischemia-reperfusion animals (P < 0.001). DMOG pretreatment reduced myocardial myeloperoxidase activity, expressed as number of PMN per gram of myocardium, to 1.43 [+ or -] 0.59 vs. 4.86 [+ or -] 1.1 (P = 0.012) in saline-treated ischemia-reperfused hearts. Both in vitro and in vivo DMOG-attenuated IL-8 production was associated with robust HO-1 expression. Thus our data show that HIF-1 activation induces substantial HOd expression that is associated with attenuated proinflammatory chemokine production by microvascular endothelium in vitro and in vivo. hypoxia-inducible factor 1; cardiac ischemia-reperfusion; interleukin-8

Das, Anindita, Ramzi Ockaili, Fadi Salloum, and Rakesh C. Kukreja. Protein kinase C plays an essential role in sildenafil-induced cardioprotection in rabbits. Am J Physiol Heart Circ Physiol 286: H1455-H1460, 2004; 10.1152/ajpheart.01040.2003.--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.sup.+] 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 iv) was given 5 min before sildenafil. Infact size (% of risk area) reduced from 33.65 [+ or -] 2.17 in the vehicle (saline) group to 15.07 [+ or -] 0.63 in sildenafil-treated groups, a 45% reduction compared with vehicle (mean [+ or -] SE, P < 0.05). Chelerythrine abolished sildenafil-induced protection, as demonstrated by increase in infarct size to 31.14 [+ or -] 2.4 (P < 0.05). Chelerythrine alone had an infarct size of 33.5 [+ or -] 2.5, which was not significantly different compared with DMSO-treated group (36.8 [+ or -] 1.7, P > 0.05). Western blot analysis demonstrated translocation of PKC-[alpha] -[theta], 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], -[theta], and -[delta], in sildenafil-induced cardioprotection in the rabbit heart. chelerythrine

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.sub.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.sub.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 [+ or -] SE) reduced from 33.8 [+ or -] 1.7 in control rabbits to 10.8 [+ or -] 0.9 during the acute phase (68% reduction, P < 0.05) and 19.9 [+ or -] 2.0 during the delayed phase (41% reduction, P < 0.05). 5-HD abolished protection with an increase in infarct size to 35.6 [+ o r -] 0.4% and 36.8 [+ or -] 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.sub.ATP] channels. ATP-sensitive potassium channel; ischemia-reperfusion; phosphodiesterase-5

Chemical preconditioning with 3-nitropropionic acid in hearts: role of mitochondrial [K.sub.ATP] channel. Am J Physiol Heart Circ Physiol 280: H2406-H2411, 2001.--We investigated the cardioprotective effect of 3-nitropropionic acid (3-NPA), an inhibitior of mitochondrial succinate dehydrogenase, and we wanted to show whether this protection is mediated by of opening mitochondrial ATP-sensitive potassium ([K.sub.ATP]) channels. Adult rabbits were treated with either 3-NPA (3 mg/kg iv) or saline (n = 6 rabbits/group). After 30 min (for early phase) or 24 h (for late phase) of the treatment, the animals were subjected to 30 min of ischemia and 3 h of reperfusion (ischemia-reperfusion). 5-Hydroxydecanoate (5-HD, 5 mg/kg iv), the mitochondrial [K.sub.ATP] channel blocker, was administered 10 min before ischemia-reperfusion in the saline- and 3-NPA-treated rabbits. 3-NPA caused a decrease in the infarct size from 27.8 [+ or -] 4.2% in the saline group to 16.5 [+ or -] 1.0% in the 3-NPA-treated rabbits during early phase and from 30.4 [+ or -] 4.2% in the saline group to 17.6 [+ or -] 1.05 in the 3-NPA group during delayed phase (P [is less than] 0.05, % of risk area). The anti-infarct effect of 3-NPA was blocked by 5-HD as shown by an increase in infarct size to 33 [+ or -] 2.7% (early phase) and 31 [+ or -] 2.4% (delayed phase) (P [is less than] 0.05 vs. 3-NPA groups). 5-HD had no proischemic effect in control animals. Also, 3-NPA had no effect on systemic hemodynamics. We conclude that 3-NPA induces long-lasting anti-ischemic effects via opening of mitochondrial [K.sub.ATP] channels. ischemia; succinate dehydrogenase; oxidative phosphorylation

Ockaili, Ramzi, Venkata R. Emani, Shinji Okubo, Michael Brown, Kavitha Krottapalli, and Rakesh C. Kukreja. Opening of mitochondrial [K.sub.ATP] channel induces early and delayed cardioprotective effect: role of nitric oxide. Am. J. Physiol. 277 (Heart Circ. Physiol. 46): H2425-H2434, 1999.--Opening of mitochondrial ATP-sensitive (mito[K.sub.ATP]) channel with diazoxide induces an early phase (EP) of cardioprotection. It is unknown whether diazoxide also induces a delayed phase (DP) of cardioprotection. Because nitric oxide (NO) modulates ATP sensitivity of the [K.sub.ATP] channel, we hypothesized that NO may play a role in diazoxide-induced cardioprotection. Diazoxide (1 mg/kg) was administered either 30 min (for EP) or 24 h (DP) before 30 min of lethal ischemia. Blockers of mito[K.sub.ATP] channel [5-hydroxydecanoate (5-HD)] or NO synthase [[N.sup.G]-nitro-L-arginine methyl ester (L-NAME)] were given 10 min before ischemia-reperfusion performed by 30 min of left anterior descending coronary artery occlusion and 3 h of reperfusion. A risk area (RA) was demarcated by Evans blue dye, and infarct size (IS) was measured by tetrazolium staining. Diazoxide caused a decrease in IS (%RA) from 27.8 [+ or -] 4.2% in the vehicle group to 12.9 [+ or -] 1.2% during EP and from 30.4 [+ or -] 4.2% in vehicle-treated rabbits to 19.6 [+ or -] 2.4% during DP (P [is less than] 0.05). IS increased to 31.3 [+ or -] 1.1% and 27.9 [+ or -] 1.0% (EP) and 29.9 [+ or -] 2.3% and 35.1 [+ or -] 1.8% (DP) with 5-HD and L-NAME, respectively (P [is less than] 0.05). 5-HD and L-NAME caused no proischemic effect in controls. Diazoxide induced both early and delayed anti-ischemic effects via opening of mito[K.sub.ATP] channels, which was NO dependent. mitochondria; adenosine 5'-triphosphate-sensitive potassium channel; ischemia-reperfusion; infarction

Maintaining physiological levels of hydrogen sulfide during ischemia is necessary to limit injury to the heart. Because of the anti-inflammatory effects of hydrogen sulfide, we proposed that the hydrogen sulfide donor, sodium sulfide (Na2S), would attenuate myocardial injury through upregulation of protective microRNA-21 (miR-21) and suppression of the inflammasome, a macromolecular structure that amplifies inflammation and mediates further injury.Na2S-induced miR-21 expression was measured by quantitative polymerase chain reaction in adult primary rat cardiomyocytes and in the mouse heart. We measured inflammasome formation and activity in cardiomyocytes challenged with lipopolysaccharide and ATP or simulated ischemia/reoxygenation and in the heart after regional myocardial ischemia/reperfusion, in the presence or absence of Na2S. To assess the direct anti-inflammatory effects of hydrogen sulfide in vivo, we used a peritonitis model by way of intraperitoneal injection of zymosan A. Na2S attenuated inflammasome formation and activity, measured by counting cytoplasmic aggregates of the scaffold protein apoptosis speck-like protein containing a caspase-recruitment domain (-57%) and caspase-1 activity (-50%) in isolated cardiomyocytes and in the mouse heart (all P<0.05). Na2S also inhibited apoptosis (-38%) and necrosis (-43%) in cardiomyocytes in vitro and reduced myocardial infarct size (-63%) after ischemia/reperfusion injury in vivo (all P<0.05). These protective effects were absent in cells treated with the miR-21 eraser, antagomiR-21, and in miR-21 knockout mice. Na2S also limited the severity of inflammasome-dependent inflammation in the model of peritonitis (P<0.05) in wild-type but not in miR-21 knockout mice.Na2S induces cardioprotective effects through miR-21-dependent attenuation of ischemic and inflammatory injury in cardiomyocytes. [ABSTRACT FROM AUTHOR]

Cinaciguat (BAY 58-2667) is a novel nitric oxide (NO)-independent activator of soluble guanylate cyclase (sGC), which induces cGMP-generation and vasodilation in diseased vessels. We tested the hypothesis that cinaciguat might trigger protection against ischemia/reperfusion (I/R) in the heart and adult cardiomyocytes through cGMP/protein kinase G (PKG)-dependent generation of hydrogen sulfide (H2S). Adult New Zealand White rabbits were pretreated with 1 or 10 μg/kg cinaciguat (iv) or 10% DMSO (vehicle) 15 min before I/R or with 10 μg/kg cinaciguat (iv) at reperfusion. Additionally, adult male ICR mice were treated with either cinaciguat (10 μg/kg ip) or vehicle 30 min before I/R or at the onset of reperfusion (10 μg/kg iv). The PKG inhibitor KT5283 (KT; 1 mg/kg ip) or DL-propargylglycine (PAG; 50 mg/kg ip) the inhibitor of the H2S-producing enzyme cystathionine-γ-lyase (CSE) were given 10 and 30 min before cinaciguat. Cardiac function and infarct size were assessed by echocardiography and tetrazolium staining, respectively. Primary adult mouse cardiomyocytes were isolated and treated with cinaciguat before simulated ischemia/ reoxygenation. Cinaciguat caused 63 and 41% reduction of infarct size when given before I/R and at reperfusion in rabbits, respectively. In mice, cinaciguat pretreatment caused a more robust 80% reduction in infarct size vs. 63% reduction when given at reperfusion and preserved cardiac function following I/R, which were blocked by KT and PAG. Cinaciguat also caused an increase in myocardial PKG activity and CSE expression. In cardiomyocytes, cinaciguat (50 nM) reduced necrosis and apoptosis and increased H2S levels, which was abrogated by KT. Cinaciguat is a novel molecule to induce H2S generation and a powerful protection against I/R injury in heart. [ABSTRACT FROM AUTHOR]

Abstract: cGMP and opening of mitochondrial KATP channel play an important role in preconditioning of the heart following ischemia/reperfusion (I/R) injury. We investigated the cardioprotective effect of vardenafil (VAR) (Levitra™), a highly selective and biochemically potent inhibitor of phosphodiesterase-5 (PDE-5) that enhances erectile function in men through up-regulation of cGMP. Rabbits were treated with VAR (0.014 mg/kg, iv) or volume-matched saline, 30 min prior to 30 min of sustained regional ischemia followed by 3 h of reperfusion. 5-hydroxydecanoate (5-HD, 5 mg/kg, iv) or HMR 1098 (HMR, 3 mg/kg, iv), the respective blockers of mitochondrial or sarcolemmal KATP channels were administered 10 min before I/R. Infarct size was measured by computer morphometry of tetrazolium stained sections. Vardenafil treatment caused decrease in mean arterial blood pressure from 93.5±2.6 to 82.2±1.5 mmHg and increase in heart rate from baseline value of 151±20 to 196±4.6 bpm (mean±standard error of mean (S.E.M.), P <0.05) within 5 min. The infarct size (% of risk area) was reduced from 33.8±1.3 in control rabbits to 14.3±2.2 (58% reduction, P <0.05). 5-HD abolished VAR-induced protection as demonstrated by increase in infarct size to 34.5±2.3 (P <0.05, N =6 per group). In contrast, HMR failed to block the protective effect of VAR (infarct size, 14.3±2.2 versus 16.3±1.0 in VAR + HMR, P >0.05). Neither inhibitors of the KATP channel influenced the infarct size in the control rabbits, as shown by infarct size of 34.9±1.1 and 33.3±1.4 in animals treated with 5-HD and HMR, respectively. For the first time, we demonstrate that VAR induces protective effect against I/R injury via opening of mitochondrial KATP channel. These results further support our hypothesis that the novel class of PDE-5 inhibitors induce protective effect in the ischemic heart, in addition to their well known clinical effects in the treatment of erectile dysfunction in men. [Copyright &y& Elsevier]

Reports that the sildenafil citrate or Viagra induces powerful cardioprotective effect through opening of mitochondrial ATP-sensitive channels in rabbits. Hemodynamics; Infarction size; Effectiveness of Viagra for the treatment of erectile dysfunction in men.

Presents information on a study which investigated the role of mitochondrial adenosine triphosphate-sensitive potassium channels in the cardioprotective effect of 3-nitropropionic acid in the heart. Methodology; Results of the study; Discussion.

Presents information on a study which examined the role of nitric oxide in the opening of mitochondrial adenosine triphosphate-sensitive channel. Methodology; Results; Discussion on the results.

Abstract: Phosphodiesterase-5 (PDE-5) inhibitors including sildenafil and vardenafil induce powerful preconditioning-like cardioprotective effect against ischemia/reperfusion injury through opening of mitochondrial KATP channels in the heart. The goal of these studies was to demonstrate the protective effect of sildenafil and vardenafil on reperfusion injury and to compare it with the antianginal vasodilator nitroglycerin (NTG). In addition, we determined the role of mitochondrial KATP channels in protection. Adult male New Zealand white rabbits were anesthetized and subjected to ischemia by 30 min of coronary artery occlusion followed by 3 h of reperfusion. Seven groups were studied. 1—Controls; 2—Sildenafil (total dose: 0.71 mg/kg; iv) infused for 65 min starting 5 min before reperfusion; 3—Sildenafil+5-hydroxydecanoate (5-HD, blocker of mitochondrial KATP channel, total dose: 5 mg/kg) administered as 2 bolus injections; 4—Vardenafil (total dose: 0.014 mg/kg; iv) administered as in group 2; 5—Vardenafil+5-HD administered as in group 3; 6—5-HD administered as two bolus injections and 7—Nitroglycerin (NTG, total dose: 2 μg kg−1 min−1) administered as in group 2. Infarct size was reduced in sildenafil (19.19±1.3%) as well as vardenafil (17.0±2.0%) treated groups as compared to controls (33.8±1.7%). However, NTG failed to confer similar cardioprotection (31.5±0.8%). 5-HD blocked the cardioprotective effects of sildenafil and vardenafil as shown by an increase in infarct size (34.0±1.1% and 28.3±1.9%, respectively). Both sildenafil and vardenafil protect the ischemic myocardium against reperfusion injury through a mechanism dependent on mitochondrial KATP channel opening. [Copyright &y& Elsevier]

Abstract: Rapamycin (Sirolimus®) is used to prevent rejection of transplanted organs and coronary restenosis. We reported that rapamycin induced cardioprotection against ischemia–reperfusion (I/R) injury through opening of mitochondrial KATP channels. However, signaling mechanisms in rapamycin-induced cardioprotection are currently unknown. Considering that STAT3 is protective in the heart, we investigated the potential role of this transcription factor in rapamycin-induced protection against (I/R) injury. Adult male ICR mice were treated with rapamycin (0.25mg/kg, i.p.) or vehicle (DMSO) with/without inhibitor of JAK2 (AG-490) or STAT3 (stattic). One hour later, the hearts were subjected to I/R either in Langendorff mode or in situ ligation of left coronary artery. Additionally, primary murine cardiomyocytes were subjected to simulated ischemia-reoxygenation (SI/RO) injury in vitro. For in situ targeted knockdown of STAT3, lentiviral vector containing short hairpin RNA was injected into the left ventricle 3weeks prior to initiating I/R injury. Infarct size, cardiac function, and cardiomyocyte necrosis and apoptosis were assessed. Rapamycin reduced infarct size, improved cardiac function following I/R, and limited cardiomyocyte necrosis as well as apoptosis following SI/RO which were blocked by AG-490 and stattic. In situ knock-down of STAT3 attenuated rapamycin-induced protection against I/R injury. Rapamycin triggered unique cardioprotective signaling including phosphorylation of ERK, STAT3, eNOS and glycogen synthase kinase-3ß in concert with increased prosurvival Bcl-2 to Bax ratio. Our data suggest that JAK2–STAT3 signaling plays an essential role in rapamycin-induced cardioprotection. We propose that rapamycin is a novel and clinically relevant pharmacological strategy to target STAT3 activation for treatment of myocardial infarction. [Copyright &y& Elsevier]

Abstract: The phosphodiesterase type-5 (PDE5) inhibitor, sildenafil, is the first drug developed for treatment of erectile dysfunction in patients. Experimental data in animals show that sildenafil has a preconditioning-like cardioprotective effect against ischemia/reperfusion injury in the intact heart. Mechanistic studies suggest that sildenafil exerts cardioprotection through NO generated from eNOS/iNOS, activation of protein kinase C/ERK signaling and opening of mitochondrial ATP-sensitive potassium channels. Additional studies show that the drug attenuates cell death resulting from necrosis and apoptosis, and increases the Bcl2/Bax ratio through NO signaling in adult cardiomyocytes. Emerging new data also suggest that sildenafil may be used clinically for treatment of pulmonary arterial hypertension and endothelial dysfunction. Future demonstration of the cardioprotective effect in patients with the relatively safe and effective FDA-approved PDE5 inhibitors such as sildenafil could have an enormous impact on bringing the long-studied phenomenon of ischemic and pharmacologic preconditioning to the clinical forefront. [Copyright &y& Elsevier]